-u- .‘-_— m. A ”1' wu— EFFECTS OF DEFAU NATION AND FAUNATION 0N NlTROGEN. METABOLISM OF RUMINANTS Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSlTY JAMES. ROBERT MALES 1:969 I I1 ESlS ~v-1 .6.- F5 in aim .o . ‘1. 2; Pa 5% . . ABSTRACT EFFECTS OF DEFAUNATION AND EAUNATION ON NITROGEN METABOLISM OF RUMINANTS By JAMES ROBERT MALES Twelve, two year old Cheviot, wethers were incorporated into four treatment combinations, which consisted of: urea infusion plus protozoa, water infusion plus protozoa, urea infusion with no protozoa and water infusion with no proto- zoa. Defaunation was accomplished using dioctyl sodium sulfosuccinate. An attempt was made to raise rumen ammonia-N levels of defaunated sheep u mg. percent above that of the protozoa water infused control animals by infusion of urea at airateof .123% of the ration fed per day. Metabolism trials of three weeks duration were initiated to study the effects of the different treatment combinations. Defaunation resulted in higher rumen dry matter percen- tages when compared to the:faunated sheep. This difference was significant (P<.Ol) at T0 sampling time. Faunated animals had rumen pH values of 6.19 and 5.66 compared to 5.80 and 5.33 for defaunated sheep at the TO and T2 sampling times, respectively (P<.lO). The rumen pH values for mean urea infusion were significantly (P<£10) higher than the values observed when water was infused. There were no differences observed in any of the nitrogen metabolism parameters for James R. Males defaunated sheep receiving either the urea infusion or the water infusion. Urea infusion greatly reduced nitrogen util— ization (33.86% vs. 53.28%) and nitrogen balance (3.06” vs. 5.200) in faunated animals and raised fecal nitrogen as a per- cent of nitrogen intake (31.31% vs. 27.61%). It was concluded‘ that with a high protein ration, the excess nitrogen supplied in the urea infusion could not be adequately used by the microbial population present in faunated sheep. Sheep with protozoa present in their rumen ecology had a higher level of rumen ammonia-N (11.6” mg./100 ml) than was observed in Sheep without protozoa (7.07 mg./100 ml). Urea infusion raised rumen ammonia-N levels above the level that was observed for the water control animals; however, this difference between water and urea infusion was more pronounced for faunated sheep (lu.58 mg.% vs. 8.71 mg.%) than for defaunated animals (7.6” mg.% vs. 6.00 mg.%). An in zi££2_fermentation was designed to further study this effect. Ammonia levels in vi£§2_were similar for all treat- ments including urea or for all treatments including only water. It is hypothesised that the greater bacterial con- centrations in defaunated ruminants have a more rapid util- ization of ammonia-N and therefore levels are observed to be lower in the protozoa free animals. Pooled data for ammonia production and acetate:propion- ate ratios, acetate:butyrate ratios, and propionatezbutyrate ratios were fitted to linear regression equations. From these regressions it was concluded that at low levels of rumen ammonia-N and molar percent propionate was high James R. Males and as rumen ammonia-N levels increased there was a shift from propionate to acetate and butyrate. This trend was observed for three different rations of varying protein levels and also for urea and water infusion for both fauna- ted and defaunated ruminants. EFFECTS OF DEFAUNATION AND FAUNATION ON NITROGEN METABOLISM OF RUMINANTS By James Robert Males A THESIS Submitted To Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1969 ACKNOWLEDGEMENTS The author is deeply grateful to Dr. Harlan D. Ritchie for his advice in planning the course of study and to Dr. D. Barrie Purser for his patience and counsel in completing the research procedures and in interpreting the experimental re- sults. The writer is further indebted to the other members of the graduate committee, Dr. Terrance R. Greathouse and Dr. Esther M. Smith for their sound advice and willing partici- pation in the authors' graduate program. The auther also wished to thank Dr. William T. Magee for his help in the statistical analysis of this project. Sincere gratitude is expressed to Dr. Ronald H. Nelson, Dr. J.A. Hoefer and the Animal Husbandry Department of Mich- igan State University for the use of facilities and animals and for financial support through an assistantship in research. American Cyanamid Company, Pearl River, New York is acknow- ledged for their generous supply of Complemix. The author extends his sincere graditude to his parents for their continued interest and encouragement throughout his life. ii TABLE OF CONTENTS Acknowledgement................ List of Tables................. List of Figures................ List of Appendices............. Introduction................... Literature Review ........... ... Growth Data... ..... .......... Bacteria Concentration.. ..... Dry Matter Digestion......... Cellulose Digestion ..... ...... Volatile Fatty Acids. ..... .... Blood Constituents........... Nitrogen Metabolism.......... Materials and Methods........... In ViVOOOI.........OOOOOOOOCO Experimental Design....... Defaunation............... urea.......OOOOOOOOOOOOOOOOO Feeding Regime...... ...... Sampling Collections...... Nitrogen Determinations... Rumen and Plasma Analyses... In Vitro .......... . ......... ... Fermentation A............ iii .vii 10 1a 1b. 1a 11+ 15 16 17 18 19 19 20 Table of Contents - Continued Materials and Methods (Continued) Page Fermentation BOOOOOOOOOOOOOOOOOOOOOO Fermentation C................. Fermentation D...... ...... .... Statistical Analyses.......... Results and Discussion..... ........ . In ViVOOOOOOOOOOOOO. ..... 0.00.0.0... . Rumen Dry Matter.............. . RumenpHOOOOIOOOOOOOOOOOOOOOOO Volatile Fatty Acids.......... Plasma Urea Nitrogen............... Dry Matter and Nitrogen Digestion... Nitrogen Utilization............... Fecal Nitrogen as Percent Nitrogen IntakeOOOOOOOOOOIO ....... ......OIOO Nitrogen Balanceoo................. Rumen Ammonia Nitrogen.............. In Vitro............................ sumaryOOOOO.........OOOOOOOOOOOOOOO Bibliography.. ................. ...... Appendix ........ . ............... ..... iv 22 23 23 23 23 26 35 38 38 no 1+0 1+0 53 5a 57 61 19.11112. 1 Ration...................................... 2 Mineral and Vitamin Mix..................... 3 Rumen Dry Matters.. ..... ..... ..... . .. ... H Rumen pH Values.......... ...... . . . ..... . 5 Volatile Fatty Acid Concentration TO........ 6 Volatile Fatty Acid Concentration T2........ 7 Volatile Fatty Acid Concentration TH....... 8 Volatile Fatty Acid As Molar Percent Total TO. 9 Volatile Fatty Acid As Molar Percent Total T2. 10 Volatile Fatty Acid As Molar Percent Total T4. 11 Volatile Fatty Acid Ratios...... ..... . 12 Plasma Urea Nitrogen............ ... . . 13 Dry Matter Digestion and Nitrogen Metabolism..... 1H Rumen Ammonia-N Levels..................... 