RELATIONSHIP OF SILAGE FERMENTATION AND ADDITIVES T0 DRY MATTER 7 p CONSUMPTION BY RUMINANTS Thesis for the Degree of Ph. D.’ MICHIGAN STATE UNIVERSITY ERSKINE HAMILTON GASI-I 1972 LIBRA RY « A o- 1‘ 15%|. n‘ : :3h‘ "'6 Univcrsi 1“] was This is to certify that the thesis entitled Relationship of Silage Fermentation and Additives to Dry Matter Consumption by Ruminants presented by Erskine Hamilton Cash has been accepted towards fulfillment of the requirements for Ph.D. dimAnimal Husbandry Date 0-7639 . BUUK MNDERYINC.r 35555:“??? I ‘amoma av HDAB & SUNS' W510 ABSTRACT RELATIONSHIP OF SILAGE FERMENTATION AND ADDITIVES TO DRY MATTER CONSUMPTION BY RUMINANTS BY Erskine Hamilton Cash Six experiments were conducted to investigate the rela- tionship of corn silage fermentation and additives to dry matter consumption by ruminants. A common objective of all six experiments was to vary the level of unidentified water soluble nitrogen fraction in corn silage and determine its effect on ruminant nutritional parameters. Experiment I was designed to measure the effects of maximizing fermentation of corn silage with limestone treat- ment and minimizing fermentation with formic acid treatment on steer performance, silage nitrogen and acid fractions, and steer metabolic parameters. The formic acid and lime- stone treated silages were compared with a control silage receiving no treatment. All three silages were fed to steer calves in a 161 — 238 day feeding trial and the silages were compared on both an all silage ration and a 60% corn silage and 40% high moisture shelled corn ration on a dry matter basis. Lactic acid levels were significantly (P < .01) increased and decreased with limestone and formic Erskine H. Cash acid treatments, respectively (control — 9.44% of DM, formic acid treated — 2.61% of DM, and limestone treated - 14.94% of DM). Formic acid treated silage contained only about 25% as much lactate as the control silage and the limestone treated silage resulted in a 74% increase in lactate content compared to the control. The unidentified nitrogen levels were not significantly different for the three treatments. The unidentified nitrogen compounds made up 41% of the total nitrogen in the control silage and 34% of the total nitrogen in the formic acid and limestone treated silages; therefore, varying the extent of fermentation did not greatly affect the silage unidentified nitrogen levels. Average daily gain was essentially identical for cattle fed the three treated silages (.80 kg). Eighty—five percent dry matter consump- tion for the three silages was control - 7.88 kg, formic acid treated - 7.78 kg and limestone treated - 7.58 kg. The decreased consumption for the limestone treated silage fed group was offset by an improvement in feed efficiency (con- trol - 9.88, formic acid treated - 9.87 and limestone treated - 9.50). Feed cost per 100 kg gain was elevated for the formic acid treated silage fed cattle compared to the other two silages due to the cost of the formic acid. Car- cass grade averaged middle to high Choice for all groups of cattle with small differences being significant (P < .01). Small differences in marbling (moderate — to slightly abun- dant) were also significant (P < .05) and differences in other'carcass traits were not significant. The nitrogen Erskine H. Cash digested (g/day), nitrogen retained (g/day) and nitrogen retained as a percent of nitrogen intake were significantly higher (P < .01) for the control silage compared to the two treated silages. Other metabolic parameters were not signi— ficantly different (as shown in Experiment III). Experiment II was designed to measure the effects of stimulating fermentation with NPN additions on steer per- formance, nitrogen balance parameters and silage nitrogen and organic acid fractions. Five silage treatments were studied: control untreated silage, Pro-Sil supplemented and treated silages, urea-mineral treated silage and urea-mineral plus formic acid treated silage. The silage treatments were com- pared on an all silage ration and a 60% corn silage and 40% high moisture shelled corn ration on a dry matter basis. The neutralizing effect of Pro—Sil and urea-mineral without for- mic acid resulted in stimulated fermentation and bacterial activity, yielding significantly (P < .01) greater lactic acid levels (control - 7.75% of DM, Pro-Sil and urea-mineral treatments — 10.79% of DM). The urea-mineral plus formic acid treated silage resulted in significantly (P < .01) less lac- tic acid than the control (5.36% of DM and 7.75% of DM). Total nitrogen was significantly (P < .01) increased approx- imately 50% by NPN additions compared to the control untreated silage. Water insoluble nitrogen levels were significantly (P < .01) higher for all NPN treated silages compared to the control; however, unidentified nitrogen levels were not significantly altered by stimulating fermentation with NPN Erskine H. Cash additions (range of unidentified nitrogen, .52% — .69% of DM). Average daily gains were significantly higher for the NPN treated silages (.88 kg) than for the control silage sup- plemented with soyumineral (.80 kg) (P < .05) or Pro-Sil (.75 kg) (P < .01). Feed consumption varied from 7.57 kg to 8.05 kg on 85% dry matter basis. Feed efficiency followed average daily gain and feed cost favored the NPN treated silages. Carcass grade for all groups of cattle averaged between low and middle Choice; however, the Pro-Sil supple- mented silage fed cattle were significantly (P < .05) lower in carcass grade and marbling than the control silage fed group. The Pro-Sil supplemented silage fed group had signi- ficantly (P < .05) less fat thickness than the control and Pro-Sil treated silage fed groups. Fat thickness was also significantly (P < .05) lower for the urea-mineral plus formic acid treated silage than the control silage fed cat- tle. Percent kidney, heart and pelvic fat significantly favored the Pro-Sil supplemented (P < .01) and the urea- mineral plus formic acid treated (P < .05) silage fed groups, compared to all other treatments. Other carcass traits were not significantly different. There were no significant dif- ferences in nitrogen balance parameters in this study (as shown in Experiment IV). Results of Experiments I and II were summarized across level of silage contained in the ration. Average daily gain was significantly (P < .01) higher for the 60% corn silage and 40% shelled corn fed cattle than for the all silage fed Erskine H. Cash cattle (.92 kg vs. .73 kg). Dry matter intake (8.13 kg vs. 7.40 kg) and feed efficiency (8.84 vs. 10.14) paralleled average daily gain. The all silage ration produced 51% more beef per hectare of corn grown (1730 kg vs. 1149 kg) and, consequently, returns were greater per hectare of corn grown ($1191 vs. $803). All cattle graded middle to high Choice; however, cutability significantly (P < .01)favored the all silage fed cattle due to less fat thickness. Experiment V was designed to monitor changes occurring during fermentation in untreated control and Pro-Sil treated corn silage. Samples were taken when fresh and on days 1 through 10, 15, 20, 30, 60 and 90 after ensiling. The pH of the control silage decreased rapidly from 5.72 in fresh mater- ial to 4.65 on day one. This was apparently due to the large increase in lactate from 0 to 1.60% of silage dry matter. Most of the initial increase in soluble nitrogen in the con- trol silage (fresh - day one) was due to an increase in unident- ified nitrogen which increased from .29% in the fresh mater- ial to .43% of dry matter on day one. The majority of the proteolytic activity occurred early in the fermentation pro- cess (fresh - day one). The insoluble nitrogen level in the Pro-Sil treated silage increased to a maximum of 1.23% of silage dry matter on day 5 when the soluble nitrogen as a percent on total nitrogen had decreased to a low level. The unidentified nitrogen was slightly higher in the Pro-Sil treated silage than in the control silage. Therefore, the increase in insoluble nitrogen was not due to a reduction in proteolysis and evidence suggests that soluble nitrogen Erskine H. Cash compounds in corn silage may be incorporated into microbial protein. The lactate content of the Pro—Sil treated silage was higher than in the control silage after day two on all but one sampling time. The pH on day 90 was 4.08 and 4.05 for the Pro-Sil treated and control silages, respectively. Experiment VI was designed to vary the resulting level of unidentified water soluble nitrogen compounds occurring in fermented silage and to determine the effects of these compounds on silage dry matter intake and metabolic and blood parameters. The four silage treatments utilized to vary the unidentified nitrogen levels were untreated control - 31.68% DM, autoclaved and reinoculated - 32.85% DM, sun dried - 52.13% DM and air dried - 84.51% DM. The unidentified nitro- gen ranged from 15.95% to 33.89% of total nitrogen for the air dried and control silages, respectively. Dry matter 75 and intake ranged from 63.47 to 68.48 g/kg body weight’ was not significantly affected by altered unidentified nitro- gen levels. Nitrogen balance parameters were not signifi- cantly different for the four silage treatments. In yitrg studies indicated that the unidentified nitrogen was con- verted to volatile base (NH3) within 12 hours. RELATIONSHIP OF SILAGE FERMENTATION AND ADDITIVES TO DRY MATTER CONSUMPTION BY RUMINANTS BY Erskine Hamilton Cash A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1972‘ \v-Auu cnUb-‘n SP" " --.~$. bu -. 'w I '3‘"? "my; Erskine Hamilton Cash candidate for the degree of Doctor of Philos0phy [HSSERTATION: Relationship of Silage Fermentation and Addi- tives to Dry Matter Consumption by Ruminants OUTLINE OF STUDIES: Major Area: Animal Husbandry (Ruminant Nutrition) Minor Subject: Biochemistry BIOGRAPHICAL ITEMS : Born: June 21, 1947; Spottswood, Virginia Undergraduate Studies: Virginia Polytechnic Institute, 1965-1969 Graduate Studies: Michigan State University, 1969-1972 EXPERIENCE: Graduate Teaching Assistant, Michigan State University, 1969-1972 BEWBER: American Society of Animal Science Alpha Zeta Phi Kappa Phi National Block and Bridle Club ii ACKNOWLEDGMENTS The author extends his appreciation to Dr. Hugh E. Henderson for his advice, guidance and patient counsel throughout his graduate program. His encouragement and enthusiasm have been greatly appreciated. Appreciation is also extended to Dr. Werner G. Bergen for his advice and assistance in interpretation of labora— tory results. The author is further indebted to the other members of his graduate committee, Dr. J. T. Huber and Dr. Richard W. Luecke, for their advice and participation in the writer's graduate program. The author also wishes to thank Dr. Ronald H. Nelson for making the facilities of Michigan State University avail- able for this research. The author extends his appreciation to Dr. William T. Magee for his assistance in the statistical analysis of data and to Mrs. Phyllis A. Whetter for her assistance in labora— tory analysis. Appreciation is also extended to Mrs. Susan B. Steiner for her assistance and typing of this manuscript. The author is grateful to his wife, Wilhelmina, for her understanding patience and encouragement throughout the author's career. Also, the author is grateful to his parents for their support. ... 111 volt— \ gnu. ...- \ net. '9 TABLE OF CONTENTS LIST OF TABLES . . . . . LIST OF FIGURES . . . . . I. II. III. INTRODUCTION . . . . . LITERATURE REVIEW . . Silage Fermentation Carbohydrate Breakdown Protein Breakdown Silage Additives . Urea . . . . . Limestone . . . Pro-Sil . . . . Formic Acid . . Mineral Acid and th Other Additives Control of Voluntary Intake Voluntary Silage Intake Summary . . . . . MATERIALS AND METHODS Experiment I — Steer Feeding Design . . . . Harvesting of Silages Feeding Trial . Silage Analysis Experiment II - Steer Feeding Trial 2 Design . . . . Harvesting of Silages . Feeding Trial . iv e AIV Method Trial Page .viii . xi . l . 3 . 4 . 7 . 10 . 15 . 16 . 20 . 23 . 25 . 30 . 30 . 30 O 38 . 46 . 47 . 47 . 47 . 47 . 50 O 50 . 52 . 52 . 52 . 