_ A. .m it. 9.. ram” u .3???» ‘ 4.4:, .. a. x a . , . V 5%.» , .. ‘ . w. z. Ix. A w k ,— wwg . «my. .5: wt... NP: .2. L ‘ a? m. , v.0. . x. .. I .w . he 3v." 3 %} 4.1::Ilfi l! EB...) z: .... um. . 9.... 35m...) _ 3.5L 5.0:: .a 1.4. diUllyva . runny): crux! 5.3:} 110.44.! a .1 av. I .l'. V 0': WI Iv it I) that}... 5‘ x!!! i... ‘ :7 tr: .. If.) v: 17.. 155 $29!! .I 1 t1 3.13. . I i... 1411:. h LIBRARY .2. C7 ’1 MiCi haul: State University * This is to certify that the dissertation entitled DEVELOPMENT OF A DIRECT-FED MICROBIAL FOR BEEF CATTLE presented by SEON-WOO KIM has been accepted towards fulfillment of the requirements for the “ETD- __ degree in ’ Animal Science -—_---——- Major Professor’s Signature 8’ ~42 7 ~ 07 Date MSU is an affirmative-action, equal-opportunity employer --— -._._._.- -A-... -—-—A-.-.-/-- PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATEDUE DAIEDUE DAIEDUE 6/07 p:/ClRC/DateDue.indd-p.1 DEVELOP“ | DEVELOPMENT OF A DIRECT-FED MICROBIAL FOR BEEF CATTLE By Seen-Woo Kim A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 2007 ABSTRACT DEVELOPMENT OF A DIRECT-F ED MICROBIAL FOR BEEF CATTLE By Seon—Woo Kim To minimize ruminal lactic acidosis in beef cattle, the development of a direct- fed microbial using lactic acid-utilizing bacteria was conducted. This study included an aerobic enrichment of rumen contents in a lactic acid medium, isolation of aero-tolerant lactic acid-utilizing bacteria, and in vitro and in vivo animal trials to evaluate isolates’ effects on lactic acid utilization in the rumen. Aerobically enriched rumen microorganisms in a lactic acid medium decreased lactic acid production in the in vitro ruminal fermentation more efliciently than anaerobic enrichment. Acre-tolerant lactic acid-utilizing microorganisms were isolated from rumen contents enriched in an aerobic lactic acid media, and identified and designated to Megasphaera elsdenii (RK02) and Enterococcus faecium (RK03). The combination of these microorganisms grew in aerobic conditions. In vitro fermentations of ground corn or readily fermentable carbohydrate using rumen fluids collected from concentrate- or hay-diet adapted cows were conducted and the addition of RK02 and RK03 into the fermentation systems decreased lactic acid concentration, increased butyric and valeric acids at the expense of acetic and propionic acids, and increased pH. However, in the study of eight steers fed a high concentrate diet, RK02 and RKO3 did not change the fermentation characteristics. The combination of RK02 and RK03 tended to increase butyric acid and tended to decrease propionic acid concentration. When eight steers were challenged with ru RK02 and RKtl3 l] concentration of ti partial pressure of steers challenged ' be required to dClt admlnlSImtinn of ln anothc DWI Was used a thmfore D114: \ and ”I. Dli421‘ PCTfOnnanCe “in carcass yield gm attire in the “m Funher isolatior [he ChOiQe 0f DI challenged with rapidly fermentable substrate (finely ground wheat), the combination of RK02 and RK03 facilitated the ruminal fermentation that resulted in greater concentration of total volatile fatty acid (V FA). This microbial treatment decreased partial pressure of carbon dioxide and increased partial pressure of oxygen in the blood of steers challenged with acidosis. DF M tended to have higher blood pH. Further study will be required to determine the appropriate dose, microbial mixture, and timing of administration of DF M to prevent acidotic conditions in the rumen. In another animal performance study, Propionibacterium acidipropionici, strain DH42 was used as a DP M. DH42 did not establish a niche in the rumen of the steer; therefore DH42 was added daily as a top dress to the diet for a 123 (I study. Between d 56 and 111, DH42 tended to decrease dry matter intake (DMI), but over the entire trial, performance was similar to the control. At the end of trial, cattle were harvested, and carcass yield grade was improved by DH42, but quality grade was similar. DH42 was active in the rumen and further research with other strains and dosages are warranted. Further isolation of lactic acid-utilizing bacteria using an aerobic enrichment may expand the choice of DFM candidates which can be used in the development of DFM. The author understanding a manuscript. Apr Yokoyama for h manuscript. A 5r assistance in dm Further app laeheung K0 for Burnett Mr. Hut Guillermo Oniz. analyses and Lini: splendid help in D" Jone K. A special thanks ACKNOWLEDGMENTS The author expresses his sincere appreciation to Dr. Steven R. Rust for his guidance, understanding and invaluable assistance in the course of this study and preparation of this manuscript. Appreciation is also extended to Dr. Daniel D. Buskirk and Melvin T. Yokoyama for helpful suggestions, providing laboratory facilities, and preparation of this manuscript. A special thanks is due to Dr. Zeynep Ustunol and Dr. Elliot T. Ryser for assistance in developing the author’s doctoral program and encouragement. Further appreciation is extended to Dr. Paul Coussens, Dr. Suil Kang and Dr. Jaeheung Ko for stimulation of new ideas and providing laboratory facilities. Mr. Bob Burnett, Mr. Hugo Roman-Rosario, Dr. Sue Hengemuehle, Dr. Emilio Ungerfeld, Dr. Guillermo Ortiz-Coldn and Mr. Ig-Seo Choi provided invaluable help in laboratory analyses and animal studies. Mr. Dave Main deserves special recognition for his splendid help in preparation of the animal study. Dr. Jong K. Ha deserves gratitude for encouragement during the course of this study. A special thanks is extended to Rev. Cho and Mrs. Cho for their guidance, prayer and love during my life in the States. The author is indebted and thankful to his brothers, sisters-in-law for support and encouragement during the study abroad. The author’s parents-in-law deserve sincere appreciation for their support and encouragement. Enough appreciation cannot be offered to the author’s wife, Myong—Hee and son, Johnson whose love, understanding and perseverance completed this dissertation. This thesis is dedicated to author’s mother and the loving memories of his father. iv LIST OF TAI? LIST OF HG KEY TO SYN lNTRODbC’I CHAPTER 1_ 1. Direct. 1.1 l 1.21 1.3] 1 . " Pmplo 2.] l 2.” 1 L) b.) 3' [ml-MC; 3.1_. -‘~. 1 4. COM“ TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... viii LIST OF FIGURES ..................................................................................................... xii KEY TO SYMBOLS AND ABBREVIATIONS ......................................................... xv INTRODUCTION ....................................................................................................... 1 CHAPTER 1. LITERATURE REVIEW 1. Direct_fed microbial (DFM) or probiotics ....................................................... 7 1.1 Definition ............................................................................................... 7 12 Criteria Of pl'0bi0tiCS and DFM .............................................................. 8 1.3 Bacterial DFM for cattle ........................................................................ 9 1.31 Bacterial DFM 0n ruminal fermentation ..................................... 10 1.3.2 Bacterial DFM on beef cattle production .................................... 14 1.3.3 Reduction of E. coli 01 57:H7 shedding in cattle ----------------------- 15 2. Propionic acid production and lactic acid utilization --------------------------------------- 18 2.1 Mechanism of propionic acid production .............................................. 19 2.2 Ruminal microbiology of propionic acid production and lactic acid utilization ..................................................................... 21 2.3 Factors modulating propionic acid production and lactic acid utilization ............................................................................ 24 3. hnplications on flew-tolerance of DFM Strains ............................................... 32 3.]. Oxygen toxicity ofruminal bacteria ..................................................... 33 3.2. Classification of maembes ................................................................... 34 4. Conclusions ...................................................................................................... 36 5. Literature Cited ................................................................................................ 38 CHAPTER 3 ' 2.11:6 Emmi] i DH43. 35 a d feedlot SW“ 2.1 Summ" 2.2 lnm’d” 2.3 MW 2.4 ResultS 2.5 Imp“Ca 2.6 Literalh CHAPTER 3 ' 3. Effects Ola“ in vitro mmine conditions " 3.1 Summar 3.2 lntrOdUC 3.3 Material 3.4 Results 3.5 Discussi 3.6 lmplicaI 3.7 Literatur CHAPTER 4 - 4. bolation. ident RK02 and E. ft 4.! Summer 4.2 lntroduc 1 .3 Material 44Remu 4.5 DiScussi 4.6 Implicat . iteratur CHAPTER 2 - 2. The growth in the rumen and effects of Propionibacterium acidipropionici, strain DH42, as a direct-fed microbial on the performance and carcass characteristics of feedlot steers .............................................................................................................. 47 2.1 Summary ........................................................................................................ 47 22 Introduction .................................................................................................... 49 2.3 Materials and Methods ................................................................................... 51 2.4 Results and Discussion .................................................................................... 57 2.5 Implications .................................................................................................... 63 26 Literature Cited .............................................................................................. 64 CHAPTER 3 - 3. Effects of aerobically enriched ruminal lactic acid-fermenting microorganisms on in vitro ruminal fermentation characteristics and bacterial changes with aerobic conditions ................................................................................................................. 74 3.1 Summary ........................................................................................................ 74 3.2 Introduction .................................................................................................... 76 3.3 Materials and Methods ................................................................................... 79 34 Results ............................................................................................................ 84 35 Discussion ...................................................................................................... 89 3.6 Implications .................................................................................................... 95 3.7 Literature Cited .............................................................................................. 96 CHAPTER 4 - 4. Isolation, identification, and effects of the combination of M. elsdenii, strain RK02 and E. faecium, strain RK03 on ruminal fermentation in vitro --------------------- 106 4.1 Summary ........................................................................................................ 106 4.2 Introduction .................................................................................................... 108 4.3 Materials and Methods ................................................................................... 111 4.4 Results ............................................................................................................ 117 4. 5 Discussion ...................................................................................................... l 3 1 4.6 Implications .................................................................................................... 136 47 Literature Cited .............................................................................................. I 37 vi C HAPTE R 5 5. EtTects of .l characterist: into acute at 5.1 Sumn 5.3 lntrod 5.3 Matcri 5.4 Result 5.5 Discus 5.6 Implic; 5.7 Literati 6. CONCLL'SH APPENDICES A' Tables d. 8' Figures ( C' Raw data CHAPTER 5 - 5. Effects of M. elsdenii, strain RK02 and E. faecium, strain RKO3 on fermentation characteristics in the rumen of steers fed a concentrate diet and of steers induced into acute acidosis .................................................................................................... 159 5.1 Smmary ........................................................................................................ 1 59 5 .2 Introduction .................................................................................................... 1 6 l 5.3 Materials and Methods ................................................................................... 164 5.4 Results ............................................................................................................ 167 5.5 Discussion ...................................................................................................... 169 5.6 Implications .................................................................................................... 177 57 Literature Cited .............................................................................................. 178 6. CONCLUSIONS ...................................................................................................... 190 APPENDICES ............................................................................................................. 1 96 A. Tables discussed in Chapters ........................................................................... 196 B. Figures discussed in Chapters ......................................................................... 206 C. Raw data .......................................................................................................... 223 vii Table ll. Compo Table 2.2. C ompo Table 2.3. The cm Table 2.4. In Vim) pH of r Table 2.5. Effects and fee Table 2.6. Effects chame1 Table 3.1. Modlll Table 3.2. Effects prodm Table 3.3. ElTect: produl Table 3.4. Effect: prOpil rUmln Table 3.5. Effcu isobul Table 3.6. Butte] em‘iel Table 2.1. Table 2.2. Table 2.3. Table 2.4. Table 2.5. Table 2.6. Table 3.1. Table 3.2. Table 3.3. Table 3.4. Table 3.5. Table 3.6. Table 4.1. Table 4.2. Table 4.3. LIST OF TABLES Composition of in vitro fermentation buffer solution and premixes ----------- 67 compOSition of the prOtein and mineral supplement .................................. 68 The concentration of P. acidipropionici strain DH42 in the rumen ------------ 69 In vitro effects of P. acidipropionici strain DH42 on VFA, lactic acid, and pH ofmmina] fermentation ........................................................................ 70 Effects of probiotic addition of P. acidipropionici strain DH42 on growth and feed conversion efficiency of growing-finishing cattle ----------------------- 71 Effects of probiotic addition of P. acidipropionici strain DH42 on carcass characteristics Of growing-finishing cattle ................................................. 72 MOdified M10 lactate medium used in enrichment .................................... 99 Effects of enriched ruminal lactic acid-utilizing bacteria on organic acid production in the in VitI'O Mina] fementation ......................................... 100 Effects of enriched ruminal lactic acid-utilizing bacteria on lactic acid production, pH and All) In the ruminal fermentation ................................. 101 Effects of enriched ruminal lactic acid-utilizing bacteria on acetic acid, propionic acid and butyric acid production (changes over time) in the ruminal fermentation in ViiIO ..................................................................... 102 Effects of enriched ruminal lactic acid-utilizing bacteria on valeric acid, isobutyric and isovaleric acid production in the in vitro fermentation ------- 103 Bacteria closely related to bacterial l6S rDNA sequences cloned from enriched rumen COHtCIltS in a lactic acid media .......................................... 104 Four in vitro studies utilizing a 2 x 2 factorial arrangement of direct-fed microbial “caants ................................................................................... 140 Changes of organic acid and populations of the co-culture of Megasphaera elsdem'i RK02 and Enterococcus faecium RK03 in a Na- lactate broth medium containing different concentration of cysteine HC1 and Na_sulfide afiel- 24 h of incubation ...................................................... 141 Effects of M elsdenii RK02 with or without E. faecium RKO3 on fermented organic matter, pH, and lactic acid production with rumen fluid from concentrate diet and ground corn as substrate (Trial 1) --------------------- 142 viii Table 4.4. Effec prop: fluid Table 4.5. Elfec isobu concl Table 4.6. Effect fenm from Table 4.7. Effect andga rumer Table 4.8. E ll‘ect isobu1 hay dl Table 4.9. Effect; fermc fTOm ( Table 4.“). Elfcc and r acid ‘ 3) Table 4.11. Effec isobu Concc Table 4.12_ Efch ft‘nm flukl Table 4.]3. Effec and r acid . Table 4.4. Effects of M. elsdenii RK02 with or without E. faecium RK03 on acetic, propionic acid, and the ratio of acetic acid to propionic acid with rumen fluid from concentrate diet and ground corn as substrate (Trial 1) ------------ 143 Table 4.5. Effects of M. elsdenii RK02 with or without E. faecium RK03 on butyric, isobutyric, valeric, and isovaleric acid production with rumen fluid from concentrate diet and ground corn as substrate (Trial 1) --------------------------- 144 Table 4.6. Effects of M. elsdenii RK02 with or without E. faecium RKO3 on fermented organic matter, pH, and lactic acid production with rumen fluid from hay diet and ground corn as substrate (Trial 2) ................................. 145 Table 4.7. Effects of M. elsdem'i RK02 with or without E. faecium RK03 on acetic and propionic acid, and the ratio of acetic acid to propionic acid with rumen fluid from hay diet and ground corn as substrate (Trial 2) ------------- 146 Table 4.8. Effects of M. elsdem'i RK02 with or without E. faecium RKO3 on butyric, isobutyric, valeric, and isovaleric acid production with rumen fluid fiom hay diet and ground corn as substrate (Trial 2) .......................................... 147 Table 4.9. Effects of M. elsdenii RK02 with or without E. faecium RKO3 on Table 4.10. fermented organic matter, pH, and lactic acid production with rumen fluid from concentrate diet and RFC as substrate (Trial 3) ................................. 148 Effects of M. elsdenii RK02 with or without E. faecium RKO3 on acetic and propionic acid production and the ratio of acetic acid to propionic acid with rumen fluid from concentrate diet and RFC as substrate (Trial 3) ............................................................................................................... l 49 Table 4.11. Effects of M. elsdenii RK02 with or without E. faecium RKO3 butyric, Table 4.12. Table 4.13. Table 4.14. isobutyric, valeric, and isovaleric acid production with rumen fluid fi‘om concentrate diet and RFC as substrate (Trial 3) ........................................ 150 Effects of M. elsdenii RK02 with or without E. faecium RK03 on fermented organic matter, pH, and lactic acid production with rumen flLlid from hay diet and RFC as substrate (Trial 4) ................................... 151 Effects of M. elsdem‘i RK02 with or without E. faecium RKO3 on acetic and propionic acid production and the ratio of acetic acid to propionic acid with rumen fluid from hay diet and RFC as substrate (Trial 4) -------- 152 Effects of M elsdenii RK02 with or without E. faecium RKO3 on butyric, isobutyric, valeric, and isovaleric acid production with rumen fluid from hay diet and RFC as substrate (Trial 4) ..................................................... 153 Table 5.1 . Effects of supplementation of M. elsdem’i RK02 with E. faecium RK03 on dry matter intake of steers fed a concentrate diet prior to acidosis induction ..................................................................................................... 1 80 ix Table 5.2. Efll con run‘ Table 5.3. Efi'c COni [0 p acnl 'hHeSA.Eflbx COHC FUNK Table 5.5. lifl‘cc conu nunc 'hMe56.Eflbm conc< propi 'hMeSJ.EflbcI COUCC HHDCI Tabh: fiwciu andpx acidos leeSfllSflbcL blood (BEN Table 5.2. Effects of supplementation of M. elsdenii RK02 with E. faecium RK03 on concentrations of total organic acid, lactic acid, total VFA, and pH in the rumen of steers fed a concentrate diet prior to acidosis induction ------------- 181 Table 5.3. Effects of supplementation of M. elsdenii RK02 with E. faecium RK03 on concentrations of acetic acid, propionic acid and butyric acid, and acetate to propionate ratio in the rumen of steers fed a concentrate diet prior to acidosis induction ....................................................................................... 1 82 Table 5.4. Effects of supplementation of M. elsdem'i RK02 with E. faecium RK03 on concentrations of butyric, isobutyric, valeric, and isovaleric acids in the rumen of steers fed a concentrate diet prior to acidosis induction ------------- 183 Table 5.5. Effects of supplementation of M. elsdenii RK02 with E. faecium RK03 on concentrations of total organic acid, lactic acid, total VFA, and pH in the rumen of steers during experimentally induced acidosis --------------------------- 184 Table 5.6. Effects of supplementation of M. elsdem'i RK02 with E. faecium RK03 on concentrations of acetic, propionic and butyric acid, and acetate to propionate ratio in the rumen of steers during induced acidosis ---------------- 185 Table 5.7. Effects of supplementation of M. elsdenii RK02 with E. faecium RK03 on concentrations of butyric, isobutyric, valeric, and isovaleric acids in the rumen of steers during experimentally induced acidosis --------------------------- 186 Table 5.8. Effects of supplementation of M. elsdenii RK02 with E. faecium RK03 on blood pH, concentrations of lactic acid and bicarbonate, and partial pressure of carbon dioxide during experimentally induced acidosis in steel-s ......................................................................................... 187 Table 5.9. Effects of supplementation of M. elsdem’i RK02 with E. faecium RK03 on Table 6.1. Table 6.2. Table 6.3. Table 6.4. Table 6.5. blood oxygen saturation ($02), partial pressure of 02, blood base excess (BEb), and blood glucose during induced acidosis in steers ---------------------- 188 Substrate utilization of M. elsdem‘i RK02 on Biolog AN plate ------------------- 197 Substrate utilization of E. faecium RK03 on Biolog GP2 plate ------------------ 198 Growth of the combination of M. elsdenii RK02 and E. faecium RK03 with difiemnt reducing agent contents ....................................................... 199 Bacteria closely related to RK02 with high scoring BLAST hits (Database: All GenBank + EMBL + DDBJ + PDB sequences; 5,750,983 sequences) .................................................................................................. 200 Bacteria closely related to RK03 with high scoring BLAST hits (Database: All GenBank + EMBL + DDBJ + PDB sequences; 5,750,983 sequences) oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo 201 Table 6.6. Eli the rut Table 6.7. HT on SIC: Table 6.8. Elli on I CillL Table 6.9. Efl‘e on f Stan duri Table 7. 1. Chap Table 7.2. C h'JT Table 7.3. C hap Table 7.4. Chap Table 7.5. Chap Table 7.6. Chap] RKO: Table 7.7. Chum TCd uC Table 6.6. Table 6.7. Table 6.8. Table 6.9. Table 7.1. Table 7.2. Table 7.3. Table 7.4. Table 7.5. Table 7.6. Table 7.7. Table 7.8. Table 7.9. Effects of supplementation of M. elsdenii RK02 with E. faecium RK03 on the minimum and maximum values of fermentation characteristics in the rumen 0f steers fed a concentrate diet ........................................................ 202 Effects of supplementation of M. elsdenii, RK02 with E. faecium RK03 on concentrations of total organic acid on hexose basis in the rumen of steers fed a concentrate diet prior to acidosis induction ----------------------------- 203 Effects of supplementation of M. elsdenii, RK02 with E. faecium, RK03 on blood concentration of ionized magnesium (MgH) and ionized calcium (CaH), sodium, and potassium during induced acidosis in steers 204 Effects of supplementation of M. elsdenii, RK02 with E. faecium, RK03 on blood hematocrit (Hct), base excess of extracellular fluid (BE-ECF), standard bicarbonate concentration (SBC), and concentration of chloride during experimentally induced acidosis in steers ....................................... 205 Chapter 2. In vitro fermentation (Data for Table 2.4) --------------------------------- 224 Chapter 2. BW, kg (Data for Table 25) ...................................................... 225 Chapter 2. Dry matter intake, kg/pen (Data for Table 2.6) ------------------------- 228 Chapter 2. Carcass characteristics (Data for Table 2.6) ------------------------------ 229 Chapter 3. In vitro fermentation (Data for Table 3.2 to 3.5) ----------------------- 232 Chapter 4. Growth of the co-culture of M. elsdenii RK02 and E. faecium RK03 (Data for Figure 4.1) ..................................................................... 234 Chapter 4. Fermentation products after 24 h of incubation with different reducing agent content (Data for Table 4.2) ............................................... 236 Chapter 4. DFM effects on in vitro fermentation (Data for Tables 4.2 to 4. 1 4) ............................................................................................................ 23 7 Chapter 5. Feed Intake, kg/d prior to acidosis trial (Data for Figure 5.1) ----- 245 Table 7.10. Chapter 5. Feed intake (kg) after feeding on d 10 (Data for Table 5.1) 247 Table 7.11. Chapter 5. D10 Rumen contents (Data for Tables 5.2 to 5.4) ------------------- 248 Table 7.12. Chapter 5. Rumen contents on acidosis induction (Data for Tables 5.5 to 5.7) ............................................................................................................ 250 Table 7.13. Chapter 5. Blood pH, gases, and metabolites on acidosis induction (Data for Tables 5.8, 5.9, 6.8, and 6.9) ................................................................ 252 xi Figure 1.1. Figure 2.1. Figure 3.1. Figure 4.1. Figure 4.2. Figure 4.3. Figure 4.4. Figure 4.5. Figure 5.1. Figure 6.1. Figure 6.2. Figure 6.3. LIST OF FIGURES Two pathways for formation of propionic acid fiom lactic acid or pyruvic acid ............................................................................................................. 20 Alignment of Taq Nuclease Assay region of 16S rDNA of DH42 and the closest strain Propionibacterium microaerophilus ----------------------------------- 73 Enrichment conditions for three microbial treatments. N6 passed only anaerobic condition, N2A2N2 passed two anaerobic, two aerobic, and two anaerobic conditions, sequentially. N2A4 treatment passed two anaerobic, and four aerobic conditions. Each pass lasted 24 h at 39°C ----- 105 Growth of the co-culture of Megasphaera elsdenii RK02 and Enterococcusfaecium RK03 in a Na—lactic acid broth with 0, 20, 40, 60, 80, or 100% of reducing agent recommended by Goering and Van Soest (1970) at 39 0C. The concentration of cysteine HCl was 0.0, 0.4, 0.8, 1.2, 1.6, or 2.0 mM and Na239H20 was 0.00, 0.26, 0.52, 0.78, 1.04, or 1.30 mM for 0, 20, 40, 60, 80, or 100% of reducing agent, respectively ---------- 154 Effects of M elsdenii RK02 + E. faecium RK03 on organic acids production and pH during 12 h of the in vitro fermentation; organic acid values are differences of concentrations between at 0 and 12 h ---------------- 155 Effects of M. elsdenii RK02 + E. faecium RK03 on acetic (A), propionic (B), and butyric acid (C) production during 12 h of the in vitro fermentation; VFA values are differences of concentrations between at 0 and 12 h ..................................................................................................... 156 Effects of M. elsdenii RK02 with E. faecium RK03 on lactic acid and VFA production, and pH during a 24 h in vitro fermentation ------------------- 157 Effects of M. elsdenii RK02 with E. faecium RK03 on VFA production during a 24 h in the in VitrO fennel-nation .................................................. 158 Dry matter intake (% BW) during the adaptation period -------------------------- 189 Partial sequence (497bp) of 16S rDNA of M. elsdenii RK02 ------------------- 207 Partial sequence (494bp) of 168 rDNA of E. faecium RK03 -------------------- 207 Alignment of RK02 with the highest scoring BLAST hit sequence, Megasphaera elsdenii strain ST 16S ribosomal RNA gene -------------------- 208 Figure 6.4. Alignment of RK03 with the highest scoring BLAST hit sequence, Enterococcusfaecium isolate F02171 16S ribosomal RNA gene ----------- 209 xii Figure 6.5. Figure 6.6. Figure 6.7. Figure 6.8. Figure 6.9. Distance tree of M. elsdem'i RK02 and bacterial DNA sequences with high scoring hit obtained from GenBank Bacterial Sequences (genbankbct0.na). Same species with low hit score was omitted from selection ..................................................................................................... 2 1 0 Distance tree of E. faecium RK03 and bacterial DNA sequences with high scoring hit obtained from GenBank Bacterial Sequences (genbankbct0.na). Same species with low bit score was omitted from SCICCtiOIl. .................................................................................................... 21 1 Effects of M. elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid from concentrate diet and ground corn as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 1; -x- Control, -O- RK02 + RK03; * Means differ at the time point marked (“p < 0.05)) .............................................................. 212 Effects of M. elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid from hay diet and ground corn as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 2; -x- Control, -0- RK02 + RK03; “ Means differ at the time point marked (P < 0.05)) .............................................................. 213 Effects of M. elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid from concentrate diet and RFC as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 3; -x- Control, .0. RK02 + RK03; * Means differ at the time point marked (p < 0.05)) .............................................................. 214 Figure 6.10. Effects of M. elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid from hay diet and RFC as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 4; -x- Control, -0- RK02 + RK03; * Means differ at the time point marked (1) < 0.05)) ............................................................................ 215 Figure 6.11. Dry matter intake in % of BW during DF M supplementation (A and B), DMI in % ofBW (C and D) and Ruminal pH (E and F) on d 10 ofDFM supplementation (-<>- 801, -o- SOS, -A- 806, -X- S09 in control and - O_ SO4,-O- 808,-A-SIO,-X- Sl2inDFM group) ................................ 216 Figure 6.12. Total organic acid concentration (A), lactic acid (B), total VFA concentration (C), acetic acid (D), propionic acid (E), and butyric acid concentration (F) on d 10 of daily DF M treatments (-<>- control and -o- DFM group) ............................................................................................... 2 1 7 Figure 6.13. Ruminal acetic acid (A and B), propionic acid (C and D) and butyric acid concentration (E and F) on d 10 of DFM supplementation ------------- 218 xiii *— Figure 6.l4. Fem! experf Mean~ Figure 615. Blue. 5 I conce} Comn Figure 6.16. OX}; 1 COHCC‘ COTlth Figure 6.17. Blow St) 1. ~ S 10. ~ Figure 7.1. 168 rl (Lane N2A4 FlgUI672.D1gc\~ l Figure 7.3. Di ges ‘ Flgure 7.4. Digest Figure 7.5. Pam; Table Figure 7.6, pm-1 for] Fighte7_7_ Pan for" ”Elite 7.8. (hr (Ni 6.1 Figure 7 9. C Figure 6.14. Fermentation characteristics in the rumen of steers during experimentally induced acidosis (-<>- control and -O— DF M group; * Means difl‘er at the time marked (P < 0.05)) .............................................. 219 Figure 6.15. Blood pH, lactic acid, bicarbonate, base excess, pCOz and p02 concentration in the steers during experimentally induced acidosis (-<>- control and _O_ DFM group) ...................................................................... 220 Figure 6.16. Oxygen saturation, glucose and cations Mg, Ca, Na, and K concentrations in the steers during experimentally induced acidosis (-<>- control and _O_ DFM group) ...................................................................... 221 Figure 6.17. Blood pH in each steer during experimentally induced acidosis (-<>- SO], -0- SOS, -A- 806, —X- 809 in control and -<>- 804, -o- SO8, -A- 310, -X- 312 in DFM group) ..................................................................... 222 Figure 7.1. 16S rDNA amplication of enriched bacteria using primers 8F and 139OR (Lanes 1 and 20, lkb ladder; 2 ~ 6, N6; 7 ~ 11, N2A2N2; 12 ~ 16, N2A4; 17, RK02; 18, RK03; 19, DH42; Data for Table 3.6) -------------------- 256 Figure 7.2. Digestion of clones with HhaI. Data for Table 3.6 ------------------------------------ 257 Figure 7.3. Digestion of clones with HaeIII. Data for Table 3.6 --------------------------------- 258 Figure 7.4. Digestion of clones with Mpsl. Data for Table 3.6 ------------------------------------ 259 Figure 7.5. Partial sequence of 16S rDNA of clones from enrichment N6 (Data for Table 3.6.) .................................................................................................. 260 Figure 7.6. Partial sequence of 16S rDNA of clones from enrichment N2A2N2(Data for Table 3.6.) ............................................................................................ 265 Figure 7.7. Partial sequence of 16S rDNA of clones from enrichment N2A4 (Data fOI' Table 3.6.) ............................................................................................ 269 Figure 7.8. Chromatogram of 16S rDNA sequences of Megasphaera elsdenii RK02 (primer: 8F, 5'-AGA GTT TGA TCC TGG CTC AG—3'; Data for Figure 6.1) ............................................................................................................. 273 Figure 7.9. Chromatogram of 16S rDNA sequences of Enterococcusfaecium RK03 (primer: 8F, 5'-AGA GTT TGA TCC TGG CTC AG-3'; Data for Figure 6.2) ..................................................................................................................... 276 xiv ATP ratio : BEb BEecf Bb' cfu CP DM D.\ll FOM HPLC MPN ND 00 PC07 § SExl 80. $01 VFA I Stand : SUll‘U- acetic : blood : extra : bod} : colon : crude I Ihresl : dry n‘ 2 dry n. iaPpar :high] 1 most 3 not (1. : Optic: : Danie I Dani; : "Ola: A/P ratio : BEb BEecf BW cfu CP CT DM DMI FOM HPLC SEM S02 802 VFA KEY TO SYMBOLS AND ABBREVIATIONS acetic acid to propionic acid ratio : blood base excess : extra cellular fluid base excess : body weight : colony forming unit : crude protein : threshold cycle : dry matter : dry matter intake : apparently fermented organic matter : high performance liquid chromatography : most probable number : not determined : optical density : partial pressure of carbon dioxide : partial pressure of oxygen : Rust and Kim : standard error of mean : oxygen saturation : sulfur dioxide : volatile fatty acids XV Conccrm- animal feed indu health and pcrf0 cultures haw bee and stressed cab eflicienq and d propionibacteriz (ADG) and imr e1a1.. 2000'. Kr the mmen of c: Ethical E. cu] Differ: The first appn INTRODUCTION Concerns regarding the use of antibiotics and other grth stimulants in the animal feed industry and interests in the effects of direct-fed microbials (DFM) on animal health and performance have increased (Krehbiel et al., 2003). For ruminants, microbial cultures have been used to potentially replace or reduce the use of antibiotics in neonatal and stressed calves, to enhance milk production in dairy cows, and to improve feed efficiency and daily gain in beef cattle. Recent studies have reported that DF M containing propionibacteria and lactobacilli generally resulted in increased average daily gain (ADG) and improved feed efficiency in feedlot cattle (Swinney-Floyd et al., 1999; Rust et al., 2000; Krehbiel et al., 2003). Megasphaera elsdenii can prevent acute acidosis in the rumen of cattle (Greening et al., 1991; Robinson et al., 1992), and Lactobacillus has reduced E. coli 0157:H7 shedding in feedlot cattle (Ohya et al., 2000; Elam et al., 2003). Different approaches have been used to develop bacterial species for use as DFM. The first approach utilized strict anaerobic bacterial species (Bug/rivibriofibrisolvens, Ruminococcus albus, R. flavefaciens, or M. elsdenit) and focused on establishment of exogenous or genetically modified strains after short-term administration (Miyagi et a1 ., 1995; Gregg et al., 1998; Klieve et al., 2003; Chiquette et al., 2007). The second approach utilized facultative or aero—tolerant anaerobic bacterial species (Propionibacterium, Lactobacillus, Enterococcus species) and focused on long-term daily supercharging rather than establishing DFM in the rumen (Rust et al., 2000, Elam et al., 2003, Beauchemin et al., 2003). Aero-tolerance allows the DF M to be added to the diet, which is more acceptable in commercial operations. Therefore, DFM candidates of ruminant animals possess two characteristics. First, they must beneficially alter the ruminal fennen niche in the run dependent on tl time or shon-te For lht’ minimize acidc bacteria. This I lactic acid-utili and in vitro C0 fermentation; ; Stems fed a C0: Ml“Mimi oft chammerlslics The h} bacteria in the ruminal fermentation, and the other is that they must be aero-tolerant or can establish a niche in the rumen with short—term administration. Development of a suitable DFM is dependent on these two strain characteristics. The chance to succeed as a DFM with one- time or short-term administration may be limited to only a few strains. For these reasons, the current study, development of a direct-fed microbial to minimize acidosis in beef cattle, started with the isolation of aero-tolerant ruminal bacteria. This thesis includes 1) development of an enrichment method for the isolation of lactic acid-utilizing bacteria; 2) isolation of lactic acid-utilizing bacteria from the rumen and in vitro confirmation of their ability to prevent lactic acid accumulation in ruminal fermentation; 3) evaluation of isolates on fermentation characteristics in the rumen of steers fed a concentrate diet and challenged with an acidosis provocative diet; and 4) evaluation of the effect of Propionibacterium on the performance and carcass characteristics of feedlot cattle. The hypotheses for the first part were that aero-tolerant lactic acid-fermenting bacteria in the rumen might be enriched in aerobic lactic acid media, and that enriched facultative or aero-tolerant anaerobic lactic acid-fermenting bacteria could prevent lactic acid accumulation during fermentation induced to produce acidotic condition. The abilities of aerobically enriched rumen microorganisms to impact lactic acid utilization duing an in vitro ruminal fermentation and microbial diversity were tested (Chapter 3). The second part was conducted with the hypotheses that isolates from enriched mixed rumen microorganisms in the aerobic lactic acid medium might possess the properties to prevent lactic acid accumulation that is observed during acidosis, and that these isolates might be used as DFM to prevent lactic acid accumulation and positively influence ferrm isolated and the Based o vitro fermentati favorably alter l of steers experic ruminal ferment induced into an Pmpimu pmpionate (Kun increase hepatic influence metal» propionic acid I: Propiom'baaeri culture [0 enhan 1998). DH43 m molds in high m ei‘fECtiveneSS ELK immuminan). a have grealer AI 20l )0). Therefor F M WQuid inc influence fermentation in the rumen. Aero-tolerant lactic acid-utilizing bacteria were isolated and their DF M effects on the in vitro ferrnentations were evaluated (Chapter 4). Based on the abilities of isolates to prevent lactic acid accumulation during the in vitro fermentations, the investigator hypothesized that feeding of isolates as a DFM will favorably alter the fermentation pattern by decreasing lactic acid production in the rumen of steers experiencing acute acidosis. The effects of the combination of isolates on ruminal fermentation characteristics of steers fed a high concentrate diet and of steers induced into acute acidosis were studied (Chapter 5) Propionibacterium species convert lactic acid and glucose to acetic acid and propionate (Kung et al., 1991). Direct-fed propionibacteria may be a natural way to increase hepatic glucose production via greater propionic acid production, and positively influence metabolism (Francisco et al., 2002). Furthermore, a decreased acetic acid to propionic acid ratio accompanies a decrease in methane production (Van Soest, 1994). Propionibacterium acidipropionici strain DH42 was previously evaluated as a starter culture to enhance bunk stability of high moisture corn (Dawson 1994; Dawson et al. 1998). DH42 was shown to improve bunk stability and reduce the number of yeasts and molds in high moisture corn (Bato, 2001). The organism also has been studied for effectiveness as a DP M to lessen lactic acid accumulation in cattle received intraruminally a slurry of ground wheat to induce acidosis (Aviles, 1999). Growing- finishing cattle receiving high moisture corn (HMC) fermented with DH42 tended to have greater ADG, and improved feed efficiencies compared to the control (Rust et al., 2000). Therefore the author hypothesized that feeding P. acidipropionici strain DH42 as a DFM would increase propionic acid production in the rumen and improve animal perfonnances. (I carcass character ln summ. 1. Develop a set real-time PCR feeding direct Steers 2' Vail.“ Whetht lactic acid ac dii'ersit} rcsi media performances. Growth in the rumen and effects of DH42 on the animal performance and carcass characteristics were examined in Chapter 2. In summary, the objectives of this dissertation were to: 1. Develop a set of primers and probe specific to DH42 for the Taq Nuclease Assay with real-time PCR, quantify DH42, evaluate its growth in the rumen, and verify effects of feeding direct-fed DH42 on the grth and carcass characteristics of growing-fmishing steers 2. Verify whether aerobically enriched lactic acid-utilizing rumen bacteria could prevent lactic acid acidosis in a ruminal fermentation system and evaluate the microbial diversity resulting from enrichments in different aerobicity conditions of lactic acid media 3. Isolate oxygen tolerant lactic acid-fermenting rumen bacteria, and evaluate the effects of these isolates on in vitro ruminal fermentation 4. Evaluate the combined effects of M. elsdem'i strain RK02 and E. faecium strain RK03 on ruminal fermentation characteristics in steers fed a high concentrate diet and prevention of lactic acid accumulation in the rumen of steers with acidosis induction Miles. 1. 19‘: acido. Bmo.R.200 Mic Beauchemin. Leedlc extent cattle. Chiquette. J._ . dosing of Con. Populu DaWSOH‘ T E ensued Ml. Dawson. T. F... Slabilit at‘itfipr Eh"hhir\-1_ and S. (51mins CaTCass Offinis FranClScO. C_ ( metab‘. Greening. R. ( Robim 12497, (Suppl GTegg K“ B ] ’nOdnL ”Vin“ jeh'e‘ A, \J 2003:l Kl LITERATURE CITED Aviles, I. 1999. The use of DH42, a propionibacterium for the prevention of lactic acidosis in cattle. MS. Thesis. Michigan State Univ. East Lansing, MI. Bato, R. 2001. Propionibacteria as inoculants to high moisture corn. Ph.D Dissertation. Michigan State Univ. East Lansing, MI. Beauchemin, K. A., W. Z. Yang, D. P. Morgavi, G R. Ghorbani, W. Kautz, and J. A. Z. Leedle. 2003. Effects of bacterial direct-fed microbials and yeast on site and extent of digestion, blood chemistry, and subclinical ruminal acidosis in feedlot cattle. J. Anim. Sci. 81 :1628-1640. Chiquette, J ., G Talbot, F. Markwell, N. Nili, and R. J. Forster. 2007. Repeated ruminal dosing of Ruminococcusflavefaciens NJ along with a probiotic mixture in forage or concentrate-fed dairy cows: Effect on ruminal fermentation, cellulolytic populations and in sacco digestibility. Can. J. Anim. Sci. 87: 237-249. Dawson, T. E. 1994. Propionic acid-producing bacteria as inoculants for preservation of ensiled, high moisture corn. Ph.D Dissertation. Michigan State Univ. East Lansing, MI. Dawson, T. E., S. R. Rust, and M. T. Yokoyama. 1998. Improved fermentation and aeobic stability of ensiled, high moisture corn with the use of Propionibacterium acidipropionici. J. Dairy Sci. 81 : 1012-1021. Elam, N. A., J. F. Gleghom, J. D. Rivera, M. L. Galyean, P. J. Defoor, M. M. Brashears and S. M. Younts-Dahl. 2003. Efi‘ects of live cultures of Lactobacillus acidophilus (strains NP45 and NP51) and Propionibacterium fieudenreichii on performance, carcass, and intestinal characteristics, and Escherichia coli strain 0157 shedding of finishing beef steers. J. Anim. Sci. 81 :2686-2698. Francisco, C. C., C. S. Chamberlain, D. N. Waldner, R. P. Wettemann, and L. J. Spicer. 2002. Propionibacteria fed to dairy cows: Effects on energy balance, plasma metabolites and hormones, and reproduction. J. Dairy Sci. 85:1738—1751. Greening, R. C., W. J. Smolenski, R. L. Bell, K. Barsuhn, M. M. Johnson, and J. A. Robinson. 1991 . Effects of inoculation of Megasphaera elsdenii strain 407A (U C- 12497) on ruminal pH and organic acids in beef cattle. J. Anim. Sci. 69 (Suppl. 1 ):5 l 8 (Abstr.). Gregg, K., B. Hamdorf, K. Henderson, J. Kopecny, and C. Wong. 1998. Genetically modified ruminal bacteria protect sheep fiom fluoroacetate poisoning. Appl. Environ. Microbiol. 64:3496—3498 Klieve, A. V., D. Hennessey, D. Ouwerkerk, R. J. Forster, R. I. Mackie, and G T. Attwood. 2003. Establishing populations of Megasphaera elsdenii YB 34 and Butyrivibrio fibrisu/ 95 :62 l Krehbiel. C. R microb Sci. 8]: Kung. L.. Jr.. :1 I’m/71'“; on Run MiYagi T.. K. 1 Ohmi}; sun-ix-a Ohm T.. T. M; Sheddir prelimi RObinSOH. J, A and J_ p lDlakc i 1):310. Rm 8. R.. s, Probim DiSCOM Steam-L C. S. COmmu Swinneyqrim‘ fleets “Club \ran 80651. P J fibrisolvens YB 44 in the rumen of cattle fed high-grain diets. J. Appl. Microbiol. 95:62 1—630. Krehbiel, C. R., S. R. Rust, G. Zhang, and S. E. Gilliland. 2003. Bacterial direct-fed microbials in ruminant diets: performance response and mode of action. J. Anim. Sci. 81(E. Suppl. 2):E120—E132. Kung, L., Jr., A. Hession, R. S. Tung, and K. Maciorowski. 1991. Effect of Propionibacterium shermanii on ruminal fermentations. Proc. 21 st Biennial Conf. on Rumen F unc., Chicago, IL, p 31. (Abstract). Miyagi T., K. Kaneichi, R. I. Aminov, Y. Kobayashi, K. Sakka, S. Hoshino, and K. Ohmiya. 1995. Enumeration of transconjugated Ruminococcus albus and its survival in the goat rumen. Appl. Environ. Microbiol. 61: 2030—2032. Ohya, T., T. Marubashi, and H. Ito. 2000. Significance of fecal volatile fatty acids in shedding of Escherichia coli 0157 from calves: experimental infection and preliminary use of a probiotic product. J. Vet. Med. Sci. 62:1151—1155. Robinson, J. A., W. J. Smolenski, R. C. Greening, M. L. Ogilvie, R. L. Bell, K. Barsuhn, and J. P. Peters. 1992. Prevention of acute acidosis and enhancement of feed intake in the bovine by Megasphaera elsdenii 407A. J. Anim. Sci. 70 (Suppl. 1):310 (Abstr.). Rust, S. R., S.-W. Kim, B. M. Ungerfeld, and M. T. Yokoyama. 2000. Efficacy of probiotics to improve growth and feed efficiency in beef cattle. Fourth ADSA Discover Conference on Food Animal Agriculture. Nashville, IN Stewart, C. S., G Fonty, and P. Gouet. 1988. The establishment of rumen microbial communities. Anim. Feed Sci. Technol. 21 :69—97. Swinney-Floyd, D., B. A. Gardiner, F. N. Owens, T. Rehberger, and T. Parrott. 1999. Effects of inoculation with either strain P-63 alone or in combination with Lactobacillus acidophilus LA53545 on performance of feedlot cattle. J. Anim. Sci. 77 (Suppl. 1):77 (Abstr.). Van Soest, P. J. 1994. Nutritional Ecology of the Ruminant, 2nd ed. Cornell University Press, Ithaca, NY, 1994. Ll. Definition Probiolll affect the host 11 definition “ELK b benefits man or (Hat'enaar and l refined to li\ing benefits beyond viable bacterial t components ot‘b 0L 1999). Rumi Chapter 1 Review of Literature 1. DIRECT-FED MICROBIALS (DFM) OR PROBIOTICS 1.1. Definition Probiotics for livestock are live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance (Fuller, 1989). This definition was broadened to a mono- or mixed-culture of live microorganisms which benefits man or animals by improving the properties of the indigenous microflora (Havenaar and Huis in’t Veld, 1992). Most recently, this definition has been fiirther refined to living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition (Guamer and Schaafsman, 1998) and similarly to viable bacterial cell preparations or foods containing viable bacterial cultures or components of bacterial cells that have beneficial effects on the health of the host (Lee et al., 1999). Ruminal probiotics are live cultures of microorganisms that are deliberately introduced into the rumen with the aim of improving animal health or nutrition (Kmet et al., 1993). The United States Food and Drug Administration (FDA) in 1989 required manufacturers to use the term direct-fed microbial (DFM) instead of probiotics. Most of the microorganisms being used as DFM are classified as being generally recognized as safe (GRAS) according to the FDA and the American Association of Feed Control Officials (Miles and Bootwalla, 1991). DFM product, defined as a source of live naturally occurring microorganisms, is regulated as food under the provisions of the Compliance Policy Guide (CPG) 689.100 (FDA, 1995). For ruminar veast and bacteria. use ofantibioties ir covvs. and to impro 1.2. Criteria of Pr Common c origin ofisolates. passage to intestir lactobacilli isolate Cell viability duri should be toleran' Sile- The minimu- (Lee ela1.. 1999) DFM [arc selection. Rumin microorganism t? maabolisrn of m Selection of mmi For ruminants, microorganisms used as DF M include viable cultures of fungi, yeast, and bacteria. These organisms have been used to potentially replace or reduce the use of antibiotics in neonatal and stressed calves, to enhance milk production in dairy cows, and to improve feed efficiency and daily gain in beef cattle (Krehbiel et al., 2003). 1.2. Criteria of Probiotics and DFM Common criteria for selection of probiotic microorganisms for human use include origin of isolates, stability during storage, tolerance to carrier molecules, stability during passage to intestinal sites, and a minimum effective dose (Lee et al., 1999). Most lactobacilli isolated from the gastro-intestinal (GI) tract are site specific within the tract. Cell viability during storage varies between species and strains. Additionally, strains should be tolerant to compounds used as a vehicle. Probiotics should be alive at the target site. The minimum dose required to establish a niche can be determined by fecal recovery (Lee et al., 1999). DFM targeted at altering ruminal metabolism may utilize the similar criteria for selection. Ruminal strains must be viable and active in the rumen. However microorganism establishing a small population would have a negligible effect on the metabolism of major substrates in feed material (Hungate 1966). One of the difficulties in selection of ruminally active DFM is the anaerobic and highly competitive environment. Anaerobic bacteria adapted to the ruminal environment are very sensitive to oxygen. If a suitable method of adding rumen anaerobes to the diet, while maintaining viability, can be found, the idea of supercharging the rumen might find application in a number of situations (N agaraja et al., 1997). Therefore, aero-tolerant anaerobic bacteria are likely candidates as l'UI ionophores. and usage. 1.3. Bacterial D Bacterial cellulolytic baen mminal malt‘unc example of a SIM lg86). Which pr. introduced into 1 Lucmhui been studied for cattle. Sole straii bacteria have be Y00“ and Stem~ Chiquette er al.. candidates as ruminally active DFM. DF M should be tolerant to dietary additives such as ionophores, and other antibiotics. The benefit of a DFM must be cost effective to ensure usage. 1.3. Bacterial DFM for Cattle Bacterial DF M resources such as lactic acid—producing, lactic acid-utilizing, or cellulolytic bacteria are being investigated to alter the ruminal fermentation, to remedy ruminal malfiinction and to improve ruminants’ performance and food safety issues. An example of a successful bacterial DF M is Synergestiesjonesii (Jones and Megaritty, 1986), which prevented toxicity from the ruminal metabolite of mimosine when introduced into the rumen of goats. Lactobacillus, Enterococcus, Streptococcus, and Bifidobacterium species have been studied for use in young calves (Newman and Jacques, 1995). For growing-finishing cattle, sole strain or combinations of lactic acid-utilizing and lactic acid-producing bacteria have been investigated in laboratory and(or) farm settings (Greening et al., 1991; Yoon and Stern, 1991; Miyagi et al., 1995; Zhao et al., 1998; Swinney-Floyd et al., 1999; Chiquette et al., 2007). Another use of DFM has been to prevent or reduce toxicological symptoms. Exogenous bacterial strains have been studied for their ability to establish a niche in the rumen (Jones and Megaritty, 1986; Gregg et al. 1998). Bacterial DFM sources studied for cattle include M. elsdenii, Propionibacterium species, Lactobacillus species, Enterococcus species, Ruminococcus albus, R. flavefaciens, Synergestiesjonesii, and Bug/rivibriofibrisolvens. 13.1. AffeC‘ 0‘ illegasl" readily ferment: utilizer (Conn01 (Russell and 8'4 microorganismS acid and 4.3 ml acid. 8.4 ml! of 1994‘). In other i fermentation an to high- concent In a stud M. elsdenii was carbohydrates. 1 1.3.1. Affect of Bacterial DFM on Ruminal Fermentation Megasplraera elsdenii. In a ruminal ecosystem adapted to a diet composed of readily fermentable carbohydrates, M. elsdenii would be the major ruminal lactic acid- utilizer (Counotte et al., 1981) and simultaneously use lactic acid, glucose, and maltose (Russell and Baldwin, 1978). M. elsdenii would compete with lactic acid-producing microorganisms for substrate. M. elsdenii fermented 8 mM of glucose to 6.8 mM of acetic acid and 4.3 mM of butyric acid and converted 40 mM of lactic acid to 13.5 mM of acetic acid, 8.4 mM of propionic acid, and 8.5 mM of butyric acid in a laboratory test (Hino, 1994). In other in vitro and in vivo studies, M. elsdenii inoculation also modified nuninal fermentation and prevent the accumulation of lactic acid during the transition from a low- to high- concentrate diet (Greening et al., 1991; Kung and Hession, 1995). In a study by Kung and Hession (1995), the minimum pH of cultures treated with M. elsdenii was 5.3, whereas the control was 4.8 during the fermentation of soluble carbohydrates. Lactic acid concentration peaked at more than 40mM in the control after 8 h and remained fairly constant thereafier. But in the M elsdenii treatment, lactic acid was less than 5 mM through incubation and lactic acid was not accumulated. Total VF A concentration in cultures treated with M. elsdenii was more than twice that of the control (131 vs. 63 mM). Acetic acid concentration was unchanged after 2 h. The concentration of propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid for the control and M. elsdenii inoculation at 6 h were 38, 47; 2, 35; l, 15; 1, 11; and 1, 2 (mM), respectively. The largest differences in individual VF A concentrations occurred in butyric acid, valeric acid, and branched-chain fatty acids. 10 Greening ('1 minced lactic acid llinimal pH for the acidosis induction m were 134. 