15 In Vitro Ammonia-N Levels..................... LIST OF TABLES PAGE 16 17 2H 25 27 28 29 30 31 32 34 36 39 42 uu LIST OF FIGURES FIGURE PAGE 1 In Vivo Plasma Urea-N Levels.. 37 2 In Vivo Ammonia-N Levels...... #3 3 Ammonia-N Levels for In Vitro fermentation with urea—added to media and water added to media......................... H5 u Regression of rumen ammonia—N with the Acetate:Propionate RatioTOOOOOOOOOOOOOOOIIOOOOOO ”7 5 Regression of rumen ammonia—N with the Acetate:ProPionate Ratio TMOCOOOOOOOOOOOOOOOIOOOO 1+8 6 Regression of rumen ammonia-N with the AcetatezButyrate Ratio TOOOOOOOOOOOOCOOIOOOOOOO [+9 7 Regression of rumen ammonia-N with the AcetatezButyrate Ratio TQOOOCOOOIOOOOOOOOOCCOIO 50 8 Regression of rumen ammonia-N with the PropionatezButyrate Ratio TOOOOOOOOOOCOOOOOOIOCOOO 51 8 Regression of rumen ammonia-N with the PropionatezButyrate RatioTMIIOOOOOOOCIOOOOOOCOOOO 52 vi TABLE 1 Pooled Data of Ammonia-N Levels and V.F.A. Ratios ......... ......OOOOOOOOOOOO0.00.0... 2 Fermentation In Vitro..................... 3 Rumen Dry Matter, rumen pH and feed diges- tibilities for faunated lambs............. u Rumen dry matter, rumen pH and feed diges- tibilities for defaunated lambs........... 5. Nitrogen metabolism data for faunated lanleOI...00............‘OOOCOOCOIOOOOOOO 6 Nitrogen metabolism data for defaunated lambBCIOOOOO......IOOOOOOOOOOOOOOIOOOOO.... 7 Rumen Ammonia-N and plasma urea-N........ 8 Total V.F.A. concentration for faunated lambs receiving urea infusion............. 9 Total V.F.A. concentration for faunated lambs receiving H20 infusion............ 10 Total V.F.A. concentration for defaunated lambs receiving urea infusion............ 11 Total V.F.A. concentration for defaunated lambs LIST OF APPENDIX TABLES receiving water infusion..... vii PAGE 61 62 63 64 65 67 68 69 70 71 AU» qu ‘h .3 1“ -\ DU I. Introduction A trend toward higher nitrogen retention and digestibil- ity by lambs with protozoa (faunated) compared to lambs that do not have protozoa (defaunated) has been observed, expec- ially when low protein rations are fed. Previous work has also indicated that the presence of protozoa in the rumen ecology results in significantly higher rumen ammonia-N levels. This suggests two possibilities for the more fav- orable nitrogen balance observed in faunated animals. The first possibility is that the presence of protozoa in the rumen could directly enhance the nitrogen retention. In other words that the protozoa are of greater nutritional value than are the bacteria. Another possibility may be that the higher ammonia-N levels in the rumen enhance greater bacterial activity and this in turn raises nitrogen reten- tion. The higher rumen ammonia-N level associated with pro- tozoa raises more interesting possibilities. It suggests that most of the proteolytic activity in the rumen is either due directly to ciliate protozoa or to bacteria that are closely associated and dependent on protozoa. The greater bacteria concentrations in protozoa free animals, on the other hand, could have a much quicker utilization of rumen ammonia-N. This study was designed to study the effect of an ele- vated rumen ammonia-N level in protozoa free ruminants. Urea was infused directly into the rumen in an attempt to raise l ammonia-N levels of defaunated lambs to a level similar to that of faunated control animals. Fermentations in_vi£rg were also initiated to study the effect of higher bacterial concentrations on digestibility and volatile fatty acid pro- duction. The true effect of protozoa on the nutrition of the ruminant has long been questioned and it is hOped that this study will further extend the nutritionist understand- ing of the effect of protozoa in ruminant nutrition. II. Literature Review Due to the problems encountered in culturing protozoa in vitrg, especially in the absence of bacteria, defaunation of ruminants has become a popular method of studying the effects of protozoa on rumen fermentation. HCL administration, starvation, milk feeding, cooper sulfate administration, overfeeding, and various combina- tions of these methods have been used to remove protozoa from ruminants (Hungate, 1966). Another method that has been used, which less drastically affects the animals health, is isolation (Akkada and el-Shazly, 196M; Bryant and Small, 1960; Eadie and Hobson, 1962). This method has a larger animal space requirement and there is some question of whether the rumen bacteria population is typical (Church, 1969). Two methods of defaunation used on adult animals, which leSs drastically affects the animalfs health, have been used.. Eadie and Oxford (1957) removed the rumen contnets and heated them at 50°C, while washing the rumen with a saline solution. Some problems have been encountered with this method in removing all protozoa. Dioctyl sodium sulfosuccinate, administered on two successive days, elimin— ated all types of ciliate protozoa without changing the para- meters of normal rumen processes which were measured (Akkada 2.1:. 31. 1968). Many parameters have been used to measure the affect of protozoa on ruminal processes. There is some discrepancy in 3 the results reported in the literature; however, these dif— ferences may be due to the method of defaunation used and to the ration being fed. In the following review, the work that has been done with protozoa free animals is summarized. An attempt will be made to associate results with the type of ration fed, level of protein in the ration, and the method of obtaining protozoa free animals that was used. Growth Data: Nearly all the growth data available in the literature comes from experiments using inoculated and isolated animals; therefore, different bacterial populations in isolated animals compared to inoculated controls may give a difference in observed results. When alfalfa hay was fed free choice with a grain ration, inoculated calves had slightly better gains, while the isolated calves had poorer hair coats and were slightly pot-bellied (Pounden and Hibbs, 1950). In a later trial at the same station (Hibbs and Conrad, 1958), when high roughage pellets were fed, slightly improved gains were obtained from isolated calves. More re- cently, significantly higher gains for inoculated lambs than for isolated lambs, which were pot-bellied and had a rough hair coat, were reported by Akkada and el-Shazly (196”). Certain large oval organisms and Oscillispira_were observed in rumen contents from isolated lambs used in this study, these organisms were not present in inoculated animals. No differences in growth data from isolated and inoculated animals have been observed in two recent studies (Bryant and Small, 1960; Chalmers g: 31. 