55 IV. Experiment III — Metabolism Study with Corn Silage Varied in Extent of Fermentation . Design . . . . . . . . . . . . . . . . . . Feeding Regime . . . . . . . . . . . . . . . Sample Collection . . . . . . . . . . . . . Laboratory Analysis . . . . . . . . . . . . Experiment IV - Nitrogen Balance Study with NPN Silage Additives . . . . . . . . . . . . . DeSign O O O O O O O O C O O O O O O O O 0 Experiment V - Silage Fermentation Study . . . Design . . . . . . . . . . . . . . . . . . Silage Analysis . . . . . . . . . . . . . . Experiment VI - Intake, Metabolic and In Vitro Studies of Corn Silage Containing Varying Levels of Unidentified Soluble Nitrogen Design . . . . . . . . . . . . . . . . . . Forage Preparation . . . . . . . . . . . . . Experimental Silos . . . . . . . . . . . . . Feeding Regime . . . . . . . . . . . . . . . Sample Collection . . . . . . . . . . . . . Intake Period . . . . . . . . . . . . . . Collection Period . . . . . . . . . . . . Laboratory Analysis . . . . . . . . . . . . Procedures for In Vitro Fermentation . . . . Statistical Analysis . . . . . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . . . . Experiment I - Feeding Trial 1 . . . . . . . . Chemical Analysis of Silage . . . . . . . . Nitrogen Fractions . . . . . . . . . . . Organic Acids . . . . . . . . . . . . . . Effect of Maximizing and Minimizing Silage Fermentation on Performance of Feedlot cattle O O O O O O I O O O O O O O O O O O 55 55 56 59 60 60 60 62 62 64 64 64 65 67 67 67 67 69 70 71 72 73 73 73 73 75 77 Experiment II - Feeding Trial 2 . . . . . Chemical Analyses of Silage Containing NPN Additions 0 o o o o o o o 0 o o o o. Nitrogen Fractions . . . . . . . . . Organic Acids . . . . . . . . . . . . Effect of Additives on Feedlot Performance All Silage Ration vs. 40% Shelled Corn and 60% Silage Ration . . . . . . . . . Experiment III - Metabolism Study with Corn Silage Varied in Extent of Fermentation Chemical Analysis of Silage . . . . . . Rumen Ammonia and Blood Urea Concentrations. Rumen VFA Concentrations . . . . . . . . Dry Matter Intake and Dry Matter Digestibility . . . . . . . . . . . . . Nitrogen Balance . . . . . . . . . . . . Experiment IV - Nitrogen Balance Study with NPN Silage Additives . . . . . . . . . . . Chemical Analysis of Silage . . . . . . Dry Matter Intake and Dry Matter Digestibility . . . . . . . . . . . . . Nitrogen Balance . . . . . . . . . . . . Experiment V - Silage Fermentation Study . Control Silage Fermentation Parameters . Pro-Sil Treated Silage Fermentation Parameters O O O O I O I O I I O O O 0 Experiment VI - Intake, Metabolic and In Vitro Studies of Corn Silage Containing Vary1ng Levels of Unidentified Soluble Nitrogen . . . . . . . . . . . . . . . . . Pilot Experiment . . . . . . . . . Chemical Analysis of Silage . . . . . . Dry matter 0 O O O O O O O O O O O I Nitrogen Fractions . . . . . . . . . Organic Acids ... . . . . . . . . . . StUdieS I—rl- Vivo O O O O O O O O O O O O Water Balance Trial . . . . . . . . . Dry Matter Intake . . . . . . . . . . vi 81 82 84 86 91 92 92 94 94 96 99 99 101 101 101 103 107 112 112 116 116 117 120 121 121 124 Dry Matter Digestibility . Nitrogen Balance . . . . Rumen Ammonia and Blood Urea Rumen pH and VFA Concentrations Rumen Dry Matter . . . . Plasma Amino Acid Analyses Studies In Vitro . . . . . CorrelatISn Coefficients . V. SUMMARY 0 O C C O O O O O C O O . BIBLIOGRAPHY . C O C C O O O C O O O . APPENDIX - Correlation Coefficients . vii 125 127 127 131 134 134 136 137 141 147 166 TABLE 10 11 12 13 14 15 LIST OF TABLES Silage Treatments Used in Experiment I - Feeding Trial I O O O O O C O O O O O O I O C Formulation of Soy-Mineral Supplement (45% CPE on DM Basis) . . . . . . . . . . . . Silage Treatments Used in Experiment II Feeding Trial II C O O O O O O O O O O O O O Formulation of Pro-Sil . . . . . . . . . . . Formulation of Urea-Mineral Silage Additive . Experiment III - Metabolic Study Design of Experiment . . . . . . . . . . . . Experiment III - Metabolic Study Treatments Utilized . . . . . . . . . . . . Experiment III - Metabolic Study Formulation of Mineral Supplement . . . . . . Experiment IV — Metabolic Study Treatments Utilized . . . . . . . . . . . . . Experiment IV - Metabolic Study Design of Experiment . . . . . . . . . . . . Experiment VI - Metabolic Study Design of Experiment . . . . . . . . . . . . Experiment VI - Metabolic Study Formulation of Mineral and Vitamin Supplement Average Chemical Analysis on Dry Matter Basis of Silages and High Moisture Corn Fed . . . . Corn Silage Additives Compared . . . . . . . Corn Silage Additives Compared . . . . . . . viii 49 53 54 54 57 57 58 61 61 68 68 76 79 80 TABLE 16 17 18 19 20 21 22 23 24 25 26 .27 2E3 £29 .30 31. 32! 323 I34 35 36 ‘Page Average Chemical Analysis on Dry Matter Basis of Silages and High Moisture Corn Fed . . Corn Silage NPN Additives Compared . . . . . . Corn Silage NPN Additives Compared . . . . . . All Silage vs. 40% Shelled Corn and 60% Silage Ration . . . . . . . . . . . . . . . . . Mean Rumen Ammonia Values . . . . . . . . . . . Mean Blood Urea Values . . . . . . . . . . . . . Mean Rumen Acetate Concentrations . . . . . . . Mean Rumen Propionate Concentrations . . . . . . Mean Rumen Butyrate Concentration . . . . . . . Effects of Silage Additives on Digestion Parameters . . . . . . . . . . . . . . . . . . Effects of Silage NPN Additives on Digestion Parameters . . . . . . . . . . . . . Means of Control Silage Fermentation Para- meters by Days 0 O C O O O O O O O O I O O O O 0 Means of Pro-Sil Treated Silage Fermentation Parameters by Days . . . . . . . . . . . . . . . Cell Wall Constituents and ADF Nitrogen values 0 O O O O O O O O C O O O I O O O O O 0 Pilot Experiment Altering Unidentified Nitrogen Levels 0 I I O O O O O O O O I O O O O O O O 0 Average Chemical Analysis on Dry Matter Basis of Silages O O O O O O O O I O C O O C O O O O O 0 Cell Wall Constituents and ADF Nitrogen Values . . . . . . . . . . . . . . . . . . . . Means for Water Balance Study . . . . . . . . . Means for Dry Matter Intake and Digestibility . Means for Nitrogen Balance Study . . . . . . . . Mean Rumen Ammonia Values . . . . . . . . . . . ix 85 88 90 93 95 95 97 97 98 100 102 105 109 113 115 122 123 126 126 128 130 TABLE 37 38 39 40 41 42 43 44 Mean Blood Urea Values . . . . . . . Mean Rumen Acetate Concentrations . Mean Rumen Propionate Concentrations Mean Rumen Butyrate Concentrations . Mean Rumen pH . . . . . . . . . . . Means for Rumen Dry Matter Determinations Mean Plasma Amino Acid Concentrations Means of Cellulose Digestibility for In Vitro Studies of Corn Silages . . . . . ‘ Page 130 132 132 133 133 135 135 138 LIST OF FIGURES FIGURE Page 1 Barnett - General scheme of fatty acid formation in silage . . . . . . . . . . . . . 9 2 Barnett - Diagram of protein breakdown in Silage O O O O O O O O O O O O O I O O O O 14 3 Experimental Silo Unit . . . . . . . . . . . 63 4 Changes in water soluble nitrogen and lactic acid concentrations during fermentation . . . . . . . . . . . . . . . . 111 5 Changes in water soluble nitrogen and volatile bases during Ig‘Vitro digestion . . 139 6 Changes in water soluble nitrogen and volatile bases during In Vitro digestion (urea added) . . . . . . . . . . . . . . . . 149 xi I INTRODUCTION Research conducted has demonstrated that no other crop will equal corn silage in energy production per acre of crop fed. However, silage rations have been shown to have a depressing effect on voluntary dry matter intake when com- pared to the same crOp harvested in another manner. Gener- ally, feed efficiency is improved on silage rations, thus partially offsetting the reduction in voluntary dry matter intake. Assuming that the reduction in dry matter intake of Silage was eliminated and feed efficiency was unchanged, it can be readily seen that animal production would be enhanced. It appears that products or a combination of products Produced or activated during fermentation may be responsible for'decreased dry matter consumption. Much research has been reported attempting to relate levels of organic acids pro- dIllced in silage during fermentation to the depression in the dRY matter intake. These results have been inconsistent. Liirtle is known relative to the effect of unidentified water SDluble nitrogen compounds on dry matter intake. Therefore, the objectives of this study were: (1) To compare and evaluate the effects of maximizing Euhfl minimizing fermentation without the confounding effect 1 Q~Ih a: I. ,. .rI.‘ ZEZEI H.“ Jbb‘ 73 V . a A I I? u. \ 4 I of added nitrogen on silage proteolysis, metabolic para— meters, dry matter consumption and performance of feedlot cattle. (2) To compare the effects of treating green corn plant material with NPN and minerals on silage fermentation parameters, metabolic parameters and evaluating the protein and mineral adequacy of the treated silages. (3) To define and compare the changes taking place in the silo during the fermentation process in untreated and treated silages. (4) To determine the cause of proteolysis in silage and evaluate the effect of experimentally altered levels of Infidentified water soluble nitrogen compounds in silage utilizing both in vivo and in_vitro systems. II LITERATURE REVIEW A review by Coppock and Stone (1968) refers to ancient methods of storing grain and ensiling crops. The practice of storing grain in pits or underground trenches dates back to the Greeks and Egyptians, as pointed out by Jenkins (1884). In 1843, Johnston described and recommended a German method lfbr harvesting green fodder by packing the direct cut mater- ial into trenches, which were then covered with boards and earth to facilitate sealing. Miles (1918) gave Reihlen, a German, credit for being the first to preserve the whole corn pflant utilizing a silo. Goffart (1877), of Burtin, France, conducted silage exper- iments as early as 1852 and was the first to describe in a practical way the important aSpects of making corn silage. He stressed the importance of reducing the length of cut from four centimeters to one centimeter and the basic need for air exclusion from the silage mass. Silage production and preservation has undergone many Changes, and many questions have been answered but an abun- dance of questions remain unanswered. Today the economic significance of silage as a method ‘Of preserving and storing feed crops is recognized in most countries of the world. Since silage is a perishable 3 4 fermentation product, storage conditions are critical to minimize loss of nutrients and assure a high quality feed- stuff. Many factors affect the establishment and maintenance of an anaerobic atmOSphere and the development of sufficient acid to preserve the ensiled material. There has been an increasing interest in additives to alter fermentation products and correct the nutritional defi- cfiencies of the ensiled feedstuff. An excellent review on silage additives has been written by Owen (1971). One of the major problems encountered in feeding ensiled material is a reduction in voluntary dry matter consumption as compared to companion forage preserved as hay or other dry forms of storage. Thomas EE.El° (1961) and McCullough (1962) suggested that the problem is due to compounds formed during the fermentation process. This can explain the in- creasing interest in additives that alter resulting ferment- ation products. The purpose of this review is to evaluate the effect Of silage additives on silage fermentation products and the resulting performance of feedlot cattle. Another objective is to consider factors affecting voluntary dry matter con- Sumption by identifying products of silage fermentation that reduce silage dry matter intake. iiiggg Fermentation It is necessary to have an understanding of the silage fermentation process and the resulting products before one can identify changes in this fermentation process that may ‘lcaa‘ “Hi 5’ i 5“ 5 be caused by silage additives. A knowledge of the origin of fermentation products is required to understand their possible relationship with a reduction in dry matter consump- tion. Annett and Russell (1907) concluded that the major changes which take place during fermentation are a large reduction in nitrogen free extract (later found to be soluble carbohydrate), an increase in nonprotein nitrogen (due to protein breakdown) and an almost complete disappearance of sugars; however, they found little change in fiber content. Barnett (1954) summarized the changes occurring during fermentation in cr0p material ensiled without additives as a four- or possibly five-phase process. Phase 1. Respiration of the plant cells results in the . production of carbon