13l.50.:1r and 870 ml! for resp Propionibaett lactic acid-fennentini: production by Pro/tin. and C03. This occurs balance. Also. produc' production and entrnp fermented 1.3 ml! of Whereas 6 ml! of lacti PIOPlOHiC acid (Johns. “WE efficient with P; reWed that in the m Pr Opitmibucterium at 'i . L Occasmns' In an ill Wt“ Very low, in. Lactation/Irv Greening et al. (1991) reported that M. elsdenii increased minimal pH and reduced lactic acid concentration (P < 0.002) in the acidosis-induced rumen of beef cattle. Minimal pH for the control and treated cattle prior to acidosis induction, 0 and 2 h after acidosis induction were 4.65, 4.73, 5.51 and 5.26 and maximal lactic acid concentrations were 124, 121, 50, and 46 mM, respectively. Accumulated total VF A were 472, 507, 910, and 870 mM for respective treatments and time points. Propionibacterium with or without lactic acid-producing bacteria. Another lactic acid-fermenting rumen bacteria is Propionibacterium species. Propionic acid production by Propionibacterium is usually accompanied by the formation of acetic acid and C02. This occurs for stoichiometric reasons and to maintain hydrogen and redox balance. Also, product ratios are controlled for thermodynamic changes, such as ATP production and entropy generation (Lewis, 1996). In a laboratory test P. shermanii fermented 1.3 mM of glucose to 0.8 mM of acetic acid and 2.3 mM of propionic acid, whereas 6 mM of lactic acid was converted to 1.7 mM of acetic acid and 3.4 mM of propionic acid (Johns, 1951). Formation of propionic acid from lactic acid or glucose is more efficient with Propionibacterium species than M. elsdenii. Mackie (1978 and 1979) reported that in the rumen of sheep during stepwise adaptation to a high-concentrate diet, Propionibacterium accounted for 40 to 50% of the lactic acid-utilizer population on 5 of 12 occasions. In an adapted ruminal ecosystem, the population of Propionibacterium is usually very low. The concept of daily or periodic supplementation of Propionibacterium may be a strategy to increase propionic acid production on high concentrate diets. Lactobacillus acidophilus supplementation has been shown to increase weight gain in young calves (Beckman et al., 1977; Gilliland et al., 1980; Cruywagen et al., 11 1996). As Nagaraja (1997) summarized, L. acidophilus appeared to reduce ruminal proteolysis in calves (Skrivanova and Machanova, 1990). Yoon and Stern (1991) found that L. acidophilus stimulated fibrolysis and reduced proteolysis in a simulated rumen fermentation. Nollet et al. (1998) reported that L. plantarum cultures increased total VFA production and decreased methane production in a rumen fermentation system. Therefore, Lactobacillus seems to have some potential as a growth promoter in cattle. Jaquette et al. (1988) and Ware et al. (1988) showed improvements in milk production with L. acidophilus supplementation. Supplementation of a combination of Propionibacterium and Lactobacillus might be a viable strategy to enhance energy efficiency in cattle fed high concentrate diets. The ability to reduce methane production by switching from acetic acid to propionic acid with the role of propionibacteria and their ability to grow on lactic acid may reduce energy lost as methane and relieve the symptoms of acidosis caused by lactic acid accumulation. Likewise, the inhibition of methane with lactic acid production by Lactobacillus may further improve energy efficiency in the rumen, and consequently animal production. In our laboratory, the effects of Propionibacterium acidipropionici supplementation on rumen fermentation in steers fed a high concentrate diet were studied (Kim et al., 2000) at different dose levels (none, 1 X 107, 1 X 108, 1 X 109, 1 X 10locolony forming unit (cfu)/animal). All dose levels and the post-test period had lower acetic acid levels than the control treatment. Acetic acid levels were greater (P < 0.01) for l x 107 cfu/animal and the post-test period than the pre-test period. Propionic acid levels were greater (P < 0.05) for all dose levels than the control treatment. Numerically, propionic acid increased as the dose increased and tended to decrease across all treatments in the 12 post-test period. ' decreased (P < 0. acidipmpirmit‘i 3 acid. Butyric acic increased. “hen near pre-test leve acid levels in the concentrations vse In vitro ex pmtiles with P. (It content was simila (P < 0.05) and the acidipropionici an treatments after 24 Ghorbani e protozoal IIUInhcrs dehydrogenase (Ll amylolytic bacteri' 3163mm” ed barley post-test period. Consequently, the acetic acid to propionic acid ratio (A/P ratio) decreased (P < 0.05) at all dosages except 1 ><108 cfu/animal. It would appear the P. acidipropionici altered ruminal metabolism toward less acetic acid and more propionic acid. Butyric acid concentration decreased (P < 0.01) as dose of P. acidipropionici increased. When P. acidipropionici was removed, butyric acid concentration returned to near pre-test levels. This suggests that P. acidipropionici did effectively reduce butyric acid levels in the rumen. Lactic acid concentration, pH, and branched-chain fatty acids concentrations were similar among dose levels. In vitro experiments (our unpublished observations) compared fermentation profiles with P. acidipropionici inoculated with or without L. plantarum. The total VFA content was similar among treatments, however propionic acid concentration was greater (P < 0.05) and the NP ratio was smaller (P < 0.05) for a combination of P. acidipropionici and L. plantarum as compared to the control or the L. plantarum treatments afier 24 h of fermentation. Ghorbani et al. (2002) documented that feeding Propionibacterium P15 increased protozoal numbers (P < 0.05), molar percentage of butyric acid and blood lactic acid dehydrogenase (LDH; P < 0.05), and decreased NH3-N concentration (P < 0.01) and amylolytic bacteria (P < 0.05) in the rumen of steers fed a concentrate diet (87% of steam-rolled barley). A combination of P15 and E. faecium EF 212 increased the acetic acid (P < 0.05), and decreased the valeric acid (P < 0.01), blood C02 concentration (P < 0.01) and blood LDH (P < 0.05). DM1 and ruminal and blood pH were similar between DFM treatments and the control group, and lactic acid was not detected in the rumen across all treatments. Recently, induced inflammatory response in feedlot steers was 13 repOned with m increased serum feeding a combi had no effect on 5AA are indieat plasma haptoglc induced during 1 feeding of DFM Reductit? from the rectum fieudenreiehii (1 Effects of DFM reported with these microbial treatments (Emmanuel et al., 2007). Feeding P15 alone increased serum amyloid A (SAA) and decreased plasma haptoglobin (P < 0.001), and feeding a combination of P15 and EF212 increased (P < 0.001) SAA concentration, but had no effect on plasma haptoglobin. The authors declared that elevated concentrations of SAA are indicative of the presence of endotoxin in the blood-stream, whereas high plasma haptoglobin indicates whether translocation of bacteria into the bloodstream was induced during feeding of DF M. The mechanisms underlying the association between feeding of DFM and hepatic release of SAA and haptoglobin are not clear at present. Reductions in prevalence and concentration of E. coli 0157:H7 in feces collected from the rectum were achieved by feeding the combination of L. acidophilus and P. fieudenreichii (Elam et al., 2003; Younts-Dahl et al., 2004; 2005; Stephens et al., 2007). Effects of DF M on E. coli 0157:H7 shedding is described in section 1.3.3. 1.3.2. Affect of Bacterial DFM on Beef Cattle Production Megasphaera elsdenii. Robinson et al. (1992) reported the effects of M. elsdenii inoculation on feed intake, ruminal pH, osmolarity, lactic acid, and VFA concentration in steers induced with acute acidosis. M. elsdenii inoculation prevented acute acidosis. Steers treated with M elsdenii consumed 24% more DM on a % BW basis than the control group. The authors suggested that inoculation with M. elsdenii accelerated bovine adaptation from forage to a grain diet. Propionibacterium with or without Lactobacillus. A study conducted at Oklahoma State University (Swinney-Floyd et al., 1999) has shown improvements in feed conversion efficiency when feedlot steers were supplemented with a combination of 14 L aeidophilus ant concentrate diet. / 17.5.32. and 4.: 1J1 freudenreichii ant were 5.17. 5.32 at respective treatmt carcass traits “he ficudenreichii str graded choice ant Six DFM LAD-'1 ) Perlbmtec dose levels of P1! finishing steers t treatments impro weight (P = 0.02 combinaimnS “i1 L. acidophilus and P. freudenreichii P-63. During the first 10 (1 receiving a high concentrate diet, ADG was 0.93, 1.11 and 1.63 kg/d, and feed conversion efficiency was 5.17, 5.32, and 4.5 for the control, P. fieudenreichii alone, and a combination of P. fi'eudenreichii and L. acidophilus, respectively. Feed efficiencies for the total 120—d trial were 5.17, 5.32 and 4.97, and liver abscesses at harvest were 8%, 8%, and 0% for the respective treatments. In another study, P. fieudenreichii P-63 was shown to improve carcass traits when fed as a DFM (Huck et al., 2000). Yield grade was similar between P. freudenreichii strain P-63 treatment and the control, and the percentage of carcasses graded choice and prime increased for treated animals. Six DFM studies of P. fi'eudenrichii (PF24) and L. acidophilus (LA45 and(or) LA51) performed in four states were summarized (McPeake et al., 2002). Various daily dose levels of PF24 and LA45 and(or) LA51 were tested on performances of growing- finishing steers. When all DFM treatments were contrasted to the control group, DFM treatments improved final BW (P < 0.01), DMI (P = 0.07), ADG (P = 0.02), carcass weight (P = 0.02) and carcass ADG (P = 0.05). The level of LA45 and LA51 in combinations with PF24 had a positive correlation with DMI (P = 0.05). Cattle receiving DFM (109 of PF24 + 10‘5 of LA45 + to6 cfu/animal of LA51) had carcasses 4.4 kg heavier than the controls. 1.3.3. DFM on food safety: reduction of E. coli 0157 shedding in cattle Reduction of E. coli 0157:H7 carriage in cattle was reported when various nonpathogenic E. coli and Proteus mirabilis species were fed as DFM’s (Zhao et al., 1998). Seventeen strainsof E. coli and one Proteus mirabilis strain showing anti-E. coli 15 0157:H7 propcm mixture of these C less expert!“e “131 controls. At necrt‘ rumen of any an” the cecum and co necropsy from th« calves. but not fit colonic contents. Ohya e! u Lacrobucillus gut Composed of equ Io calves sheddin LCBll’ mixture i the DFM leIurt inhibit E. coli 0] higher level of m may have acted a administraii0n of A group r‘; acidophilus and I _ fcattle (Elam, 1.. acrdophi/us (’Nl 0157:H7 properties were isolated from cattle feces or cattle gastrointestinal tissue. A mixture of these organisms was used as a DFM treatment. Calves that received DF M had less experimentally induced E. coli 0157:H7 present in the rumen and feces than the controls. At necropsy, 30 d after treatment, E. coli 0157:H7 was not recovered fi'om the rumen of any animals treated with DFM, however, E. coli 0157:H7 was recovered from the cecum and colon of one of the six treated animals. DF M bacteria were recovered at necropsy from the contents of the rumens, reticulum, omasa, and colons of five of six calves, but not from the calf from which E. coli 0157:H7 was isolated from cecal and colonic contents. Ohya et al. (2000) isolated lactic acid-producing Streptococcus bovis, LCB6 and Lactobacillus gallinarum, LCB12 from feces of adult cattle, and administered a mixture composed of equal proportions of each organism at the daily rate of 2 x 1011 cfu/animal to calves shedding experimentally induced E. coli 0157:H7. Feeding the LCB6 and LCB12 mixture inhibited fecal shedding of E. coli 0157:H7. Fecal VFA increased when the DF M mixture was fed, and 70% of VFA was acetic acid. Acetic acid is known to inhibit E. coli 0157:H7; however, the DFM mixture might not directly contribute to the higher level of acetic acid. Therefore, the authors suggested that other unknown bacteria may have acted antagonistically against E. coli 0157:H7 in response to the administration of DFM. A group from Texas Tech University evaluated the effect of DF M containing L. acidophilus and Propionibacteriumfieudenreichii on prevalence of E. coli 0157:H7 in beef cattle (Elam et al., 2003; Younts-Dahl et al., 2004; 2005). Cattle receiving 109 cfu of L. acidophilus (NPS 1; also known as NPC747) plus 109 cfu of P. freudenreichii (NP24) l6 were less likely Dahl 8101.. 200 prevalence in fl Cattle receiving than were cattle 0157:H7 at bar among control :- feeding combin. NPZS. or NPS 1a 0157:H7 in fee, 11% for the Con (“Oh-0157:H7 in (also knoun as l Ofteces‘ regPcct were less likely to shed detectable E. coli 0157:H7 than the controls by 58% (Younts- Dahl et al., 2004) or 77% (Younts-Dahl er al., 2005). At harvest, E. coli 0157:H7 prevalence in feces was 32 and 8% for cattle receiving the control and DFM, respectively. Cattle receiving DF M were 62% less likely to be carrying E. coli 0157:H7 on their hides than were cattle fed the control diet (P = 0.12). The proportion of cattle carrying E. coli 0157:H7 at harvest, based on either fecal or hide culture results, were 38% and 10% among control and DFM, respectively. In a more recent study (Stephens et‘ al., 2007), feeding combinations of P. fi'eudenreichii (N P24) and various L. acidophilus (NP51, NP28, or NP51and NP35) reduced (P < 0.05) the prevalence and concentration of E. coli 0157:H7 in feces of feedlot cattle. Prevalence of E. coli 0157:H7 was 26, 13, l 1, and 11% for the control, NP51, NP28, or NP51 + NP35, respectively. Concentrations of E. coli 0157:H7 in positive animals receiving the control, NP51, NP28, or NP51 + NP35 (also known as NPC750) were 3.2, 0.9, 1.1, and 1.7 log most probable number (MPN)/g of feces, respectively. 17 2. PROPIO Propltml production of Pr fermentation em substrate of glUC important single hormonal releast For grout release (Hunting gluconeogenesis insufficient for g glucogenic amin 2. PROPIONIC ACID PRODUCTION AND LACTIC ACID UTILIZATION Propionic acid is the sole glucogenic precursor among VF As, and increased production of propionic acid is beneficial to the animal through increased capture of fermentation energy (decreased methane production) in the rumen and serving as a substrate of glucose synthesis in the liver. Propionic acid is quantitatively the most important single precursor of glucose synthesis, and therefore, it has a major impact on hormonal release and tissue distribution of nutrients (N agaraja et al., 1997). For growing and lactating cattle, propionic acid accounts for 67% of glucose release (Huntington, 2000). Propionic acid may spare glucogenic amino acids fi'om gluconeogenesis in the liver. When acetic acid ratios are high, propionic acid may be insufficient for gluconeogenesis and glucogenic amino acids are required. Use of glucogenic amino acids for glucose synthesis will detract from their use in protein synthesis and increase the maintenance cost of metabolizable protein and the output of ammonia (Van Soest, 1994). Nutrient intake lags behind nutrient demand during early lactation, especially in dairy cows, therefore, the ruminal supply of propionic acid may not be sufficient (Overton et al., 1999). Lipolysis in adipose tissue was initiated when blood glucose level was low. Although it is not clear whether increased propionic acid drives the decrease in methane or vice versa (Russell, 1998), it is certain that a decreased A/P ratio accompanies the decrease in methane production (Van Soest, 1994). When the NP ratio is decreased, methane production declined, and energy retention by the cattle increased (Wolin, 1960). When A/P = 1, one mole of methane would be produced from 3 moles of glucose based on the following stoichiometric equation: 18 3C0H1300 I N30 i llhen M) = 3‘ three SChHlIOI’ + 6“:0 + —+ 6;" 2.1. Mechanism of The dicarbt“. pathway. and acryl are the two known pyruvate (Fig. 1.1 lactic acid remain pathway involves subsequently deer by the fennentati. Propionic Propionic acid ft) PFOpionic acid. T Presence of ”co dioactivity a and EISden‘ 1970 3C6H1206 + 2H20 + 4C02 + 4HCOOH + 12H —> 2Ac + Bt + 2Pr + 7C02 + 2H20 + 4HCOOH + CH4 + 12H. When A/P = 3, three moles of methane would be produced from 5 moles of glucose: 5 C6H1206 + 6H20 + 12C02 + 12HCOOH + 28H —-) 6Ac + Bt + 2Pr + 17C02 + 21-120 + 12HCO0H + 3CH4 + 28H. 2.1. Mechanism of propionic acid production The dicarboxylate pathway referred to as the random pathway or succinic acid pathway, and acrylate pathway referred to as the non-random pathway or direct reduction are the two known mechanisms for formation of propionic acid from lactic acid or pyruvate (Fig. 1.1). In the acrylate pathway (Baldwin et al., 1962), the carbon skeleton of lactic acid remains intact when it is converted to propionic acid. The succinic acid pathway involves heterotrophic fixation of CO2 to form succinic acid and this is subsequently decarboxylated to propionic acid. These two pathways were distinguished by the fermentation end products from [2-14C] lactic acid. Propionic acid formed by the acrylate pathway is [2-14C] propionic acid, whereas propionic acid formed by the dicarboxylate pathway is a mixture of [2-14C] and [3-14C] propionic acid. The pathways can be also identified by fermentation of lactic acid in the presence of 14C02. The dicarboxylate pathway produced [1-14C] propionic acid, whereas no radioactivity appeared in propionic acid produced from the acrylate pathway (Paynter and Elsden, 1970). 19 SCoA CHOH CH3 Lacty|~ Filo SCoA C=O CH CH. Acrylyl. FiSure ],1_] mid SCoA COOH C O OH A COOH COOH C=O _ C=0 CHOH CHOH -\ €30” ' \ ' €90 f' CH2 7 CH2 CH3 CoASH Lacatate 2” 3 t C02 COOH 2H COOH Lactyl-CoA Pyruva e Oxalacetate Malate H O F‘HZO h 2 CH COOH 3 CH SC A -0 2H CH2 COOH | | 8:? \ r—w 0° CH: CH I l 300/} CH3 COOH CH2 Biotin-C02 Propronyl—CoA Propionate Fumarate Acrylyl-CoA L— 1 2H CoASH COOH C00“ COOH HC-CH CHZ CH co 3 H CHZ ‘ CH2 SCoA ggoA 002011 Methylmalonyl-CoA Succinate SuccinyI-CoA Figure 1.1. Two pathways for formation of propionic acid from lactic acid or pyruvic acid. A. dicarboxylate pathway (random pathway and succinic acid pathway); B. acrylate pathway (non-random pathway and direct reduction). Several ruminal microorganisms can produce propionic acid. In the studies reported by Counotte et al. (1981; 1983), Megaspaera elsdenii, which used the acrylate pathway, metabolized 74% of the lactic acid produced in the rumen. Another organism capable of producing propionic acid is the anaerobic, alanine-fermenting bacterium, Closm'dium propionicum, which produces acetic acid, propionic acid and CO2 from alanine and lactic acid (Johns, 1951a). The organism was also shown to rapidly convert 20 acrylate IO Pmp propionic acid F (Stewart and BI". When p} ATP yield from transcarboxylatit reduction yields 1 Russell and War 2.2. Ruminal mi Succinie acid. succinic aci hemicellulose an succinic acid. 01 flavefueiens. F ih. dexrrinosolvens. Stevvan er al., 19 acrylate to propionic acid (Cardon and Barker, 1947). Prevotella species is not a primary propionic acid producer but, does produce propionic acid via the acrylate pathway (Stewart and Bryant, 1988). When pyruvate is converted to propionic acid by the dicarboxylate pathway, the ATP yield from propionic acid can be as great as from acetic acid. The transcarboxylation and CoA transferase reactions do not require ATP, and furnarate reduction yields ATP formation. In the acrylate pathway, ATP synthesis does not occur (Russell and Wallace, 1997). 2.2. Ruminal microbiology of propionic acid production and lactic acid utilization Succinic acid producers. Bacteroides ferments various carbohydrates to acetic acid, succinic acid, formate, and propionic acid. Prevotella ruminicola ferments hemicellulose and starch to succinic acid. Ruminobacter amylophilus ferments starch to succinic acid. Other minor producers of succinic acid include: Ruminococcus flavefaciens, F ibrobacter (Formerly Bacteroides) succinogenes, Succinivibrio dextrinosolvens, Succinomonas amylolytica and species of spirochaetes (Wolin, 1975; Stewart et al., 1997). Succinic acid is readily decarboxylated to propionic acid and carbon dioxide and does not accumulate in the rumen contents (Blackburn and Hungate, 1963). Succinic acid utilizers. Selenomonas ruminatium can account for 22 to 51% of the total viable bacterial counts in the rumen and appears to be responsible for most of the propionic acid produced (Caldwell and Bryant, 1966). S. ruminatium produce propionic acid directly by fermentation of carbohydrate and lactic acid. Resting cells decarboxylate succinic acid and a cell density of 1.2 X 109/mL could account for succinic acid 21 disappfiarance in vivo if reducing equivalent from S. ruminantium 1 .llegusphueru acid. formic acid. and l i’tl/one/la spc alculescerrs produced it producers. S. bows and lllarounelt and Barrios. isolated from cattle are "a“ Gilsvvylr er al.. N Lactic acid pm Bull'ril‘ihrio. Succinit‘r lionentation products. lactic acid and S. rumii (Ride at al.. l99tst. 1. portion of the lactic a particularly promine‘ containing large amt proliferate in com a n Wad/e after; rapid , i a . Ic’toferant. i is gran disappearance in vivo (Strobel and Russell, 1991). Martin and Park (1996) suggested that if reducing equivalents (H2) accumulate in the growth medium, lactic acid dehydrogenase from S. ruminantium cannot convert lactic acid to pyruvate. Megasphaera elsdenii with B. fibriosolvens or P. rumim'cola produced butyric acid, formic acid, and acetic acid instead of propionic acid Warounek and Bartos, 1987). Veillonella species isolated from sheep can also decarboxylate succinic acid. V. alcalescens produced more propionic acid than acetic acid in co-culture with lactic acid producers, S. bovis and P. fibrosolvens and succinic acid producer B. ruminicola (Marounek and Bartos, 1987). Succiniclasticum ruminis and Schwartzia succinivorans isolated from cattle are also succinic acid utilizers (Gylswyk and Van Gylswyk, 1995; van Gylswyk et al., 1997). Lactic acid producers. Most amylolytic bacteria produce lactic acid. Bacteroides, Butyrivibrio, Succinivibrio, and Ruminicoccus species produce lactic acid as minor fermentation products. Selenomonas ruminantium ssp. ruminantium produces and utilizes lactic acid and S. ruminantium ssp. Iactilytica does not produce but utilizes lactic acid (Ricke et al., 1996). Lactobacillus and Streptococcus are species that account for a large portion of the lactic acid produced in the rumen, especially at lower pH. Lactobacillus is particularly prominent in the microflora of young ruminants. In animals fed rations containing large amounts of readily fermentable carbohydrate, lactobacilli often proliferate in company with streptococci, thus creating highly acidic conditions. Streptococcus bovis ferments starch to lactic acid, the main end product. S. bovis is capable of very rapid growth (Stewart et al. 1997). Although S. bovis is considered to be acid tolerant, its grth rate is reduced if the pH is less than 6.0 (Wells et al., 1997). 22 Ruminal lactol Lactic 0 their lactic ElCld dry weight basis reported for Pr”) l'er‘llonclla ( SC h“ repression (Russc Gilchrist. 1979). l 'er'l/(mc/lc. but not sugars. Ma acid. C03. and H: to propionic acid. 5 fennents lactic acid Jl/t’gusphun. fe rmented mainly to Ruminal lactobacilli are more resistant to low pH than S. bovis, and are the primary producers when pH is less than 5.6 (Nagaraja and Titgemeyer, 2007). Lactic acid utilizers. Both subspecies of S. ruminantium ferment lactic acid and their lactic acid fermentation varies between strains. The decarboxylating activity on a dry weight basis of a S. ruminantium HD4 cell suspension was 87 times greater than reported for Propionibacterium and one-third as much as the reported value for Veillonella (Scheifinger and Wolin, 1973). S. ruminantium undergoes catabolite repression (Russell and Baldwin, 1978) and is relatively acid-intolerant (Mackie and Gilchrist, 1979). Veillonella parvula ferments lactic acid, pyruvate, malate, fumarate, and tartrate, but not sugars. Major products from lactic acid fermentation are acetic acid, propionic acid, CO2, and H2 (Stewart et al., 1997). Anaerovibrio lipolytica also ferments lactic acid to propionic acid, succinic acid, and acetic acid. Propionibacterium as described above ferments lactic acid to propionic acid via the succinic acid pathway (Johns, 195 la). Megasphara elsdenii grows on glucose, fructose and lactic acid. Lactic acid is fermented mainly to butyric acid, propionic acid, isobutyric acid, valeric acid, CO2 and H2. Glucose is fermented mainly to caproate and formate with some acetic acid, propionic acid, butyric acid, and valeric acid. This species was isolated from the rumen of sheep on lactic acid agar plates in the 1950’s by S.R. Elsden. It is a large gram-negative cocci that occurs in pairs and chains. The fermentation products from a culture containing 129 mM of lactic acid were 2.04, 2.43, 2.04, 2.58, 0.15, 7.45, 0.47 mM of acetic, propionic, butyric, valeric, caproic acid, carbon dioxide, and hydrogen, respectively (Elsden et al., 1956). Based on its shape and fermentation characteristics, it was 23 originally classl Rogosa(197l )1 positive. but .11. production is dit -lI. elsdenii strait -ll. elsdenii femii utilization. The l' for approximatel ) grain. .l‘l. elsdenii amino acids Such Provided. Some st chain VFA. but th Fermentatir which convert COm Protein B‘Vllamine host animal (0%,” m 2. an) factors such - C 1976 USSel] and l~ originally classified as Peptostreptococcus elsdenii (Gutierrez et al., 1959). However, Rogosa (1971) noted that it had uncharacteristic features. Peptostreptococci are gram- positive, but M. elsdenii has an outer membrane. Because the pathway of propionic acid production is different from that of Veillonella, a new genus, Megasphaera, was created. M. elsdenii strains are gram-negative, but its closest relatives are gram-positive species. M. elsdenii ferments a variety of simple sugars but its primary niche is lactic acid utilization. The 13C-labeling studies of Counotte et al. (1981) indicated that it accounted for approximately 80% of the lactic acid turnover in cattle fed large amounts of cereal grain. M. elsdenii produces ammonia from protein hydrolysate, but it only uses a few amino acids such as serine and threonine, and grows slowly if carbohydrates are not provided. Some strains can deaminate branched chain amino acids and produce branched chain VFA, but this catabolism provides insufficient energy for growth (Russell, 2002). 2.3. Factors modulating propionic acid production and lactic acid utilization Fermentation in the rumen is the result of physical and microbiological activities which convert components of the diet to products which are useful (V FA, microbial protein, B-vitamins), useless (methane, CO2) or even harmful (ammonia, nitrate) to the host animal (Owens and Goetsch, 1988). Fermentation end products are dependent on many factors such as diet, ruminal pH, and feed additives (Blaxter, 1962; Raun et al., 1976; Russell and Hino, 1985; Sutton et al., 2003; Seymour et al., 2005). 2.3.1. Feed composition The A/P ratio is generally lower for cereal grains than forage (Blaxter, 1962). As 24 the proportion of for lesser amounts of {if high concentrate die post feeding were 3 grain and molasses concentration of hit diet. In another stut acid and butyric a. 60% concentrate d Bauman 'df Propionic acid pro dlSCStible energy Pfllduction per un Latham 3; fed [lime diets; he com. The numbe barley, The mm“ high and Showed the ”umber of 131. Sn- eP’Oc 00Gb: 5‘ T rolled barley Fati ' i .i legQSPhaera (”I . IS ac . tena rm flak Ci the proportion of forage in the diet increases, the NP ratio tends to increase because of lesser amounts of propionic acid (Bauman et al., 1971). During stepwise adaptation to a high concentrate diet in the rumen of sheep (Mackie and Gilchrist, 1979), A/P ratios 2 h post feeding were 3.17, 3.22, 2.84, 2.63, and 2.22 on 10, 24, 44, 60, and 71% of corn grain and molasses diets, respectively. The pH declined from 6.7 to 5.8 and the concentration of lactic acid increased from 0.7 to 4.26 mM as forage decreased in the diet. In another study (Sutton et al., 2003), the production levels of acetic acid, propionic acid, and butyric acid were 57, 17, and 7 mon, respectively, in the rumen of cattle fed a 60% concentrate diet, and 49, 36, and 5 mon, respectively, in a 90% concentrate diet. Bauman and coworkers (1971) suggested that the major factor affecting ruminal propionic acid production was energy intake in relation to animal requirements. When digestible energy intake is restricted to near maintenance requirements, propionic acid production per unit of digestible energy intake was higher than at free choice intake. Latham and coworkers (1971) measured the microbial flora in non-lactating cows fed three diets; hay alone, 20% hay and 80% rolled barley; and 20% hay and 80% flaked corn. The number of Lactobacillus was greater with flaked corn than either hay or rolled barley. The numbers of total organisms growing on the lactic acid medium were always high and showed little change with diet. The cereals caused a considerable reduction in the number of Butyrivibrios but an increase in the numbers of Selenomonas and Streptococcus. The increases in Selenomonas and Streptococcus were significant for the rolled barley ration. Total lactic acid-utilizing bacteria were similar. The proportion of Megasphaera elsdenii and Selenomonas were 8%, 2%; 0%, 12%; and 16%, 5% of total bacteria for flaked corn, rolled barley, and hay, respectively. 25 Mackie an occurred durintI 5‘ concentrate diets ( amylohtic and lat corn grain. The ar the diets. Lactic at until the final higl they formed 20° 0 lactic acid produc propionic acid. ar Bucreror'des predt bl" Lactobucillus Among the lactic ”Ughage contain Anaerovibrio ant Mackie and coworkers (1978; 1979) characterized the bacterial changes that occurred during stepwise adaptation of sheep from low (10% of molasses)- to high concentrate diets (10% of molasses and 60.8% of corn grain). The mean number of amylolytic and lactic acid-utilizing bacteria increased with each incremental addition of corn grain. The amylolytic bacteria were greater than 6% of the total viable counts in all the diets. Lactic acid-utilizing bacteria comprised less than 2% of the total viable count until the final high concentrate diet was fed. It was only after 21 d on the final diet that they formed 20% of the total viable count. Most of the amylolytic bacteria (92%) were lactic acid producers. Isolates of lactic acid-utilizers fermented lactic acid to acetic acid, propionic acid, and small amounts of succinic acid. In the case of amylolytic bacteria, Bacteroides predominated throughout the whole adaptation period, but was superseded by Lactobacillus and Eubacterium on the final diet containing 71 % grain and molasses. Among the lactic acid utilizers, VeiIlonelIa and Selenomonas predominated in the high roughage containing diets, but disappeared after the 24% concentrate was reached. Anaerovibrio and Propionibacterium were observed throughout the experiment and usually formed a large proportion of the isolates. Propionibacterium accounted for 40 to 50% of the lactic acid utilizers on 5 of 12 occasions. Megasphaera appeared intermittently throughout the experiment and usually formed less than 22% of the lactic acid utilizer population. Goad and coworkers (1998) reported that the A/P ratio decreased over time in both grain-adapted (3.8 to 0.9) and hay-adapted (5 .5 to 1.4) steers when acidosis was induced. Total viable anaerobic and amylolytic bacterial counts showed the interactions of diet and time, but increased over time in both groups. Initially there were no 26 dill‘erences berm higher counts oft was strongly inlli adapted steers th; Lactic acid-utili/ tended to have hi 2.3.2. Ruminal l Russell ( ruminal bactcri a crack“ com) an were Used fOr i‘c decrease. Differ. lower Arp ratio differences between hay-adapted and grain adapted, but by 60 h, grain-adapted steers had higher counts of total anaerobic and amylolytic bacteria. The Lactobacillus population was strongly influenced by diet, with lO-fold higher numbers initially present in grain- adapted steers than in hay-adapted steers. The counts increased over time in both groups. Lactic acid-utilizing bacteria increased over time in both groups, but grain-adapted steers tended to have higher counts than hay-adapted steers. 2.3.2. Ruminal pH Russell (1998) studied the effects of different initial and final culture pH of ruminal bacteria on fermentation characteristics. Two fermentation substrates (hay and cracked corn) and two ruminal bacteria sources (concentrate-fed cow and hay-fed cow) were used for fermentation, and sodium tricarballylate was added to prevent drastic pH decrease. Differences in initial and final pH were about 0.25 units. He reported that the lower A/P ratio was observed with the lower culture pH until the pH was 5.3. A/P ratios of cracked corn incubations were 1.2 and 0.6 when the pH values were 6.3 and 5.3, respectively. When pH was less than 5.3, the NP ratio of cracked corn incubations increased dramatically (P < 0.05), and the author suggested that propionic acid-producing ruminal bacteria could be even more sensitive to pH than acetic acid-producing bacteria (Russell, 1998). Methane as a percentage of total VFA production also was lower as pH was lower. Over the pH range of 6.3 to 5.3, methane production was highly correlated with A/P ratio. With a pH decline from 6.5 to 5.8, Russell (1998) suggested 25% of the decrease in A/P ratio could be explained by pH effects. 27 SeynlOUT treatment means. propionic acid (r acid”: -0.2l l a 2.3.3. lonophore The most with ionophore 5 decreased propor The magnitude 0 related to energ} high energy fee-c cattle consuming rumen Ofionopl Caused by lower Seymour et al. (2005) summarized data from 20 research studies with 92 treatment means. Rumen pH was most strongly correlated with rumen concentration of propionic acid (r = -0.45) and total VF A (r = -0.40) and slightly associated with acetic acid (r = -O.21) and butyric acid (r = -0.185). 2.3.3. Ionophores The most consistent and well documented fermentation alternations associated with ionophore supplementation are increased molar proportion of propionic acid and decreased proportion of acetic acid and butyric acid (Raun et al., 1976; Nagarja, 1995). The magnitude of increase in the molar percent of propionic acid generally is inversely related to energy density of the diet. Relative enhancement is lower in cattle consuming a high energy feed that already yields large amounts of propionic acid in the rumen than in cattle consuming a lower energy feed. Increased propionic acid accumulation in the rumen of ionophore-fed animals may be a consequence of redirected hydrogen utilization caused by lower methane production. However, monensin can shift the A/P ratio even in cultures not producing methane, suggesting that part of the increase in propionic acid is independent of its effects on methane production (N agarja, 1995). Lasalocid or monensin has been shown to inhibit most of the lactic acid- producing bacteria in the rumen (Butyrivibriofibrosolvens, Eubacterium cellulosolvens, E. ruminantium, Lachnospira multiparus, Lactobacillus ruminis, L. virtulinus, Ruminococcus albus, R. flavefaciens, Streptococcus bovis; Dennis et al., 1981). Among the lactic acid producers, those that produce succinic acid as a major end product (Bacteroides, Selenomonas, Succinimonas, Succinivibrio) are not inhibited by lasalocid 28 or monensin. Also, none of the major lactic acid fermenters (A naerovibrio, Megasphaera, Selenomonas) was inhibited (Dennis et al., 1981). Propionibacterium acne was inhibited by lasalocid or monensin, whereas Veillonella alcalescens and Veillonella parvula were not inhibited (Watanabe et al., 1981). The reported increase of propionic acid in lasalocid or monensin fed cattle may result from selection for succinic acid producers and lactic acid fermenters (Dennis et al., 1981). 2.3.4. Acidosis Ruminal acidosis continues to be a common ruminal digestive disorder in beef cattle and can lead to marked reductions in cattle production (N agaraja and Titgemeyer, 2007). Acidosis is generally related to the amount, frequency, and duration of grain feeding. Two management practices to prevent acidosis are diluting the diet with roughage or modulating intake of starch (Owens et al., 1998). However, the cost per unit of net energy (N E) of feed ingredients favors feeding higher concentrate diets, and handling characteristics of dry forages also favors minimizing forage inclusion (Brown et al., 2006). Therefore, reduction of the grain proportion in feedlot cattle diets may not be acceptable, whereas demand for acidosis prevention may increase. A ruminal pH range of 5.2 to 5 .6 is regarded as subacute or chronic acidosis; and a pH below 5.2 is considered acute acidosis (Owens et al., 1998; Brown et al., 2000; Krause and Oetzel, 2006; Nagaraja and Titgemeyer, 2007). A drop in pH below 5.6 in subacute acidosis apparently results from total accumulation of VF A alone, and not from lactic acid accumulation (Krause and Oetzel, 2006). This results from a combination of overproduction and decreased absorption of VFA (N agaraja and Titgemeyer, 2007). 29 Lactic acid does ni fermenting bacteri. however. a transiei (Kennelly et al.. 1‘. below 5.0 even “it responsible for acit Bacteria in utilizers. Balance t End products of ba culture conditions sensitive to lo“ p} Conditions. pyruyg Norman.“ lactic a Lactic acid does not accumulate in the rumen during subacute acidosis, because lactate- fermenting bacteria remain active (Goad et al., 1998) and rapidly metabolize it to VF A; however, a transient increase in ruminal lactic acid up to 20 mM has been reported (Kennelly et al., 1999). In some studies (Britton and Stock, 1987), ruminal pH falls below 5.0 even without lactic acid being present. Total acid load, not lactic acid alone, is responsible for acidosis, particularly with chronic acidosis. Bacteria in the rumen often are classified as lactic acid-producers or lactic acid- utilizers. Balance between these two groups determines whether lactic acid accumulates. End products of bacterial strains may change depending on substrate availability and culture conditions (Russell and Hino, 1985). Most lactic acid-utilizing microbes are sensitive to low pH, whereas most lactic acid producers are not. Under anaerobic conditions, pyruvate is converted to lactic acid to regenerate the NAD used in glycolysis. Normally, lactic acid does not accumulate in the rumen at concentrations above 5 mM. In contrast, ruminal concentrations exceeding 40 mM are indicative of severe acidosis. Ruminal and silage microbes produce two forms of lactic acid, the D and L form. The L form, identical to that produced from glucose by exercising muscle, can be readily metabolized by liver and heart tissue. In contrast, the D isomer, typically 30 to 38% of the total lactic acid found in the rumen, is not produced by mammalian tissues, and is not absorbed from the gut. In addition to D-lactic acid and VFA being involved with acidosis, other microbial products including ethanol, methanol, histamine, tyramine, and endotoxins often are detectable during acidosis and can exert systemic effects (Koers et al., 1976; Slyter, 1976). In low to moderate energy diets, VFAs do not accumulate to sufficient concentrations in the rumen to reduce pH drastically. However, in high 30 concentrate diets accumulate in th ruminal stasis or carbohydrates rc: pH decreases. rut total VFA concei decline because t influx of fluids In Titgemeyer. 200'. is inhibited. and 1 has the potential (Nagaraja and Ti The mecl nuninal lactobae ”“3 S‘eMp perio Inhibits the 2 T0 \\ Tllgemeyer. zoo Ht concentrate diets, the rate of acid production can exceed the rate of acid absorption, and accumulate in the rumen. This can occur due to rapid production, inhibited absorption, ruminal stasis or a combination of all three. An excessive intake of readily fermentable carbohydrates results in a sudden and uncompensated drop in ruminal pH, and as ruminal pH decreases, ruminal lactic acid concentrations increase (Krause and Oetzel, 2006). The total VF A concentration initially increases; however as pH declines, VF A concentrations decline because of destruction of the normal bacterial flora and ruminal dilution from an influx of fluids to compensate for increased osmolality (Huber, 1976; Nagaraja and Titgemeyer, 2007). As the pH nears 5.0 or below, the growth of lactate-utilizing bacteria is inhibited, and lactate begins to accumulate in the rumen. Therefore, subacute acidosis has the potential to become lactic acidosis if the pH of 5.0 is sustained for a time (Nagaraja and Titgemeyer, 2007). The mechanism of ruminal lactic acidosis involves Streptococcus bovis and ruminal lactobacilli (Owens et al., 1998). When cattle are not adapted to grain or during the step-up period, Streptococcus bovis initiates lactic acid production, and the low pH inhibits the growth of lactic acid-utilizing bacteria in the rumen (N agaraja and Titgemeyer, 2007). Consequently, acid-tolerant ruminal lactobacilli predominate in the acidotic rumen. It is a widely practiced management strategy to introduce cattle to grain over a number of weeks with the proportion of grain in the diet increasing over that period (Klieve et al., 2003). This is to allow time for the resident populations of lactic acid-utilizing and other starch-fermenting bacteria to keep up with the growth of S. bovis and prevent acidosis. Alternative preventative strategies include the use of antibiotics, immunization against S. bovis and probiotic bacteria (Klieve et al., 2003). 31 3. [MPH There are 11 a DFM. The first a establishment of er administration (M; 1993: Miyagi et a} approach utilizes f long-term daily su Floyd er al.. 1999: 2003). Any dosing acceptable as a ge dosing Beef cattl 18.17.11. 18 am 8.000 ~ 15.999. 1 3. IMPLICATIONS ON AERO-TOLERANCE OF DFM STRAINS There are at least two different approaches to develop bacterial species for use as a DFM. The first approach utilizes strict anaerobic bacterial species and focuses on establishment of exogenous or genetically modified strains after short-term administration (Mann and Stewart, 1974; Jones and Megaritty, 1986; Robinson et al., 1992; Miyagi et al., 1995; Gregg et al., 1998; Chiquette et al., 2007). The second approach utilizes facultative or aero-tolerant anaerobic bacterial species and focuses on long-term daily supercharging rather than establishing DF M in the rumen (Swinney- Floyd et al., 1999; Ohya et al., 2000; Rust et al., 2000; Elam et al., 2003; Krehbiel et al., 2003). Any dosing method other than adding rumen anaerobes to the diet is unlikely to be acceptable as a general on—farm practice (Nagaraja et al., 1997), especially as a daily dosing. Beef cattle feedlot sizes (animals per feedlot) have been increasing and in 2000, 18, 17, 11, 18 and 36 % of cattle were fed on farms of less than 1,000, 1,000 - 7,999, 8,000 - 15,999, 16,000 - 31,999 and 32,000 animals or more, respectively (USDA, 2000). Individual administration may be labor- and time-intensive and therefore, prohibitive for large feedlots. Therefore, drenching, an available method for strict anaerobes, is usually only performed during times of critical need, such as immediately after the animals arrive at the feedlot or when an animal is obviously sick (Pratt, 2001). To maintain the viability of anaerobes, delivery of strict anaerobes via feed ingestion requires a suitable method like capsular freeze drying. In addition to cultivation under anaerobic conditions, preparation of ready to use anaerobes may be cost-prohibitive; however, comparison of cost between facultative anaerobic and anaerobic preparation was unavailable in the literature. 32 Several in the ruminal r in the rumen of hvlegaritty. l98t metabolite of m the rumen ecos) C hiquette et al. and Young calve bacterial Popular Concentrate diet, h after dosing T introduced in the d). Therefore, re] chance to SUCcec Str'clins. Several researches have attempted to establish specific strains of microorganisms in the ruminal ecosystem. Mann and Stewart (1974) attempted to establish Ruminococcus in the rumen of gnotobiotic lambs but were unsuccessful. Synergestiesjonesii (Jones and Megaritty, 1986) introduced into the rumen of goats prevented toxicity from the ruminal. metabolite of mimosine. Miyagi et al. (1995) added Ruminococcus albus strain A3 into the rumen ecosystem in a goat and it survived at a very low level (102 cells/mL). Chiquette et al. (2007) added Ruminococcusflavefaciens NJ to the rumen of dairy cows and young calves. After repeated dosing, NJ modified the abundance of other cellulolytic bacterial populations and improved in sacco digestibility of timothy hay with a high concentrate diet. However, NJ declined rapidly in the rumen fi'om 106 to 102 cells/mL 24 h after dosing. The persistence of NJ improved after many weeks of dosing or when introduced in the rumen of young calves (105 cells/mL after 48 h and 102 cells/mL after 7 (1). Therefore, repeated dosing may be required to modify the ruminal ecosystem. The chance to succeed as a DFM with one-time administration may be limited to only a few strains. 3.1. Oxygen toxicity of ruminal bacteria By the early 1900’s, microbiologists knew that some bacteria were sensitive to oxygen, as these species could not be cultivated on an agar plate unless flushed with nitrogen or carbon dioxide and placed in an incubator. Most rurrrinal microorganisms are strict anaerobes and cannot sustain viability or grow if the oxygen concentration is greater than 1 part per million and the oxidation-reduction potential is greater than -300 millivolts (Russell, 2002). 33 The oxygc reactive oxygen n“ viith protons to fo peroxide. but man Oxygen reacts vv it Superoxide can or llavoproteins. thit dismutase and cat 2006). Because l'L metabolism. thev 3.2. Classificatio LOCC he ( 1 The oxygen sensitivity of ruminal microorganisms is due to the presence of reactive oxygen molecules and the lack of a detoxification enzyme system. Oxygen reacts with protons to form peroxide. Most aerobes have catalase, an enzyme that detoxifies peroxide, but many ruminal bacteria lack this enzyme or have low enzyme activity. Oxygen reacts with free electrons to form an even more reactive species, superoxide. Superoxide can oxidize a variety of cellular components (reduced flavins, quinines, flavoproteins, thiols and iron sulfur proteins). The combined action of superoxide dismutase and catalase detoxifies oxygen, superoxide and peroxide (Brioukhanov et al. , 2006). Because ruminal bacteria need SH' containing molecules (thiol) for their metabolism, they are more sensitive to oxygen than aerobes. 3.2. Classification of anaerobes Loeche (1969) classified anaerobes into three groups; 1) strict anaerobes which grow on spread plates only when the atmosphere contained less than 0.5% 02 e.g. Selenomonas ruminantium, Clostridium haemolyticum; 2) moderate anaerobes which would generally grow on plates when the atmosphere contained less than 10% O2 e.g. Peptostreptococcus elsdenii (Megasphaera elsdenii); and 3) microaerophiles which require a low concentration of 02 for optimal growth but are still unable to grow in air. Individual cells in a pure culture, however, can vary tremendously in their oxygen sensitivities. If viable counts are performed on a culture of Propionibacterium acnes using strictly anaerobic procedures, a thousand times greater recovery will be obtained than if the count is performed by surface inoculating blood agar plates and then incubating these anaerobically. It is not uncommon for organisms grown under strictly 34 anaerobic condition 1969). In batch cultt oxygen sensitive the. anaerobic conditions to become oxygen tolerant after two or three subcultures (Willis, 1969). In batch culture, cells harvested from exponential phase cultures may be more oxygen sensitive than those taken from the succeeding stationary phase (Morris, 1976). 35 Organisms t‘ acetic acid and prop natural way to incre; and positively intluc acetic acid to propit (Van Soest 1994 t. DH42. has been sit in cattle challengec finishing cattle rec improved feed em P. acidiprupjun I-L. t rumen 311d lmpl'O‘ BOlll lnuc microorganisms ; population bUI a 4. CONCLUSIONS Organisms fiom the Propionibacterium species convert lactic acid and glucose to acetic acid and propionate (Kung et al., 1991). Direct-fed Propionibacteria may be a natural way to increase hepatic glucose production via greater propionic acid production, and positively influence metabolism (Francisco et al., 2002). Furthermore, a decreased acetic acid to propionic acid ratio accompanies the decrease in methane production (Van Soest, 1994). A facultative anaerobe, Propionibacterium acidipropionici strain DH42, has been studied for effectiveness as a DFM to lessen the lactic acid accumulation in cattle challenged with an adidotic slurry of ground wheat (Aviles, 1999). Growing- fmishing cattle receiving HMC fermented with DH42 tended to have greater ADG, and improved feed efficiencies compared to the control (Rust et al., 2000). Therefore, feeding P. acidipropionici, strain DH42 as DF M may increase propionic acid production in the rumen and improve animal performances (Chapter 2). Both introduction of exogenous or genetically modified indigenous microorganisms and supercharging the rumen with a microorganism which has a low population but a high capability to beneficially modify the ruminal fermentation have been shown to be successful in cattle. Inoculation of Synergestiesjonesii prevented toxicity from the ruminal metabolite of mimosine when introduced into the rumen of goats (Jones and Megaritty, 1986). Feeding of propionibacteria and lactobacilli improved body weight (BW), dry matter intake (DMI), average daily gain (ADG), carcass weight, and carcass ADG in beef cattle (McPeake et al., 2002). Inoculation of M. elsdenii prevented the accumulation of lactic acid and increased DMI during ration transition from a forage diet to a concentrate diet (Greening et al., 1991; Robinson et al., 1992). 36 Feeding of lactoba shedding in feces t Stephens er al.. 20 adaptation to high Lactic acic species. lei/lone]! propionic acid. Br may benefit the m 2006,). The balane the lactic acid ace bacteria and acid- rumen resulting n Aeroqoler commercial SCttin delivery to Ih e ani tolerant icroOrg here fibre. urilizin men b Feeding of lactobacilli and propionibacteria decreased prevalence of E. coli 0157:H7 shedding in feces of beef cattle (Elam et al., 2003; Younts-Dahl et al., 2004; 2005; Stephens et al., 2007). DFM may be an alternative feeding strategy for ruminal adaptation to high concentrate diets or modification of ruminal fermentation outcomes. Lactic acid—utilizing bacteria, Selenomonas ruminantium, Propionibacterium species, Veillonella parvula, Anaerovibrio lipolytica, and Megasphara elsdenii produce propionic acid. Both characteristics, lactic acid utilization and propionic acid production, may benefit the ruminal ecosystem and animal by improving metabolism (Stein et al., 2006). The balance between lactic acid-producing and -utilizing bacteria may determine the lactic acid accumulation in the rumen. Declined pH depletes the lactic acid-utilizing bacteria, and acid-tolerant lactic acid-producing bacteria accumulates lactic acid in the rumen resulting ruminal lactic acidosis. Aero-tolerance of DFM is an advantage over strict anaerobes in application on commercial settings. Aero-tolerance allows the addition of DFM into the feed prior to delivery to the animal. One of the difficulties in selection of a ruminally active DFM is the anaerobic and highly competitive ruminal environment. Anaerobic bacteria adapted to the ruminal environment are sensitive to oxygen. Therefore, ruminally adapted aero- tolerant microorganisms may be preferred as DFM sources. Therefore, supercharging the rumen with aero- and acid-tolerant lactic acid- utilizing rumen bacteria may be a strategy to minimize the lactic acidosis in cattle, and using an aerobic lactic acid medium and targeting rumen microorganisms may be the most practical method of enrichment and isolation of lactic acid-utilizing bacteria for used as a DP M in cattle diets. 37 Baldwin. R. L.. propion Bauman. D. E.. rumen 0 54: 1282 Bechman. T. J .. Lac‘mhu. 60( Supp Blackburn, T_ H Productii Blaxter. K. L. 19 Britton, R A. an Agric, E) Brioukhan0\’_ j\_ Superoxi, stress in t l52:167] Caldwell. D. R. a enumem Cardon. B_ p_ am propiorfic ChiQUene‘ J“ G _ dOSlng of OT Colleen] pODUlatiOr LITERATURE CITED Baldwin, R. L., Wood, W. A., and Emery, R. S. 1962. Conversion of lactate-14C to propionate by the rumen microflora. J. 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Influence of various levels of Lactobacillus acidophilus supplementation on fermentation by rumen microorganisms in continuous culture. p 35 in Proc. let Bienn. Conf. Rumen Funct. Chicago, IL. Younts-Dahl, S. M., G. D. Osborn, M. L. Galyean, J. D. Rivera, G. H. Loneragan, and M. M. Brashears. 2005. Reduction of Escherichia coli 0157 in finishing beef cattle by various doses of Lactobacillus acidophilus in direct-fed microbials. J. Food Prot. 68:6-10. 45 Younts-Dahl, S. M., M. L. Galyean, G. H. Loneragan, N. A. Elam, and M. M. Brashears. 2004. Dietary supplementation with Lactobacillus- and Propionibacterium-based direct-fed rrricrobials and prevalence of Escherichia coli 0157 in beef feedlot cattle and on hides at harvest. J. Food Prot. 67:889-893. Zhao, T., M. P. Doyle, B. G. Harmon, C. A. Brown, P. O. E. Mueller, and A. H. Parks. 1988. Reduction of carriage of enterohemorrhagic Escherichia coli 0157:H7 in cattle by inoculation with probiotic bacteria. J. Clin. Microbiol. 36:641-647. 46 Growth in the "l as a direct-fed "1 Rumen via using Taq NUCICB.‘ primers and probe ruminal microorgt ecosystem was ne level of propionic rumen of a steer. l longer detected in may have ban s1.- DH42 II] the Fume A fourteei growing-finishin, medium was dilu' Chapter 2 Growth in the rumen and effects of Propionibacterium acidipropionici strain DH42 as a direct-fed microbial on the performance and carcass characteristics of feedlot steers SUMMARY Rumen viability of Propionibacterium acidipropionici strain DH42 was evaluated using Taq Nuclease Assay with quantitative real-time PCR. A set of Taq Nuclease Assay primers and probe enabled the detection and quantification of DH42 in a mixture of ruminal microorganisms. Initial numbers of DH42 added to the in vitro ruminal microbial ecosystem was nearly doubled after 24 h of incubation. DH42 treatment had a higher level of propionic acid (P < 0.05) than the control group. When added directly into the rumen of a steer, DH42 populations fluctuated and decreased over time with DH42 no longer detected in the rumen after 4 d. DH42 grew in the rumen, however, its growth rate may have been slower than the turnover rate of rumen contents. To maintain a level of DH42 in the rumen, daily dosage might be required. A fourteen pen study was conducted to evaluate the effects of feeding DH42 to growing-finishing cattle fed high concentrate diets. Fresh grown DH42 in a broth medium was diluted with tap water and administrated on the top of feed so that each animal received daily dose of 109 cfu. Average daily gain (ADG) (P = 0.28), dry matter intake (DMI) (P = 0.27) and feed conversion efficiency (P = 0.45) were similar between treatments over the 123 d study. For the interim period, (I 56 to 111, DH42 treatment decreased DMI (P < 0.05) and consequently lower BW at d 84 (P = 0.07) and d 111 (P = 0.06). Lower BW in the DH42 treatment was compensated by a greater ADG (P = 0.03) 47 duringd 112 to 12 Yield grade vvas ir similar to the cont and active in the r beneficially alter r optimal dose for a and feed additives heiia'ords: Pro/iii Fermi during d 112 to 123 compared to the control. Cattle were harvested after 123 d on feed. Yield grade was improved (P = 0.04) by the DH42 treatment, but quality grade was similar to the control. Based on these studies, P. acidipropionici strain DH42 was viable and active in the rumen, and changed animal performance at the dosage used. To beneficially alter ruminal fermentation and animal performance by feeding DH42, the optimal dose for animal growth phase and interactions of DH42 with feed components and feed additives need to be studied. Keywords: Propionibacterium, Direct-F ed Microbial, F eedlot, Steer, Ruminal Fermentation, Growth, Carcass. 48 Propiuni/ starter culture to Davvson er a1. 19 of yeasts and mo effectiveness as 2 diets (Aviles. 19‘ tended (P = 0.10 gain‘lcg DMI vs. the mechanism i cells or DH42 in Direct-fe PF'Oduction Via u & (Francisco 8! a] INTRODUCTION Propionibacterium acidipropionici strain DH42 was previously evaluated as a starter culture to enhance bunk stability of high moisture corn (HMC) (Dawson 1994; Dawson et al. 1998). DH42 was shown to improve bunk stability and reduce the number of yeasts and molds in HMC (Bato, 2001). The organism also has been studied for effectiveness as a DFM to lessen the lactic acid accumulation in cattle fed high grain diets (Aviles, 1999). Growing-finishing cattle receiving HMC fermented with DH42 tended (P = 0.10) to have greater ADG, and improved (P = 0.04) feed efficiencies (151 g gain/kg DMI vs. 145 g gain/kg DMI) compared to the control (Rust et al., 2000). While the mechanism is not entirely clear, DH42 may alter ruminal fermentation directly as live cells or DH42 improve the nutritional value of the HMC. Direct-fed Propionibacteria may be a natural way to increase hepatic glucose production via greater propionic acid production and positively influence metabolism (Francisco et al., 2002). In short term studies, DH42 increased propionic acid levels in the rumen (Kim et al., 2000). An essential requirement when evaluating the efficacy of a DFM organism is an ability to both reliably enumerate the population of the organism in the rumen and to then monitor establishment and stability of this population (Ouwerkerk et al., 2002). Microbial growth and viability can be verified by quantifying the microorganisms through incubation time, however, counting and detection of propionibacteria in the environment is complicated because the media currently being used for their isolation are not sufficiently selective (Thierry and Madec, 1995). Specific bacteria] populations in the rumen have been enumerated using labeled hybridization probes complementary to rRN A 49 genes (Stahl er U1- Russell 1996) and quantification of I 1' the research group including non-radii primers for PC R (F 103 cfu’mL and 10 quantification of C quantification of h Nuclease Assay (l genes (Stahl et a1. 1988; Briesacher et al. 1992; McSweeney et al. 1993; Krause and Russell 1996) and by competitive PCR (Reilly and Attwood 1998). For detection and quantification of Propionibacterium DH42 isolated from fermented high moisture com, the research group of Dr. Rust at Michigan State University has been developing methods, including non-radioactive probes for genomic DNA (Dawson, 1994) and species specific primers for PCR (Romanov et al., 2004), and was able to detect DH42 at levels as low as '102 cfu/mL and 103 cfu/mL in silage and rumen contents, respectively. However, quantification of DH42 in a mixed bacterial ecosystem was not yet possible. For quantification of bacteria at the species or strain level in mixed environments, Taq Nuclease Assay (TNA) might be the most efficient technique, and recently M. elsdenii could be quantified in the rumen at the species level (Ouwerkerk et al., 2002). To apply the TNA to P. acidipropionici, strain DH42 in the rumen, primers and probes specific to DH42 need to be developed. To evaluate the potential of DH42 as a DFM for beef cattle, growth of DH42 in the rumen and effects of feeding DH42 on animal performance were evaluated in the current study. The objectives were to 1) develop a set of primers and probe specific to DH42 for TNA with real-time PCR, 2) quantify DH42 and evaluate its growth in the rumen, and 3) verify effects of feeding direct-fed DH42 on the grth performance and carcass characteristics of growing-finishing steers. 