1968); however, Chalmers 33 El. (1968) did observe a significantly greater body girth measure- ment from ciliate free lambs. Using copper sulfate as the defaunation agent, Christiansen a: El' (1965) observed higher gains and better feed efficiency with faunated lambs. Bacteria Concentration: The removal of ciliate proto- zoa from the rumen alters the bacterial concentration in the rumen. Higher bacteria counts were obtained from isolated calves (Bryant and Small, 1960), except when a cellulose mediumwas used to cultivate bacteria and the highest counts were obtained from inoculated calves. Bacteria concentra— tions in protozoa free lambs have been found to be nearly two times greater than bacteria concentrations from faunated animals (Eadie and Hobson, 1962; Klopfenstein g: 31., 1966). Hungate (1966) suggests that this difference in bacterial numbers can be attributed to either competition for food or to consumption of bacteria by protozoa. Very little work has been done to identify bacterial species in defaunat- ed ruminants; therefore, it is not known how the bacteria pop- ulation in the presence of protozoa compares to the bacteria population when protozoa are not in the rumen ecology. This lack of information as to the bacterial species present in defaunated animals could be an important key to better under- standing the effect of protozoa on ruminant nutrition. Dry Matter Digestion; Dry matter digestion is usually higher when protozoa are present; most deviations from this trend can be attributed to either ration composition or to the amount of ration fed. In two trials using inoculated and isolated dairy calves, the inoculated calves had dry matter digestibilities 3% higher than those obtained with isolated calves (Conrad and Hibbs, 1953; Hibbs and Conrad, 1958). Dry matter digestibilities of 67.1% for faunated and 65.7% for defaunated lambs on a soybean meal treatment and 66.0% for faunated and 64.1% for defaunated lambs on a urea treatment were obtained by Luther and Perkins (1968). When 1200 g. per day of a semipurified diet were fed, Yoder at El. (1969) obtained dry matter digestibilities of 69.0% for faun- ated lambs and 58.3% for defaunated lambs; however, these differences were not observed when only 720 g. of the same ration were fed. When a ration consisting of 500 g. of cot- tonseed cake and rice bran, 3,500 g. of berseem and 250 g. of wheat straw were fed, dry matter digestibilities of 69% for inoculated lambs and 66.5% for uninoculated lambs were observed (Akkada and el-Shazly, 1965). There is an indica- tion that the differences in dry matter digestibility be- tween faunated and protozoa free animals is dependent on the amount of energy present and independent of the amount of protein in the diet. A high protein, low energy diet gave no differences in dry matter digestion, but a low protein, high energy diet gave dry matter digestibilities of 73.7% for faunated lambs compared to 6U.2% for defaunated lambs, while a high protein, high energy diet gave dry matter diges- tibilities of 78.0% for lambs with protozoa and 73.7% for ciliate free lambs (Klopfenstein e£_al. 1966). Cellulose Digestion: Cellulose digestion has been higher with protozoa present in the rumen ecology. Cell- ulose digestion, of 64.4% and 50.5% for inoculated calves compared to 61.3% and 49.3% for isolated calves, was observed by Conrad and Hibbs in two trials (Conrad and Hibbs, 1953; Hibbs and Conrad, 1958). Higher cellulose digestion, when protozoa were present, 53.5% for inoculated vs. 49.3% for isolated lambs, was observed by Akkada and el—Shazly (1965). A considerably greater cellulose digestion from lambs with protozoa compared to lambs without protozoa, 43.3% and 30.1% respectively, were observed by Yoder 3: El. (1964). As was the case for dry matter digestion, these differences were only measured at higher feed intakes, suggesting that cellu- lose digestion may also be dependent on energy levels of the diet. The increased cellulose digestion observed for faunated animals may not be due directly to the protozoa, but may be as a result of bacteria closely associated and dependent on protozoa. As was previously reported when a cellulose medium was used to cultivate bacteria, the highest bacteria counts‘ were obtained from animals with protozoa (Bryant and Small, 1960). Volatile Fatty Acids: The effects of protozoa on vol- atile fatty acid production has produced considerable discre- pancy in the literature; however, there seems to be a trend towards lower prOpionate and higher butyrate when protozoa are part of the rumen ecology. Conrad 31 a1, (1958) obtained a higher propionate and lower butyrate level for isolated calves, while inoculated animals had lower propionate and higher butyrate levels. Using an in zitrg_fermentation system, Luther g: 31. (1964) obtained significantly (P<.01) lower acetate and propionate when protozoa were added to the fermentation system and significantly (P<.01) increased levels of butyrate, valerate and branched chained fatty acids. No differences in total volatile fatty acid production, but an increased proportion of butyrate and a higher acetate to propionate ratio when protozoa were present was observed by Klopfenstein g: 21. (1966). Higher levels of propionate have been obtained from lambs with protozoa (Akkada and el-Shazly, 1964; Christiansen gt 21.,1965; Luther and Perkins, 1968), and these lambs have had a higher total volatile fatty acid production (Christiansen e: 31., 1965; Luther and Perkins, 1968). Blood Constituents: Blood urea levels have been higher when protozoa are present. There are some exceptions to this trend due to the type of protein supplement used or to the level of protein in the diet. Slightly higher blood urea levels from faunated lambs when a urea supplemented diet was used, were obtained by Luther and Perkins (1968); however, when they fed a soybean meal diet there were no differences in blood urea levels observed. Higher plasma urea levels from faunated lambs on a high protein ration and higher plasma urea levels from ciliate free lambs on a low protein ration have been reported (Klopfenstein e£_al., 1966). Iso- lated lambs had higher blood urea nitrogen levels when they were on a low protein ration (Akkada and el-Shazly, 1965). Very little work has been done concerning the effect of defaunation on any other blood constituents. Faunation has been shown to increase oleic acid and decrease linoleic and stearic acid (KlOpfenstein gt 31., 1966), while also significantly changing the steariczoleic and palmitic: stearic acid ratios from those of protozoa free lambs. These results were corroborated by Lough (1968). Protozoa free lambs were suggested to have lysine as a limiting amino acid, while faunated lambs were suggested to have no amino acid consistently limiting (Klopfenstein g: 31.,1966). Nitrogen Metabolism Nitrogen and Crude Protein Digestion: There are consid- erable differences in nitrogen digestibilities as reported in the literature. Nitrogen digestibility is quite dependent on the level of protein in the ration, the amount of energy in the ration