dioxide, the utilization of simple car- bohydrates and a flow of water from the mass due to these biochemical processes and the mechanical compression of the crop are accompanied by these events. Phase 2. Acetic acid is produced in small quantities by organisms of the coliform group. This phase is short and merges into the third phase. Phase 3. Lactic acid organisms, lactobacilli and strep- tococci, supported by adequate carbohydrate, initiate a lactic acid fermentation. Phase 4. The mass reaches a period of quiescence during Which the lactic acid accumulation peaks resulting in a pH 0f less than 4.2. m‘ Ur oin- . ., (I. H :5 4' (I a t.) (I? U. F‘- (.8 6 The four phases require about three weeks for completion with the first 3 being complete after three days. If there is not a sufficient amount of lactic acid to give a pH of approximately 4.2 or less or if air is allowed to penetrate the mass then the fifth phase may result. Phase 5. Butyric acid-producing organisms attack both residual soluble carbohydrate and the lactic acid which has been formed. In extreme cases, there may be deamination of amino acids with the formation of higher volatile fatty acids and ammonia as well as decarboxylation leading to the formation of amines and carbon dioxide. Peterson, Hastings and Fred (1925) noted that after four to five hours, oxygen disappeared and carbon dioxide increased at a rapid rate for about forty-eight hours. They reported large numbers of lactic acid producing bacteria twenty-four to forty-eight hours after ensiling. Utilizing antiseptics, Russell (1907) demonstrated that the living maize cell and plant enzymes are primary and essential during Phase 1 of the ensiling process. He con- sidered microorganisms secondary and nonessential during Phase 1. Peterson, Hastings and Fred (1925), utilizing sterilized Corn inoculated with bacteria to make silage, demonstrated that plant cell respiration was nonessential. They con- cluded that bacteria are mainly responsible for the produc- tion of acids from sugar and starches. Kempton (1958) found that less than 0.1% of the bacteria 3:. the . u 1...»!- ‘A. I: .u‘unvvb - v jwr-a "~H v C)“ (I) (”f 7 on the cr0p at time of ensiling were capable of growing on lactobacillus selection medium. He also found no relation— ship between the initial number of bacteria on the fresh silage crop and final silage quality. Kroulik, Burkey and Wiseman (1955) found few organisms on green plants charac- teristic of those in silage and none were typical of silage lactobacilli. Carbohydrate Breakdown Hunter (1921) proved that the carbohydrate breakdown was due to bacterial action. Watson and Nash (1960) stated that the formation of organic acids is the most striking feature of the silage making process. They considered the action of the microorganisms on carbohydrates of the crop to be primarily responsible for the organic acid formation. These researchers also pointed out that some organic acids do result from respiratory activity which gives rise to alcohol. Geasler (1970) reported a significant positive corre- lation between soluble carbohydrate content and organic acid content of corn silage. He also reported that increasing maturity of the corn plant significantly reduced soluble carbohydrate content of a silage sample taken at 12 days after ensiling and significantly reduced lactic acid content 0f the silage. Hawkins also reported (1969) a negative correlation between total organic acid production and silage dry matter. When studying the degradation of carbohydrates, it is .d v a: HIT.» n: In. C ‘. 'fi necessary to divide organic acids into volatile and non- volatile acids. The major nonvolatile acid, lactic acid, is present in the largest quantity (Barnett, 1954) and is more important in silage fermentation than the volatile acids (Watson and Nash, 1960). The volatile acids in well- fermented silage include acetate with traces of propionic and butyric acids (Barnett, 1954). A general scheme of the formation of organic acids in lsilage was put forth by Barnett (1954) as shown in Figure l. The fate of starch during the ensiling process is unclear. Dox and Yoder (1920) reported a 10 percent loss in starch during fermentation and Peterson, Hastings and Fred (1925) obtained a loss of 26 to 29 percent. Other work showed little degradation of starch (Dexter, Huffman and Benne, 1959). Morgan and Pereira (1962) showed that steam distillates 0f grass-legume and corn silages contained C2 - C6 isobutyric, a and B methyldentyric acids, furfural, phenylacetaldehyde, benzaldehyde, butanone, acetone, C2 - C6 aldehydes, 2 and 3 meflhylbutanal and 2 methylpropanal. Generally a pH of less than about 4.5 is indicative of a desirable silage fermentation. Kroulik it; al. (1955a) found a-SiJMgle pH determination at the end of the storage period to be! unreliable as a measure of acid production because he Obtairned erratic pH values in normal alfalfa silage during “Wage. Watson and Nash (1960) disagree and report that pH;u3 a: satisfactory index of the course of fermentation process. pH Carbohydrates (Lactic acid organisms) 4 (coliforms, etc.) .2 \ Lactic Acid Acetic Acid Propionic Acid III-II... III-III-I III-IIIII’ III-II..- .2 4 (Clostridia) Butyric Acid Figuxne 1. Barnett — General scheme of fatty acid formation in silage. 10 Protein Breakdown The occurrence of proteolysis during the ensiling pro- cess has been well established, but its extent varies greatly. Brody (1965) showed that from 18% to 29% of the total nitro- gen underwent proteolysis depending upon the dry matter of the ensiled crop. Others (Bentley 22.21:! 1955 and Hender— son, gt al., 1970a and 197ld) have demonstrated that nearly half of the crude protein of the fresh corn plant was degraded to ammonia and other NPN during fermentation. En- siling of grass resulted in 65 percent of the total nitro- gen appearing as soluble nonprotein nitrogen compared to 20% in the fresh grass (Hughes, 1970a). Hawkins (1969) reported that 54% of the total nitrogen was soluble non- protein nitrogen in 22% dry matter alfalfa silage. Accord- ing to Fatianoff gt 31. (1966), proteolysis during ensiling is generally very extensive and irregular. Russell (1908), using maize, reported that tryptic enzymes are responsible for the breakdown of protein to andno acids. His work indicates that these changes in pro- tein are functions of living cell protoplasm and plant pro- teolytic enzymes. The microorganisms may attack the nitro- genous products and carry them beyond the stage reached by the enzymes of the cell (Russell, 1908). Kirsch (1930) repeated the work of Russell using red clover. He reported protein breakdown did not occur in antoclaved silage, a process that denatures proteins, even after inoculation with lactic acid bacteria. In silage treated with toluene, at levels to inhibit any possible .4 'l ll bacterial activity, degradation was the same as in the con- trol silage. Kirsch (1930) concluded that proteolysis to the stage of amino acids is due to plant enzymes and further changes are not necessarily due to plant enzymes but may be caused by bacteria. Hunter (1921) and Mabbit (1951) also reported plant enzymes were the cause of proteolysis. Hughes (1970a) working with grass silage reported the percent of nonprotein nitrogen made up by amino acids, vola- tile basic nitrogen (ammonia), and nonvolatile amines to be 63.0, 17.2 and 14.4, respectively. This was from silage ensiled for two months. Amino acids decreased to 50.3 percent and ammonia increased to 25.8 percent by 18 months. The water soluble nitrogen fraction in the initial grass con- tained only 7.3 percent amino acids. Hughes (1970b) reported that high pH spoiled silage resulted in a loss of amino acids and a proportionate increase in volatile basic nitrogen (ammonia). In overheated perennial ryegrass-cocksfoot silage, the ammonia nitrogen was high with complete destruction of certain amino acids. No putrefaction products appeared in this overheated silage (Hughes, 1970b). . Earlier work at Jealott's Hill reviewed by Watson and Nash (1960) indicated that grassland silage in the pH range of 4.0 to 4.49 contained 21.26 percent of the crude protein as amino acids. Hawkins (1969) reported that a amino nitrogen represented approximately 20% of the total nitrogen in 22% dry matter alfalfa silage. Amino acid expressed as a percent of total nitrogen decreased as dry matter content increased. 12 Other research with corn silage (Bergen and Henderson, unpub- lished) indicates that less than .1 percent of the silage dry matter was recovered as amino acids. Geasler (1970) reported that total nitrogen (expressed as a percent of dry matter) in corn silage decreased with maturity as did water soluble nitrogen (expressed as a per— cent of total nitrogen). Protein degradation during ensiling of alfalfa measured by the increase in water soluble NPN (expressed as a percent of total nitrogen) was increased with decreasing dry matter content (Hawkins, 1969). Comparing 40% and 51% dry matter ryegrass wilted silage, Brady (1965) reported less proteo- lysis and amino acid metabolism in the 51% dry matter silage. Proteolysis is very rapid when sufficient moisture is present and the degree of proteolysis is influenced by the time taken in the removal of the moisture (MacPherson, 1952b). Protein degradation in direct cut silage and a slow dried cr0p is very similar with one major exception. Amides, espec- ially asparagine, accumulate rapidly during wilting but a low level of amides and a high ammonia content is observed in silage (MacPherson, 1952b). MacPherson (1962) suggested specific bacterial decar— bOxylases form amines. Gale (1941) demonstrated that bac- terial decarboxylases could produce Y amino - n - butyric acid from glutamic acid. Neumark (1962) showed that many Plants may contain tyrosine decarboxylase, therefore, the Presence of tyramine in silage may be only partially due to 13 bacterial activity; however, tyramine recovered in corn sil- age is not present in the corn plant (Neumark, 1961). Accord- ing to Hughes (1970a), Voss (1966) reported that plant decar- boxylases were active during early stages of ensiling and bac- terial decarboxylases became active during later stages. Hughes (1970a) reported that observations of Voss (1966) could imply that losses of amino acids were principally due to the action of bacterial rather than plant decarboxylases. Nonvolatile amines accounted for some 14 percent of the nonprotein nitrogen in grass silage (Hughes, 1970a). Putres- cine and cadaverine were the main amines present and histamine formed only a minor portion. The concentrations of cadaverine, tyramine and putrescine were sufficient to account for a high proportion of the losses of the respective parent amino acids while the amounts of ethanolamine, B phenylethylamine, trypta- mine and histamine were not. Watson and Nash (1960) state that, "Protein breakdown to simpler nitrogenous substances is obtained from all types of silage. The degree of proteolysis is variable, and cannot be used by itself as a guide to the quality of the silage since the nature of the breakdown products is also of supreme impor- tance." Barnett (1954) has a comprehensive diagram of protein breakdown as shown in Figure 2. Virtanen (1934) showed that protein degradation is al- most inhibited below pH 4.0 and that ammonia nitrogen increa- ses with the pH value. MacPherson (1952a) reported the break- down of protein is extremely rapid but slows down at a pH of 14 Plant Protein (Plant Enzymes) / I Amino Acids Amides (Stable phase at sustained low pH) pH <4.2 L — — _ >4.2 I Amino Acid Breakdown / Decarboxylation Deamination (and oxidation) (and reduction) Amides C0 + Amiges Keto Acids Fatty Acids + NH3 Figure 2. Barnett — Diagram of protein breakdown in silage. 