50 Quantification Q Prime“ U Taq Nuclease As Foster City. CA) AY360222'). Seq Biotechnology Ir Regions apparen regions were des MATERIALS AND METHODS Quantification of DH42 Using T aq Nuclease Assay Primers and Probe Design and Real-time PCR. The primers and probe for the Taq Nuclease Assay were designed using Primer Express Software (Applied Biosystems, Foster City, CA) from alignment of the 16S rRNA of DH42 (accession number AY3 60222). Sequences restricted by primers were blasted to the National Center for Biotechnology Information (NCBI)database (http://www.ncbi.nlm.nih.gov/BLAST/). Regions apparently unique to DH42 were selected, and primers complementary to these regions were designed. The sequence of forward primer, dh42f, was 5’- gacatggattggtaacggtcagag-3’ (976-999 of DH42 AY360222 and 993-1016 of E. coli numbering system), and reverse primer, dh42r, was 5’-agccatgcaccacctgtgaa—3’ (1022- 1043 DH42 and 1042-1060 E. coli). The sequence of probe, dh42p, was 5’- atggccgccccccttgtgg-3’ (1000-1018 DH42 and 1017-1035 E. coli). Primers were synthesized at the Macromolecular Structure Facility at MSU and stored at —20°C until use. TAMRA and 6-FEM were used in the probe as the 5’ fluorescent reporter dye and 3’ quencher, respectively (Holland et al., 1991; Higuchi et al., 1993). Quantitative real- time PCR was performed using the ABI Prizm 7700 Sequence Detection System (Applied Biosystems) up to 40 cycles with denaturation at 95°C for 15 sec, and annealing and extension at 60°C for 1 min. DH42 was obtained from the culture collection at the MSU Dr. Yokoyama’s laboratory. Culture of DH42 grown for 24 h in a Na-lactate broth (N LB; 1% yeast extract (Difco, Detroit, MI), 1% trypticase soy broth (Difco), 1% sodium lactate syrup (Sigma, St Louis, MO); Hoflrerr et al., 1983) was decirnally serially diluted to 1:1010 in 0.1% (w/v) 51 proteose pepton plates containin. and incubated at to NLB incubate and the culture g was serially dilu dilutions (1:10I containing 1% (i incubated at 39 ‘ culture was direc serially diluted tr centrifugation at viable plate cour levels of DH42 v DNA Exri r1lthinaI contents Solana Beach, C Using a Bechman era1.. I982). proteose peptone (Difco) solution, and then spread on Purple Broth Base (Difco) agar plates containing 1% (w/v) i-erythritol (Sigma). Plates were placed in an incubation jar and incubated at 39 0C for 3 d and stored at room temperature. A colony was transferred to NLB incubated at 39 °C for 24 h. 0.5 mL of culture was transferred to 9.5 mL of NLB and the culture grown for 24 h used to calibrate the real-time PCR. One mL of culture was serially diluted to 1:10'0 in 0.1% (w/v) proteose peptone solution, and then all dilutions (1 :101 to 1:10”) were spread to Purple Broth Base (Difco) agar plates containing 1% (w/v) i-erythritol (Sigma). Plates were placed in an incubation jar and incubated at 39 °C. After 3 d of incubation, colonies were counted. One 1 mL of DH42 culture was directly used for DNA extraction. An additional one mL of DH42 culture was serially diluted to 1:10lo in the supernatant of autoclaved rumen fluid prepared by centrifugation at 24,000 x g for 20 rrrin and then DNA was extracted. Populationst fi'om viable plate counts were compared to the threshold cycle (CT) of real-tirne PCR, and the levels of DH42 were expressed as both CT and equivalent cfu/mL. DNA Extraction. DNA was extracted fiom pure bacterial cultures and from ruminal contents using a Ultr'aCleanTM Soil DNA Isolation Kit (Mo Bio Laboratories Inc., Solana Beach, CA). DNA extracts were stored at -20°C until use. DNA was quantified using a Beckman DU-600 spectrophotometer (Beckman Coulter Inc., Fullerton; Maniatis et al., 1982). Growth of DH42 in the Rumen In Vitro Fermentation. The in vitro fermentation was performed to evaluate the growth of DH42 and its effects on ruminal fermentation. Procedures of Goering and Van 52 Soest ( 1970) were from two steers fc through 4 layers 0 rumen fluid and 5t- :50 mL round bot ' glucose (26%). mt (3%) on a wt’wt h: | Ten mL of resuspended to 10 fementation soluti 39°C for 24 h. am free CO2. Treatm collected at 0 anc incubation. Soest (1970) were used for the basic in vitro fermentation. Rumen fluid was collected from two steers fed a high concentrate diet through canula, composited and strained through 4 layers of cheese-cloth while gassing with O2-free CO2. Fifty mL of strained rumen fluid and 50 mL of in vitro fermentation solution (Table 2.1) were placed into a 250 mL round bottom flask. Three g of fermentable carbohydrates [soluble starch (55%), glucose (26%), methyl cellulose (6%), cellobiose (7%), tryptone (3%), proteose peptone (3%) on a wt/wt basis; modified from Kung and Hession (1995)] were added to the flask. Ten mL of DH42 incubated in NLB for 24 h was pelleted by centrifugation and resuspended to 10 mL of fermentation solution before inoculation. For the control, pure fementation solution was added. Flasks were prepared anaerobically and incubated at 39°C for 24 h, and 3 mL of culture media were removed while gassing flasks with 02- free CO2. Treatments were in triplicate. Real time PCR was performed using cultures collected at 0 and 24 h. Lactic acid and VF A were analyzed after 0, 6, 12, and 24 h of incubation. Ruminal pH was recorded immediately after sample collection and incubation fluid was centrifuged at 24,000 x g for 20 min and 25 mL of supernatant was acidified with 12 N H2SO4 for organic acid analysis. Lactic acid and VFA contents were determined by ion exchange exclusion HPLC (Aminex HPX-87 h; Bio-Rad, Richmond, CA) following the general procedures of Canale et al. (1984). The mobile phase consisted of 0.005N H2SO4 at a flow rate of 0.6 ml/min. Column temperature was maintained at 65°C by an external column heater (Waters Associates, Milford, MA). Fifteen uL of samples were injected by an autoinjector (WISP 712, Waters Associates), and analytes were detected by refractive index (Waters 410 refractive index detector, 53 Waters Associate package ( TWPOCI standards of lacta‘ Enumerur: canulated steer vi; Center (f BC TRC l was fed a high cor 5% protein supple Sampled at 1. 7. 1 24h was mixed v through a canula counting as desei supplementation supplementation colleeted from d Waters Associates). Peak heights were quantified by a commercial HPLC software package (Turbochrom 3; PE Nelson, Cupertino, CA) and compared with external standards of lactate and VFA. Enumeration of DH42 in the Rumen of a Steer. One permanently ruminally canulated steer was allotted to a pen at the MSU Beef Cattle Teaching and Research Center (BCTRC) and kept alone for the entire experimental period. Once a day the steer was fed a high concentrate diet containing 85% high moisture com, 10% corn silage, and 5% protein supplement. Two d before the first dose of DH42, the ruminal contents were sampled at 1, 7, 13, and 19 h after feeding. Ten mL of DH42 cultured in NLB at 39°C for 24 h was mixed with 40 mL of distilled water, and administrated directly into the rumen through a canula after feeding. DH42 in the culture was enumerated using colony counting as described above. Dose level was 2 x 1010 cfu/d. On d 6 and 7 of DH42 supplementation, rumen contents were sampled at 1, 7, 13, 19 h after feeding. DH42 supplementation was stopped on d 7. The 100 mL sample of the ruminal contents were collected from different locations in the rumen on d 4, 6, and 9 after the last dose, composited, strained through four layers of cheese-cloth, and then analyzed for DH42. The rumen was then evacuated with the rumen contents being 72 L and 59.4 kg on 1 d post-trial, and 40 L and 38.5 kg on d 9 of post-trial. Effects of DH42 as a DF M on Steer Performance Experimental Design of Feeding Trial. One hundred and twelve steers were randomly allotted to 14 pens with 8 animals in each pen at the MSU BCTRC. Fourteen 54 pens were rar and 2) DH42. Cattle 449/3 HMC. ar adjusted to a c after 10 d. The Supplement. R 250 mg."'anima Shifting COnsecutiv-e d3 Ill. and 123 d hanvest facility. plant. Were Few Prepare Containing 0.5 r miCmCentiit‘uge inoculated in a 9 culture was tran. Sevv en subcultum eac h subculture ‘ pens were randomly assigned to one of two treatments: 1) control (uninoculated buffer) and 2) DH42. Cattle were started on a diet containing 10% ground hay, 41% corn silage (CS), 44% HMC, and 5% of a protein-mineral supplement (Table 2.2) for the first 4 d and then adjusted to a diet containing 41% CS, 54% HMC, and 5% protein-mineral supplement after 10 d. The final diet included 16% CS, 79% HMC, and 5% protein-mineral supplement. Rurnensin (Elanco Animal Health, Greenfield, IN) was added at a level of 250 mg/animal for the first 16 d, and excluded thereafter. Starting and final weights were the average of two full weights measured on consecutive days before the morning feeding. Interim weights were taken on 27, 55, 84, 111, and 123 d on trial. After 123 d on feed, all steers were shipped to a commercial harvest facility, at which quality and yield grade as assigned by USDA personnel, in the plant, were recorded. Preparation and Administration of BF M. Two hundred frozen stocks of DH42 containing 0.5 mL of 109 cfu/mL in NLB with 30% (v/v) glycerol were prepared in microcentrifuge tubes and stored at -20°C. For each day, 0.5 mL of frozen stock was inoculated in a 9.5 mL of NLB medium. After 1 d of incubation at 39°C, 0.5 mL of each culture was transferred to seven 25-m] culture tubes containing 9.5 mL of NLB. These seven subcultures were incubated for 1 d at 39°C and delivered to BCTRC. Ten mL of each subculture was added to 1 L of tap water and poured on the top of the feed of each pen for DF M treatment. For the control groups, 10 mL of uninoculated NLB was added to 1 L of tap water and poured on the top of feed. DH42 in the culture was enumerated using colony counting as described above. Ten mL of DH42 culture contains 2.5 i 0.75 x 55 1010 cfu and was cfut’animal. Statistical Analyst“ The correl standard DH42 D NC). In vitro ferr procedure. and tl‘ a classification e time. and interac the model when COVariance strut Squares means i Overall Carcass quality performance 10l0 cfu and was given to 8 steers in each pen, with a theoretical daily dosage of 3.1 x 109 cfu/animal. Statistical Analysis The correlation (Table 2.3) of populations of DH42 and threshold cycle of standard DH42 DNA extracts was analyzed using a GLM procedure (SAS Inst. Inc., Cary, NC). In vitro fermentation data were analyzed with repeated measures using the MIXED procedure, and the experimental unit was the incubation flask. Incubation time served as a classification effect. Initially, the model contained microbial treatments, incubation time, and interaction of treatment with time (Table 2.4). Interaction was removed from the model when P > 0.10. Auto-regression was determined as the most appropriate covariance structure using the Schwarz Baysean criterion (Littell et al., 1998). Least squares means within time were compared using the Bonferroni t-test. Overall BW, ADG, DMI, feed conversion efficiency for 123 (1 (Table 2.5), and carcass quality (Table 2.6) were analyzed as a completely randomized design. Animal performance, measured at 28 (1 intervals (Table 2.5), was treated as a repeated measures using the AR(I) error structure and pen as the experimental unit. Period (days on feed) served as a classification effect. Model included microbial treatment, period, and interaction of treatments with period. Treatment means within period (Table 2.5) were compared with the Bonferroni t—test. Significance and tendency were declared at P < 0.05 and P < 0.10, respectively. 56 Growth of DH43 When regi rRNA ofDH42 r Propirmihacteriu differing by one n Pure cultu Threshold cycles 3.28.an intereep1 contents before 4.00. respectiV less sensitive f after 4'0 Cycles eqUi\'alent 10 “men fluicL 1 time PCR an RESULTS AND DISCUSSION Growth of DH42 in the rumen When regional 68 bp nucleotide sequences (AY360222 number 976-1043) of 16S rRNA of DH42 restricted by primers were blasted to the NCBI database, Propionibacterium microaerophillus (Koussemon et al., 2001) had the closest sequences differing by one nucleotide in the TaqMan probe region (Fig. 2.1). Pure culture samples of DH42 had detectable limit as low as 10 cfu/mL. Threshold cycles and logarithmic cfu were a linearly related (P < 0.01) with a slope of - 3.28,an intercept of 40.12 and an R2 value of 0.989. When DH42 was mixed with rumen contents before DNA extraction, the intercept and slope of the regression were 50.62 and -4.00, respectively, with an R2 value of 0.995 (P < 0.01). The Taq Nuclease Assay was less sensitive for the rumen sample and was unable to detect levels of 10 or 100 cfu/mL after 40 cycles. The lowest level of standard DH42 detected was 103 cfu/mehich was equivalent to 38.54 of CT. Therefore, threshold for quantification was set at 103 cfu/mL of rumen fluid. Ouwerkerk et al. (2002) developed M. elsdenii specific probes using real- time PCR and set the threshold for enumeration arbitrarily at 104 cells/mL of sample, because a ruminal population below 104 cells/mL would have a negligible effect on the metabolism of major substrates in feed material and this threshold would exclude any ambiguity caused by CT values obtained from unrelated bacteria due to non-specific amplification in the last few cycles of the PCR reaction. The set of primers and probe was tested for specificity using the mixed rumen microorganisms collected from a steer which had not received DH42. Eight rumen samples were collected every 6 h for 48 h and 7 of them did not show any amplification 57 through 40 cycles of real-time PCR. Only one of eight rumen samples had detectable threshold cycles (CT) of 39.59 which is equivalent to 575 cfu/mL which is outside the standard threshold set for quantification. Therefore, the set of primers and probe used in this study was specific to DH42. DH42 grew in an in vitro ruminal fermentation system inoculated to contain 10,565 cfu/mL (Table 2.3). After 24 h of incubation, DH42 populations nearly doubled to 19,916 cfu/mL. Fermentation profiles also were changed with DH42 administration increasing the propionic acid concentration (P < 0.05) in the culture between 6 and 24 h of incubation (Table 2.4). Consistent with this result, in the rumen of steers fed a concentrate diet, administration of DH42 increased the propionic acid concentration at the expense of acetic acid and consequently decreased the acetic acid/propionic acid ratio (Kim et al., 2000). These differences in microbial numbers and fermentation profile in an in vitro fermentation might indicate the viability and growth of DH42 in the rumen. When added directly into the rumen of a steer, the concentration of DH42 decreased over time (Table 2.3). Cattle received DH42 for 7 d, and rumen contents were collected on d 6 and 7. On (1 6 of administration, the concentration decreased after administration, and increased between 13 h and 19 h after administration. On (1 7, the DH42 concentration did not increase with administration at 1 h. The concentration of DH42 was greater at 7 h and decreased thereafter. Four (I post administration, DH42 was not detected in the rumen. The small increase in concentration of DH42 after 19 h on d 6 may imply growth of in the rumen. However, it is more likely that DH42 does not establish a niche in the rumen. Bacterial growth should be greater than turnover rate to maintain a certain level in the rumen; otherwise bacteria would disappear from the 58 environmc DH42 int Effect of 0 Table 2.5 treatment Conversi. EITOUp. I days On greater 1 numeric COmPEn DH42 c Value e betwee “ere fe reSports high VE environment. Therefore, daily administration is required to maintain a certain level of DH42 in the rumen. Effect of DH42 as a DFM on Animal Performance Overall animal growth and feed efficiency during 123 d feeding trial are shown in Table 2.5. Both initial (P = 0.55) and final BW (P = 0.29) were similar between treatments. Throughout the 123 d feeding trial, ADG (P = 0.27), DMI (P = 0.27) and feed conversion efficiency (P = 0.45) were similar between the DH42 treated and control group. Pair-wise comparison between DH42 and the control group was evaluated within days on feed for growth performance (Table 2.5). The control group tended to have greater BW than DH42 treated on d 84 (P = 0.07) and d 111 (P = 0.06). DH42 had numerically greater ADG (P = 0.30) than the control during (1 112-123. This compensation in gain lessened the differences in final BW between treatments. Cattle fed DH42 consumed less (P < 0.05) than the control group during (1 56-111. These low DMI value explains the low BW on d 84 and d 111. Feed conversion efficiency was similar between DH42 treated and control group for all periods. It appears that the longer cattle were fed DH42, DMI and resulting growth were reduced. It is unknown whether this response is due to P. acidipropionici or some other factor in the added culture. Dressing percentage and quality grade were similar between treatments (Table 2.6). Yield grade values were lower (P = 0.04) for DH42 (2.39) compared to the control (2.60). Yield grades estimate the amount of boneless, closely trimmed retail cuts from the high value parts of the carcass including the round, loin, rib, and chuck. The USDA yield 59 grades are rated 1 carcass and yield ofless DMI and In the in t This is in agreen‘. administration 01 Propionibacterit. major precursor 1939). The theor compared With E ngingfinishir high moisture c( Propioniba‘vwi Positively infiue DH42 m the CU! grades are rated numerically 1 through 5. Yield grade 1 denotes the highest yielding carcass and yield grade 5, the lowest. The lower yield grade for DH42 is likely the result of less DMI and ADG during the last two periods of the study. In the in vitro fermentation study, DH42 was viable, and increased propionic acid. This is in agreement with previous published data (Kim et al., 2001) where in vivo administration of DH42 increased propionic acid and decreased the A/P ratio. Propionibacteria are natural inhabitants of the rumen and produce propionic acid, a major precursor for glucose production through hepatic gluconeogenesis (Sauer et al., 1989). The theoretical efficiency of propionic acid as a source of energy for ATP is 108% compared with glucose (McDonald et al., 2002). Increased ADG and feed efficiency in growing-finishing steers that received DH42 via inoculation before the fermentation of high moisture corn (Rust et al., 2000) may support the hypothesis that directly feeding Propionibacteria would be a natural way to increase hepatic glucose production and positively influence metabolism (Francisco et al., 2002; Stein et al., 2006). However, DH42 in the current animal study did not improve grth performance. Reduction of DMI observed in this study with direct-fed Propionibacteria administration did not occur in other studies (Swinney-Floyd et a1 ., 1999; Huck et al., 1999; Ghorbani et al. 2002). However administration of Propionibacterium strain P169 decreased DMI per kg of BW of lactating cows from 3 to 7, 10 and 12 wk postpartum, and increased the plasma leptin level (Francisco et al., 2002). Leptin has been implicated. as a modulator of feed intake (Ingvartsen and Andersen, 2000; Meister, 2000). Early lactating Holstein cows fed monensin, an ionophore that increases ruminal propionic acid, had reduced DMI when expressed per kg of BW (Ramanzin et al., 1997). It was reported 60 that propionic acid infusion reduced feed intake of Holstein cows in midlactation (Sheperd and Combs, 1998). Improvement of carcass traits with administration of direct-fed P. freudenreichii strain P-63 has been reported (Huck et al., 1999). Although yield grade was similar between P. fieudenreichii strain P-63 treatment and the control, administration of P. fieudenreichii strain P-63 at the daily rate of 1 x 109 cfu/animal for 126 (I provided a higher percentage of choice and prime heifer carcasses. However, in another 120 d feeding trial (Swinney-Floyd et al., 1999), dosing the same strain P-63 as a DFM at the daily rate of 3 x 1011 cfu/animal did not alter carcass traits. The inconsistency of animal performance and fermentation characteristics with the same species or strain is common in DFM studies. Using Propionibacterium strain P169, Stein et al. (2006) reported that daily doses of 6 x 1010 and 6 x 10ll cfu/animal increased 4% fat corrected milk in lactating cows. However, only the high-dose changed the ruminal propionic acid level, A/P ratio, and ruminal pH. The low-dose of 6 x 1010 cfu/animal did not change any ruminal fermentation characteristics, and is comparable to the dose of Francisco et al. (2002), which reduced DMI and increased leptin level. The mode of action for changes in milk yield, DMI, and leptin level without alteration of ruminal fermentation is uncertain. Ghorbani et al. (2002) reported that Propionibacterium strain P15 increased protozoal numbers (P < 0.05) with concomitant increases in ruminal NH3-N concentration (P < 0.10), butyric acid (P < 0.05) and a decrease in the number of amylolytic bacteria (P < 0.05) compared with the control. Concentration of acetic acid or propionic acid was not altered by Propionibacterium strain P15 in the rumen of feedlot 61 cattle. The autho‘ butyric acid and i and engulf starch. increase in proto,I Ruminal I with direct-fed Pr al.. 2006) and str' direct-fed Prnpim other feed additiV When var ILA45 and(or) L Sleers (McPeake iImproved final E 0-03) and careae; ofPF24. LA45 .- combination of Efi‘eet n Vitro (K 84 fe “latio cattle. The authors suggested the increased protozoal numbers increased NH3-N and butyric acid and decreased amylolytic bacteria because protozoa are predators of bacteria, and engulf starch and produce butyric acid in the rumen. However, the mechanism of increase in protozoal numbers with direct-fed Propionibateria administration is unknown. Ruminal fermentation discrepancies and inconsistencies in animal performance with direct-fed Propionibacteria may result from dose variations between trials (Stein et al., 2006) and strains of Pr0pionibacteria. To define optimal dose, the interactions of direct-fed Propionibacteria with animals including growth phase, feed components and other feed additives must be considered. ‘ When various daily dose levels of P. fieudenrichii (PF 24) and L. acidophilus (LA45 and(or) LA51) were tested in combinations for performance of growing-finishing steers (McPeake et al., 2002), the combinations of PF 24 and LA45 and(or) LA51 improved final BW (P < 0.01), DMI (P = 0.07), ADG (P = 0.02), carcass weight (P = 0.02) and carcass ADG (P = 0.05). The dose levels of LA45 and LA51 in combinations of PF 24, LA45 and LA51 had a positive correlation with DMI (P = 0.05). The combination of Propionibacterium species with lactate-producing bacteria may have an advantage over single strains. Effects of P. acidipropionici strain DH42 as a DFM fluctuated in previous studies in vitro (Kim et al., 2001) and in vivo (Kim et al., 2000). DH42 did not alter the ruminal fermentation significantly in vitro (Kim et al., 2001), whereas DH42 administration increased propionic acid and decreased the NP ratio in the rumen (Kim et al., 2000). Growth of DH42 in fermentation vessels or the rumen was not confirmed. Therefore, it was difficult to conclusively infer that DH42 was unsuccessful because it was unknown 62 whether live cc enumeration SL propagate in th rates sufficient 1997). Thus dir cfir'animal. Fee except yield gr; COnsequently d. with and likely DH43 adminigr “as active in II”. To build the pn whether live cells were present in the rumen or not. The cru'rent in vitro study and enumeration suggest that P. acidipropionici strain DH42 may grow too slow to self propagate in the rumen. Organisms surviving in significant numbers must have growth rates sufficient to counteract dilution due to turnover of rumen contents (Stewart et al., 1997). Thus direct-fed DH42 was given to feedlot steers daily at the dose of 3 x 109 cfu/animal. Feeding of direct-fed DH42 did not change the overall animal performance except yield grade. During the intermediate period, DH42 treatment reduced DMI, and consequently decreased the live weight for that period, and these results are not consistent with and likely opposite to the improved performances (Rust et al., 2000) observed with DH42 administration via fermented HMC. However, this change may suggest that DH42 was active in the rumen and further research with other strains and dosages are warranted. To build the proper foundation for use of DH42 as a DFM, further studies are need. IMPLICATION P. acidipropionici strain DH42 was viable and grew in an in vitro ruminal ecosystem, but failed to establish a niche in the rumen. Daily feeding of DH42 at the rate of 3 x 109 cfu/animal slightly reduced DMI after 56 d which resulted in lower ADG and less fat accumulation (lower yield grade score). DH42 has the potential to alter runrinal fermentation and animal performance as a DP M. To improve animal production with direct-fed DH42, the optimal dose of DH42 for the animal growth phase needs to be defined. 63 miles. I. 1999. 'l acidosis i Bato. R. 2001. P; Michigan Briesacher. S. L. 1992. £51 and Fifth Dawson. T. E.. S aeobic st at‘idipr()p Dawson. T. E. 1C. ensiled. h Lansing. FIanCisco. C. c_ 2002. p, melaboli Ghorbani‘ G R bacteria microbi CKfiring, H. K. Proced, \Xyagh“ I' LITERATURE CITED Aviles, I. 1999. The use of DH42, a propionibacterium for the prevention of lactic acidosis in cattle. MS. Thesis. Michigan State Univ. East Lansing, MI. Bato, R. 2001 . Propionibacteria as inoculants to high moisture corn. Ph.D Dissertation. Michigan State Univ. East Lansing, MI. Briesacher, S. L., T. May, K. N. Grigsby, M. S. Kerly, R. V. Anthony, and J. A. Paterson. 1992. Use of DNA probes to monitor nutritional effects on ruminal prokaryotes and F ibrobacter succinogenes S85. J. Anim. Sci. 70:289-295. Dawson, T. E., S. R. Rust, and M. T. Yokoyama. 1998. Improved fermentation and aeobic stability of ensiled, high moisture corn with the use of Propionibacterium acidipropionici. J. Dairy Sci. 81:1012-1021. Dawson, T. E. 1994. Propionic acid-producing bacteria as inoculants for preservation of ensiled, high moisture corn. Ph.D Dissertation. Michigan State Univ. East Lansing, MI. Francisco, C. C., C. S. Chamberlain, D. N. Waldner, R. P. Wettemann, and L. J. Spicer. 2002. Propionibacteria fed to dairy cows: Effects on energy balance, plasma metabolites and hormones, and reproduction. J. Dairy Sci. 85: 1 738—1 75 1. Ghorbani, G. R., D. P. Morgavi, K. A. Beauchemin, and J. A. Z. Leedle. 2002. Effects of bacterial direct-fed microbials on ruminal fermentation, blood variables and the microbial populations of feedlot cattle. J. Anim. Sci. 80:1977—1986. Goering, H. K. and P. J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agric. Handbook 379. ARS, USDA, Washington, DC. Higuchi, R., C. F ockler, G. Dollinger, and R. Watson. 1993. Kinetic PCR analysis: real- time monitoring of DNA amplification reactions. Biotechnol. 11:1026—1030. Hoflrerr, L. A., B. A. Glatz, and E. G. Hammond. 1983. Mutagenesis of strains of Propionibacterium to produce cold-sensitive mutants [Starter cultures, Swiss cheese]. J. Dairy Sci. 66: 2482-2487. Holland, P. M., R. D. Abramson, R. Watson, and D. H. Gelfand. 1991. Detection of specific polymerase chain reaction product by utilizing the 5’ to 3’ exonuclease activity of T hermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA. 88:7276—7280. Huck, G. L., K. K. Kriekemeier, and G. A. Ducharme. 1999. Effect of feeding Lactobacillus acidophilus BG2F04 (Micro cell) and Propionibacterium freudenrechii P-63 (MicroCell PB) on grth performance of finishing heifers. J. Anim. Sci. 77(Suppl. 1):264 (Abstract). 64 Ingvartsen, K. L. and J. B. Andersen. 2000. Integration of metabolism and intake regulation: a review focusing on periparturient animals. J. Dairy Sci. 83:1573- 1597. Kim, S.-W., D. G. Standorf, H. Roman-Rosario, M. T. Yokoyama, and S. R. Rust. 2000. Potential use of Propionibacterium acidipropionici, strain DH42, as a direct-fed microbial for cattle. J. Anim. Sci. 78(Suppl. 1.): 292 (Abstr.). Kim, S.-W., S. R. Rust, H. Roman-Rosario, and M. T. Yokoyama. 2001. In vitro effects of Propionibacterium acidipropionici, strain DH42 on fermentation characteristics of rumen microorganisms. p 36 in Beef Cattle, Sheep and Forage Systems. Res. Report. Mich. Sta. Univ, E. Lansing, MI. Koussemon, M., Y. Combet-Blanc, B. K. Patel, J. L. Cayol, P. Thomas, J. L. Garcia and B. Ollivier. 2001. Propionibacterium microaerophilum sp. nov., a microaerophilic bacterium iSolated from olive mill wastewater. Int. J. Syst. Evol. Microbiol. 51:1373-1382. Krause, D. O. and J. B. Russell. 1996. How many ruminal bacteria are there? J. Dairy Sci. 79:1467—1475. Kung, L., Jr. and A. O. Hession. 1995. Preventing in vitro lactate accumulation in ruminal fermentations by inoculation with Megasphaera elsdenii. J. Anim. Sci. 73:250-256. Littell, R. C., P. R. Henry, and C. B. Ammerrnan. 1998. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. 76:1216-1231. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular Cloning (A Laboratory Manual). Cold Spring Harbor Laboratory. Cold Spring Harbor, NY. McDonald, P., R. A. Edwards, J. F. D. Greenhalgh, and C. A. Morgan. 2002. Animal Nutrition. 6th ed. Pearson Education Limited, Harlow, UK. McSweeney, C. S., R. I. Mackie, A. A. Odenyo, and D. A. Stahl. 1993. Development of an oligonucleotide probe targeting 16S rRNA and its application for detection and quantitation of the ruminal bacterium Synergistes jonesii in a mixed-population chemostat. Appl. Environ. Microbiol. 59:1607—1612. Meister, B. 2000. Control of food intake via leptin receptors in the hypothalamus. Vit. Hormones. 59:265—304. Ouwerkerk, D., A. V. Klieve, and R. J. Forster. 2002. Enumeration of Megasphaera elsdenii in rumen contents by real-time Taq nuclease assay. J. Appl. Microbiol. 92:753-758. 65 Ramanzin, M., L. Bailoni, S. Schiavon, and G. Bittante. 1997. Effect of monensin on milk production and efficiency of dairy cows fed two diets differing in forage to concentrate ratios. J. Dairy Sci. 80:1136—1142. Reilly, K. and G. T. Attwood. 1998. Detection of Clostridium proteoclasticum and closely related strains in the rumen by competitive PCR. Appl. Environ. Microbiol. 64:907—91 3. Romanov, M. N., R. V. Bato, M. T. Yokoyama, and S. R. Rust. 2004. PCR detection and 16S rRNA sequence-based phylogeny of a novel Propionibacterium acidipropionici applicable for enhanced fermentation of high moisture corn. J. Appl. Microbiol. 97:38—47. Rust, S. R., S.-W. Kim, E. M. Ungerfeld, and M. T. Yokoyama. 2000. Efficacy of probiotics to improve growth and feed efficiency in beef cattle. Fourth ADSA Discover Conference on Food Animal Agriculture. Nashville, IN. Sauer, F. D., J. K. G. Kramer, and W. J. Cantwell. 1989. Antiketogenic effects of monensin in early lactation. J. Dairy Sci. 72:436—442. Sheperd, A. C. and D. K. Combs. 1998. Long-term effects of aceate and propionic acid on voluntary feed intake by nridlactation cows. J. Dariy Sci. 81 :2240-2250. Stahl, D. A., B. Flesher, H. R. Mansfiel, and L. Montgomery. 1988. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54:1079—1084. Stein, D. R., D. T. Allen, E. B. Perry, J. C. Bruner, K. W. Gates, T. G. Rehberger, K. Mertz, D. Jones, and L. J. Spicer. 2006. Effects of feeding propionibacteria to dairy cows on milk yield, milk components, and reproduction. J. Dairy Sci. 89:111—125 Stewart, C. S., H. J. Flint, and M. P. Bryant. 1997. The rumen bacteria. In The Rumen Microbial Ecosystem; P. N. Hobson and C. S. Stewart, Eds; Blackie Academic & Professional, London; New York. pp 10-74. Swinney-Floyd, D., B. A. Gardiner, F. N. Owens, T. Rehberger, and T. Parrott. 1999. Effects of inoculation with either strain P-63 alone or in combination with Lactobacillus acidophilus LA53545 on performance of feedlot cattle. J. Anim. Sci. 77 (Suppl. 1):77 (Abstr.). Thierry, A. and M. N. Madec. 1995. Enumeration of propionibacteria in raw milk using a new selective medium. Lait. 75:315—323. 66 Table 2.1. CO" Component 4 thicase, g Distilled 11:0 ‘ Micro 501mm” Buffer Solmion' Macro solution- Rresazmin 0- 19 Reducing ascn' Component Distilled H20. CaC122H20. g MnCl2.4H20. ‘ C0C12.6H20. g FeC13.6HgO. t: 234loset’ing and remixet Table 2.1 . Composition of in vitro fermentation buffer solution and premixes In vitro Component Buffer Solutionl Commnent Buffer solution3 Trypticase, g 0.24 Distilled H20, mL 100 Distilled H2O, mL 47.19 NH4HCO3, g 0.4 Micro solution, mL2 0.01 NaHC03, g 3.5 Buffer solution, mL3 23.72 Macro solution, mL4 23.72 Component Macro solution4 Rresazurin 0.1%, mL 0.12 Distilled H20, mL 100 Reducing agent, mL5 5.0 Na2HPO4, g 0.57 KH2PO4, g 0.62 MgSO4.7H2O, g Component Micro solution2 Component Reducing agent5 Distilled H2O, mL 100.00 Cysteine HCl, g 0.625 CaCl2.2H2O, g 13.20 Distilled H20, mL 94.76 MnC12.4H2O, g 10.00 1N NaOH, mL 3.99 CoCl2.6H2O, g 1.00 Na2S9H20, g 0.625 FeCl3.6H2O, g 8.00 lGoering and Van Soest, 1970. 2 3 4 5 Premixed solution and corresponding composition. 67 Table 2.2. Com ; Component W Calcium carbon: Trace-mineralizt Urea Potassium Chlori Dicalcium phosfi Ground com Selenium2 Vitamin A3 Rulnensin 804 ,COmpositiOn (°o): :COmPOSllion (00-): :Contains 30.000 IL Contains 176 g 0f] Table 2.2. Composition of the protein and mineral supplement Component Amount, % Dry matter (DM) Soybean meal 46,10 Calcium carbonate 22,87 Trace-mineralized saltl 10,40 Urea 7.77 Potassium chloride 5,80 Dicalcium phosphate 2. 30 Ground corn 3.22 Selenium2 1.09 Vitamin A3 0.16 Rurnensin 804 0.28 1 Composition (%): NaCl, 96—985; Zn, > 0.35; Mn, > 0.2; Fe, > 0.2; Cu, > 0.03; l, > 0.007; Co, > 0.005. 2 Composition (%): Ca, > 28.5; Se, 0.02. 3 Contains 30,000 IU of vitamin A per gram. 4 Contains 176 g of monensin per kg; Excluded after d 16. 68 f ‘ Table 2.3. The r Time after dosing .I “. In vitro: 0 h In vitro: 24 h M.— Trial (dosage) Day 6 ‘DH42: l H ‘DH42: 7 H ‘DH42: 13H ‘DH42: 19 H Day 7 ‘DHJQ: l H ‘DHQ; 7 H *DH42: 13 H TDH42: 19 H PW‘M'aI ~DH42: + 4 d -DHQ. + 6 d DH42: + g d ZThreshold CECIQ. 3 CT : ‘4-00 x \DTotal DNA “01 det ‘ 0m 0ft '03 C01]. ected hreShO'd Table 2.3. The population of P. acidipropionici, strain DH42 in the rumen Time after dosing CT 1 DH42 (%uantity, Total lgNA Conc., cfu/mL ug/mL In Vitro In vitro: 0 h 34.53 10565 30.5 In vitro: 24 h 33.43 19916 39.9 In Situ Trial (dosage) Day 6 +DH42: 1 H 34.29 12129 58.9 +DH42: 7 H 36.74 2962 21.5 +DH42: 13 H 37.69 1713 27.8 +DH42: 19 H 36.00 4534 31.3 Day 7 +DH42: 1 H 36.14 4180 48.0 +DH42: 7 H 35.94 4708 35.0 +DH42: 13 H 38.27 1226 20.9 +DH42: 19 H 38.70”“ 0 31.3 Post-trial -DH42: + 4 d ND 0 119.1 -DH42: + 6 d 39.78“ 0 33.7 -DH42: + 9 d ND 0 30.9 IThreshold cycles of Taq Nuclease Assay real-time PCR. 2 CT = -4.00 x loglo cfu/mL + 50.62 (R2=0.995; P < 0.01). 3 Total DNA concentration in DNA extract. ND not detected * out ofthreshold set ( CT < 38.53, cfu/mL > 103). 69 Table 24. In \‘il ‘r. lacti Time. ll 3 ,.J 0" O f‘») u—l J.- Table 2.4. In vitro effects of P. acidipropionici strain DH42 on volatile fatty acids (V F A), lactic acid, and pH of ruminal fermentation Treatments P-value3 Time, h SEMl P-value2 , Trt Control DH42 Trt Time x Time Total VFA (mM) 69.2 69.4 3.80 0.94 0.41 < 0.001 0.69 105.7 105.4 0.46 12 206.4 211.7 0.24 24 244.4 246.9 0.55 Lactic acid (mM) 3.17 4.17 0.38 0.02 0.58 < 0.001 < 0.01 2.1 1 2.62 0.39 12 1.44 1.16 0.74 24 2.95 2.33 0.19 Acetic acid (mM) 32.90 32.56 1.61 0.72 0.88 < 0.001 0.91 48.30 46.92 0.96 12 83.81 83.84 0.87 24 96.07 95.07 0.74 Propionic acid (mM) 15.39 15.73 0.89 0.60 0.01 < 0.001 0.34 30.22 31.07 0.03 12 68.51 70.34 0.04 24 76.40 78.77 0.02 NP ratio 2.15 2.06 0.02 < 0.01 < 0.001 < 0.001 < 0.05 1.58 1.48 < 0.01 12 1.22 1.20 0.29 24 1.25 1.21 0.10 pH 6.68 6.68 0.03 0.93 0.39 < 0.001 0.53 6.56 6.54 0.46 12 5.34 5.30 0.15 24 5.05 5.05 0.87 l Standard error of mean. P-value assocrated wrth treatment wrthln incubation time. 3 P-value associated with fixed effects. 70 Table 2.5. Elfec feed No. of pen No. of cattle‘pen Overall perform an Initial BW, kl: Final BW. kg ADG. kg DMI. kg d Feed gain. kg Periodic Performa BW. kg 7 5 84 d l l 1 d 123 ADG. kg (1 02 I 'Jt [J d d d Table 2.5. Effects of probiotic addition of P. acidipropionici, strain DH42 on growth and feed conversion efficiency of growing-finishing cattle Treatments 1 P-value Control DH42 SEM P'value T” Fem“ T". xPerrod No. of pen 7 7 No. of cattle/pen 8 8 Overall performance, 123 d Initial BW, kg 413.2 412.7 0.45 0.55 Final BW, kg 595.8 587.9 4.95 0.29 ADG, kg 1.49 1.42 0.04 0.27 DMI, kg/d 10.54 10.29 0.15 0.27 Feed/gain, kg feed/ kg gain 7.12 7.27 0.14 0.45 Periodic performances BW, kg d 27 461.9 462.2 7.32 0.60 0.17 <0.001 0.18 d 55 500.7 494.8 0.60 d 84 541.0 527.1 0.07 d 111 586.3 572.1 0.06 d 123 597.5 586.2 0.16 ADG, kg (1 0- 27 1.80 1.83 0.19 0.86 0.54 <0.001 0.28 d 28- 55 1.39 1.16 0.24 d 56- 84 1.39 1.11 0.15 (1 82-111 1.68 1.67 0.95 d 112-123 0.94 1.18 0.30 DMT,kg/d d 0- 27 10.14 10.36 0.32 0.51 0.18 <0.001 0.10 d 28- 55 11.13 10.99 0.66 d 56- 84 9.47 8.74 0.03 d 82-111 11.33 10.66 0.04 d112-123 11.31 11.01 0.36 Feed/gain, kg feed/ kg gain (I 0- 27 5.75 5.73 1.73 0.99 0.30 <0.001 0.61 d 28— 55 8.08 10.70 0.14 d 56- 84 7.12 8.84 0.32 d 82-111 6.83 6.52 0.86 d 112-123 12.93 12.48 0.80 I Standard error of mean. P-value assocrated wrth treatment wrthrn an item and(or) a period. 3 P-value associated with fixed effects. 71 Table 2.6. Etfe characteristics Carcass trails * No. ofcarcasses Hot carcass uei; Dressing percen Yield grade - 3 Quallty grade Standard error 0] , , P~Value associate .r_> . . ¢4=ane :23 17:36I6C10; l6-~‘f Table 2.6. Effects of probiotic addition of P. acidipropionici, strain DH42 on carcass characteristics of growing-finishing cattle Treatments 1 2 Carcass traits SEM P-value Control DH42 No. of carcasses 56 56 Hot carcass weight, kg 346.5 342.9 3.54 0.49 Dressing percent, % 62.41 62.41 < 0.01 0.34 Yield grade 2.60 2.39 0.06 0.04 Quality grade3 19.3 19.1 0.19 0.56 1 Standard error of mean. 2 P-value associated with treatment. 3 24 = Prime+; 23 = Primeo; 22 = Prime'; 21 = Choice+; 22 = Choiceo; 21 = Choice'; 18 = Select+; 0 - 17=Select ; 16=Select . 72 DH42 (d1 P. microue 131142 (an P. mlCl'Odt' D H 42: P. mlcn nu DH42 (d P' "”Crfldfl Fig. 2-1. Alignn 1043); al.201 and rei Sequen DH42 (dh421): 976 gacatggattggtaacggtcagag 999 llllllllllllllllllllllll P. microaerophilus: 975 gacatggattggtaacggtcagag 998 DH42 (dh42p): 1000 atggccgcccccc-ttgtgg 1018 11111111111” llllll P. microaerophilus: 999 atggccgcccccctttgtgg 1018 DH42: 1019 gccggt 1024 l 1 I I l I P. microaerophilus: 1019 gccggt 1024 DH42 (dh42r): 1025 tcacaggtggtgcatggct 1043 lllllllllllllllllll P. microaerophilus: 1025 tcacaggtggtgcatggct 1043 Fig. 2-1. Alignment of Taq Nuclease Assay region (68 bp) of 16S rDNA of DH42 (976 — 1043) and the closest strain Propionibacterium microaerophilus (Koussemon et al., 2001). Sequences of forward primer (dh42f , 976-999 of DH42 AY360222) and reverse primer (dh42r, 1022-1043) are same to P. microaerophilus, and the sequence of probe (dh42p, 1000—1018) differs fi'om reference. 73 Effects of scroll vitro rumin; An expel mixed lactic acit fermentable earl collected from 1,- 3910bicall}; To to through two ; A Strict anaerol conditions. An then 2 anaerob fennentarion 3 LI anEaled CO n Chapter 3 Effects of aerobically enriched ruminal lactic acid-fermenting microorganisms on in vitro ruminal fermentation characteristics and bacterial changes with aerobic conditions SUMMARY An experiment was conducted to evaluate the effects of aerobically enriched mixed lactic acid-fermenting rumen bacteria on in vitro ruminal fermentation of readily fermentable carbohydrates when added as a direct-fed microbial. Ruminal contents were collected from lactating dairy cattle and enriched in lactic acid media anaerobically or aerobically. To exclude transient aerobic microorganisms, all enrichments were subjected to through two anaerobic transfer (N2). Each subculture was incubated at 39°C for 24 h. A strict anaerobic preparation (N6) was enriched through 4 subcultures in anaerobic conditions. An aero-tolerant preparation (N 2A2N2) passed 2 aerobic subculturing and then 2 anaerobic enrichments. An aerobic preparation (N 2A4) passed 4 aerobic enrichments. One mL of each enrichment was added as a DFM to an in vitro ruminal fermentation system containing 50 mL of rumen fluid and 50 mL of buffer solution. Untreated control cultures produced little VFA after 6 h of incubation with readily fermentable carbohydrates but produced increasing amounts of lactic acid with time. Acute acidosis was induced in the control group. Both the anaerobic preparation (N 6) and the aero-tolerant preparation (N 2A2N2) had higher total VFA production (P < 0.01), lower lactic acid accumulation (P < 0.01), and higher levels of fermented organic matter (P < 0.05) than the control. When used as a DFM, the aerobic preparation (N 2A4) had 74 greater effects 0‘ production was and pH's were respectively. all and 0 ml! at 9. continued throul increased to 4.9 prevented lactic Additionally. a< acid-utilizers b' evaluated by c‘ that N2A4 hac NlAlNl but Aql’H-‘Ords; R greater effects on ruminal fermentation than other treatments and the control. Total VFA production was 23, 57, 61, and 90 mM, lactic acid content was 129, 99, 98, and 0 mM, and pH’s were 4.4, 4.5, 4.5, and 4.9 for the control, N6, N2A2N2, and N2A4, respectively, afier 24 h of incubation. In N2A4, lactic acid concentrations were 78, 57, and 0 mM at 9, 12, and 24 h of incubation, respectively. Fermentation of organic matter continued throughout incubation. The minimum pH of 4.7 was observed at-12 h, and increased to 4.9 at 24 h. Ruminal microorganisms enriched in aerobic lactic acid media prevented lactic acid accumulation more effectively than anaerobic enrichments. Additionally, aerobic enrichment may increase the chance to isolate aero-tolerant lactic acid-utilizers by reducing strict anaerobes in the culture. However, microbial diversity evaluated by cloning and sequencing of 16S rDNA of bacteria in enrichments showed that N2A4 had a higher proportion of Provotella and Synergistes species than N6 and N2A2N2, but a similar proportion of lactic acid-utilizers. Keywords: Rumen Microorganisms, Direct-F ed Microbial, Ruminal Fermentation, Aerobic Enrichment, Lactic Acid— Utilizer 75 A prob animal by imp} broadened to 3 animals by iml Veld. 1992). M microorganism inherent basic r cultures of micr of improving ar Drug Administl (DFM) instead 1 Occurring micrc as being genera Association of I 218 DFM for run Concerr feed indUStl-y h: placed on iSea 7 's -005). AS a reg Increased‘ or r r reduce the Use ( INTRODUCTION A probiotic is a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance (Fuller 1989). This definition was broadened to a mono- or mixed-culture of live microorganisms which benefits man or animals by improving the properties of the indigenous rrricroflora (Havenaar and Huis in’t Veld, 1992). Most recently, this definition has been further refined to living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition (Guamer and Schaafsman 1998). Ruminal probiotics are live cultures of microorganisms that are deliberately introduced into the rumen with the aim of improving animal health or nutrition (Kmet et al., 1993). The United States Food and Drug Administration in 1989 required manufacturers to use the term direct-fed microbial (DFM) instead of probiotics. The FDA defines DFM as a source of live naturally- occurring microorganisms. Most of the microorganisms being used as DFM are classified as being generally recognized as safe (GRAS) according to the FDA and the American Association of Feed Control officials (Miles and Bootwalla, 1991). Microorganisms used as DFM for ruminants include viable cultures of fturgi and bacteria (Krehbiel et al., 2003). Concern regarding the use of antibiotics and other growth stimulants in the animal feed industry has increased in recent years, and there has been increasing emphasis placed on disease prevention as a means of reducing the use of antibiotics (Krehbiel et al., 2003). As a result, interest in the effects of DF M on animal health and performance has increased. For ruminants, microbial cultures have been used to potentially replace or reduce the use of antibiotics in neonatal and stressed calves, enhance milk production in 76 dairy COWS- an 2003). Krehbil and Laclobucil 2% improyenn Propionibactel convert lactic 2 major propionz microorganisrr pan‘ula. Anael Comml Origin of isolal to intestinal sil PrObiotics User animal and Sill Additionally, 5 alive at the construction 0] Can be deter-mi IDOcula prEVQnt min in cattle (Hibb hung and }{es dairy cows, and improve feed efficiency and daily gain in beef cattle (Krehbiel etal., 2003) Krehbiel et al. (2003) suggested that feeding a DP M containing Propionibacteria and Lactobacilli generally resulted in a 2.5 to 5% increase in ADG and an approximate 2% improvement in feed efficiency in feedlot cattle. Organisms from the Propionibacterium species may be useful in ruminant diets because these organisms convert lactic acid and glucose to acetic acid and propionate (Kung et al., 1991). The major propionate-producing bacteria in the rumen that ferment lactic acid are microorganisms such as Selenomonas ruminantium, Megasphaera elsdenii, Veillonella parvula, Anaerovibrio lipolytica, and Propionibacterium species. Common criteria for selection of probiotic microorganisms for human use include origin of isolates, stability during storage, tolerance to additives, stability during passage to intestinal sites, and minimum effective dose. Lactobacilli are the most common probiotics used in human diets. Most of the lactobacilli isolated from the GI tract are animal and site specific. Cell viability during storage varies between species and strains. Additionally, strains should be tolerant to additives used in the vehicle. Probiotics should be alive at the target site, the large intestine. Fecal recovery of live organisms reflects the construction of a niche in the intestine. The minimum dose required to establish a niche can be determined by fecal recovery (Lee et al., 1999). Inoculation of M. elsdenii as a DFM into the ruminal system has been shown to prevent ruminal acidosis. Supplementation with M. elsdenii prevented lactic acid acidosis in cattle (Hibbard et al., 1993) and in vitro ruminal fermentations (Robinson et al., 1992; Kung and Hession, 1995) during transition from a low- to a high-concentrate diet. 77 Hibbard et at. l l Viability of Stl‘iL‘ on-farm dOSlmé facultative anae Therefore. aero- lnfloyy 1 feed and water probably trans must haye grc (Stewart et a1 specialized r1 total microbi The PODulat Hibbard et al. (1993) administered M. elsdenii through the mouth (drenching) to maintain viability of strict anaerobes. However, drenching is unlikely to be acceptable as a general on-farm dosing method (Nagaraja et al., 1997), especially for daily dosing. Aero-tolerant, facultative anaerobic species such as P. fi'eudenreichii and L. acidophilus have been used as DF M in large scale animal feeding trials. Top-dressing on the feed was the method of delivery for those facultative anaerobes and is acceptable as a daily dosing method. Therefore, aero-tolerance is an important characteristic for a DFM to be used. Inflow of foreign microorganisms and nutrients into the rumen with ingestion of feed and water results in the recovery of many bacterial species, some of which are probably transients rather than residents. Organisms surviving in significant numbers must have grth rates sufficient to counteract dilution due to turnover of rumen contents (Stewart et al., 1997). For some ecosystems, particularly dominated by slow-growing or specialized microorganisms, it has become clear that only a very small fraction of the total microbial diversity has been recovered by cultural methods (Amann et al., 1995). The populations of microorganisms with slow growth rates may need to be added daily. The hypotheses for the current study were that aero-tolerant lactic acid-fermenting bacteria in the rumen might be enriched in aerobic lactic acid media, and that enriched facultative or aero-tolerant anaerobic lactic acid-fermenting bacteria could prevent lactic acid accumulation in a lactic acidosis induced fermentation system. The objectives of the current study were 1) to verify whether aero-tolerant lactic acid-fermenting rumen bacteria could prevent lactic acid acidosis in a rurrrinal fermentation system and 2) to evaluate the microbial diversity resulting from enrichments in different aerobicity conditions of lactic acid media. 78 Preparation of Enrichmi‘ N6 (strictly anal N3“ (aerobic - lactating dairy ii into 225 mL of Caldwell and Bi incubation. 1 m After 2 Passes 1 anaerobic IN6. cultures Were ‘ subcultured in enrlChmenI “'I In Vitro Fer, The 1‘ afld Van Soc a)’ diet fOr 1 cloth While 1 Fifty (Table 2‘]; 1hr eegofj MATERIALS AND METHODS Preparation of the Three Microbial Treatments Enrichment procedures are shown in Figure 3-1. The four treatments were control, N6 (strictly anaerobic), N2A2N2 (aero-tolerant organisms grown anaerobically), and N2A4 (aerobic grown). Rumen contents were collected through the canula from two lactating dairy cows fed a high concentrate diet, mixed, and then 25 mL was inoculated into 225 mL of M10 media containing 2% (v/v) of DL-lactic acid (modified from Caldwell and Bryant, 1966; Table 3.1) while gassing with O2 free—CO2. Afier 24 h of incubation, 1 mL of enrichment was transferred to 10 mL of the identical fresh medium. After 2 passes under strict anaerobic conditions, 1 mL of enrichment was transferred to anaerobic (N 6, strict anaerobic enrichment) or aerobic lactic acid-M10 medium. Aerobic cultures were transferred to anaerobic media afier 2 passes in aerobic media, and subcultured in anaerobic media for N2A2N2. One mL of the aero-tolerant anaerobic enrichment was transferred to aerobic media and subcultured in aerobic media for N2A4. In Vitro Fermentation Treatments The in vitro fermentation was performed according to the procedures of Goering and Van Soest (1970). Rumen fluid was collected from two Holstein heifers fed only a hay diet for more than 2 months, composited, and strained through 4 layers of cheese- cloth while gassing with 02 free-CO2. Fifty mL of strained rumen fluid and 50 mL of in vitro fermentation buffer (Table 2.1; Goering and Van Soest, 1970) were placed into a 250 mL round bottom flask. Three g of fermentable carbohydrates [soluble starch (55%; Difco, Detroit, MI), glucose 79 (26%: Sigma. 1 tryptone (3%: Z flask. Ten mL for 10min and to the respcclir Flasks were pr. were removed incubation. Tre Lactic acid, VI Rumina was centrifuge N H2804 for or exchange excll conditions des< Appare stoichiometry instead of capr bull'rate T 1801 DNA Burden“, GEDOH (26%; Sigma, St Louis, MO), methyl cellulose (6%; Sigma), cellobiose (7%; Sigma), tryptone (3%; Difco), proteose peptone (3%; Difco) on wt/wt basis] were added to the flask. Ten mL of each microbial enrichment was pelleted by centrifugation at 6,600 x g for 10 min and resuspended to 10 mL of the in vitro buffer solution, and 1 mL was added to the respective flasks. For the control, sterile buffer was added without microorganisms. Flasks were prepared anaerobically and incubated at 39°C. Three mL of culture media were removed while gassing flasks with O2 free-CO2 at 0, 3, 6, 9, 12, and 24 h of incubation. Treatments were conducted in triplicate. Lactic acid, VFA, and pH Analysis Ruminal pH was recorded immediately after sample collection and rumen fluid was centrifuged at 24000 x g for 20 min and 25 mL of supernatant was acidified with 12 N H2SO4 for organic acid analysis. Lactic acid and VFA contents were determined by ion exchange exclusion HPLC (aminex HPX-87 h; Bio-Rad, Richmond, CA) with the same conditions described in Chapter 2. Apparently fermented organic matter (FOM) was calculated from the VFA stoichiometry (Demeyer and Van Nevel, 1979), but including lactic acid and isobutyrate instead of caproate: F OM (mmol of hexose) = (lactic acid + acetic acid + propionate)/2 + butyrate + isobutyrate + isovalerate + valerate, with all VFA expressed in mmol produced. DNA Extraction and Cloning Genomic DNA of microorganisms in each enrichment was extracted with UltraClean Fecal DNA kit (Mo Bio Laboratories Inc., Solana Beach, CA). Community 80 16S rDNAs were PCR amplified in a reaction mixture containing 1 x PCR buffer, 200 mM dNTPs, 500 nM of each forward and reverse primer, and 0.05 U of T aq polymerase (Promega, Madison, WI) per pl. The PCR program for 16S rDNA amplification was 96°C for 4 min (for initial denaturation), followed by 32 cycles at 94°C for 1.5 min, 42°C for 1 min, 72°C for 4 min and a final extension at 72°C for 10 rrrin. PCR products were purified using a DNA purification kit (Promega). One bacteria clone library was prepared for each enriched community. For all clone libraries, rDNA’s were amplified with forward primer 8F (specific for bacteria, 5'-AGA GTT TGA TCC TGG CTC AG-3') and universal reverse primer 1390R (5'-CG GTG TGT ACA AGG CCC-3'). PCR products (5 1.11) were run on a 0.8% agarose gel to confirm the size and purity of the DNA before they were used for cloning. Cloning was performed using a cloning kit (Invitrogen Corp. Carlsbad, CA) following the manufacturer's instructions. Plasmids were purified from E. coli transformants using the Minipreps system (Promega). Screening Clones by Restriction Fragment Length Polymorphism (RF LP) Analysis and Sequencing Inserts of 16S rDNA in recombinant clones were re-amplified ham 2 ul of plasmid DNA by PCR using primers 8F and 1492R as previously described. The re- arnplified inserts were run on a 0.8% agarose gel to check the size and purity before doing used for digestion. Aliquots of crude PCR products from each clone were separately digested with four tetrameric restriction enzymes, Mspl, Hhal, and HaeIII (New England BioLabs Inc., Ipswich, MA). For Mspl, 5 ul aliquots of PCR products were digested in mixtures containing 1x NEB (New England BioLabs Inc.) buffer 2 and 81 10 l ol‘P rest sep: 3 I: ban clo Phi 10 U of restriction enzymes in a final volume of 20 pl at 37°C for 4 h. For Tsp509 I, 5 pl of PCR products were digested in mixtures containing 1x NEB buffer 1 and 10 U of restriction enzymes in a final volume of 20 pl at 65°C for 1.5 h. DNA fragments were separated on a 2% agarose gel, which was stained with 0.5 pg/mL of ethidium bromide in a 1x TBE buffer. Two ladders (50 bp and 1 kb) were used as standards to determine the band sizes. RFLP patterns of each clone library were grouped visually, and representative clones from each group (libraries) were selected for sequencing. Partial l6S rDNA of clones was sequenced with primer 8F using ABI 3730x1 high through-put capillary DNA sequencers at the Genomics Technology Support Facility at Michigan State University. Phylogenic Analysis Using each 16S rDNA clone sequence as a query sequence, a local alignment was performed by means of the blastn (nucleotide-nucleotide BLAST, Basic Local Alignment Search Tool, http://www.ncbi.nlm.nih.gov/BLAST/). As references, closest bacterial sequences to query sequences were downloaded, and deposited in Biology WorkBench 3.2 database at San Diego Supercomputer Center at UCSD (http://workbench.sdsc.edu/). Multiple alignments of the clones and reference sequences were performed using the ClustalW method as described in Thompson et al. (1994). A phylogenetic tree was generated by Phylip's Drawgram (Felsenstein, 1993). The phylogenetic tree represented either a phenogram with balanced branches that averaged the distances between ancestors in the tree or a cladogram with unbalanced branches that forced branch distances to correspond to sequence divergence. 82 Stan's MIXI fenne treatn remor appro 1998 l Home ICSpel' Statistical Analysis In vitro fermentation data were analyzed with repeated measures using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC), with the subject defined as fermentation tube, the experimental unit. Initially, model variables were microbial treatment, incubation time, quadratic effect of time and all interactions. Interactions were removed fi'om the model when P > 0.10. Auto-regression was determined as the most appropriate covariance structure using the Schwarz Baysean criterion (Littell et al., 1998). Least squares means within time or treatments were compared by t-test using the Bonferroni unequality. Significance and tendency were declared at P < 0.05 and P < 0.10, respectively. 