and on the amount of ration that is fed. When feeding a low protein diet, inoculated lambs and calves had slightly higher crude protein digestibilities (Akkada and el-Shazly, 1965; Conrad and Hibbs, 1953); how- ever, this same trend was observed by Hibbs and Conrad (1958) when they fed high roughage pellets and obtained crude protein digestibilities of 65.2% for inoculated calves, 10 while isolated calves digested 63.1% of the crude protein in their ration. Feeding 1200 g. of a semipurified diet gave nitrogen digestibilities of 62.8% for faunated lambs and 55.6% for protozoa free lambs; however, these differences were not obtained when only 720 g. of the same ration were fed (Yoder E: 21., 1964). No differences in nitrogen digestibility were observed when a high protein low energy diet was fed (Klopfenstein 33.31" 1966), but a low protein, high energy feed and a high protein, high energy ration gave digestibilities of 64.3% and 73.0% for faunated lambs and 61.7% and 63.0% for protozoa free lambs, respectively. No differences in nitro- gen digestibilities between faunated and ciliate free lambs were observed by Chalmers g: 31. (1968). Nitrogen Retention: Higher nitrogen retentions from faunated lambs have been obtained on low protein rations, while high protein rations have tended to give higher nitro- gen retention in protozoa free lambs. On a low protein ration, Akkada and el-Shazly (1965) obtained significantly higher nitrogen retention from inoculated lambs and Klopfen- stein g£_al. (1966) observed a significantly (P<.05) higher nitrogen retention from lambs with protozoa. On high protein rations ciliate free lambs had higher nitrogen retention (Klopfenstein E: 31.,1966); however, these differences were not significant. Ammonia: Ammonia constitutes the main source of non- protein nitrogen in the rumen (McDonald, 1952) and comes from the breakdown of dietary protein to ammonia, carbon ll dioxide, and volatile fatty acids (Bryant and Robinson, 1963). A high correlation between rumen ammonia and the concentration of the combined branched chain fatty acids was observed by Jamieson (1959). Seven species of bacteria have been found to be proteolytic (Akkada and Blackburn, 1963) and at least one species of protozoa, Entodinium Qaudatum, has been shown to hydrolyse amide groups of case- in to ammonia (Akkada and Howard, 1962). Warner (1956) suggests that half the proteolytic activity in the rumen is due to bacteria and further postulates that much of the con- tinuing ammonia production in the rumen, in the absence of readily attacked protein, might be due to the endogenous metabolism of rumen protozoa. Ammonia production is of a greater magnitude in the presence of bacteria with protozoa absent. The greatest production of ammonia-N from glutamine was obtained using a bacteria rich in vitrg inoculum (Hoshino g: 31., 1966). Warner (1956) also observed an in— creased ammonia concentration from bacteria. Ammonia in the presence of carbohydrate can be utilized for production of bacterial protein or it can be absorbed through the rumen wall and then excreted as urea (Hobson, 1959). McDonald (1948) suggests that the absorption of ammonia into the blood stream may be of the magnitude of 4-5 g. per day; however, this absorption is probably dependent on the level of ammonia in the rumen. The highest blood urea levels were obtained with rations that caused a high rumen ammonia-N level (Klopfenstein gt_al., 1966) and a low plasma urea level was 12 observed when a low protein ration was fed and rumen ammonia- N level in turn was low. Many species of rumen bacteria require ammonia and are inefficient in utilizing amino acid carbon (Bryant and Robinson, 1963). When given the choice between ammonia and preformed amino acids, rumen bacteria preferentially utilize ammonia (Akkada and Blackburn, 1963). Removal of protozoa from the rumen ecology lowers the ammonia- N levels (Akkada and el-Shazly, 1964; Chalmers 33 51., 1968; Christiansen g: 21., 1965; Klopfenstein 3: al., 1966; Luther and Perkins, 1968). This lower ammonia level from ciliate free animals has been shown regardless of the type ration fed, the amount of ration, Or the method of defaunation used; however, the magnitude of the difference between faunated and defaunated animals varies with the amount of protein. Urea: Feeding urea has long been of interest, due to its availability and economy as a source of nitrogen. An excellent review on non-protein nitrogen as the primary source of nitrogen has been recently published (Oltjen, 1969), and urea as a protein supplement is reviewed by Briggs (1967). This short review is designed to only touch on effects of urea supplementation associated with this work and is far from all inclusive. Feeding urea has been shown to give higher rumen ammonia levels (Hoshino 33 31., 1966; Hemsley and Moir, 1963; Johnson and McClure, 1964), and a higher concentration of total volatile fatty acids (Hemsley and Moir, 1963). As early as 1939, urea was shown to gives 13 A nearly as good gains as did casein and that the most efficient utilization of urea was obtained when some soluble sugars were included in the diet (Hart 2: 31., 1939). Adding urea to the diet has increased dry matter digestion, cellulose digestion, and nitrogen digestion (Johnson and McClure, 1964). Adding urea to a basal ration significantly increased dry matter intake, increased nitrogen digestion and retention, but lowered dry matter digestion (Hemsley and Moir, 1963). III Materials and Methods A. Experiment In Vivo Experimental Design The original experimental design of this project was a double 4 x 4 latin square with the four variables consisting of urea supplementation vs. a water control and the presence or absence of protozoa. Due to problems encountered with the original eight sheep, only three of these lambs completed the entire experiment. Consequently, a total of twelve sheep were used; six with each of the water infusion treatments and seven with each of the urea treatments, and a lease squares analysis of variance was used to determine statistical signi- ficance. The treatments used consisted of urea infusion plus protozoa, water infusion plus protozoa, urea infusion with no protozoa and water infusion with no protozoa. During each feeding period the sheep were allowed a thirteen day adjust- ment period. After one day in metabolism stalls, feed in- take, urine production and fecal output were measured and sampled for analysis. This sampling period lasted for six days; two days later rumen samples and jugular blood samples were obtained at feeding and at two and four hours post- feeding. In later references these times will be referred to as T0, T2 and T4, respectively. Defaunation Removal of protozoa was accomplished, using a slightly modified method of Akkada E£.E£3’ (1968). Dioctyl sodium sulfosuccinate, under the trade name Complemix and furnished 11+ 15 by American Cyanamid Company, was used as the defaunation agent. Twelve c.c. of this compound were infused directly into the rumen on three successive days. Feed was withheld., at each administration of Complemix. On the second day of treatment, the rubber cannula was removed, completely cleaned and then disinfected and replaced. At this same time all wool was clipped from around the cannula and this area was disin- fected. After two to four days the sheep were consuming their entire ration and were free of ciliate protozoa. These animals were then kept isolated from all other ruminants and the rumen contents were regularly checked for the absence of protozoa. 