15 about 5. Proteolysis appears difficult to control except by treat- ment with mineral acids. The theory behind the Virtanen (A.I.V.) process of ensiling is that adding mineral acid should immediately decrease the pH, thus preventing protein break- down without using up the carbohydrates to produce organic acids (Peterson 33 a1., 1936). Virtanen (1934) showed res- piration of the plant cells to be suppressed as acidity rose, being only 20 percent of normal at pH 3.5 and ceasing com- pletely at pH 3.0. The A.I.V. process will be discussed in more detail later. Silage Additives In this section attention will be given to additives directed toward controlling the rate and level of endproducts of fermentation and to those designed to improve the nutri— tive value of corn silage, particularly, by elevating the crude protein content. Major consideration will be given to those that are considered to have the greatest potential and economic significance. Other reviews covering silage addi- tives were written by Watson and Nash (1960), Barnett (1954), COppock and Stone (1968), and Owen (1971). Many additives contribute to the nutritive value and directly or indirectly affect the products of fermentation. Legume forages normally contain excess levels of protein, ‘larotene, and calcium. Legume forage nutrient additives itre generally high in fermentable carbohydrates. The high cEarbohydrate containing additives serve as a supplemental 16 energy source and stimulate lactic acid fermentation which is often inadequate in high moisture legume silages. Grain crop silages are high in available energy and fermentable carbohydrate, but low in protein and minerals. Therefore, the addition of nitrogen and mineral containing additives at time of ensiling has become widely studied and practiced. Urea Urea added to corn silage is hydrolyzed by urease con- tained in the fresh chOpped corn plant resulting in higher levels of ammonia than the control untreated corn silage (Hastings, 1944 and Karr 3E 31., 1965). The later workers reported that 28 to 50 percent of the urea added to corn silage at ensiling was hydrolyzed. Most of the hydrolyzed urea is recovered in the form of ammonia (Huber 33 31., 1968b; Henderson 33 31., 1970a; and Lopez 31 31., 1970). Urea recov- ery from treated silage has varied from 4% to 84% (Bentley E3 31., 1955; Hatfield 3E 31, 1966; and Huber 3E 31., 1968b); however, it is generally agreed that about 50% of the urea applied remains as urea for silage in the dry matter range of 28 to 40 percent. Urea treated silage was found to have a higher pH than untreated silage as reported by Davis (1944) and Cullison (1944). Klosterman 3E 31. (1963b) reported that the buffer- in'g capacity of ammonia arising from urea hydrolysis pro- duced a higher pH and increased levels of lactate and acetate in_ urea treated silage. Formation of ammonium salts result- 1n€g from the combination of ammonia and organic acids 17 produced in the silage has been suggested by Bentley 31 31. (1955), Henderson, 33 31, (1969) (1970a), (197ld) and Johnson 33 31. (1967). In the rumen the ammonia from ammonium salts is released more slowly than ammonia from urea and this may facilitate rumen microbial growth (Wetterau and Holzchub, 1960 and 1961), as reported by Coppock and Stone (1968). Belasco (1954), utilizing 13_!1E£3 studies, showed that the nitrogen utiliza- tion from ammonium salts is greater than that from urea. Ammonium lactate and ammonium succinate were especially higher than urea. According to Belasco, ammonium salts have a stim- ulatory effect on nitrogen fixation by rumen micro flora. Varner 3E 31. (1971) reported no difference in digesti- bility of dry matter, organic matter, cellulose or crude pro- tein when rations were supplemented with either ammonium salts, urea or soybean meal. Higher rumen ammonia levels were ob- tained with urea and ammonium salts. Steers fed soybean meal or ammonium salts either in mixtures or separately retained more of their dietary nitrogen daily as compared to steers fed urea. Using feedlot cattle, Varner 3E 31. (1968) found that mixtures of ammonium salts high in ammonium propionate were superior to urea and ammonium acetate, and equal to soy- bean meal as measured by cattle performance. High levels of acetate in the mixture of ammonium salts resulted in perfor- Imnnce that approached or equaled urea. Allen 33 31. (1971) compared ammonium acetate, ammonium lactate, urea and soybean meal as sources of supplemental nitrogen for feedlot steers receiving a high concentrate l8 ration. The ammonium lactate supplemented group gained sig— nificantly (P < .05) faster than the urea and soybean meal supplemented steers. The ammonium acetate supplemented group gained significantly faster than the urea supplemented group. Feed efficiency followed average daily gain. Allen 33 31. (1972) observed no significant increase in gain when ammonium salts were compared to soybean meal and urea as sources of supplemental nitrogen for feedlot steers. It is well documented that urea treated silage results in higher levels of water insoluble protein than the control (Rayetskaya 3E 31., 1964; Johnson 33 31., 1967; and Henderson 313 31., 197ld). Rayetskaya 3E 31. (1964) utilizing N15 labeled urea reported that higher true protein content of urea treated silage is due to both a protein Sparing effect and increased bacterial synthesis. A comparative feeding trial with urea treated corn sil- age and untreated corn silage utilizing lambs indicated that apparent dry matter digestibility was similar and apparent crude protein digestibility was improved (Bentley, Klosterman and Engle, 1955, and Wetterau, 1959). Bentley 3E_31. (1955) also reported increased 13_vivo cellulose digestibility for the urea treated silage. Gorb and Lebedinsky, 1960, (as reported by Owens, 1968) found small increases in apparent digestibility of dry matter and crude protein for urea theated silage as compared to control corn silage when fed to lambs. Schmutz (1966) utilizing wethers reported that urea treated corn silage when compared to urea-limestone treated l9 silage resulted in a slight depression in digestibility of dry matter, crude protein, and ash, with a significant reduc- tion in crude fiber digestibility at 0.5 percent urea. Lambs were reported to retain more nitrogen when urea was added at time of ensiling (Karr 33 31, 1965). Hatfield 3E 31. (1966) concluded that supplemental energy was more effectively utilized when urea was added at ensiling. Hender- son and Purser (1968) reported reduced dry matter consumption of beef heifer calves when fed urea additions added to silage at time of feeding versus urea added at ensiling and control supplemented with soybean meal, the latter two were similar. Differences in gain were small for heifers receiving urea treated and urea supplemented silages. They also reported reduced daily gain of beef heifers receiving urea treated silage in comparison to control silage supplemented with soy- bean meal. Urea reduced feed cost, however. McClure 3E 31. (1972) observed similar daily gain and feed efficiency values for steers fed urea treated and urea supplemented silages. Grain was fed at approximately 1 percent of body weight. Early work with urea showed reduced ration acceptability but no reduction in milk production (Woodward and Sheperd, 1944, and Wise, 1944). Huber, Thomas and Emery (1968a) found no difference in average production of cows fed urea treated silage as compared to untreated silage. These authors suggest that high heat of fermentation may render nitrogen unavail- able since the persistency of lactation was lower for cows on urea treated high dry matter corn silage (44 - 45%). When corn silage is treated with 0.5 percent urea, a 20 concurrent reduction in the natural protein content of the concentrate from about 18 to 13 percent did not depress milk yield, whereas yields are reduced without the urea (Huber 33 31., 1967). Huber 3E 31. (1967) reported that it may be possible to increase the level of urea in silage to 0.85 per- cent when urea is not present in the grain ration; however, when the ration contained 0.5% urea treated silage and a 1% urea grain mixture, milk yields were depressed (Owen, 1968). Limestone Klosterman 33 31. (1961a) suggested that the high feeding value of corn silages might be due to their organic acid con- tent. They also found that the amount of acetic and lactic acids in these silages could be markedly increased by the addition of neutralizing material at the time of ensiling. Treating whole plant corn silage of about 30 percent dry matter with 1 percent high-calcium limestone (36.7% Ca and 0.29% Mg) or 0.5 percent high—calcium limestone and 0.5 percent urea produced increases up to 100 percent in the acetic and lactic acid content (Klosterman 3E 31., 1961b, and Klosterman 3E 31., 1963b). Johnson 33 31. (1967) con- firmed the previous findings with limestone-urea treated Silage and also reported that the amount of acids (lactic and acetic) produced, decreased as dry matter content of the Silage increased. In eight experiments utilizing 600 steers and heifers, Klosterman 33.31, (1963b) reported that the addition of 1 Percent high-calcium limestone or 0.5 percent limestone and 21 0.5 percent urea to either whole plant corn silage or ground ear corn silage increased feed efficiency. Cattle fed the treated silages gained significantly faster in four of the eight experiments. Immature corn silage (20% dry matter) treated with 1 percent high—calcium limestone was higher in pH and lactate content but resulted in lower intake and rate of gain in growing heifers (Nicholson and Cunningham, 1964). Harvey 33 31. (1963) found no difference in feed effi- ciency or gain of beef calves fed corn silage treated with 0.5% limestone and those fed the control corn silage. Schmutz (1966), using 28 percent dry matter corn silage, compared silages containing (a) no additive, (b) 0.5 percent urea, (c) 0.75 percent urea, (d) 0.5 percent limestone, (e) 0.5 percent urea plus 0.5 percent limestone, and (f) 0.75 percent urea and 0.5 percent limestone. All rations were isonitrogenous. Significantly higher gains were reported for heifers fed a, b and c as compared to those fed d and f. Limestone treated corn silage fed to lactating dairy cows has given inconsistent results with reSpect to intake and milk production. There was no difference in silage dry matter intake or in milk yield of cows fed either untreated silage, silage treated with 0.5 or 1.0% limestone, or un— treated silage plus 1 percent limestone added at feeding (Byers, 1964; Huber, 1966; and Schmutz, 1966). Kesler 33 31. (1964), McCullough 33 31. (1964) and Simkins (1965a) reported greater dry matter consumption by cows fed untreated silage as compared to the limestone treated silage. McCullough 22 33 31. (1964) also reported a decrease in milk production for cows receiving limestone treated silage. A summary of urea and limestone treatments prepared by Essig (1968) and reviewed by Owen (1971) reached the fol- lowing conclusions: 1) Limestone addition between 0.5% and 1.0% increased the total organic acid production especially lactic acid. Limestone additions greater than 0.5% reduced total titra- table acidity and increased pH as the level of limestone increased. It appears necessary to limit limestone to 1% or less in order to maintain a pH of 4.5 or less. 2) Limestone generally increased acceptability of the silage or had no effect, and urea addition improved intake when compared with untreated silage fed without added pro- tein. Compared with natural protein supplements; silages treated with high levels of urea were consumed in lower amounts but usually had no effect when a level of no more than 0.5% was included. 3) Average daily gain was not affected by silage treated with 0.5% to 1.0% limestone. 4) The addition of up to 1.0% of limestone generally improved feed efficiency. 5) Limestone additions had no effect on digestibility Of organic matter, cellulose or protein of whole plant or ground ear corn silage. 6) Ensiling losses of dry matter were increased by the addition of 0.5% to 1.0% urea to corn at ensiling time. Increased gas production and lactate levels were associated 23 with these losses. Ammonia levels were elevated six to nine times the amount in untreated silage. 7) A combination of urea and limestone addition pro- duced effects similar to those produced when each was added separately. 