83 RESULTS Treatment, time and the interaction of treatment and time affected for total VF A production (P < 0.01). Treatment N2A4 had the greatest total VFA production (P < 0.05) among treatments after 9 h of incubation (Table 3.2), and afier 24 h, was 9 and 3 times greater than the control or the other microbial treatments, respectively. Both N6 and N2A2N2 had a higher total VFA from 12 to 24 h over the control (P < 0.05). Total VF A production was similar between N6 and N2A2N2 throughout the 24 h incubation (P < 0.01). Total VFA production in the control group was not changed after 6 h, while production in all microbial treatments increased (P < 0.05) until the end of incubation. Interaction between treatment and quadratic time was observed for the amount of fermented organic matter (FOM; Table 3.2). N2A4 had a greater FOM than the control at all time points between 6 and 24 h of incubation (P < 0.01). N2A4 also had greater FOM than N6 (P < 0.01) between 9 and 24 h, and N2A2N2 (P < 0.01) at 12 h and 24 h. Both N6 and N2A2N2 had higher F OM at 6 h (P < 0.01) and at 24 h (P < 0.05) over control. FOM increased over time for all treatments (P < 0.05). The three microbial treatments had a greater sum of total VFA and lactic acid production than the control at 6 h (P < 0.01; Table 3.2). The pattern of differences between treatments was similar to FOM. N2A4 had a greater (P < 0.01) content of total organic acids than other treatments after 12 h. In all treatments, the sum of total VFA and lactic acid increased (P < 0.01) as incubation time increased. All microbial treatments had greater lactic acid production (P < 0.01; Table 3.3) than the control at 6 h of incubation. N2A4 had the least lactic acid production (P < 0.01) among treatments from 9 h to 24 h. At 9 h and 12 h, the amount of lactic acid in the 84 control was similar to N6 and N2A2N2. At 24 h, the control had the greatest lactic acid content (P < 0.01). N6 and N2A2N2 showed similar amounts and patterns of lactic acid accumulation throughout 24 h of incubation. Lactic acid in both N6 and N2A2N2 increased by 12 h, and decreased thereafter (P < 0.01). Lactic acid in the control group increased throughout the 24 h of incubation (P < 0.01). In N2A4, lactic acid increased to 9 h, and then decreased (P < 0.01). Twenty-one and 57 mmol/L of lactic acid in N2A4 disappeared between 9 - 12 h, and 12 - 24 h, respectively (P < 0.01). At the end of incubation, lactic acid production levels were 125.7, 99.1, 97.9, and 0.1 mM for the control, N6, N2A2N2, and N2A4, respectively. At 6 h of incubation, pH was higher in the control than other treatments (P < 0.01). However, at 24 h, N2A4 had the highest pH (p < 0.01). N2A4 had a higher pH (P < 0.05) than the other microbial treatments at 12 h. Both N6 and N2A2N2 had a lower pH (P < 0.01) than the control at 6 h, but similar thereafter. In the control group, pH decreased at each time point (P < 0.05). For the microbial treatments, the overall trend was for pH to decrease (P < 0.01) through 12 h and remain relatively constant thereafter. The final pH of the cultures were 4.44, 4.50, 4.52 and 4.92 for the control, N6, N2A2N2, and N2A4, respectively. Interaction of treatment and quadratic time (time x time) existed for the ratio of acetic acid to propionic acid (A/P; Table 3.3). A/P was greater than 1 at 3 h, but less than 1 thereafter for all cultures (P < 0.01). The control group had a smaller ratio at 3 h than N2A2N2 and N2A4. Between 9 and 24 h, N2A4 had the lowest (P < 0.01) values over the control, N6, and N2A2N2. From 6 h through 24 h of incubation, the control, N6, and N2A2N2 had similar A/P ratios. In the control group, the A/P ratio after 24 h of 85 incubation was 0.75 and this value was smaller (P < 0.01) than the level observed at 3 and 6 h of incubation. N6 and N2A2N2 showed similar values and trends in A/P ratio. In general, the NP ratio decreased across time (P < 0.01). Effects of quadratic time and interaction of treatment and time were observed on acetic acid production (P < 0.01; Table 3.4). N2A4 had the highest acetic acid level (P < 0.01) among treatments from 9 h to 24 h. N6 and N2A2N2 had a higher acetic acid production level than the control (P < 0.01) at 24 h. In the control, acetic acid content was unchanged after 6 h. N6 and N2A2N2 increased acetic acid throughout the 24 h incubation period (P < 0.05), and N2A4 increased the amount until 12 h (P < 0.01). After 24 h, control had the lowest acetic acid content. The time effects was quadratic (P = 0.01) and the interaction (P < 0.01) of treatment and time were observed on propionic acid production (Table 3.4). N2A4 showed the largest (P < 0.01) propionic acid production level among treatments after 6 h. N6 and N2A2N2 had greater levels at the end of the incubation than the control (P < 0.01). Propionic acid content in the control group was unchanged after 3 h. Microbial treatment groups increased propionic acid over the incubation period (P < 0.01). This increase was more striking for N2A4 than N6 or N2A2N2. Interaction of treatment and quadratic time was seen for butyric acid production 0’ < 0.01; Table 3.4). N2A4 had greater butyric acid production than the control (P < 0.05) from 6 h to 24 h of incubation and was greatest among treatments (P < 0.01) after 9 h. N6 and N2A2A3 had similar butyric acid levels throughout incubation. N2A2N2 had a greater butyric acid production (P < 0.05) than the control at 24 h. Butyric acid levels were similar at all time points for the control treatment. In N6, butyric acid levels were 86 reduced at 9 h (P < 0.01) but similar at the other endpoints. This reduction is unlike the overall trend and likely to be an analysis error. Butyric acid levels at 24 h were higher (P < 0.01) than at 3 h of incubation for N2A2N2. N2A4 showed a continuous increase in butyric acid levels after 6 h (P < 0.01). N2A4 had higher valeric acid production levels than the control at 9, 12, and 24 h (P < 0.05; Table 3-5). At 24 h of incubation, N2A4 also had a greater valeric acid content (P < 0.01) than N6 and N2A2N2. Both N6 and N2A2N2 had higher amounts of valeric acid than the control (P < 0.01). In the control, valeric acid levels were similar throughout incubation. N6 had greater valeric acid levels at 24 h than earlier time points. Valerie acid levels increased (P < 0.01) throughout incubation for N2A4, but a large increase occurred at 24 h. Only small differences were noticed in isobutyric acid and isovaleric acid levels over the various time endpoints (Table 3.5). The microbial treatments had greater isobutyric acid levels at the end of the incubation. Isobutyric acid levels were negative until the last collection which would suggest a net utilization of the acid. In N2A4, isovaleric acid amount was greatest (P < 0.01) between the 12 - 24 h time period. Microbial diversity in the enrichment was evaluated using phylogenetic analysis of 16S rDNA sequence (Table 3.6). A total of 84 clones were obtained from 3 enrichments; 28, 29, and 27 clones for N6, N2A2N2, and N2A4, respectively. A genotyping, RF LP, resulted in 24, 19, and 16 patterns for N6, N2A2N2, and N2A4, respectively (Appendix C. Figures 7.2 to 7.4). A total of 59 recovered 16S rDNA fragments from clones were sequenced using the 8F bacterial specific primer (Appendix C. Figures 7.5 to 7.7). Multiple alignment of sequences of clones within the enrichment 87 revealed that 9 different sequences were in N6, and 11 in N2A2N2, and 6 in N2A4. A local alignment of each sequence was performed using BLASTN (Altschul et al., 1997) against sequences on the database from GenBank, EMBL, DDBJ, and PDB which contained 4,084,561 sequences through National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/ BLAST/). Eight, 10, or 5 different genus or species were close to N6, N2A2N2, or N2A4 enrichments, respectively (Table 3.6). Bacteroides species, Prevotella species, Entercoccus species, and M. elsdenii were matched in all 3 enrichments. Desulfovibrio species, Eubacterium species, and Holdemaniafiliformis were observed in N6 and N2A2N2, but not in N2A4. Synergistes species were observed in N2A2N2 and N2A4, but not in N6. Bulleidia species was matched in only N6. Acidaminococcusfermentans and Spirocliaeta species were unique in N2A2N2. N2A4 had more clones (9 of 27) of Prevotella species than N6 (4 of 28) and N2A2N2 (4 of 29). N2A4 also had higher number (10 of 27) of Synergistes species clones than N2A2N2 (5 of 29). However, there was no clone unique to only N2A4. 88 DISCUSSION Propionic acid-producing bacteria are the sugar- or lactate-fermenting microorganisms such as Propionibacterium spp., Veillonella spp., Selenomonas ruminatium, and Anaerovibrio lypolytica (Stewart et al., 1997), therefore enrichment and isolation of propionic acid-producing bacteria are based on their ability to use lactic acid. To enrich Propionibacterium spp. 1.6% (v/v) lactic acid (60% syrup; Dawson, 1994) or 2.0% (w/v) lactic acid (50%; Britz and Riedel, 1991) media were used. In the current study, a 2.0% (v/v) lactic acid (60% syrup) medium was used to enrich lactic acid-fermenting rumen bacteria. Because of the poor viability of strict anaerobic bacteria in a commercial setting, development of an aero-tolerant species is needed. To apply a strict anaerobic microorganism to feed as a DFM, a suitable method maintaining viability is required (N agaraja et al., 1997). Facultative or aero-tolerant anaerobic bacteria have an advantage as a DP M over strict anaerobes in cultivation, storage, and delivery to the animal. The current experiment used enriched ruminal bacteria that were shown to grow in anaerobic lactic acid medium (N2) and exposed them to different aerobic conditions to develop cultures that contained strictly anaerobic, facultative or aero-tolerant anaerobic bacteria. After 2 subculturing at a 1:10 inoculation rate, the microorganisms which did not grow in the enrichment condition would be diluted to 1:102 or destroyed, whereas microorganisms growing in the enrichment would establish a population in the culture. The strict anaerobic condition (N6) will enrich lactic acid-utilizing rumen bacteria, regardless of microorganisms’ aero-tolerance, and may contain both strict and facultative anaerobes. Two transfers in aerobic conditions following the initial two passes in 89 anaerobic enrichment (N 2A2) may enable the aero-tolerant or facultative anaerobic microorganisms predominate in the culture. This enrichment was split to anaerobic (N2A2N2) or aerobic (N 2A4) conditions. Anaerobic following aerobic enrichment (N 2A2N2) may increase the populations of aero-tolerant microorganisms that grow faster in anaerobic environment. Aerobic enrichment (N 2A4) may allow aero-tolerant anaerobic microorganisms of which grth is not affected by aerobicity predominate in the culture. One mL of each enrichment was inoculated into 100 mL of an in vitro anaerobic fermentation system containing buffer, ruminal fluid, and substrates to evaluate the DF M effect of enriched mixed microorganisms. F OM increased throughout the incubation period for all treatments which suggests a vigorous fermentation continued. The total organic acid also increased as incubation time increased, however the proportion of products was different among treatments. The control group produced only lactic acid but not VFA after 6 h, while other microbial treatments produced VFA continuously throughout incubation and turned metabolism toward the utilization of lactic acid after certain time points. Between bacterial treatments, N2A4 showed the highest VFA levels and FOM, and completely utilized the accumulated lactic acid. Both N6 and N2A2N2 had greater VFA levels and FOM and a lower lactic acid concentration than the control. However, no difference in fermentation characteristics was seen between N6 and N2A2N2. Two passes in an aerobic condition in the middle of enrichment did not appear to influence microbial diversity, at least in terms of effective microorganisms and end product levels. All microbial treatments were enriched in lactic acid media, however, both N6 and N2A2N2 showed less lactic acid utilization in the culture than N2A4. Enriched lactic 90 acid-utilizing bacteria in N6 or N2A2N2 seemed to differ from N2A4 in species or in number. Whether those effects resulted from the microbial species or number, N2A4 should be the appropriate resource for selection and isolation of DF M candidates. Above all, aerobic enrichment (N2A4) seems to provide bacteria that have a higher efficiency in prevention of lactic acid accumulation than anaerobic enrichment. This may support the possible existence of aero-tolerant lactic acid-utilizing bacteria in the rumen. After 12 h of incubation, pH in all treatments was similar, but VFA and lactic acid levels between microbial treatments differed. The difference might result from microbial composition among the treatments. Even in N2A4, the patterns of fermentation before and after12 h seemed to be different. Different lactic acid-fermenting bacteria could predominate for each period. Between 9 and 12 h, 2.3 mmol of OM (hexose) and 2.1 mmol of lactic acid were fermented to 1.3 mmol of acetic acid, 4.0 mmol of propionate, 0.6 mmol of butyrate, and 0.1 mmol of valerate. After 12 h, 5.7 mmol of lactic acid and 4.3 mmol of OM (hexose) were fermented to 2.5, 4.1, and 1.5 mmol of propionate, butyrate, and valerate, respectively. Propionate and acetic acid were main products between 9 - 12 h, and butyrate, propionate, and valerate were obviously high in production after 12 h. Acetic acid was not produced after 12 h. Previous reports have shown different fermentation endproducts from different propionate-producing bacteria. P. shermanii produced 0.8 mM of acetic acid and 2.3 mM of propionic acid from 1.3 mM of glucose; and 1.7 mM of acetic acid and 3.4 mM of propionate from 6mM of lactic acid (Johns, 1951). M. elsdenii (Hino, 1994) produced 6.8 mM of acetic acid and 4.3 mM of butyrate and negligible caproate and other VFA from 8 mM of glucose; and 14 mM of acetic acid and 8 mM of propionate and 8.5mM of butyrate from 40 mM of lactic acid 91 media. In another study (Elsden et al., 1956) M. elsdenii produced 2.0 mM of acetic acid, 2.4 mM of propionate, 2.0 mM of butyrate, 2.6 mM of valerate, 0.2 mM of caproate, 7.5 mM of CO2, and 0.5 mM of H2 from 129 mM of lactic acid medium. Additionally, M. elsdenii (Waldrip and Martin, 1993) was reported to produce 1.5, 1.4, 7.4, 0.5, 3.3, 0.8 mM of acetic acid, propionate, butyrate, isobutyrate, valerate, and isovalerate, respectively. When Kung and Hession (1995) inoculated M. elsdenii into an in vitro ruminal fermentation, the concentrations of propionate, butyrate, valerate, isobutyrate, and isovalerate for the control and M. elsdenii inoculation at 6 h were 38, 47; 2, 35; l, 15; l, 11; and 1, 2 (mM), respectively. Acetic acid was not different after 2 h. The lowest pH in N2A4 was 4.7 at 12 h, which increased slightly to 4.9 at 24 h. The largest amount of lactic acid was 78 mM at 9 h which was entirely utilized by 24 h. A similar trend was reported by Kung and Hession (1995). They reported that the pH in untreated in vitro runrinal culture decreased to approximately 4.8. The pH of cultures treated with the low dose of M. elsdenii decreased to approximately 5.0 but then gradually increased and reached a plateau of approximately 5.4. After a slight initial drop, the pH of cultures treated with the high dose of M. elsdenii remained fairly constant at approximately 5.3. Lactic acid concentration was less than 2 mM at any sampling time with the high dose of M. elsdenii. Total lactic acid concentration peaked at more than 40 mM in the control while the current study showed 126 mM of lactic acid production in the control treatment. The elevated pH and lower lactic acid accumulation after 24 h in the current study was less than that reported for the single microorganism (Kung and Hession, 1995). The N2A4 organisms may have a potential for use as a DFM. The utilization of 7.8 mmol/100mL of lactic acid and 6.5 mmol (hexose)/100mL of OM after 92 rec Ad .lIc mil I'll" enn' Iran cnrj lmo the g anae dil‘l‘ej Hung [he fir. the culture pH reached 4.8, suggests that the organisms in N2A4 may be acid-tolerant lactic acid-utilizing bacteria that can utilize readily fermentable substrates. Loesche (1969) classified anaerobes into three groups; 1) strict anaerobes which grow on spread plates only when the atmosphere contained less than 0.5% 02 (e.g. Selenomonas ruminantium, Clostridium haemolyticum); 2) moderate anaerobes which would generally grow on plates when the atmosphere contained less than 10% 02, (e. g. Peptostreptococcus elsdenii (Megasphaera elsdenii»; and 3) microaerophiles which require a low concentration of 02 for optimal growth and are still unable to grow in air. Additionally, facultative anaerobes can grow either aerobically or anaerobically. Moderate anaerobes could be considered aero-tolerant anaerobes. Aerobicity of microorganisms, however, can vary tremendously even within a species. Although S. ruminantium is generally a strict anaerobe, some strains are reported to tolerate exposure to small amounts of oxygen (Samah and Wimpenny, 1982). In the current study, all enrichments passed two anaerobic conditions initially to exclude or dilute the aerobic transients. N6 which was enriched in only anaerobic conditions represents the traditional enrichment method, and may include strict (obligate), facultative and aero-tolerant (moderate) anaerobes as discussed above. The last two passes in anaerobic condition in N2A2N2 were utilized to enhance the growth rate of facultative and aero-tolerant anaerobes of which growth is faster in anaerobic environment. There is evidence that propionate-producing species grown in different aerobicity environments can have different recovery rates (Morris, 1976). The Hungate roll-tube (strict anaerobic) technique had a thousand times greater recovery than the anaerobic blood agar plate (microaerobic) technique in viable counting of 93 Propionibacterium acnes. However, in this study, N6 and N2A2N2 appeared to have a similar levels of active lactic acid-fermenting organisms. The results of this study suggest oxygen exposure for the last two passes changed the microbial population and amplified active lactic acid-fermenting organisms in N2A4 relative to N6 or N2A2N2. Less lactic acid-utilization in N6 or N2A2N2 than in N2A4 implies that lactic acid-utilizing microorganisms in N2A4 may be dominated by other microorganisms in a lactic acid- rich anaerobic environment. Furthermore, the ruminal ecosystem may be similar to N6 enrichment rather than N2A4. Therefore, lactic acid-utilizing microorganisms in N2A4 may have a small population in the rumen. In this study, the supercharging an in vitro ruminal fermentation system with the lactic acid-utilizing microorganisms in N2A4 prevented lactic acid accumulation more efficiently than N6 or N2A2N2. Microbial diversity was investigated using cloning and sequencing of mixed bacterial DNA extracts from each of the enrichments. Common microorganisms in the three enrichments were Bacteroides sp., Prevotella sp., Entercoccus sp., and M. elsdenii. Species found in N6 and N2A2N2, but not in N2A4 were Desulfovibrio sp., Eubacterium sp., and Holdemaniafiliformis. Synergistes sp. was observed in N2A2N2 and N2A4, but not in N6. Bulleidia sp. was matched in only N6. Acidaminococcusfermentans and Spirocliaeta sp. were unique in N2A2N2. N2A4 had a higher number of clones closely related to Synergistes species and Prevotella species than N6 and N2A2N2. However, cloning was based on the PCR product from a mixed bacterial extract, and the number of clones would not represent the concentration of the bacteria in the culture. There was no species or genus unique to N2A4. Among the ruminal lactic acid-fermenting bacteria, only M. elsdenii was found. Microbial diversity data does not support the microbial 94 C0 change in the enrichment except that aerobic enrichment (N 2A4) reduced the variety of microbial species. IMPLICATIONS F acultative or aero-tolerant anaerobes have an advantage when used as a DFM over strict anaerobes in easier cultivation, storage, and delivery to the animal. The present study assessed the combination of anaerobic and aerobic conditions in addition to using substrate selective media to exclude transient microorganisms and amplify tentative aero- tolerant or facultative anaerobic lactic acid-fermenting microorganisms. Aerobically enriched mixed lactic acid-fermenting organisms prevented lactic acid accumulation and facilitated in vitro fermentation of readily fermentable substrates in rumen fluid from hay fed animals. Furthermore, aerobic enrichment showed a significantly greater DF M effect than anaerobic enrichment. Therefore, aerobic enrichment has an advantage over anaerobic enrichment of lactic acid utilizing microorganisms in viability and growth rate. The present study provided strong evidence supporting the concept of adopting aerobic conditions in enrichment for DF M candidates. 95 LITERAURE CITED Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. Amann, R. I., W. Ludvig, and K. H. Schleifer. 1995. Physiological identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169. Britz T. J. and K. H. J. Riedel. 1991. A numerical taxonomic study of Propianibacterium strains from dairy sources. J. Appl. Bacteriol. 71 1407-416. Bryant, M. P. and L. A. Burkey. 1953. Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36:205-217. Caldwell, D. R. and M. P. Bryant. 1966. Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 14:794-801. Dawson T. E., S. R. Rust, and M.T. Yokoyama. 1998. Improved fermentation and aeobic stability of ensiled, high moisture corn with the use of Propionibacterium acidipropionici. J. Dairy Sci. 81:1015-1021 Demeyer, D. I. and C. J. Van Nevel. 1979. Effect of defaunation on the metabolism of rumen microorganisms. Brit. J. Nutri. 42:515-524 Elsden, S. R., B. E. Volcani, F. M. C. Gilchrist, and D. Lewis. 1956. Properties of a fatty acid forming organism from the rumen of sheep. J. Bacteriol. 72:681—689. Felsenstein, J. 1989. PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics. 5: 164-166. Fuller R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66:365-378. Goering, H. K. and P. J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agric. Handbook 379. ARS, USDA, Washington, DC. Guarner, F. and G. J. Schaafsm. 1998. Probiotics. Int. J. Food Microbiol. 39:237-238. Havenaar, R. and J. H. J. Huis in’t Veld. 1992. Probiotics: a general view. pp 209-224 in The Lactic Acid Bacteria; Vol. 1: The Lactic Acid Bacteria in Health and Disease. Wood B.J.B. ed. Chapman & Hall, New York, NY. 96 Hibbard, B., J. A. Robinson, R. C. Greening, W. J. Smolenski, R. L. Bell, and J. P. Peters. 1993. The effect of route of administration of isolate 407A (UC-12497) on feed intake and selected ruminal variables of beef steers in an acute acidosis inappetance model. Proc. 22nd Biennial Conf. Rumen F unc., Chicago, IL. p 19. (Abstr.). Hino, T., K. Shimada, and T. Maruyama. 1994. Substrate preference in a strain of Megasphaera elsdenii, a ruminal bacterium, and its implications in propionate production and growth competition. Appl. Environ. Microbiol. 60:1827-1831. Johns, A.T. 1951. Isolation of a bacterium, producing propionic acid, from the rumen of sheep. J Gen Microbiol. 52317—325. Kmet, V., H. J. Flint, and R. J. Wallace. 1993. Probiotics and manipulation of rumen development and function. Arch. Anim. Nutr. 4421—10. Krehbiel, C. R., S. R. Rust, G. Zhang, and S. E. Gilliland. 2003. Bacterial direct-fed microbials in ruminant diets: performance response and mode of action. J. Anim. Sci. 81(E. Suppl. 2):E120—E132. Kung, L., Jr. and A. O. 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Fifth Stenhous-Williams memorial lecture. Oxygen and the obligate anaerobe. J. Appl. Bacteriol. 40:229—244. Nagaraja, T. G., C. J. Newbold, C. J. Van Nevel and D. I. Demeyer. 1997. Manipulation of ruminal fermentation. In The Rumen Microbial Ecosystem; P. N. Hobson and C. S. Stewart, Eds.; Blackie Academic & Professional: London; New York; pp 523-632. 97 Robinson, J. A., W. J. Smolenski, R. C. Greening, M. L. Ogilvie, R. L. Bell, K. Barsuhn, and J. P. Peters. 1992. Prevention of acute acidosis and enhancement of feed intake in the bovine by Megasphaera elsdenii 407A. J. Anim. Sci. 70 (Suppl. 1):310 (Abstr.). Russell, J. B., M. A. Cotta, and D. B. Dombrowski. 1981. Rumen bacterial competition in continuous culture: Streptococcus bovis versus Megasphaera elsdenii. Appl. Environ. Microbiol. 41 :1394-1399. Samah, O. A. and J. W. T. Wimpenny. 1982. Some effects of oxygen on the physiology of Selenomonas ruminantium WPL 151/1 grown in continuous culture. J. Gen. Microbiol. 128:355—360. 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Modified M10 lactate medium used in enrichment Medium 10, modified‘ Ingredient 2 Anaerobic Aerobic Mineral 14, m1, 17.00 17.00 Mineral 115, mi, 17.00 17.00 dH20, mL 55.89 55.89 Trypticase, g 0.23 0.23 Yeast extract, g 0.06 0.06 Lactate (60%), mL 2.00 2.00 VFA mixture6, mL 0.35 0.35 Reducing agent7, mL 0-57 0 Hemin sln.8, mL 1.13 1.13 Na2C03 (8%) 5.67 5.67 Resazurin (0.1%), mL 0.1 0 Ingredient VF A mixture6 Acetic acid, mM 29 Propionic acid, mM 8 Butyric acid, mM 4.3 IsoButryric acid, mM 1.1 n-valeric acid, mM 0.9 isovaleric acid, mM 0.9 DL-a-methylbutyrate 0.9 Ingredient Mineral l & II Mineral 14 KZHPO4, g 0.60 dH20, mL 100.00 Mineral [15 NaCl, g 1.20 (NH4);SO4, g 1.20 KH2P04, g 0.60 CaClz, g 0.12 MgSO47H20, g 0.25 dH20, mL 100.00 Ingredient Reducin7g agents Cysteine-HG] H20, g 2.50 dH20, mL 50.00 NaOH sln (10%) to pH 10 Na2S9H20, g 2.50 dH20, mL to 200mL I Glucose, cellobiose and soluble starch (Caldwell and Bryant, 1966) were replaced with lactate. 2 Medium except reducing agent and NaZCO3 boiled and cooled twice while bubbling with Oz-free C02. 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Bacteria closely related by means of blastn to bacterial 16S rDNA sequences cloned from enriched rumen contents in a lactic acid media Enrichment Clones| Closest bacteria2 N6 93, 75, 101, 80 Bacteroides sp., mm;3 N6 9] Bacteroides sp., URB4 N2A2N2 293, 295 Bacteroides sp. N2A4 204, 210, 215, 229, 234 Bactemides Sp“ [1111133 N6 92, 64, 97 Prevotella rumincola N6 3' Prevotella sp., URB4 N2A2N2 30], 290, 302 Prevotella rumim'cola, P. species N2A2N2 282 Prevotellasp., URB“ N2A4 205, 209, 226 Prevotella sp. N2A4 200, 201, 252, 254, 253, 230 prevotella sp., URB“ N6 63 Enterococcus sp N2A2N2 299, 305, 284, 294 Enterococcusfaecium, N2A4 214 Enterococcusfaecium N6 61, 74, 104 M. elsdenii N2A2N2 298, 31 l, 313 M. elsdenii. N2A4 244, 236 M. elsdenii N6 54 H. filiformis, 11111133 N2A2N2 291, 303 H filifomis, UHIB3 N6 111, 76, 55, 110, 100, 73, 79, 78 Eubacterium pyruvivorans N2A2N2 285, 308, 292, 315, 310 Eubacterium pyruvivorans, Eubacterium sp. N6 59, 70, 7|, 99 Desulfovibrio sp. N2A2N2 306, 314 Desulfovibrio sp., UHIB3 N2A2N2 286, 276, 275, 287, 288 Synergistes sp. N2A4 225, 216, 220, 228, 241, 243, 248, synergism sp., mm“ 233, 251, 232 N2A2N2 273 Uncultured Synergistes, Acidaminococcusfermentans N2A2N2 281 Spirocliaeta sp., Treponema sp. N6 95, 103 Bulleidia sp. , 11111133 Clones from ennched rumen contents in a lact1c ac1d medla usmg d1fferentaerob1c1t1es; N6, 6 passes 1n anaerobic media; N2A2N2, 2 passes in anaerobic, 2 passes in aerobic, and 2 passes in anaerobic media; N2A4, 2 passes in anaerobic and 4 passes in aerobic media. 2 Bacteria closely related to query clone sequence with highest score of BLAST hits. 3 . . . . Uncultured human mtestmal bacteria. Uncultured rumen bactena. I Anaerobic @H I Anaerobic (Nfl I Anaerobic (N )7 r Aerobic (A) I I Anaerobic (N) I I Aerobic (A) j I Anaerobic (Nfl I Anaerobic (N) I I Aerobic (A) 7 LAnaerobic (N) I I Anaerobic (N) I I Aerobic (Mi N6 N2A2N2 N2A4 Figure 3.1. Enrichment conditions for three microbial treatments. N6 passed only anaerobic condition, N2A2N2 passed two anaerobic, two aerobic, and two anaerobic conditions, sequentially. N2A4 treatment passed two anaerobic, and four aerobic conditions. Each pass lasted 24 h at 39°C. 105 Chapter 4 Isolation, Identification, and Effects of the Ctr-Culture of M. Elsdenii, Strain RK02 and E. Faecium, Strain RK03 on Ruminal Fermentation In Vitro SUMMARY Rumen fluid collected from cows fed a high concentrate diet was enriched in aerobic lactic acid media, and a co-culture of two bacteria was isolated. This co-culture was screened and purified through a series of cross-passes in aerobic and anaerobic conditions. One was closely related to M. elsdenii and the other matched E. faecium through carbon substrate utilization tests using GP2 and AN MicroPlateTM and phylogenic analysis of 16S rDNA. Isolates were designated M. elsdenii RK02 and E. faecium RK03. The combination of RK02 and RK03 grew faster in a highly reduced condition than in an aerobic medium; however after 24 h of incubation, bacterial numbers and fermentation profiles were similar between the reduced and aerobic conditions. RK02 and RK03 could maintain viability during storage in aerobic conditions. Four ruminal in vitro fermentation experiments were performed with a 2 x 2 factorial arrangement of RK02 and RK03. In experiments 1 and 2, ground corn was the substrate and in experiments 3 and 4, readily fermentable carbohydrates served as the Substrate. Ruminal fluid sources were from cows adapted to a hay (experiments 1 and 3) or concentrate (experiments 2 and 4) diets. In trial 1, lactic acid did not accumulate in any treatment and addition of RK02 with RK03 increased fermented organic matter, butyric acid, valeric acid, isovaleric acid productions and pH of the culture. In trial 2, the cOmbination of RK02 and RK03 prevented lactic acid accumulation, increased total VFA, 106 butyric acid, valeric acid, isobutyric acid, isovaleric acid production and pH but, decreased acetic acid and propionic acid production. Supplementation of RK02 and RK03 decreased lactic acid production and increased total VF A and butyric acid, valeric acid and isovaleric acid production and pH in trial 3. Vigorous lactic acid production occurred in trial 4, and addition of RK02 and RK03 reduced lactic acid and acetic acid production, and increased total VFA, FOM, butyric acid, valeric acid, isovaleric acid production and pH of the culture. The co-culture of RK02 and RK03 was viable and active and resulted in modifications of the ruminal fermentation characteristics in all four in vitro fermentations. RK02 decreased the lactic acid concentration and increased pH when lactic acid accumulated in the culture. Therefore, when cattle are fed a high concentrate diet or are in a transition phase from hay- to concentrate-diets, the aero- tolerant combination of RK02 and RK03 as a direct-fed microbial (DFM) may decrease lactic acid production, increase ruminal pH and improve overall fermentation of the ruminal ecosystem. Keywords: Direct fed microbials, Rumen, Fermentation, Megasphaera elsdenii, Enterococcusfaecium, Aero-tolerance, Concentrate, Hay. 107 INTRODUCTION The term probiotic has been defined as a live microbial feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance (Fuller, 1989). This definition was broadened to a mono- or mixed-culture of live microorganisms which benefits man or animals by improving the properties of the indigenous microflora (Havenaar and Huis in’t Veld, 1992). Most recently, this definition has been further refined to living microorganisms, which upon ingestion at certain numbers, exert health benefits beyond inherent basic nutrition (Guamer and Schaafsman 1998). Ruminal probiotics may include live cultures of microorganisms that are deliberately introduced into the rumen with the aim of improving animal health or nutrition (Kmet et al., 1993). Microorganisms used as a direct-fed microbial (DFM) for ruminants include viable cultures of fungi, yeast and bacteria (Krehbiel et al., 2003). The United States Food and Drug Administration (FDA, 1988) required manufacturers to use the term DFM instead of probiotics. The FDA defines a DFM as a source of live naturally-occurring microorganisms. Most of the microorganisms being used as a DP M are classified as being generally recognized as safe (GRAS) according to the FDA and the American Association of Feed Control Officials (Miles and Bootwalla, 1991). Concern regarding the use of antibiotics and other growth stimulants in the animal feed industry has increased in recent years. As a result, interest in the effects of DFM on animal health and performance has increased. For ruminants, microbial cultures have been used to potentially replace or reduce the use of antibiotics in neonatal and stressed calves, to enhance milk production in dairy cows, and to improve preharvest safety, feed efficiency and daily gain in beef cattle (Krehbiel et al., 2003). 108 Inoculation of M. elsdenii as a DFM into the ruminal system has shown positive effects on fermentation that prevents ruminal acidosis characterized by low ruminal pH and high ruminal concentrations of lactic acid. Supplementation with M. elsdenii prevented lactic acid accumulation in laboratory fermentation studies during simulated of transition from a low— to a high-concentrate diet (Robinson et al., 1992; Kung and Hession, 1995). Oral drenching of this microorganism improved feed intake and prevented lactic acidosis in cattle switched from a 50 to 90% concentrate diet (Hibbard et al., 1993). However, drenching is unlikely to be acceptable as a general on-farm dosing method. A suitable method of adding ruminal anaerobes to the diet, while maintaining viability, is required before the idea of supercharging the rumen can be accomplished on a commercial basis (N agaraja et al., 1997). Therefore, in addition to common criteria for selection of probiotic microorganisms, aero—tolerance should be considered a necessary trial for DFM targeting the ruminal ecosystem. In accordance with the importance of aero-tolerance, facultative anaerobic species P. fieudenreichii and L. acidophilus have been used as DFM in large scale animal feeding trials. DF M containing Propionibacteria and Lactobacilli generally result in a 2.5 to 5% increase in ADG and an approximate 2% improvement in feed efficiency in feedlot cattle (Krehbiel et al., 2003). The belief that colonization and growth in the gut were prerequisites for probiotic activity prompted the use of intestinal isolates (Rettger et al., 1935). Common criteria for selection of probiotic microorganisms include origin of isolates as well as viability at the target site (Lee et al., 1999). Therefore, microorganisms isolated from the rumen may have an advantage over microbes from other origins in viability and metabolic activity in the rumen. 109 In Chapter 3, an enrichment method was developed to amplify the ruminal oxygen-tolerant lactic acid-fermenting microorganisms. Ruminal contents collected from cattle fed a concentrate diet were enriched in a lactic acid medium and incubated anaerobically (N6), anaerobically following aerobic conditions (N2A2N2), or under aerobic conditions only (N 2A4). Each of the enrichments was inoculated into in vitro ruminal fermentation system containing readily fermentable carbohydrates. Lactic acid concentration was 126, 99, 98, and 0.1 mM; and pH was 4.4, 4.5, 4.5, and 4.9 after 24 h of incubation for the untreated control, anaerobic enrichment (N 6), anaerobic following aerobic enrichment (N 2A2N2), or aerobic enrichment (N 2A4), respectively. In the aerobic enrichment, lactic acid concentrations were 78, 57, and 0 mM at 9, 12, and 24 h of incubation, respectively. The minimum pH, 4.7, was observed at 12 h, and increased to 4.9 at 24 h. Total VF A increased continuously during incubation. The lactic acid- fermenting microorganisms present in the aerobic enrichment appeared to be aero- tolerant. Therefore, exposure to aerobic conditions during enrichment appeared to develop aero—tolerant or facultative anaerobes and amplify the lactic acid-fermenting anaerobes. The hypotheses for the current study were that some isolates fiom the enriched mixed rumen culture in the aerobic lactic acid medium might possess properties to prevent lactic acid accumulation that is observed during acidosis, and that these isolates might be used as a DP M to prevent lactic acid accumulation and positively influence fermentation in the rumen. The objectives were to isolate oxygen tolerant lactic acid- ferrnenting rumen bacteria, and evaluate the effects of these isolates on in vitro ruminal fermentation. 110 MATERIALS AND METHODS Isolation and Identification Enrichment and Isolation. Rumen fluid was collected from two ruminally canulated dairy cows fed a high concentrate diet and enriched with M10 media containing 2% (v/v) of DL—lactic acid (modified from Caldwell and Bryant, 1966; Table 3.1). Enrichment included two passes in anaerobic condition and four passes in aerobic condition sequentially (similar to treatment N2A4 as described in Chapter 3). One mL of the enrichment culture was diluted with 9 mL of anaerobic dilution solution (Bryant and Burkey, 1953) and then serially diluted up to 1:10”). One mL of each dilution was inoculated onto Na—lactic acid agar medium prepared with the roll tube method (Hungate, 1966) or on the lactic acid agar plate placed in an incubation jar filled with C02 gas. After 3 d of incubation at 39°C, colonies were transferred to anaerobic NLB. Selection and Purification. To select the aero-tolerant anaerobic bacteria, both aerobic and anerobic NLB was prepared. Anaerobic NLB was boiled, bubbled with 02 free-CO; and added with reducing agent (Table 3.1), capped with rubber stopper while gassing Oz free-C02, and autoclaved at 121 °C for 15 min. Aerobic NLB was agitated to dissolve component and autoclaved. Colonies on the Hungate roll tubes or on the lactic acid agar plates were transferred into anaerobic NLB and incubated at 39°C. Afier 24 h of incubation until 3 d, colony cultures showing growth by optical density change were selected for chemical analysis. Lactic acid-utilizing colonies were transferred to aerobic NLB to confirm oxygen tolerance. Isolates that grew in NLB under aerobic conditions were purified through repetitions of colony picking from the roll-tubes and reculturing in 111 aerobic NLB. When repetition of dilution, roll-tube culturing and colony picking was not effective in purifying isolates, a modified purification method was developed. Colonies were transferred directly to anaerobic dilution solution. Dilution tubes for 1:101 to 1:103 contained 2 mL of glass beads (~2mm ¢)- For each dilution, tubes were vortexed for 30 s and serially diluted to 1:10'°. Na-lactic acid agar roll tubes were made with these diluted inoculants. Afier repetition of modified purification, uniform colonies and microscopic morphology were obtained for each isolate. Carbon Substrate Utilization. Microscopic morphology was observed after gram staining. Catalase activity was assessed by dropping 3% hydrogen peroxide on colonies on the plate. GP2 and AN MicroPlateTM were used to analyze the carbon substrate utilization according to the manufacturer’s recommendations (Biolog, Hayward, CA). Each test was duplicated for each isolate. The ability of isolates to oxidize 95 substrates was compared to identification databases using MicroLog3 4.01c. Phylogenetic Analysis. Genomic DNA of isolates was extracted using a UltraClean Fecal DNA kit (Mo Bio Laboratories Inc., Solana Beach, CA). The 16S rDNAs were PCR amplified in a reaction mixture containing 1 x PCR buffer, 200 mM dNTPs, 500 nM of each forward and reverse primer, and 0.05 U of T aq polymerase (Promega, Madison, WI) per al. The PCR program for 168 rDNA amplification was 96°C for 4 rrrin (for initial denaturation), followed by 32 cycles at 94°C for 1.5 min, 42°C for 1 min, 72°C for 4 min and a final extension at 72°C for 10 min. Primers were 8F (specific for bacteria, 5'-AGA GTT TGA TCC TGG CTC AG-3') and 1390R (5'-CG GTG TGT ACA AGG CCC-3'). PCR products were purified using a DNA purification kit (Promega). Partial 16S rDNA of clones was sequenced with primer 8F using ABI 112 3730xl high through-put capillary DNA sequencers at the Genomics Technology Support Facility at Michigan State University. Using each 16S rDNA isolate sequences as a query sequence, a local alignment was performed by means of the blastn (nucleotide-nucleotide BLAST (Basic Local Alignment Search Tool; http://www.ncbi.nlm.nih.gov/BLAST/). Sequences were aligned to Phylip format using ReadSeq.pl v.1.2 (http://www0.nih.go.jp/~jun/cgi-bin/readseq.pl) and Seqboot from the Phylip package (Felsenstein, 1993). Matrix pairwise comparisons were corrected for multiple base substitutions by the method of Juke and Cantor (Juke and Cantor, 1969). Phylogenetic trees were constructed by the neighbor-joining method (Saitou and Nei, 1987). A bootstrap confidence analysis was performed with 1000 replicates to determine reliability of the tree topology obtained (Felsenstein, 1985). Growth of Isolates in Aerobic Condition Aerobic growth of isolates RK02 and RK03 was evaluated through the incubation in NLB media with different concentration of reducing agents containing cysteine HC1 and sodium sulfide (Table 2.1.). Regarding the concentration (2.0 mM cysteine HC1 and 1.3 mM Na289HzO) recommended by Goering and Van Soest (1970) as a 100% of reducing agents, NLB with 100, 80, 60, 40, 20, or 0% of reducing agent were prepared (Table 4.2). The concentration of cysteine HCl was 0.0, 0.4, 0.8, 1.2, 1.6, and 2.0 mM and Na2S9H20 was 0.00, 0.26, 0.52, 0.78, 1.04, and 1.30 mM for O, 20, 40, 60, 80, or 100% of reducing agent, respectively. Co-culture of isolates RK02 and RK03 was incubated in NLB for 24 h at 39°C, and 0.1 mL of culture was transferred to 10 mL of NLB media containing 0, 20, 40, 60, 80, or 100% of the reducing agent. Each treatment 113 was conducted in triplicate. For each culture, optical density at 600 nm was measured every hour during 24 h of incubation. At 24 h, afier measuring OD values, cultures were used for organic acid analysis and colony counting. Five mL of culture were centrifuged at 24,000 x g for 20 min and 3 mL of supernatant were acidified with 12 N H2SO4 for organic acid analysis. One mL of each culture was transferred to 9 mL of anaerobic dilution solution (Bryant and Burkey, 1953) and then serially diluted up to 1:101°. One mL of each dilution from 1:106 to 1:10” was inoculated onto Na-lactic acid agar prepared with the roll tube method (Hungate, 1966) or on plates of Brain-Heart infusion agar (Difco, Sparks, MD) plate. Roll tubes were prepared and incubated anaerobically, and agar plates were aerobically incubated. Colonies on tubes or plates were counted after 3 d of incubation. Effects of Isolates as DF M ’s on In Vitro Ruminal Fermentation Experimental Design. A repeated measures design of an in vitro fermentation study included a 2 x 2 factorial arrangement of microbial treatments consisting of RK02 and RK03 (Table 4-1). Four trials were conducted with the four treatments. The treatments were tested in two ruminal fluid sources (concentrate vs. roughage diets) and two in vitro fermentation substrates (ground corn vs. readily fermentable carbohydrates (RFC); Table 4.1). Microbial Preparation. RK02 was grown in anaerobic NLB, and RK03 in Brain- Heart infusion broth at 39°C. Ten mL of each 24 h culture was pelleted by centrifugation at 6000 x g for 10 min and resuspended to 10 mL of in vitro buffer solution (Table 2.1; Goering and Van Soest, 1970). 114 T reatmenm. Rumen fluid was collected from two ruminally canulated lactating Holstein cows that were fed a high concentrate diet for trials 1 and 3, or from two Holstein heifers that were fed only a hay diet for more than 2 months for trials 2 and 4. Fermentation substrates were ground corn or RFC. RFC was comprised of soluble starch (5 5%), glucose (26%), cellulose (ground filter paper, 6%), cellobiose (7%), tryptone (3%), and proteose peptone (3%) on wt/wt basis (modified from Kung and Hession, 1995) Fifty mL of buffer solution was placed into a 250 mL round bottom flask containing 3 g of ground corn, then 50 mL of strained rumen fluid was pipetted into the buffer solution for trial 1 and 2. RFC except cellulose (ground filter paper) was dissolved in buffer solution, then 50 mL of buffer solution with substrates was pipetted into a 250 mL round bottom flask containing cellulose for trials 3 and 4. The final amount of RFC was 3 g/flask. Fifty mL of strained rumen fluid was pipetted into the buffer solution. Treatment RK02 received 1 mL of RK02 suspension and 1 mL of pure buffer solution, and treatment RK03 received RK03 suspension and 1 mL of pure buffer solution. Treatment RK02 + RK03 received 1 mL of RK02 and 1 mL of RK03, and the control treatment received 2 mL of pure buffer solution. Treatments were conducted in triplicate. All procedures were performed anaerobically as described by Goering and Van Soest (1970). Flasks were incubated at 39°C, and 5 mL of culture media were removed while gassing flasks with 02 free-C02 at 0, 6, 12, 18, and 24 h of incubation. Lactic acid, VFA, and pH Analysis. Ruminal pH was recorded immediately after 115 sample collection and incubation fluid was centrifuged at 24,000 x g for 20 min and 25 mL of supernatant was acidified with 12 N H2804 for organic acid analysis. Lactic acid and VFA contents were determined by ion exchange exclusion HPLC (Aminex HPX-87 h; Bio-Rad, Richmond, CA) as described in Chapter 2. Apparently fermented organic matter (F OM) was calculated from VF A stoichiometry (Demeyer and Van Nevel, 1979) with a slight modification where caproate was replaced by lactic acid and isobutyric acid: F OM (moles of hexose) = (lactic acid + acetic acid + propionic acid)/2 + butyric acid + isobutyric acid + isovaleric acid + valeric acid, with all VFA expressed in mmoles produced. Statistical Analysis Changes of organic acid concentration and pH between sampling times were analyzed with repeated measures using the MIXED procedure (SAS Inst. Inc., Cary, NC), with fermentation tube as the experimental unit. Auto-regression was determined as the most appropriate covariance structure using the Schwarz Baysean criterion (Littell et al., 1998). Incubation time served as a classification effect. The model included microbial treatment, time, and the resulting interaction. Initial concentrations of lactic acid and VF A and pH of the fermentation system were analyzed using GLM procedure of SAS. When a main treatment effect existed, means separation was performed according to Fisher's Protected LSD test (P < 0.05). Significance was declared at P < 0.05. 116 RESULTS Isolation and Identification The new isolates were cultivated in anaerobic and aerobic NLB. Colonies in anaerobic roll-tubes showed two kinds of morphology. Both 1~2 mm round and pin-point shaped colonies were observed at dilution levels of 1:107, 1:108, and 1:109. The pin-point size colony did not utilize lactic acid, and the large round-shaped colony utilized lactic acid in both aerobic and anaerobic NLB. However, this large round isolate still contained pin-point colony after several repetitions of roll-tube formation or streaking on slant agar medium. A modified purification method was developed in this study using an anaerobic roll-tube procedure. Dilution solution tubes for 1:101 to 1:103 contained 2 mL of glass beads (~2mm (b) to allow separation of bacterial clones in the colony. For each dilution, tubes were vortexed for 30 s and transferred serially up to 1:1010 dilutions. Na—lactic acid agar roll-tubes were made with these diluted inoculants. After repetition of modified purification, uniform colonies and microscopic morphology were obtained for each isolate. The purified large round colony did not grow in aerobic NLB, but anaerobic NLB, and was a Gram negative coccus, designated as strain RK02. The small pin-point colony grew in both aerobic and anaerobic NLB, and was a Gram positive coccus, designated as strain RK03. RK02 was tested for its ability to utilize various carbon substrates with the Biolog AN MicroPlateTM system (Appendix Table 6.1) and compared to identification databases using Biolog MicroLog3 4.01c. RK02 matched Megasphaera elsdenii with 100% probability and 0.867 similarity. RK03 was tested using GP MicroPlateTM (Appendix A. Table 6.2) and matched Enterococcusfaecium with 97% probability and 117 0.96 similarity. Isolates were re-designated M. elsdenii RK02 and E. faecium RK03. To confirm the identification of isolates, partial sequences of 16S rDNA were obtained (Appendix B. Figures 6.1 and 6.2). A local alignment of each sequence was performed using BLASTN (Altschul et al., 1997) against sequences in the database from GenBank, EMBL, DDBJ, and PDB which contained 4,084,561 sequences through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). Based on the 497-nucleotide sequence obtained for isolate RK02 (Figure 6.1), BLAST generated 66 hits having an E-value of 0.0 including 14 strains of M elsdenii, one M. homis, and uncultured bacteria (Appendix A. Table 6.4). The highest BLAST scores (939~901) were found for the species M. elsdenii (Appendix B. Figures 6.3 and 6.5). BLAST search generated more than 100 hits having an E-value of 0.0 based on the partial 494-nucleotide sequence of isolate RK03 (Appendix B. Figure 6.2). The highest BLAST scores (944~902) were found for the five species: Enterococcusfaecium. E. azikeevi, E. hirae, E. durans, and E. lactis (Appendix A. Table 6.5). This demonstrates that the Enterococcus genus is closely related to isolate RK03 based on the 16S rRN A sequences (Appendix B. Figures 6.4 and 6.5). Growth of Isolates in Aerobic Conditions To confirm aerobic growth and evaluate the effects of aerobicity on growth of the combination of RK02 and RK03, these microorganisms were inoculated into the NLB containing different reducing equivalents. Growth was measured as optical density at 600 nm (Figure 4.1). The lag phase was longer in 0% and shorter in 80% and 100% than the other reducing equivalents (P < 0.05; Appendix A. Table 6.5). Because the OD values 118 represented growth of the mixture and the objective was to verify growth of isolate RK02, viable counts and fermentation products at 24 h of incubation were analyzed. Levels of fermentation products were -114.6 i 1.88, 15.57 :1: 1.07, 34.74 i 3.40, 25.30 i 1.96, 24.37:l: 1.72, 2.11:t 0.12, 9.42 :1: 0.20, 111.5 :1: 5.5 mM for lactic acid, acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, isovaleric acid, and total VFA, respectively (Table 4.2). Viable numbers of RK02 and RK03 were 31.5 :1: 2.42 x 107 and 49.6 i: 3.25 x 10° cfu/mL, respectively (Table 4.2). VFA production, lactic acid utilization and microbial numbers were similar between reducing agent proportions after 24 h of incubation. The combination of RK02 and RK03 in aerobic conditions may require a longer adaptation period than under anaerobic conditions. The reducing agent facilitated growth of the combination; however, after 24 h of incubation, their growth and fermentation characteristics were similar. RK02 grew successfully in aerobic conditions when RK03 was present. Trial I . Ground Corn as Substrate and Rumen Fluid from Cows Fed a Concentrate Diet Changes in fermentation product concentrations and pH at 6 h interval over 24 h of incubation are shown in Tables 4.3 to 4.14. The values in the table represent the changes between 0 and 6, 6 and 12, 12 and 18, and 18 and 24 h of incubation. Concentrations of organic acids and pH at time 0 are shown on the left column of each table. Positive values represent the production and negative values mean the utilization of the organic acids. Microbial treatment effects on in vitro ruminal fermentation using rumen fluid from concentrate diet adapted cows and ground corn as substrate are shown 119 on Tables 4.3 to 4.5. Fermented organic matter was greatest at 6 h for all treatments and decreased thereafter (P < 0.05; Table 4.3). RK02 had a greater increase in FOM (P < 0.05) between treatments at 6 h and 18 h. RK03 had a greater FOM (P < 0.05) than the control at 18 h and 24 h. RK02 + RK03 had a greater F OM (P < 0.05) than the control at 24 h. The control had a greater FOM (P < 0.05) than the other treatments at 12 h. The average increase in F OM over time was smaller for the control than the other treatments (P < 0.05). The average initial pH was 6.58 (Table 4.3). RK02, RK03 and control had larger (P < 0.05) decreases in pH at 6 h than the combination. RK02 + RK03 exhibited the greatest change (P < 0.05) in pH at 12 h. The average reduction in pH was smaller in RK02 + RK03 than the other treatments (P < 0.05). The concentration of lactic acid was greater in RK03 and less in RK02 initially than the control or the combination treatment (P < 0.05; Table 4.3). This likely reflects differences in concentration of the inoculation volumes and(or) the early fermentation processes during preparation of treatments before initial sampling at O h. Changes in lactic acid concentration were negative during the first (0 to 6 h) and last period (1 8 to 24 h), and positive between 6 and 18 h in all treatments. Rumen fluid used in this trial may have contained populations of lactic acid-utilizing bacteria to ferment initial lactic acid contents in the fermentation vessels. At 6 h, RK03 exhibited the greatest (P < 0.05) and RK02 (P < 0.05) the smallest decrease in lactic acid compared to other treatments, however the extent of the change was limited to initial concentrations. RK02 + RK03 had the smallest (P < 0.05) increase in lactic acid production at 12 h, and RK02 had less lactic 120 acid increase (P < 0.05) than the control and RK03. The control and RK03 produced more (P < 0.05) lactic acid than RK02 + RK03 at 24 h. The net accumulation of lactic acid over 24 h was negative for treatments containing RK02 (P < 0.05). The initial concentration of total VFA was greater in RK02 than the other treatments (P < 0.05) and lowest in RK02 + RK03 (P < 0.05; Table 4.3). Total VFA production rate decreased over time (P < 0.01) for all treatments. RK02 and RK03 had a greater increase in total VFA than the combination treatment at 6 h. Less (P < 0.05) increase in total VFA was seen for all microbial treatments compared to the control at 12 h. RK02 exhibited the greatest increase in VFA (P < 0.05) among treatments after 18 h and RK03 was greater (P < 0.05) than the control. Afier 24 h, the combination treatment had a greater increase in total VF A compared to the control and RK02. RK03 had a greater increase than the control at 24 h (P < 0.05). The average production level for total VFA across all periods was greater in RK02 and RK03 than the control (P < 0.05). The initial acetic acid concentration was less in RK02 + RK03 (P < 0.05; Table 4.4) than the other treatments. During incubation, the rate of acetic acid production decreased over time (P < 0.01). Acetic acid production was greater in RK03 and less in RK02 + RK03 than the control or RK02 at 6 h (P < 0.05). After 12 h, RK02 exhibited the greatest acetic acid production among all treatments, and RK03 had greater production than the control (P < 0.05). RK02 and RK03 had smaller production rates than the control or the combination treatment at 18 h (P < 0.05). For the last period, acetic acid served as a substrate for all treatments. Utilization was highest in the control and lowest in RK02 + RK03 (P < 0.05). The initial concentration of propionic acid was lowest in the combination 121 treatment (P < 0.05; Table 4.4). Propionic acid production was greatest during the first period (between 0 and 6 h), and decreased over time in all treatments (P < 0.01). RK03 and the control had greater (P < 0.05) changes in propionic acid concentration than RK02 and RK03 + RK03. Likewise, RK02 had a greater production rate than the combination treatment (P < 0.05). RK02 + RK03 had a smaller change in propionic acid than the control at 12 h (P < 0.05). The change in propionic acid was greater in RK02 and RK03 (P < 0.05) than the control at 18 h. RK02 + RK03 had greater propionic acid production than the control at 24 h. The average periodic increase in propionic acid was smaller in RK02 + RK03 than the other treatments (P < 0.05). The ratio of acetic acid and propionic acid was decreased over time (P < 0.01; Table 4.4). After 18 h, the change in the ratio was greater (P < 0.05) with the combination treatment than RK02 or RK03. Because of the negative values of acetic acid, the ratio at 24 h was not calculated. Butyric acid concentration was greater in RK02 than the other treatments initially (P < 0.05; Table 4.5). Butyric acid production was greater in RK02 and RK02 + RK03 than the control at 6 h (P < 0.05). By 12 h, the greatest greater (P < 0.05) production rate was in the control treatment and the lowest with RK02 (P < 0.05). A similar pattern in production rate was observed at 6 and 18 h where RK02 exhibited the greatest and the control the least (P < 0.05). The average across periods demonstrated that control produced lower amounts of butyric acid compared to the other treatments (P < 0.05). Isobutyric acid production during the first 6 h was greater in RK02 and RK02 + RK03 than the control and RK03 (P < 0.05; Table 4.5). The combination treatment and control had greater production than RK02 and RK03 at 12 h (P < 0.05). Some isobutyric 122 acid was utilized between 6 and 12 h for RK02 and RK03 (P < 0.05). Conversely, an opposite trend was observed at 18 h. Utilization occurred from the control and RK02 + RK03 and a net production occurred for RK02 and RK03 (P < 0.05). Isobutyric acid production was smallest in the combination treatment at 24 h (P < 0.05; Table 4.5). Across time periods, RK02 had a greater isobutytic acid production rate than the other treatments (P < 0.05). Initial valeric acid concentration was greater in RK02 and RK02 + RK03 than the control or RK03 (P < 0.05; Table 4.5). Production of valeric acid was also greater in RK02 and RK02 + RK03 than the control or RK03 at 6 h (P < 0.05). RK02 had a greater increase than the control or RK03 at 18 h (P < 0.05). The production rate was lower (P < 0.05) for the control at 24 h than RK03 or RK02 + RK03. Averaged across times, the control had the smallest production rate and RK02 and the combination the greatest (P < 0.05). The initial concentration of isovaleric acid was greater in RK02 than the control and RK02 + RK03 (P < 0.05; Table 4.5). RK02 and the combination treatment had a greater increase in isovaleric acid production than the control and RK03 at 6 h (P < 0.05). During the second 6 h period (12 h), isovaleric acid was utilized in RK02 and RK03 (P < 0.05). A net accumulation occurred for the control and the combination treatments. At 18 h, the reverse was observed where a net utilization occurred for the control and the combination, but a net accumulation for RK02 and RK03 (P < 0.05). A greater production rate of isovaleric acid was observed for RK03 and the combination treatment than the control and RK03 at 24 h (P < 0.05). The average increase in isovaleric acid was smaller in the control than the other treatments (P < 0.05). 123 Trial 2. Ground Corn as Substrate and Rumen Fluid from Cows Fed a Hay Diet Effects of microbial treatments on in vitro ruminal fermentation using rumen fluid from hay diet-adapted cows and ground corn as substrate are shown in Tables 4.6 to 4.8 (A - C). The time by treatment interaction was not significant for F OM in this experiment (P = 0.25). RK02 and the combination had greater fermented organic matter than the control and RK03 (Table 4.6). The initial pH of RK02 was higher than RK03 or the combination treatment, and the control had a higher pH than the combination treatment (P < 0.05; Table 4.6). The combination had the smallest decrease in pH among treatments at 6 h (P < 0.05). However, at 12 h, RK02 had a smaller decrease than RK03 or RK02 + RK03 (P < 0.05). Decrease in pH was less for RK02 and the combination treatment than the control or RK03 at 18 h (P < 0.05). During the last period (between 18 and 24 h), pH changed less (P < 0.05) for RK02 and the combination treatment than the control and RK03. The average pH dr0p was less (P < 0.05) for RK02 and RK02 + RK03 than the other treatments. The initial concentration of lactic acid was greater in RK03 than the other treatments, and greater in RK02 + RK03 than the control and RK02 (P < 0.05; Table 4.6). A net utilization of lactic acid occurred by 6 h for all treatments. The lowest utilization rate was observed in the control treatment (P < 0.05). Production rates were less for RK02 and the combination at 12 h (P < 0.05) than the control or RK03. During the last two periods from 12 to 24 h, a net utilization of lactic acid occurred for RK02 and the combination treatment, whereas a net production was observed in the control and RK03 treatments. Over the entire 24 h period, there was a net lactic acid utilization in RK02 and 124 RK02 + RK03 and a net production in the control and RK03 (P < 0.05). A treatment by time interaction was non-significant in the model for total VF A. Averaged across time, RK02 and the combination produced more total VFA than the control or RK03 (P < 0.05; Table 4.6). The treatment by time interaction term was non-significant in the models for acetic acid, propionic acid and the resulting ratio (P < 0.05; Table 4.7). Acetic acid and propionic acid production rates were smaller for RK02 and the combination (P < 0.01) than the control or RK03. The A/P ratios were similar for all treatments. Initial butyric acid concentration was greater in RK02 + RK03 than the control and RK03, and greater in RK02 than RK03 (P < 0.05; Table 4.8). RK02 and the combination treatment had greater butyric acid production rates than the control and RK03 at 12, 18, and 24 h (P < 0.05). The change was greater in RK02 than the combination treatment at 18 h (P < 0.05). Over the incubation period, butyric acid production rates were greater in RK02 and RK02 + RK03 than the control and RK03 (P < 0.05; Table 4.8). Isobutyric acid production rates were similar between treatments at 6, 12, and 24 h (Table 4.8). Isobutyric acid production rates were similar between 12 and 18 h in RK02 and the control, but lower (P < 0.05) in RK03 and RK02 + RK03. Initial concentration of valeric acid was greater in RK02 and RK02 + RK03 than the control and RK03 (P < 0.05; Table 4.8). During every time period, RK02 and the combination had greater (P < 0.05) rates of valeric acid than RK03 and control. Over the entire incubation period, valeric acid production rates were greater in RK02 and RK02 + RK03 than the control and RK03 (P < 0.05; Table 4.8). 