922%. An objective of this project was to raise the rumen ammonia-N level of protozoa free wethers 4 mg. percent above that of the control animals. The estimated average rumen size of the sheep used was four liters. In a rumen this size, the desired increase in ammonia-N would require an increase of .16 grams, which could be accomplished with .32 grams of urea. Urea nitrogen was assumed to have a half life of two hours and it was assumed that urea would be recycled at a level of 30 to 50 percent. From this it was estimated that an average of 1.00 gram of urea per day should be adequate to raise rumen ammonia-N levels the desired 4 mg. percent. This Was calculated to represent .123 percent of the ration fed per day to a particular sheep and therefore when urea was infused into the animals it was always at this propor- tion of the ration fed. Sodium sulfide was added to the warm ~ 16 urea solution at a ratio of fifteen parts urea nitrogen to one part of sulfur. This mixture of urea and sodium sulfide was infused in 480 ml. of water each day. The control animals received an infusion of 480 ml. of water. A model 954, four channel, infusion/withdrawal pump (Harvard.Apparatus Company) was used to make the urea infusion. Sheep .fifi The ruminants used in this experiment were two-year-old Cheviot wethers that averaged 32 kg. prior to the first two treatment periods and 28 kg. prior to the final two perids. All animals used in this project were fitted with rumen cann- ulas (Jarrett, 1948) prior to the first experimental period. FeedingfiRegime The sheep were fed twice daily, at 8 a.m. and 5 p.m., receiving six percent of their metabolic body weight (B.WT‘75) per day. The ration composition (Table l) was similar to that fed by Klopfenstein E: 31. (1966). .Table 1.1 Ration Ingredient Kg. per 100 kg. of Mix Alfalfa Meal 38.00 Corn Cobs 7.80 Ground Shelled Corn 47.00 Cerelose 4.55 Minerals 2.40 Vitamins 0.25 17 The minerals and vitamins were supplied in a mix (Table 2). Table 2. Mineral and Vitamin Mix Ingredient Kg. per 100 kg. of Mix Dicalcium Phosphate (26.5% Ca-20.5% P) 47.38 Trace Mineralized Salt (High Zinc) 47.42 Sodium Sulfate (25.5% Sulfur) 4.78 q Vitamin A (10,000 IU/g) .32 L» Vitamin D (9,000 IU/g) .10 5 Only sheep that consumed their entire ration were used in the statistical analysis. Daily' portions were weighed at one week intervals and a sample was taken each week for dry matter determinations. This sample was oven dried at 105°C for 24 hours and saved for later analysis. Sample Collegtigns Total fecal collections were made using a canvas bag collection harness. The daily feces were weighed and a sample was taken for dry matter and nitrogen determinations. Urine was collected in two liter glass bottles. Each bottle contained 25 ml. of 20 percent sulfuric acid. The total volume was measured, then the collection was diluted up to two liters and one sixth of this dilution was retained for further analysis. 18 Rumen contents (Purser and Moir, 1959) and jugular blood were sampled at T0, T2 and T4 as previously described. Rumen samples were measured for pH using a Corning model 12 pH meter; a portion of the rumen sample was then oven dried at 105°C for 24 hours to determine rumen dry matter. Eleven g. of the TO rumen sample were diluted in 99 m1. of an anaerobic dilution solution, under anaerobic conditions. Further dilutions to 10"8 were made and bacterial counts were measured using the method of Hungate (1966) and the media of Bryant and Robinson (1961). Twenty ml. of whole rumen contents were mixed with twenty ml. of a 50 percent formaldehyde solution and retained for protozoa counts (Purser and Moir, 1959). The T0, T2 and T4 samples of whole rumen contents were strained through two layers of cheesecloth and 19 ml. of the resulting fluid was mixed with 1 m1. of saturated mercuric chloride. This mix- ture was retained for volatile fatty acid and rumen ammonia—N analyses. The whole blood was centrifuged at 12,100 x g for fifteen minutes and the plasma was retained for plasma urea determinations. Nitrogen Determinations The dried feces retained from each day's sample was thoroughly mixed and then ground through a 20 mesh screen in a Wiley mill. Total nitrogen was determined on this ground sample by the micro-Kjeldahl method. Oven dried feed samples were analyzed for total nitrogen by the same method. The urine was thoroughly mixed and a subsample was used to l9 determine total urine nitrogen using the micro-Kjeldahl pro- cedure. Rumen_and Plasma Analyses Rumen volatile fatty acid concentrations were deter- minded on a Packard Gascnummmtograph. Five m1. of rumen fluid were mixed with one ml. of 25 percentmetaphosphoric acid, centrifuged at 12,100 x g for 10 minutes and the supernatant retained. The peak heights were converted to micromoles of acid with the aid of standards. Rumen ammonia-N concentra- tions were determined using the micro-diffusion method of Conway (1950). Plasma urea concentrations were also deter- mined using the micro-diffusion method. Jackbean urease was used to release the urea and samples were corrected for ammonia levels in the plasma. B;_Experiments In Vitro The fermentation system used was an adaption of the Ohio system (Karn, £3 31. 