8) The combination of urea and limestone addition did not affect gain, but feed efficiency was improved by about 5% over the control. Pro-Sil Pro-Sil is a suspension of ammonia, minerals, and mol- asses formulated to supply all supplemental protein and min- erals needed to make corn silage a balanced ration for feed- lot cattle (Henderson 33 31., 1970a), non-lactating dairy cattle or cows producing below 40 pounds of milk (Huber 33 31., mimeo D-236). Henderson 33 31. (1971e) reported that corn silage treated with Pro-Sil significantly (P < .01) increased total nitrogen, water insoluble nitrogen, nonprotein nitrogen, ammonia nitrogen, lactic acid and pH when compared to the control silage. Acetic acid content in Pro-Sil treated silage decreased significantly (P < .01). This is in agree- ment with other work (Beattie 33 31q.1971). Beattie 33 31. (1971) reported that nitrogen fraction- ization of the Pro-Sil treated silage revealed 21% of the increase in total crude protein was in the form of water insoluble protein, 58% in the form of ammonium salts, and 21% remained as unidentified nitrogen compounds. Approximately 24 95% of the nitrogen added as Pro-Sil was accounted for by the increased crude protein content of the Pro-Sil treated silage. Pro-Sil treated corn silage has given results equal to control silage supplemented with soybean meal and urea treated silage for feedlot cattle receiving an all silage ration and a 60% corn silage - 40% shelled corn ration on a dry matter basis (Beattie 33 31., 1971; Henderson 33 31., 1971e; and Henderson 33 31., 1971b). Henderson 33 31. (1971c) and (197ld) reported no significant difference in daily gains due to nitro- gen source when yearling steers and steer calves were fed varying concentrate levels in combination with either Pro-Sil treated silage or control silage supplemented with soybean meal. Little difference in feed consumption was reported for the various protein sources.) Feed cost favored the NPN treated silages in all cases. Pro-Sil treated rye silage fed steers gained faster and more efficiently than steers receiving urea supplemented sil- age and feed costs were lower for the Pro-Sil silage fed steers (Henderson 33 31., 1970b). Higher milk yields, less average change per day and greater milk persistencies have been noted for high producing cows fed Pro-Sil treated silage than those receiving control silage or silage treated with urea or urea plus minerals (Huber 33_31., 1971a; Huber 33 31,, 1971b; and Huber 33 31., mimeo D-236). Their data also indicates that Pro-Sil will maintain produc- tion even on silage of high dry matter content (40% DM) which is an advantage over higher dry matter corn silage treated with urea. Huber 33 31. (1968b) reported poor results with 25 high dry matter, urea treated silage (40% dry matter). Beattie (1970), using steers, reported no significant difference in apparent dry matter digestibility, nitrogen digestibility or nitrogen retention when comparing control silage supplemented with soybean meal, Pro-Sil treated silage and urea-mineral treated silage. Henderson 33 31. (1970a) reported dry matter digestibility to be essentially identical in sheep fed Pro-Sil, urea plus minerals and urea alone treated silages. The nitrogen from the ammonia treated silages was used as effectively as nitrogen from urea treated silage and the inclusion of minerals at ensiling appeared to be advan- tageous to the utilization of silage nitrogen. Lichtenwaler (1971) observed only small differences in dry matter digesti- bility and nitrogen digestibility with lactating cows fed con- trol, Pro-Sil treated and urea treated silages. Formic Acid Much of the work using formic acid as a silage additive has been done in Norway where weather conditions make it dif- ficult to wilt grasses and legumes to a dry matter level cap— able of undergoing a desirable fermentation. Also, wilting often results in heat damage which depresses protein digesti- bility, consequently there was a need for a silage making alter- native other than wilting. The addition of formic acid to grass direct cut, high moisture grass and legume silage has markedly influenced fermentation and improved nutritive quali- ty (Saue and Breirem, 1969a). Norwegian experiments demon— strated that formic acid treated silage was equal to 26 artificially dried grass and better than untreated silages or hay when fed in mixed rations for milk production and growth (Saue and Breirem, 1969a and Saue and Breirem, 1969b). Waldo 33 31. (1969) conducted experiments with Holstein heifers fed formic acid treated (0.5%) unwilted silage. Daily gain and digestibility of energy of the formic acid treated unwilted silage were increased by 12% and 14%, respectively, when compared to control untreated hay. The crOps utilized were orchardgrass and alfalfa. The formic acid treated silages were lower in pH, butyric acid, acetic acid and ammonia nitrogen but higher in lactic acid level when compared to untreated unwilted silages. More rapid gains were made on the formic acid silage from equal digestible energy intake, which indicated greater efficiency of utilization of digestible energy. Waldo 33 31. (1971a) compared both direct-cut alfalfa and sudan-sorghum hybrid ensiled untreated or treated with 0.5% of a 90% solution of formic acid. Formic acid treatment reduced silo storage losses of dry matter, energy, nitrogen and sugar. The reduced dry matter loss is consistent with results of Derbyshire 33 31. (1969). The formic acid treated silage was lower in pH and butyric acid content and similar in acetate level when compared to untreated silage. Although lactate in formic acid treated silages was only about half that in untreated silages; in one experiment, formic acid treated alfalfa silage contained twice as much as its control. .Holstein heifers fed formic acid treated silage gained signi- ficantly more than those fed untreated silage. The intake of 27 dry matter or energy was generally greater when the silage was treated with formic acid. Feed efficiency was improved in all experiments (Waldo 33_31., 1971a). In two experiments using wilted orchardgrass silage, for— mic acid treatment increaséd silage intake and dry matter pre- servation and reduced ammoniacal nitrogen and pH (Derbyshire 33 31., 1969 and Derbyshire 33 31., 1970). The treated silage had a higher lactate content and gave a slight increase in milk yield when compared to the control silage in the second experiment. Derbyshire 33 31. (1971) treated orchardgrass and alfalfa- orchardgrass silage with formic acid. Formic acid treated silages were significantly lower in pH, ammoniacal nitrogen as a percent of total nitrogen, acetic acid and lactic acid. When a ratio of 60 : 4O forage to grain was fed, there was a slight reduction in milk yield on the formic acid treated silage; however, the average daily change in fat corrected milk favored formic acid addition. No effect of formic acid on milk yield was noted on a 70 : 30 ratio. These workers also report an increase in daily gain and greater feed effi- ciency for the treated silage when fed to heifers. Waldo 33 31. (1970) compared a 19% dry matter alfalfa silage preserved with formic acid with the same forage wilted to 35% dry matter and untreated. Storage losses were 20% for the direct cut treated forage and only 4% for the wilted untreated silage; however, sugar losses were greater in the wilted untreated material. The low dry matter treated silage was fed to Holstein heifers and resulted in lower digestibility, 28 similar intakes and greater gains and feed efficiency when compared to the wilted untreated material. Results of Thomas 33 31. (1969) indicated that milk yields, intake and digestibility of formic acid treated silage was similar to a control hay ration. Lessard 33 31. (1970) found dry matter intake, digesti- bility and milk fat test were reduced when direct cut, formic acid treated sudan-sorghum silage was fed, but milk yields were maintained slightly better With the treated silage. Wilted, untreated silage resulted in performance similar to the direct cut formic acid treated silage. Recent work (Fisher 33_31., 1970) indicated that wilting sudan-sorghum increased dry matter intake when fed to lactat- ing cows. The addition of formic acid to the direct cut material resulted in lower dry matter digestibility, but the efficiency of energy utilization in terms of milk yield and body weight change was improved. Castle 33 31. (1970a) and (1970b), using Ayrshire cows, reported increased digestibility, intake and milk production with formic acid treated direct cut grass silage. In the first experiment, lactate levels were increased 2 to 3 times by treating a mixture of direct cut timothy and ryegrass with formic acid. No such increase in lactate occurred in the latter experiment when the formic acid treated silage consisted of meadow fescue and meadowgrass. There does not seem to be a consistent trend in lactate cOntent in formic acid treated silage. Wilkins and Wilson (1970) reported that lactic acid content of formic acid treated 29 silage is directly related to the water-soluble carbohydrate level in the plant. Formic acid apparently increased lactic acid concentrations in high moisture grass silages according _ to Saue 33 31. (1969a). Huber (Mimeo D-235) treated 44% dry matter corn silage with formic acid and received an increase in milk yield which he attributed to the increase in silage intake. Formic acid treatment resulted in reduced organic acid levels, NPN concen- trations and ammonia levels. A smaller increase in perform— ance was noted with a 28% dry matter silage; however, the organic acid levels, NPN concentrations and ammonia levels were reduced. Huber (Mimeo D-235) suggested that the reduced NPN concentrations in the formic acid treated silage may have been related to higher intakes of the treated silage. Waldo 33 31. (1971b) reviewed research relating to for- mic acid treatment of hay crop silage and concluded that formic acid treated direct cut grass or legume silage will reduce silo storage loss, proteolysis, total organic acid levels, ammoniacal nitrogen, and pH when compared to untreated wilted silages, untreated direct cut silage and hay. Per- formance traits that are usually improved with formic acid treatment when compared to untreated wilted silage, direct cut silage and hay include: gain, milk production, feed intake, feed efficiency and dry matter digestibility. Additional work needs to be done before definite con- clusions can be drawn concerning formic acid treatment of corn silage. 30 Mineral Acid and the AIV Method Archibald 33 31. (1960) summarized the effects of acid- ifying silage additives over a number of silage parameters. Lactic acid levels were higher, but butyric acid and vola- tile bases were lower compared to control silages. Owen (1971) reported that the corrosive nature of mineral acids is a serious problem which tends to limit their use. Other Additives The use of bacterial cultures, molasses, whey, sterilants and etc. as additives to properly ensiled corn silage appears to be of little or no value. For a review of these additives, see Owen (1971) and Watson and Nash (1960). Control 33_Voluntary Intake The voluntary intake of feed is often the main factor limiting animal production. Consequently, it is of consider— able interest to researchers and has been under investigation for some time. Factors affecting voluntary feed consumption were reviewed by Balch and Campling (1962) and Baile (l968d) and will be of primary concern in this review. Physical limi- tations of the digestive tract and the physiological processes of ruminant digestion and metabolism will be consicbred as fac- tors controlling intake. Components of silage that affect con- sumption will also be considered. A quantitative similarity between sheep and cattle in relation to their voluntary consumption of roughages of dif- ferent apparent digestibilities was reported by Blaxter and Wilson (1962). Differences between sheep and cattle in 31 fermentation,digestion, and utilization of a ration of dried grass and cats were minor (Blaxter and Wainman, 1961a). Therefore, this review will not make reference to the Species of ruminant from which the data were obtained. Balch and Campling (1962) report that voluntary intake of diets consisting mainly of roughages was limited by the capacity of the reticulorumen and by the rate of disappearance of digesta from this organ. The reticulorumen contains appro- ximately 70% of the total gut contents (Coombe and Kay, 1965); therefore, its role in control of intake appears to be signi- ficant. ' The addition of materials (digesta or chopped roughage) given intra—ruminally has been shown to reduce oral intake (Campling and Balch, 1961b and Weston, 1966). Intra-ruminal addition of water in adult sheep and cattle did not affect intake since water rapidly leaves the rumen (Campling and Balch, 1961b). Water bladders placed in the rumen decreased intake (Campling