125 Production of isovaleric acid was greater (P < 0.05; Table 4.8) for RK02 than the other treatments at 18 h. By 24 h, the combination treatments exhibited the greatest production rate (P < 0.05). Treatments that contained RK02 had greater production rates than RK03 or the control over the entire incubation period. Trial 3. RFC as Substrate and Rumen Fluid from Cows Fed a Concentrate Diet Effects of microbial treatments on in vitro ruminal fermentation using rumen fluid from concentrate diet-adapted cows and readily fermentable carbohydrates (RFC) as substrate are shown on Table 4.9 to 4.11. The change in fermented organic matter was similar among treatments (Table 4.9). The average initial pH was 6.67 (Table 4.9). The decrease in pH during the first 6 h was greatest for RK03 and least for RK02 (P < 0.05). The control exhibited the greatest decrease in pH among treatments at 12 h (P < 0.05). RK02 had a greater decrease in pH than RK03 or RK02 + RK03 at 12 h (P < 0.05). The rate of pH change was similar among treatments at 18 and 24 h. Treatments that contained RK02 had smaller decreases in pH (P < 0.05) than the control or RK03 over 24 h of incubation. Initial lactic acid concentration was greater in the control than other microbial treatments (P < 0.05; Table 4.9). The combination treatment had the lowest average production rate (P < 0.05) over 24 h period compared to the other treatments. The rates for production of total VFA (Table 4.9), acetic acid (Table 4.10) and propionic acid were similar among treatments. The ratio of acetic acid to propionic acid was also similar among treatments (Table 4.10). Butyric acid concentration increased more rapidly in RK02 and the combination 126 than the control and RK03 at 6 h (P < 0.05; Table 4.11). A net utilization of butyric acid occurred in RK03 and control at 12 h, whereas a net accumulation of acid occurred for RK02 (P < 0.05). At 18 h, the combination treatment exhibited the greatest production rate (P < 0.05). The average production rate across periods was greater for RK02 and the combination treatment than the control or RK03 (P < 0.05). Isobutyric acid production was greater in RK03 than the control or the combination treatment during the first 6 h (P < 0.05; Table 4.11). Utilization of isobutyric acid occurred in the second 6 h period for all treatments. Initial concentration of valeric acid was greater in RK02 than the control and RK03 (P < 0.05; Table 4.11). RK02 and the combination of treatment had a greater increase in valeric acid production than the control and RK03 at 6 h (P < 0.05). At 12 h, RK02 exhibited the greatest production rate for valeric acid among treatments and RK02 + RK03 was greater than the control (P < 0.05). Over the entire incubation, valeric acid production rates were greatest for RK02 and the combination treatment (P < 0.05). The average production rate for isovaleric acid was greater in RK02 and RK02 + RK03 than the control (P < 0.05). Trial 4. RFC as substrate and rumen fluid from cows fed a hay diet Effects of microbial treatments on in vitro ruminal fermentation using rumen fluid fiom hay diet-adapted cows and readily fermentable carbohydrates as substrate are shown in Table 4.12 to 4.14. The increase in FOM was greater for RK02 and the combination treatment than the control or RK03 at 6 and 18 h (P < 0.05; Table 4.12). RK02 + RK03 exhibited the 127 greatest FOM increase among all treatments, and RK02 had a greater increase than the control at 12 h (P < 0.05). RK03 had greater (P < 0.05) FOM than RK02 at 24 h. RK02 + RK03 had the lowest amount of OM fermented at 24 h (P < 0.05). Overall, RK02 and RK02 + RK03 had greater amounts of FOM than the control and RK03 (P < 0.05). Initial pH was highest in the control and lowest in the combination treatments (P < 0.05; Table 4.12). During the first 6 h, pH declined by 2 units for all treatments. Decreases in pH were smaller in RK02 and RK02 + RK03 than the control and RK03 at 6 and 24 h (P < 0.05). pH decreased in RK03 at 18 h (P < 0.05), but increased in RK02 + RK03 (P < 0.05). The average pH decline across periods was smaller for treatments containing RK02 (P < 0.05). Initial lactic acid concentration was greatest in RK03 and was greater in RK02 + RK03 than the control (P < 0.05; Table 4.12). The increase in lactic acid concentration was smaller in RK02 and the combination treatment than the control and RK03 at 6 and 18 h (P < 0.05). At 18 h, a net utilization of lactic acid occurred in RK02 and the combination treatments (P < 0.05), whereas lactic acid accumulated in the other two treatments. By 24 h, a net utilization was maintained by the combination treatment (P < 0.05), whereas lactic acid accumulated in the other three treatments with the largest increase in RK03. The average lactic acid level was lower for RK02 and the combination treatment (P < 0.05) than RK03 and control during 24 h of incubation. Initial total VF A concentration was greater in RK02 than the control and RK03 (P < 0.05; Table 4.12). The increase in total VFA was greater in RK02 and the combination treatment than the control and RK03 at 6 and 18 h (P < 0.05). At 12 h, RK02 + RK03 exhibited the greatest increase in total VFA and control the least (P < 0.05). Increase in 128 concentration was greater in the combination than RK02 at 18 h (P < 0.05). A net utilization of VFA occurred at 24 h for all treatments except the control. Average of total VFA production was greater in RK02 and RK02 + RK03 than the control and RK03 (P < 0.05) over 24 h of incubation. Acetic acid concentration was less in the control than other treatments initially (P < 0.05; Table 4.13). Acetic acid production was less in RK02 and the combination treatment than the control and RK03 at 6 h (P < 0.05). The 24 h average followed a similar pattern with treatments containing RK02 having lower production rates than RK03 and the control (P < 0.05). Propionic acid production rates were greater in RK02 and RK02 + RK03 than the control and RK03 at 12 and 18 h (P < 0.05; Table 4.13). A net utilization of propionic acid occurred at 18 h for the control and RK03. The increase in propionic acid concentration was smallest in RK03 at 24 h (P < 0.05). Over the 24 h incubation period, propionic acid concentration was greater in RK02 and RK02 + RK03 than the control and RK03 (P < 0.05). The ratio of acetic acid and propionic acid production was similar between treatments (Table 4.13). 3 Initial butyric acid concentration was greater in RK02 than the other treatments, and in RK02 + RK03 than the control (P < 0.05; Table 4.14). Butyric acid production rates were greater in RK02 and RK02 + RK03 than the control or RK03 at 6, 12, and 18 h (P < 0.05). The combination treatment had a greater increase than RK02 at 6 h (P < 0.05). A net utilization of butyric acid occurred in the control and RK03 at 12 and 18 h. The combination treatment exhibited the greatest utilization rate (P < 0.05) of all treatments at 24 h. Overall, the average production rate of butyric acid was greater for RK02 and RK02 129 + RK03 (P < 0.05) than the control or RK03. Isobutyric acid concentration changes were similar among treatments (P < 0.05; Table 4.14). Initial valeric acid concentration was greatest in RK02, and greater in RK02 + RK03 than the control or RK03 (P < 0.05; Table 4.14). Valeric acid production rates were greater in RK02 and RK02 + RK03 than the control or RK03 at 6 and 18 h, and in the overall average (P < 0.05). Isovaleric acid utilization during the second 6 h period was greater in RK02 and RK02 + RK03 than the control or RK03 (P < 0.05; Table 4.14) with production greater in RK02 and RK02 + RK03 than the control or RK03 at 18 h (P < 0.05). Isovaleric acid concentration decreased in all treatments and RK02 had a greater decrease than RK03 and RK02 + RK03 at 24 h (P < 0.05). The average isovaleric acid production rate was greater in RK02 + RK03 than the control or RK03 (P < 0.05). 130 DISCUSSION Anaerobes may be classified into three groups; 1) strict anaerobes which grow on spread plates only when the atmosphere contains less than 0.5% Oz, 2) moderate anaerobes which would generally grow on plates when the atmosphere contains less than 10% Oz, and 3) microaerophiles which require a low concentration of O; for optimal growth, but are unable to grow in air. Using these classification, M. elsdenii was classified as a moderate anaerobe (Loeche, 1969). Facultative anaerobes are more tolerant to oxygen because they have the capacity to detoxify the superoxide ion. In this study, M elsdenii RK02 was isolated from an enrichment exposed to four passes in aerobic culture conditions, but failed to grow aerobically. When RK02 was combined with the co-isolate, E. faecium RK03, RK02 grew in aerobic lactic acid media and fermented lactic acid. Each colony of RK02 in the lactic acid agar roll-tubes contained RK03. RK03 did not grow in lactic acid media. These two isolates may have a symbiotic relationship in an aerobic environment that contains lactic acid. Co-isolates grew faster in a highly reduced environment than under aerobic condition (Table 4—2); however microbial numbers and fermentation products were similar between the aerobic and reduced conditions after 24 h of incubation. Therefore, RK02 with RK03 could be maintained in an aerobic media. Bacteria in the rumen are often classified as lactic acid-producers or lactic acid- utilizers, and the balance between these two groups determines whether lactic acid accumulates (Russell and Hino, 1985). Most amylolytic bacteria produce lactic acid. Bacteroides, Butyrivibrio, Succinivibrio, and Ruminicoccus species produce lactic acid as a minor fermentation product whereas Lactobacillus and Streptococcus species account 131 for a large portion of the lactic acid produced in the rumen, especially at lower pH. Lactobacillus is particularly prominent in the microflora of young ruminants and at rumen pH below 5.0. In animals fed rations containing large amounts of readily fermentable carbohydrate, lactobacilli often proliferate in company with Streptococcus species, thus creating highly acidic conditions. Streptococcus bovis ferments starch to lactic acid as a main end product and is capable of very rapid grth (Stewart and Bryant, 1988). M. elsdenii is mainly found in the rumen of young animals and in animals receiving high grain rations and its growth usually occurs on glucose, fructose and lactic acid. Lactic acid is fermented by M. elsdenii mainly to butyric acid, propionic acid, isobutyric acid, valeric acid, CO2 and H2. This same organism ferments glucose to caproate and formate with some acetic acid, propionic acid, butyric acid, and valeric acid. In the current study, FOM increased throughout the incubation period in all in vitro fermentations which suggests that a fermentation continued. Supplementation with RK02 + RK03 increased F OM in Trials 1, 2, and 4 (Figures 4.2 and 4.4). In Trial 4, FOM increased rapidly until 6 h, and plateaued thereafter in the control. RK02 + RK03 increased FOM throughout the incubation. Greater FOM may result from a greater proportion of butyric acid in RK02 + RK03 than the control when total organic acid concentrations were similar (trials 1 and 2). Final pH was lower when RFC was used as substrate than ground corn, and RK02 + RK03 increased the pH in all trials (Figures 4.2 and 4.4). RK02 + RK03 had a higher pH than the control from 6 to 24 h in trials 1, 2, and 4; and from 12 h to 24 h in Trial 3. 132 Lactic acid accumulated in trials 2, 3, and 4 (Figures 4.2 and 4.4). When ground corn was fermented with rumen fluid from concentrate diet adapted cows (Trial 1), lactic acid did not accumulate in the culture during the 24 h of incubation. Rumen fluid from concentrate diet may have sufficient populations of lactic acid-fermenting bacteria to prevent lactic acid accumulation and(or) contain other microorganisms that are more competitive than lactic acid-producing bacteria for the substrate, ground corn. RK02 + RK03 successfully removed all lactic acid in the media in Trial 2, and 29 and 34% of the lactic acid in trial 3 and 4, respectively. A drastic decrease in pH and accumulation of lactic acid during the first 6 h of fermentation of readily fermentable carbohydrates in trial 3 and 4 may have depressed lactic acid utilization by RK02 + RK03. Consistent with the current study, M. elsdenii inoculation modified ruminal fermentation and prevented lactic acid accumulation in in vitro fermentation using rumen fluid from hay adapted cows with RFC as a substrate (Kung and Hession, 1995). RK02 + RK03 increased total VF A production in trials 2 and 4 when the rumen fluid was from cows fed a hay diet (Figure 4.2 and 4.4). In trial 4, VFA concentration plateaued after 6 h similar F OM in the control treatment, whereas RK02 + RK03 continued to increase total VFA accumulated. In trials 3 and 4, lactic acid accumulated in RK02 + RK03 after 6 h, and pH dropped to below 5. At pH value below 5.0 in trials 3 and 4, RK02 + RK03 decreased lactic acid and increased total VF A. This observation demonstrates that RK02 + RK03 was acid-tolerant and has the potential to moderate ruminal lactic acidosis. Acetic acid concentrations decreased with RK02 + RK03 in trials 2 and 4, and butyric acid increased with RK02 + RK03 in trials 2 and 4 (Figures 4.3 and 4.5). 133 Propionic acid concentrations decreased with RK02 + RK03 addition in trials 1 and 2, whereas it increased in trial 4 (Figures 4.3 and 4.5). Propionyl CoA may be diverted toward the production of valeric acid (Marounek et al., 1989), thereby this may explain the decrease in propionic acid and increase in valeric acid production. RK02 + RK03 increased butyric acid and valeric acid in all trials, and both butyric acid and valeric acid were greatest in trial 2. In a previous trial, giving of E. faecium to steers fed a high concentrate diet decreased minimum pH, increased propionic acid, decreased butyric acid and NP ratio (Beauchemin et al., 2003). In trial 3, the combination of M. elsdenii (RK02) and E. faecium (RK03) yielded a higher propionic acid concentration than RK02. However, in trial 1, E. faecium (RK03) produced more butyric acid than the control. RK03 may have different metabolic activities than strain EF212 used by Beauchemin et al. (2003). Consistent with the current study, M. elsdenii inoculation modified the ruminal fermentation and prevented the accumulation of lactic acid during the transition from a low- to high- concentrate diet in in vitro and in vivo studies (Greening et al., 1991; Robinson et al., 1992; Kung and Hession, 1995). Greening et al. (1991) reported that inoculation of M elsdenii enhanced minimum pH and reduced lactic acid concentration (P < 0.002) in the acidotic rumen of beef cattle. Robinson et al. ( 1992) reported the effects of M elsdenii inoculation on feed intake, ruminal pH, osmolarity, lactic acid, and VFA concentration in steers with induced acute acidosis. M elsdenii inoculated steers had 24 % more DMI. In the study by Kung and Hession (1995), the pH of cultures treated with M elsdenii decreased to 5.3, while the control decreased to 4.8. Lactic acid concentration peaked at more than 40 mM in the control after 8 h and remained fairly 134 constant thereafter, but in the M elsdenii treatment, lactic acid was less than 5 mM throughout the incubation. Total VF A concentration of cultures treated with M elsdenii was more than twice that of the control (131 vs. 63 mM). The largest differences in individual VFA concentrations occurred in butyric acid, valeric acid, and branched-chain fatty acids. M elsdenii is an anaerobic bacterium and administration of this microorganism into the animal was performed by oral drenching to maintain viability (Hibbard et a1 ., 1993). However, drenching is unlikely to be acceptable as a general on-farm dosing method (Nagaraja et al., 1997), especially for daily dosage. In the current study, the co- isolates, RK02 and RK03 could grow in an aerobic environment with its aero-tolerance perhaps enhancing the commercial application of this technology. 135 IMPLICATIONS The combination of M elsdenii (RK02) and E. faecium (RK03) was isolated from aerobically enriched rumen fluid, and screened through cross-passes under aerobic and anaerobic conditions. The combination of RK02 and RK03 maintained viability in an aerobic environment. The aero-tolerance of this combination may provide an advantage of these bacteria over strict anaerobes in application to animals as a DFM. M elsdenii (RK02) with or without E. faecium (RK03) was viable and changed the VFA profiles in all ruminal fermentations in this study. RK02 decreased the level of lactic acid and increased pH when lactic acid accumulated in the culture. 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A neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 44:406—425. Samah, O. A. and J. W. T. Wimpenny. 1982. Some effects of oxygen on the physiology of Selenomonas ruminantium WPL 151/1 grown in continuous culture. J. Gen. Microbiol. 128:355-360. Stewart, C. S., H. J. Flint, and M. P. Bryant. 1997. The rumen bacteria. In The Rumen Microbial Ecosystem; P. N. Hobson and C. S. Stewart, Eds.; Blackie Academic & Professional: London ; New York; pp 10-74. Thompson J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. Verbeke, G. and G. Molenberghs. 2000. Linear Mixed Models for Longitudinal Data. Springer-Verlag, NY. 139 Table 4.1. Four in vitro studies utilized a 2 x 2 factorial arrangement of direct-fed microbial treatments Four in vitro studies Microbial treatments2 Substrate Ground 1 RK02 . . . . Rumen fluid source corn RFC M No addrtron Addition Concentrate diet Trial 1 Trial 3 No addition Control RK02 . . . . . RK02 + Hay dret Trial 2 Trial 4 Addition RK03 RK03 I Readily fermentable carbohydrates; 55% soluble starch, 26% glucose, 6% cellulose (ground filter paper), 7% cellobiose, 3% tryptone, and 3% proteose peptone on wt/wt basis (modified from Kung and Hession, 1995). 2 Megasphaera elsdenii RK02 and Enterococcusfaecium RK03 140 Table 4.2. Changes of organic acid and populations of the co-culture of Megasphaera elsdenii RK02 and Enterococcusfaecium RK03 in a Na-lactate broth medium containing different concentration of cysteine HC1 and Na-sulfide after 24 h of incubation Reducing agent 3 Change of concentration after 24 h of incubation, mmol/L RA, 0/0] concentration, Cysteine HCl Na2S9l-120 Lactic Total VFA Acetic Propionic Butyric 0 % 0.0 0.00 -1 16.5 100.6 14.73 30.97 22.29 20 % 0.4 0.26 -1 13.6 106.6 14.49 33.69 24.73 40 % 0.8 0.52 -113.5 108.6 15.75 31.17 25.16 60 % 1.2 0.78 -115.5 116.7 15.24 36.65 25.77 80 % 1.6 1.04 -115.5 123.0 17.71 40.15 26.64 100% 2.0 1.30 -113.0 113.7 15.52 35.81 27.19 Mean -114.6 1 1 1.5 15.57 34.74 25.30 SEM“ 1.9 5.5 1.07 3.40 1.96 p - val“? 0.72 0.13 0.38 0.42 0.58 Reducing agent . 3 ' 6 RA, %1 concentration, m Change of concentration, mmoVL Populatron at 24 h, cfu/mL Cysteine HCl Na2S9H2O Isobutyric Valerie Isovaleric RK02 (x 107) RK03 (x 106) 0 % 0.0 0.00 1.91 21.51 9.20 35.7 47.3 20 % 0.4 0.26 2.04 22.22 9.38 37.3 49.7 40 % 0.8 0.52 2.41 24.35 9.79 28.7 57.4 60 % 1.2 0.78 2.14 27.82 9.05 35.0 40.7 80 % 1.6 1.04 2.16 26.87 9.42 22.3 51.7 100 % 2.0 1.30 1.99 23.46 9.69 30.0 51.0 Mean 2.1 1 24.37 9.42 31.5 49.6 SEM4 0.12 1.72 0.20 2.5 3.2 p - van“? 0.14 0.13 0.16 0.56 0.83 1 The ratio (%) of reducing agents contained in the medium to that recommended by Goering and Van Soest (1970). Concentration of reducing agents (cysteine HC1 and sodium sulfide) in Na-lactate medium. 3 Difference of organic acid concentration between 24 and 0 h ([value] = [24 h] - [0 h]). Negative value of lactic acid represents the net utilization, positive volatile fatty acids the net production. 4 Standard error of mean. 5 . . . . P-value assoc1ated wrth reducrng agent concentration. 6 Colony forming unit at 24 h in NLB with different reducing agents concentrations. Colony counting was performed on lactic acid roll-tubes for RK02 and on plates of Brain-Heart infusion agar for RK03 after 3 d incubation at 39°C. 141 Table 4.3. Effects of M elsdenii RK02 with or without E. faecium RK03 on fermented organic matter, pH, and lactic acid production with rumen fluid from concentrate diet and ground corn as substrate (TrialD lnitial Changes between incubation time, h2 Trt 3 P-Value4 Treatments Contents] 6 12 18 24 Mean SEM Trt Time if." x 11116 Fermented organic matter (hexose) mmol/L Control 61.24 a 45.72 b 20.79 a 11.05 a 34.70 a 1.28 < 0.01 < 0.01 < 0.01 RK02 68.19 b 37.86 a 30.98 ° 13.69 a 37.68 ° RK03 63.86 a 39.72 a 26.08 b 17.62 ° 36.82 ° RK02 + RK03 64.23 a 40.53 a 24.30 3° 17.66 b 36.68 b Time Mean 64.38 40.96 25.54 15.00 pH Control 6.59 -092 a -054 b -0.10 -0.02 -039 a 0.03 0.01 < 0.01 < 0.01 RK02 6.60 -090 a -055 3" -0.09 -0.03 -o.39 8‘ RK03 6.58 0.95 a -051 ° 0.09 0.02 .039 a RK02+RK03 6.56 066° 41,63“ .012 —0.03 .035" Time Mean 6.58 -0.86 -0.56 -0.10 0.02 Lactic acid, mmol/L Control 3,79 b -3,79 b 201 ° 0.85 -127 b -0.55 b 0.21 < 0.01 < 0.01 < 0.01 RK02 2.52 a -252 c 1.35 b 0.49 071 “° -0.35 °° RK03 5.20 ° -503 a 2.18 c 0.39 -0.98 b -0.86 a RK02 + RK03 3.86 b -3.61 ° 0.45 a 0.83 -0.33 a -0.67 ab Time Mean 3.84 -3.74 1.50 0.64 -0.82 Total VFA, mmol/L Control 92.90 3° 94.09 ab 64.46 b 28.58 a 10.78 a 49.48 a 1.65 0.03 < 0.01 < 0.01 RK02 95.88 c 98.89 b 56.88 a 38.71 ° 14.57 8° 52.26 b RK03 93.15 ° 98.65 b 58.42 a 33.89 ° 18.59 °° 52.39° ab RK02 + RK03 90.49a 92.68 a 58.63 a 32.74 8° 19.77 c 50.95 Time Mean 93.10 96.08 59.60 33.48 15.93 I Concentration of contents in the fermentation vessels at time 0. 2 Differences of concentrations of organic acids and pH value for 6 h period. 3 Standard error of mean. 4 P-value associated with main effects; time, treatment, and interaction of time and treatment. a b 6 Means in a column within an item with unlike superscripts differ (P < 0-05)' 142 Table 4.4. Effects of M elsdenii RK02 with or without E. faecium RK03 on acetic, propionic acid, and the ratio of acetic acid to propionic acid with rumen fluid from concentrate diet and ground corn as substrate (Trialfl Initial Changes between incubation time, h2 Trt 3 P-value Treatments Contents1 6 12 18 24 Mean SEM Trt Time 11.“ x rme Acetic acid, mmoVL Control 43.79 b 32.22 b 15.78 a 6.97 ° -3.71 a 12.82 0.49 0.06 < 0.01 < 0.01 RK02 42.92 b 31.38° 18.45 c 4.82 a -1.94 ° 13.18 RK03 43.38 b 34.10° 17.59b 4.89 a -1.73 b 13.71 RK02 + RK03 40.73 a 28.463 16343" 7.81 ° -0.27 c 13.09 Time Mean 42.70 31.54 17.04 6.12 -1.91 Propionic acid, mmol/L Control 24.75 b 2989" 23.70b 9.44a 1.89“ 18.18bc 0.47 < 0.01 < 0.01 < 0.01 RK02 25.31 b 27.50b 20.95 ab 11.13 b 2.99” 15.65 ° RK03 25.07 b 30.45 c 21.98 ab 11.12 b 2.68 a 16.560 RK02 + RK03 23.80 a 24.81 a 20.31 a 9.90 3° 4.15 ° 14.80 a Time Mean 24.73 28.12 21.74 10.40 2.93 Acetate : Propionate Control 1.77 1.08 0.67 o_74b° -5 -5 0.11 < 0.01 < 0.01 < 0.01 RK02 1.72 1.14 0.88 0.43 a - - RK03 1.71 1.12 0.80 0.44 ab - - RK02 + RK03 1.71 1.15 0.80 0.71;c Time Mean 1.73 1.12 0.79 0.60 l . . . . Concentration of contents 1n the fermentation vessels at time 0. N Differences of concentrations of organic acids for 6 h period. Standard error of mean. P-value assoc1ated w1th main effects; time, treatment, and mteractron of time and treatment. DJ 5 . . The ratio was negative and means are not presented. a b c Means in a column within an item with unlike superscripts differ (P < 0.05). 143 Table 4.5. Effects of M elsdenii RK02 with or without E. faecium RK03 on butyric, isobutyric, valeric, and isovaleric acid production with rumen fluid from concentrate diet and ground corn as substrate (Trial 1L Treatments 1nitial 1 Changes between incubation time, h2 Trt SEM3 P-value4 Tn Contents 6 12 18 24 Mean Trt Time Tim: Butyric acid, mmol/L Control 18.17 a 24.38 a 19.94 c 12.53 a 6.29 a 15.78 a 0.44 0.01 < 0.01 < 0.01 RK02 19.56 ° 27.81 c 15.83 a 17.04 d 6.34 a 16.76 ° RK03 18.48 a 25.54 3° 17.55 b 15.11 c 8.36 b 16.64 ° RK02 + RK03 18.64 a 26.51 b 18.03 b 13.87 ° 7.79 b 16.55 b Time Mean 18.71 26.06 17.84 14.64 7.19 Isobutyric acid, mmol/L Control 1.61 2.50 a 1.68 b -1.61a 2.94 b 1.38 a 0.12 0.01 < 0.01 < 0.01 RK02 1.68 3.47 ° -0.38 a 0.55 ° 2.70 b 1.58 b RK03 1.81 2.55 a -0.36 a 0.42 ° 2.91 ° 1.38 “ RK02+RK03 1.72 324° 140° -1.15° 2.27a 1.44a Time Mean 1.71 2.94 0.58 0.45 2.71 Valerie acid, mmol/L Control 2.06 a 1.95 a 2.01 2,17 8" 153 8 1,93 3 0.40 < 0.01 0.02 0.01 RK02 3.09 b 4.19 b 2.84 3.75 ° 2.48 8° 3.31 c RK03 2.23 a 2.33 a 1.99 2.03 a 3.27 b 2.41 ° RK02 + RK03 2.98 b 4.78 b 1.75 3.20 °° 2.96 b 3.17 c Time Mean 2.59 3.31 2.15 2.79 2.57 Isovaleric acid, mmol/L Control 2.52 a 3.36 a 1.36 ° -0.93 a 1.78 a 1.39 a 0.25 < 0.01 < 0.01 < 0-01 RK02 2.86 b 4.55 b -0.81 a 1.42 ° 2.00 a 1.79 ° RK03 2.65 ab 3.67 a 0.34 a 0.32 b 3.09 ° 1.68 b RK02 + RK03 2.61 a 4.87 b 0.80 b -0.88 a 2.86 ° 1.91 b Time Mean 2.66 4.11 0.25 -0.02 2.44 I . . . . Concentratron of contents 1n the fermentatron vessels at trrne 0. 2 . . . . . Differences of concentratrons of orgaruc acrds for 6 h period. 3 Standard error of mean. P-value assocrated w1th main effects; time, treatment, and mteractron of time and treatment. abc Means in a column within an item with unlike superscripts differ (P < 0.05). 144 Table 4.6. Effects of M elsdenii RK02 with or without E. faecium RK03 on fermented organic matter, pH, and lactic acid production with rumen fluid from hay diet and ground corn as substrate (Tiial 2) Initial Changes between incubation time, h2 Trt 3 P-value4 Treatments Contents1 6 12 18 24 Mean SEM Trt Time git x lme Fermented organic matter (hexose) mmol/L Control 39.90 53.98 24.55 18.90 3433 a 6.53 < 0.01 < 0.01 0.25 RK02 47.90 64.91 51.79 23.72 47.08b RK03 51.33 51.95 15.26 17.79 34,113 “ RK02 + RK03 52.69 58.95 38.62 28.58 4471b Time Mean 47.96 57.45 32.55 22.25 pH Control 6.71 °° -O.58 a 090 3° -0.34 a 0.10 a -0.48 a 0.02 < 0.01 < 0.01 < 0-01 RK02 6.72 c -0.56 a -0.80 ° -0.11 b -0.03 b -0.38 b RK03 6.66 3° 0.56“ -092“ -032“ .009“ .047“ RK02 + RK03 6.64 a -0.42 b -0.84 b .010 ° 004 3° .035 ° Time Mean 6.68 .053 -0.87 0.22 -0.06 Lactic acid, mmol/L Control 4.18 a -o. 16 b 16.97 b 34.41 b 15.38 b 16.65 ° 1.24 < 0.01 < 0.01 < 0.01 RK02 4.14a 4.14“ 4.73a -182“ -0.813 0.51“ RK03 12.14 c -303 3° 19.70 b 35.68 b 12.80 b 16.29b RK02 + RK03 7.40 ° -6.42 a 4.28 a -3.37 a -0.63 a -1.53 a Time Mean 6.96 -3.44 11.42 16.23 6.69 Total VFA, mmol/L Control 97.66 62.24 69.07 8.61 13.83 31144 a 9.88 < 0.01 < 0.01 0.22 RK02 96.20 70.33 86.71 44.61 17.46 54,73b RK03 100.31 81.47 60.87 -2.88 14.90 33598 RK02 + RK03 105.02 78.23 77.21 34.08 23.60 5328 b Time Mean 99.80 73.07 73.47 21.10 17.45 1 Concentration of contents in the fermentation vessels at time 0. 2 Differences of concentrations of organic acids and pH value for 6 h period. 3 Standard error of mean. 4 P-value associated with main effects; time, treatment, and interaction of time and treatment. a b 0 Means in a column within an item with unlike superscripts differ (P < 0'05)' 145 Table 4.7. Effects of M elsdenii RK02 with or without E. faecium RK03 on acetic and propionic acid, and the ratio of acetic acid to propionic acid with rumen fluid from hay diet and ground corn as substrate (Trial 2) Initial Changes between incubation time, h Trt 3 P-value4 Treatments Contents.l 6 12 18 24 Mean SEM Trt Time 1?." x lme Acetic acid, mmol/L Control 50.84 20.15 22.03 0.82 4.30 11,33 b 4.15 < 0.01 < 0.01 0.17 RK02 50.59 18.67 23.26 -9.93 -720 15,20a RK03 50.81 27.53 15.55 -0.11 5.73 1217 b RK02 + RK03 51.96 21.11 17.88 -6.18 -6.24 6,64a Time Mean 51.05 21.86 19.68 -3.85 -0.85 Propionic acid, mmoVL Control 22.50 24.35 25.12 1.70 0.96 13,03 b 2.86 < 0.01 < 0.01 0.40 RK02 22.24 22.06 25.06 —6.23 -6.13 3,159a RK03 22.08 29.72 21.98 0.49 1.28 13,13 1’ RK02 + RK03 23.59 23.54 22.92 -6.27 -4.34 3,91;al Time Mean 22.61 24.92 23.77 -2.82 -2.06 Acetate : Propionate Control 2.26 0.76 0.84 -5 -5 -5 1.56 0.20 0.34 0.39 RK02 2.28 0.84 0.90 - - - RK03 2.30 0.92 0.70 - - - RK02 + RK03 2.20 0.83 0.74 - - - Time Mean 2.26 0.84 0.80 - - - I . . . . Concentratlon of contents 1n the fermentation vessels at time 0. N Differences of concentrations of organic acids for 6 h period. U Standard error of mean. 4 P-value associated with main effects; time, treatment, and interaction of time and treatment. The ratio was negative and means are not presented. abc Means in a column within an item with unlike superscripts differ (P < 0.05). 146 Table 4.8. Effects of M. elsdenii RK02 with or without E. faecium RK03 on butyric, isobutyric, valeric, and isovaleric acid production with rumen fluid from hay diet and gound corn as substrate (Trial 2) initial Changes between incubation time, h2 Trt 3 P-value4 Tremems Contents1 6 12 18 24 Mean SEM Trt Time l." x mm Butyric acid, mmol/L Control 16,59 3" 13.13 19,73 a 6.86 a 3.41 a 10.78 a 2.66 < 0.01 < 0.01 < 0.01 RK02 18.04 bc 19.96 29.82 b 47.63 c 20.36 b 29.44 b RK03 16.08 a 17.39 20.88 a 1.02 a 3.62 a 10.73 a RK02 + RK03 19.01 ° 22.95 28.95 b 37.14" 22.12 b 27.79b Time Mean 17.43 18.36 24.85 23.16 12.38 Isobutyric acid, mmoVL Control 2.40 0.77 0.61 -0.40 1” 1.76 0.69 0.35 0.19 < 0.01 0.01 RK02 2.17 1.91 0.87 006 ° 0.77 0.91 RK03 2.25 1.32 1.44 -1 .66 a 1.82 0.73 RK02 + RK03 2.49 1.94 1.71 -120 ab 1.62 1.02 Time Mean 2.33 1.49 1.16 -0.80 1.49 Valerie acid, mmol/L Control 2.92 a 1.72 a 1.55 a 0.23 a 1.35 “ 1.10a 0.72 < 0.01 < 0.01 < 0.01 RK02 4.80 b 4.32 b“ 7.66 c 13.15 b 6.64 b 7.94 b RK03 2.71 a 2.68 ab 0.43 a -0.07 a 0.56 a 0.90 “ RK02 + RK03 5.14 b 5.09 ° 5.23 b 12.27 b 6.88 b 7.37 b Time Mean 3.89 3.45 3.72 6.28 3.86 Isovaleric acid, mmol/L Control 2.41 2.11 0.03 -0.15 b 207 8 1,02 3 0.39 < 0.01 < 0.01 < 0.01 RK02 2.47 3.41 0.05 .0.06 b 3.02 ab 1.61 b RK03 2.28 2.84 0.59 -1.57 a 1.88 a 0.94 a RK02 + RK03 2.83 3.60 0.51 -168 a 3.55 b 1.50 b Time Mean 2.50 2.99 0.30 -0.87 2.63 I . . . . Concentration of contents 1n the fementatlon vessels at time 0. 2 . . . . . le’ferences of concentratlons of organic ac1ds for 6 h period. 3 Standard error of mean. P-value assoc1ated w1th mam effects; time, treatment, and mteractlon of time and treatment. ab 147 c Means in a column within an item with unlike superscripts differ (P < 0.05). Table 4.9. Effects of M. elsdenii RK02 with or without E. faecium RK03 on fermented organic matter, pH, and lactic acid production with rumen fluid from concentrate diet and RFC as substrate (Trial 3) Initial Changes between incubation time, 1? Trt 3 P-value4 Treatments Contents1 6 12 18 24 Mean SEM Trt Time g.“ x 1me Fermented organic matter (hexose) mmol/L Control 57.14 33.29 20.99 8.94 30.09 8.93 0.63 < 0.01 0.27 RK02 74.42 43.76 18.49 5.64 35.58 RK03 67.60 13.79 34.85 8.94 31.30 RK02 + RK03 69.98 18.61 42.02 6.10 34.18 Time Mean 67.29 27.36 29.09 7.40 pH Control 6.69 4.75 b -0.58 a -0.06 -0.05 .06] a 0.02 < 0.01 < 0.01 < 0.01 RK02 6.66 -1.68 c -053 b -0.08 -0.02 .653 b RK03 6.68 -1.80 a -0.48 c -0.09 -0.04 .650 a RK02+RK03 6.66 -1.73b -0.48° -0.04 -0.01 .056" Time Mean 6.67 -1.74 -0.52 -0.07 -0.03 Lactic acid, mmoVL Control 3.05 b 38.27 49.48 18.03 -3.31 2562" 5.21 0.02 <0.01 0.23 RK02 1.26 a 30.73 43.60 11.42 -0.14 21,40ab RK03 1.98 a 50.18 34.57 27.04 -3.33 2711" RK02 + RK03 152 a 42.30 25.99 13.91 -9.32 1322" Time Mean 1.96 40.37 38.41 17.60 -4.03 Total VFA, mmol/L Control 85.10 64.52 21.43 21.42 16.84 31.05 10.45 0.36 <0.01 0.13 RK02 79.04 87.28 31.81 19.03 10.82 37.24 RK03 78.31 70.27 -0.32 38.40 16.81 31.29 RK02 + RK03 82.66 72.00 11.38 56.08 16.84 39.08 Time Mean 81.28 73.52 16.08 33.74 15.33 T Concentration of contents in the fermentation vessels at time 0. 2 Differences of concentrations of organic acids and pH value for 6 h period. 3 Standard error of mean. 4 P-value associated with main effects; time, treatment, and interaction of time and treatment. a b c Means in a column within an item with unlike superscripts differ (P < 0.05). 148 Table 4.10. Effects of M elsdenii RK02 with or without E. faecium RK03 on acetic and propionic acid production and the ratio of acetic acid to propionic acid with rumen fluid fi'om concentrate diet and RFC as substrate (Trial 3) Initial Changes between incubation time, h2 Trt P-value Treatments Contentsl 6 12 18 24 Mean SEM3 Trt Time .331 x 1me Acetic acid, mmol/L Control 36.31 27.56 12.02 7.42 8.84 13.96 4.13 0.90 < 0.01 0.21 RK02 35.37 29.92 8.26 4.27 8.05 12.63 RK03 33.48 28.97 0.58 15.65 8.89 13.52 RK02 + RK03 36.19 24.53 1.29 17.59 7.37 12.70 Time Mean 35.34 27.75 5.54 11.23 8.29 Propionic acid, mmol/L Control 24.97 25.46 13.75 11.48 3.65 13.59 3.73 0.45 < 0.01 0.17 RK02 23.52 26.54 11.44 8.23 2.17 12.10 RK03 22.41 26.54 5.77 18.49 3.52 13.58 RK02 + RK03 24.13 21.81 10.24 24.46 4.78 15.32 Time Mean 23.76 25.09 10.30 15.67 3.53 Acetate : Propionate Control 1.45 1.10 0.91 0.65 2.48 1.29 0.37 0.37 < 0.01 0.05 RK02 1.50 1.13 0.72 0.52 4.04 1.60 RK03 1.51 1.09 1.18 0.78 2.85 1.48 RK02+RK03 1.50 1.12 1.24 0.64 1.61 1.15 Time Mean 1.49 1.1 1 1.01 0.65 2.74 T . . . . Concentration of contents in the fermentatlon vessels at time 0. 2 Differences of concentrations of organic acids for 6 h period. 3 Standard error of mean. P-value assoc1ated w1th main effects; time, treatment, and interactlon of time and treatment. ab 149 c Means in a column within an item with unlike superscripts differ (P < 0.05). Table 4.11. Effects of M elsdenii RK02 with or without E. faecium RK03 butyric, isobutyric, valeric, and isovaleric acid production with rumen fluid from concentrate diet and RFC as substrate (Trial 3) Treatments Initial | Changes between incubation time, h2 Trt SEM} P-value4 Trt x Contents 6 12 18 24 Mean Trt Time Time Butyric acid, mmoVL Control 17.37 997 a -1 . 1 5 a 133 a 0.23 2,72 3 2.17 < 0.01 < 0.01 < 0.01 RK02 15.33 23.94 b 10.84b 5.45 a 4.68 9.64b RK03 15.81 11.08 a -3.55 a 4.07 a 0.68 3.07 a RK02 + RK03 16.32 20.32 b 0.51 a 12.06 b 1.09 8.49b Time Mean 16.21 16.33 1.66 5.85 0.08 Isobutyric acid, mmoVL Control 1.55 1,21 3 -125 3" 0.11 2.24 0.52 0.18 0.96 < 0.01 0.01 RK02 1.19 1,63 3" -100 b -0.03 1.59 0.55 RK03 1.08 199 b -1.70 a 0.16 1.84 0.57 RK02 + RK03 1.22 1,27 8 41,96 b 0.31 1.63 0.56 Time Mean 1.26 1.53 -123 0.08 1.82 Valerie acid, mmol/L Control 2,03 a 011 a .055 a 0.58 0.14 0,07 3 0.47 < 0.01 < 0.01 < 0.01 RK02 3.68 b 3.80 b 3.01 c 1.01 -0.94 1.72 b RK03 1.87 a 0.41 a -0.28 ab -0.02 0.43 0.13 a RK02 + RK03 2.99 ab 2.85 b 0.91 b 1.64 0.03 135 b Time Mean 2.65 1.79 0.77 0.80 0.09 Isovaleric acid, mmol/L Control 2.82 0.20 -1.38 0.22 1.73 0,211 a 0.30 0.01 < 0.01 0.09 RK02 1.75 1.45 .075 0.10 1.63 061 b RK03 1.86 1.28 -1. 13 0.06 1.45 0,41 3" RK02 + RK03 1.82 1.22 -0.60 0.02 1.93 0,64 b Time Mean 2.06 1.04 .0.97 0.10 1.69 1 . . . . Concentration of contents in the fermentation vessels at time 0. 2 Differences of concentrations of organic acids for 6 h period. 3 Standard error of mean. P-value assoc1ated w1th main effects; time, treatment, and interaction of time and treatment. abc Means in a column within an item with unlike superscripts differ (P < 0.05). 150 Table 4.12. Effects of M elsdenii RK02 with or without E. faecium RK03 on fermented organic matter, pH, and lactic acid production with rumen fluid from hay diet and RFC as substrate (Trial 4) Initial Changes between incubation time, h Trt 3 P-valuer Tremems Contents1 6 12 18 24 Mean SEM Trt Time g.“ x 11118 Fermented organic matter (hexose) mmol/L Control 35,15 a 339‘51 -023 3 9,19 bc 2433 a 2.40 < 0.01 < 0.01 < 0.01 RK02 107.59 b 12.85 b 24.23 b 2.71 b 36.85 b RK03 81.02 a 7.64 ab 2.74 a 9.94 ° 25.33 a RK02 + RK03 1 12.59 b 15.00 c 24.45 b -9.00 a 35.76 b Time Mean 96.59 9.72 12.80 3.21 pH. Control 665 ° -203 a -0.15 .0112 b .009 a -1159 a 0.01 < 0.01 < 0.01 < 0.01 RK02 6.61 ab -1.95 b 41.15 0.01 be 0.00 b -0.52 b RK03 6.63 b -207 a -0. 12 -0.05 a -0.06 a -0.58 a RK02 + RK03 6.61 a -197 b 41.13 004° 0.00b .0.52 b Time Mean 6.62 -2.02 -0.14 -0.01 -0.04 Lactic acid, mmol/L Control 1,458 103,17b 14.37 752° 9,21b 33,57c 1.75 <0.01 <0.01 <0.01 RK02 1.93 “b 87.57 a 14.99 -1.07 b 8.06 b 27.39 b RK03 113° 101,39b 17.04 644° 22.66c 37.01 c RK02 + RK03 226 b 33,01 3 13.55 -6.55 a -1.22 a 22.20 a Time Mean 2.20 93.91 14.99 1.59 9.68 Total VFA, mmol/L Control 75.89 a 53.48 a -3.28 a -3.88 a 6.38 b 13.17 a 3.39 < 0.01 < 0.01 < 0.01 RK02 87.89b 78.69 b 8.51 ”c 26.99b .0.21 ab 28.50 b RK03 79.30a 48.71 a 0.38 ab -0.49 a -3.20 ab 11.35 a RK02 + RK03 81.66 3" 85.46 b 11.88 ° 31.24 c -8.22 a 30.09 b Time Mean 81.19 66.59 4.37 13.46 -1.32 l . . . . Concentration of contents in the fermentation vessels at time 0. 2 Differences of concentrations of organic acids and pH value for 6 h period. 3 Standard error of mean. P-value assoc1ated w1th main effects; time, treatment, and interaction of time and treatment. ab 151 c Means in a column within an item with unlike superscripts differ (P < 0.05). Table 4.13. Effects of M elsdenii RK02 with or without E. faecium RK03 on acetic and propionic acid production and the ratio of acetic acid to propionic acid with rumen fluid from hay diet and RFC as substrate (Trial 4) Initial Changes between incubation time, h2 Trt 3 P-value7r Treatments Contents1 6 12 18 24 Mean SEM Tit Time :[f-rt x mm Acetic acid, mmol/L Control 4004 a 1332 b 1.17 2.16 0.69 5.59 c 0.92 < 0.01 < 0.01 < 0.01 RK02 42.50 b 9.87 a 2.02 0.65 0.41 324 a RK03 41.17 b 16.28 b 2.06 1.54 -2.05 4,415b RK02 + RK03 41.69b 8.41 a 1.73 1.29 -1.31 253 a Time Mean 41.49 13.22 1.75 1.41 -0.57 Propionic acid, mmol/L Control 18.39 21.51 41,115 a -194 a 2.90 b 553 a 1.00 < 0.01 < 0.01 < 0.01 RK02 19.04 19.90 4.30 b 3.81 b 1.80b 7.45 b RK03 20.49 20.98 0.47 a -1.57 a -1.57 a 4.58 8‘ RK02 + RK03 19.30 20.33 5,511 b 5.75 b 1.66b 8.33 b Time Mean 19.30 20.68 2.55 1.51 1.20 Acetate : Propionate Control 2.19 0.85 7.84 -5 -5 -5 1.72 0.30 0.01 0.12 RK02 2.07 0.50 0.48 - - - RK03 2.19 0.78 7.77 - - - RK02 + RK03 2.16 0.41 0.33 - - - Time Mean 2.15 0.64 4.10 - - - 1 Concentration of contents in the fermentation vessels at time 0. 2 Differences of concentrations of organic acids for 6 h period. 3 Standard error of mean. 4 P-value associated with main effects; time, treatment, and interaction of time and treatment. 5 The ratio was not calculated when the change of acetic acid or propionic acid was negative. abc 152 Means in a column within an item with unlike superscripts differ (P < 0.05). Table 4.14. Effects of M elsdenii RK02 with or without E. faecium RK03 on butyric, isobutyric, valeric, and isovaleric acid production with rumen fluid from hay diet and RFC as substrateiTrial 4L Treatments Initial 1 Changes between incubation time, h Trt SEM3 Trt P1311184 Contents 6 12 13 24 Mean lme Trt x Time Butyric acid, mmol/L Control 13.77 a 8.64 a -1 .51 a -322 a -0.11b 0.95 a 1.39 < 0.01 < 0.01 < 0.01 RK02 17.76° 33.85 b 6.23 b 16.17 b -0.83 b 13.85 b RK03 14.35 ab 7.62 a -0.34 a -2.85 a 2.21 b 1.66a RK02 + RK03 15.59b 39.75 ° 8.16b 16.72 b -7.53 a 14.27 b Time Mean '5-37 22.47 3.13 6.70 -1.56 Isobutyric acid, mmol/L Control 0.74 2.32 -0.49 -0.96 1.02 0.47 0.38 0.36 < 0.01 0.05 RK02 1.13 2.18 -0.70 0.50 -0.62 0.34 RK03 1 .26 1.90 O -0.29 -0.39 -041 0.20 RK02 + RK03 1.19 2.26 -0.61 0.81 -0.57 0.47 Time Mean 1-03 2.16 -0.52 -0.01 -0.15 Valerie acid, mmol/L Control 133 3 1,30 8 -1.26 0,12 a 0.68 0.21 a 0.57 < 0.01 < 0.01 < 0.01 RK02 4.48 ° 8.81 b -1.20 3.22 b 1.08 2.98 b RK03 1.91 a 0.87 a -0.72 1.30 a -0.24 0.30 a RK02 + RK03 2.72 b 10.34 b -0.86 4.10 b 0.68 3.56b Time Mean 2-75 5.33 -1.01 2.18 0.55 Isovaleric acid, mmol/L Control 1.07 1.40 -103 b .005 a 1.21 c 0.38 a 0.28 0.03 < 0.01 < 0.01 RK02 1.16 4.07 -213 a 2.65 ° -205 a 0.64 3" RK03 1.41 1.07 .079" 1.48b -1.14b 0.15a RK02 + RK03 1.18 4.38 -213 “ 2.58c -1.15 b 0.92b Time Mean 1.20 2.73 -1.52 1.67 -0.78 l . . . . Concentration of contents in the fermentation vessels at time 0. 2 Differences of concentrations of organic acids for 6 h period. 3 Standard error of mean. P—value assoc1ated w1th main effects; time, treatment, and mteractlon of time and treatment. ab 153 6 Means in a column within an item with unlike superscripts differ (P < 0.05). 10 ifli-iA2 fi.*___i _7__.2_._**_~2”__2fin.7.wfl.7. OD 600nm 0.01 ., W ~ W W— W- W4 WW ._2._._2 . . J O 3 6 9 12 15 18 21 24 Incubation time, h 535261832 % 349622—2993;- 327—5925 —-e— t 00%1 Fig. 4.1. Growth of the co-culture of Megasphaera elsdenii RK02 and Enterococcus faecium RK03 in a Na-lactic acid broth with 0, 20, 40, 60, 80, or 100% of reducing agent recommended by Goering and Van Soest (1970) at 39 °C. The concentration of cysteine HCl was 0.0, 0.4, 0.8, 1.2, 1.6, and 2.0 mM and NazS9H20 was 0.00, 0.26, 0.52, 0.78, 1.04, and 1.30 mM for 0, 20, 40, 60, 80, or 100% of reducing agent, respectively. 154 200 150 2 E 100 50 220 180 140 2 E 100 60 20 A. FOM a b if??? 2 4 - 554.; % 3 A” l 1 Trial 4 l 2 Trial 3 4 C. Lactic acid 200 P D- Total VFA 160 l- 5120 1 I, f _ 5 a .1 If; . 80 '- 3] if; W”,- 9 5 4° ' 3‘42}, 4 0 ' ' - ’1 T 1 Trial 3 4 1 2 Trial 3 4 Figure 4.2. Effects of M elsdenii RK02 + E. faecium RK03 on organic acids production and pH during 12 h of the in vitro fermentation; organic acid values are differences of concentrations between at 0 and 12 h (Trial 1 concentrate fed cow and ground corn; Trial 2 hay fed cow and ground corn; Trial 3 concentrate fed cow and RFC; and Trial 4 hay fed cow and RFC; 1:1 Control and - RK02 + RK03; 1* " Values in a trial with unlike superscripts differ (P < 0.05)). 155 120 80 120 80 mM 40 A. Acetic acid C. Butyric acid \\."\.‘ i 7. 7 /, Vi.“ \ \ \\1\:\\ \_‘-1 ‘-.\\ <. {)4 ' .\ 120 801- mM 401 B. Propionic acid Figure 4.3. Effects of M elsdenii RK02 + E. faecium RK03 on acetic (A), propionic (B), and butyric acid (C) production during 12 h of the in vitro fermentation; VFA values are differences of concentrations between at 0 and 12 h (Trial 1 concentrate fed cow and ground corn; Trial 2 hay fed cow and ground corn; Trial 3 concentrate fed cow and RFC; and Trial 4 hay fed cow and RFC; 1:1 Control and I RK02 + RK03; ’ b Values in a trial with unlike superscripts differ (P < 0.05)). 156 A no a ,. ...... 4 w u. m r. a , .......... 3 D 8| 2 8| r _ 5 In 3 m w al 31 c al m b 2 a I N w AWN—E oo 4 and pH during 24 h of the in vitro fermentation; organic acid values are differences of concentrations between at 0 and 24 h (Trial 1 concentrate fed cow and ground corn; Trial 2 hay fed cow and ground corn; Trial 3 Figure 4.4. Effects of M elsdenii RK02 + E. faecium RK03 on organic acids production and I RK02 + RK03; a b Values in a trial with unlike superscripts differ (P < concentrate fed cow and RFC; and Trial 4 hay fed cow and RFC; [1 Control 0.05)). 157 120 120 A. Acetic acid B. Propionic acid 80 80 - a E E '. E E 40 40 - g 52% 0 0 " 1 1 Trial 3 4 1 2 Trial 4 120 30 C. Butyric acid D. Isobutyric acid 80 20 - a 2 E // 40 g/g 4/ g/ 0 :49”: 1 L 1 Trial 4 l 2 Trial 3 4 30 .1, a 30 E. Valerie acid F. Isovaleric acid 20 £7.75} 20 - 4?} 55/ a 2 E / E 10 / 10 \\\\\ \ \x.\ \x“'\\ ~\\.\‘ \I: ‘ “-.\ \>-:_~~ ‘ " \ \\\ “\Q‘\\‘\.\f\\\{ l 1’57: ///. 0 . 3 4 l 2 . 3 4 Trial Trial Figure 4.5. Effects of M elsdenii, RK02 + E. faecium, RK03 on VFA production during 24 h of the in vitro fermentation; VFA values are differences of concentrations between at 0 and 24 h (Trial 1 concentrate fed cow and ground corn; Trial 2 hay fed cow and ground corn; Trial 3 concentrate fed cow and RFC; and Trial 4 hay fed cow and RFC; 1:1 Control and I RK02 + RK03; “b Values in a trial with unlike superscripts differ (P < 0.05)). 158 Chapter 5 Effects of Megasphaera elsdenii (RK02) with Enterococcusfaecium (RK03) on fermentation characteristics in the rumen of steers fed a concentrate diet and of steers induced into acute acidosis SUMMARY Eight ruminally canulated Holstein steers (613 :1: 17 kg) were randomly assigned to one of two treatments; the control and microbial treatment (4 steers/treatment). Steers were adapted to an experimental diet and metabolism facility prior to initiation of the study for 2 mo, and fed 83% concentrate diets. Steers were offered 14 g DM/kg BW once daily. Steers received microbial treatments intraruminally immediately after feeding. Daily dose rate was 1.2 x 1010 cfu/animal ofM elsdenii (RK02) and 6.4 x 109 of E. faecium (RK03). Afier 10 d, rumen fluid was collected every 3 h for 24 h. On d 14, steers were fasted for 24 h, and received an intraruminal slurry of 1 part finely ground wheat to 2 parts warm tap water. Slurry was given at a rate of 1.33% BW on a DM basis. After administration of the wheat slurry, steers received the microbial treatment. Rumen fluid and jugular vein blood were collected at 0, 2, 4, 6, 8, 10, 12, 15, 18 and 21 h after acidosis challenge. DMI was similar between treatments during the 10 d microbial treatment. Effects of treatment and the interaction of treatment with time were similar for all variables measured on d 10 of the trial. During acidosis challenge, total organic acid concentrations were greater for the microbial treatment at 2 (P = 0.02) and 4 h (P = 0.03). Ruminal pH dropped to 5.0 at 4 h in the microbial treatment and at 6 h in the control. Total VFA concentrations were 159 greater for the microbial treatment at 2 (P = 0.01) and 4 h (P = 0.05). Lactic acid concentration was similar between treatments. Acetic acid concentration was greater for the microbial treatment at 2 (P < 0.01) and 4 h (P = 0.03). The microbial treatment facilitated continued fermentation of ground wheat during acidosis induction and did not prevent a drastic decrease in ruminal pH or ruminal lactic acid accumulation. Blood pH was similar between treatments. Blood partial pressure of C02 was less for the microbial treatment at 8 (P < 0.10) and 21 h (P < 0.01) than the control. Partial pressure of 02 was greater (P < 0.01) for the microbial treatment than the control at 21 h. Blood lactic acid, glucose, bicarbonate, cations, base excess concentrations, and hematocrit percentage were similar between treatments. The microbial treatment seemed to increase the animals physiological ability to compensate during acidosis. Keywords: Beef cattle, Direct-fed microbials, Megasphaera elsdenii, Enterococcus faecium, High concentrate diet, Acidosis. 160 INTRODUCTION Ruminal acidosis continues to be a common ruminal digestive disorder in beef cattle and can lead to marked reductions in cattle production (N agaraja and Titgemeyer, 2007). Acidosis is generally related to the amount, frequency, and duration of grain feeding. Two management practices to prevent acidosis are diluting the diet with roughage or modulating intake of starch (Owens et al., 1998). However, the cost per unit of net energy (NE) of feed ingredients favors feeding higher concentrate diets, and handling characteristics of dry forages also favors minimizing forage inclusion (Brown et al., 2006). Therefore, reduction of the grain proportion in feedlot cattle diets may not be acceptable, whereas demand for acidosis prevention may increase. A ruminal pH range of 5.2 to 5.6 is regarded as subacute or chronic acidosis; and a pH below 5.2 is considered acute acidosis (Owens et al., 1998; Brown et al., 2000; Krause and Oetzel, 2006; Nagaraja and Titgemeyer, 2007). A drop in pH below 5.6 in subacute acidosis apparently results from total accumulation of VFA alone, and not from lactic acid accumulation (Krause and Oetzel, 2006). This results from a combination of overproduction and decreased absorption of VFA (N agaraja and Titgemeyer, 2007). Lactic acid does not accumulate in the rumen during subacute acidosis, because lactate- fermenting bacteria remain active (Goad et al., 1998) and rapidly metabolize it to VFA; however, a transient increase in ruminal lactic acid up to 20 mM has been reported (Kennelly et al., 1999). In acute ruminal acidosis, an excessive intake of readily fermentable carbohydrates results in a sudden and uncompensated drop in ruminal pH, and as ruminal pH decreases, ruminal lactic acid concentrations increase (Krause and Oetzel, 2006). The 161 total VFA concentration initially increases; however as pH declines, VFA concentrations decline because of destruction of the normal bacterial flora and ruminal dilution from an influx of fluids to compensate for increased osmolality (Huber, 1976; Nagaraja and Titgemeyer, 2007). As the pH nears 5.0 or below, the growth of lactate-utilizing bacteria is inhibited, and lactate begins to accumulate in the rumen. Therefore, subacute acidosis has the potential to become lactic acidosis if the pH of 5.0 is sustained for a time (N agaraja and Titgemeyer, 2007). The mechanism of ruminal lactic acidosis involves Streptococcus bovis and ruminal lactobacilli (Owens et al., 1998). When cattle are not adapted to grain or during the step-up period, Streptococcus bovis initiates lactic acid production, and the low pH inhibits the growth of lactic acid-utilizing bacteria in the rumen (Nagaraja and Titgemeyer, 2007). Consequently, acid-tolerant ruminal lactobacilli predominate in the acidotic rumen. It is a widely practiced management strategy to introduce cattle to grain over a number of weeks with the proportion of grain in the diet increasing over that period (Klieve et al., 2003). This is to allow time for the resident populations of lactic acid-utilizing and other starch-fermenting bacteria to keep up with the grth of S. bovis and prevent acidosis. Alternative preventative strategies include the use of antibiotics, immunization against S. bovis and probiotic bacteria (Klieve et al., 2003). Previous in vitro studies (Chap. 4) have shown that M elsdenii (RK02) with or without E. faecium (RK03) may favorably alter the ruminal fermentation as a microbial treatment. The combination of RK02 and RK03 increased lactic acid utilization and pH in the culture of mixed ruminal microorganisms when the rumen fluid donor was adapted to either hay or high concentrate diets. Increases in butyrate and valerate production with 162 these organisms and their subsequent absorption may moderate ruminal acidity. Furthermore, the combination of RK02 and RK03 will grow in aerobic conditions, and this aero-tolerance enables the combination to be used in commercial settings. Therefore, we hypothesize that supplementation of M elsdenii RK02 with E. faecium RK03 will favorably alter the fermentation pattern in the rumen of steers in subacute or acute acidosis conditions. The current study was designed to evaluate the combined effects of M elsdenii strain RK02 and E. faecium strain RK03 on ruminal fermentation characteristics of steers fed a high concentrate diet and prevention of lactate accumulation in the rumen of steers with acidosis induction. Our objectives were to 1) evaluate the changes in pH and VFA concentrations with microbial treatment in the rumen, and 2) verify the decrease in lactic acid concentration and consequent acidity moderation in an acidosis provocative condition. 163 MATERIALS AND METHODS Experimental Design and Animal Feeding The animal studies protocol was approved by the All-University Committee on Animal Use and Care (AUF # 07/03-081-00) of Michigan State University. Eight ruminally canulated Holstein steers (613 :t 17 kg) were randomly allotted to individual metabolism stalls at the Beef Cattle Teaching and Research Center. Steers were adapted to experimental feed and facility for 2 mo, and fed 83% concentrate diets containing 11% CP. The diet included on a DM basis 39% high moisture com, 39% dry com, 12% corn silage, 5% hay, 5% of a protein-mineral supplement, and 0.3% urea. Steers were given a 14 g DM/kg BW ration once daily at 0830, and remainder was measured and discarded prior to the next feeding. Steers were randomly assigned to one of two treatments; the control and microbial treatment (a combination of M elsdenii strain RK02 and E. faecium strain RK03). Steers received microbial treatments immediately after feeding. Preparation and Treatments of Direct-F ed Microbials M elsdenii strain RK02 stored in anaerobic Na—lactate broth (N LB; Hoflierr et al., 1983) with 30% (v/v) glycerol was transferred to anaerobic NLB and cultured at 39°C for 24 h and E. faecium strain RK03 stored in Brain Heart Infusion (Difco) broth with 30% (v/v) glycerol was inoculated to aerobic Brain Heart Infusion (Difco) broth and incubated at 39°C for 24 b. Each 0.5 mL of RK02 and RK03 was inoculated to 9 mL of anaerobic NLB, and incubated at 39 °C for 24 h. The populations of RK02 and RK03 was measured using anaerobic NLB agar roll—tube or aerobic Brain Heart Infusion agar plate, 164 respectively. Five 10 mL of co-culture were pelleted by centrifiigation at 6,600 x g for 10 min and resuspended to 250 mL of 0.1% proteose peptone (Difco). Fifty mL of 0.1% proteose peptone or 50 mL of microbial suspension was intraruminally administrated to each steer for the control or microbial treatment, respectively. Daily dose rate was 1.2 x 10'0 cfu/animal of RK02 and 6.4 x 109 cfu/animal of RK03. Microbial treatment was supplemented for 14 d and at acidosis induction. After 10 d of daily dosing, rumen fluid was collected every 3 h for 24 h. Ruminal pH, lactic acid and VFA concentrations were analyzed. On (1 14, steers were fasted for 24 h, and acidosis was induced. Acidosis Induction Jugular catheters (Rouleau etal., 2003) were placed in all steers prior to acidosis induction, and flushed before and after sampling with a sterile heparinized solution (200 U/mL heparin, 0.9% NaCl, 1% benzyl alcohol). Steers were fasted for 24 h prior to the acidosis challenge. A slurry of 1 part finely ground wheat to 2 parts warm tap water was administered intraruminally to each steer. Slurry was given at a rate of 1.33% BW on a DM basis (Aviles, 1999). After administration of wheat slurry, steers received their microbial treatment intraruminally as described above. When a pH value less than 5.0 was observed at 4 h post challenge, all steers were offered 1 kg of hay. After 12 h, another 1 kg of hay was given to all steers. Rumen fluid and jugular vein blood were collected fiom each steer at 0, 2, 4, 6, 8, 10, 12, 15, 18 and 21 h after wheat slurry administration. 165 Chemical Analyses Ruminal pH was recorded immediately after sample collection with a combination electrode (6002; Cole-Farmer, Vernon Hills, IL) attached to a digital pH meter (pH Vision 05669-20; Cole-Parmer,) that had been standardized from pH 4 to 7 using commercial buffers (Curtin Mattheson, Wood Dale, IL). Rumen fluid was centrifuged at 24,000 x g for 20 min and 5 mL of supernatant was acidified with 12 N H2804 for organic acid analysis. Lactic acid and VFA contents were determined by ion exchange exclusion HPLC (aminex I-IPX-87H; Bio-Rad, Richmond, CA) with the same conditions described by Dawson et al. (1998). Venous blood was collected into heparinised syringes from the jugular catheter, placed immediately into an ice bath and analysed within 15 min using a blood gas analyser (Stat Profile M; Nova Biomedical, Waltharn, MA). This machine underwent calibration with reagent pack and gas standards before initiation of the study and every 2 to 3 h during use. All blood gas analyses were performed in duplicate. Statistical Analysis Data were analyzed with repeated measures using the MDIED procedure of SAS (SAS Inst. Inc., Cary, NC), with steer defined as the experimental unit. Model variables were microbial treatment, incubation time, and the resulting interaction. Auto-regression was determined as the most appropriate covariance structure using the Schwarz Baysean criterion (Littell et al., 1998). When main treatment effects existed, means separation was performed according to F isher's Protected LSD test. Significance was declared at P < 0.05. 166 RESULTS Rumen contents were collected every 3 h on d 10. Effects of treatment and the interaction of treatment with time were similar for all variables measured (Tables 5.1 to 5.4). All steers were oflered DM equivalent to 1.4% of BW, and five steers consumed all that was offered whereas 3 steers left remainder. The control treatment steers ate 68 and 83% of feed offered by 6 and 9 h, respectively, and the microbial treatment group consumed 71 and 81% by 6 and 9 h, respectively (Table 5.1). Total organic acid concentration was similar between treatments. The average ruminal concentrations were 209.7 and 206.2 mM for the control and microbial treatment, respectively (Table 5.2). Ruminal pH was similar between treatments (Table 5.2). Minimum pH was 5.72 and 5.56, and maximum pH was 6.99 and 6.86 for the control and microbial treatment, respectively (Appendix A. Table 6.6). Lactic acid and total VFA concentration were similar between treatments (Table 5.2). Concentrations of acetic, propionic and butyric acid and the ratio of acetic acid to propionic acid (A/P) were similar between treatments (Table 5.3). The average propionic acid concentrations were 67 and 59 mM, and maximum concentrations (Appendix Table 6.7) were 92 and 77 mM for the control and microbial treatment, respectively. The average A/P ratios were 1.41 and 1.58 for the control and microbial treatment, respectively. Butyric acid concentration was 33 and 41 mM, and maximum concentration was 42 and 60 mM for the control and microbial treatment respectively. Isobutyric acid, valeric acid and isovaleric acid concentrations were similar between treatments (Table 5.4). During induced acidosis, total organic acid concentrations were greater for the microbial treatment than the control at 2 (P = 0.02; Table 5.5) and 4 h (P = 0.03). This 167 results from greater total VFA for microbial treatment at 2 h (P = 0.01) and 4 h (P = 0.05). Lactic acid concentration was numerically greater in microbial treatment after 2 h (Table 5.5). The overall ruminal pH was 5.16 and 5.00 for the control and microbial treatment, respectively. Ruminal pH dropped to 5.0 at 4 h in microbial treatment and at 6 h in the control, and then remained below 5.0 for both treatments. Acetic acid concentration was greater for the microbial treatment at 2 (P < 0.01; Table 5.6) and 4 h (P = 0.03). Propionic acid concentration (P = 0.83) and the A/P ratio (P = 0.69) were similar between treatments. The ruminal A/P ratios were 1.84 and 2.01 for the control and microbial treatment, respectively (Table 5.6). A microbial treatment effect was not observed for butyric, isobutyric, valeric or isovaleric acid concentration (Table 5.7). Blood pH, lactic acid and bicarbonate concentrations were similar between treatments (Table 5.8). Partial pressure of carbon dioxide was lower for the microbial treatment at 8 (P < 0.10) and 21 h (P < 0.01; Table 5.8). Partial pressure of oxygen was greater (P < 0.01) for the microbial treatment than the control at 21 h (Table 5.9). Blood oxygen saturation percentage was higher (P < 0.01) for the microbial treatment than the control at 21 h (Table 5.9). Blood base excess (P = 0.66) and glucose concentrations (P = 0.50) were similar between treatments. Ionized calcium, ionized magnesium, sodium and potassium concentrations were similar between treatments (Appendix A. Table 6.8). A microbial treatment effect was not observed for blood hematocrit percentage, base excess of extracellular fluid, standard bicarbonate concentration, or chloride concentration (Appendix A. Table 6.9). 