1967). Two grams of the ration, used in vizg and ground through a 40 mesh screen in a Wiley mill, was used as the substrate. The media, a mixture of biotin, para- amino-benzoic acid, valeric acid, urea, and mineral mix was similar to that used by Dehority (1961). Rumen fluid in- oculum, obtained from the same lambs used in viva, was strained through two layers of cheesecloth. The strained fluid was then either used as whole contents or centrifuged at 250 x g for 3 minutes and the supernatant used as the in- oculum. In all further discussion the inoculum will be 20 referred to as whole contents or supernatant. The amount of inoculum used varied depending on the experiment. The total volume of each fermentation bottle was 100 ml., with the difference between inoculum levels made up with media. Car- bon dioxide was continually bubbled through the system. Every three hours for the first twelve hours and each twelve hours thereafter, pH was adjusted to 6.7. Dry matter digestion was determined by centrifuging sub- samples from each fermentation system in 40 ml. pyrex tubes at 5,000 x g for fifteen minutes. In the last two experiments) the supernatant was saved for volatile fatty acid determin- ations. The sediment remaining in the centrifuge tube was washed twice with distilled water and then oven dried at 105°C for 24 hours. The dried material was then weighed to determine the percent dry matter in the fermentation system. The dried sample was then subjected to cellulose analyses by the method of Crampton and Maynard (1938). Egrmentation_A This experiment was designed to determine the extent of ammonia-N production from urea with defaunated and faunated lambs. Urea was omitted from the media in half the fermen- tation bottles; these bottles were used as a control. Treat- ment combinations consisted of: urea plus whole content in— oculum, no urea plus whole content inoculum, urea plus super- natant inoculum and no urea plus supernatant inoculum. Inoc- ulum was obtained from both faunated and defaunated lambs. A total of 32 fermentation bottles were used. Subsamples were 21 taken at zero time, and at three hours, six hours, nine hours and twelve hours after inoculum was added. At T6, pH was ad— justed to 6.7; in all other experiments pH was adjusted every three hours for the first twelve hours and thereafter every twelve hours Until the end of the experiment. Each subsample was analyzed for ammonia—N using the micro-diffusion method of Conway (1950). Dry matter digestion was determined for the twelve hour period. Fermentation B The second fermentation in vitae was designed to deter- mine the effect of varying inoculum levels on dry matter and cellulose digestion. Urea was used in all media for this experiment. The treatments consisted of whole contents and supernatant of rumen fluid from faunated and ciliate free sheep and inoculations of 12.50 m1. and 25.00 ml. were used. Eight sheep were used as inoculum donors and the fermenta- tion system consisted of 32 bottles. Dry matter and cellu— lose digestion were determined at 24 and 48 hours after the fermentation began. Pigmentation_g This experiment was a repeat of the previous experiment. Only faunated lambs were used as inoculum source. Total volatile fatty acid production was determined in this exper- iment. 22 Fermentation D This fermentation in vitrg was a continuation of experi- ment B and C. Inoculum levels were halved to 6.25 ml. and 12.50 m1. Subsamples were taken at 12, 24 and 48 hours. Inoculum was obtained from only two faunated and protozoa free lambs. Thirty-two fermentation bottles were used so there was a replicate of each variable. Volatile fatty acids, dry matter digestion and cellulose digestion were analyzed. C. Statistical Analyses The data from i2_vivg experiments were analyzed on the IBM 3600 computer at the Michigan State University Computer Laboratory. Least squares analyses of variance was employed to define the significant relationships in this study. Data from experiments in zitrg were not an— alyzed due to small numbers and differences between exper- iments. IV Results and Discussion A. In Vivo Rumen Dry Matter: Rumen dry matter was higher in pro- tozoa free sheep' at all sampling times when compared to sheep. with protozoa (Table 3). Faunated sheep had rumen dry matters of 12.22% compared to 16.52% for defaunated sheep, at the TO sampling time (P<.01). Rumen dry matter values were also observed to be greater for defaunated sheep at T2 and T4; however, these differences were not significant. There was no significant interaction between the presence or absence of protozoa and urea or water infusion; however, the mean water values for rumen dry matter were slightly higher than those observed for the urea treated animals. At the TO sampling time this difference approached significance (P<.10). Rumen pH: Rumen pH was slightly higher for faunated sheep and for the animals receiving the urea infusion (Table 4). The rumenpr for faunated ’sheep was 6.19 and 5.66 compared to 5.80 and 5.33 for protozoa free sheep at the TO and T2 sampling times respectively. These differences only approached significance (P<.10) and the T4 values were not significantly different between faunated and defaunated animals. At T2 and T4 there were differences approaching significance (P<.10) between the mean values for animals receiving the water infusion and those .‘sheep receiving the urea infusion. There were no significant interactions between the presence or absence of protozoa and the urea or water in- fusions. 23 24 .AoH.vmv pampmmmao >HPGMUMMHCMHm ohm mpmflnomthSm pampmMMHo mafl>mn modam> .Aao.vmv PcmsmMMfiU havcmofimwcmwm mam mpmwhomprSm pcmhmmmwo wcw>ms mosam> n.m m.< momma may mo nogam UQMUGMPMN mcowpm>hmmno mm Mo cmmzH om.H mm.hH mm.>H mo.mH mm.bH :m.mH mm.mH mm.mH OH.mH :5 mm. :m.oH mN.mH mH.mH om.bH mh.ma ::.mH mH.mH o>.bH NB mm. Amm.mH Moo.mH mNm.mH hm.mH hm.ha H9:0owM9:mfim 090 09:990090a30 9:090mmwp m:9>0: m0sa0> a.m. 0:008 0:9 mo 90990 0900:09mm 0:09p0>90m:o mm mo :00ZH mo. nos.m m~:.m m:.m m:.m m:.m Nk.m Na.m 9m.m :9 mo. 0Hm.m Amm.m mm.m sm.m mm.m 0mm.m mm.m m:.m NE NH. nm.m mm.m om.m mm.m mu.m 0ma.m om.m so.m OB mm.w 0092 Cam :00: 0093 Cam :00: 0093 0mm .1 1|r. :00: W090:30m0ml. U090::0m 0599 I, .L: w:HHmE0m #:0590099 . Hw0sa0> mm :065m .: 0HA0H 26 Volatile Fatty Acids: The effect of the absence of pro- tozoa from the rumen ecology, on volatile fatty acid (V.F.A.) concentrations is outlined in Tables 5, 6 and 7. There was only a slight difference in total V.F.A. concentration ob— served at any of the sampling times; however, at T0 the total V.F.A. concentration was 66.64 micromoles per mil- Liliter for defaunated sheep and 57.15 micromoles per mil- liliter for faunated sheep (Table 5). This difference ap- proached significance (P‘910). At T2 the difference was re- versed and faunated wethers showed a higher V.F.A. concentra— tion than did defaunated sheep (Table 6). This slight differ- ence in total V.F.A. concentration is in agreement with most of the work that has been done comparing ruminants with pro— tozoa and those without protozoa. Acetate concentration was also only slightly different, although at T2 (Table 6) the difference between faunated and defaunated sheep approached significance (P<.10). This difference in the level of acetate at T2 is also shown in the molar percent acetate (Table 9). The higher molar percent acetate for faunated wethers is significantly different (P‘<05)1from the molar percent acetate for defaunated wethers. The greatest and most consistent difference observed in V.F.A. concentration, between faunated and defaunated sheep, was in the level of propionate. Pro- pionic .acid was consistently lower in sheep with protozoa, this difference was highly significant (P<.01) at T0 and .Aoa.vmv #:0909990 mapamofimflamflm 090 mpm99omnmasm 9cm909990 mc9>mn mmsam>.an .90 .Amo.vmv #:0909990 >H#:00999:m#m 090 m#m#9om90::m.