and Balch, 1961b). Pregnancy has been shown to decrease dry matter intake especially during late gestation (Campling, 1966a) and Graham and Williams (1962) observed an increase in passage through the digestive tract as the gestation period progressed. Hulton (1963) as reported by Campling (1966b) used mono- zygotic twin cattle and obtained a 47 percent increase in intake with the lactating twins. Taylor (1959) observed an inverse relationship between internal fat and fill weight in Steers. Differences in physiological volume of the reticulorumen 32 in sheep was reported to affect voluntary intake on an indi- vidual basis (Purser and Mois, 1966). Another factor respon— sible for differences between animals in intake is variation of retention time between animals (Campling et al., 1961a). Differences in retention time may be due to variation in the efficiency of chewing during eating and ruminating or in the amount of movement of the digestive tract or both. The rate of disappearance of digesta depends on its rate of breakdown in the reticulorumen by microbial and mechanical processes (Campling, 1969). The rate of outflow through the reticulo-omasal orifice is dependent upon a reduction in par- ticle size and possibly particle density (Montogomery and Baumgardt, 1965a). Campling and Freer (1962a) found the mean retention time of particles is inversely related to their specific gravity within the range 1.02 to 1.21 g/cm3 and directly related to particle size within a range of 4.8 to 3.2 mm in diameter. ‘King and Moore (1957) reported particles of approximately l.Zg/cm3 in density and 20 to 30 x 10'3 cm3 in size resulted in maximum rate of passage. Pearce and Moore (1964) observed an increased retention time of particles in the rumen due to restricting rumination. Digestibility affects the rate of breakdown to small particle size. Conrad 33 31. (1964) demonstrated a positive relationship between dry matter digestibility and voluntary intake in predominantly roughage diets ranging in digesti- bility from 52 percent to 80 percent; however, above 65 per- cent, intake decreased with increasing digestibility of the 33 diet. When cell wall constituents compose 50 to 60 percent of the forage dry matter, they appear to limit intake (Van Soest, 1965). Alterations in the physical form of roughages has gained popularity as a method of reducing particle size thus reduc- ing retention time in the digestive tract and thereby increas- ing voluntary intake. Three experiments conducted with dried grass, hay and oat straw fed as such or after grinding and pelleting resulted in an appreciable increase (26%) in vol- untary intake of straw only (Campling 33_31., 1963). Grinding a roughage may increase voluntary dry matter intake up to 50% (Weston and Hogan, 1967b), increase daily gain up to 100% (Beardsley, 1964), increase the rate of pas- sage, decrease the retention time (Keith 33 31, 1961), and decrease dry matter digestibility (Rodrigue and Allen, 1960). The inclusion of grain in the diet will remove the effect on dry matter digestibility due to modification of physical form of the roughage (Johnson 33 31., 1964). Passage through the intestines is rapid compared to the rumen (Castle, 1956a and 1956b). Campling (1969) reports that fill in the abomasum and intestines seems unlikely to restrict intake of long roughages; however, ground and pel- leted hay-may reduce voluntary intake. On pelleted hay, large amounts of digesta in the abomasum and intestines inhibit the flow of digesta from the reticulorumen (Campling 33 31., 1966b and Campling 33 31., 1963). In order to account for intake differences due to 34 physical form of diets and thus to refine the relationship between ration nutritive value and intake, Baumgardt (1970) proposed including density in addition to energy measures in the description of ration nutritive value. The term calo- ric density has unit of kcal/ml. Palatability is often considered to be a factor influ- encing intake; however, it is difficult to assess. Green- halgh and Reid (1967) equalized dry matter digestibility and showed a significant difference in intake which they conclu— ded was due to palatability. Their results indicate that palatability and digestibility are of approximately equal magnitude in influencing voluntary dry matter intake. An important factor in the control of voluntary intake that is often overlooked is the nitrogen status of the ani- mal. Egan (1965b) increased dry matter intake of a low nitro- gen chaffed oaten hay by 42% and 12% with duodenal infusions of casein and urea, respectively. Egan (1965a) reported elevated blood urea levels, ruminal ammonia levels and in- creased cellulose digestion from duodenal infusion of casein when a low protein cereal hay was fed. Weston (1967a) in- creased voluntary feed consumption of a wheaten hay by infus— ing protein abomasally or increasing the crude protein con- tent of the diet to 7% or 15% with gluten. Henderson 33 31. (1971f) and Beattie 33 31. (1971) using corn silage rations not supplemented with protein for negative control rations received significantly lower ave— rage daily gains by steers on these rations due to reduced daily dry matter consumption, when compared to protein 35 supplemented rations. There is evidence that ruminants will eat to a constant dry matter rumen fill (Blaxter 33 31., 1961b; Ullyatt 33 31., 1967; and Freer 33 31., 1963). Other work suggests that on some diets ruminants do not eat to a constant fill (Campling 33 31., 1961b and Montgomery 33 31., 1965a). Waldo 33 31. (1965) and Campling (1966b) found a higher percent of dry matter in ruminal digesta in the animals fed hay than silage. The inclusion of concentrate in roughage rations has been shown to increase the dry matter digestibility (Mont- gomery and Baumgardt, 1965b and Bloom 33 31., 1957) and decrease cellulose digestibility (Montgomery and Baumgardt, 1965b and Conrad 33 31., 1963). Increased retention time for the roughage portion of a concentrate roughage ration was reported by Montgomery and Baumgardt (1965b) and Eng 33 31. (1964). Utilizing isonitrogenous rations varying in concentrate to roughage ratios, Cowsert and Montgomery (1969) reported increased apparent digestibility of dry matter, crude protein and energy. Dry matter intake decreased as the percent of concentrate in the ration increased. Montgomery and Baumgardt (1965a) reported that the vol- untary intake of sheep fed completely ground and pelleted mixtures of different proportions of lucerne and maize declined with increasing digestibility so that with each diet the voluntary intake of digestible energy was about the same. Therefore, they hypothesized that ruminants adjust voluntary food intake according to the physiological demand for energy if fill or rumen load does not limit their 36 consumption. Campling 33 31. (1962b) and Hemsley 33 31. (1963) observed increased cellulolytic activity of the rumen microflora, faster disappearance and a resulting increase in intake due to the addition of urea or protein to a low pro- tein roughage ration. Baile and Pfander (1964) suggested that with highly digestible diets distension of the gut was not important in controlling voluntary intake. Perhaps some products of digestion limited the intake of the more digestible diets by ruminants. Mayer (1955), now refuted, postulated that the blood glucose concentration was a factor controlling intake of food in non-ruminants. The theory of the glucostatic con- trol was based on chemoreceptors in the hypothalamus to mon- itor blood glucose levels. In ruminants, peripheral blood glucose concentration is low and infusions of glucose have not generally affected voluntary intake (Manning 33 31., 1959; Holder, 1963; Simkins 33 31., 1965b; and Baile and Mayer, 1968c). Manning 33 31. (1959) suggested that blood acetate concentration in ruminants may act in a similar way to that proposed by Mayer for glucose in non-ruminants. Acetic acid is produced in large amounts in the reticulorumen and is absorbed; it is the only fatty acid normally found in significant amounts in peripheral blood and the concentration changes with time after feeding (Balch and Campling, 1962, and Campling, 1966b). Intravenous infusion of sodium acetate, acetic acid 37 and propionic acid depressed intake in cattle (Dowden and Jacobson, 1960). Glucose, butyrate, valerate, hexanoic acid and lactate intravenous infusions did not alter intake. Holder (1963) reported that intravenous infusions of either glucose or acetate did not affect intake in sheep. Intraruminal infusions of acetate have been shown to decrease intake (Simkins 33 31., 1965b; Weston, 1966; Rook 33 31., 1963; and Baile 33 31., 1965). Montgomery 33 31. (1963) observed a greater effect due to acetate than pro- pionate or butyrate. The regulation of intake by volatile fatty acids appears to be in the rumen since no depression was noted in duodenal infusions of propionate (Egan and Moir, 1965c) or abomasal infusions of acetate (Baile and Mayer, 1967c). Acetate receptors are more likely to be located on the lumen side of the reticulorumen than in an area where they respond to blood acetate since intraruminal infusions of acetate depress intake and intravenous infusions do not (Baile and Mayer, 1968b). These authors stated that the feed intake depression following an acetate infusion is related to sat- iety. The rumen pH is influenced by the buffering action of saliva; however, no consistent effect upon voluntary feed intake has been shown due to dietary buffers (Bhattacharya andWarner, 1968; Kromann and Mayer, 1966; and Huber 33 31., 1969). The extra heat realized during the assimilation of flmad designated as heat increment or specific dynamic effect 38 may be another controlling mechanism of food intake. Brobeck (1948) and (1955) proposed that animals may eat in reSponse to a fall in heat production to keep warm and stop eating when heat production rises to prevent hyperthermia. Work with ruminants has given support to this theory (Balch and Campling, 1962; Simkins 33 31., 1965b; and Conrad, 1966). Other researchers have disputed the theory (Baile 33 31., 1967c and Baile and Mayer, 1968a). There are many changes that occur within the body that directly or indirectly affect centers within the hypothala- mus which in turn control eating behavior (Brobeck, 1955). Baile 33 31. (1967c and 1968d) reported that lesions in the ventromedial area of the hypothalamus produced sustained hyperphagia and subsequent rapid weight gain in ruminants. This review has discussed some of the factors that may act as signals to the hypothalamic centers which include distension of the digestive tract, changes in the concentra- tion of metabolites in the blood resulting from digestion, and a rise in heat production, etc. Balch and Campling (1962) reviewed these and other factors affecting intake of feed by ruminants. More research is needed in the area concern- ing the effect on intake of signals to the central nervous System. Zgluntarnyilage Intake Conrad 33 31, (1964) concluded that factors affecting a ration low in digestibility (52% to 66%) were such things as lxody weight, reflecting roughage capacity, and undigested 39 residue per unit of body weight per day, reflecting rate of passage. Production of the animal, digestibility of the ration and metabolic body size are factors affecting intake of highly digestible rations (67% to 80%). Since corn silage falls in the high digestibility range, factors other than capacity for consumption must be explored. Balch and Campling (1962) reported that intake of a wide range of dried forages was limited by bulk of ingesta within the rumen; however, the quantity of dry matter within the'rumen was less for animals fed silage 33 libitum than for those fed hay 33 libitum (Thomas 33 31., 1961 and Waldo 33 31., 1965). Campling (1966c) found no relationship between the digestibility and the intakes of silages. It appears unlikely that bulk factors limit intake of all silages. Ensiling has received widespread acceptance as an excellent method of forage preservation; however, the ensiling process was shown to reduce the feeding value of the corn plant pri- marily by decreasing the voluntary intake by growing dairy heifers (Noller 33 31., 1963). They reported average dry matter intakes of 2.39 and 1.71 kg per 100 kg body weight for green plant material and silage, respectively. Increases in