168 DISCUSSION Eflects on Fermentation Characteristics in the Rumen of Steers fed a Concentrate Diet In the current study, steers received 1.6% of BW of an 83% concentrate diet for 24 d and 1.4% for the remainder of the adaptation and test period. Feed intake fluctuation had been observed in steers throughout the experimental period (Figure 5.1). To reduce DMI variation, feed offered was reduced to 1.4% of BW on d -52, and the half of the high moisture corn in the diet was replaced with dry corn on d -36. Klieve et a1 . (2003) reported that one time-inoculation established M elsdenii the niche in the rumen during a step-up adaptation period to concentrate diets. Without inoculation (the control group), M elsdenii was detected in the rumen at > 104 cell/mL after 5 d on grain diets and reached a similar level (~ 108 cell/mL) in the inoculated group after 13 d on the grain diet. Klieve et al. (2003) suggested that M elsdenii inoculation reduced the adaptation period by 5 to 7 d to establish a lactic acid-utilizing bacterial population in the rumen of cattle fed grain diets. Mackie and Gilchrist (1979) also reported that during changes from hay to concentrate diets, lactate-utilizing bacteria established populations in the rumen. However, similar ruminal fermentation patterns during subacute acidosis also had been reported regardless of whether steers were adapted or unadapted to a high concentrate diet (Goad et al., 1998). In this study, a lactic acid-utilizing bacterial population was likely established in the rumen of steers during more than 2 mo of adaptation to concentrate diets. Increased DMI with supplementation of M elsdenii has been reported in cattle that were switched from a 50 to 90% concentrate diet (Hibbard et al., 1993) and in steers induced with acute acidosis (Robinson et al., 1992). M elsdenii is acid-tolerant (Therion et' al., 1982) and its lactic 169 acid fermentation is not subject to catabolite repression by glucose or maltose (Russell and Baldwin, 1978; Hino etal., 1994). M elsdenii ferments 60 to 80% of the lactic acid in the rumen (Counotte et al., 1981). In the current study, administration of RK02 and RK03 did not change DMI (P = 0.57) during the 10 (1 experimental period with DMI fluctuating in steers in both treatments (Appendix B. Figure 6.11). DMI measured every 3 h on d 10 showed different individual feed intake patterns among steers (Appendix B. Figure 6.11), however DMI values every 3 h or overall were similar between treatments (Table 5.1). Average ruminal pH was similar between treatments prior to acidosis induction and the values were 5.97 and 5.95 for the control and microbial treatment, respectively. These values are typical (5.8 to 6.2) in beef cattle fed high-grain diets (N agaraja and Titgemeyer, 2007). Ruminal pH values were higher than 5.6 for both treatments (Table 5.2), so by definition, the cattle in the adaptation period were not experiencing subacute acidosis. However, individual animals differed between the the control and microbial treatments, and did have ruminal pH values below 5.6 during the 24 h sampling period (the control; steer id 801; average pH = 5.64; Figure 6.11, and microbial treatment; steer id 812; average pH = 5.43; Figure 6.11). Steer SOl consumed all of the diet (1 .4% BW) offered by 9 h and steer 812 consumed 1.2% BW. Although steer 801 had a ruminal pH in the range of subacute acidosis (below 5.6) for a period during the feeding cycle, feed intake for this animal remained constant at 1.4% of BW during the adaptation (Figure 5.1) and experimental periods (Figure 5.2). Failure of steer 812 to consume the entire feed offered during both adaptation and experimental periods (Figures 5.1 and 6.11) may have resulted fi'om subacute acidosis associated with low ruminal pH (Table 5.2). 170 Differences in individual acidity tolerance have been reported (Dougherty et al., 1975; Brown et al., 2000) for cattle fed similar diets. Variation in feed intake between days has been used as an index of subclinical or chronic acidosis based on the concept that an increased variability from day to day in feed intake by individual animals is associated with acidosis (Britton et al., 1991 cited in Nagaraja and Titgemeyer, 2007). Steer 812 was on the microbial treatment which may suggest that acidosis was not the cause of reduced intake or the microbial treatment was inefl‘ective to the control acidosis for that steer. Microbial treatment had a lower total organic acid concentration (252 vs 204 mM; Table 5-1 B) and total VFA (237 vs 191 mM; Table 5.2) than the control at 15 h, with these differences resulting from a decrease in acetic acid (96 vs 76 mM) and propionic acid concentration (83 vs 58 mM; Tables 5.2 and 5.3). However, the overall means for total organic acid (210 vs 206 mM) and total VFA concentrations (198 vs 195 mM) throughout the 24 h period were similar between treatments (Table 5.2). When total organic acid concentrations were converted to a hexose basis (Appendix Table 5-1F; [total] = ([acetate] + [propionate] + [lactate]) /2 + [butyrate] + [isobutyrate] + [valerate] + [isovalerate]), the patterns of fermentation were similar to total organic acid, and the averages were similar between treatments (128 vs 130 mM). Lactic acid concentration was similar between treatments (Table 5.2 and Figure 6.12) and contributed 1.4 to 7.3 % of the total organic acid concentration. Under normal conditions, lactic acid does not accumulate in the rumen at concentrations above 5 W, and ruminal concentrations exceeding 40 mM are indicative of severe acidosis (Owens et al., 1998). However, in the study of carbohydrate challenge (Brown et al., 2000), peak ruminal lactic acid concentrations of steers having an average daily ruminal pH above 5.61 (not affected) 171 were between 4 to 67 mM, whereas peak concentrations were 15 to 61 mM and 92 to 123 mM for steers experiencing subacute acidosis and acute acidosis, respectively. Distinct differences between treatments were observed in propionic acid and butyric acid concentration (Figure 6.12). Although propionic acid levels were not different (67 vs 59 mM, P = 0.52), numerically, the the control group had a higher propionic acid concentration than the microbial treatment between 9 and 21 h. At 15 h, the numerical difference of propionic acid (25 mM) was greater than that of acetic acid (20 mM). The minimum and maximum values of fermentation characteristics were compared between treatments (Appendix A. Table 6.6), and no differences were detected. In conclusion, overall fermentation was similar between treatments, however the microbial treatment tended to have greater butyric acid and lower propionic acid concentrations than the control. In the previous in vitro studies (Chapter 4), microbial treatment (RK02 and RK03) increased butyric acid concentration in all conditions. Propionic acid concentration decreased with the microbial treatment when ground corn was used as substrate with either rinnen fluid adapted to a concentrate- or hay-diet (Trial 1 and 2 in Chapter 4), and was similar when rumen fluid adapted to a concentrate diet and readily fermentable carbohydrates as substrate were used (Trial 3) and increased when rumen fluid adapted to hay-diet and readily fermentable carbohydrates as substrate were used (Trial 4). Therefore, changes in fermentation characteristics observed in the current study are consistent with in vitro Trial 1 from Chapter 1 which used rumen fluid adapted to a high concentrate diet and ground corn as substrate. Adaptation to the high concentrate diet involves the establishment of lactic acid-utilizing bacteria (Mackie and Gilchrist, 172 1979; Klieve et al., 2003) including Anaerovibrio Iipolytica, F usobacterium necrophorum, M elsdeni i, Peptostreptococcus asaccharolyticus, S. ruminantium, Propionibacterium acnes, and Veillonella parvula (Nagaraja and Tigemeyer, 2007). M elsdenii and S. ruminantium ssp. lactilytica are the predominant lactate-fermenting bacteria in concentrate-fed animals (Huber et al., 1976; Mackie et al., 1978). Since some changes were observed in fermentation endproducts, M elsdenii strain RK02 and E. faecium RK03 likely establish a niche in the ruminal ecosystem. Isobutyric acid, valeric acid, and isovaleric acid concentrations were similar between treatments. In the previous in vitro studies, isobutyric acid concentrations were similar between the control and microbial treatment, and valeric acid and isovaleric acid concentrations were greater for the microbial treatment in all trials reported in Chapter 4. Possibly propionyl CoA was diverted toward the production of valerate (Marounek et al., 1989) resulting in decreased propionic acid and increased valeric acid production. Lactic acid concentrations also were not consistent with previous in vitro studies. M elsdenii is dependent on the amylolytic activities of other bacteria to obtain energy substrates, because it does not utilize starch (Marounek et al., 1989), therefore it would not compete with amylolytic microorganisms for substrates. However, when lactic acid accumulated in the culture (Trial 2, 3 and 4 in Chapter 4), microbial treatment decreased lactic acid concentrations. In the in vivo study, lactic acid concentrations were similar between treatments (Table 5.2 and Appendix B. Figure 6.12). Further studies will be required to define the appropriate dose level and circumstances to utilize the microbial treatment to enhance ruminal efficiency. 173 Eflects on Fermentation Characteristics in the Rumen of Steers with Induced Acidosis In the enrichment study reported in Chapter 3, the lactic acid-utilizing ability of a mixture of ruminal microorganisms increased by continually growing (enrichments through six passes) in 2% (v/v) lactic acid media, and when the enriched microorganisms were added as a microbial treatment, lactic acid concentrations decreased in the in vitro ruminal fermentation. However, aerobicities of enrichment processes have shown different efficiencies in the prevention of lactic acid accumulation. Aerobically enriched microorganisms (N 2A4 in Chapter 3) completely utilized lactic acid after 24 h of incubation, whereas enrichments in anaerobic (N 6) or ending in anaerobic conditions (N2A2N2) decreased lactic acid only 21 to 22%. These observations imply that microbial adaptation to high grain diets may not be sufficient to utilize all lactic acid produced during ruminal acidosis. Artificial supercharging of lactic acid-utilizing bacteria by addition of a microbial treatment may prevent lactic acidosis. Therefore, the combination of M elsdenii RK02 and E. faecium RK03, which showed acidosis preventive capabilities in Chapter 4, was administered to the rumen of steers experiencing acidosis. In the current study, the rate of pH decrease was slower during the first 2 h than in a previously reported study (Aviles, 1999), but was faster thereafter and reached 4.6 at 10 h and plateaued until 18 h. This range of pH has been associated with acute acidosis (Owens et al., 1998; Brown et al., 2000; Nagaraja and Titgemeyer, 2007). The numerically lower pH in microbial treatment at 4 h might result fi'om the greater total organic acid at 2 (101 vs 157 mM) and 4 h (173 vs 224 mM). Among the individual organic acids, only acetic acid showed a significant decrease in the microbial treatment at 2 (76 vs 113 mM, P = 0.01) and 4 h (100 vs 126 mM, P = 0.05). However, small, non- 174 significant increases in lactic, propionic and butyric acid may have contributed to diflerences in total organic acid and pH at 4 h (Table 5.5 and Appendix B. Figure 6.14). In previous in vitro studies, microbial treatments decreased lactic acid concentration (Trial 2, 3 and 4 in Chapter 4) and increased pH in all trials. The current study was not consistent with those observations. In in vitro fermentations, endproducts accumulate and are subsequently metabolized or remain in solution. In the ruminal fermentation environment, a continual influx of nutrients and buffers occurs plus removal of end products via absorption or passage. Additionally, a more robust population of microorganisms to include protozoa likely exists in the ruminal system as compared to the in vitro fermentation system. Clinical diagnosis of acidosis requires blood pH to fall below 7.35 (Owens et al., 1998). Two steers in the the control group experienced systemic acidosis as defined by blood pH (Appendix B. Figure 6.17). Steer SO6 had a pH below 6.35 between 8 and 15 h, and steer 809 from 12 h until the end of the experiment. The average blood pH tended to be greater for the microbial treatment than the control (7.40 vs 7.44, P = 0.12). The microbial treatment had a numerically higher blood pH throughout experimentally induced acidosis (Appendix B. Figure 6.15). Opposite to the trends seen in blood pH, microbial treatment had a small numerical increase in blood lactic acid concentration (Figure 6.15). In both treatments, as time increased blood lactic acid concentration increased. Blood bicarbonate concentration was similar between treatments (26.30 vs 26.52 mM) and decreased over time (Figure 6.15). Decreases in blood bicarbonate and base excess (Appendix B. Figure 6.15) in both groups may be indicative of some degree of 175 physiological compensation for increased absorption of ruminal VFA (Goad et al., 1998) and lactic acid. The microbial treatment had a smaller C02 partial pressure than the control at 21 h (41.29 vs 32.19 mmHg, P < 0.05; Table 5 .8). Carbon dioxide depresses blood pH unless bicarbonate (HC03) compensates sufficiently (Owens, 1998). Oxygen saturation was numerically greater in the microbial treatment at 21 h (63.50 vs 85.81%) although the overall average was similar between treatments (73.33 vs 73.37%). Partial pressure of oxygen was also greater (P < 0.01) for the microbial treatment at 21 h (Table 5.9). In the present study, cations did not decrease during acute acidosis (Appendix B. Figure 6.16), however, the 21 h observation period may have been too short to detect these differences. From 58 to 120 h after carbohydrate loading, decreased Ca, K, and Mg associated with acidosis has been reported (Irwin et al., 1979; Patra et al., 1993). All differences between treatments in blood partial pressure of C02 (pC02) and 02 (p02) were observed at 21 h and one steer (809) in the the control group had clinical acidosis at the same time. When 809 was removed from the the control, the microbial treatment still had lower pC02 (P < 0.01) and higher p02 (P = 0.01) and 02 saturation percentage (P = 0.01) at 21 h. The microbial treatment group may have a greater physiological compensatory capacity for acid load than the control. Addition of the microbial treatment appears to improve the animals ability to withstand rapid accumulation of acids in the rumen. 176 IMPLICATIONS Daily supplementation of a microbial treatment consisting of M elsdenii (RK02) and E. faecium (RK03) for 10 d did not change total fermentation in the rumen of steers fed a 83% concentrated diet. However, microbial treatment numerically increased butyric acid concentration and numerically decreased propionic acid. These differences are consistent with the in vitro fermentation finding reported in Chapter 4 when rumen fluid from cows adapted to a high-concentrate diet and ground corn as substrate were used. The effect of increased butyric acid at the expense of propionic acid or acetic acid would suggest a lower energetic efficiency and reduced animal performance. Further study will be required to determine the appropriate dose, microbial mixture, and timing of administration of microbial treatment to prevent acidotic conditions in the rumen. The microbial treatment facilitated the fermentation of ground wheat during acidosis induction, and tended to have low ruminal pH earlier than the the control. The microbial treatment did not prevent a drastic decrease in ruminal pH or ruminal lactic acid accumulation. Blood pH, gases and metabolites were indicative of compensation of acid load in both treatments. The microbial treatment increased partial pressure of oxygen, and decreased partial pressure of carbon dioxide for a period during acidosis. The microbial treatment seemed to increase physiological ability to compensate for the acid loads during systemic acidosis. 177 LITERATURE CITED Aviles, I. 1999. The use of DH42, a propionibacterium for the prevention of lactic acidosis in cattle. MS. Thesis. Michigan State Univ. East Lansing, MI. Brown, M. S., C. H. Ponce, and R. Pulikanti. 2006. Adaptation of beef cattle to high- concentrate diets: Performance and ruminal metabolism. J. Anim. Sci. 2006. 84(E. Suppl.):E25—E33. Brown, M. S., C. R. Krehbiel, M. L. Galyean, M. D. Remmenga, J. P. Peters, B. Hibbard, J. Robinson, and W. M. Moseley. 2000. Evaluation of models of acute and subacute acidosis on dry matter intake, ruminal fermentation, blood chemistry, and endocrine profiles of beef steers. J. Anim. Sci. 78:3155—3168. Counotte, G. H. M., R. A. Prins, R. H. A. M. Janssen, and M. J. A. deBie. 1981. Role of Megasphaera elsdenii in the fermentation of DL-2-13C lactate in the rumen of dairy cattle. Appl. Environ. Microbiol. 42:649-655. Dougherty, R. W., K. S. Coburn, H. M. Cook, and M. J. Allison. 1975a. Preliminary study of appearance of endotoxin in circulatory system of sheep and cattle after induced grain engorgement. Am. J. Vet. Res. 36:831—832. Dougherty, R. W., J. L. Riley, A. L. Baetz, H. M. Cook, and K. S. Coburn. 1975b. Physiologic studies of experimentally grain-engorged cattle and sheep. Am. J. Vet. Res. 36:833—835. Goad, D. W., C. L. Goad, and T. G. Nagaraja. 1998. Ruminal microbial and fermentative changes associated with experimentally induced subacute acidosis in steers. J. Anim. Sci. 76:234-241. Hibbard, B., J. A. Robinson, R. C. Greening, W. J. Smolenski, R. L. Bell, and J. P. Peters. 1993. The effect of route of administration of isolate 407A (U C-12497) on feed intake and selected ruminal variables of beef steers in an acute acidosis inappetance model. Proc. 22nd Bienn. Conf. on Rumen Funct., Chicago, IL. p l 9. (Abstr.). Hino, T., K. Shimada, and T. Maruyama. 1994. Substrate preference in a strain of Megasphaera elsdenii, a ruminal bacterium, and its implications in propionate production and grth competition. Appl. Environ. Microbiol. 60: 1827-1831. Hofherr, L. A., B. A. Glatz, and E. G. Hammond. 1983. Mutagenesis of strains of Propionibacterium to produce cold-sensitive mutants [Starter cultures, Swiss cheese]. J. Dairy Sci. 66: 2482-2487. Huber, T. L., J. H. Cooley, D. D. Goetsch, and N. K. Das. 1976. Lactic acid-utilizing bacteria in ruminal fluid of a steer adapted from hay feeding to a high-grain ration. Amer. J. Vet. Res. 37:611-613. 178 Kennelly, J. J ., B. Robinson, and G. R. Khorasani. 1999. Influence of carbohydrate source and buffer on rumen fermentation characteristics, milk yield, and milk composition in early-lactation Holstein cows. J. Dairy Sci. 82:2486—2496. Klieve, A. V., D. Hennessey, D. Ouwerkerk, R. J. Forster, R. I. Mackie, and G T. Atwood. 2003. Establishing populations of Megasphaera elsdenii YB 34 and Butyrivibrio fibrosolvens YB 44 in the rumen of cattle fed high-grain diets. J. Appl. Microbiol. 95:621—630. Krause, K. M. and G. R. Oetzel. 2006. Understanding and preventing subacute ruminal acidosis in dairy herds: A review. Anim. Feed Sci. 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S. Secrist, W. J. Hill, and D. R. Gill. 1998. Acidosis in cattle: A review. J. Anim. Sci. 76:275—286. Rouleau, G, M. Babkine, and P. Dubreuil. 2003. Factors influencing the development of jugular thrombophlebitis in cattle and comparison of 2 types of catheter. Can. Vet. J. 44:399-404. Robinson, J. A., W. J. Smolenski, R. C. Greening, M. L. Ogilvie, R. L. Bell, K. Barsuhn, and J. P. Peters. 1992. Prevention of acute acidosis and enhancement of feed intake in the bovine by Megasphaera elsdenii 407A. J. Anim. Sci. 70 (Suppl. 1):310 (Abstr.). Russell, J. B. and R. L. Baldwin. 1978. Substrate preferences in rumen bacteria: evidence of catabolite regulatory mechanisms. Appl. Environ. Microbiol. 36:319—329. Therion, J. T., A. Kistner, and J. H. Komelius. 1982. Effect of pH on growth rates of rumen amylolytic and lactilytic bacteria. Appl. Environ. Microbiol. 44:428—434. 179 Table 5.1. Effects of supplementation of M elsdenii RK02 with E. faecium RK03 on dry matter intake of steers fed a concentrate diet prior to acidosis induction Time post Control Microbial Time Mean Control Microbial Time Mean feeding, h treatment treatment DMI, %BW DMI, kg 3 0.51 0.50 0.51 ° 3.08 3.05 3.06c 6 0.44 0.49 0.47 ° 2.68 3.03 2.85 ° 9 0.21 0.14 0.18b 1.25 0.85 1.05 b 12 0.03 0.05 0.04 'b 0.23 0.33 0.28 'b 15 0.02 0.03 0.03 ‘ 0.15 0.20 0.18 ‘ 18 0.03 0.04 0.03 ‘ 0.18 0.23 0.20 "’ 21 0.04 0.07 0.06 'b 0.28 0.45 0.36 'b 24 0.00 0.02 0.01 1 0.00 0.10 0.05 ' Trt Mean 0.16 0.17 0.98 1.03 Effects Time Trt Trt x Time Time Trt Trt x Time SEM' 0.05 0.02 0.07 0.30 0.13 0.42 Prob.2 < 0.01 0.82 1.00 < 0.01 0.78 1.00 ------------ - Total DMI, %BW Total DMI, kg ---------- Trt Mean 1.29 1.34 1.32 7.86 8.22 8.04 SEM‘ 0.09 0.51 Prob.2 0.68 0.64 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. a b c Means in a column with unlike superscripts differ (P < 0'05)' 180 Table 5.2. Effects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on concentrations of total organic acid, lactic acid, total VFA, and pH in the rumen of steers fed a concentrate diet prior to acidosis induction Time post Control Microbial Time Mean Control Microbial Time Mean feeding, h treatment treatment ------- Total organic acid, mmol/L pH 0 130.00 133.00 131.50 a 7.01 6.86 6.93 ° 3 163.25 186.00 174.63 b 6.45 6.38 6.41 d 6 215.00 236.75 225.88 “ 5.76 5.81 5.79 “b 9 267.00 244.50 255.75 ° 5.63 5.67 5.65 “b 12 246.00 250.25 248.13 ° 5.75 5.68 5.72 ”b 15 251.75 204.00 227.88 “C 5.65 5.67 5.66 “b 18 213.75 198.25 206.00 °“ 5.57 5.67 5.62 a 21 222.25 208.25 215.25 °“ 5.87 5.84 5.85 b 24 178.50 195.00 186.75 b° 6.04 6.00 6.02 ° Trt Mean 209.72 206.22 5.97 5.95 Effects Time Trt Trt x Time Time Trt Trt x Time SEM 11.24 5.98 15.90 0.13 0.15 0.19 Prob. < 0.01 0.68 0.35 < 0.01 0.93 0.98 ------------ Lactic acid, mmol/L Total VFA, mmoVL-—---—- 0 1.88 2.48 2.18 a 128.25 130.75 129.50 a 3 9.33 9.48 9.40 b 154.00 176.50 165.25 b 6 15.60 15.38 15.49 d 199.50 221.50 210.50 4° 9 1 1.94 13.04 12.49 b° 255.25 231.25 243.25 f 12 12.40 11.59 12.00 b° 233.50 238.50 236.00 °f 15 14.13 13.02 13.57 “d 237.50 191.25 214.38 M 18 12.13 10.19 11.16bc 201.75 188.25 195.00bed 21 14.42 10.61 12.51 b“ 207.75 197.25 202.50 °d 24 10.46 12.55 11.51 b° 168.25 182.50 175.38 “ Trt Mean 11.36 10.93 198.42 195.31 Effects Time Trt Trt x Time Time Trt Trt x Time SEMI 1.61 1.52 2.27 11.25 6.59 15.92 Prob.2 < 0.01 0.84 0.65 < 0.01 0.74 0.39 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. a b c d e fMeans in a column within an item with unlike superscripts differ (P < 0435)- 181 Table 5.3. Effects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on concentrations of acetic acid, propionic acid and butyric acid, and acetate to propionate ratio in the rumen of steers fed a concentrate diet prior to acidosis induction Time post Control Microbial Time Mean Control Microbial Time Mean dosing, h treatment treatment ------- - Acetic acid, mmol/L Propionic acid, mmol/L 0 65.95 64.31 65.13 a 29.80 31.27 30.53 a 3 68.61 81.83 75.22 a“ 47.62 47.96 47.79" 6 90.37 97.33 93.85 “c 67.32 64.01 65.66 ° 9 105.14 92.71 98.92 ° 91.61 74.51 83.06 d 12 95.31 93.89 94.60 d‘ 86.10 76.10 81.10 d 15 96.29 75.56 85.93 “ 83.14 58.00 70.57 °“ 18 80.76 74.02 77.39““ 73.40 60.73 6707" 21 88.27 76.64 82.46“" 66.92 58.45 62.69“ 24 68.11 75.76 71.94 a” 57.43 59.75 58.59 “ Trt Mean 84.31 81.34 67.04 58.97 Effects Time Trt Trt x Time Time Trt Trt x Time SEM 5.37 2.57 7.60 8.06 8.90 1 1.40 Prob. < 0.01 0.42 0.40 0.00 0.52 0.56 --------- Acetic acid : Propionic acid Butyric acid, mmoVL ----—---- 0 2.20 2.05 2.12 ° 23.92 26.45 25.19 a 3 1.51 1.79 1.65 d 25.47 32.52 29.00 “b 6 1.42 1.65 1.53 °" 26.41 44.52 35.46 a“ 9 1.24 1.35 1.29 a‘ 39.23 46.79 43.01 °"° 12 1.21 1.38 1.29ab 37.48 54.36 45.92° 15 1.27 1.48 1.38““ 40.93 43.45 42.19Ode 18 1.24 1.41 1.33 a“ 32.73 39.29 36.01 ““ 21 1.36 1.58 1.47 “d 38.68 49.43 44.05 mi 24 1.24 1.53 1.38 a“ 30.96 33.13 32.04ab Trt Mean 1.41 1.58 32.87 41.10 Effects Time Trt Trt x Time Time Trt Trt x Time SEM' 0.18 0.23 0.25 4.27 4.79 6.04 Prob.2 < 0.01 0.61 0.34 < 0.01 0.23 0.35 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. abcde Means in a column within an item with unlike superscripts differ (P < 0.05). 182 Table 5.4. Effects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on concentrations of butyric, isobutyric, valeric, and isovaleric acids in the rumen of steers fed a concentrate diet prior to acidosis induction Time post Control Microbial Time Mean Control Microbial Time Mean feeding, h treatment treatment ------- lsobutyric acid, mmol/L Valerie acid, mmol/L 0 1.45 1.60 1.52 a 2.43 2.23 2.33 a 3 2.41 2.91 2.66 “ 3.16 3.07 3.11 “b 6 3.86 3.85 3.85 °° 4.39 3.57 3.98 “ 9 4.28 4.12 4.20 ° 6.10 3.66 4.88 ° 12 1.94 2.86 2.40 b 5.61 4.02 4.81 ° 15 3.99 3.82 3.91 °° 5.40 3.92 4.66 ° 18 3.04 3.69 337°d 5.18 3.21 4.19"c 21 2.70 3.08 2.89 “ 4.27 3.77 4.02 “ 24 2.26 3.54 2.90 “ 3.49 3.36 3.42 °“ Trt Mean 2.88 3.27 4.45 3.42 Effects Time Trt Tit x Time Time Trt Trt x Time mm1 0.29 0.22 0.41 0.69 0.75 0.97 Prob.2 < 0.01 0.21 0.48 0.06 0.34 0.53 ------- Isovaleric acid, mmol/L --- 0 4.56 4.80 4.68 a 3 6.67 8.15 7.41 b 6 6.90 8.12 7.51 " 9 8.86 9.50 9.18 ° 12 7.12 7.41 7.26 " 15 7.83 6.23 7.03 b 18 6.74 7.20 6.97 " 21 7.00 6.04 6.52b 24 5.88 6.79 6.33 b Trt Mean 6.84 7.14 Effects Time Trt Trt x Time SEMl 0.93 1.12 1.31 Prob.2 < 0.01 0.85 0.39 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. a b ° Means in a column within an item with unlike superscripts differ (P < 0.05)- 183 Table 5.5. Effects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on rumen of steers during experimentally induced acidosis concentrations of total organic acid, lactic acid, total VFA, and pH in the Time after Control Microbial Time Mean Control Microbial Time Mean challenge, h treatment treatment ------- Total organic acid, mmoVL pH 0 44.72 40.1 1 42.50 A 7.08 7.04 7.06 ° 2 100.71 A 156.69 B 128.50 b 6.62 6.18 6.40 d 4 173.01 A 224.27 B 198.75 ° 5.54 5.00 5.27 ° 6 207.85 241.15 224.38 d 4.98 4.62 4.80 b 8 223.58 258.56 241.13 “ 4.69 4.38 4.53 “b 10 235.18 262.28 248.88 A” 4.57 4.36 4.47 A 12 256.41 262.99 259.75 ° 4.55 4.44 4.50 “b 15 248.52 250.10 249.38 ° 4.61 4.77 4.69 A” 18 266.91 237.60 252.25 ° 4.60 4.63 4.61 “b 21 259.44 240.83 250.25 ° 4.39 4.61 4.50 A” Trt Mean 201.63 217.46 5.16 5.00 Effects Time Trt Trt x Time Time Trt Trt x Time SEM‘ 1 1.62 1 1.40 16.43 0.14 0.13 0.20 Prob? < 0.01 0.33 0.03 < 0.01 0.40 0.45 ---------- Lactic acid, mmol/L Total VF A, mmoVL—m 0 4.88 1.50 3.19 a 39.78 38.87 42.45 A 2 24.43 43.43 33.93 " 76.38 A 113.23 B 128.55 b 4 72.70 98.30 85.50 ° 100.19 126.02 198.75 ° 6 99.60 122.53 111.06 d 108.16 118.56 224.39 d 8 108.90 127.55 1 18.23 d 1 14.91 130.98 241.05 °° 10 119.62 148.35 133.99 ° 115.71 114.04 248.83 °° 12 136.87 143.83 140.35 ° 119.32 119.23 259.74 ° 15 132.27 128.75 130.51 °° 116.45 121.62 249.40 ° 18 135.27 1 14.90 125.09 d° 131.61 122.80 252.26 ° 21 128.45 118.25 123.35 °° 130.86 122.76 250.15 ° Trt Mean 96.30 104.74 105.33 1 12.81 Effects Time Trt Trt x Time Time Trt Trt x Time SEMl 9.93 1 1.08 14.04 1 1.64 5.26 8.95 Prob? < 0.01 0.59 0.18 < 0.01 0.32 0.05 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. A 3 Means in a row within an item with unlike superscripts differ (P < 0.05). abcde 184 Means in a column within an item with unlike superscripts differ (P < 0.05). Table 5.6. Effects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on concentrations of acetic, propionic and butyric acid, and acetate to propionate ratio in the rumen of steers during induced acidosis Time after Control Microbial Time Mean Control Microbial Time Mean challenge, h treatment treatment ------- -- Acetic acid, mmol/L Propionic acid, mmol/L 0 18.10 16.30 17.20 a 9.23 8.75 8.99 ‘ 2 39.28 A 64.48 B 51.88 b 21.75 29.33 25.54 b 4 54.35 A 70.75 B 62.55 ° 29.15 33.60 31.38 ° 6 59.18 62.90 61.04 ° 32.18 34.88 33.53 ° 8 60.93 67.75 64.34 ° 34.93 37.43 36.18 ° 10 58.33 60.53 59.43 “ 36.95 34.60 35.78 ° 12 58.23 60.90 59.56 “ 40.60 36.73 38.66 ° 15 56.93 62.43 59.68 “ 40.63 34.65 37.64 ° 18 67.80 63.43 65.61 ° 41.33 32.80 37.06 ° 21 66.73 62.15 64.44 ° 42.15 32.73 37.44 ° Trt Mean 53.98 59.16 32.89 31.55 Effects Time Trt Trt x Time Time Trt Trt x Time SEMI 3.73 2.99 5.28 4.23 4.36 5.98 Prob? < 0.01 0.23 0.02 < 0.01 0.83 0.89 --------- Acetic acid : Propionic acid Butyric acid, mmol/L -- 0 2.03 1.91 1.97 6.70 7.90 7.30 A 2 1.84 2.39 2.12 10.20 13.83 12.01 b 4 1.86 2.23 2.04 11.78 16.20 13.99 ““ 6 1.90 1.88 1.89 11.03 14.90 12.96 “ 8 1.88 1.90 1.89 12.10 18.25 15.18“‘l 10 1.74 1.86 1.80 13.93 14.23 14.08 “" 12 1.98 1.74 1.86 14.48 16.63 15.55 ““ 15 1.53 2.02 1.78 12.75 18.13 15.44 ““ 18 1.86 2.12 1.99 15.00 20.35 17.68 d 21 1.81 2.02 1.92 15.43 18.70 17.06 °“ Trt Mean 1.84 2.01 12.34 15.91 Effects Time Trt Trt x Time Time Trt Trt x Time SEMl 0.26 0.29 0.36 1.91 1.86 2.70 Prob? 0.94 0.69 0.62 0.01 0.18 0.75 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. a b Means in a row within an item with unlike superscripts differ (P < 0.05). a b c d Means in a column within an item with unlike superscripts differ (P < 0'05)" 185 Table 5.7. Effects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on concentrations of butyric, isobutyric, valeric, and isovaleric acids in the rumen of steers during experimentally induced acidosis Time after Control Microbial Time Mean Control Microbial Time Mean challenge, h treatment treatment ------ lsobutyric acid, mmol/L Valerie acid, mmol/L 0 1.45 1.48 1.46 a“ 1.56 1.67 1.61 2 1.13 1.05 1.09“” 1.14 1.18 1.15 4 1.03 0.88 0.95 a 1.26 1.32 1.30 6 1.38 1.98 1.68“ 1.51 0.88 1.18 8 2.28 2.68 2.48 d 1.78 1.73 1.76 10 1.58 1.18 1.38““ 1.86 1.11 1.49 12 1.30 1.23 1.26 a“ 1.97 1.05 1.51 15 1.25 1.50 1.38““ 1.81 1.81 1.83 18 1.53 2.08 1.80 °“ 2.04 1.34 1.68 21 1.80 2.00 1.90 °" 1.75 3.87 2.80 Trt Mean 1.47 1.60 1.67 1.60 Effects Time Trt Trt x Time Time Trt Trt x Time mm1 0.25 0.12 0.35 0.49 0.33 0.69 Prob? 0.00 0.45 0.92 0.43 0.88 0.38 ------- Isovaleric acid, mmol/L .. 0 2.69 2.78 2.74 2 2.87 3.41 3.14 4 2.65 3.25 2.95 6 2.92 3.04 2.98 8 2.86 3.11 2.99 10 3.11 2.40 2.74 12 2.75 2.71 2.75 15 3.10 3.08 3.10 18 3.89 2.80 3.35 21 3.04 3.32 3.15 Trt Mean 2.99 2.99 Effects Time Trt Trt x Time SEMl 0.36 0.30 0.50 Prob? 0.88 1.00 0.40 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. a b c d c fMeans in a column within an item with unlike superscripts differ (P < (105)- 186 Table 5.8. Eflects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on blood pH, concentrations of lactic acid and bicarbonate, and partial pressure of carbon dioxide during experimentally induced acidosis in steers Time after Control Microbial Time Mean Control Microbial Time Mean challenge, h treatment treatment Time pH Lactic acid, mmol/L------— 0 7.44 7.47 7.45 0.30 0.35 0.33 a 2 7.45 7.46 7.45 0.30 0.29 0.29al 4 7.43 7.47 7.45 0.60 0.81 0.71 “b 6 7.41 7.43 7.42 0.44 0.48 0.46 a 8 7.39 7.42 7.40 0.44 0.73 0.58 a 10 7.38 7.42 7.40 0.54 0.86 0.70 “b 12 7.38 7.41 7.40 0.75 1.02 0.88 a” 15 7.39 7.41 7.40 0.84 1.57 1.21 “ 18 7.38 7.43 7.40 1.30 1.75 1.53 ° 21 7.38 7.49 7.44 1.34 1.15 1.24 “ Trt Mean 7.40 7.44 0.68 0.90 Effects Time Trt Trt x Time Time Trt Trt x Time SEMl 0.02 0.02 0.02 0.25 0.20 0.31 Prob? 0.14 0.12 0.44 0.01 0.44 0.81 ----------- HC03, mmol/L pC02, mmHg-«--—---- 0 31.10 31.38 31.24f 45.18 43.66 44.42f 2 28.38 30.60 29.49 ° 41.19 42.20 41.69 d° 4 27.44 29.10 28.27 A 41 . 19 40.25 40.72 °“ 6 29.01 27.78 28.39 d° 45.34 41.44 43.39 °‘ 8 26.88 26.24 26.56 ° 44.41 40.06 42.23 M 10 25.18 26.04 25.61 ° 42.63 40.03 41.33 d° 12 24.54 23.70 24.12 “b 40.93 37.43 39.18“ 15 23.71 22.24 22.98 8" 38.44 35.55 37.00 3“ 18 21.39 24.15 22.77 a 35.21 36.31 35.76 a 21 25.43 23.98 24.70 “ 41.29 32.19 36.74 3" Trt Mean 26.30 26.52 41.58 38.91 Effects Time Trt Trt x Time Time Trt Trt x Time 513Ml 1.29 1.50 1.76 1.43 1.28 1.81 Prob? < 0.01 0.92 < 0.01 < 0.01 0.14 0.01 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. abcdef 187 Means in a column within an item with unlike superscripts differ (P < 0.05). Table 5.9. Effects of supplementation of M elsdenii, RK02 with E. faecium, RK03 on blood oxygen saturation (S02), partial pressure of 02 (p02), blood base excess (BEb), and blood glucose concentration during acidosis in steers Time after Control Microbial Time Mean Control Microbial Time Mean challenge, h treatment treatment 502, % P025 "111ng ' 0 74.45 73.55 74.00 39.84 38.30 39.07 2 75.20 70.68 72.94 39.50 35.95 37.73 4 74.33 74.16 74.24 39.55 38.29 38.92 6 72.48 73.96 73.22 39.22 38.94 39.08 8 75.18 69.25 72.21 41.80 36.45 39.13 10 70.39 66.74 68.56 39.10 35.74 37.42 12 73.64 73.61 73.63 40.81 39.18 40.00 15 74.45 70.87 72.66 41.20 38.04 39.62 18 79.74 75.08 77.41 44.95 39.69 42.32 21 63.50 85.81 74.66 35.25 49.35 42.30 Mean 73.33 73.37 40.12 38.99 Effects Time Trt Trt x Time Time Trt Trt x Time SEM‘ 2.83 1.97 3.38 2.09 1.07 2.43 Prob? 0.39 0.99 0.00 0.41 0.45 0.02 --------------- BEb, mmol/L Glucose, mg/ lOOmL -- 0 6.93 7.51 7.22 I 83.63 88.50 86.06 2 4.58 6.88 5.73 ° 88.75 84.38 86.56 4 3.50 5.60 4.55 d‘ 93.50 90.13 91.81 6 4.50 3.80 4.15 d 88.88 87.00 87.94 8 2.16 2.36 2.26 ° 91.13 89.25 90.19 10 0.53 2.1 l 1.32 “ 93.00 84.25 88.63 12 0.05 -001 0.02 “b 86.63 89.80 88.21 15 -0.50 -1.31 091 a 96.38 85.00 90.69 18 -2.58 0.66 -0.96 A 112.25 84.13 98.19 21 0.83 1.86 1.35 “ 122.46 98.00 110.23 Mean 2.00 2.95 95.66 88.04 Effects Time Trt Trt x Time Time Trt Tit x Time SEM' 1.33 1.52 1.81 8.74 7.94 11.38 Prob? < 0.01 0.66 0.04 0.45 0.50 0.79 I Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. abcdef Means in a column within an item with unlike superscripts differ (P < 0.05). 188 O 1.5 9 n K: . III‘. g'l‘;~‘ :, 1‘ . 1.111" ' sI' 25“.“ 1” .. '. 1:" 1‘11"; 74'? “01¢ 3 . “9 1:1“ °\ I _. ‘9 ié . )k . .' 2 ' I.’ Q I .- v u . ' Eb x z.- . I I g . u x I I . I. : 0 ~ :I o l i l A l l -50 -40 -30 -20 -10 Days before DFM treatment, (1 Figure 5-1. Dry matter intake (DMI, % of BW) during adaptation period. Steers received 1.6% DM of BW between 12/26 and 1/20, and 1.4% from 1/21 throughout the rest of adaptation and experimental period (-<>- SOl , -o- 804, -A- $06, - Cl- 809, -X- 812 are marked for the discussion). 189 Chapter 6 Conclusions and Future Studies The hypotheses for this study, development of a direct-fed microbial for beef cattle, were that l) aero-tolerant lactic acid-fermenting bacteria in the rumen might be enriched in aerobic, lactic acid media, and that enriched facultative or aero-tolerant anaerobic lactic acid-fermenting bacteria could prevent lactic acid accumulation; 2) isolates from enriched mixed rumen microorganisms in the aerobic lactic acid medium might possess the properties to prevent lactic acid accumulation that is observed during acidosis, and that these isolates might be used as a direct-fed microbial (DF M) to prevent lactic acid accumulation and positively influence fermentation in the rumen; 3) based on the abilities of isolates to prevent lactic acid accumulation during the in vitro fermentations, isolates, Megasphaera elsdenii RK02 and Enterococcusfaecium RK03, as a DP M will favorably alter the fermentation pattern by decreasing lactic acid production in the rumen of cattle experiencing acute acidosis; and 4) Propionibacterium acidipropionici strain DH42, previously isolated and evaluated as a starter culture to enhance bunk stability of high moisture corn (Dawson 1994) and tended to have greater ADG and improved feed efficiencies in beef cattle as a high moisture corn additive(Rust et al., 2000), would increase propionic acid production in the rumen and improve animal performance when used as a DP M. With these hypotheses, studies were designed to 1) develop a set of primers and probe specific to DH42 for the Taq Nuclease Assay with real-time PCR, quantify DH42, evaluate its growth in the rumen, and verify effects of feeding direct-fed DH42 on the growth and carcass characteristics of growing-finishing steers; 2) verify whether 190 aerobically enriched lactic acid-utilizing rumen bacteria could prevent lactic acid acidosis in a ruminal fermentation system and evaluate the microbial diversity resulting fiom enrichments in different aerobicity conditions of lactic acid media; 3) isolate oxygen tolerant lactic acid—fermenting rumen bacteria, and evaluate the effects of these isolates on in vitro ruminal fermentation; 4) evaluate the combined effects of M elsdenii strain RK02 and E. faecium strain RK03 on ruminal fermentation characteristics in steers fed a high concentrate diet and prevention of lactic acid accumulation in the rumen of steers with acidosis induction This dissertation included 1) development of an enrichment method for the isolation of lactic acid-utilizing bacteria (Chapter 3); 2) isolation of lactic acid-utilizing bacteria from the rumen and in vitro confirmation of their ability to prevent lactic acid accumulation in an in vitro ruminal fermentation (Chapter 4); 3) evaluation of isolates, M elsdenii RK02 and E. faecium RK03, on fermentation characteristics in the rumen of steers fed a concentrate diet and challenged with an acidosis provocative diet (Chapter 5); and 4) evaluation of effects of another isolate, P. acidipropionici DH42, on performance and carcass characteristics of feedlot cattle. Rumen microorganisms were enriched in anaerobic or aerobic conditions, and then aerobic enrichment was split to anaerobic and aerobic conditions. Anaerobic enrichment (N 6) was assumed to contain all ruminal lactic acid-utilizing microorganisms, regardless of aero-tolerance. The strategy of an anaerobic following aerobic enrichment (N 2A2N2) was designed to facilitate aero-tolerant anaerobes growing faster in anaerobic condition. Aerobic enrichment (N 2A4) should develop a mixture of anaerobic lactic acid- utilizing microorganisms that are dominated by aero-tolerance and their growth 191 unaffected by aerobicity (facultative). During the 24 h in vitro fermentation, aerobic enrichment (N 2A4) completely prevented lactic acid accumulation, whereas anaerobic enrichment or anaerobic enrichment following aerobic enrichment reduced lactic acid by 21 to 22%. This result implies that lactic acid-utilizing bacteria in N2A4 may be more active in lactic acid utilization in an aerobic environment than organisms grown anaerobically. The concept of using a DFM is to supercharging the rumen with a microorganism which has a low population but a high capability to beneficially modify the ruminal fermentation may apply to the lactic acid-utilizing bacteria, N2A4. The aero- tolerance of N2A4 provides huge advantage for practical use in commercial settings (Chapter 3). RK02 and RK03 were isolated from rumen contents using aerobic enrichment (N 2A4) and the co-culture of these microorganisms grew in aerobic conditions. This combination of RK02 and RK03 decreased lactic acid concentration, and increased butyric and valeric acids at the expense of acetic and propionic acids in the ruminal fermentation in vitro (Chapter 4). The combination of RK02 and RK03 completely prevented lactic acid accumulation when ground corn was used as a substrate in experiment 2, and by 29% (experiment 3) or 34% (experiment 4) when readily fermentable carbohydrates were used as substrates (Chapter 4). The pH values in the control and the combination were 4.79, 5.23; 4.25, 4.40; and 4.31, 4.55 in trials 2, 3, and 4, respectively. Lactic acid production in the control and the combination were 67, -6; 102, 73; and 134, 89 mM, respectively, for trials 2, 3, and 4. Lactic acid utilization by RK02 and RK03 may have diminished at low pH and(or) lactic acid production may exceed the capability of microbial treatment. 192 These reasons may explain the failure of RK02 and RK03 to prevent lactic acid accumulation and a pH drop below 5.2 in these trials. Other potential reasons for failure to prevent accumulation of lactic acid with RK02 and RK03 were inadequate dose levels used in the current study, RK02 might not be the most active lactic acid utilizer in the aerobic enrichment and(or) RK03 might increase lactic acid in the rumen. In the rumen of steers fed a 83% high concentrate diet, the combination of RK02 and RK03 did not change the fermentation characteristics. The combination numerically increased butyric acid and decreased propionic acid concentration (Chapter 5). In the rumen of steers challenged with an acidosis provocative diet, the combination of RK02 and RK03 facilitated the use of a rapidly fermentable substrate (finely ground wheat) that resulted in a greater concentration of total organic acids. The microbial treatment decreased partial pressure of carbon dioxide and increased partial pressure of oxygen in the blood of steers challenged with acidosis. The microbial treatment had a numerically higher blood pH (Chapter 5). Acidosis induction method used in this study may have created severe acidotic conditions and may have overwhelmed the lactic acid utilization. A less severe challenge is more frequent in commercial settings. A less severe acidosis induction that resulted in subacute acidosis may have allowed RK02 and RK03 to provide a significant improvement in lactic acid utilization. An effective dose level was not identified in this study. Further isolation of lactic acid-utilizing bacteria using an aerobic enrichment may expand the choice of DFM candidates which can be used in the development of DF M. The role of RK03 in the rumen is unlikely to cause varied responses between the in vitro and in vivo studies in this thesis. 193 In the animal performance study, Propionibacterium acidipropionici, strain DH42 was used as a DF M (Chapter 2). A set of Taq Nuclease Assay primers and probe was designed and used to quantify DH42 in the rumen. Growth of DH42 was confirmed in an in vitro ruminal microbial ecosystem; however DH42 did not establish a population in the rumen of the steer. To maintain a level of DH42 in the rumen, DH42 was supplemented daily on the top of feed for 123 d. During d 56 to 111, DH42 decreased DMI and had the lower ADG at d 84 and d 111. The reduced ADG with the DH42 treatment was compensated with greater ADG during d 112 to 123. Carcass yield grade was improved by DH42, but quality grade was similar to the control (Chapter 2). P. acidipropionici strain DH42 has the potential to alter the ruminal fermentation in vitro and animal performance when used as a fermentation additive to high moisture corn. Ruminal fermentation discrepancies and inconsistencies in animal performance with the previous studies may result from variations in dose between trials. To improve animal production with direct-fed DH42, the optimal dose of DH42 for animal growth needs to be defined. The rationale for using aerobic lactic acid media to enrich rumen microorganisms was that aero-tolerant lactic acid-utilizing bacteria may be developed. The reason for development of aero-tolerant bacteria as a DFM was that strict anaerobic strains fail to maintain viability under the conditions in a commercial setting. Studies utilizing strict anaerobic bacterial species have focused on establishment of populations in the rumen after short-term administration. The chance to succeed as a DFM with one-time or short- terrn administration may be limited to only a few strains. Studies using facultative or aero-tolerant anaerobic bacterial species require continuous daily supercharging. Aero- 194 tolerance allows DFM to be added to the diet, which is acceptable in a commercial setting. Therefore, DF M candidates should have at least two capabilities. One is to beneficially alter the ruminal fermentation, and the other is to be aero-tolerant or an ability to establish a population in the rumen after short-term administration. Development of a suitable DF M is dependent on the isolation of strains possessing these two capacities. RK03 was required for acre-tolerance of the strict anaerobe, RK02. It is likely that RK03 depleted oxygen to tolerable levels for growth of RK02. Peroxide and superoxide, reactive oxygen molecules, are toxic to anaerobic microorganisms due to the lack of the main enzymes of antioxidative defense: superoxide dismutase (SOD) and catalase. Most aerobes have catalase, an enzyme that detoxifies hydrogen peroxide, but many ruminal microorganisms lack this enzyme or have low enzyme activity. Generally, microbial species with high SOD activity have high or moderate aero-tolerance in comparison to species with low or no SOD activity. The combined action of SOD and catalase detoxifies oxygen, superoxide and peroxide. SOD and catalase are encoded by sod and kat genes, respectively. Genetic manipulation of M. elsdenii with insertion of sod and kat genes may expand the choice of DF M candidates. A transformed strict anaerobic DFM can be added to the diet and used for long-term daily supercharging of the rumen. It is unknown if RK03 was a necessary member of the DFM treatment. It is shown to be necessary in an in vitro experiment. The current study did not demonstrate the ability of RK03 to maintain activity of the strict anaerobic RK02 during delivery of feed to the feed bunk. By introducing sod and kat genes, DFM candidates would need only one capacity, to beneficially alter the ruminal fermentation. The author expects the transformation of anaerobic DFM with sod and kat genes to be reported in the literature soon. 195 APPENDIX A. TABLES DISCUSSED IN CHAPTERS 196 Table 6.1. Substrate utilization of M elsdenii RK02 on Biolog AN plate Substrates RK02 RK02 RK02 Water D-Mannose + Pyruvic Acid + N-Acetyl-D- D-Melezitose Pyruvic Acid + Galactosamine Methyl Ester N-Acetyl-D- D-Melibiose D-Saccharic Acid Glucosamine N-Acetyl-b-D- 3-Methyl-D- + Succinarnic Acid Mannosamine Glucose Adonitol a-Methyl-D- Succinic Acid Galactoside Amygdalin b—Methyl-D- Succinic Acid Galactoside Mono-Methyl Ester D-Arabitol a-Methyl-D- m-Tartaric Acid Glucoside Arbutin b-Methyl-D- Urocanic Acid Glucoside D-Cellobiose Palatinose + L-Alaninamide a-Cyclodextrin D-Raffinose L-Alanine + b-Cyclodextrin L-Rhamnose L-Alanyl-L- + Glutamine Dextrin + Salicin L-Alanyl-L- + Histidine Dulcitol D-Sorbitol L-Alanyl-L- + Threonine i-Erythritol + Staehyose L-Asparagine + D-Fructose + Sucrose L-Glutamic Acid + L-Fucose + D-Trehalose + L-Glutamine + D-Galactose Turanose + Glycyl-L- Aspartic Acid D-Galacturonic acid + Acetic Acid Glycyl-L-Glutamine Gentibiose F onnic Acid Glycyl-L Methionine + D-Gluconic acid + F umaric Acid Glycyl-L-Proline D-Glucosaminic acid Glyoxylic Acid + L-Methionine + a-D-Glucose + a-Hydroxybutyric Acid + L-Phenylalanine + Glucose- 1 -Phosphate b-Hydroxybutyric Acid + L-Serine + Glucose-6-Phosphate + Itaconic Acid L-Threonine + Glycerol + a-Ketobutyric Acid + L-Valine + D,L-a-Glycerol + a-Ketovaleric Acid + L-Valine plus + Phosphate L-Aspartic Acid m-Inositol D,L-Lactic Acid + 2’-Deoxy Adenosine + a-D-Lactose L-Lactic Acid + Inosine + Lactulose D-Lactic Acid + Thymidine Methyl Ester Maltese D—Malic Acid Uridine + Maltotriose + L-Malic Acid Thymidine-5’- + Monophosphate D—Mannitol Propionic Acid Uridine-5’- Monophosphate + Microorganism utilized corresponding substrate. 197 Table 6.2. Substrate utilization of E. faecium RK03 on Biolog GP2 plate Substrates RK03 Water a-Methyl-D-Galactoside L-Malic Acid a-Cyclodextrin b-Methyl-D-Galactoside Pyruvic Acid Methyl Ester b-Cyclodextrin 3-Methyl-D-Glucose Succinic Acid Mono-Methyl Ester Dextrin a-Methyl-D-Glucoside Propionic Acid Glycogen b-Methyl-D-Glucoside Pyruvic Acid lnulin a-Methyl-D-Mannoside Succinamic Acid Mannan Palatinose Succinic Acid Mono-Methyl Ester Tween 40 D-Psicose N-Acetyl-L— Glutamic Acid Tween 80 D—Raflinose L-Alaninamide N-Acetyl-D- L-Rhamnose D-Alanine Glucosamine N-Acetyl-b-D- D-Ribose L-Alanine Mannosamine Amygdalin Sal icin L-Alanyl-Glycine L-Arabinose Sedoheptulosan L-Asparagine D-Arabitol D—Sorbitol L-Glutamic Acid Arbutin Stachyose Glycyl-L- Glutamic Acid D-Cellobiose Sucrose L-Pyroglutamic Acid D-Fructose D—Tagatose L-Serine L-Fucose D-Trehalose Putrescine D—Galactose Turanose 2,3-Butanediol D-Galacturonic Acid Xylitol Glycerol + Gentiobiose D-Xylose Adenosine + D-Gluconic Acid Acetic Acid 2’-Deoxy Adenosine + a-D-Glucose a-Hydroxybutyric Acid Inosine + m-Inositol b-Hydroxybutyric Acid Thymidine + a-D-Lactose g-Hydroxybutyric Acid Uridine + Lactulose p-Hydroxy- Adenosine-5'- + phenylacetic Acid Monophosphate Maltose a-Ketoglutaric Acid Thymidine-5'- Monophosphate Maltotriose a-Ketovaleric Acid Uridine-5'- Monophosphate D-Mannitol Lactamide D-Fructose-6- Phosphate D-Mannose D-Lactic Acid a-D-Glucose-l- Methyl Ester Phosphate D-Melezitose L-Lactic Acid D-Glucose-6- Phosphate D-Melibiose D-Malic Acid D-L-a-Glycerol Phosphate + Microorganism utilized corresponding substrate. 198 Table 6.3. Growth of the combination of M elsdenii RK02 and E. faecium RK03 with different reducing agent contents1 Reducing agent2 Lag phase, h3 ODmax - OD04 Max growth rate5 ODmin6 0% 14.98d 1.04al 0.41“ 0.0408 20% 13.60° 1.04a 0.41“ 0.0433” 40% 9.86b 1.10b 0.378 0.043Ab 60% 9.19b 1.08b 0.38“ 0.044ab 80% 7.79al 1.05a 042° 0.046b 100% 7.76a 1.06a 0.39““ 0.046b 512M7 0.36 0.01 0.01 0.002 I Data obtained from growth experiments were modeled to a modified Gombertz equation (Zwieteri'ng et al., 1990)"; on = ODmin + (ODmax - 01),) exp (-exp (-Max growth rate(Time-Lag phase))). Parameters were analyzed using a Gauss—Newton algorithm in SAS (SAS Institute Inc., Cary, NC, USA) and the PROC NLIN procedure. 2 Corresponding amount of reducing agent recommended by Goering and Van Soest (1970) was added to Na-lactic acid broth medium. Time at which the absolute growth rate is maximum. Difference between initial and maximum density. Maximum relative grth rate. Minimum density, which is the lower asymptote value in the model. \IQUIthW Standard error of mean. 8 Zwietering, M. H., I. Jongenburger, F. M. Rombouts, and K. Van't Riet. 1990. Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56: 1875—1881. a b c Means in a column within an item with unlike superscripts difl°er (P < 0'05)' 199 Table 6.4. Bacteria closely related to RK02 with high scoring BLAST hits (Database: All GenBank+EMBL+DDBJ+PDB sequences; 5,750,983 sequences) Total Query E Max Accession Description score coverage value ident DQl46765.l Megasphaera elsdenii strain ST 168 rRNA gene 896 100% 0.0 98% AY03 8995.1 Megasphaera elsdenii strain T81 l6S rRNA gene 894 99% 0.0 98% AY03 8994.1 Megasphaera elsdenii strain AW106 168 rRNA gene 894 99% 0.0 98% AY03 8993.1 Megasphaera elsdenii strain J L1 168 rRNA gene 894 99% 0.0 98% EF445217.1 Uncultured bacterium clone NED4A5 16S rRNA gene 893 99% 0.0 98% AF371696.1 Uncultured bacterium clone p-1934-s962-3 16S rRNA gene 893 99% 0.0 98% M26493] M.elsdenii small subunit rRN A 885 99% 0.0 97% AF445204.1 Swine fecal bacterium F PC23B 16S rRNA gene 880 99% 0.0 97% U95027.l Megasphaera elsdenii ATCC 25940 16S rRNA gene 880 100% 0.0 97% AB034085.1 Uncultured rumen bacterium 3C3d-7 gene for 16S rRNA 876 99% 0.0 97% AY038996.] Megasphaera elsdenii strain YJ-4 l6S rRNA gene 874 99% 0.0 97% AB034086.1 Uncultured rumen bacterium 5C3d-9 gene for 16S rRNA 869 99% 0.0 97% AF445203.1 Swine fecal bacterium FPC65 16S rRNA gene 861 99% 0.0 97% AF283 705.1 Megasphaera elsdenii strain L303 16S rRNA gene 859 96% 0.0 98% AY196919.1 Megasphaera elsdenii strain 7-11 168 rRNA gene 859 95% 0.0 98% AF261784.1 Uncultured swine feces bacterium F10 l6S rRNA gene 843 99% 0.0 96% EF 593066.] Uncultured Megasphaera sp. clone 31c l6S rRNA gene 841 100% 0.0 96% EF053126.2 Bacterium LBO] l6S rRNA gene 839 99% 0.0 96% AY196918.1 Megasphaera elsdenii strain 4-13 168 rRNA gene 839 95% 0.0 97% DQ326498] Uncultured bacterium clone C607 16S rRNA gene 837 95% 0.0 97% DQ326271.1 Uncultured bacterium clone C220 l6S rRNA gene 832 95% 0.0 97% DQ326091.1 Uncultured bacterium clone 3330 168 rRNA gene 832 95% 0.0 97% L79909.l Megasphaera hominis l6S rRNA gene 828 99% 0.0 96% U95028.] Megasphaera elsdenii $2 168 rRNA gene 826 100% 0.0 96% DQ326342] Uncultured bacterium clone C333 l6S rRNA gene 826 95% 0.0 97% DQ326210] Uncultured bacterium clone C129 16S rRNA gene 826 95% 0.0 97% DQ326203] Uncultured bacterium clone C118 l6S rRNA gene 826 95% 0.0 97% 200 Table 6.5. Bacteria closely related to RK03 with high scoring BLAST hits (Database: All GenBank+EMBL+DDBJ+PDB sequences; 5,750,983 sequences) Total Query E Max Accession Description score coverage value ident EF510700.1 Uncultured bacterium clone P2D15-567 16S rRNA gene 894 100% 0.0 98% AM157434.1 Enterococcus faecium l6S rRNA gene, clone 8C4 894 100% 0.0 98% DQ329145] Enterococcus faecium isolate F0217] 16S rRNA gene 89] 99% 0.0 98% 00329141.] Enterococcus faecium isolate F00287 16S rRNA gene 891 99% 0.0 98% EF5 10798.1 Uncultured bacterium clone P2D15-558 16S rRNA gene 889 100% 0.0 98% EF5 10753.1 Uncultured bacterium clone P2D15-719 16S rRNA gene 889 100% 0.0 98% EF510737.1 Uncultured bacterium clone P2D15-561 16S rRNA gene 889 100% 0.0 98% EF510706.1 Uncultured bacterium clone P2015-759 16$ rRNA gene 889 100% 0.0 98% AJ309563.] Enterococcus azikeevi partial 16S rRNA gene, strain IB-A35 889 100% 0.0 98% EU003447.1 Enterococcus faecium strain IDCC 2103 16S rRNA gene 887 99% 0.0 98% DQ467844.1 Enterococcus hirae isolate F01959 16S rRNA gene 887 99% 0.0 98% DQ467839.1 Enterococcus hirae isolate F01695 16S rRNA gene 887 99% 0.0 98% DQ467838] Enterococcus hirae isolate F00449 16S rRNA gene 887 99% 0.0 98% 004678371 Enterococcus hirae isolate F00399 16S rRNA gene 887 99% 0.0 98% ABZ46407.1 Enterococcus faecium gene for 168 rRNA , strain: C228 887 99% 0.0 98% AY172570.1 Enterococcus faecium 168 rRNA gene, complete sequence 887 99% 0.0 98% AY97]749.1 Enterococcus faecium 16S rRNA gene 887 99% 0.0 98% DQ329144.1 Enterococcus faecium isolate F01260 16S rRNA gene 887 99% 0.0 98% DQ329143.1 Enterococcus faecium isolate F0055] 16S rRNA gene 887 99% 0.0 98% DQ329142.1 Enterococcus faecium isolate F00447 16S rRNA gene 887 99% 0.0 98% DQ329140.1 Enterococcus faecium isolate F00243 l6S rRNA gene 887 99% 0.0 98% DQ329146] Enterococcus faecium isolate F02250 168 rRNA gene 885 99% 0.0 98% AB062562.1 Enterococcus durans gene for 16S rRNA, strainzMR103 885 99% 0.0 98% EUOO3448.] Enterococcus faecium strain IDCC 2104 16S rRNA gene 883 99% 0.0 98% EF510797.1 Uncultured bacterium clone P2D15-724 16S rRNA gene 883 100% 0.0 98% EF510750.1 Uncultured bacterium clone P2D15-563 16S rRNA gene 883 100% 0.0 98% EF510364.1 Uncultured bacterium clone P2D1-721 l6S rRNA gene 883 100% 0.0 98% 201 Table 6.6. Effects of supplementation of M. elsdenii RK02 with E. faecium RK03 on the minimum and maximum values of fermentation characteristics in the rumen of steers fed a concentrate diet Minimum ' Maximum 2 Control DFM Mean SEM3 Control DFM Mean SEM3 Total organic acid, mmol/L 1 13.50 1 14.98 114.24 7.59 279.70 278.66 279.18 7.84 Total organic acid, mmol (hexose)/L 71.50 73.29 72.39 5.36 170.96 179.29 175.12 4.54 Total VFA, mmol/L 1 11.63 ] 12.50 1 12.06 7.30 262.83 262.74 262.79 8.72 pH 5.72 5.56 5.64 0.30 6.99 6.86 6.92 0.12 Lactic acid, mmol/L 1.87 2.48 2.17 0.37 16.86 15.92 16.39 1.63 Acetic acid, mmol/L 52.33 51.16 51.75 3.50 108.26 105.45 106.86 3.93 Propionic acid, mmol/L 29.80 29.73 29.77 1.86 92.34 77.38 84.86 13.97 Butyric acid, mmol/L 21.89 23.14 22.52 2.87 41.96 60.32 51.14 7.93 lsobutyric acid, mmol/L 1.38 1.60 1.49 0.16 4.68 5.04 4.86 0.39 Valerie acid, mmol/L 2.19 2.06 2.13 0.20 6.57 4.69 5.63 1.31 Isovaleric acid, mmol/L 4.04 4.80 4.42 0.67 9.02 9.87 9.44 1.70 1 Least square means of minimum values of steers within treatment. 2 Least square means of maximum values of steers within treatment. 3 Standard error of mean. 202 Table 6.7. Effects of supplementation of M. elsdenii, RK02 with E. faecium RK03 on concentrations of total organic acid on hexose basis in the rumen of steers fed a concentrate diet prior to acidosis induction Time, h Control DFM Mean -- Total organic matter, mmol (hexose)/L - 0 81.25 84.00 82.63 3 100.50 1 16.25 108.37 6 128.00 148.25 138.12 9 162.75 154.25 158.50 12 149.25 159.50 154.37 15 155.00 130.75 142.87 18 130.75 126.00 128.37 21 137.50 135.25 136.38 24 110.75 120.75 115.75 Mean 128.42 130.56 Effects Time Trt Trt x Time SEM] 5.37 2.57 7.60 Prob.2 < 0.01 0.42 0.40 I Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. 203 Table 6.8. Effects of supplementation of M. elsdenii, RK02 with E. faecium, RK03 on sodium, and potassium during induced acidosis in steers blood concentration of ionized magnesium (MgH) and ionized calcium (CaH), Time, h Control DFM Time Mean Control DF M Time Mean .......... — Ca”, mg/100mL MgH,mg/100mL 0 4.69 4.87 4.78 b“ 1.21 1.19 4.78 b° 2 4.75 4.56 4.65 “b 1.22 1.06 4.65 “b 4 4.85 4.79 4.82 b“ 1.35 1.25 4.82 d° 6 4.93 4.87 4.90 °" 1.43 1.27 4.90 d‘ 8 4.99 4.94 4.96 d 1.48 1.29 4.96 ° 10 4.77 4.84 4.81 b“ 1.33 1.22 4.81 “k 12 4.74 4.85 4.80 b“ 1.29 1.21 4.80 b“ 15 4.89 4.91 4.90 °" 1.39 1.24 4.90 d‘ 18 4.70 4.21 4.45 " 1.25 0.93 4.45 ‘ 21 4.80 4.67 4.73 b“ 1.37 1.14 4.73 °d Trt Mean 4.8103 4.75 4.81 4.75 Effects Time Trt Trt x Time Time Trt Trt x Time sam' 0.10 0.05 0.11 o. 10 0.05 0.07 Prob.2 0.00 0.39 0.18 < 0.01 0.02 0.13 Na, mmol/L K, mmol/L 0 141.21 141.24 141.23“ 3.31 3.56 3.44abc 2 141.30 141.51 141.41 a 3.24 3.47 3.36“” 4 141.36 141.23 141.29a 3.39 3.68 3.53”“ 6 143.61 145.48 144.54 b 3.50 3.74 3.62 °" 8 144.02 146.09 145.06 b“ 3.67 3.89 3.78 d 10 143.05 145.85 144.45b 3.51 3.90 3.71 °" 12 145.80 148.24 147.02 ° 3.49 3.71 3.60 b“ 15 144.75 147.50 146.12 b° 3.59 3.55 3.57 “d 18 143.80 146.01 144.91 b 3.24 3.36 3.30al 21 143.99 144.30 144.14 b 3.59 3.45 3.52 3"“ Trt Mean 143.29 144.74 3.45 3.63 Effects Time Trt Trt x Time Time Trt Trt x Time SEMI 0.88 0.56 1.06 0.15 0.16 0.20 Prob.2 < 0.01 0.07 0.85 0.00 0.44 0.58 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. abcde 204 Means in a column within an item with unlike superscripts differ (P < 0.05). Table 6.9. Effects of supplementation of M. elsdenii, RK02 with E. faecium, RK03 on blood hematocrit (Hct), base excess of extracellular fluid (BE-ECF), standard bicarbonate concentration (SBC), and concentration of chloride during experimentally induced acidosis in steers Time, h Control DFM Time Mean Control DFM Time Mean Hct, % BE-ECF, mmoVL—--- 0 29.75 30.13 29.94 b 6.78 7.35 7.06 ° 2 29.25 28.63 28.94 a 4.06 6.61 5.34 d 4 30.00 30.63 30.31 b 2.89 5.15 4.02 d 6 31.75 31.63 31.69 ° 4.21 3.26 3.74 d 8 31.63 33.63 32.63 ° 1.64 1.60 1.62 ° 10 33.13 34.25 33.69 d 41.23 1.36 0.57 b° 12 33.88 34.93 34.40 d -0.81 -1.06 -o.94 “" 15 36.38 35.20 35.79 ° -1.51 -2.67 -2.09 " 18 37.00 34.75 35.88 ° -3.95 .043 -219 a 21 38.04 35.75 36.89 ° 0.21 0.43 0.32 b‘ Trt Mean 33.08 32.95 1.33 2.16 Effects Time Trt Trt x Time Time Trt Trt x Time SEM' 0.97 1.14 1.31 1.48 1.71 2.02 Prob.2 < 0.01 0.94 0.11 < 0.01 0.73 0.01 SBC, mmol/L Cl, mmol/L 0 30.39 30.88 30.63 1 103.13 103.21 103.17 a 2 28.19 30.26 29.23 ° 104.06 104.25 104.16 ‘ 4 27.15 29.09 28.12 ° 104.24 104.18 104.21 a 6 28.00 27.40 27.70 C 105. 