#:090mmwp m:#>0: w0:a0>n.0 m.< 0:005 mo 90990 U90p:0#mm QQHHHflHHHSC mmHOEOQOHE gun GOHPMQPfimUEOON m0SHO> mm mo :00:H .Aao.vmv #:090mmwp >H#:00999:m90 090 m#mw9om90mzm #:0909990 w:9>0: 003H0> 27 :H. mm. :m. Ho.H mm. mo.H on. on. :m. 0#090H0> so. mm.w mm.m nam.: mo.m m9.: mam.e mk.m m:.m 0909992m mm.H mm.:H m:.mH m::.om mm.md HN.HN :00: U0#0:30m0a p0#0:1mm 1+ #:ME#009B .111. 09 N.Hcowpmnpamocoo s9o< 9990: 0999090> .m 09009 28 .909.vmv #:0909990 >9#:00999:w9m 090 m#m99om90050 #:0909990 m:9>0: 00:90>.9: .90 9099999995\m090509095 :9 :09#09#:00:oo 0:005 90 90990 0900:0#m m0:90> mm 90 :00: .Amo.vmv #:0909990 >9#:00999:w9m 090 m#a990090950 #:0909990 m:9>0: 00DH0> Q0 00 (U N H l 09. 99.9 90.9 000.9 00.9 99.9 090. 09. 90. 0909090> 90. 00.0 09.9 09.0 90.9 00.0 99.0 09.0 00.0 09099900 00.9 09.99 90.09 00.09 00.09 00.09 00.99 00.09 99.99 0909090090 09.9 09.90 00.90 9000.00 09.00 00.00 9009.00 09.90 09.00 090900< 90.0 90.90 00.00 90.00 00.90 00.00 90.90 00.90 00.00 90909 .... ........ ... .... ..0090....... ...0, .0099... ... ...... ll. .mm05#009h .lI 099.99099099900000 090< 9990: 0999090> .mlmmmmm 29 .Amo.vmv #:0909990 93”#:00.90.03.u9.9m90 090 0#m990090m:0 9:0909990 w:9>0: 00SH0> 0.0 0:005 90 90990 0900:0#mm 90#9999995\0090509095 :9 :09#09#:00:ooN 00SH0> mm 90 :00:9 99. 90.9 90.9 00.9 00.9 09.9 09. 00. 00. 09090909 00.9 00.0 09.0 00.0 00.9 99.0 09.0 00.0 90.0 09099900 99.9 99.09 00.99 90.09 00.09 00.09 000.99 09.09 00.09 0900090090 00.9 09.00 00.00 90.00 00.00 09.90 00.00 09.00 00.00 090900< 90.0 09.09 90.99 00.90 00.90 90.90 00.09 90.09 00.09 90909 .0.0 0090 09m 0002 0090 09m 000: 0090 09m .<.9.> :00: 00#0::090Q 00#0:d09 PamEwarHH. 099.90099099000000 090< 99900 0999090> .9 09009 30 .Amo.vmv 9:0909990 >9#:00999:m90 090 099990090930 9:0909990 m:9>0: 00:90> :90 0:005 90 90990 0900:0#mN 00:90> mm 90 :00:9 00. 99.: 99.: mm.: 9m.: mm.: 99.: mm.m 9a.: :90:o :0:09m 00. 09.9 00.9 00.9 m:.9 00.9 09.9 09.9 NN.9 0909090> 9o. 9m.99 00.0 :No.9 mm.9 09.0 0:m.m9 09.09 09.99 0#09>#:m mo. m:.mm 90.0w 000.mm 00.09 m9.9m 009.09 99.09 90.99 0#0:09:09: 90. 00.00 09.00 09.90 09.90 09.00 mm.mm 90.90 99.o0 0#0#00< I1, 0 w w w w w w w mm.m 009: ON: :00: 009: ON: :00: 0093 ON: .<.9.> :00: 00#0::090Q 00#0:d09 #:0590099 1| o9 990#09 9o #:0090m 9090: 0< 090< 99##09 099#09o> .0 09:09 31 .909.V9V #:0909990 >9#:00999:w90 090 0#99900909d0 #:0909990 m:9>0: 00999097999 .90 .A0od.9v #:0909990 >9#:00999:m90 090 0#9990090930 #:0909990 m:9>0: 00:90> 0.0 mCmmE .HO 9099.090 UQMUCMPmN 00:90> 09 9o :00: 9 00. 33.9 00.9 00.9 00.9 99.9 93.9 00.9 09.9 09000 000090 00. 00.9 09.9 099.9 00.9 03.9 090. 90. 90. 09090900 00. 09.09 00.0 00.0 90.0 90.0 90.09 00.09 09.0 09099900 00. 00.09 00.09 9030.00 00.09 90.90 9030.09 30.99 30.09 0900090090 90. 30.90 03.00 000.00 90.90 30.00 090.90 00.00 00.90 090900< 0 0 0 0 0 0 I 0 0 I .0.0 009: .090 000z[l 0090, 090 000:, 0090 090 .<.9.> 9 :00: U0#0::090Q 00#0::09 #:0590099 All 99 990909 90 9000900 90902 0< 090< 99909 0999090> .0 09009 32 .9099v9v #:0909990 >9#:00999:m90 090 0#99900909:0 #:0909990 m:9>0£ 00:90>.9n .90 0:000 90 90990 0900:0#0 .90oav9v #:0909990 >9#:00999:w90 090 0#99900909:0 #:0909990 m:9>0: 00:90> 00:90> 09 90 :002 n90 9 I. 9 00. 00.9 00.9 90.9 30.9 00.0 00.9 00.9 99.9 09000 000090 00. 00.9 30.9 00.9 30.9 03.9 00.9 00. 09.9 0909090> 90. 00.09 09.0 000.9 09.0 99.0 009.99 90.09 09.99 09099900 90. 00.09 90.09 9000.00 33.09 09.90 9000.09 39.99 00.39 0900090090 90. 09.00 99.00 00.90 93.90 00.90 93.90 00.90 90.00 0909000 9 w w w w w w .i W! 90.0 0090, .090 000:1 009:. 090 000:, 0092. 0mm .<.0.> :00: 0090::0909 00#0::09 #:0E#009B II .I .l 39 990909 90 9000900 90902 00 0900 99900 09990909 .09 09009 33 significant (P<.05) at Tu. This difference is also shown in the tables on molar percent (Tables 8, 9,10), as the molar per- cent propionate is significantly higher (P<.05) for defaunated sheep at T0 and approaching a significant difference (P<.10) at T2 and TH. Butyrate was consistently lower when protozoa were not in the rumen ecology; however, this difference was only significant (P‘QOS) at T0 (TableS). The molar percent butyrate was also significantly (P<.05) lower for defaunated wethers at this sample time (Table 8). The concentration of valeric acid was higher for defauna- ted sheep (Table 6) at the T2 sampling time (P<.05). The molar percent valerate was also significantly (P<.05) higher at T2 (Table 9). Branched chain volatile fatty acids as a molar percent of total showed no difference between treatments. There were also no observed differences in any volatile fatty acid parameters between mean water and mean urea treatments. The acetate:propionate ratio was lower for defaunated sheep (Table 11). The difference at T0 was highly signifi- cant (P'fiOl) and at T2 and TH the lower acetate:propionate ratio for wethers without protozoa was significant (P<.05). This is in agreement with the findings of Klopfenstein 33 El' (1966). The acetatezbutyrate ratio is higher for sheep with- out protozoa; however, these differences are not significant. The acetate:butyrate ratio for the defaunated water infused animals was higher than any of the other acetatezbutyrate ratios for other treatments. . . iA-Et'fl i .900.v9v #:0909990 >9#:00999:m90 090 0#99900909:0 #:0909990 w:9>0: 00:90> 34 9.0 .990.V9v #:0909990 99#:00999:w90 090 0#9990090950 #:0909990 w:9>0: 00390>m.< 0:005 9o 90990 0900:0#09 00:90> 09 90 :0029 090. 009.9 990.3 000.3 003.0 039.0 099.9 030.9 090.0 39 Dm\09 930. 900.0 030.9 900.9 900.0 000.0 099.0 990.0 090.0 39 Dm\o< 300. 300.9 090.9 0939.9 009.9 000.9 0000.0 009.0 900.9 39 M9\o< 009. 900.9 090.3 000.3 093.0 090.0 300.9 030.9 000.0 99 Dm\M9 000. 909.0 900.0 900.9 990.0 990.0 009.0 909.0 990.9 99 Dm\o< 009.. 000.9 003.9 0930.9 399.9 090.9 0000.0 900.9 000.0 99 09\o< 090. 093.9 990.3 909.3 900.0 309.3 000.9 000.9 900.0 09 Dm\09 900. 000.0 300.9 039.0 900.9 900.0 930.0 000.3 003.0 09 Dm\o< 909. 339.0 030.9 0000.9 399.9 000.9 <939.0 090.3 993.0 09 m9\u< .m.0 0093 09m :00: .m093 own :00: 0090 on I. :00: 00#0::0909 00#0::mM1 0599 O9#0m #:05#0099 .I 0:999500 9009900 0900 99900 09990909 .99 09009 35 The urea infusion into defaunated animals raised the buty- rate concentration to a magnitude similar to that observed for faunated animals; therefore, the acetate:butyrate ratio from defaunated sheep receiving the urea infusion was lower and nearly equal to the ratio for faunated animals. The propionatezbutyrate ratio was higher for sheep with- out protozoa; however, as was the case for the acetatezbuty- rate ratios these differences were not significant. The same trend for urea infused defaunated wethers to have a lower ratio of a magnitude similar to that of the faunated animals was observed and was caused by higher butyrate and lower propionate from sheep that had no protozoa and which received the urea infusion. There were no significant differences observed for the mean water values compared to the mean urea values for the three ratios that were analyzed. .Elasma Urea Nitrogen; Plasma urea nitrogen was higher for sheep with protozoa (Table 12), which is in agreement with the previous work. The biggest difference in plasma urea nit- rogen was at TH when faunated wethers had 9.17 mg. per 100 ml. plasma and defaunated wethers had 5.95 mg. per 100 ml. of plasma. This difference was highly significant (P<£Ol); however, at the other sampling times no significant differ- ences were observed. Urea infusion increased the plasma urea nitrogen levels when compared to the water infusion controls. The T0 values were not significantly different; however, at T” the urea infusion increased plasma urea nit- rogen significantly (P‘EOS) over the water control and at T2 36 .909.V9V 990909990 9999009999090 090 099990090990 990909990 w99>09 00990>n99 n90 .900.V9V 990909990 9999009999090 090 099990090990 990909990 m99>09 00990>990 0.