consumption of dry matter as the plant matured were obser- ved for both the green and the ensiled plant. Dinus 33 31. (1968) utilizing Holstein and Red Danish heifers, reported average dry matter intakes of 1.99 and 1.78 kg per 100 kg for green ch0p and the corn plant material after it had been ensiled. It is well documented that voluntary intake of silage ’_ , 40 by ruminants is lower than that of hay made from the same crop (Moore 33_31., 1960; Hawkins 33 31., 1970; and Gordon 33 31., 1961). Mackenzie (1967) in a review article concluded that, when milk production or weight gains were compared, or both, in ruminants fed silage or hay made from the same plant mater- ial, slightly smaller dry matter intakes of silage were off- set by higher production per unit of dry matter.' Huber 33 31. (1965) utilizing corn silage of 25, 30 and 33 percent dry matter reported dry matter intakes of 1.95, 2.13 and 2.31 kg per 100 kg of body weight. Johnson and McClure (1968) and Klosterman 33 31. (1963a) reported increased dry matter intake with increasing dry matter of the silage. Feed efficiency was poorer on the more mature silage (Klosterman, 1963). Henderson 33 31. (1971a) compared the performance of steers full fed all corn silage rations consisting of 35 and 46 percent dry matter corn silage and observed increased dry matter intake and poorer feed efficiency on the higher dry matter material. Gordon 33 31. (1966) and Byers and Ormiston (1966) reported no significant difference in voluntary dry matter intakes of lactating cows fed corn silage between 27.6 per- cent and 55 percent dry matter. The effect of silage dry matter and consumption was confounded by grain additions. Johnston and Cook (1970) reported a significant cor- relation of 0.65 between corn silage dry matter and dry matter intake. Hawkins (1969) using alfalfa reported a Significant correlation of 0.45. 41 Moisture level of silage or forage does not appear to be responsible for the association between voluntary intake and silage dry matter. Water additions to the rumen (Camp— ling and Balch, 1961b) and increasing the moisture level of alfalfa hay to 65 percent (Mahapatro and Leffel, 1964) pro- duced no consistent effect on dry matter intake. Others (Hillman 33 31., 1958; Hillman, 1959; and Thomas 33 31., 1961) have changed the dry matter of hay and silage and received no effect on dry matter consumption. Thomas 33 31. (1961) concluded that the differences in dry matter intake were due to products of fermentation. The products of silage fermentation resulting from car- bohydrate and protein degradation were reviewed earlier in this review. Possible relationships of some of these pro- ducts and voluntary dry matter intake will be considered here. Many researchers have attempted to relate volatile fatty acids present in the silage to a reduction in consump- tion. Dinius 33 31. (1968) added acetic acid at levels from 0 percent to 6 percent on a dry matter basis to green chop and corn silage. Acetic acid did reduce dry matter intake, but no significant effect on caloric intake was observed. Wilkins 33 31. (1971) reported a negative correlation between the acetic acid content of 70 grass and legume silages and time voluntary consumption by sheep. McDonald and Whit- tenbury (1967) reported that acetic acid is formed in silage mainly by heterolactic fermentation of sugars and organic acids and later through the breakdown of amino acids during 42 secondary anaerobic fermentation as reported by Hutchinson 33 31. (1971). Wilkins 33 31. (1971) reported a positive correlation between acetic acid and ammonia content. It is possible that the low intake was due to low fermentation quality which was characterized by high levels of acetic acid resulting from degradation of amino acids. Hutchinson 33 31. (1971) added acetic acid to ryegrass silage at two levels above that in the control silage. . ; Silage pH and dry matter were held constant using potassium Jana aura-n- hydroxide and water. The percent acetate in the three silages was 2.0%, 5.0% and 8.8% of silage dry matter. When the three silages were fed to sheep, intake over a 24—hour per- iod was unaffected. These researchers also reported that free acetic acid infused into the rumen reduced silage intake; however, the infused level of acetate was similar to the quantity of acetate consumed by the sheep fed the silage containing the high level of acetate. Senel and Owen (1967) observed no reduction in volun- tary intake when 2 percent acetate, 1 percent butyrate, or a combination of these acids were added to a hay-concentrate ration. However, a mixture of 4 percent acetate and 2 per- cent butyrate appeared to cause nasal irritation and reduced intake. Later work by Senel and Owen (1966) using sorghum silage showed increased dry matter intake when acetate was added at levels up to 2.8% of the ration dry matter. Lac— tate addition to sorghum silage at'a low level (5.90% DM) decreased intake but at the high level (9.03% DM) intake 43 higher than the control sorghum silage. Acetate and .ate improved feed efficiency, when compared to the con- ,. They concluded that something other than acetate and .ate was depressing the consumption of silage. Allen 33 31. (1971) observed no reduction in dry mat- intake when acetic or lactic acid was added to a ration isting of 75 percent concentrate and 25 percent corn ge.. Feed efficiency values were similar to the control. Emery 33 31. (1961) reported that lactic acid addition orn silage reduced appetite in proportion to its con— .ration when fed to growing heifers. Corn silage and were fed to appetite. Feed efficiency increased in ct prOportion to the lactic acid intake. This work cates that the increased efficiency will in some cases than compensate for the depressed feed intake. Johnson 33 31. (1962) observed that steers fed a puri- ration supplemented with lactic acid salivated abnor- y; this appeared to be an attempt to neutralize the acid the bicarbonate of the saliva. There are conflicting Opinions regarding the effect of ic acid content of grass silage on dry matter consump- . McLeod 33 31. (1970) reported that sodium bicarbonate tion to grass silage increased the pH from about 4.0 to and resulted in significant increases in dry matter ke ranging from 9.7 to 20.7 percent. The addition of e levels of lactate (77.3% L(+) lactate and 22.7% D(-) ate) to stepwise reduce the pH of a silage from 5.4 to 44 resulted in a maximum decrease in dry matter intake of ercent. Reductions in dry matter intake were propor- al to the amount of lactic acid added and the resulting ease in pH. Other researchers (Harris 33 31., 1966; , 1943; and McCarrick 33 31., 1966) attribute the reduced rted ruminal osmolality elevated above 400 m Osm/kg ilted in decreased feed intake in sheep. This study > indicated that ruminal osmolalities seldom reach this :1 on a high roughage or alfalfa silage ration. ~ I The relationship of reduced dry matter consumption and I :eolytic products will be considered now. Neumark (1964) suggested that histamine may limit the i . ‘ i- Ike of silage by ruminants since amines have been shown Lave pharmacological effects in nonruminants (Neumark, .). In overfed sheep, Dain 33 31. (1955) identified :amineand tyramine as toxic constituents in the rumen. [ark (1964) established a correlation between trypta- : content and silage palatability; however, none of the .es tested were found to directly affect appetite. His- ne enhanced the appetite depressing effect of formalde- :. McDonald 33 31. (1963) found that the addition of :amine to silage did not affect its level of consumption. IOtO 33_31. (1964) and Wrenn 33 31. (1964) confirmed the lings of McDonald 33 31. (1963). Amphetamine has received consideration since it is 'n to decrease food intake in monogastrics. Intraruminal .nistration of d-amphetamine sulfate had no effect on dry :er consumption of sheep and goats (Baumgardt, 1967). He :rved a significant reduction in dry matter intake with 46 intravenous injection of dl—amphetamine phosPhate. Bhatta- charya and Warner (1967) observed similar effects with sub— cutaneous injections of an aqueous solution of amphetamine. Attempts to link amines to the control of dry matter intake have been inconsistent. Further research in this area is needed to clarify the relationship of amines to the voluntary feed intake control by ruminants. . 4 l Summary The explanation of reduced voluntary dry matter con- _ sumption by ruminants fed silage compared to the same har— » I I vested in another manner continues to elude researchers. Perhaps a combination of products of fermentation is respons- ible for the depression in silage dry matter intake. Many silage additives have been utilized to improve the nutri- tive value of the silage and directly or indirectly alter the products of fermentation (mainly lactate and acetate); however, dry matter intake has not consistently been affected. Most of the research completed has attempted to relate acid concentrations in silage to reduced dry matter intake; however, results have been variable. Few attempts have been made to relate the form of the silage nitrogen or unidentified water soluble nitrogen com— pounds in corn silage to the depression in silage voluntary dry matter intake. III 1 MATERIALS AND METHODS Six experiments -— two feeding trials; two metabolic studies; a silage fermentation study; and an intake, meta- bolic and 13 31333 study combined -- are included in this dissertation. All silages for all experiments, unless otherwise stated, were characterized by the methods descri- bed in Experiment I. Experiment I - Steer Feeding Trial 1 Design A 3 x 2 factorial design was utilized to compare the effects of maximizing and minimizing fermentation without the confounding effect of added nitrogen on silage proteoly- sis, dry matter consumption and performance of feedlot cattle. The silage treatments shown in Table 1 were compared on an all corn silage ration and a 60% corn silage and 40% high moisture shelled corn ration on a dry matter basis. garvest13g333 Silages All silages were harvested between August 26 and Septem- ber 23, 1970, from a stand of hybrid corn averaging approxi— mately 35 metric tons of 35% dry matter (DM) silage or 5 metric tons of shelled corn per hectare. All silage was stored in Concrete silos fitted with metal roofs and top unloaders. 47 48 Control silage, which received no additive, was harvested .ng a two week period and stored in a 9.1 m x 18.3 m silo averaged 36.7% DM at harvest. Silage treated with 1.5% (of silage DM) formic acid was rested over a two day period and stored in a 4.9 m x 15.2 m > and averaged 34.1% DM. Silage treated with 13.6 kg of limestone per metric ton 55% dry matter silage was harvested over a two day period stored in a 3.7 m x 15.2 m silo, and averaged 36.4% DM. Limestone was applied by evenly spreading the required int over the top of each self-unloading wagon load of silage : prior to blowing into the silo. Formic acid was applied by pumping the required amount of 11d material directly into the blower housing as each load silage was blown into the silo. The average chemical composition of the corn plant mater- for each silo during harvest and feeding is shown in Table All silages were sampled every Monday, Wednesday and Fri- during the feeding experiment for a dry matter determina- 1. A composite sample of each silage was analyzed every weeks during the feeding trial for nitrogen and organic 1 fractions. (11 silages were supplemented at feeding time with soy—mineral >lement (Table 2) (45% CPE on DM basis) were mixed prior to l feeding at a ratio of 4.5 kg of supplement per 100 kg of ige on a 35% DM basis. All rations were calculated to be isonitrogenous at 13% ie protein on a dry matter basis. 