14 106.74 105.94 b‘ 8 25.98 25.96 25.97 d 106.44 107.70 107.07 °" 10 24.44 25.70 25.07 1"” 106.24 107.15 106.69 b“ 12 24.15 23.93 24.04 ’1” 107.71 109.89 108.80 d 15 23.79 22.73 23.26 a 106.20 109.22 107.71 °" 18 22.11 24.64 23.38 a” 106.19 107.84 107.01 °" 21 24.64 25.85 25.25 °“ 103.79 106.13 104.96 8" Trt Mean 25.88 26.64 105.31 106.63 Effects Time Trt Trt x Time Time Trt Trt x Time SEMl 1.1868 1.3425 1.61 1.13 1.18 1.51 Prob.2 < 0.01 0.69 0.08 0.00 0.43 0.90 1 Standard error of mean of time, treatment, and overall. 2 Probabilities associated with fixed effects; time, treatment, and interaction of time and treatment. abcdef 205 Means in a column within an item with unlike superscripts differ (P < 0.05). APPENDIX B. FIGURES DISCUSSED IN CHAPTERS 206 TAGAGTTTGATCHTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACAT GCAAGTCGAACGAGAAGAGATGAGAAGCTTGCTTCTTATYRATTCGAGTG GCAAACGGGTGAGTAACGCGTAAGCAACCTGCCCTTCAGATGGGGACAAC AGCTGGAAACGGCTGCTAATACCGAATACGTTCTTTTTGTCGCATGGCAG AGRGAAGAAAGGGAGGCTCTTCGGAGCTTTCGCTGAAGGAGGGGCTTGCG TCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTA GCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGA CTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTG AC6GAGCAGACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGUTST GTTATACGGGACGAATGGCGTAGCGGTCAATACCCGTACGAGTGACG Figure 6.]. Partial sequence (497bp) of 16S rDNA of M. elsdenii RK02. TAGAGTTTGATCHTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACAT GCAAGTCGAACGCTTCTTTTTCCAYCGGAGCTTGCTCCACCGGAAAAAGA GGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGG GATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCAT GGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCG GTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATA GCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAA CTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTG ACCGARSAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACKCTG TTGTTAGAGAAGAACAAGGATGAGAGTAACTGTCATCCCTGACG Figure 6.2. Partial sequence (494bp) of 16S rDNA of E. faecium RK03. 207 RK02 Sbjct RK02 RK02 RK02 Sbjct RK02 Sbjct RK02 Sbjct RK02 Sbjct RK02 Sbjct RK02 Sbjct RK02 Sbjct 1 121 131 181 191 241 25] 30] 311 36] 371 421 430 481 490 TAGAGTTTGATCMTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAA l||||l|||||l ||||||||l|||||||||||||||||||||||||||||||1|||||| TAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAA CGAGAAGAGATGAGAAGCTTGCTTCTTATYRATTCGAGTGGCAAACGGGTGAGTAACGCG ||l||||||||||||||||||l||l||1| ||||||||||||||||||l|l|||||||| CGAGAAGAGATGAGAAGCTTGCTTCTTATCGATTCGAGTGGCAAACGGGTGAGTAACGCG TAAGCAACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATACG ||||l||||||||||llllllllllllll||||l||||||||||||||||||||||||1| TAAGCAACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATACG TTCIIIl1GTCGCATGGCAGAGRGAAGAAAGGGAGGCTCTTCGGAGCTTTCGCTGAAGGA |||||||||||||||||||||| ||||Ill|||||||1||||||||||||||||||||l| TTCIIIl|GTCGCATGGCAGAGGGAAGAAAGGGAGGCTCTTCGGAGCTTTCGCTGAAGGA GGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTA ||||||||||||||1|1|||||||||||l|l||||||||||||ll||l|||||||||||| GGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTA GCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGG ||||1||||||||ll|Illl|l||||||1||||||||||||ll||||||||||||||||| GCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGG AGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAGACGCCGCGTGA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIII AGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCA—ACGCCGCGTGA ACGATGACGGCCTTCGGGTTGTAAAGWTSTGTTATACGGGACGAATGGCGTAGCGGTCAA |||||||||||||||||||||||||| | llllllll|||||||l||||||||||1|||| ACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATACGGGACGAATGGCGTAGCGGTCAA TACCCGT-ACGAGTGACG 497 IIIIIII IIIIIIIIII TACCCGTFACGAGTGACG 507 Figure 6.3. Alignment of RK02 with the highest scoring BLAST hit sequence, 60 70 120 130 180 190 240 250 310 360 370 420 429 489 Megasphaera elsdenii strain 5T 16S ribosomal RNA gene (DQ146765.1; Score = 896 bits (485), Expect = 0.0, Identities = 490/498 (98%)). 208 RK03 1 TAGAGTTTGATCMTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAA 6O |||||||||||l |||||||||||||l|l|||||||||||||||||||||1|||||l||| Sbjct 1563 TAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAA 1504 RK03 61 CGCTTCIIIIICCAYCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGGGTGAGT 120 llllllllllllll |||||||||||||||||||||||||||ll|||||||||||||||| Sbjct 1503 CGCTTCIIIIICCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGGGTGAGT 1444 RK03 121 AACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCG 180 |||||||l|||l|l|||||||ll||||l|||||l||||||||l||||||||||||||||| Sbjct 1443 AACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCG 1384 RK03 181 TATAACAATCGAAACCGCATGGIII1GATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGA 240 I||||11|11|||1|||||||l|||||||||||||||||||llll||||||||||||||| Sbjct 1383 TATAACAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGA 1324 RK03 241 TGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATA 300 ||||lllllllll|||||||||||l|||||||||||||||||||||||||||l||||||| Sbj ct 1323 TGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATA 1264 RK03 30] GCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGG 360 |||||||||||||||||l||||||||l||||||||||||||||||||||||||||||||| Sbjct 1263 GCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGG 1204 RK03 361 AGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGARSAACGCCGCGTGAG 420 ||||lllllllll|||||||||I||||||||||||||l||||||| ||||||||||||| Sbjct 1203 AGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAG 1144 RK03 421 TGAAGAAGGIII|CGGATCGTAAAACKCTGTTGTTAGAGAAGAACAAGGATGAGAGTAAC 480 |1|l|l|l|||||l||||||||1||l ||||||l||||||||||||||||||l||||||| Sbjct 1143 TGAAGAAGGI||ICGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAAC 1084 RK03 481 TGT-CATCCCT-GACG 494 III IIIIIII IIII Sbjct 1083 TGTTCATCCCTTGACG 1068 Figure 6.4. Alignment of RK03 with the highest scoring BLAST hit sequence, Enterococcusfaecium isolate F02171 16S ribosomal RNA gene (AM157434.1; Score = 894 bits (484), Expect = 0.0, Identities = 489/496 (98%)). 209 Megasphaera elsdenii strain Megasphaera elsdenii strain Megasphaera elsdenii strain Megasphaera elsdenii strain Megasphaera elsdenii strain Megasphaera elsdenii strain Megasphaera elsdenii strain Swine Fecal Bacterium FPC RKO — Megasphaera elsdenii strain Swine Fecal Bacterium FPC Firmicutes bacten’um EG24 Allisonella histaminiforman Dialister sp. Oral strain GB Dialisterinvisus 16$ rDNA Selenomonas mminantium E Selenomonas ruminantium F Selenomonas ruminantium Selenomonas ruminantium l Mitsuokella jalaludinii strain Veillonella parvula Veillonella dispar Veillonella parvula Veillonella caviae Veillonella atypica Veillonella ratii Veillonella sp. 2001-1 12662 Figure 6.5. Distance tree of M. elsdenii RK02 and bacterial DNA sequences with high scoring hit obtained using FAST from GenBank Bacterial Sequences (genbankbct0.na). Same species with low hit score was omitted from selection. 210 (- Entemcoccus durans CK1106 Enterococcus hirae C17456 Enterococcus hirae Enterococcus hirae LMG6399 Enterococcus durans PSM20633 __LEntemcoccus lactis CK1 114 Enterococcus lactis CK1025 Enterococcus faecium 27368 Enterococcus faecium 84778 Enterococcus hirae CECT 279T Enterococcus azikeevi lB—A3 Enterococcus azikeevi lB-A3 RK03 Enterococcus durans OK220 Enterococcus durans GM097 Enterococcus durans MR103 Enterococcus faecium 16$ rDNA Enterococcus faecium BC Enterococcus faecium 8B Enterococcus hirae MP1 Enterococcus durans MY411 Enterococcus faecium 110815 Enterococcus faecium uc824 Enterococcus faecium u0804 b Enterococcus faecium uc829 Figure 6.6. Distance tree of E. faecium RK03 and bacterial DNA sequences with high scoring hit obtained using FAST from GenBank Bacterial Sequences (genbankbct0.na). Same species with low hit score was omitted fi'om selection. 21] A. FOM ,, O k l 1 0 6 12 18 24 Incubation time, h C. Lactic acid Incubation time, h P E. Acetic acid 240 20 0 l l l 0 6 12 18 24 Incubation time, h l- G. Butyric acid 0 6 1 2 18 24 Incubation time, h l 0 6 12 1 8 24 Incubation time, h 20° F D. Total VFA I_ I 0 6 12 18 24 Incubation time, h F. Propionic acid L l 0 6 12 1 8 24 Incubation time, h 30 H. Valerie acid 0 6 12 18 24 Incubation time, h Figure 6.7. Effects of M. elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid fiom concentrate diet and ground corn as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 1; -x- Control, -0- RK02 + RK03; * Means differ at the time point marked (P < 0.05)). 212 200 150 €100 t 10 -10 60 40 mM 20 120 l 00 80 60 40 20 mM 0 6 12 18 24 Incubation time, h " C. Lactic acid * 1- l. P k (T 6 12 . I8 24 Incubation time. h P E. Acetic acid i l 1 O 6 12 18 24 Incubation time, h l- G. Butyric acid 1- f r t l_ 0 6 12 18 24 Incubation time, h L 1 0 6 12 1 8 24 Incubation time, h 20" ‘ D. Total VFA 150 r E 100 - 50 '- 0 4 L l 0 6 12 18 24 Incubation time, h 60 P F. Propionic acidu 1:, *l 240 ’ b E 20 '- 0 l i l 0 6 12 18 24 Incubation time, h H. Valeric acid 0 6 12 18 24 Incubation time, h Figure 6.8. Effects of M. elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid from hay diet and ground corn as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 2; -x- Control, -0- RK02 + RK03; * Means differ at the time point marked (P < 0.05)). 150 100 E E 50 0 0 6 12 18 24 Incubation time, h 120 C. Lactic acid * 80 2 b E 40 0 l l 0 6 12 18 24 Incubation time, h 60 P E. Acetic acid 240 E 20 0 ., 0 6 12 18 24 Incubation time, h 60 G. Butyric acid , 40 1- =1: 2 D E 20 - 0 . . ’l‘ 0 6 12 1 8 24 Incubation time, h l L 0 6 12 1 8 24 Incubation time, h 160 2 E 80 0 ., 0 6 12 18 24 Incubation time, h 0 6 12 18 24 Incubation time, h 20 H. Valeric acid ] 0 6 12 18 24 Incubation time, h Figure 6.9. Effects of M elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid from concentrate diet and RFC as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 3; -x- Control, -0- RK02 + RK03; * Means differ at the time point marked (P < 0.05)). 4 l J 12 18 Incubation time, h 24 6 12 18 Incubation time, h 24 6 12 18 Incubation time, h 6 24 1 2 Incubation time, h 18 40 F E. Acetic acid 40 r F. Propionic acid * * D E20 r 3 1",: ":2 41‘ E20 ~ x *___..——x ,2: . .. , O L l l 0 l 41 P 0 6 12 18 24 0 6 12 18 24 Incubation time, h Incubation time, h 80 30 G. Butyric acid H. Valeric acid 60 P D 2 220 h * E40 E10 D 20 0 T H 0 0 6 12 18 24 0 6 12 18 24 Incubation time, h Incubation time, h Figure 6.10. Effects of M. elsdenii, RK02 with E. faecium, RK03 on fermentation characteristics with rumen fluid from hay diet and RFC as substrate; Organic acids values are differences of concentrations between at 0 and 24 h (Trial 4; -x- Control, -O- RK02 + RK03; * Means differ at the time point marked (P < 0.05)). 215 A. Control 2 a a\° a\“ E E D D 0.2 + 02 1 0 5 10 0 5 10 Days on trial Days on trial C. Control 1.4 4 5 5 ‘ 1.4 1.2 1.2 a n a . ,,\° 0.8 °\° 0.8 -=‘ 0.6 ._r 0.6 2 Q 0-4 E 0.4 0.2 0.2 0 ‘: 0 03691215182124 03691215182124 Time after feeding, h Time after feeding, h E. Control F. DFM 03691215182124 03691215182124 Time after feeding, h Time after feeding, h Figure 6.11. Dry matter intake in % of BW during DFM supplementation (A and B), DMI in % ofBW (C and D) and Ruminal pH (E and F) on d10 ofDFM supplementation (-<>- S01, -0- S05, -A- SO6, -X- SO9 in control and -<>- 804, -O- S08, -A- 810, -X- S12 in DFM group). 216 A. Total organic acid B. Lactic acid 260 220 E 180 140 100 0 l l l l l L 4* 03691215182124 03691215182124 Time afier feeding, h Time after feeding, h C. Total VFA D. Acetic acid 240 I E 200 100 a E 160 120 60 ‘ 03691215182124 03691215182124 Time after feeding, h Time after feeding, h E. Propionic acid F. Butyric acid 100 2 E 60 20 ‘ L + 03691215182124 03691215182124 Time after feeding, h Time after feeding, b Figure 6.12. Total organic acid concentration (A), lactic acid (B), total VFA concentration (C), acetic acid (D), propionic acid (E), and butyric acid concentration (F) on d 10 of daily DFM treatments (-<>- control and -O- DF M group). 217 A. Acetic acid, Control B. Acetic acid, DFM 120 E 80 40 ' 03691215182124 03691215182124 Time after feeding, h Time after feeding, h C. Propionic acid, Control D. Propionic acid, DFM 120 I t ' 120 100 100 80 80 E 60 E 60 40 . 40 20 20 03691215182124 03691215182124 Time after feeding, h Time after feeding, h E. Butyric acid, Control F. Butyric acid, DFM 03691215182124 03691215182124 Time after feeding, h Time after feeding, h Figure 6.13. Ruminal acetic acid (A and B), propionic acid (C and D) and butyric acid concentration (E and F) on d 10 of DF M supplementation (-<>- S01, -0- SOS, -A- S06, -X- S09 in control and -<>- S04, -0- $08, -A- S10, -X- S12 in DF M group). 218 7 A. Ruminal pH 6 l- :1: o. 5 l— 4 #Q 1 I i i O 3 6 912151821 Time after inoculation, h C. Total VFA 150 * 120 - E 90 " 60 30 0 l l L 4 j 4 036912151821 Time after inoculation, h E. Propionic acid 03691215182] Time after inoculation, h Figure 6.14. Fermentation characteristics in the rumen of steers during experimentally induced acidosis (-<>- control and -0- DP M group; * Means differ at the time marked (P < 0.05)). B. Lactic acid 1 50 --. 120 90 in 30 0 annirm 03 691215182] Time after inoculation, h D. Acetic acid 03691215182] Time after inoculation, h F. Butyric acid 2L 4 L 1 1 03691215182] Time after inoculation, h 219 A. Blood pH B. Lactic acid 7.3 l J L jL l l O l [L 1 l L L 0 3 6 9 12 15 18 2] 0 3 6 9 12 15 18 21 Time after inoculation, h Time afier inoculation, h C. Bicarbonate D. Base excess 036912151821 Time afier inoculation, h E. pCOz F- P02 50- 036912151821 036912151821 Time after inoculation, h Time after inoculation, h Figure 6.15. Blood pH, lactic acid, bicarbonate, base excess, pCOz and p02 concentration in the steers during experimentally induced acidosis (-<>- control and -O- DFM group) 220 F. O; Saturation (1 Glucose 120 E o 30 100 E 80 03691215182] 03691215182] Time after inoculation, h Time afier inoculation, h C.Mg++ D. Ca” 5 . a ._1 ._1 E E o o 2 2 45 E) E) E E 4 L L l J l l 036912151821 036912151821 Time after inoculation, h Time after inoculation, h E. Na F. K 150 - 036912151821 036912151821 Time after inoculation, h Time after inoculation, h Figure 6.16. Oxygen saturation, glucose and cations Mg, Ca, Na, and K concentrations in the steers during experimentally induced acidosis (-<>- control and -O- DFM group) 221 B. Blood pH - DFM 03691215182] 03691215182] Time after inoculation, h Time after inoculation, h Figure 6.17. Blood pH in each steer during experimentally induced acidosis (-<>- SO], - 0- S05, -A- SO6, -X- SO9 in control and -<>- S04, -O- S08, -A- S10, -X- 812 in DFM group). 222 APPENDIX C. RAW DATA 223 Table 7.1. Chapter 2. In vitro fermentation (Data for Table 2.4) Trt Time Tube LAC AC PR iBUT BUT iVAL VAL Total A/P pH FOM Control 0 l 3.29 33.17 15.28 1.08 16.31 1.35 1.92 69.12 2.17 6.70 0 Control 0 2 3.14 32.99 15.39 1.08 15.76 1.58 2.77 69.56 2.14 6.66 0 Control 0 3 3.04 32.62 15.50 1.23 15.94 1.34 2.63 69.26 2.10 6.66 0 Control 0 4 2.79 33.70 15.31 1.10 16.03 1.56 2.53 70.23 2.20 6.71 0 DH42 0 5 4.37 32.63 15.66 1.1 1 15.70 1.55 2.75 69.41 2.08 6.71 0 DH42 0 6 4.12 32.53 16.13 1.22 15.88 1.28 2.45 69.48 2.02 6.65 0 DH42 0 7 3.97 32.48 15.79 1.23 15.64 1.40 2.75 69.29 2.06 6.71 0 DH42 O 8 3 .83 32.45 15.78 1.08 15.37 1.33 2.75 68.75 2.06 6.65 0 Control 6 1 2.65 49.01 30.30 0.98 22.00 1.30 2.99 106.58 1.62 6.53 21.7 Control 6 2 2.39 47.69 30.15 1.00 23.23 1.41 3.13 106.62 1.58 6.60 21.9 Control 6 3 1.57 47.59 30.13 0.95 21.67 1.5] 2.98 104.83 1.58 6.60 20.0 Control 6 4 1.05 51.10 33.33 1.10 21.28 1.57 3.03 111.41 1.53 6.52 22.6 DH42 6 5 2.48 47.17 31.43 1.00 21.44 1.56 3.14 105.74 1.50 6.59 20.2 DH42 6 6 1.89 53.02 37.05 1.12 25.22 1.53 2.72 120.67 1.43 6.45 29.4 DH42 6 7 2.75 46.66 30.70 0.98 21.92 1.69 3 .05 105.00 1.52 6.57 20.6 DH42 6 8 1.92 48.91 33.09 1.02 21.80 1.70 3.16 109.68 1.48 6.55 23.] Control 12 1 1.92 86.41 69.26 2.45 48.28 2.00 3.58 211.98 1.25 5.30 88.6 Control 12 2 1.36 83.84 68.60 2.36 46.66 2.27 4.57 208.31 1.22 5.33 85.8 Control 12 3 0.96 81.20 67.75 2.41 42.66 2.09 4.70 200.82 1.20 5.40 80.1 Control 12 4 1.09 83 .91 68.62 2.46 45.21 2.05 4.94 207.19 1.22 5.34 84.3 DH42 12 5 1.11 84.08 70.27 2.53 48.42 2.32 5.09 212.71 1.20 5.31 88.6 DH42 12 6 1.46 86.33 71.56 2.62 48.59 2.15 5.14 216.39 1.21 5.27 91.0 DH42 12 7 1.21 83.59 70.41 2.64 46.95 2.11 5.0] 210.7] 1.19 5.30 87.2 DH42 12 8 1.01 82.48 69.54 2.30 45.52 2.24 5.17 207.25 1.19 5.31 85.2 Control 24 1 4.04 100.85 76.96 2.62 64.04 2.59 9.06 256.12 1.31 4.97 122.7 Control 24 2 2.86 97.28 76.96 2.87 60.75 2.66 8.40 248.92 1.26 5.03 1 16.3 Control 24 3 1.86 91.28 75.84 2.80 53.23 2.30 7.22 232.68 1.20 5.12 103.3 Control 24 4 2.12 95.30 77.09 2.89 57.31 2.33 7.50 242.42 1.24 5 .08 1 10.2 DH42 24 5 2.09 94.23 78.67 2.98 59.78 2.37 7.74 245.77 1.20 5.06 112.9 DH42 24 6 2.66 99.04 79.66 2.97 61.57 2.42 8.72 254.38 1.24 5.02 119.1 DH42 24 7 2.56 95.91 78.87 3.07 59.41 2.68 8.03 247.98 1.22 5.03 1 14.7 DH42 24 8 1.47 93.32 78.42 3.18 55.87 2.68 8.01 241.49 1.19 5.07 109.8 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVa], isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 224 Table 7.2. Chapter 2. BW, kg (Data for Table 2.5) Initial P1 P2 P3 P4 Final TRT Pen ID 10/24 10/25 1 1/21 12/19 1/17 2/13 2/24 2/25 Control 18 123 441 445 493 534 554 591 612 603 Control 18 126 466 461 535 575 619 664 686 678 Control 18 127 402 398 460 504 534 573 592 586 Control 18 17 1 390 393 465 486 522 567 583 585 Control 18 188 453 456 495 535 575 612 628 623 Control 18 193 355 365 432 485 525 578 593 599 Control 1 8 194 408 414 469 502 5 14 548 565 557 Control 18 21 1 394 395 444 481 495 545 560 556 Control 19 114 448 444 493 506 520 557 566 563 Control 19 147 440 431 471 476 514 556 565 562 Control 19 157 471 465 512 545 585 635 636 636 Control 19 162 395 393 457 507 541 590 603 604 Control 19 199 359 364 412 481 526 593 614 613 Control 19 210 394 387 460 499 520 573 583 581 Control 19 2] 8 409 409 457 489 5 15 562 569 561 Control 19 224 387 395 444 497 533 599 605 598 Control 22 130 405 409 478 537 596 638 662 651 Control 22 145 484 481 562 593 650 675 682 677 Control 22 146 386 386 448 485 533 559 570 572 Control 22 152 445 442 5 14 553 604 641 656 649 Control 22 170 435 429 494 554 600 645 663 654 Control 22 1. 75 412 41 1 466 514 582 628 645 644 Control 22 176 367 377 414 457 500 528 538 532 Control 22 213 396 395 452 499 548 600 618 614 Control 24 128 455 450 487 535 571 625 638 641 Control 24 159 430 426 457 500 530 565 569 567 Control 24 169 454 441 502 520 599 649 654 658 Control 24 177 414 417 454 492 513 553 564 565 Control 24 186 392 385 439 483 528 571 586 584 Control 24 219 405 398 439 471 510 564 571 576 Control 24 226 385 381 429 461 507 564 576 565 Control 24 228 365 359 427 448 491 538 559 555 Control 26 121 457 455 51 1 547 610 655 664 655 Control 26 137 400 398 448 475 498 538 555 554 Control 26 141 376 366 395 442 489 556 569 564 Control 26 155 45 1 450 465 493 539 585 607 594 Control 26 181 41 l 416 457 497 537 576 592 592 Control 26 214 395 389 425 470 523 570 585 581 Control 26 2 1 7 429 426 485 506 562 602 582 582 Control 26 227 385 378 425 465 500 553 569 565 Control 27 1 17 425 424 477 510 562 591 605 598 225 Table 7.2. (Cont’d) Initial P1 P2 P3 P4 Final TRT Pen ID 10/24 10/25 11/21 12/19 1/17 2/13 2/24 2/25 Control 27 138 451 449 492 535 572 613 635 636 Control 27 163 460 462 515 555 610 660 680 676 Control 27 167 415 409 447 474 515 557 575 582 Control 27 189 402 400 433 462 508 545 555 553 Control 27 197 378 383 436 487 557 605 625 627 Control 27 198 400 405 449 500 544 595 605 61 1 Control 27 216 385 385 425 456 508 548 562 555 Control 30 1 18 422 422 456 498 523 576 587 587 Control 30 132 465 458 506 551 610 662 676 670 Control 30 134 395 389 416 460 498 532 545 535 Control 30 160 449 447 493 525 561 603 61 l 609 Control 30 174 422 427 482 514 468 505 500 496 Control 30 183 401 402 436 486 531 589 602 605 Control 30 196 380 386 416 453 501 550 564 557 Control 30 201 383 378 416 476 517 577 587 584 DH42 17 131 407 412 457 444 522 565 565 559 DH42 17 133 388 386 435 418 496 552 563 603 DH42 17 151 412 412 459 494 519 569 577 573 DH42 17 164 444 436 501 533 569 628 647 642 DH42 17 192 448 445 494 499 595 655 663 652 DH42 17 208 401 399 453 434 495 556 568 564 DH42 17 221 465 459 545 602 644 704 716 707 DH42 17 222 345 349 397 448 485 528 542 544 DH42 20 135 398 398 450 491 527 575 578 578 DH42 20 142 415 416 472 510 533 591 591 589 DH42 20 149 439 440 481 538 571 616 637 640 DH42 20 158 474 474 523 588 626 683 692 691 DH42 20 195 394 396 455 493 520 565 579 578 DH42 20 200 387 380 455 532 569 642 645 643 DH42 20 205 445 449 503 547 584 618 631 619 DH42 20 207 359 350 397 425 443 486 495 486 DH42 21 1 16 436 435 485 509 543 573 580 577 DH42 21 150 394 394 435 471 487 517 527 529 DH42 21 156 477 479 527 568 589 655 672 666 DH42 21 166 387 379 427 465 499 552 557 549 DH42 21 190 445 448 494 535 550 600 621 616 DH42 21 206 403 405 470 504 536 585 612 606 DH42 21 212 365 365 401 446 466 514 529 521 DH42 21 223 394 393 451 538 536 570 582 580 DH42 23 1 15 366 365 427 452 463 500 523 520 DH42 23 122 386 384 448 472 477 515 546 536 226 Table 7.2. (Cont’d) Initial P1 P2 P3 P4 Final TRT Pen 1D 10/24 10/25 11/21 12/19 1/17 2/13 2/24 2/25 DH42 23 165 432 434 484 518 535 578 608 600 DH42 23 168 414 409 466 503 526 565 601 594 DH42 23 179 397 404 450 483 505 552 566 573 DH42 23 187 487 492 546 595 613 653 675 662 DH42 23 191 39 '1 395 449 486 507 539 568 566 DH42 23 209 443 435 504 529 546 607 637 640 DH42 25 120 448 449 501 515 514 514 505 502 DH42 25 124 456 456 502 521 573 61 1 623 623 DH42 25 125 415 405 453 475 509 556 564 557 DH42 25 140 430 424 475 470 51 1 539 552 550 DH42 25 148 396 395 445 464 497 532 532 525 DH42 25 178 368 367 409 44] 492 527 531 532 DH42 25 203 385 3 86 441 475 510 539 546 545 DH42 25 225 402 395 452 494 529 575 585 581 DH42 28 129 379 377 415 458 480 527 544 547 DH42 28 136 384 385 436 448 487 536 557 555 DH42 28 143 450 445 484 510 542 589 605 606 DH42 28 144 395 393 423 455 473 51 1 533 528 DH42 28 153 401 393 443 473 506 552 562 554 DH42 28 154 462 456 489 545 563 615 637 634 DH42 28 204 417 413 462 493 514 568 583 582 DH42 28 220 423 418 458 506 554 608 645 645 DH42 29 1 19 423 419 474 5 '12 539 579 593 595 DH42 29 161 463 455 504 51 1 529 573 594 591 DH42 29 173 385 385 423 460 486 527 547 540 DH42 29 180 401 405 446 476 509 545 562 56] DH42 29 182 418 421 465 512 558 603 628 632 DH42 29 184 380 385 425 467 502 551 573 579 DH42 29 202 449 436 468 459 508 555 583 585 DH42 29 215 395 381 455 501 556 594 614 607 227 Table 7.3. Chapter 2. Dry matter intake, kg/pen (Data for Table 2.6) 10/25/00 11/21/00 12/19/00 01/17/01 02/13/01 Treatment Pen Animal 1 1/20/00 12/18/00 01/16/01 02/12/01 02/24/01 Control 18 8 2295 2548 2182 2465 1 158 Control 19 8 2282 2544 2142 2421 1065 Control 22 8 2272 2719 2356 2515 1099 Control 24 8 2218 2425 2160 2522 1122 Control 26 8 1901 2277 2279 2498 1070 Control 27 8 2247 2534 2275 2407 1095 Control 30 8 21 14 2407 1988 2316 998 DH42 17 8 2316 2669 2194 2449 1064 DH42 20 8 2253 2597 2188 2462 1088 DH42 2] 8 2278 2337 2054 2309 1070 DH42 23 8 2312 2550 1902 2189 1072 DH42 25 8 2247 2323 1 860 2168 876 DH42 28 8 2208 2391 2032 2395 1 13 1 DH42 29 8 2078 2345 1948 2158 1 104 228 Table 7.4. Chapter 2. Carcass characteristics (Data for Table 2.6) HCW Live F at Rib eye Yield USDA TRT Pen 1D %KPH Marbling Choice ,kg wt.,kg depth area grade grade Control 18 123 372 596 0.30 14.7 2.00 2.05 2 5150 Sel+ Control 18 126 415 664 0.60 12.6 2.00 3.83 3 sm30 Ch- Control 18 127 351 562 0.15 13.0 1.50 1.95 ] 5140 Se]- Control 18 171 338 542 0.50 12.2 2.00 3 .07 3 md70 Ch0 Control 18 188 383 614 0.50 13.3 2.00 3.10 3 sm60 Ch- Control 18 193 349 560 0.35 12.9 2.50 2.67 2 sm30 Ch- Control 18 194 351 563 0.50 12.5 2.00 3.09 3 sm60 Ch- Control 18 21 1 337 540 0.50 12.8 2.50 2.97 2 sm30 C11- Control 19 1 14 342 549 0.50 1 1.8 2.00 3.24 3 sm30 Ch- Control 19 147 340 545 0.30 1 1.4 2.50 2.95 2 sm60 Ch- Control 19 157 375 602 0.50 10.8 2.50 3.93 3 md30 Ch+ Control 19 162 361 579 0.50 10.9 2.00 3.68 3 sm40 Ch- Control 19 199 346 554 0.40 12.9 2.00 2.66 2 $150 Se1+ Control 19 210 358 574 0.40 1 1.6 2.00 3.18 3 sm70 Ch- Control 19 218 354 567 0.30 14.0 2.50 2.23 2 sm80 Ch- Control 19 224 360 577 0.30 13.7 2.50 2.38 2 $160 Sel+ Control 22 130 392 628 0.30 12.1 2.00 3 .05 3 md70 Ch0 Control 22 145 424 680 0.45 13.5 2.00 3.25 3 sm20 Ch- Control 22 146 346 554 0.60 12.4 1.50 3 .22 3 mt70 Ch+ Control 22 152 370 594 0.40 12.2 2.00 3.09 3 5130 Se]- Control 22 170 395 634 0.30 13.7 1.50 2.47 2 sm30 Ch- Control 22 175 395 634 0.40 13.8 2.00 2.79 2 $150 Se1+ Control 22 176 323 518 0.20 12.9 2.00 1.97 1 sm10 Ch- Control 22 213 354 567 0.20 12.7 2.00 2.29 2 $120 Sel- Control 24 128 388 622 0.60 15.6 2.00 2.65 2 sm50 Ch- Control 24 159 348 558 0.20 14.2 2.00 1.77 1 5140 Se]- Control 24 169 395 633 0.90 13.2 2.50 4.33 4 md ab 60 Pr 0 Control 24 177 349 559 0.05 13.6 2.50 1.69 1 dark Ct Dark ct Control 24 186 340 544 0.40 1 1.4 2.00 3.09 3 md60 ChO Control 24 219 329 526 0.10 1 1.7 2.00 2.15 2 sm50 Ch- Control 24 226 33 5 538 0.30 13.3 2.00 2.20 2 sm20 Ch- Control 24 228 329 527 0.10 1 1.2 1.50 2.22 2 sm30 Ch- Control 26 121 397 636 0.40 13 .3 2.00 2.96 2 5140 Se]- Control 26 137 334 535 0.40 12.3 2.00 2.76 2 $150 Sel+ Control 26 141 341 546 0.50 12.4 2.00 3 .03 3 sm50 Ch- Control 26 155 365 585 0.20 14.8 3.00 1.92 1 dark ct Dark ct Control 26 181 357 572 0.70 12.5 2.00 3.64 3 5140 Se]- Control 26 214 341 546 0.40 12.6 2.00 2.72 2 mt50 Ch+ Control 26 217 349 560 0.30 12.4 2.50 2.70 2 sm40 Ch- Control 26 227 337 540 0.30 13.5 2.00 2.15 2 sm40 Ch- Control 27 1 17 369 590 0.30 12.3 2.00 2.80 2 mt30 Ch+ 229 Table 7.4. (Cont’d) HCW Live Fat Rib eye Yield USDA TRT Pen ID %KPH Marbling Choice ,kg wt.,kg depth area grade grade Control 27 138 376 602 0.35 12.7 2.00 2.85 2 sm10 Ch- Control 27 163 391 627 0.50 12.] 2.00 3.55 3 $190 Sel- Control 27 167 365 585 0.40 12.6 2.00 2.92 2 sm40 Ch- Control 27 189 323 517 0.20 1 1.0 2.00 2.58 2 sm30 Ch- Control 27 197 36] 579 0.30 1 1.4 2.00 3.02 2 md90 Ch0 Control 27 198 356 570 0.50 1 1.0 2.00 3.61 3 sm40 Ch- Control 27 216 342 548 0.20 12.6 2.00 2.23 2 sm30 Ch- Control 30 1 18 302 485 0.40 10.0 2.00 3.23 3 sm40 Ch- Control 30 132 408 653 0.70 13.4 2.00 3.77 3 sm50 Ch- Control 30 134 323 518 0.30 10.3 2.00 3.06 3 sm20 Ch- Control 30 160 354 567 0.30 l 1.7 2.50 2.97 2 sm60 Ch- Control 30 174 300 481 0.30 12.6 2.00 2.13 2 sm50 Ch- Control 30 183 359 575 0.30 1 1.5 2.00 2.85 2 sm40 Ch- Control 30 196 331 53 1 0.40 1 1.6 2.50 3.06 3 md80 Ch0 Control 30 201 343 550 0.20 10.4 2.00 2.94 2 md40 Ch0 DH42 17 13 1 355 569 0.40 l 1.9 2.00 3.06 3 sm230 Ch- DH42 17 133 334 535 0.30 12.1 2.50 2.67 2 sm50 Ch- DH42 17 151 365 585 0.30 14.4 2.50 2.19 2 sm60 Ch- DH42 17 164 387 620 0.50 12.6 2.00 3.36 3 sm40 Ch- DH42 17 192 395 632 0.20 14.8 1.50 1.86 1 sm60 Ch- DH42 17 208 333 533 0.10 12.1 2.00 2.06 2 DH42 17 221 439 703 0.60 13.7 3.00 3.88 3 sm50 Ch- 2 3 2 2 3 2 3 2 2 3 2 2 3 2 2 2 3 2 3 DH42 17 222 332 532 0.20 12.5 2.00 2.18 sm10 Ch- DH42 20 135 336 539 0.30 10.1 2.00 3.23 sm40 Ch- DH42 20 142 33 1 53 1 0.40 13.3 2.00 2.41 sm70 Ch- DH42 20 149 295 472 0.30 12.6 2.00 2.08 DH42 20 158 408 654 0.30 12.6 2.00 3 .03 DH42 20 195 346 555 0.10 12.1 3.00 2.37 DH42 20 200 377 605 0.30 11.8 2.50 3.13 DH42 20 205 389 624 0.40 1 3 .0 2.00 2.99 DH42 20 207 353 566 0.30 14.1 2.00 2.09 DH42 21 116 353 566 0.45 12.3 2.00 3.04 DH42 21 150 320 514 0.60 13 .2 2.50 2.96 DH42 2 '1 156 398 638 0.20 13 .7 1.50 2.24 DH42 21 166 348 557 0.40 l 1.3 2.00 3.19 DH42 21 190 380 608 0.30 14.1 2.00 2.31 DH42 2] 206 354 567 0.20 12.0 2.00 2.52 DH42 21 212 309 495 0.20 12.2 2.50 2.16 DH42 2] 223 348 558 0.40 1 1.7 2.00 3.07 DH42 23 115 321 514 0.40 13.7 1.50 2.2] DH42 23 122 331 531 0.50 9.6 2.00 3.85 md50 ChO md40 Ch0 sm30 Ch- sm20 Ch- s120 Sel- sm40 Ch- sl60 Sel+ 3130 Se]- mt40 Ch+ $120 Sel- md50 Ch0 sm20 Ch- sl30 Sel- $140 sel- 230 Table 7.4. (Cont’d) HCW Live Fat Rib eye Yield USDA TRT Pen ID %KPH Marbling Choice ,kg wt.,kg depth area grade grade DH42 23 165 377 604 0.40 12.4 2.00 3.08 3 $130 Sel- DH42 23 168 353 565 0.10 12.0 2.00 2.26 2 sm20 Ch- DH42 23 179 340 545 0.60 12.3 3.00 3.51 3 sm40 Ch- DH42 23 187 41 1 659 0.20 15.3 2.50 2.04 2 sm70 Ch- DH42 23 191 334 535 0.10 13.9 1.00 1.29 1 5130 Se]- DH42 23 209 379 607 0.40 12.] 2.00 3.19 3 sm50 Ch- DH42 25 120 300 480 0.30 14.1 2.00 1.64 1 Noroll No Roll DH42 25 124 388 622 0.30 1 1 .9 2.00 3.09 3 mt20 Ch+ DH42 25 125 338 542 0.30 12.1 2.00 2.61 2 sm60 Ch- DH42 25 140 33 1 53 1 0.20 9.8 2.00 3.03 3 $140 Sel- DH42 25 148 314 503 0.30 12.1 2.00 2.40 2 $150 Se1+ DH42 25 178 351 563 0.30 13.8 1.50 2.07 2 $120 Sel- DH42 25 203 333 534 0.40 1 1.4 2.00 3.04 3 sm40 Ch- DH42 25 225 340 545 0.20 1 1.7 2.50 2.60 2 sl60 Se1+ DH42 28 129 330 528 0.30 14.6 1.50 1.63 1 sm20 Ch- DH42 28 136 328 526 0.20 12.8 2.00 2.05 2 sm30 Ch- DH42 28 143 361 579 0.15 14.9 1.50 1.43 2 sm30 Ch- DH42 28 144 31 1 498 0.30 12.2 2.00 2.35 2 sm80 Ch- DH42 28 153 342 548 0.30 10.9 2.00 3.02 3 sm60 Ch- DH42 28 154 3 87 620 0.40 14.4 2.00 2.53 2 sm50 Ch- DH42 28 204 350 560 0.30 13 .4 1.50 2.18 2 md80 ChO DH42 28 220 3 71 594 0.40 13 .6 2.00 2.65 2 sm40 Ch- DH42 29 1 19 365 584 0.20 13.5 1.50 2.03 2 sm40 Ch- DH42 29 161 341 547 0.40 1 1.6 2.00 3.04 3 5160 Se1+ DH42 29 173 323 517 0.30 1 1.4 2.00 2.70 2 sm40 Ch- DH42 29 180 34] 546 0.40 13.9 2.00 2.30 2 3110 Sel- DH42 29 182 362 580 0.30 10.8 2.00 3.22 3 sm50 Ch- DH42 29 184 342 548 0.20 1 1.0 2.00 2.74 2 sm30 Ch- DH42 29 202 352 565 0.30 14.3 2.00 2.02 2 sm50 Ch- DH42 29 215 360 577 0.20 13.4 2.00 2.12 2 md40 ChO 231 Table 7.5. Chapter 3. In vitro fermentation (Data for Table 3.2 to 3.5) Trt Time Tube Lac Ac Pr iBut But iBut Val AP VFA pH F OM Control 0 a 1.41 36.36 14.76 1.10 7.70 0.91 0.84 2.5 61.7 6.7] Control 0 b 1.16 36.47 14.92 1.15 7.44 0.82 0.95 2.4 61.7 6.7] Control 0 c 1.14 36.25 14.56 0.92 7.19 0.87 0.90 2.5 60.7 6.64 N6 0 d 1.12 36.39 14.73 1.39 7.64 0.89 0.84 2.5 61.9 6.76 N6 0 e 1.24 36.14 14.62 1.30 7.19 0.89 0.95 2.5 61.1 6.71 N6 0 f 1.13 35.87 14.62 1.31 7.25 0.82 0.89 2.5 60.8 6.66 N2A2N2 0 g 1.12 36.05 14.71 1.36 7.20 0.80 0.95 2.5 61.1 6.70 N2A2N2 0 h 1.14 35.96 15.08 1.56 7.47 0.86 0.25 2.4 61.2 6.66 N2A2N2 0 i 1.14 36.22 14.69 0.99 7.35 0.76 0.88 2.5 60.9 6.66 N2A4 0 j 1.08 36.35 14.94 1.41 7.41 0.90 0.81 2.4 61.8 6.67 N2A4 0 k 1.07 35.89 14.54 1.00 7.05 0.81 0.78 2.5 60.1 6.57 N2A4 0 1 1.08 35.98 14.69 1.35 7 .26 0.78 0.90 2.4 61.0 6.66 Control 3 a 8.31 41.42 19.54 0.30 7 .50 0.47 1.23 2.1 70.5 6.57 7.3 Control 3 b 8.16 40.84 19.1 1 0.43 7.69 0.62 0.92 2.1 69.6 6.52 7.1 Control 3 c 8.50 41.24 19.53 0.53 7.67 0.78 0.99 2.1 70.7 6.51 8.8 N6 3 d 1 1.17 41.49 19.64 0.25 7.38 0.33 1.16 2.1 70.2 6.52 8.4 N6 3 e 10.86 41.46 19.97 0.27 7.39 0.82 0.84 2.1 70.7 6.52 9.] N6 3 f 10.2] 41.65 19.27 0.37 8.23 0.76 0.28 2.2 70.5 6.48 9.1 N2A2N2 3 g 10.19 41.76 19.26 0.53 9.24 1.10 1.13 2.2 73.0 6.61 11.4 N2A2N2 3 h 10.54 41.87 19.27 0.65 9.03 0.95 0.97 2.2 72.8 6.56 1 1.2 N2A2N2 3 i 12.95 42.06 19.70 0.40 8.96 1.01 0.90 2.1 73.0 6.56 12.6 N2A4 3 j 9.36 41.71 19.17 0.83 10.20 1.18 1.29 2.2 74.4 6.58 11.9 N2A4 3 k 9.98 41.59 19.17 0.61 9.33 1.07 1.27 2.2 73.0 6.45 12.3 N2A4 3 l 12.12 41.90 19.65 0.32 8.95 1.18 1.49 2.1 73.5 6.44 12.6 Control 6 a 52.08 45.1 1 24.27 0.37 8.36 0.98 0.90 1.9 80.0 5.80 34.5 Control 6 b 51 .22 45.01 23.67 0.40 9.00 0.89 1.45 1.9 80.4 5.86 35.] Control 6 c 51.15 45.50 24.29 0.56 8.44 1.1 1 0.98 1.9 80.9 5.82 35.7 N6 6 d 65.17 45.02 24.05 0.41 9.14 0.83 1.00 1.9 80.5 5.57 41.6 N6 6 e 74.47 45.67 25.60 0.42 9.72 1.18 1.78 1.8 84.4 5.22 49.6 N6 6 f 63.79 45.65 25.06 0.52 8.95 0.25 0.99 1.8 81.4 5.49 41.9 N2A2N2 6 g 65.78 45.54 24.33 0.51 10.58 0.99 1.19 1.9 83.1 5.51 44.8 N2A2N2 6 h 65.25 45.78 24.41 0.43 10.88 0.98 1.02 1.9 83.5 5.48 44.8 N2A2N2 6 i 64.76 45.53 25.1 1 0.52 10.74 0.57 1.17 1.8 83.6 5.47 44.7 N2A4 6 j 63.26 45.01 25.1 1 0.37 13.05 1.16 1.71 1.8 86.4 5.52 46.2 N2A4 6 k 62.01 44.81 25.12 0.93 12.6] 1.14 1.76 1.8 86.4 5.52 47.0 N2A4 6 1 65.36 45.57 26.43 0.95 12.91 1.19 1.40 1.7 88.4 5 .37 49.0 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 232 Table 7.5. (Cont’d) Trt Time Tube Lac Ac Pr iBut But iBut Val AP VFA pH FOM Control 9 a 95.85 46.71 27.02 0.29 9.86 0.20 0.28 1.7 84.4 4.85 58.6 Control 9 b 97.64 45.96 25.81 0.60 9.30 0.66 0.71 1.8 83.0 4.82 59.3 Control 9 c 95.20 46.35 26.74 0.84 9.84 0.92 1.01 1.7 85.7 4.82 60.9 N6 9 d 97.43 45.71 25.97 0.64 10.09 0.97 1.07 1.8 84.5 4.81 60.4 N6 9 e 95.81 47.41 28.43 0.57 10.87 1.05 1.60 1.7 89.9 4.79 63.6 N6 9 f 95.17 47.06 27.32 0.73 1.11 1.03 1.15 1.7 78.4 4.81 52.7 N2A2N2 9 g 96.00 46.78 27.03 0.38 1 1.08 1.07 0.56 1.7 86.9 4.80 61.8 N2A2N2 9 h 95.24 46.97 26.43 0.29 10.49 0.91 1.28 1.8 86.4 4.81 61.1 N2A2N2 9 i 94.99 47.54 27.66 0.40 10.19 1.] 1 1.56 1.7 88.5 4.80 62.3 N2A4 9 j 80.40 50.41 37.58 0.65 15.57 1.00 1.83 1.3 107.0 4.85 66.5 N2A4 9 k 78.28 49.37 37.51 0.23 16.09 1.36 2.18 1.3 106.7 4.86 67.1 N2A4 9 l 79.08 50.80 38.89 0.69 15.65 1.23 2.0] 1.3 109.3 4.85 67.8 Control 12 a l 13.49 45.69 25.60 0.77 9.54 0.76 0.89 1.8 83.2 4.60 67.5 Control 12 b 1 12.23 46.79 27.61 0.13 9.17 0.69 0.78 1.7 85.2 4.6] 67.5 Control 12 c 1 13 .05 48.45 29.99 0.76 10.28 0.83 1.33 1.6 91.6 4.60 73.] N6 12 d 108.80 49.78 34.53 0.13 11.08 0.30 1.93 1.4 97.8 4.58 73.] N6 12 e 109.39 49.22 30.21 0.75 10.66 1.16 1.69 1.6 93 .7 4.55 72.3 N6 12 f 110.10 49.68 32.98 0.13 11.20 0.75 1.81 1.5 96.5 4.59 74.2 N2A2N2 12 g 1 10.33 49.20 30.88 0.21 l 1.57 0.94 1.90 1.6 94.7 4.56 73.6 N2A2N2 12 h 109.41 49.48 31.65 0.43 10.87 0.77 1.60 1.6 94.8 4.58 72.7 N2A4 12 j 58.59 63.93 77.37 0.88 23.02 1.1 1 2.90 0.8 169.2 4.70 91.2 N2A4 12 k 56.90 63.28 79.25 0.13 21.70 0.47 2.96 0.8 167.8 4.71 89.6 N2A4 12 l 59.01 62.93 75.99 0.13 21.62 0.72 2.92 0.8 164.3 4.7] 88.2 Control 24 a 124.42 45.21 27.30 1.43 9.65 1.03 1.08 1.7 85.7 4.43 74.8 Control 24 b 125.49 44.13 25.56 1.38 9.08 0.97 0.84 1.7 82.0 4.51 73.2 Control 24 c 131.00 45.32 25.66 0.76 10.02 0.94 1.25 1.8 83.9 4.37 78.] N6 24 d 107.70 51.65 40.48 1.57 10.69 1.03 4.59 1.3 1 10.0 4.46 80.9 N6 24 e 92.97 56.39 50.83 1.37 13.55 1.21 4.59 1.1 127.9 4.53 84.5 N6 24 f 100.00 54.39 46.30 1.35 1 1.27 1.08 3.53 1.2 1 17.9 4.52 81.5 N2A2N2 24 g 95.55 56.65 49.09 1.49 14.36 1.07 4.22 1.2 126.9 4.53 85.5 N2A2N2 24 h 100.73 53.80 43.93 1.29 14.95 1.07 3.89 1.2 1 18.9 4.53 84.2 N2A2N2 24 1 100.66 54.18 46.04 1.52 12.92 1.06 3.48 1.2 1 19.2 4.51 83.4 N2A4 24 j 2.58 62.64 101.60 2.41 62.86 1.47 18.99 0.6 250.0 4.90 132.4 N2A4 24 k 0.78 62.22 101.88 2.21 64.27 1.64 19.07 0.6 251.3 4.93 134.2 N2A4 24 1 0.28 63.85 103.89 1.95 61.20 1.38 17.95 0.6 250.2 4.93 130.3 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Va], valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 233 - .4 d“ IPA-L; Table 7.6. Chapter 4. Growth of the co-culture of M elsdenii RK02 and E. faecium RK03 (Data for Figure 4.1) Reducing 0% 20% 40% Agent Tube 1 2 3 4 5 6 7 8 9 Time, h -0 0.005 0.008 0.004 0.005 0.004 0.003 0.005 0.004 0.004 0 0.037 0.040 0.036 0.037 0.036 0.037 0.039 0.039 0.036 1 0.035 0.038 0.034 0.036 0.034 0.037 0.039 0.038 0.033 2 0.035 0.037 0.033 0.035 0.035 0.034 0.041 0.039 0.035 3 0.036 0.039 0.035 0.036 0.037 0.037 0.048 0.042 0.035 4 0.038 0.038 0.035 0.037 0.039 0.039 0.059 0.054 0.047 5 0.039 0.041 0.035 0.037 0.042 0.044 0.076 0.063 0.053 6 0.041 0.042 0.038 0.040 0.047 0.049 0.103 0.083 0.071 7 0.046 0.044 0.040 0.044 0.052 0.057 0.162 0.125 0.098 8 0.051 0.043 0.042 0.047 0.061 0.070 0.241 0.183 0.147 9 0.062 0.053 0.049 0.055 0.079 0.097 0.395 0.294 0.233 10 0.082 0.062 0.060 0.067 0.104 0.124 0.582 0.499 0.394 1 1 0.095 0.073 0.068 0.078 0.144 0.186 0.685 0.612 0.531 12 0.130 0.088 0.087 0.099 0.204 0.287 0.800 0.714 0.671 13 0.219 0.136 0.135 0.168 0.367 0.497 0.924 0.866 0.818 14 0.372 0.223 0.220 0.270 0.570 0.688 0.960 0.938 0.908 15 0.537 0.343 0.358 0.417 0.714 0.808 1.012 0.974 0.940 16 0.726 0.532 0.548 0.615 0.824 0.872 1.060 1.028 1.008 17 0.820 0.672 0.690 0.728 0.870 0.916 1.069 1.054 1.044 18 0.882 0.808 0.802 0.816 0.894 0.950 1.080 1.075 1.052 19 0.898 0.858 0.862 0.876 0.950 1.004 1.1 15 1.105 1.085 22 1.024 1.008 1.008 1.010 1.048 1.080 1.135 1.150 1.135 23 1.058 1.039 1.044 1.048 1.085 1.105 1.135 1.155 1.140 24 1.080 1.075 1.075 1.062 1.095 1.110 1.135 1.155 1.155 Colony counts RK02 (x 107) 28 37 42 27 37 48 36 26 24 RK02 (x 103) 7 2 0 1 3 3 7 3 6 Colony counts RK03 (x 106) 34 61 47 28 59 62 64 61 47 RK03 (x 107) 5 2 9 3 3 7 5 9 14 234 Table 7.6. (Cont’d) fig?“ 60% 80% 100% Tim“ “be 10 11 12 13 14 15 16 17 18 -0 0.000 0.000 0.012 0.002 0.000 -0.001 -0.001 -0001 0.000 0 0.031 0.029 0.048 0.040 0.035 0.035 0.034 0.037 0.034 1 0.031 0.029 0.052 0.043 0.037 0.035 0.038 0.041 0.037 2 0.033 0.032 0.054 0.050 0.042 0.040 0.043 0.049 0.043 3 0.044 0.044 0.064 0.071 0.053 0.049 0.057 0.064 0.054 4 0.052 0.049 0.068 0.086 0.071 0.066 0.073 0.083 0.074 5 0.065 0.063 0.088 0.1 19 0.097 0.088 0.106 0.1 16 0.100 6 0.094 0.087 0.118 0.183 0.150 0.138 0.166 0.191 0.165 7 0.151 0.141 0.197 0.324 0.272 0.239 0.288 0.336 0.288 8 0.236 0.223 0.280 0.517 0.451 0.390 0.460 0.507 0.429 9 0.389 0.355 0.448 0.712 0.690 0.595 0.644 0.671 0.602 10 0.603 0.568 0.625 0.840 0.812 0.786 0.802 0.798 0.764 1 1 0.708 0.663 0.740 0.856 0.850 0.826 0.840 0.852 0.806 12 0.832 0.786 0.844 0.904 0.874 0.882 0.888 0.902 0.866 13 0.888 0.896 0.918 0.938 0.946 0.934 0.948 0.958 0.946 14 0.949 0.952 0.976 1.000 1.004 0.982 0.990 0.994 0.974 15 1.004 1.008 1.020 1.032 1.030 1.060 1.040 1.048 1.040 16 1.044 1.052 1.065 1.065 1.058 1.065 1.085 1.065 1.052 17 1.052 1.065 1.065 1.075 1.065 1.065 1.052 1.075 1.065 18 1.065 1.075 1.080 1.085 1.085 1.075 1.075 1.100 1.085 19 1.080 1.085 1.115 1.085 1.075 1.095 1.070 1.105 1.110 22 1.095 1.135 1.145 1.130 1.105 1.095 1.080 1.120 1.115 23 1.120 1.145 1.155 1.120 1.105 1.105 1.085 1.115 1.115 24 1.105 1.140 1.155 1.120 1.110 1.105 1.080 1.115 1.120 Colony counts RK02 (x 107) 40 23 42 10 31 26 48 16 26 RK02 (x 108) 2 l 0 2 3 0 4 4 1 Colony counts RK03 (x 10‘) 59 32 31 57 62 36 55 34 64 RK03 (x 107) 18 6 7 28 5 5 6 19 16 235 Table 7.7. Chapter 4. Fermentation products after 24 h of incubation with different reducing agent content (Data for Table 4.2) RA Tube LAC AC PR iBut But iVal Val A/P Total 0% 1 -1 14.39 14.01 34.86 2.01 22.28 9.09 19.29 0.40 101.54 0% 2 —] 18.40 14.15 26.26 1.69 22.81 9.01 22.99 0.54 96.92 0% 3 -l 16.63 16.02 31.78 2.02 21.77 9.51 22.24 0.50 103.34 20% 4 -l 15.96 13.23 35.59 1.70 29.22 9.02 24.25 0.37 113.03 20% 5 -l 14.36 14.52 28.58 2.26 25.72 9.68 23.18 0.51 103.96 20% 6 -] 10.58 15.72 36.89 2.17 19.24 9.43 19.24 0.43 102.68 40% 7 -1 14.89 16.83 27.25 2.28 26.05 9.73 25.35 0.62 107.49 40% 8 -1 12.43 14.68 35.24 2.36 25.29 9.96 21.89 0.42 109.43 40% 9 -1 13.05 15.74 31.01 2.60 24.15 9.68 25.80 0.51 108.99 60% 10 -l 19.97 14.06 33.42 1.89 21.07 9.45 22.03 0.42 101.91 60% 1 l -1 13.94 15.91 44.23 2.29 26.03 8.97 29.02 0.36 126.45 60% 12 -1 12.72 15.74 32.31 2.24 30.21 8.73 32.42 0.49 121.66 80% 13 -1 1 1.47 17.78 46.50 2.26 24.89 9.45 26.90 0.38 127.79 80% 14 ~121.92 21.23 42.45 2.04 30.64 9.01 27.28 0.50 132.65 80% 15 -l 13.03 14.13 31.49 2.19 24.40 9.81 26.42 0.45 108.43 100% 16 -1 l 1.42 17.40 32.65 2.12 24.39 10.15 22.00 0.53 108.71 100% 17 -l 15.02 15.01 43.91 1.72 30.49 9.75 27.02 0.34 127.90 100% 18 -112.44 14.15 30.88 2.14 26.70 9.18 21.36 0.46 104.41 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio. 236 Table 7.8. Chapter 4. DFM effects on in vitro fermentation (Data for Tables 4.2 to 4.14) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VFA FOM pH lcontrol 0 a1 4.0 45.0 24.4 1.6 18.5 2.5 2.5 1.8 94.5 6.63 lcontrol 0 a2 4.2 43.5 25.3 1.7 18.4 2.6 2.0 1.7 93.6 6.55 lcontrol 0 a3 3.2 42.9 24.5 1.6 17.6 2.4 1.7 1.8 90.7 6.58 1RK02 0 a4 2.5 43.6 25.5 1.7 19.8 3.0 3.2 1.7 96.8 6.55 1RK02 0 a5 2.6 43.2 25.2 1.7 19.5 2.8 3.1 1.7 95.4 6.64 1RK02 0 a6 2.5 43.3 25.3 1.6 19.4 2.8 3.0 1.7 95.5 6.61 1RK03 0 a7 5.8 42.7 25.0 1.7 18.4 2.6 2.2 1.7 92.5 6.67 1RK03 0 a8 4.6 43.4 25.3 1.7 18.6 2.7 2.4 1.7 94.2 6.56 1RK03 0 a9 5.2 42.7 24.9 2.0 18.5 2.6 2.1 1.7 92.8 6.51 1RK0203 0 a10 3.9 41.1 24.2 1.9 19.1 2.8 3.2 1.7 92.2 6.56 1RK0203 0 a1 1 3.8 40.4 23.6 1.6 18.4 2.5 2.9 1.7 89.5 6.56 1RK0203 0 312 3.9 40.6 23.6 1.6 18.5 2.5 2.9 1.7 89.7 6.56 lcontrol 6 al 0.0 76.6 55.1 4.0 42.5 6.0 3.9 1.4 187.9 61.3 5.66 lcontrol 6 a2 0.0 74.8 53.7 4.1 42.3 6.0 4.0 1.4 184.9 60.4 5.70 lcontrol 6 a3 0.0 76.6 54.6 4.2 42.9 5.6 4.1 1.4 188.1 64.9 5.63 1RK02 6 a4 0.0 73.7 51.5 5.0 46.5 7.4 7.4 1.4 191.4 66.5 5.76 1RKO2 6 a5 0.0 75.7 53.7 5.2 48.1 7.7 7.2 1.4 197.6 71.4 5.67 1RK02 6 a6 0.0 74.8 53.2 5.3 47.5 7.2 7.3 1.4 195.3 70.2 5.68 1RK03 6 a7 0.5 77.2 55.7 4.3 43.9 6.3 4.6 1.4 191.9 65.3 5.61 1RK03 6 a8 0.0 76.1 54.1 4.2 43.1 6.2 4.4 1.4 188.1 62.1 5.63 1RK03 6 a9 0.0 77.8 56.8 4.6 45.1 6.4 4.8 1.4 195.4 67.8 5.64 1RK0203 6 310 0.0 69.5 48.9 5.0 45.0 7.9 7.8 1.4 184.1 65.2 5.95 1RK0203 6 al 1 0.7 68.9 48.4 5.1 45.5 7.7 7.9 1.4 183.4 67.7 5.89 1RK0203 6 a12 0.1 69.2 48.5 4.8 45.0 6.8 7.6 1.4 182.0 65.0 5.87 lcontrol 12 al 2.8 91.7 79.4 5.8 63.0 7.1 5.8 1.2 252.8 108.0 5.10 lcontrol 12 a2 1.5 91.4 76.7 5.5 61.4 7.2 5.9 1.2 248.1 105.0 5.16 lcontrol 12 a3 1.8 92.3 78.3 6.0 63.0 7.4 6.3 1.2 253.3 1 11.9 5.11 1RK02 12 a4 1.4 93 .2 72.9 4.9 63.0 6.4 9.4 1.3 249.9 106.1 5.16 1RK02 12 35 1.3 93.2 74.3 4.6 63.5 6.7 10.4 1.3 252.7 109.1 5.14 1RK02 12 a6 1.4 93.2 74.2 4.8 63.1 6.8 10.5 1.3 252.4 109.5 5.15 1RK03 12 a7 2.2 94.6 77.4 4.1 61.2 6.0 6.5 1.2 249.8 105.0 5.12 1RK03 12 a8 2.4 93.7 76.0 3.9 61.1 5.8 6.9 1.2 247.3 103.6 5.13 1RK03 12 a9 2.4 95.5 79.2 4.0 62.5 6.1 6.2 1.2 253.5 107.6 5.10 1RK0203 12 a10 0.6 85.3 68.9 6.3 62.6 7.8 8.9 1. .2 239.8 103.8 5.28 1RK0203 12 a1 1 0.8 85.7 69.1 6.4 63.9 8.5 9.6 1.2 243.1 108.9 5.27 1RK0203 12 al 2 0.6 85.6 68.8 6.4 63.1 8.6 10.0 1.2 242.5 107.7 5.28 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 237 Table 7.8. (Cont’d) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VFA F OM pH lcontrol 18 a1 3.0 99.6 88.9 4.1 75.6 6.6 8.5 1.1 283.3 130.5 5.01 lcontrol 18 a2 2.5 98.4 87.1 4.2 74.5 6.3 8.1 1.1 278.7 127.9 5.05 lcontrol 18 a3 3.1 98.3 86.7 4.2 74.9 6.1 7.9 1.1 278.1 130.4 5.02 1RK02 18 a4 1.7 98.9 85.0 5.2 81.0 7.8 14.0 1.2 291.9 140.2 5.07 1RK02 18 a5 1.5 97.5 84.8 5.2 80.0 7.6 13.6 1.1 288.7 138.7 5.05 1RK02 18 a6 2.3 97.7 84.9 5.6 79.7 8.7 14.0 1.2 290.5 141.0 5.07 1RK03 18 a7 2.6 100.0 88.4 4.3 76.3 6.7 8.1 1.1 283.8 131.6 5.04 1RKO3 18 a8 2.7 98.8 87.7 4.3 76.8 6.4 8.5 1.1 282.6 130.6 5.04 1RK03 18 a9 2.9 99.8 89.7 4.6 76.9 5.8 9.1 1.1 285.9 133.0 5.01 1RK0203 18 810 1.3 94.1 79.8 5.3 77.9 7.6 13.1 1.2 277.9 133.3 5.16 1RK0203 18 all 1.9 93.9 78.9 5.3 77.5 7.5 13.0 1.2 276.0 130.8 5.15 1RK0203 18 312 1.4 92.0 77.8 5.1 75.7 7.1 12.0 1.2 269.7 127.6 5.17 lcontrol 24 a1 1.7 95.3 90.9 7.3 82.7 8.2 9.9 1.0 294.2 141.5 4.98 lcontrol 24 a2 1.4 95.0 89.3 7.1 79.9 8.1 9.6 1.1 289.0 137.9 5.05 lcontrol 24 a3 1.7 94.9 88.2 7.0 81.3 8.0 9.7 1.1 289.2 141.3 5.00 1RK02 24 a4 1.2 96.8 87.6 7.8 86.6 9.3 14.5 1.1 302.6 149.5 5.04 1RK02 24 a5 1.1 96.3 88.0 8.2 87.3 10.3 17.6 1.1 307.7 155.7 5.03 1RK02 24 a6 1.1 95.2 88.0 8.1 85.8 10.5 16.8 1.1 304.5 153.5 5.04 1RK03 24 a7 1.5 98.0 92.0 7.4 84.9 9.5 l 1.6 1.1 303.4 149.2 5.02 1RK03 24 a8 2.0 97.8 90.5 7.4 85.3 9.5 11.8 1.1 302.4 149.1 5.03 1RK03 24 a9 1 .8 97.4 91.4 7.2 84.9 9.2 12.1 1.1 302.3 148.9 4.98 1RK0203 24 a10 1.3 94.6 83.9 7.7 85.5 10.6 15.6 1.1 297.9 150.4 5.12 1RK0203 24 al 1 1.3 93.1 83.4 7.5 86.0 10.3 16.2 1.1 296.5 152.0 5.12 1RK0203 24 312 1.0 91.5 81.6 7.2 83.1 9.9 15.2 1.1 288.5 144.8 5.14 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 238 Table 7.8. (Cont’d) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VFA FOM pH 2control 0 b1 3.8 50.3 22.5 2.4 16.4 2.3 3.1 2.2 96.9 6.72 2control 0 b2 4.6 51.5 22.9 2.6 17.2 2.7 3.0 2.3 99.9 6.69 ZControl 0 b3 4.1 50.7 22.2 2.3 16.2 2.2 2.6 2.3 96.2 6.71 2RK02 0 b4 4.5 53.6 22.3 1.8 17.7 2.1 4.8 2.4 102.3 6.78 2RK02 0 b5 4.2 52.4 23.9 2.5 19.4 2.9 5.1 2.2 106.2 6.71 2RK02 0 b6 3.7 45.7 20.6 2.2 17.0 2.5 4.5 2.2 92.5 6.68 2RK03 0 b7 12.2 52.4 22.9 2.4 16.8 2.3 2.9 2.3 99.7 6.66 2RK03 0 b8 12.9 53.1 23.0 2.3 16.8 2.6 2.9 2.3 100.8 6.69 2RK03 0 b9 1 1.3 46.9 20.3 2.0 14.7 1.9 2.3 2.3 88.0 6.63 2RK0203 0 bl 0 7.8 52.9 23.9 2.4 19.1 2.6 5.1 2.2 106.1 6.64 2RK0203 0 b1 1 7.5 53.0 24.1 2.5 19.3 2.8 5.2 2.2 106.9 6.64 2RK0203 0 bl 2 6.9 49.9 22.8 2.6 18.6 3.1 5.2 2.2 102.1 6.64 2control 6 b1 2.9 78.5 52.7 3.5 33.0 4.9 5.1 1.5 177.6 52.6 6.13 Zoontrol 6 b2 6.5 78.1 50.9 3.4 32.8 5.0 5.1 1.5 175.2 50.4 6.10 2control 6 b3 2.6 56.4 37.0 2.6 23 .4 3 .7 3.7 1.5 126.8 20.5 6.15 2RK02 6 b4 0.0 74.8 47.9 4.6 39.6 6.1 9.6 1.6 182.6 56.2 6.17 2RK02 6 b5 0.0 75.2 48.5 4.2 41.9 6.5 10.2 1.6 186.5 56.2 6.15 2RK02 6 b6 0.0 57.8 36.5 3.4 32.6 5.0 7.6 1.6 142.8 35.5 6.16 2RK03 6 b7 8.9 79.5 53.2 3.4 35.1 5.2 5.5 1.5 181.9 53.7 6.10 2RK03 6 b8 9.1 77.9 51.6 3.7 33.1 5.2 5.4 1.5 176.9 49.0 6.10 2RK03 6 b9 9.3 77.6 50.6 3.6 32.2 5.0 5.2 1.5 174.2 56.2 6.1 1 2RK0203 6 b10 1.7 76.7 50.3 4.9 45.2 6.9 10.9 1.5 194.9 62.4 6.16 2RK0203 6 b1 1 1.2 60.5 38.3 3.6 35.4 5.4 8.5 1.6 151.8 32.1 6.30 2RK0203 6 b12 0.0 82.0 52.7 4.8 45.3 7.0 1 1.2 1.6 203.0 68.5 6.20 2contIol 12 b1 17.4 92.6 72.3 3.9 49.8 4.5 5.9 1.3 228.9 94.2 5.25 2control 12 b2 28.6 93.3 71.1 3 .8 47.5 4.9 6.4 1.3 227.0 95.6 5.20 Zeontrol 12 b3 17.0 93 .2 72.6 3.7 51.1 4.3 6.2 1.3 231.0 96.5 5.24 2RK02 12 b4 4.9 92.1 68.7 4.7 63.4 5.7 15.5 1.3 250.2 107.3 5.38 2RK02 12 b5 4.9 91.5 70.0 5.0 72.6 6.0 18.8 1.3 263.9 117.3 5.33 2RKO2 12 b6 4.4 93.9 69.4 5.2 67.5 6.1 16.0 1.4 258.1 119.3 5.36 2RKO3 12 b7 33.4 92.8 72.6 4.6 54.1 5.4 5.6 1.3 235.1 102.4 5.15 2RK03 12 b8 25.0 94.2 74.7 5.2 55.8 5.9 6.0 1.3 241.9 101.9 5.21 2RK03 12 b9 28.0 94.7 74.0 5.2 53.1 5.8 5.8 1.3 238.6 109.5 5.20 2RK0203 12 b 10 5.4 90.2 70.4 6.2 74.9 6.9 16.3 1.3 265.1 1 17.4 5.35 2RK0203 12 bl 1 5.5 90.3 70.3 6.4 73.3 7.1 16.1 1.3 263.5 115.4 5.37 2RK0203 12 b12 4.9 92.3 69.4 5.8 64.5 6.8 13.9 1.3 252.8 106.7 5.41 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; F OM, apparently fermented organic matter. 239 Table 7.8. (Cont’d) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VFA F OM pH 2control 18 bl 50.6 93.7 73 .6 3.3 56.9 4.6 6.2 1.3 238.3 1 19.4 4.92 2control 18 b2 66.1 92.1 71.4 3.0 51.5 4.0 5.4 1.3 227.3 114.7 4.84 ZControl 18 b3 49.5 95.8 76.1 3.8 60.6 4.6 6.3 1.3 247.2 124.7 4.91 2RK02 18 b4 3.0 84.7 64.8 4.7 104.8 5.7 26.9 1.3 291.5 153.4 5.26 2RK02 18 b5 1.8 77.9 60.9 4.8 125.2 5.7 33.4 1.3 307.9 171.1 5.24 2RK02 18 b6 4.0 85.2 63.7 5 .5 1 16.3 6.3 29.5 1.3 306.6 175.5 5.25 2RK03 18 b7 70.3 94.5 71.9 3.7 54.8 4.6 6.3 1.3 235.8 121.2 4.84 2RK03 18 b8 58.3 95.9 74.8 3.4 58.3 4.1 5.6 1.3 242.2 1 18.1 4.88 2RK03 18 b9 64.8 90.9 73.2 2.9 53.1 3.6 5.3 1.2 229.0 120.5 4.87 2RK0203 18 b10 2.4 80.1 60.6 4.1 113.8 4.5 29.1 1.3 292.2 151.5 5.26 2RK0203 l8 b11 1.7 82.8 62.6 5.5 115.6 5.2 30.1 1.3 301.8 159.3 5.26 2RK0203 18 b 12 1. .5 91.4 68.1 5.2 94.7 6.1 24.0 1.3 289.6 143.9 5.30 2control 24 b1 64.5 95 .9 74.3 5.1 60.0 6.2 7.0 1.3 248.4 134.6 4.83 2control 24 b2 80.2 98.5 73.5 5.0 55.5 6.3 7.0 1.3 245.8 136.4 4.76 2control 24 b3 67.7 100.1 76.2 5.3 63.6 6.9 7.8 1.3 260.0 145.3 4.79 2RK02 24 b4 3.0 78.7 59.7 5.6 126.2 8.8 33.2 1.3 312.2 180.1 5.21 2RK02 24 b5 0.9 71.9 55.5 6.1 142.8 8.9 39.6 1.3 324.7 194.0 5.22 2RK02 24 b6 2.4 75.6 55.8 5.6 138.5 9.0 36.8 1.4 321.4 198.4 5.24 2RK03 24 b7 79.7 97.8 73.2 5.0 59.8 5.9 6.9 1.3 248.6 136.5 4.75 2RK03 24 b8 73.7 100.1 75 .7 5.1 61 . 1 6.2 6.3 1.3 254.4 135.6 4.79 2RK03 24 b9 78.4 100.7 74.8 5.4 56.1 5.9 5.8 1.3 248.6 141.6 4.77 2RK0203 24 b10 2.6 76.5 58.2 6.4 137.0 9.0 36.9 1.3 324.0 189.0 5.21 2RK0203 24 b1 1 1.2 72.6 56.2 6.5 138.9 8.8 38.4 1.3 321.4 188.2 5.23 2RK0203 24 b12 0.0 86.4 63.9 6.7 1 14.6 8.7 28.6 1.4 309.0 166.9 5.26 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 240 Table 7.8. (Cont’d) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VFA F OM pH 3control 0 CI 3.6 36.5 25.3 1.5 17.6 2.6 2.0 1.4 85.5 6.73 3control 0 02 3.1 37.1 25.2 1.9 18.1 4.0 2.7 1.5 89.0 6.68 3control 0 c3 2.4 35.4 24.4 1.3 16.4 1.8 1.6 1.5 80.8 6.66 3RK02 0 c4 1.2 31.1 20.4 1.0 14.6 1.9 3.2 1.5 72.4 6.68 3RK02 0 CS 1.1 30.9 20.3 0.8 13.8 1.4 3.0 1.5 70.2 6.68 3RK02 0 06 1.5 38.4 26.6 1.4 19.1 2.3 4.7 1.4 92.4 6.63 3RK03 0 c7 1.5 31.4 20.2 0.8 12.7 1.2 1.2 1.6 67.5 6.72 3RK03 0 c8 2.1 37.1 24.9 1.3 16.7 1.9 2.1 1.5 84.1 6.63 3RK03 0 09 2.3 37.6 25.4 1.4 16.6 2.2 2.3 1.5 85.6 6.69 3RK0203 0 c10 1.5 37.5 25.1 1.1 16.7 1.8 2.6 1.5 84.7 6.71 3RK0203 0 c1 1 1.8 37.7 25.0 1.3 17.3 1.9 3.2 1.5 86.4 6.64 3RK0203 0 c12 1.3 33.3 22.3 1.3 15.0 1.8 3.2 1.5 76.8 6.62 3control 6 CI 40.7 63.7 47.8 2.9 27.0 3.1 2.2 1.3 146.7 54.3 4.97 3control 6 c2 41.6 67.7 54.5 2.8 28.3 3.2 2.2 1.2 158.7 58.2 4.89 3control 6 03 41.7 64.0 48.9 2.6 26.8 2.8 2.1 1.3 147.2 60.2 4.96 3RK02 6 c4 31.7 62.8 48.0 2.7 39.5 3.5 7.7 1.3 164.2 77.4 5.01 3RK02 6 c5 31.9 63.6 49.0 2.7 40.3 3.5 7.8 1.3 166.8 81.0 4.97 3RK02 6 c6 32.3 63.8 49.8 2.7 39.4 2.9 7.0 1.3 165.7 63.8 4.96 3RK03 6 c7 52.2 64.3 49.2 3 .4 26.8 3.2 2.3 1.3 149.1 75.6 4.87 3RK03 6 c8 52.7 65.0 51.5 3.1 26.5 3.0 2.3 1.3 151.5 65.0 4.87 3RKO3 6 c9 51.6 63.7 49.5 3.0 26.0 2.8 2.3 1.3 147.3 60.9 4.89 3RK0203 6 c10 43.8 60.8 45.6 2.5 37.4 3.3 6.1 1.3 155.7 69.7 4.93 3RK0203 6 cl 1 44.0 60.5 45.5 2.5 36.4 2.9 5.8 1.3 153.5 66.1 4.93 3RK0203 6 012 43.7 60.9 46.7 2.5 36.1 2.9 5.7 1.3 154.8 72.3 4.92 3control 12 cl 90.5 77.4 66.1 1.6 26.4 1.7 1.7 1.2 174.7 91.3 4.35 3control 12 c2 88.5 76.4 64.9 1.5 26.7 1.8 1.8 1.2 173.2 86.7 4.36 3control 12 c3 93.4 73.9 61.6 1.4 25.5 1.5 1.4 1.2 165.2 92.6 4.37 3RK02 12 c4 76.4 71.2 59.9 1.4 48.8 2.1 9.6 1.2 193.1 118.6 4.44 3RK02 12 CS 75.2 71.4 59.9 1.8 51.0 2.5 10.4 1.2 196.9 123.5 4.45 3RK02 12 c6 75.2 72.3 61.4 1.9 52.0 3.1 11.5 1.2 202.2 112.1 4.46 3RK03 12 c7 96.0 73.6 63.8 1.6 26.3 2.0 2.2 1.2 169.5 106.3 4.39 3RK03 12 c8 98.6 71.9 60.9 1.8 25.2 2.3 2.3 1.2 164.3 92.4 4.40 3RK03 12 c9 65.6 49.3 42.9 1.0 17.1 1.4 1.5 1.1 113.2 43.8 4.39 3RK0203 12 c10 78.0 70.3 63.3 1.8 42.4 2.7 7.5 1.1 188.1 105.8 4.46 3RK0203 12 cll 82.3 71.7 65.0 1.6 42.6 2.7 7.8 1.1 191.5 108.2 4.44 3RK0203 12 c12 49.2 44.1 40.1 1.2 26.4 1.9 4.9 1.1 118.6 49.9 4.44 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 241 .' v7- au‘uv‘ Table 7.8. (Cont’d) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VF A F OM pH 3control 18 cl 1 13.0 82.9 74.0 1.5 27.8 2.0 2.5 1.1 190.6 111.9 4.30 3control 18 c2 103.3 85.5 79.1 1.5 29.0 2.2 2.4 1.1 199.5 108.7 4.30 3control 18 c3 1 10.3 81.6 74.0 1.2 27.3 1.4 1.8 1.1 187.2 113.2 4.29 3RK02 18 c4 88.1 76.0 68.6 1.8 55.4 2.8 11.6 1.1 216.3 140.7 4.37 3RK02 18 c5 88.4 75.5 67.2 1.7 55.4 2.6 11.2 1.1 213.6 140.6 4.37 3RK02 18 c6 84.5 76.3 70.1 1.6 57.3 2.6 11.7 1.1 219.5 127.4 4.38 3RK03 18 c7 109.7 82.6 78.3 1.4 28.3 2.2 2.0 1.1 194.9 125.9 4.31 3RK03 18 c8 1 14.3 79.6 72.2 2.2 26.0 1.8 1.9 1.1 183.8 109.9 4.31 3RK03 18 c9 117.3 79.4 72.5 1.3 26.4 1.8 2.0 1.1 183.5 110.4 4.30 3RK0203 18 c 1 0 79.0 79.6 80.5 2.1 50.2 2.5 8.8 1.0 223.7 128.4 4.43 3RK0203 l8 c1 1 91.5 78.9 79.4 1.6 47.9 2.5 8.0 1.0 218.4 128.8 4.39 3RK0203 18 c 12 80.7 80.4 82.0 1.8 49.5 2.4 8.4 1.0 224.4 132.9 4.41 Boontrol 24 c1 1 10.3 91.7 76.9 3.6 27.8 3.9 2.5 1.2 206.3 120.4 4.23 3control 24 c2 96.6 93.4 83.1 3.8 29.2 3.4 2.4 1.1 215.3 1 15.2 4.28 3control 24 c3 109.7 91.4 78.0 3.5 27.8 3.5 2.2 1.2 206.3 125.1 4.24 3RK02 24 c4 89.7 84.5 71.2 3 .2 52.5 4.3 10.2 1.2 225.9 145.8 4.34 3RK02 24 c5 87.9 83.2 68.6 3.2 53.7 4.1 10.5 1.2 223.2 146.0 4.36 3RK02 24 c6 82.9 84.2 72.7 3.4 56.8 4.5 10.9 1.2 232.7 134.5 4.36 3RK03 24 c7 104.3 90.5 80.1 3.5 28.7 3.2 2.3 1.1 208.2 132.5 4.28 3RK03 24 c8 1 1 1.0 88.4 76.3 3.4 26.9 3.3 2.3 1.2 200.6 1 19.0 4.27 3RK03 24 09 115.9 89.5 77.1 3.5 27.3 3.7 2.6 1.2 203.8 122.6 4.25 3RK0203 24 c10 70.3 86.4 85.2 3.7 51.3 4.4 8.7 1.0 239.7 134.4 4.42 3RK0203 24 cl 1 82.3 86.9 83.2 3.2 47.9 4.4 8.1 1.0 233.7 133.5 4.37 3RK0203 24 c12 70.6 87.7 87.9 3.4 51.7 4.3 8.5 1.0 243.5 140.6 4.41 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; FOM, apparently fermented organic matter. 242 Table 7.8. (Cont’d) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VFA FOM pH 4control 0 d1 1.8 41.2 19.3 1.1 14.1 0.9 1.6 2.1 78.2 6.65 4control 0 d2 1.8 40.8 19.7 1.1 15.4 1.9 2.5 2.1 81.4 6.64 4control 0 d3 0.7 38.1 16.2 0.0 11.8 0.4 1.5 2.3 68.1 6.65 4RK02 0 d4 1.9 42 .4 20.6 1.4 18.0 1.4 4.2 2.1 88.0 6.60 4RK02 0 d5 1.9 42.5 20.5 1.2 17.6 1.4 4.5 2.1 87.8 6.62 4RK02 0 d6 1.9 42.6 20.3 1.2 17.7 1.4 4.7 2.1 87.9 6.61 4RK03 0 d7 3.0 42.0 19.2 1.2 14.5 1.3 2.1 2.2 80.3 6.62 4RK03 0 d8 3.3 41.6 19.0 1.1 14.3 1.1 1.8 2.2 78.9 6.63 4RK03 0 d9 3.3 41.6 19.0 1.0 14.2 1.1 1.8 2.2 78.7 6.63 4RK0203 0 d10 2.3 41.7 19.4 1.2 15.7 1.2 2.8 2.2 82.0 6.62 4RK0203 0 d1 1 2.2 41.9 19.2 1.4 15.6 1.1 2.8 2.2 81.9 6.62 4RK0203 0 d12 2.3 41.4 19.3 1.0 15.5 1.2 2.6 2.1 81.1 6.60 4control 6 d1 104.7 58.2 39.2 2.8 22.7 2.7 3.3 1.5 128.8 84.0 4.54 4control 6 d2 104.7 58.6 40.5 3.3 22.4 2.5 3.5 1.4 130.8 81.8 4.60 4control 6 d3 104.4 58.4 40.0 3.1 22.1 2.2 2.8 1.5 128.5 90.7 4.57 4RK02 6 d4 85.9 51.4 40.6 3.3 53.7 5.6 13.8 1.3 168.4 108.9 4.69 4RK02 6 d5 90.8 52.6 40.1 3 .5 50.9 5.3 12.9 1.3 165.4 107.7 4.66 4RK02 6 d6 91.8 53.1 40.4 3.5 50.2 5.6 13.2 1.3 166.0 108.0 4.64 4RK03 6 d7 104.9 57.7 39.4 2.9 21.4 2.2 2.3 1.5 126.0 78.7 4.57 4RK03 6 d8 104.8 57.9 39.8 2.9 21.8 2.1 2.8 1.5 127.3 81.2 4.55 4RK03 6 d9 105.4 58.4 40.8 3.3 22.6 2.5 3.2 1.4 130.8 84.1 4.54 4RK0203 6 d10 85.0 50.1 40.1 3.4 54.6 5.7 13.1 1.2 167.1 112.6 4.64 4RK0203 6 d1 1 80.7 48.4 39.6 3.5 61.5 5.6 14.6 1.2 173.3 118.1 4.66 4RK0203 6 d12 90.0 51.8 39.2 3.4 49.9 5.3 1 1.4 1.3 161.0 109.8 4.63 4control 12 d1 116.0 58.5 38.4 2.4 20.6 1.6 1.9 1.5 123.5 84.4 4.41 4control 12 d2 121.0 60.4 40.6 2.6 21.0 1.3 1.8 1.5 127.7 85.9 4.42 4control 12 d3 1 19.9 59.8 40.2 2.7 21.0 1.4 2.0 1.5 127.1 96.2 4.42 4RK02 12 d4 103.0 53.6 46.0 2.8 61.1 3.4 13.2 1.2 180.2 125.1 4.51 4RK02 12 d5 104.4 54.4 44.2 2.7 57.9 3 .5 1 1.9 1.2 174.5 120.6 4.52 4RK02 12 d6 106.1 55.2 43.8 2.8 54.5 3.1 11.3 1.3 170.6 116.8 4.52 4RK03 12 d7 122.5 60.2 40.1 2.7 21.7 1.4 2.2 1.5 128.3 88.2 4.44 4RK03 12 d8 122.1 59.7 40.4 2.7 21.1 1.4 2.2 1.5 127.5 88.6 4.43 4RK03 12 d9 121.7 60.3 41.0 2.8 22.0 1.5 1.8 1.5 129.3 89.9 4.43 4RK0203 12 d10 97.5 51.5 45.3 2.9 62.9 3.3 11.9 1.1 177.9 126.0 4.52 4RK0203 12 d1 1 93.5 50.3 46.9 3.0 70.5 3.7 13.9 1.1 188.3 134.7 4.54 4RK0203 12 d12 105.5 53.7 43.5 2.6 57.1 3.2 10.7 1.2 170.9 124.0 4.49 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; F OM, apparently fermented organic matter. 243 Table 7.8. (Cont’d) Trt Time Tube LAC AC PR iBut BUT iVal VAL AP VFA FOM pH 4control 18 d1 126.4 61.3 37.1 1.5 17.3 1.3 2.0 1.7 120.4 85.7 4.42 4control 18 d2 126.7 62.1 38.5 1.6 18.0 1.3 2.1 1.6 123.6 84.8 4.39 4control 18 d3 126.4 61.7 37.9 1.7 17.7 1.6 2.0 1.6 122.7 95.3 4.39 4RK02 18 d4 97.3 54.2 51.1 3.5 79.0 6.2 16.5 1.1 210.4 150.0 4.55 4RK02 18 d5 106.2 55.3 47.2 3.1 71.7 6.2 14.9 1.2 198.4 143.8 4.52 4RK02 18 d6 106.7 55.6 47.2 3.1 71.4 5.6 14.5 1.2 197.4 142.5 4.51 4RK03 18 d7 128.8 61.6 38.6 2.3 18.7 2.6 2.9 1.6 126.7 89.9 4.38 4RK03 18 d8 129.0 61.5 38.9 2.2 18.6 3.0 3.1 1.6 127.2 91.7 4.38 4RK03 18 d9 127.9 61.7 39.3 2.5 19.1 3.1 4.0 1.6 129.7 93.6 4.38 4RK0203 18 d10 91.2 52.2 50.9 3.3 80.1 6.1 15.6 1.0 208.2 150.6 4.55 4RK0203 18 d1 1 81.6 50.5 53.7 3.8 93.0 7.3 20.9 0.9 229.2 166.6 4.60 4RK0203 18 d12 104.0 56.7 48.2 3.9 67.5 4.6 12.4 1.2 193.3 142.2 4.51 4control 24 d1 134.6 62.7 40.0 2.4 17.5 2.6 2.5 1.6 127.7 94.9 4.30 4control 24 d2 135.9 62.2 41.3 2.7 17.5 2.5 2.7 1.5 128.8 93.3 4.31 4control 24 d3 136.6 62.3 40.9 2.9 17.7 2.7 2.9 1.5 129.3 105.2 4.31 4RK02 24 d4 105.3 54.8 52.8 2.8 77.5 3.9 17.4 1.0 209.2 151.6 4.54 4RK02 24 d5 115.0 55.6 48.9 2.5 71.3 3.8 15.7 1.1 197.8 146.7 4.52 4RK02 24 d6 1 14.1 56.0 49.1 2.6 70.7 4.1 16.1 1.1 198.7 146.3 4.53 4RK03 24 d7 151.5 59.9 37.9 2.1 23.0 2.2 3.9 1.6 129.0 104.9 4.33 4RK03 24 d8 150.8 58.8 36.8 1.9 20.7 1.5 2.9 1.6 122.7 100.5 4.32 4RK03 24 d9 151.3 59.8 37.3 1.8 19.2 1.6 2.6 1.6 122.3 99.8 4.32 4RK0203 24 d10 97.0 54.4 55.3 4.2 71.7 5.8 17.2 1.0 208.6 150.6 4.53 4RK0203 24 d1 1 73.0 51.2 59.2 4.3 87.8 6.3 22.3 0.9 231.2 161.4 4.64 4RK0203 24 d12 103.1 49.8 43.3 0.8 58.6 2.4 11.4 1.2 166.2 120.3 4.49 Lac, lactic acid; Ac, acetic acid; Pr, propionic acid; But, butyric acid; iBut, isobutyric acid; Val, valeric acid; iVal, isovaleric acid; Total, total VFA; A/P, acetic acid to propionic acid ratio; F OM, apparently fermented organic matter. 