< 09009 90 90990 090090900 000090 00 .99 oo9\zumz .9900v9v 990909990 9999009999090 090 099990090990 990909990 m99>09 00990> 9 090990>90090 09 90 90029 00.9900.0 099.0 000.0 00.0 00.0 <99.0 00.09 00.9 39 00.9990.0 9000.0 00.0 00.9 99.0 03.0 90.99 00.9 99 00. 99.09 00.9 00.0 00.09 99.0 99.0 00.09 00.0 09 09.0 0090 09m 9002 0093 09m 900: 0093 093 AM002 l10090900909 0090990M1. m9MEM%0 990990099 .9 m 9.990009992 009: 090099 .99 09909 37 ”$1.9“?- . ,. |"|I|l‘.| ........"'l 1, ||| ... z. . If d ? 0:: ‘02:- '0 ‘0 .. .r ,.g‘~= Mt.» : ' h i V O 099.090.4002." Plasma Uroo-N (mm/100 ml.) 0- $9990; ., ——Wotor I: Protozoa 2:" alumni-coll. Protozoa “‘Wotor ‘ w/aUroa Figure l. _I_rl vivo plasma urea-N levels. 38 this difference approached significance (P<.10). There were no significant interactions between urea infusion and the pre- sence or absence of protozoa. The plasma urea nitrogen results for the four treatment combinations are displayed graphically in Figure 1. Dry Matter and Nitrogen Digestion: Dry matter digestion was not significantly different when the treatment effects were analyzed (Table 13). Urea infusion slightly decreased dry matter digestion for both faunated and defaunated sheep when compared to the water controls; however, this difference was not significant. Nitrogen digestion was similar for all de- faunated animals. The mean nitrogen digestion value for fau- nated sheep was not signifcantly different from the mean value for defaunated sheep; however, urea infusion significantly (P‘QOS) lowered nitrogen digestion of the faunated wethers when compared to the water infusion controls. Nitrogen Utilization: The nitrogen utilization figures that were observed for this experiment were extremely high for sheep that were on a maintenance ration; however, there were significant differences observed. Nitrogen utilization for defaunated sheep was significantly (P<.Ol) higher than utilization from sheep with protozoa. In defaunated animals the infusion of urea only slightly increased nitrogen util- ization over that of the water infused sheep; however, in faunated animals the urea infusion decreased utilization significantly (P<.10). It is postulated that the greater bacterial concentration in defaunated sheep was better able to utilize the excess nitrogen supplied by the urea. 9 3 .909«.9v 990909990 9999009999090 090 099990090990 990909990 9993 00990>. .900.V9V 990909990 9999009999090 090 099990090990 990909990 9993 00990> .990.V9v 990909990 9999009999090 090 099990090990 990909990 9993 00990> 90 .90 0.0 0.... 09005 90 90990 090090909 00990> 09 90 9002 9 033. 90. 00.0 90. 00. 9.0.0 900.0 09.00 03.30 00.00 99.09 9002 009D 009.0 00.09 90.90 09.99 00.09 9002 090 9000.0 09.00 030.09 00.09 99.99 9002 900009.0 09.00 99.09 00.09 00.09 009: 900000.0 09.00 00.99 00.09 90.99 092 0090990909 0909.0 00.09 <90.00 00.00 00.99 9002 99000.0 990.90 9900.00 00.00 09.09 0093 90009.0 090.99 9009.00 M09.99 00.09 092 00909909 0 9 F 0..I 1, lirul 0090900 000999 90009992 9099099999: 909900090 909900090 990590099 90009992 9000999z 9000“ 90009992 90009992 909902 999 95099090902 90009992 090 900900099 909902 999 .09 09909 40 Fecal NitrogenAs Percent Nitrogen Intake: There were no significant differences observed for the mean values (de- faunated vs. faunated) of fecal nitrogen as a percent of nitro- gen intake (F.N./N.I.); however, an interesting trend was ob- served (Table 13). Regardless of whether urea or water was in- fused, the F.N./N.I. for defaunated sheep was 30.19%. The fa- unated wethers that received the urea infusion had a mean. F.N./N.I. value of 31.31% compared to 27.61% from faunated sheep receiving the water infusion (P<.05). This figure further magnifies the nitrogen inbalance observed with fa- unated wethers receiving the urea infusion. Nitrogen Balance: The highest nitrogen balance values were observed in defaunated sheep (P<.05); however, these values were also very high for adult sheep on a maintenance ration (Table 13). As was the case for other nitrogen metab- olism data, the urea infusion into defaunated wethers did not greatly effect the nitrogen balance results. The lowest nitrogen balance was observed when faunated sheep were in- fused with urea, a difference that approached significance (P 9.0 .990.V9v 990909990 9999009999090 090 099990090990 990909990 9993 00990>0.< 09005 90 90990 090090909 00990> 09 90 90029 90.9 009.3 009.0 9009.9 099.9 009. 0099.0 090.0 990.0 39 99.9 99009.0 90009.0 0900.0 000.0 999.9 <009.99 900.99 000.0 99 00.9 009.99 390.9 900.9 000.9 003.0 030.99 090.39 999.0 09 w0.0 0090 090 9002 009: 092 9002 009: 09 9002 0090990909 00909909 LH1 0599 990590099 tl 09999500 9.95 009\.050 9090>09 2:099055< 90590 .09 09909 I4 V b ' a lumen Ammonia-N (mg/100 ml., 0 0 1+3 —Wotor& Protozoa mun Drool P009".- I“WO’OI W4 Una Figure 2. In vivo rumen ammonia-N levels. nu 00990> 90 90 9002 9 00.0: 90.00 99.00 00.0: 00.00 95090590950 0090 09.00 00.90 99.00 09.90 09.0: 09509500 09053 0095 00.0 00.0 09.9 00.9 90.0 95090590050 005 90.0 90.0 00.9 00.9 99.0 09509500 09053 005 900909.950me 00.00 00.00 90.00 00.00 00.90 95090590950 0095 99.00 00.0: 00.0: 00.0: 00.0: 09509500 09052 0000 00. 90.9 00. 00. 00.9 95090590550 005 99.0 09.0 00.0 00.0 00.0 09509500 09053 0 5 005 005 005 005 005 00905509 099 09 09 09 09 950990099 0599 'klr Ill 9090>09 5-0950555 09990 59 .09 09509 40 «IQSQJ u o Annonlo—N us “‘ Protozoa Wholocontont — Profoxoo Sdpornotont so". No ProtoxooWholo Contont uum No Protozoo Soporuofont 1'! mo Ammonia-N levels for in vitro fermen- tation with urea added-00 media and water added to media. us Data for rumen ammonia-N levels and for the acetate:pro- pionate, acetate:butyrate and propionate:butyrate ratios at T0 and TH from this experiment were combined with similar data from Klopfenstein 33 El' (1966) (Appendix Table l). Regresr» sions for each ratio and rumen ammonia-N levels were calculated. The acetate:propionate ratio at T0 and TM is plotted in Figure u and Figure 5. 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