244 Table 7.9. Chapter 5. Feed Intake, kg/d prior to acidosis trial (Data for Figure 5.1) Trt Control DFM Steer S01 SOS SO6 SO9 804 S08 S10 812 12/26/04 1.91 8.84 7.52 8.30 8.98 8.60 2.82 7.27 12/27/04 2.73 3.97 3.64 6.41 8.11 6.20 1.58 2.29 12/28/04 6.17 6.47 6.12 5.82 6.60 8.05 0.00 4.62 12/29/04 3.29 5.33 6.73 2.71 4.76 7.96 1.20 4.38 12/30/07 3.98 7.86 6.06 4.38 5.73 7.33 8.18 5.18 12/31/04 5.16 8.25 7.08 3 .21 6.53 8.37 9.55 5.96 01/01/05 5.85 8.67 6.47 5 .47 4.40 8.27 3.82 4.40 01/02/05 6.75 9.68 6.92 9.02 7.72 7 .33 0.32 6.72 01/03/05 7.50 7.33 5.70 6.42 7.15 7.00 6.97 4.95 01/04/05 8.77 8.77 6.95 7.60 7.25 6.15 6.90 4.95 01/05/05 9.81 9.61 9.05 8.69 8.59 9.69 9.15 6.83 01/06/05 8.74 9.48 8.39 7 .93 8.49 9.00 7.17 4.82 01/07/05 7.88 9.46 7.14 6.89 8.34 6.76 8.16 6.03 01/08/05 7.17 9.58 7.73 7.67 6.66 7.27 9.81 5.61 01/09/05 8.21 9.23 6.96 5.05 6.45 6.61 9.10 3.50 01/10/05 8.69 9.46 7.57 8.23 8.59 9.30 9.91 4.93 01/ 1 1/05 7.29 9.33 7.27 9.44 6.20 7.55 9.28 6.83 01/12/05 8.33 8.89 7.49 7.93 8.38 8.86 9.33 5.68 01/13/05 8.43 8.87 3.88 8.28 8.64 8.86 9.30 5.38 01/14/05 8.43 8.87 2.56 7.06 8.38 8.86 9.38 0.00 01/15/05 8.15 8.63 0.00 7.70 8.28 8.58 9.35 4.02 01/16/05 8.10 8.71 7.05 7.77 8.46 8.46 9.33 5.35 01/17/05 8.05 8.46 1.81 7.98 8.48 6.83 8.36 4.15 01/18/05 8.17 8.74 7.63 7.70 8.69 8.25 8.92 3.45 01/19/05 8.17 8.71 9.02 7 .03 7.87 7.82 8.53 8.17 01/20/05 8.17 8.74 7.33 7.47 8.00 7.95 8.97 5.92 01/21/05 7.91 8.32 6.40 8.39 8.21 8.26 8.77 6.11 01/22/05 7.91 8.22 6.40 7.93 8.03 8.24 8.77 5.53 01/23/05 7.91 8.37 6.98 8.44 7.88 8.39 8.89 5.53 01/24/05 7.89 8.35 7.56 8.49 8.47 8.26 8.84 6.1 1 01/25/05 7 .91 8.35 8.73 8.01 8.44 8.26 8.84 7.37 01 /26/05 7.91 8.15 6.92 6.71 8.26 8.06 8.84 4.52 01/27/05 7.91 8.27 5.80 5.33 5.13 7.55 8.87 5.89 01/28/05 7.9] 8.37 6.95 7.17 4.36 7.65 8.82 5.21 01/29/05 7 .91 7.97 7 .38 7.93 6.30 7.88 8.84 6.66 01/30/05 7.91 8.35 6.79 6.35 7.67 7.98 8.82 5.95 0‘1/31/05 7.91 8.35 7.25 4.56 6.60 7.12 8.84 5.99 245 Table 7.9. (Cont’d) Trt Control DFM Steer SO 1 SOS SO6 SO9 804 S08 ' S 10 S l 2 02/01/05 7.91 8.35 7.45 8.31 8.31 8.08 8.84 7.28 02/02/05 7.94 8.35 7.81 8.15 6.02 7.93 8.84 7.85 02/03/05 7.91 8.35 6.97 8.44 5.56 7.24 8.84 7.85 02/04/05 7.91 8.35 7.33 8.00 7.01 6.05 8.84 4.85 02/05/05 7.91 8.35 5.57 8.15 7.49 7.98 8.84 6.43 02/06/05 7.91 8.35 7.28 8.87 8.23 7.50 8.84 5.41 02/07/05 8.15 8.47 7.13 8.15 8.11 7.60 8.84 6.55 02/08/05 8.15 8.47 8.04 5.82 6.73 7.93 9.19 7.19 02/09/05 8.15 8.47 7.03 6.89 6.48 7.25 9.19 5.79 02/10/05 8.15 8.47 4.53 6.49 5.21 8.14 9.19 6.25 02/1 1/05 8.15 8.06 5.65 3.23 6.28 8.49 9.19 6.91 02/12/05 8.15 8.47 4.17 Out 6.12 8.49 9.19 7.21 02/13/05 8.15 8.47 4.28 Out 6.00 8.49 9.19 6.88 02/14/05 8.15 8.47 6.08 Out 4.90 8.49 9.19 7.37 02/15/05 8.17 8.47 8.27 Out 6.25 8.47 9.17 6.99 02/16/05 8.12 8.49 8.35 Out 6.12 8.49 9.17 6.60 02/17/05 8.12 8.49 8.20 Out 5.61 8.49 8.91 7.39 02/18/05 8.15 8.49 7.3 1 Out 6.43 8.44 9.07 7.27 02/19/05 8.09 8.47 6.92 Out 5.92 8.37 9.07 7.21 02/20/05 8.15 8.49 7.76 Out 6.07 8.39 9.17 2.76 02/21/05 8.15 8.52 8.17 Out 6.30 8.42 9.14 6.37 02/22/05 8.17 8.52 8.12 Out 8.1 l 8.49 9.17 7.75 02/23/05 8.17 8.52 8.27 Out 1.85 8.49 9.17 7.88 02/24/05 8.17 8.52 8.20 Out 7.88 8.49 9.14 7.80 02/25/05 8.15 8.52 8.43 4.91 6.76 8.49 9.14 7.65 02/26/05 6.95 3.09 5.91 6.03 4.32 6.28 8.99 7.06 02/27/05 8.15 8.52 8.25 9.14 8.34 8.49 9.14 7.62 02/28/05 8.15 8.47 8.50 8.35 8.41 8.49 9.14 7.93 03/01/05 8.15 8.52 8.32 6.69 8.24 8.49 9.14 7.93 03/02/05 8.17 8.52 8.53 7.56 7 .75 8.49 9.14 7.95 03/03/05 8.15 8.52 8.48 5.12 6.89 8.49 9.14 7.98 03/04/05 8.15 8.52 8.45 5.37 8.21 8.16 9.14 7.93 03/05/05 8.15 6.17 7.99 5.04 5.21 7.07 8.96 6.04 03/06/05 8.15 8.26 8.09 5.83 7.12 8.49 8.71 4.24 03/07/05 8.47 8.52 8.12 5.88 8.49 8.47 8.41 7.04 246 Fuzmxmxtr ‘ . Table 7.10. Chapter 5. Feed intake (kg) after feeding on d 10 (Data for Table 5.1) Trt Steers Time after feeding, b Total 3 6 9 12 15 18 21 24 Control 501 3.4 2.9 1.9 0.0 0.0 0.0 0.0 0.0 8.3 Control 505 2.2 4.7 1.3 0.0 0.3 0.0 0.0 0.0 8.6 Control 506 5.5 1.7 0.4 0.0 0.0 0.6 0.0 0.0 8.2 Control 509 1.2 1.4 1.4 0.9 0.3 0.1 1.1 0.0 6.4 DFM 504 2.3 2.9 2.1 0.0 0.1 0.5 0.6 0.0 8.5 DFM 508 2.5 4.8 0.4 0.8 0.0 0.0 0.0 0.0 8.5 DFM 510 3.5 3.5 0.3 0.0 0.6 0.0 1.2 0.0 9.2 DFM 512 3.9 0.9 0.6 0.5 0.1 0.4 0.0 0.4 6.7 247 Table 7.11. Chapter 5. D10 Rumen contents (Data for Tables 5.2 to 5.4) Trt Steer Time LAC AC PR iBut But iVal Val A/P VFA Org pH Control S01 0 0.5 42.8 26.9 1.6 17.5 4.1 2.6 1.6 95.5 96.0 7.33 Control SOS 0 2.0 69.2 32.0 1.2 23.2 4.7 2.4 2.2 132.8 134.8 6.97 Control SO6 0 2.2 67.0 29.9 1.2 19.6 3.3 1.9 2.2 122.9 125.2 7.05 Control SO9 0 2.8 84.7 30.4 1.8 35.3 6.1 2.8 2.8 161.1 163.9 6.70 DFM $04 0 2.2 43.3 28.6 1.4 20.6 3.7 2.0 1.5 99.7 101.9 7.01 DF M S08 0 2.8 73.9 30.0 1.3 27.1 5.0 2.1 2.5 139.5 142.3 6.85 DF M 810 0 2.7 68.4 30.5 1.8 24.2 5.2 2.2 2.2 132.4 135.0 6.88 DFM $12 0 2.2 71.5 36.0 1.8 33.9 5.2 2.6 2.0 151.1 153.3 6.68 Control 801 3 5.7 56.8 57.4 2.0 13.4 3.2 4.3 1.0 137.1 142.9 6.00 Control SOS 3 13.1 75.2 53.2 3.1 25.7 8.2 2.2 1.4 167.6 180.7 6.61 Control SO6 3 9.1 64.8 43.4 1.8 25.8 6.2 3.4 1.5 145.4 154.5 6.61 Control SO9 3 9.4 77.6 36.5 2.8 37.0 9.0 2.7 2.1 165.6 175.0 6.59 DF M 804 3 7.3 56.2 32.3 3.4 22.0 5.2 2.1 1.7 121.3 128.6 6.75 DFM $08 3 10.5 88.6 38.7 2.4 39.3 9.6 3.1 2.3 181.6 192.1 6.61 DF M S10 3 1 1.3 87.5 47.6 2.5 40.4 10.2 3.5 1.8 191.7 203.0 6.58 DF M 812 3 8.9 95.0 73.1 3.3 28.3 7.7 3.6 1.3 211.1 220.0 5.56 Control S01 6 12.3 77.5 84.8 2.8 17.0 3.6 5.4 0.9 191.1 203.4 5.38 Control SOS 6 17.0 1 12.4 73.3 4.0 30.9 8.2 3.8 1.5 232.6 249.6 5.74 Control SO6 6 14.9 84.6 66.9 3.4 26.5 5.2 3.2 1.3 189.7 204.6 5.90 Control SO9 6 18.1 87.0 44.3 5.3 31.3 10.6 5.1 2.0 183.6 201.7 6.03 DFM S04 6 15.5 92.1 69.9 4.1 30.0 6.5 3.1 1.3 205.7 221.1 6.00 DFM 808 6 17.5 87.4 43.3 3.8 44.0 8.4 3.0 2.0 190.0 207.5 5.88 DF M S10 6 18.0 97.8 47.2 4.0 59.3 10.4 3.9 2.1 222.7 240.7 6.19 DF M 812 6 10.6 112.0 95.6 3.5 44.7 7.2 4.2 1.2 267.2 277.8 5.16 Control 801 9 1.1 97.6 125.9 4.7 25.6 3.8 1 1.3 0.8 269.0 270.1 5.17 Control S05 9 16.6 121.0 87.8 4.7 46.6 10.5 4.9 1.4 275.4 291.9 5.51 Control S06 9 14.6 96.2 91.7 3.9 40.9 7.6 4.6 1.0 244.8 259.4 6.00 Control SO9 9 15.5 105.8 61.0 3.8 43.9 13.6 3.6 1.7 231.7 247.1 5.85 DF M 804 9 10.9 107.5 96.6 5.1 37.8 8.2 4.5 1.1 259.7 270.6 5.45 DF M S08 9 18.9 72.5 51.0 3.8 39.9 13.2 2.0 1.4 182.4 201.3 5.86 DFM S 10 9 16.6 90.7 48.9 3.9 67.3 9.9 3.5 1.9 224.2 240.8 6.28 DFM 812 9 5.8 100.1 101.6 3.7 42.1 6.7 4.6 1.0 258.8 264.6 5. 10 Control 801 12 0.4 101.3 128.9 1.9 25.0 2.1 11.6 0.8 270.7 271.2 5. 18 Control SOS 12 18.0 101.5 76.5 1.7 44.1 9.8 4.1 1.3 237.6 255.6 5.99 Control SO6 12 13.5 93 .0 86.9 1.7 40.7 6.5 4.7 1.1 233.4 247.0 6.00 Control SO9 12 17.7 85.4 52.1 2.5 40.2 10.1 2.1 1.6 192.5 210.1 5.84 DFM $04 12 1 1.5 95.9 99.8 2.3 44.7 7.5 4.2 1.0 254.4 265.9 5.68 DFM S08 12 18.3 104.4 57.9 2.3 82.6 7.2 3.4 1.8 257.9 276.2 5.59 DFM 810 12 11.2 78.6 43.7 2.9 51.7 9.0 2.1 1.8 188.1 199.3 6.30 DFM 812 12 5.4 96.7 102.9 3.9 38.4 5.8 6.3 0.9 254.1 259.5 5.15 248 Table 7.11. (Cont’d) Trt Steer Time LAC AC PR iBut But iVal Val A/P VFA Org pH Control SO 1 15 1.4 94.1 123.7 4.9 25.4 2.1 9.9 0.8 260.0 261.4 5.23 Control $05 15 18.9 93.8 68.7 3.9 44.4 10.4 3.9 1.4 225.2 244.1 6.10 Control 806 15 17.4 87.6 82.0 2.8 39.5 6.9 4.3 1.1 223.1 240.5 5.74 Control SO9 15 18.9 109.7 58.1 4.4 54.5 1 1.9 3.4 1.9 242.0 260.9 5.52 DFM 804 15 11.9 57.8 59.3 2.0 30.7 5.3 3.4 1.0 158.5 170.4 5.69 DFM 808 15 16.8 55.3 37.1 2.1 44.3 5.2 2.2 1.5 146.1 162.9 5.54 DFM $10 15 12.4 91.1 37.2 5.1 57.2 7.9 3.2 2.5 201.5 213.9 6.31 DFM $12 15 10.9 98.1 98.5 6.1 41.6 6.5 7.0 1.0 257.8 268.8 5.12 Control S01 18 6.8 83.3 1 10.7 3.4 22.4 2.0 9.8 0.8 231.7 238.5 4.92 Control SOS 18 18.8 100.0 76.6 3.9 39.9 10.8 4.2 1.3 235.4 254.2 5.72 Control S06 18 15.0 83.8 75.2 2.6 31.4 6.7 4.5 1.1 204.2 219.2 6.10 Control S09 18 8.0 55.9 31.0 2.2 37.3 7.4 2.2 1.8 136.0 144.0 5.54 DFM 804 18 10.8 67.7 84.3 3.7 24.1 8.0 3.4 0.8 191.3 202.1 5.58 DF M 808 18 17.3 90.7 49.8 2.9 65.8 6.8 2.6 1.8 218.7 236.0 5.67 DFM S 10 18 8.9 83.7 36.8 4.0 46.5 8.0 2.5 2.3 181.5 190.4 6.13 DFM S 12 18 3.8 53.9 72.0 4.1 20.7 6.0 4.3 0.7 161.0 164.8 5.30 Control 801 21 10.6 71.2 74.5 2.1 25.9 2.8 6.1 1.0 182.5 193.0 5.71 Control SOS 21 18.8 74.4 56.7 2.6 37.6 7.4 3.0 1.3 181.7 200.5 5.97 Control SO6 21 13.6 101.0 81.3 2.2 40.7 6.5 4.5 1.2 236.3 249.8 5.92 Control SO9 21 14.8 106.4 55.3 3.9 50.5 1 1.3 3.5 1.9 230.9 245.6 5.87 DFM S04 21 1 1.3 102.0 80.2 3.1 39.9 7.4 4.0 1.3 236.6 247.9 5.75 DF M 508 21 15.5 81.2 38.0 3.0 59.4 5.7 2.2 2.1 189.4 204.9 5.96 DF M S 10 21 4.4 52.2 24.4 2.4 69.3 5.4 2.6 2.1 156.3 160.7 6.38 DF M S 12 21 1 1.3 71.2 91.3 3.7 29.1 5.7 6.4 0.8 207.4 218.7 5.27 Control 801 24 9.5 71.7 84.3 1.9 19.8 2.4 6.6 0.9 186.8 196.3 5.83 Control SOS 24 1 1.0 61.9 43.3 2.8 42.7 6.7 2.2 1.4 159.6 170.5 6.23 Control SO6 24 5.0 48.7 45.9 1.0 24.9 3.5 2.5 1.1 126.4 131.4 6.39 Control SO9 24 16.4 90.1 56.2 3 .4 36.4 10.8 2.7 1.6 199.7 216.0 5.71 DFM 804 24 11.0 77.3 68.5 2.3 25.7 6.3 2.6 1.1 182.8 193.8 5.97 DF M 808 24 14.8 59.8 32.3 3.7 36.8 6.5 1.5 1.9 140.5 155.3 6.24 DFM S 10 24 14.3 86.3 36.9 4.2 46.3 7.9 3.0 2.3 184.6 198.8 6.31 DFM 812 24 10.2 79.6 101.3 3.9 23.7 6.5 6.3 0.8 221.4 231.5 5.49 249 Table 7.12. Chapter 5. Rumen contents on acidosis induction (Data for Tables 5.5 to 5.7) Trt Steer Time Lac Ac Pr iBut But iVal Val VFA Org A/P pH Control 501 O 1.5 13.7 8.7 0.8 7.3 1.0 1.1 32.8 34.3 1.6 7.17 DFM $04 0 0.0 12.7 9.4 0.9 5.8 1.2 1.3 31.2 31.2 1.4 7.06 Control 505 O 4.3 16.9 11.8 1.3 6.5 3.3 1.8 41.6 45.9 1.4 7.11 Control 506 0 1.6 19.5 8.2 1.4 5.9 2.8 2.3 40.1 41.7 2.4 7.06 DFM 308 O 2.8 19.9 8.2 1.7 7.6 3.8 1.7 42.9 45.7 2.4 6.93 Control SO9 O 12.4 22.3 8.2 2.3 7.1 3.7 1.0 44.6 57.0 2.7 6.98 DFM 510 O 0.6 17.6 7.5 2.1 6.5 2.6 1.7 38.0 38.7 2.3 7.14 DFM 312 O 1.6 15.0 9.9 1.2 11.7 3.5 2.0 43.3 44.9 1.5 7.03 Control 501 2 3.7 23.7 16.7 0.7 5.1 1.3 2.0 49.5 53.2 1.4 7.11 DFM $04 2 22.7 51.2 18.0 0.9 8.1 2.2 0.7 81.1 103.8 2.8 6.43 Control 505 2 20.0 44.7 30.7 1.4 11.3 3.7 1.2 93.0 113.0 1.5 6.74 Control 306 2 22.1 45.0 20.9 0.4 9.2 2.3 1.3 79.2 101.4 2.1 6.68 DF M 508 2 57.0 70.9 21.9 1.2 17.2 4.0 1.2 116.5 173.5 3.2 5.81 Control 509 2 51.5 43.7 18.7 2.0 15.2 4.2 0.0 83.7 135.3 2.3 5.96 DFM s 1 0 2 27.8 60.2 36.4 1.2 17.2 4.3 1.4 120.7 148.5 1.7 6.66 DFM 512 2 66.3 75.6 41.0 0.9 12.8 3.1 1.4 134.7 201.0 1.8 5.81 Control 501 4 19.8 29.7 20.9 1.0 4.4 1.6 1.1 58.6 78.3 1.4 6.46 DFM 504 4 86.2 69.5 35.5 1.2 11.7 2.8 1.3 121.9 208.2 2.0 5.07 Control 505 4 78.5 57.0 36.0 1.2 11.7 3.2 1.4 110.4 188.9 1.6 5.29 Control 506 4 92.4 66.8 33.4 1.0 10.7 2.6 1.2 115.7 208.1 2.0 5.20 DFM 308 4 110.8 66.0 21.1 1.0 18.1 3.5 1.2 110.9 221.6 3.1 4.78 Control 309 4 100.6 63.9 26.3 0.9 20.3 3.2 1.3 116.0 216.6 2.4 5.22 DFM $10 4 79.6 69.5 33.3 1.3 20.6 3.9 1.4 130.0 209.6 2.1 5.14 DFM $12 4 1 16.4 78.0 44.5 0.0 14.4 2.8 1.5 141.3 257.7 1.8 5.00 Control 501 6 47.4 39.0 28.1 1.8 4.3 1.3 3.1 77.7 125.0 1.4 5.45 DFM 504 6 95.1 79.8 47.8 1.2 12.6 2.6 1.2 145.3 240.4 1.7 4.63 Control 805 6 101.3 50.6 36.7 1.6 13.9 3.6 1.3 107.6 208.9 1.4 4.82 Control 306 6 115.7 76.5 39.2 1.3 11.1 2.6 0.9 131.5 247.2 2.0 4.72 DF M 508 6 143.4 44.2 20.0 1.2 10.2 4.2 0.3 80.1 223.5 2.2 4.59 Control 509 6 134.4 70.6 24.7 0.8 14.8 4.1 0.7 115.8 250.2 2.9 4.92 DFM 310 6 1 10.0 62.9 30.2 1.8 24.0 3.1 0.8 122.7 232.7 2.1 4.57 DFM 512 6 142.0 64.7 41.5 3.7 12.8 2.3 1.1 126.1 268.1 1.6 4.67 Control 501 8 65.6 46.5 48.1 1.5 7.6 1.4 3.6 108.8 174.5 1.0 5.36 DFM 504 8 115.2 78.4 43.2 2.7 13.0 2.9 1.6 141.8 257.0 1.8 4.42 Control 505 8 106.5 61.2 31.0 5.0 12.2 3.3 1.3 114.0 220.6 2.0 4.58 Control 506 8 130.9 60.5 33.6 2.6 12.1 2.6 1.5 112.9 243.8 1.8 4.47 DFM $08 8 127.8 48.2 22.9 2.1 25.7 3.0 1.1 102.9 230.7 2.1 4.36 Control 509 8 131.7 75.5 27.0 0.0 16.5 4.2 0.7 123.9 255.6 2.8 4.35 DFM $10 8 123.9 79.6 33.2 1.8 19.4 3.6 0.7 138.3 262.2 2.4 4.39 DFM 512 8 143.5 64.8 50.4 4.1 14.9 2.9 3.6 140.9 284.3 1.3 4.34 250 Table 7.12. (Cont’d) Trt Steer Time Lac Ac Pr iBut But iVal Val VFA Org M? pH Control 501 10 85.5 48.2 50.3 1.5 9.7 1.3 3.7 114.6 200.1 1.0 5.20 DF M 504 10 154.4 66.9 39.6 0.9 5.7 1.7 0.5 115.5 269.9 1.7 4.45 Control 505 10 91.6 66.8 30.7 1.6 23.1 4.0 1.1 127.3 218.8 2.2 4.65 Control 506 10 146.0 52.1 40.6 1.6 13.0 2.9 1.6 111.8 257.8 1.3 4.28 DFM 508 10 152.6 46.2 22.5 1.4 20.9 2.6 0.8 94.3 247.0 2.1 4.38 Control SO9 10 154.8 66.2 26.2 1.6 9.9 4.2 1.0 109.1 263.9 2.5 4.14 DF M 310 10 111.4 66.8 27.8 1.5 19.1 3.0 0.9 119.1 230.5 2.4 4.37 DFM 512 10 174.5 62.2 48.5 0.9 11.2 2.2 2.3 127.3 301.8 1.3 4.25 Control 301 12 105.5 45.0 58.8 1.8 12.0 1.5 3.0 122.1 227.5 0.8 4.61 DF M 504 12 1 19.3 48.0 40.4 1.5 10.3 3.6 1.1 104.9 224.2 1.2 4.65 Control 805 12 131.0 62.7 50.5 1.5 17.6 4.1 1.8 138.3 269.3 1.2 5.19 Control 506 12 135.4 57.4 37.7 1.0 15.4 2.9 1.8 116.2 251.6 1.5 4.39 DFM $08 12 154.5 66.5 26.2 1.4 31.3 2.9 1.2 129.5 283.9 2.5 4.58 Control $09 12 176.5 67.8 15.4 0.9 12.9 2.6 1.2 100.7 277.2 4.4 4.01 DFM 510 12 112.5 71.5 38.0 1.5 19.7 3.4 1.5 135.6 248.1 1.9 4.51 DFM 512 12 188.8 57.6 42.3 0.5 5.2 1.0 0.5 107.0 295.8 1.4 4.02 Control 501 15 103.1 68.5 62.5 1.0 9.6 1.0 2.5 145.0 248.1 1.1 4.48 DFM 304 15 108.0 51.9 46.6 1.4 10.1 3.7 1.8 115.4 223.4 1.1 5.36 Control 505 15 123.6 53.4 27.4 1.2 13.9 3.2 1.2 100.4 224.0 1.9 4.94 Control 506 15 112.9 51.3 44.5 1.3 14.8 3.2 2.3 117.3 230.2 1.2 4.98 DFM 508 15 1 18.8 64.7 20.5 1.4 27.8 2.7 1.0 1 18.1 236.8 3.2 4.87 Control 509 15 188.6 54.5 28.1 1.5 12.7 5.0 1.3 103.1 291.7 1.9 4.02 DFM 510 15 104.6 69.7 31.1 1.8 21.9 3.4 1.1 129.0 233.6 2.2 4.89 DFM $12 15 182.5 63 .4 40.4 1.4 12.7 2.6 3.4 124.0 306.5 1.6. 3.96 Control $01 18 123.1 76.3 69.0 1.8 9.5 4.3 2.8 163.7 286.8 1.1 4.40 DFM $04 18 100.6 53.1 43.6 2.0 16.4 3.2 1.2 119.5 220.1 1.2 5.12 Control 505 18 138.2 55.7 23.6 1.8 23.7 1.6 1.5 107.9 246.1 2.4 4.48 Control 306 18 98.8 77.8 45.6 1.3 16.0 4.8 2.7 148.2 247.0 1.7 5.38 DF M 508 18 101.8 79.1 22.5 1.7 28.0 2.0 1.8 135.1 236.9 3.5 4.70 Control 509 18 181.1 61.4 27.1 1.2 10.8 4.9 1.1 106.5 287.6 2.3 4.14 DFM sl 0 18 88.9 60.2 30.9 1.9 23.9 2.7 1.5 121.2 210.0 1.9 4.90 DFM 512 18 167.9 61.3 34.2 2.7 13.1 3.3 0.8 115.5 283.4 1.8 3.79 Control 501 21 133.8 78.8 74.1 1.4 10.6 3.7 2.5 171.1 304.9 1.1 4.29 DFM $04 21 101.0 51.2 40.7 1.7 16.8 3.5 2.1 116.0 217.0 1.3 4.87 Control 505 21 109.4 55.5 25.9 1.4 21.3 2.0 1.1 107.2 216.6 2.1 4.46 Control $06 21 97.6 70.0 42.5 1.7 17.3 3.3 2.2 137.0 234.6 1.6 4.75 DFM 508 21 104.8 72.8 23.8 1.7 23.6 3.6 1.6 127.2 232.0 3.1 4.82 Control $09 21 173.5 62.6 26.1 2.7 12.5 3.0 1.2 108.2 281.7 2.4 4.06 DFM 510 21 93.9 61.1 32.3 2.1 20.8 3.0 11.0 130.3 224.2 1.9 5.02 DFM $12 21 172.6 63.5 34.1 2.5 13.6 3.1 0.7 117.5 290.1 1.9 3.71 251 Table 7.13. Chapter 5. Blood pH, gases, and metabolites on acidosis induction (Data for Tables 5.8, 5.9, 6.8, and 6.9) Trt Cow Time pH PC02 P02 802 Hct Na K C1 control 1 O 7.44 43.1 43.8 80.6 28.5 140.8 2.9 104.0 control 5 0 7 .46 49.1 37 .5 70.2 30.0 142.5 3.7 101.9 control 6 O 7 .44 47.8 41.4 76.3 29.0 141.3 3.0 103.2 control 9 O 7.43 40.8 36.8 70.8 31.5 140.3 3.7 103.6 DFM 4 O 7.46 47.6 34.7 65.8 30.0 140.6 3.4 101.7 DF M 8 O 7.48 41.2 39.6 77 .4 32.5 142.0 3.6 100.9 DFM 10 O 7.47 44.8 41.6 78.4 30.0 140.9 3.4 102.9 DFM 12 O 7.45 41.2 37.4 72.7 28.0 141.6 3.8 107.5 control 1 2 7 .45 40.6 41.7 78.8 28.0 141.0 2.6 103.3 control 5 2 7.46 42.2 37.7 72.5 29.0 141.6 3.7 103.4 control 6 2 7.43 40.8 40.2 75.3 29.0 142.1 3.0 105.9 control 9 2 7.44 41.2 38.5 74.3 31.0 140.7 3.7 103.8 DFM 4 2 7.47 44.4 34.6 68.2 28.5 141.6 3.4 103.4 DFM 8 2 7.46 42.6 38.5 75.0 29.5 141.9 3.4 102.9 DFM lO 2 7.48 41.7 35.0 69.6 29.5 141.7 3.4 103.5 DFM 12 2 7.44 40.2 35.8 69.9 27.0 141.0 3.6 107.3 control 1 4 7.44 42.2 42.8 79.5 28.0 140.4 2.9 102.7 control 5 4 7.43 42.5 37.2 70.3 31.0 143.5 3.9 103.9 control 6 4 7.41 41.8 40.8 75.2 29.0 141.9 2.9 106.1 control 9 4 7.43 38.4 37.5 72.4 32.0 139.8 3.8 104.3 DF M 4 4 7.47 44.6 32.7 64.3 33.0 139.4 3.9 100.1 DFM 8 4 7 .44 41.5 39.2 74.6 31 .0 143.2 3.6 104.8 DFM lO 4 7.51 36.2 39.1 78.9 31.0 143.4 3.7 105.5 DFM 12 4 7.44 38.8 42.3 79.0 27.5 139.0 3.6 106.4 control 1 6 7.45 45.0 41.7 78.2 30.0 142.0 3.2 101.7 control 5 6 7.41 46.8 39.9 73.0 32.0 142.1 4.0 104.1 control 6 6 7.38 44.4 37.2 67.1 32.0 147.6 3.0 109.8 control 9 6 7.41 45.2 38.1 71.7 33.0 142.8 3.8 105.0 DFM 4 6 7 .46 42.8 36.4 71.2 32.0 143.5 3.9 104.0 DFM 8 6 7.39 42.8 39.9 73.2 34.0 149.4 3.9 108.0 DFM 'lO 6 7.45 40.4 39.8 76.1 32.5 145.5 3.8 107.2 DFM 12 6 7.43 39.8 39.7 75.4 28.0 143.7 3.4 107.9 control 1 8 7.42 46.6 41.6 76.6 30.0 143.3 3.3 103.2 control 5 8 7.38 46.7 40.6 72.5 32.0 145.6 4.2 106.5 control 6 8 7.34 43.1 43.4 75.2 31.0 143.1 3.3 108.6 control 9 8 7.40 41.4 41.6 76.5 33.5 144.1 3.9 107.6 252 Table 7.13. (Cont’d) Trt Cow Time pH PC02 P02 802 Hct Na K Cl DF M 4 8 7 .43 43.9 33.0 62.1 34.0 145.1 3.9 105.1 DF M 8 8 7.40 37.8 37.2 70.4 37.0 146.8 4.4 108.2 DFM 10 8 7.43 38.5 37.1 71.1 33.0 148.0 3.8 110.1 DF M 12 8 7.42 40.2 38.6 73.5 30.5 144.5 3.5 107.5 control 1 10 7.44 43.8 42.3 78.7 31.0 137.6 3.2 99.1 control 5 10 7.38 43.9 40.4 72.2 31.5 144.1 3.8 106.3 control 6 10 7.33 42.5 39.5 68.6 34.0 146.5 3.1 1 10.8 control 9 10 7.36 40.4 34.3 62.1 36.0 144.1 3.9 108.8 DFM 4 10 7.41 45.0 28.6 50.3 36.0 145.0 3.9 104.3 DF M 8 10 7.37 40.8 36.0 65.6 36.0 146.3 4.2 107.5 DFM IO 10 7.49 33.7 35.8 72.8 36.0 148.0 3.9 109.1 DF M 12 10 7.41 40.7 42.6 78.3 29.0 144.1 3.5 107.8 control 1 12 7.45 42.7 41.8 78.6 29.0 144.6 3.0 104.4 control 5 12 7.39 43.3 42.8 76.4 33.0 144.8 3.6 105.0 control 6 12 7.33 39.7 41.4 72.0 35.5 146.8 3.4 1 10.7 control 9 12 7.35 38.1 37.3 67.6 38.0 147.1 4.0 110.9 DF M 4 12 7.41 40.3 36.1 68.2 37.0 148.8 3.8 108.2 DF M 12 12 7.41 39.0 40.7 75.8 30.0 146.3 3.5 109.8 control 1 l 5 7 .49 41 .5 38.4 76.2 34.0 142.9 3.2 99.7 control 5 15 7.40 41.2 46.4 81.3 33.0 143.4 3.3 104.3 control 6 15 7.33 36.3 43.6 75.7 36.5 146.1 3.7 110.6 control 9 15 7 .32 34.9 36.5 64.7 42.0 146.7 4.1 1 10.3 DFM 4 15 7.42 37 .2 41.2 77.2 37.0 148.3 3.5 107.5 DFM 12 15 7.40 37.8 33.1 62.5 31.0 145.7 3.7 109.4 control 1 18 7.42 38.2 42.3 78.7 33.5 140.1 2.4 102.0 control 5 18 7.44 37.1 46.3 83.2 35.5 142.9 3.1 102.6 control 6 18 7.38 34.3 43.9 78.4 36.0 145.2 3.4 109.7 control 9 18 7.28 31.4 47.4 78.8 43.0 147.1 4.0 1 10.5 DF M 4 18 7.46 40.5 41.3 78.6 35.5 145.8 3.5 106.0 DFM 8 18 7.40 32.3 42.7 78.6 36.0 144.5 3.2 107.9 DF M 10 18 7.43 36.6 35.4 68.2 35.5 148.8 3.2 109.2 DFM 12 18 7.42 35.9 39.5 75.0 32.0 145.0 3.6 108.4 control 5 21 7.47 41.2 39.9 77.2 37.0 142.0 3 .5 98.4 control 6 21 7 .39 40.1 35.2 66.3 37.0 145.2 3.8 108.0 control 9 21 7.25 40.9 29.8 45.2 43.0 146.7 4.2 108.5 DFM 4 21 7.44 39.1 39.7 75.9 35.0 145.8 3.3 106.3 DFM 8 21 7.57 20.9 41 .O 87.9 38.0 139.6 3 .9 104.9 DFM 10 21 7.54 29.0 73.0 100.0 37.0 145.2 3.3 105.7 DF M 12 21 7.41 39.9 43.8 79.5 33.0 146.7 3.4 107.7 253 Table 7.13. (Cont’d) Trt Cow Time Ca Mg Glu Lac BEECF BEB SBC HCO3 control 1 O 4.6 1.1 84.5 0.2 5.5 5.8 29.4 29.8 control 5 O 4.5 1.3 85.5 0.3 10.7 10.3 33.6 34.8 control 6 O 4.8 1.3 83.0 0.2 8.1 8.1 31.5 32.5 control 9 0 4.9 1.1 81.5 0.6 3.0 3.6 27.2 27.5 DFM 4 O 4.6 1.1 90.5 0.4 9.8 9.7 32.8 33.9 DFM 8 O 4.8 1.2 81.5 0.5 6.8 7.1 30.5 30.6 DFM 10 O 5.0 1.2 93.5 0.4 8.5 8.5 32.0 32.5 DFM 12 O 5.0 1.2 88.5 0.2 4.3 4.8 28.3 28.6 control 1 2 4.9 1.3 90.5 0.3 4.2 4.7 28.4 28.4 control 5 2 4.7 1.3 95.0 0.3 5.8 6.1 29.5 29.9 control 6 2 4.5 1.2 84.5 0.3 2.3 3.0 26.8 26.9 control 9 2 4.9 1.1 85.0 0.3 4.0 4.6 28.1 28.4 DF M 4 2 4.3 1.0 80.5 0.2 9.0 9.0 32.3 32.8 DFM 8 2 4.6 1.1 82.5 0.3 6.9 7.1 30.5 30.8 DFM 10 2 4.6 1.1 87.0 0.4 7.6 7.8 31.1 31.4 DFM l2 2 4.8 1.1 87.5 0.3 3.1 3.7 27.3 27.4 control 1 4 4.9 1.5 98.5 0.4 4.6 5.0 28.6 28.9 control 5 4 4.6 1.4 96.0 1.3 4.2 4.7 28.1 28.7 control 6 4 4.9 1.4 97 .5 0.4 1.6 2.3 26.1 26.5 control 9 4 5.0 1.2 82.0 0.3 1.3 2.2 25.9 25.8 DFM 4 4 4.8 1.3 91.5 1.2 8.8 8.7 31.8 32.7 DFM 8 4 4.8 1.2 94.5 0.9 3.8 4.3 27.9 28.2 DF M 10 4 4.8 1.2 88.5 0.7 6.2 6.8 30.3 29.4 DF M 12 4 4.8 1.3 86.0 0.5 1.8 2.6 26.4 26.2 control 1 6 4.9 1.5 91.5 0.6 6.9 7.0 30.5 31.1 control 5 6 4.8 1.5 90.0 0.4 4.9 5.1 28.5 29.7 control 6 6 4.7 1.4 89.5 0.5 1.0 1.6 25.2 26.3 control 9 6 5.3 1.3 84.5 0.3 4.2 4.4 27.9 29.0 DFM 4 6 4.9 1.3 93.0 0.4 6.8 7.0 30.3 30.8 DF M 8 6 5.0 1.3 84.5 0.6 0.8 1.4 25.2 26.0 DFM lO 6 5.1 1.4 94.5 0.4 3.8 4.4 27.9 28.1 DF M 12 6 4.5 1.1 76.0 0.5 1.7 2.5 26.3 26.3 control 1 8 5.0 1.6 91.0 0.5 5.6 5.7 29.2 30.3 control 5 8 5.0 1.7 93.5 0.5 2.3 2.7 26.4 27.7 control 6 8 4.8 l .4 86.5 0.4 -2.3 -l .4 22.9 23.6 control 9 8 5.1 1.2 93.5 0.4 1.0 1.7 25.5 26.0 DFM 4 8 4.8 1.2 91 .O 0.5 4.8 5.2 28.4 29.3 DFM 8 8 5.1 1.4 79.5 0.6 -1.2 -O.l 23.8 23.7 DFM 10 8 4.8 1.2 86.5 0.5 1.3 2.1 25.8 25.8 DFM l2 8 5.0 1.4 100.0 1.3 1.6 2.3 26.0 26.2 254 Table 7.13. (Cont’d) Trt Cow Time Ca Mg Glu Lac BEECF BEB SBC HCO3 control 1 10 4.6 1.5 76.0 0.4 5.3 5.6 29.1 29.7 control 5 10 4.4 1.3 84.5 0.4 0.5 1.2 25.0 25.9 control 6 10 5.2 1.5 92.5 0.4 -3.8 -2.8 21.5 22.4 control 9 10 4.9 1.1 1 19.0 1.0 -2.9 -1.8 22.2 22.9 DF M 4 10 4.8 1.2 98.5 1.1 4.1 4.3 27.3 28.9 DFM 8 10 4.8 1.2 70.5 0.8 -2.1 -1.2 22.9 23.5 DFM 10 10 4.8 1.2 81.0 1.0 2.1 3.2 26.7 25.7 DF M 12 10 5.0 1.3 87.0 0.6 1.4 2.2 26.0 26.2 control 1 12 4.2 1.2 69.5 0.3 5.5 5.8 29.4 29.7 control 5 12 5.0 1.5 95.0 1.1 1.0 1.6 25.4 26.2 control 6 '12 4.9 1.4 84.0 0.6 -5.2 -4.0 20.7 21.0 control 9 12 4.8 1.0 98.0 1.1 -4.5 -3.2 21.2 21.3 DFM 4 1.2 4.8 1.2 104.0 1.0 1.1 1.8 25.4 25.9 DFM 12 12 4.9 1.2 96.5 0.5 -O.l 0.9 24.8 24.8 control 1 15 5.0 1.6 94.0 0.5 8.2 8.3 31.6 31.8 control 5 15 4.8 1.4 81.5 0.7 0.6 1.3 25.3 25.6 control 6 15 4.8 1.4 85 .5 0.7 -6.6 -5.1 19.8 19.5 control 9 15 5.0 1.1 124.5 1.5 -8.2 -6.5 18.5 18.1 DFM 4 15 4.8 1.1 117.0 1 .2 ~02 1.0 24.8 24.5 DF M 12 15 5.0 1.2 79.5 0.7 -1.6 -O.5 23.4 23.5 control 1 18 4.4 1.2 89.5 0.4 0.6 1.5 25.4 25.2 control 5 18 4.7 1.3 62.5 1.1 0.9 1.9 25.8 25.3 control 6 18 4.7 1.3 102.0 0.5 -5 .2 -3.7 21.1 20.3 control 9 18 5.1 1 1.2 195.0 3.3 -12.1 -1.0.0 16.3 14.9 DFM 4 18 4.8 1.1 109.5 0.8 4.8 5.2 28.8 28.9 DFM 8 18 3.9 0.8 50.5 1.8 -4.7 -3.1 21.5 20.3 DFM 10 18 3.8 0.8 83.5 3.8 -O.3 0.8 24.5 24.3 DFM 12 18 4.3 1.0 93.0 0.6 -1.5 -O.3 23.8 23.3 control 5 21 4.6 1.4 82.0 0.9 6.5 6.7 30.1 30.3 control 6 21 4.9 1.4 105.5 0.8 -O.7 0.2 24.0 24.5 control 9 21 4.9 l. .2 197.0 3.0 -9.2 -7.9 17.1 18.2 DFM 4 21 4.7 1.0 85.5 '1 .O 2.4 3.1 26.8 26.8 DFM 8 21 4.5 1.2 87.5 1.0 -3.2 -0.5 23.8 19.1 DFM 10 21 4.8 1.2 98.5 1.9 2.1 3.6 27.7 24.8 DFM 12 21 4.7 1.2 120.5 0.7 0.5 1.3 25.2 25.3 255 Lane Figure 7.1. 16S rDNA amplication of enriched bacteria using primers 8F and 139OR (Lanes 1 and 20, lkb ladder; 2 ~ 6, N6; 7 ~ 11, N2A2N2; 12 ~ 16, N2A4; 17, RK02; 18, RK03; 19, DH42; Data for Table 3.6). 256 Lane Rowl Row2 Row3 Row4 Row5 Row6 Row7 Row8 Row9 Clone# Rowl Row2 Row3 Row4 RowS Row6 Row7 Row8 Row9 Lanel Marker Marker Marker Marker Marker Marker Marker Marker Marker Lane2 18 47 74 104 149 199 241 286 311 Lane3 l 37 61 92 134 165 216 273 298 Lane4 19 49 75 110 153 200 243 288 314 LaneS 4 38 63 93 135 166 220 274 299 Lane6 20 50 76 1 1 1 154 201 244 290 315 Lane7 6 40 64 95 136 170 223 275 301 LaneS 25 54 78 125 157 204 248 291 316 Lane9 7 41 65 96 140 173 225 276 302 Lane 10 26 55 79 127 158 205 254 292 P1 Lane 1 1 8 42 67 97 141 179 228 281 303 Lane12 27 S8 80 129 blank 210 251 293 P2 Lane 13 9 44 70 100 142 182 230 282 305 Lanel4 28 59 81 130 161 211 252 294 P3 LanelS 10 45 71 101 143 183 233 284 306 Lane16 29 60 91 131 162 215 253 295 P4 Lane 1 7 15 46 73 103 144 184 234 285 308 Lane18 Marker Marker Marker Marker Marker Marker Marker Marker Marker Figure 7.2. Digestion of clones with Hhal. Data for Table 3.6. 257 Lane Rowl Row2 Row3 Row4 Row5 Row6 Row7 Row8 Row9 Clonefi Rowl Row2 Row3 Row4 RowS Row6 Row7 Row8 Row9 Lanel Markerl Markerl Markerl Markerl Markerl Markerl Markerl Markerl Markerl Lane2 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Lane3 18 47 74 104 149 199 241 286 31 1 Lane4 l 37 61 92 134 165 216 273 298 LaneS 19 49 75 1 10 153 200 243 288 314 Lane6 4 38 63 93 135 166 220 274 299 Lane7 20 50 76 1 1 l 154 201 244 290 315 Lane8 6 40 64 95 136 170 223 275 301 Lane9 25 54 78 125 157 204 248 291 316 Lane 1 0 7 41 65 96 140 173 225 276 302 Lanel 1 26 55 79 127 158 205 254 292 P1 Lane 1 2 8 42 67 97 141 179 228 281 303 Lane 1 3 27 58 80 129 blank 210 251 293 P2 Lanel4 9 44 70 100 142 182 230 282 305 Lane15 28 59 81 130 161 211 252 294 P3 Lanel6 10 45 71 101 143 183 233 284 306 Lane 1 7 29 60 91 131 162 215 253 295 P4 Lane 1 8 15 46 73 103 144 184 234 285 308 Lanel9 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Marker2 Lane20 Markerl Markerl Markerl Markerl Markerl Markerl Markerl Markerl Markerl Figure 7.3. Digestion of clones with HaeIII. Data for Table 3.6. 258 Lane Rowl Row2 Row3 Row4 RowS Row6 Row7 Row8 Row9 \OWQO‘MAWN— Clone# Rowl Row2 Row3 Row4 RowS Row6 Row7 Row8 Row9 Lanel Marker Marker Marker Marker Marker Marker Marker Marker Marker Lane2 18 47 74 104 149 199 241 286 31 1 Lane3 1 37 61 92 134 165 216 273 298 Lane4 19 49 75 1 10 153 200 243 288 314 Lane5 4 38 63 93 135 166 220 274 299 Lane6 20 50 76 1 1 1 154 201 244 290 3 15 Lane7 6 40 64 95 136 170 223 275 301 Lane8 25 54 78 125 157 204 248 291 316 Lane9 7 41 65 96 140 173 225 276 302 Lane 1 0 26 55 79 127 158 205 254 292 P1 Lanel 1 8 42 67 97 141 179 228 281 303 Lane 1 2 27 58 80 129 blank 210 251 293 P2 Lane 1 3 9 44 70 100 142 182 230 282 305 Lanel4 28 59 81 130 161 211 252 294 P3 Lane15 10 45 71 101 143 183 233 284 306 Lane 16 29 60 91 131 162 215 253 295 P4 Lane 1 7 15 46 73 103 144 184 234 285 308 Lanel8 Marker Marker Marker Marker Marker Marker Marker Marker Marker Figure 7.4. Digestion of clones with MpsI. Data for Table 3.6. 259 Figure 7.5. Partial sequence of 16S rDNA of clones from enrichment N6 (Data for Table 3.6.) > N6—U'13 ATACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGCAGGCCTGAACCAGCCAAGTA GCGTGAAGGATGACTGCCCTATGGGCTGTAAACTTCTTTTATATGGGAATAAAGTTTTCC ACGTGTGGAATTTTGTATGTACCATATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCC GCGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGT GGACAGTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATA(TGG CTGTCTTGAGTACAGTAGAGGTGGGCGGAATTCGTGGTGTAgCGGTGAAATGCTTAGATA TCACGAAgAACTCCGATTGCGAAGGCAGCTCACTGGACTGCAACTGAcACTGATGCTCGA AAGTGTGGGTATCAAACAGGATAGAtACCCTGGTAGTCCACACAGtA > N6-O75 TACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGCAGGCCTGAACCAGCCAAGTAG CGTGAAGGATGACTGCCCTATGGGTTGTAAACTTCTTTTATATGGGAATAAAGTTTTCCA (GTGTGGAATTTTGTATGTACCATATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCG CGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTG GACAGTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGC TGTCTTGAGTACAGTAgAGGTGGGCGGAATTCGTGGTGTAgCGgTGAAATGCTTAgATAT CACgAAgAACTCCgATTGCgAAGGCAGCTCACTGGACTGCAACTGACACTGATGCTC > N6-050 GCAGAACGGCTCTCTGTACGCGCCATTGTAACACGNTGTGTAGCCCCGGTACGTAAGGGC CGTGCTGATTTGACGTCATCCCCACCTTCCTCACACCTTACGGTGGCAGTGTCCGCAGAG TGCCCAGCTTAACCTGATGGCAACTACGGAGAGGGGTTGCGCTCGTTATGGCACTTAAGC CGACACCTCACGGCACGAGCTGACGACAACCATGCAGCACCTTCACAGATGCCCCGAAGG GCGTCACCATCTCTGGATCCTTCATCTGCAATTCAAGCCCGGGTAAGGTTCCTCGCGTAT CATCGAATTAAACCACATGTTCCTCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTT CACCGTTGCCGGCGTACTCCCCAGGTGAGATGCTTAATGCTTTCGCTTAGCCCCGGCCGC CAGGCGGCCAGAGCGGGCATCCATCGTTTACCGTGCGGACTACCAGGGTATCTAATCCTG TTCGATACCCGCACTTTCGAGCTTCAGCGTCGGTTGTGCTCCCGCAAGCTGCCTTCGCAA TCGGAGTTCTTCGTCATATCTAAGCATTTCACCGCTACACGACGAATTCCACCTGCGTTG CGCACACTCAAGACCTCCAGTTCGCGCTGCAATTCAgACGTTGAGCGTCTACATTTCACA ACACGCTTAAAGGCCGGCCTACgCTCCCTTTAAACCCAATAAATCCGGATACgCCtGACT TCCtATT > 146-nan GCAGAACGGCTCTCTGTACGCGCCATTGTAACACGNTGTGTAGCCCCGGTACGTAAGGGC CGTGCTGATTTGACGTCATCCCCACCTTCCTCACACCTTACGGTGGCAGTGTCCGCAGAG TGCCCAGCTTAACCTGATGGCAACTACGGAGAGGGGTTGCGCTCGTTATGGCACTTAAGC CGACACCTCACGGCACGAGCTGACGACAACCATGCAGCACCTTCACAGATGCCCCGAAGG GCGTCACCATCTCTGGATCCTTCATCTGCAATTCAAGCCCGGGTAAGGTTCCTCGCGTAT CATCGAATTAAACCACATGTTCCTCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTT CACCGTTGCCGGCGTACTCCCCAGGTGAGATGCTTAATGCTTTCGCTTAGCCCCGGCCGC CAGGCGGCCAGAGCGGGCATCCATCGTTTACCGTGCGGACTACCAGGGTATCTAATCCTG TTCGATACCCGCACTTTCGAGCTTCAGCGTCGGTTGTGCTCCCGCAAGCTGCCTTCGCAA TCGGAGTTCTTCGTCATATCTAAGCATTTCACCGCTACACGACGAATTCCACCTGCGTTG CGCACACTCAAGACCTCCAGTTCGCGCTGCAATTCAgACGTTGAGCGTCTACATTTCACA ACACGCTTAAAGGCCGGCCTACgCTCCCTTTAAACCCAATAAATCCGGATACgCCtGACT TCCtATT > N6—D‘11 ATTAGCTAGTTGGCGGGGCAACGGCCCACCAAGGCGACGATGTCTAGGGGTTCTGAGAGG AAGATCCCCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGAGG AATATTGGACAATGGAGGCAACTCTGATCCAGCCATGCCGCGTGAAGGACGAAGGCCCTA TGGGTCGTAAACTTCTTTTGTCAGGGAGCAATAAGGGCCACGCGTGGTCCGACGAGAGTA CCTGGCGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGA GCGTTATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGACGATTAAGTCAGCTGTGA AATGCCCGCGCTTAACGCGGGAAGTGCAGTTGATACTGGTTGTCTGGAGTGCCGTTGCCG T6 260 *1 musk,“ 4.".4 ‘9'.“ K (.‘I‘ij.'.‘».l' Figure 7.5. (cont’d) > 196-0‘12 GGCTAAGGGATAACCCGTAGAAATGCGGCCTAATACCTTATGATCTCCGAAGAAGACATC TGACTTGGAGTAAAGATTTATCGGCGAGGGATGGGGATGCGTCTGATTAGGCAGTCGGCG GGGTAACGGCCCACCGAGCCGACGATCAGTAGGGGTTCTGAGAGGAAGGTCCCCCACATT GGAACTGAGACACGGTCCAAACTCNTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGG GCGTGAGCCTGAACCAGCCAAGTAGCGTGCAGGAAGACGGCTCTATGGGTTGTAAACTGC TTTTGTATGGGGATAAAGTGCTCCACGTGTGGGGTATTGCAGGTACCATACGAATAAGGA CCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAAGGTCCAGGCGTTATCCGGATTT ATTGGGTTTAAAGGGAGCGTAGGCCGGCCTTTAAGCGTGTTGTGAAATGTAGACGCTCAA CGTCTGAATTGCAGCGCGAACTGGAGGTCTTGAGTGTGCGCAACGCAGGTGGAATTCGTC GTGTAGCGGTGAAATGCTTAGATATGACGAAGAACTCCGATTGCGAAGGCAGCTTGCGGG AGCACAACCGACGCTGAAGCTC > N6—Dlall GGCCTAATACCTTATGATCTCCGAAGAAGACATCTGACTTGGAGTAAAGATTTATCGGCG AGGGATGGGGATGCGTCTGATTAGGCAGTCGGCGGGGTAACGGCCCACCGAGCCGACGAT CAGTAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGACACGGTCCAAACTCCT ACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGTGAGCCTGAACCAGCCAAGTAGC GTGCAGGAAGACGGCTCTATGGGTTGTAAACTGCTTTTGTATGGGGATAAAGTGCTCCAC GTGTGGGGTATTGCAGGTACCATACGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCGC GGTAATACGGAAGGTCCAGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTA > N6-O‘l? CCGTAGAAATGCGGCCTAATACCTTATAATCTCCGAAGAAGACATCTGACTTGGAGTAAA GATTTATCGGTTATGGATGGGGATGCGTCTGATTAGATTGTTGGCGGGGCAACGGCCCAC CAAGTCTGCGATCAGTAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGACACG GTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGAGCCTGAAC CAGCCAAGTAGCGTGCAGGACGACGGCCCTATGGGTTGTAAACTGCTTTTGTATGGGAAT AAAGTGCTCCACGTGTGGAGTTTTGTAGGTACCATACGAATAAGGACCGGCTAATTCCGT GCCAGCAGCCGCGGTAATAC > N64161: CACCAAGGCGACGATCGGTAGGGGTTCTGAGAGGAAGGCCCCCCACAATGGAACTGAGAC ACGGTCCATACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGGAAGCCTG AACCAGCCATGCCGCGTGCAGGAATGAGGCCCTACGGGTCGTGAACTGCTTTTCGCGCGG AGCAATAAGGGGGGTTCGCCCCTCCGATGAGAGTACGCGCGGAATAAGCATCGGCTAACT CCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTATCCGGATTCATTGGGTATA AAGGGTGCGTAGGCGGC >N6flba ATTGGAAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTTGATTTGAAAGG CGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACG GCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGA GACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGT CTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAG AGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACG GCTAACTACGTGCCAGCAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATTACT GGGCGTAAAGAGTGCGTAGGGGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAACCG TGAACCGCCCTGCGAACTGTCTAGCTTGAGTACAGGAGAGGAAGGCGGAATTCCTAGTGT AGCGGTGAAATGCGTANATATTAGGAGGAACACCGGTGGCGAAGCGGCCTTCTGGACTGA AACTGACACTGAGGCACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCA CGC 261 Figure 7.5. (cont’d) > N6-Dlal CCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATACGTTCTTTT TGTCGCATGGCAGAGGGAAGAAAGGGAGGCTCTTCGGAGCTTTCGCTGAAGGAGGGGCTT GCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGTC TGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC AGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAACGATGAC GGCCTTCGGGTTGTAAAGTTCTGTTATACGGGACGAATGGCGTAGCGGTCAATACCCGTT ACGAGTGACGGTACCGTAAGAGAAAGCCATGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCGTCGT AAGTCGGTCTTAAAAGTGCGGGGCTTAACCCCGTGAGGGGACCGAAACTGCGATGCTAGA GTATCGGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGA ACACCAGTGGCGAAAGCGGCTTTCTGGACGACACTGACGCTGAGGCgC > N6-D7'l aaggagggGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGAT CAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCT ACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGC GTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATACGGGACGAATGGCGTAGCGG TCAATACCCGTTACGAGTGACGGTACCGTAAGAGAAAGCCACGGCTAACTACGTGCCAGC AGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGC AGGCGGCGTCGTAAGTCGGTCTTAAAAGTGCGGGGCTTAACCCCGTGAGGGGACCGAAAC TGCGATGCTAGAGTATCGGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAQ ATATTAGGAGGAACACCAGTGGCGAAAGCGGCTTTCTGGACQACAACTGACNCTGAGGC > N6-DS'I AACCTGCCCCTCACAGGGAGATAGCCGAGGGAAACTTCGAGTAATATCCCATGATGCCTC ATGATCACATGATCGAGAGGCCAAAGATTTATCGGTGAGGGATGGGCCCGCGTCCGATTA GCCAGTTGGCAGGGTAGCGGCCTACCAAAGCGATGATGCGTAGCCGGCCTGAGAGGGTGA ACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATT TTCGGCAATGGGGGGAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGAT TGTAAAGCTCTGTTGCAAGGGAAGAACGGCACATAGAGGATATGTGAGTGACGGTACCTT GCGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGT TATCCGGAATTATTGGGCGTAAAGGGTGCGTAGGCGGCCTTTTAAGTTTAAGGTGAAAGC GTGGGGCTTAACCCCATAAAGCCTTAGATACTGGGAGGCTAGAGTACTGGAGAGGAAAGT AGAATTCCATGTGTAGCGGTAAATGCGTAGATATATGGAGGAATACCGGTGGCGAAGGCG GCTTTCTAGACAGTAACTGACGCTGAGGCACgAAAGCGTGGGGAGCAAATAGGATTAGAT ACCCTAgTAGTCCAC9CTGTAACCGATGAATGCTAAQTATC > N64111:]- GTGGGCAACCTGCCCCTCACAGGGAGATAGCCGAGGGAAACTTCGAGTAATATCCCATGA TGCCTCATGATCACATGATCGAGAGGCCAAAGATTTATCGGTGAGGGATGGGCCCGCGTC CGATTAGCCAGTTGGCAGGGTAGCGGCCTACCAAAGCGACGATCGGTAGCCGGCCTGAGA GGGCGGACGGCCACATTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGG GGGATATTGCACAATGGAGGAAACTCTGATGCAGCAACGCCGCGTGAAGGAGGAAGGCCT TCGGGTCGTAAACTTCTGTCCTCAGGGAAGAACAAAATGACGGTACCTGAGGAGGAAGCC CCGGCTAACTACGTGCCAGCAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATT ACTGGGCGTAAAGAGTGCGTAGGTGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAA CCGTGAACCGCCCTGCGAACTGTCTAGCTTGAGTACAGGAGAGGAAGGCGGAATTCCTAG TGTAGCGGTG > N6-D7I. CGAGAGGCCAAAGATTTATCGGTGAGGGATGGGCCCGCGTCCGATTAGCCAGTTGGCAGG GTAGCGGCCTACCAAAGCGACGATCGGTAGCCGGCCTGAGAGGGCGGACGGCCACATTGG AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAG GAAACTCTGATGCAGCAACGCCGCGTGAAGGAGGAAGGCCTTCGGGTCGTAAACTTCTGT CCTCAGGGAAGAACAAAATGACGGTACCTGAGGAGGAAGCCCCGGCTAACTACGTGCCAG CAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATTACTGGGCGTAAAGAGTGCG TAGGTGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAACCGTGAACCGCCCTGCGAA CTGTCTAGCTTGAGTACAGGAGAGGAAGGCGGAATCCCTAGTGTAGCGGTGAAATGCGTA gATATTA 262 Figure 7.5. (cont’d) >N6«DSS AACCCTGCCCCTCACAGGGAGATAGCCGAGGGAAACTTCGAGTAATATCCCATGATGCCT CATGATCACATGATCGAGAGGCCAAAGATTTATCGGTGAGGGATGGGCCCGCGTCCGATT AGCCAGTTGGCAGGGTAGCGGCCTACCAAAGCGACGATCGGTAGCCGGCCTGAGAGGGCG GACGGCCACATTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGGAT ATTG(ACAATGGAGGAAACTCTGATGCAGCAACGCCGCGTGAAGGAGGAAGGCCTTCGGG TCGTAAACTTCTGTCCTCAGGGAAGAACAAAATGACGGTACCTGAGGAGGAAGCCCCGGC TAACTACGTGCCAGCAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATTACTGG GCGTAAAGAGTGCGTAGGTGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAACCGTG AACCGCCCTGCGAACTGTCTAGCTTGAGTACAGGAGAGGAAGGCGGAATTCCTAGTGTAG CGGTGAAATGCGTAGATATTAGGAGGAACACCGGTGGCGAAGGCGGCCTTCTGGACTGAA ACTGACACTGAGGCACGAAAGCGTGGGTAGCAAACAGGATTAGATACCTGGTAGTCACGC GTAACGATGAGCACTAGTGTCGGGCTCG > N6-110 GTGGGCAACCTGCCCCTCACAGGGAGATAGCCGAGGGAAACTTCGAGTAATATCCCATGA TGCCTCATGATCACATGATCGAGAGGCCAAAGATTTATCGGTGAGGGATGGGCCCGCGTC CGATTAGCCAGTTGGCAGGGTAGCGGCCTACCAAAGCGACGATCGGTAGCCGGCCTGAGA GGGCGGACGGCCACATTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGG GGGATATTGCACAATGGAGGAAACTCTGATGCAGCAACGCCGCGTGAAGGAGGAAGGCCT TCGGGTCGTAAACTTCTGTCCTCAGGGAAGAACAAAATGACGGTACCTGAGGAGGAAGCC CCGGCTAACTACGTGCCAGCAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATT ACTGGGCGTAAAGAGTGCGTAGGTGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAA CCGTGAACCGCCCTGCGAACTGTCTAGCTTGAGTACAGGAGAGGAAG > N640!) AAAGCGACGATCGGTAGCCGGCCTGAGAGGGCGGACGGCCACATTGGAACTGAGACACGG TCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGC AGCAACGCCGCGTGAAGGAGGAAGGCCTTCGGGTCGTAAACTTCTGTCCTCAGGGAAGAA (AAAATGACGGTACCTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAAC ACGTAGGGGGCGAGCGTTATCCGGAATTACTGGGCGTAAAGAGTGCGTAGGTGGCTAA > N6—O73 AGGGAAACTTCGAGTAATATCCCATGATGCCTCATGATCACATGATCGAGAGGCCAAAGA TTTATCGGTGAGGGATGGGCCCGCGTCCGATTAGCCAGTTGGCAGGGTAGCGGCCTACCA AAGCGACGATCGGTAGCCGGCCTGAGAGGGTGGACGGCCACATTGGAACTGAGACACGGT CCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCA GCAACGCCGCGTGAAGGAGGAAGGCCTTCGGGTCGTAAACTTCTGTCCTCAGGGAAGAAC AAAATGACGGTACCTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAACA CGTAGGGGGCGAGCGTTATCCGGAATTACTGGGCGTAAAGAGTGCGTAGGTGGCTAAGCA AGCGCGGGGTTTAATTTCACGGCCCAACCGTGAACCGCCCTGCGAACTGTCTAGCTTGAG TACAGGAGAGGAAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAG >N6053 ATACCGGATACGCTTAAGTTTTACCTCTTAAGGAAAGATGACCTCTATTTATAAGTTATC GTGCAGAGATGAGTCCGCGTCCCATTAGCTTGTTGGCGGGGTAACGGCCCACCAAGGCGA CGATGGGTAGCCGATTTAAGAGGATGATCGGCCACATTGGAACTGAAACACGGTCCAAAC TCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGAAAGCCTGACGCAGCGACG CC6CGTGAGGGATGAAGGTCTTCGGATCGTAAACCTCTGTCAGAAGGGAAAAATGTACaG TGCCCCAATCAACATTGTATTGATGGTACCTTCAGAGGAAGCACCGGCTAACTCCGTGCC AGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCG CGTAGGTTGTTTTGTAAGTCAGAGGTGTAATCCCACGGCTTAACCGTGGAACTGCCTTTG ATACTGCATAACTTGGATC(GGGAGAGGACAGCGGAATTCCAGGTGTAGGAGTGAAATCC GTAGATATCTGGAGACATCAGTGGCGAGCGGCTGTCTGGACCGGTATTGACGCTGAGCGC GAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCC 263 Figure 7.5. (cont’d) > N64371:] GCGTCCCATTAGCTTGTTGGCGGGGTAACGGCCCACCAAGGCGACGATGGGTAGCCGATT TAAGAGGATGATCGGCCACATTGGAACTGAAACACGGTCCAAACTCCTACGGGAGGCAGC AGTGGGGAATATTGCGCAATGGGCGAAAGCCTGACGCAGCGACGCCGCGTGAGGGATGAA GGTCTTCGGATCGTAAACCTCTGTCAGAAGGGAAAAATGTACAGTGCTCCAATCAACACT GTATTGATGGTACCTTCAGAGGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATA CGGAGGGTGCAAGCGTTAATCGGAATTACTGGGGCGTAAAGCGCGCGTAGGTTGTTTTGT AAGTCAGAGGTGTAATCCCACGGCTTAACCGTGGAACTGCCTTTGATACTGCATAACTTG GATCCGGGAGAGGACAGCGGAATTCCAGGTGTAGGAGTGAAATCCGTAGATATCTGGAAG AACATCAGTGGCGAAGGCGGCTGTCTGGACCGGTATTGACGCTGAGGCGCGAAAGCGTGG GTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAAC > N6-071 TCTTAAGGAAAGATGACCTCTATTTATAAGTTATCGTGCAGAGATGAGTCCGCGTCCCAT TAGCTTGTTGGCGGGGTAACGGCCCACCAAGGCGACGATGGGTAGCCGATTTAAGAGGAT GATCGGCCACATTGGAACTGAAACACGGTCCAAACTCCTACGGGAGGCAGCAGTGGGGAA TATTGCGCAATGGGCGAAAGCCTGACGCAGCGACGCCGCGTGAGGGATGAAGGTCTTCGG ATCGTAAACCTCTGTCAGAAGGGAAAAATGTACAGTGCCCCAATCAACACTGTATTGATG GTACCTTCAGGGGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTG CAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTTGTTTTGTAAGTCAGAGG TGTAATCCCACGGCTTGACCGTGGAACTGCCTTTGATACTGCATAACTTGGATCCGGGAG AGGACAGCGGAATTCCAGGTGTAGGANTGAAATCCGTAGATATCTGGAAGAACATCANTG GCGAAGGCGGCTGTCTGGACCGGTATTGACGCT > N6-D‘15 tgtaAGGACCGGGATAATGCCTGGAAACGGGTACTAAAACCGGATAGGCATAGATGGGGC ATCCCATTTATGTTAAAGGTGAGAGACACAAACAGATGGGCTTATGGCGCATTAGCCAGT TGGTGAGGTAACGGCCCACCAAAGCGATGATGCGTAGCCGGCCTGAGAGGGTGAACGGCC ACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCGGC AATGGGGGGAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATTGTAAA GCTCTGTTGCAAGGGAAGAACGGCACATAGAGGATATGTGAGTGACGGTACCTTGCGAGG AAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTTATCCG GAATTATTGGGCGTAAAGGGTGCGTAGGCGGCCTTTTAAGTTTAAGGTGAAAGCGTGGGG CTTAACCCCATAAAGCCTTAGATACTGGGAGGCTAGAGTACTGGAGAGGAAAGTAGAATT CCTGGTGTAGGGGTGAAATCTGTAGATATCAGGAAGAACACCGATGGC > N6-103 TACCGGATAGGCATAGATGGGGCATCCCATTTATGTTAAAGGTGAGAGACACAAACAGAT GGGCTTATGGCGCATTAGCCAGTTGGTGAGGTAACGGCCCACCAAAGCGATGATGCGTAG CCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGA GGCAGCAGTAGGGAATTTTCGGCAATGGGGGGAACCCTGACCGAGCAACGCCGCGTGAGT GAAGAAGGTTTTCGGATTGTAAAGCTCTGTTGCAAGGGAAGAGCGGCACATAGAGGATAT GTGAGTGACGGTACCTTGCGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGTGGCGAGCGTTATCCGGAATTATTGGGCGTAAAGGGTGCGTAGGCGGCCTTTT AAGTTTAAGGTGAAAGCGTGGGGCTTAACCCCATAAAGCCTTAGATACTGGGAGGCTAGA GTACTGGAGAGGAAAGTAGAATTCCATGTGTAGCGGTAAAATGCGTAQATATATGGAGGA ATACCGGTGGCGAAGGCGGCTTTCTAGACAGTAACTGACGCTGA 264 Figure 7.6. Partial sequence of 16S rDNA of clones from enrichment N2A2N2(Data for Table 3.6.) > N2A2N2-3‘1 3 TACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGCAGGCCTGAACCAGCCAAGTAG CGTGAAGGATGACTGCCCTATGGGTTGTAAACTTCTTTTATATGGGAATAAAGTTTTCCA CGTGTGGAATTTTGTATGTACCATATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCG CGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTG GACAGTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGC TGTCTTGAGTACAGTAQ > N2A2N2-2‘1 S TACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGCAGGCCTGAACCAGCCAAGTAG CGTGAAGGATGACTGCCCTATGGGTTGTAAACTTCTTTTATATGGGAATAAAGTTTTCCA CGTGTGGAATTTTGTATGTACCATATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCG CGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTG GACAGTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGGC TGTCTTGAGTACAG > N2A2N2-31] 1 TACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGTGAGCCTGAACCAGCCAAGTAG CGTGCAGGAAGACGGCTCTATGGGTTGTAAACTGCTTTTGTATGGGGATAAAGTGCTCCA CGTGTGGGGTATTGCAGGTACCATACGAATAAGGACCGGCTAATTCCGTGCCAGCAGCCG CGGTAATACGGAAGGTCCAGGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCC GGCCTTTAAGCGTGTTGTGAAATGTAGACGCTCAACGTCTGAATTGCAGCGCGAACTGGA GGTCTTGAGTGTGCGCAACGCAGGTGGAATTCGTCGTGTAGCGGTGA > N2A2N2-308 CCCGTAGAAATGCGGCCTAATACCTTATGATCTCCGAAGAAGACATCTGACTTGGAGTAA AGATTTATCGGCGAGGGATGGGGATGCGTCTGATTAGGCAGTCGGCGGGGTAACGGCCCA CCGAGCCGACGATCAGTAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGACAC GGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGTGAGCCTGAA CCAGCCAAGTAGCGTGCAGGAAGACGGCTCTATGGGTTGTAAACTGCTTTTGTATGGGGA TAAAGTGCTCCACGTGTGGGGTATTGCAGGTACCATGCGAATAAGGACCGGCTAATTCCG TGCCAGCAGCCGCGGTAATACGGAAGGTCCGGGCGTTATCCGGATTTATTGGGTTTAAAG GGAGCGTAGGCCGGCCTTTAAGCGTGTTGTGAAATGTAGACGCTCAACGTCTGAATTGCA GCGCGAACTGGAGGTCTTGAGTGTGCGCAACGCAGGTGGAATTCGTCGTGTAGCGGTGAA ATGCTTAGATATGACGA > N2A2N2-EOE CCTAATACCCCATATGAGGCCAGGCCACATGGCCGGGCCTTGAAAGTCAAGGCGGTGACG GATTGGCTCGCGTCCGATTAGCCAGTTGGCGGGGCAACGGCCCACCAAGGCGACGATCGG TAGGGGTTCTGAGAGGAAGGCCCCCCACAATGGAACTGAGACACGGCCCATACTCCTACG GGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGGAAGCCTGAACCAGCCATGCCGCGTG CAGGAATGAGGCCCTACGGGTCGTGAACTGCTTTTCGCGCGGAGCAATAAGGGGGGTTCG CCCCTCCGATGAGAGTACGCGCGGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGG TAATACGGAGGATGCGAGCGTTATCCGGATTCATTGGGTATAAAGGGTGCGTAGGCGGCC CCCCAAGTCAGCGGTGAAATGTCCGGGCCCAACCCGGAGGGTGCCGTTGATACTGGGGGG CTGGAGTACGGACGCCGGCGGAGGAATGAGTGGTGTAGCGGTGAAATGCATAGATATCAC TCAGA > N2A2N2-2‘1“! AAACAGGTGCTAATACCGTATAACAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTT TCGGGTGTCNCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCA CCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACAC GGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGAC CGAGCAAC6CCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAG AACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAA CTACGTGCCAGCAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATTACTGGGCG TAAAGAGTGCGTAGGGGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAACCGTGAAC CGCCCTGCGAACTGTCTAGCTTGAGTACAG 265 Figure 7.6. (cont’d) > N2A2N2-3'15 ACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATACGTTCTTT TTGTCGCATGGCAGAGGGAAGAAAGGGAGGCTCTTCGGAGCTTTCGCTGAAGGAGGGGCT TGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGT CTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG CAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAACGATGA CGGCCTTCGGGTTGTAAAGTTCTGTTATACGGGACGAATGGCGTAGCGGTCAATACCCGT TACGAGTGACGGTACCGTAAGAGAAAGCCATGGCTAACTACGTGCCAGCAGCCGCGGTAA TACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCGTCG TAAGTCGGTCTTAAA > N2A2N2-E "I 1 ACCTGCCCCTCACAGGGAGATAGCCGAGGGAAACTTCGAGTAATATCCCATGATGCCTCA TGATCACATGATCGAGAGGCCAAAGATTTATCGGTGAGGGATGGGCCCGCGTCCGATTAG CCAGTTGGCAGGGTAGCGGCCTACCAAAGCGATGATGCGTAGCCGGCCTGAGAGGGTGAA CGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTT TC66CAATGGGGGGAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATT GTAAAGCTCTGTTGCAAGGGAAGAACGGCACATAGAGGATATGTGAGTGACGGTACCTTG CGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTT ATCCGGAATTATTGGGCGTAAAGGGTGCGTAGGCGGCCTTTTAAGTTTAAGGTGAAAGCG TGGGGCTTAACCCCATAAAGCCTTAGATACTGGGAGGCTAGAGTACTGGAGAGGAAAGTA GAATTCCATGTGTAGCGGTAAAATGCGTAGATATATGGAGGAATACCGGTGGCGAAGGCG 6CTTTCTAGACAGTAACTGACGCTGAGGCACGAAAGCGTGGGGAGCAAATAGGATTAGAT ACCCTAGTAGTCCACGCTGTAACGATGAa > N2A2N2-3 El 3 AAAACCGGATAGGCATAGATGGGGCATCCCATTTATGTTAAAGGTGAGAGACACAAACAG ATGG6CTTATGGCGCATTAGCCAGTTGGTGAGGTAACGGCCCACCAAAGCGATGATGCGT AGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGG GAGGCAGCAGTAGGGAATTTTCGGCAATGGGGGGAACCCTGACCGAGCAACGCCGCGTGA GTGAAGAAGGTTTTCGGATTGTAAAGCTCTGTTGCAAGGGAAGAGCGGCACATAGAGGAT ATGTGAGTGACGGTACCTTGCGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA ATACGTAGGTGGCGAGCGTTATCCGGAATTATTGGGCGTAAAGGGTGCGTAGGCGGCCT > N2A2N2-E 6 5 AGGGAAACTTCGAGTAATATCCCATGATGCCTCATGATCACATGATCGAGAGGCCAAAGA TTTATCGGTGAGGGATGGGCCCGCGTCCGATTAGCCAGTTGGCAGGGTAGCGGCCTACCA AAGCGACGATCGGTAGCCGGCCTGAGAGGGCGGACGGCCACATTGGAACTGAGACACGGT CCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCA GCAACGCCGCGTGAAGGAGGAAGGCCTTCGGGTCGTAAACTTCTGTCCTCAGGGAAGAAC AAAATGACGGTACCTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAACA CGTAGGGGGCGAGCGTTATCCGGAATTACTGGGCGTAAAGAGTGCGTAGGTGGCTAAGCA AGCGCGGGGTTTAATTTCACGGCCCAACCGTGAACCGCCCTGCGAACTGTCTAGCTTGAG TACAGGAGAGGAAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAA CACCGGTGGCGAAGGCGGCCTTCTGGACTGAAACTGACACTGAGGCACGAAAGCGTGGGT AGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGCACTAGGTGTCGG GCTCGAGAGAGTTCGGTGCCGAAGCAACGCATTAAGTGCTCCGCCTGGGAGTACCCACGC AGTGTAAACTCAAQG 266 Figure 7.6. (cont’d) > N2A2N2-306 CGAGAGGCCAAAGATTTATCGGTGAGGGATGGGCCCGCGTCCGATTAGCCAGTTGGCAGG GTAGCGGCCTACCAAAGCGACGATCGGTAGCCGGCCTGAGAGGGTGGACGGCCACATTGG AACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAG GAAACTCTGATGCAGCAACGCCGCGTGAAGGAGGAAGGCCTTCGGGTCGTAAACTTCTGT CCTCAGGGAAGAACAAAATGACGGTACCTGAGGAGGAAGCCCCGGCTAACTACGTGCCAG CAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATTACTGGGCGTAAAGAGTGCG TAGGTGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAACCGTGAACCGCCCTGCGAA CTGTCTAGCTTGAGTACAGGAGAGGAAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTA GATATTAGGAGGAACACCGGTGGCGAAGGCGGCCTTCTGGACTGAAACTGACACTGAGGC ACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGATGAG CACTAGGTGTCGGGCTCGA > N2A2N2-2‘13 NATGGGCCCGCGTCCGATTAGCCAGTTGGCAGGGTAGCGGCCTACCAAAGCGACGATCGG TAGCCGGCCTGAGAGGGCGGACGGCCACATTGGAACTGAGACACGGTCCAGACTCCTACG GGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCAGCAACGCCGCGTG AAGGAGGAAGGCCTTCGGGTCGTAAACTTCTGTCCTCAGGGAAGAACAAAATGACGGTAC CTGAGGAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAACACGTAGGGGGCGAG CGTTATCCGGAATTACTGGGCGTAAAGAGTGCGTAGGTGGCTAA > N2A2N2-315 tggAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATG GAGGAAACTCTGATGCAGCAACGCCGCGTGAAGGAGGAAGGCCTTCGGGTCGTAAACTTC TGTCCTCAGGGAA6AACAAAATGACGGTACCTGAGGAGGAAGCCCCGGCTAACTACGTGC CAGCAGCCGCGGTAACACGTAGGGGGCGAGCGTTATCCGGAATTACTGGGCGTAAAGAGT GCGTAGGTGGCTAAGCAAGCGCGGGGTTTAATTTCACGGCCCAACCGTGAACCGCCCTGC GAACTGTCTAGCTTGAGTACAGGAGAGGAAGGCGGAATCCCTAGTGTAGCGGTGAAATGC GTAGATATTAGGAGGAACACCGGTGGCGAAGGCGGCCTTCTGGACTGAAACTGACACTGA GGCACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGA TGAGCACTAGGTGTCGGGCTC > N2A2N2-301: CAATCGGGAATAACGACTGGAAACGGTCGCTAATACCGGATACGCTTAAGTTTTACCTCT TAAGGAAAGATGACCTCTATTTATAAGTTATCGTGCAGAGATGAGTCCGCGTCCCATTAG CTTGTTGGCGGGGTAACGGCCCACCAAGGCGACGATGGGTAGCCGATTTAAGAGGATGAT CGGCCACATTGGAACTGAAACACGGTCCAAACTCCTACGGGAGGCAGCAGTGGGGAATAT TGCGCAATGGGCGAAAGCCTGACGCAGCGAC6CCGCGTGAGGGATGAAGGTCTTCGGATC GTAAACCTCTGTCAGAAGGGAAAAATGTACAGTGCCCCAATCAACACTGTATTGATGGTA CCTTCAGGGGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAA GCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTTGTTTTGTAAGTCAGAGGTGT AATCCCACGGCTTGACCGTGGAACTGCCTTTGATACTGCATAACTTGGATC > N2A2N2-EBB CAAGGACTTGTCC6AAAGAGAGGGACACCTTCGGGAAACCGGAGCTAATACCTCATAAGC CGGAAGGTGAAAAGCAGAGATGCGCTTTTGGAGAGACTTGTGTCCTATCAGGCAGTTGGT GAGGTGAAAGCTCACCAAACCGAAGACGGGTAGCCGGACTGAGAGGTCGACCGGCCACAT TGGAACTGAGAGACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGGCAATG GGCGGAAGCCTGACCCAGCGACGCCGCGTGAGGGAAGACAGCCTTCGGGTTGTAAACCTC TGTTGCAGGGGAAGAAGGAAGTGACGGTACCCTGCGAGGAAGCTCCGGCTAACTACGTGC CAGCAGCCGCGGTAATACGTAGGGAGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGC GCGTAGGCGGCGCTTCAAGTCGTCTGTCAAATGGAAGGGCTTAACCCTTTTTCGCAGACG AAACTGGAGAGCTTGAGAAGCAGAGAGGCAAACAGAATTCCTGGTGTAGCGGTGAAATGC GTAGATATCAGGAAGAATACCAGTGGCGAAGGCGGTTTGCTGGCTGCATACTGACGCTGA AGCGCGAAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCAgTaAACGA TGTATGCTG 267 Figure 7.6. (cont’d) > N2A2N2-B71: gAAACCGGAGCTAATACCTCATAAGCCGGAAGGTGAAAAGCAGAGATGCGCTTTTGGAGA GACTTGTGTCCTATCAGGCAGTTGGTGAGGTGAAAGCTCACCAAACCGAAGACGGGTAGC CGGACTGAGAGGTCGACCGGCCACATTGGAACTGAGAGACGGTCCAGACTCCTACGGGAG GCAGCAGTGGGGAATATTGGGCAATGGGCGGAAGCCTGACCCAGCGACGCCGCGTGAGGG AAGACAGCCTTCGGGTTGTAAACCTCTGTTGCAGGGGAAGAAGGAAGTGACGGTACCCTG CGAGGAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGAGCGAGCGTT GTCCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCGCTTCAAGTCGTCTGTCAAATGG AAGGGCTTAACCCTTTTTCGCAGACGAAACTGGAGAGCTTGAGAAGCAGAGAGGCAAACA GAATTCCTGGTGTAGCGGTGAAATGCGTAGATATCAGGAAGAATACCAGTGGCGAAGGCG GTTTGCTGGCTGCATACTGACGCTGAAGCGCGA > N2A2N2-E 7 S GAAACCGGAGCTAATACCTCATAAGCCGGAAGGTGAAAAGCAGAGATGCGCTTTTGGAGA GACTTGTGTCCTATCAGGCAGTTGGTGAGGTGAAAGCTCACCAAACCGAAGACGGGTAGC CGGACTGAGAGGTCGACCGGCCACATTGGAACTGAGAGACGGTCCAGACTCCTACGGGAG GCAGCAGTGGGGAATATTGGGCAATGGGCGGAAGCCTGACCCAGCGACGCCGCGTGAGGG AAGACAGCCTTCGGGTTGTAAACCTCTGTTGCAGGGGAAGAAGGAAGTGACGGTACCCTG CGAGGAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGAGCGAGCGTT GTCCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCGCTTCAAGTCGTCTGTCAAATGG AAGGGCTTAACCCTTTTTCGCAGACGAAACTGGAGAGCTTGAGAAGCAGAGAGGCAAACA GAATTCCTGGTGTAGCGGTGAAATGCGTANATATCAGGAANAATACCAGTGGCGAAGGCG GTTTGCTGGCTGCATACTGACNCTGAANCGCGAAAGCCAGGGGANCGAACGGGATTAGAT ACCCCGGTAGTCCTGGCA > N2A2N2—B 7 3 ggttgggGACAACATTCCGAAAGGGATGCTAATACCGAATGTGCTCACACTTCCGCATGG AGGTGTGAGGAAAGATGGCCTCTACTTGTAAGCTATCGCCAGAAGATGGGCCTGCGTCTG ATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGATGATCAGTAGCCGGTCTGAGAGG ATGAACGGCCGCATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGG AATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTC GGATTGTAAAACTCTGTTGTCAGGGACGAATGTACTGATTTATAATACACTTCGGTATTG ACGGTACCTGACGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGG TGGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCGCTTCAAGTCGT CTGT > N2A2N2-E 6 1 agaCGGAGCGGGGGCGGCCCTGTTCCGGGAAAGCGCCTGCGGGCGCGCCGGGGGACGGGT CTGCGTCNCATCAGCTAGACGGCGGGGTGAAGGCCCACCGTGGCGACGACGGGTAGCCGG CCTGAGAGGGTGGACGGCCACATTGGGACTGGGAACGGCCCAGACTCCTACGGGAGGCAG CAGCTAAGGATCTTCCGCAATGGGCGAAAGCCTGACGGAGCGACGCCGCGTGGACGATGG AGGCCGGAAGGTTGTGAAGTCCTTTTCTCGGGGGGGAACAACCGTGCCAGGGAATGGGCG CGGGGTGACGCGGACCGAGGAATAAGCCCCGGCAAACTACGTGCCAGCAGCCGCGGTAAC ACGTAGGGGGCGAGCGTTGTTCGGATTCATTGGGCGTAAAGGGCGCGCGGGCGGCCGGGC GAGCCTGCCGTGAAATCCCCGTGCTCAACACG 268 Figure 7.7. Partial sequence of 16S rDNA of clones from enrichment N2A4 (Data for Table 3.6.) > N2A4-2011 ACTTGGAGTAAAGATTTATCGGCGAGGGATGGGGATGCGTCTGATTAGGCAGTCGGCGGG GTAACGGCCCACCGAGCCGACGATCAGTAGGGGTTCTGAGAGGAAGGTCCCCCACATTGG AACTGAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGC GTGAGCCTGAACCAGCCAAGTAGCGTGCAGGAAGACGGCTCTATGGGTTGTAAACTGCTT TTGTATGGGGATAAAGTGCTCCACGTGTGGGGTATTGTAGGTACCATACGAATAAGGACC GGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGTTATCCGGATTTAT TGGGTTTAAAGGGAGCGTAGGTGGACAGTTAAGTCAGTTGTGAAAGTTTGCGGCTCAACC GTAAAATTGCAGTTGATACTGGCTGTCTTGAGTACAGTAGAGGTGGGCGGAATTCGTGGT GTAGCGGTGAAATGCTTAGATATCACGAANAACTCCGATTGCGAAGGCAGCTCACTGGAC TGCAACTGACACTGATGCTCGAAAGTGTGGGTATCAAACAGGATTAGATACCCTGGTAGT CCACACAGTA > N2A4-B 1.0 TCCTGTCACCAGGTAGCTGCCTTCTGTACCCCCCATTGTAACACGTGTGTAGCCCCGGAC GTAAGGGCCGTGCTGATTTGACGTCATCCCCACCTTCCTCACATCTTACGACGGCAGTCT CTCTAGAGTCCTCAGCATGACCTGTTAGTAACTAAAGATAAGGGTTGCGCTCGTTATGGC ACTTAAGCCGACACCTCACGGCACGAGCTGACGACAACCATGCAGCACCTTCACATTTGT CTTACGACTATACTGTTTCCAATATATTCAAATGCAATTTAAGCCCGGGTAAGGTTCCTC GCGTATCATCGAATTAAACCACATGTTCCTCCGCTTGTGCGGGCCCCCGTCAATTCCTTT GAGTTTCACCGTTGCCGGCGTACTCCCCAGGTGGAATACTTAATGCTTTCGCTTGGCCGC TTACTGTATATCGCAAACAGCGAGTATTCATCGTTTACTGTGTGGACTACCAGGGTATCT AATCCTGTTTGATACCCACACTTTCGAGCATCAGTGTCAGTTGCAGTCCAGTGAGCTGCC TTCGCAATCGGAGTTCTTCGTGATATCTAAGCATTTCACCGCTACACCACGAATTCCGCC CACCTCTACTGTACTCAAGACAGCCAGTATCAACTGCAATTTTACGGTTGAGCCGCAAAC TTTCACAACTGACTTAACTGT > N2A4-r".| 15 AGTTTGCTTGCAAACTGGAGATGGCGACCGGCGCACGGGTGAGTAACACGTATCCAACCT GCCGATAACTCGGGGATAGCCTTTCGAAAGAAAGATTAATACCCGATGGTATAATTAGAC CGCATGGTCTTGTTATTAAAGAATTTCGGTTATCGACGGGGATGCGTTCCATTAGGCAGT TGGTGAGGTAACGGCTCACCAAACCTTCGATGGATAGGGGTTCTGAGAGGAAGGTCCCCC ACATTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCAC AATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAA GTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGC AGAAGAAGTACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGTT ATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTGGACAGTTAAGTCAGTTGTGAAAGTT TGCGGCTCAACCGTAAAATTGCAGTTGATACTGGCTGTCTTGAGTACAGTAGAGGTGGGC GGAATTCGCGGTGTAGCGGTGAAATGCTTA > N2A4-EDS CGTTCGACCGGCGCACGGGATGAGTAACGCGGTATCCAACCATGCCCCTGGCTAAGGGAT AACCCGCTAGAAATGCGGCCTAATACCCTATGATCTCCGAAGAAGACATCTGACTTGGAG TAAAGATTTATCGGCGAGGGATGGGGATGCGTCTGATTAGGCAGTCGGCGGGGTAACGGC CCACCGAGCCGACGATCAGTAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGA CACGGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGTGAGCCT GAACCAGCCAAGTAGCGTGCAGGAAGACGGCTCTATGGGTTGTAAACTGCTTTTGTATGG GGATAAAGTGCTCCACGTGTGGGGTATTGCAGGTACCATACGAATAAGGACCGGCTAATT CCGTGCCAGCAGCCGCGGTAATACGGAAGGTCCA66CGTTATCCGGATTTATTGGGTTTA AAGGGAGCGTAGGCCGGCCTTTAAGCGTGTTGTGAAATGTAGACGCTCAACGTCTGAATT GCAGCGCGAACTGGAGGTCTTGAGTGTGCGCAACGCAGGTGGAATTCGTCGTGTAGCGGT GAAATGCTTAGATATGACGAAGAACTCCGATTGCGAAGGCAGCTTGCGGGAGCACAACTG ACGCTGAAGCTCGAAAGTGCGGGTATCGAACAGGATTAGATACCCTGGTAGTCCGCACGG TAAACGATGGATGCCCGCTCTGGCCGCNTGGCGGCCGGGGCTAANCNAAGCATNAGCATC CCCCTGGGGAGTACCCCGGCAACGGTGAACTCAAG 269 Figure 7.7. (cont’d) > N2A4-EDD GGGGTAGCAATACCCTGGCGGCGACCGGCGTAAAGGGTGCGTAACGCGTGAGCGACATGC CCGTCACAGGGGGATAACCGGCGGAAACGCCGCCTAATACCCCATATGAGGCCAGGCCAC ATGGCCGGGCCTTGAAAGTCAAGGCGGTGACGGATTGGCTCGCGTCCGATTAGCCAGTTG GCGGGGCAACGGCCCACCAAGGCGACGATCGGTAGGGGTTCTGAGAGGAAGGCCCCCCAC AATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAA TGGGCGGAAGCCTGAACCAGCCATGCCGCGTGCAGGAATGAGGCCCTACGGGTCGTGAAC TGCTTTTCGCGCGGAGCAATAAGGGGGGTTCGTCCCTCCGATGAGAGTACGCGCGGAATA AGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTATCCGG ATTCATTGGGTATAAAGGGTGCGTAGGCGGCCCCCCAAGTCAGCGGTGAAATGTCCGGGC CCAACCCGGAGGGTGCCGTTGATACTGTGGGGCTGGAGTACGGACGCCGGCGGAGGAATG AGTGGTGTAGCGGTGAAATGCATAGATATCACCCAGAACACCGATTGCGAAGGCATCCGC CGAGGCCGTTACTGACGCTGAgGCACGAAAGCGTGGGATAGAACAGGATTAGATACCTGG TATTCCACGCCGTAAACGATGATGACTAACC > N2A4-Bl] 1 GGGGTAGCAATACCCTGGCGGCGACCGGCGAAAGGGTGCGTAACGCGTGAGCGACATGCC CGTCACAGGGGGATAACCGGCGGAAACGCCGCCTAATACCCCATATGAGGCCAGGCCACA TGGCTGGGTCTTGAAAGTCAAGGCGGTGAC66ATTGGCTCGCGTCCGATTAGCCAGTTGG CGGGGCAACGGCCCACCAAGGCGACGATCGGTAGGGGTTCTGAGAGGAAGGCCCCC(ACA ATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAAT GGGCGGAAGCCTGAACCAGCCATGCCGCGTGCAGGAATGAGGCCCTACGGGTCGTGAACT GCTTTTCGCGCGGAGCAATAAGGGGGGTTCGCCCCTCCGATGAGAGTACGCGCGGAATAA GCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTATCCGGA TTCATTGGGTATAAAGGGTGCGTAGGCGGCCCCCCAAGTCAGCGGTGAAATGTCCGGGCC CAACCCGGAGGGTGCCGTTGATACTGGGGGGCTGGAGTACGGACGCCGGCGGAGGAATGA GTGGTGTAGCGGTGAAATGCATAGATATCACTCAGAACACCGATTGCGAAGGCATCCGCC GAGGCCGTTACTGACGCTGAGGCACGAAAGCGTGGGGATAGAACAGGATTAGATACCCTG GTATTCCACGCCGTAAACGATGATGACTAACCGCC > N2A4-352 ATGCCCGTCACAGGGGGATAACCGGCGGAAACGCCGCCTAATACCCCATATGAGGCCAGG CCACATGGCCGGGCCTTGAAAGTCAAGGCGGTGACGGATTGGCTCGCGTCCGATTAGCCA GTTGGCGGGGCAACGGCCCACCAAGGCGACGATCGGTAGGGGTTCTGAGAGGAAGGCCCC CCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTGAGGAATATTGG TCAATGGGCGGAAGCCTGAACCAGCCATGCCGCGTGCAGGAATGAGGCCCTACGGGTCGT GAACTGCTTTTCGCGCGGAGCAATAAGGGGGGTTCGCCCCTCCGATGAGAGTACGCGCGG AATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTAT CCGGATTCATTGGGTATAAAGGGTGCGTAGGCGGCCCCCCAAGTCAGCGGTGAAATGTCC GGGCCCAACCCGGAGGGTGCCGTTGATACT > N2A4-2511 CCCGTCACAGGGGGATAACCGGCGGAAACGCCGCCTAATACCCCATATGAGGCCAGGCCA CATGGCCGGGCCTTGAAAGTCAAGGCGGTGACGGATTGGCTCGCGTCCGATTAGCCAGTT GGCGGGGCAACGGCCCACCAAGGCGACGATCGGTAGGGGTTCTGAGAGGAAGGCCCCCCA CAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCA ATGGGCGGAAGCCTGAACCAGCCATGCCGCGTGCAGGAATGAGGCCCTACGGGTCGTGAA CTGCTTTTCGCGCGGAGCAATAAGGGGGGTTCGCCCCTCCGATGAGAGTACGCGCGGAAT AAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTATCCG GATTCATTGGGTATAAAGGGTGCGTAGGCGGCCCCCCAAGTCGGCGGTGAAATGTCCGGG CCCAACCCGGAGGGTGCCGTTGATACTGCGGGGCTGGAGTACGGACGCCGGCGGAGGAAT GAGTGGTGTAGCGGTGAAATGCATAGATATCACTCAGAACACCGATTGCGAAGGCATCCG CCGAGGCCGTTACTGACGCTGAGGCACGAAAAGCGTGGGGATAGAACAGGATTAGATACC CTGGTATTCCACGCCGTAAACGATGATGACTAACCGCCGGGGGGTAGACTTCCGGCAGCC AAGCGAAAGCGATAAGTCATCCCCTTG 270 Figure 7.7. (cont’d) > N2A4-E '1 '1 CTTGCTAATACCGAATACGTTCTTTTTGTCGCATGGCAGAGGGAAGAAAGGGAGGCTCTT CGGAGCTTTCGNTGAAGGAGGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCC ACCAAGGCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGATA TGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGA CGGAGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATACGGGAC GAATGGCGTAGCGGTCAATACCCGTTACGAGTGACGGTACCGTAAGAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGA CGTAAAGGGCGCGCAGGCGGCGTCGTAAGTCGGTCTTAAAAGTGCGGGGCTTAACCCCGT GAGGGGACCGAAACTGCGATGCTAGAGTATCGGAGAGGAAAGCGGAATTCCTAGTGTAGC GGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAAGCGGCTTTCTGGACGACAA CTGACGCTGAGGCGCGAAAGCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGTCCTGG CCGTAAACGATGGATACTAGGTGTAGGAGTATCGACCC > N2A4-E E S ATAAGCCGGAAGGTGAAAAGCAGAGATGCGCT TTTGGAGAGACTTGTGTCCTATCAGGCAGTTGGTGAGGTGAAAGCTCACCAAACCGAAGA CGGGTAGCCGGACTGAGAGGTCGACCGGCCACATTGGAACTGAGAGACGGTCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGGCAATGGGCGGAAGCCTGACCCAGCGACGCCG CGTGAGGGAAGACAGCCTTCGGGTTGTAAACCTCTGTTGCAGGGGAAGAAGGAAGTGACG GTACCCTGCGAGGAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGAG CGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCGCTTCAAGTCGTCTG TCAAATGGAAGGGCTTAACCCTTTTTCGCAGACGAAACTGGAGAGCTTGAGAAGCAGAGA GGCAAACAGAATTCCTGGTGTAGCGGTGAAATGCGTAGATATCAGGAAGAATACCAGTGG CGAAGGCGGTTTGCTGGCTGCATACTGACGCTGAAGCGCGAAAGCCAGGGGAGCGAACGG GATTAGATACCCCGGTAGTCCTGGCAGTAAACGATGTATGCTGGGTGTGAGACTAGCGAT AGTTTCGTGCCgAANTTAACGCGATAAGCATACCGCCTG > N2A4-BIB ACTCCTACGGGAGGCAGCAGTGGGGAATATTGGGCAATGGGCGGAAGCCTGACCCAGCGA CGCCGCGTGAGGGAAGACAGCCTTCGGGTTGTAAACCTCTGTTGCAGGGGAAGAAGGAAG TGACGGTACCCTGCGAGGAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA GGGAGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCGCTTCAAGTC GTCTGTCAAATGGAAGGGCTTAACCCTTTTTCGCAGACGAAACTGGAGAGCTTGAGAAGC AGAGAGGCAAACAGAATTCCTGGTGTAGCGGTGAAATGCGTAGATATCAGGAAGAATACC AGTGGCGAAGGCGGTTTGCTGGCTGCATACTGACGCTGAAGCGCGAAAGCCAGGGGAGCG AACGGGATTAGATACCCCGGTAGTCCTGGCAGTAAACGATGTATGCTGGGTGTGA > N2A4-EEO AGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGGCAATGGGCGGAAGCCTGACCCAGC GACGCCGCGTGAGGGAAGACAGCCTTCGGGTTGTAAACCTCTGTTGCAGGGGAAGAAGGA AGTGACGGTACCCTGCGAGGAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACG TAGGGAGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCGCTTCAAG TCGTCTGTCAAATGGAAGGGCTTAACCCTTTTTCGCAGACGAAACTGGAGAGCTTGAGAA GCAGAGAGGCAAACAGAATTCCTGGTGTAGCGGTGAAATGCGTAGATATCAGGAAGAATA CCAGTGGCGAAGGCGGTTTGCTGGCTGCATACTGACGCTGAAGCGCGAAAGCCAGGGGAG CGAACGGGATTAGATACCCCGGTAGTCCTGGCAGTAAACGATGTATGCTGGGTGTGAGAC TAGCGATAGTTTCGTGC 271 Figure 7.7. (cont’d) > N2A4-EEG ~ GAGCTAATACCTCATAAGCCGGAAGGTGAAAAGCAGAGATGCGCTTTTGGAGAGACTTGT GTCCTATCAGGCAGTTGGTGAGGTGAAAGCTCACCAAACCGAAGACGGGTAGCCGGACTG AGAGGTCGACCGGCCACATTGGAACTGAGAGACGGTCCAGACTCCTACGGGAGGCAGCAG TGGGGAATATTGGTCAATGGGCGGAAGCCTGAACCAGCCATGCCGCGTGCAGGAATGAGG CCCTACGGGTCGTGAACTGCTTTTCGCGCGGAGCAATAAGGGGGGTTCGCCCCTCCGATG AGAGTACGCGCGGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGG ATGCGAGCGTTATCCGGATTCATTGGGTATAAAGGGTGCGTAGGCGGCCCCCCAAGTCAG CGGTGAAATGTCCGGGCCCAACCCGGAGGGTGCCGTTGATACTGCGGGGCTGGAGTACGG ACGCCGGCGGAGGAATGAGTGGTGTAGCGGTGAAATGCATAGATATCACTCAGAACACCG ATTGCGAAGGCATCCGCCGAGGCCGTTACTGACGCTGAGGCACGAAAGTGTGGGGATAGA ACAGGATTAGATACCCCTGGTATTCCACGCCNTAAACGATGATGACTA > N2A4-E ll 1. TAATACCCCATATGAGGCCAGGCCACATGGCTGGGTCTTGAAAGTCAAGGCGGTGACGGA TTGGCTCGCGTCCGATTAGCCAGTTGGCGGGGCAACGGCCCACCAAGGCGACGATCGGTA GGGGTTCTGAGAGGAAGGCCCCCCACAATGGAACTGAGACACGGTCCATACTCCTACGGG AGGCAGCAGTGAGGAATATTGGTCAATGGGCGGAAGCCTGAACCAGCCATGCCGCGTGCA GGAATGAGGCCCTACGGGTCGTGAACTGCTTTTCGCGCGGAGCAATAAGGGGGGTTCGCC CCTCCGATGAGAGTACGCGCGGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTA ATACGTAGGGAGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCGCGTAGGCGGCGCT TCAAGTCGTCTGTCAAATGGAAGGGCTTAACCCTTTTTCGCAGACGAAACTGGAGAGCTT GAGAAGCAGAGAGGCAAACAGAATTCCTGGTGTAGCGGTGAAATGCGTAGATATCAGGAA GAATACCAGTGGCGAAGGCGGTTTGCTGGCTGCATACTGACGCTGAAGCGCGAAAGCCAG GGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCAGTAAACGATGTATGCTGGGTGT GA 272 26 £5me 8m San— m.m.0< 0H0 OOH 00,—.