49 Table l ilage Treatments Used in Experiment I - Feeding Trial Additive Added at Supp. Added at of Silage1 Ensiling kg/MT2 Feeding kg/MT2 Control + soy-mineral3 added at feeding 45.0 Formic acid treated + soy-mineral added at . feeding 5.3 45.0 Limestone treated + soy—mineral added at feeding ' 13.6 45.0 . three treatments were compared on all silage rations I 60% corn silage and 40% high moisture corn on a dry .ter basis. - .ograms of supplement or additive added per metric ton ') of 35% DM silage. : Table 2 for formulation of soy—mineral supplement. Table 2 Formulation of Soy-Mineral Supplement (45% CPE on DM Basis) l‘ h; 'edient Percent of Mixture ;lcium phosPhate (20% Ca - 18.5% P) 3.45 (um sulfate (22.5% S) 3.07 :e mineral salt (high Zn) 3.45 Tean meal (50% CPE) 90.04 .L 100.00% I— 50 Feeding Trial Choice Angus steer calves, averaging 229 kg, when pur— chased in September, 1970 were used in this trial. They were acclimated on a full feed of regular corn silage, hay and one pound of 50% soybean meal per head daily until placed on exper— iment October 22, 1970. They were weighed on two consecutive days and the average of the two weights was used as the ini- tial and final weight. The steers were assigned to blocks on the basis of the first-day weight and randomly assigned to the one of the six treatments following the second-day weight. Steers were terminated from the experiment when they reached Choice slaughter grade. Therefore, the number of days on feed varied as did the final weights. While on experiment, all lots of steers were fed 33 libitum twice daily, and water was available at all times. Immediately following the final individual weight, all cattle were trucked 100 miles for slaughter. They were killed upon arrival or early the next morning. After 48 hours in the cooler, carcasses were ribbed, graded by a federal grader, and carcass measurements taken. Kidney, heart and pelvic fat was estimated by the federal grader and fat and lean tracings were made of the 13th rib for accuracy in determining cutability grade, fat thickness and rib eye area. Silage Analysis Composite samples of moist silage were analyzed for nitro- gen and organic acid fractions and expressed as a per cent of dry matter. 51 Total nitrogen was determined by macrOeKjeldahl proce- and percent dry matter determined by oven drying for urs at 550 C. Silage extracts were prepared by homogenizing a 25 g ot of the sample in a Sorvall Omni-mixer with 100 m1 of lled water for two minutes and straining through two 8 of cheesecloth. The pH of the homogenate was deter- by using a Corning Model 12 pH meter. A 27 ml sample of the extract was deproteinized using .1 of 50% sulfosalicylic acid (SSA) per nine ml of (ct. The sample was then centrifuged at 18,000 rpm for nutes and stored in a refrigerator for lateranalysis. I water soluble nonprotein nitrogen was determined by I-Kjeldahl procedures using the deproteinized homogenate. .ia nitrogen in the water soluble nonprotein fraction Letermined by the method of Conway (1950). Volatile ' acid content of the silage was determined by injecting .es of the deproteinized silage homogenate into a .rd gas chromatograph. The column packing used was IOSOIb 101 with a 1.98 in x 0.05 cm teflon column. Car- flow rate was 40 ml per minute N2 and column oven tem- .ure was 1880 C. The peak areas were converted to (grams per 100 ml by comparing with standard solutions .latile fatty acid analyzed at the same time. Color- .c procedures of Barker and Summerson (1941) were used :termine lactic acid content of the deproteinized sample. we W—“r‘” 52 Experiment II - Steer Feeding Trial 2 3133 A 5 x 2 factorial design was utilized to compare Pro- L and urea-mineral for stimulating fermentation and cor- :ting protein and mineral deficiencies of corn silage an fed to feedlot cattle. These NPN treated corn silages it b :e compared with corn silage not treated but supplemented A L“: :h soybean meal at feeding time. All five treatments shown in Table 3 were compared at ‘ E a two levels of concentrate feeding used in Experiment I. . 3 rvesting 33 Silages Silage yield per hectare, storage facilities and har- st dates were identical to those described in Experiment Control silage, which received no additive, was har- sted during a two week period and stored in‘a 9.1 m x .3 m silo and averaged 36.7% DM at harvest. Silage treated with 22.5 kg of Pro-Sil (Table 4) per tric ton of 35% DM silage was harvested over a two day riod, stored in three 4.9 m x 15.2 m silos, and averaged .5% DM. Silage treated with 20.6 kg of urea-mineral (Table 5) r metric ton of 35% DM silage was harvested over a two day riod, stored in a 4.9 m x 15.2 m silo, and averaged 36.1% during harvest. Silage treated with urea-mineral and formic acid was har— sted over a two day period, stored in a 3.7 m x 15.2 m silo, 53 Table 3 Llage Treatments Used in Experiment II - Feeding Trial Additive Added at Supp. Added at a of Silage Ensiling kg/MT2 Feeding kg/MT2 9 Control + soy—mineral3 added at feeding 45.0 Pro-Sil treated“ 22.5 Control + Pro-Sil added at feeding 22.5 Urea—mineral treated5 20.6 Urea-mineral + formic acid treated 20.6 + 5.3 L five treatments were compared on all silage rations i 60% corn silage and 40% high moisture corn on a dry :ter basis. Lograms of supplement or additive added per metric ton P) of 35% DM silage. a Table 2 for formulation of soy-mineral supplement. 2 Table 4 for formulation of Pro-Sil. : Table 5 for formulation of urea-mineral supplement. 54 Table 4 Formulation of Pro-Sil1 lent Percent 18888 16.54 :r and inert ingredients 61.15 :ogen 13.60 :ium .7936 sphorus .4850 .um 2.0450 >rine 3.8420 fur .9371 lesium .4886 : .0597 >er .0088 llt .0002 -ne .0530 XL 100.00% :ent applied for by Michigan State University. Table 5 Formulation of'Urea-Mineral Silage Additive :edient l_‘ 1 grade urea (45% N) ilcium phosphate (20% Ca - 18. .um sulfate (22.5% S) :e mineral salt (high Zn) 1nd shelled corn I_ U; # Percent of Mixture 30.35 5% P) 6.80 6.05 6.80 50.00 100.00% wmmf ~ 55 and averaged 36.8% DM. Both additives were added at levels described previously. Control silage was supplemented at feeding time and mixed in a horizontal mixer with an equivalent of 22.5 kg of Pro-Sil per metric ton of 35% DM silage. Urea-mineral was applied by evenly spreading the required amount over the top of each self-unloading wagon load of silage just prior to blowing into the silo. The average chemical composition of the corn plant mater- ial for each silo during harvest and feeding is shown in Table 16. All silages were sampled for dry matter determina- tion and lab analysis as described in Experiment 1. Control silage supplemented at feeding time with soy- mineral supplement (Table 2) (45% CPE on DM basis) was mixed prior to each feeding at a ratio of 4.5 kg of supplement per 100 kg of silage on a 35% DM basis. I All rations were calculated to be isonitrogenous at 13% crude protein on a dry matter basis. Feeding Trial Procedures described for the feeding trial in Experiment I were followed in Experiment II. Experiment III - Metabolism Study with Corn Silage Varied in Extent of Fermentation Design A 3 x 3 Latin Square design was utilized in this experi- ment. Three all silage rations were fed to three 18-month 56 Hereford steers fitted with permanent rumen cannulas : three 28-day periods. No supplemental nitrogen was fed. atments were randomized by time and animals as shown in Le 6. Out of each 28-day period, 21 days were allowed for ars to adjust to the new ration before being placed in Lection stalls. After an adjustment period of 14 hours arnight) in the stalls, feed intake, fecal output, and i 3 1e production were measured and sampled for chemical analy- over a period of six days. During the day following col- tion, jugular blood and rumen fluid samples were secured ‘ ’.w adiately before feeding and at two hour intervals there- 2 2r up to 10 hours, post-feeding. The experiment was ini— ted on January 17, 1971 and completed on April 10, 1971. ding Regime Steers in the collection stalls were fed twice daily at .m. and 5 p.m. The silage treatments utilized are shown Table 7. All rations were supplemented at feeding with a aral mixture (Table 8) and thoroughly mixed. The steers 3 fed 33_1ibitum during the acclimation period and collec- 1 period. The respective silages were removed from the as just prior to each feeding. Representative samples of all rations were taken just or to feeding for laboratory analysis and dry matter deter- ation. Feed not consumed was weighed, sampled and dis- ied prior to the 8 a.m. feeding. 57 Tflfle6 Experiment III - Metabolic Study Design of Experiment Steer No. Period 1 2 3 -------- Ration--—------ 1 A B C 2 B C A 3 C A B Table 7 Experiment III - Metabolic Study Treatments Utilized Additive Added Ration Silage Treatment kg/MTI A Formic acid 5.3 B Control - C Limestone 13.6 lKilograms of additive added per metric ton (MT) of 35% DM silage. 58 Table'8 Experiment III - Metabolic Study Formulation of Mineral Supplement Ingredient Mineral Supplement1 Dicalcium - phosphate (20% Ca — 18.5% P) 3.89 Sodium sulfate (22.5% S) 3.46 Trace mineral salt (high Zn) 3.89 Ground shelled corn 88.76 TOTAL 100.00% 1Supplement added at rate of 10.4 kg per 100 kg of silage DM to all rations. 59 Sample Collection Total feces were allowed to pass through a wide—space steel grid in the floor immediately behind each steer and were collected in large plastic containers in a pit below the col- lection stalls. Feces were removed once daily and total out- put was weighed. A 5% aliquot was retained each day for nitro- gen determination, a 100 9 sample was analyzed daily for dry matter content and the remaining feces were discarded. At the end of the sixéday collection period, all samples were thor- oughly mixed and a composite 200 g sample was taken for immed- iate total nitrogen determination. Total urine was collected in a plastic carboy (in the pit below the collection stalls) which contained 200 ml of 6 N sul- furic acid. The carboy was emptied daily and urine volume was measured, then diluted to 12 litres with water and an aliquot of 10% stored in a cooler. The remaining diluted urine was discarded. After the six-day collection period, all urine samples were thoroughly mixed and a one-litre composite sample was taken for immediate nitrogen determination. Samples of whole rumen contents were taken through the per— manent rumen cannulas fitted to the steers. Rumen samples were strained through two layers of cheesecloth and 1 ml of mecuric chloride (saturated) was added to 19 ml of the strained rumen fluid in a test tube and retained for rumen ammonia determination. Five ml of the above 20 ml mixture were added to 1 ml of meta- phosphoric acid and centrifuged at 10,000 rpm for 10 minutes. The supernatant was retained for volatile fatty acid analysis. 60 Jugular vein blood samples (10 ml) were taken with a 16 gauge needle into a heparinized test tube and retained for plasma urea analysis. LaboratoryrAnalysis Dry matter of feed and feces samples was determined daily by oven-drying at 600 C for 24 hours. Total nitrogen contents of feed, feces, and urine were analyzed by macro-Kjeldahl procedures on well-mixed wet samples. Rumen volatile fatty acid concentrations were determined. Blood samples were centrifuged at 6,000 rpm for 10 min- utes, and the plasma recovered with a Pasteur pipette. Urea content of the plasma was determined by the micro-diffusion method of Conway (1950). Experiment IV - Nitrogen Balance Study with NPN Silage Additives Design A 4 x 4 Latin Square design was utilized in this experi- ment. The silage treatments utilized are shown in Table 9. Treatments were randomized by time and animal as shown in Table 10. No supplemental nitrogen or minerals were fed. Four Angus steer calves without cannulas were used to study parameters of nitrogen metabolism on four all-silage rations. The experiment was initiated on January 24, 1970 and termina- ted on May 15, 1970. All procedures and analysis for this experiment were identical to those in Experiment III except blood and rumen samples were not taken. 61 Table‘9 Experiment IV - Metabolic Study Treatments Utilized Additive Added Ration Silage Treatment kg/MT‘ A Urea-mineral + Formic acid 20.6 + 5.3 B Urea-Mineral 20.6 C Pro-Sil Supplemented at Feeding3 22.5 D Pro-Sil 22.5 1Kilograms of additive added per metric ton (MT) of 35% DM silage. 2See Table 5 for formulation of urea-mineral supplement. 3See Table 4 for formulation of Pro-Sil. Table 10 Experiment IV - Metabolic Study Design of Experiment Steer No. Period 1 . 2 3 4 ------------ Ration--——--------- 1 B C D A 2 C D A B 3 D A B C 62 Experiment V - Silage Fermentation Study Design A 16 x 2 factorial design was utilized to study the para- meters of corn silage fermentation over time comparing untreated and Pro-Sil treated silages. The silage was harvested Septem— ber 8, 1971 and a fresh sample was taken for laboratory analysis at the time of filling the experimental silos. One gallon glass jars with rubber seal metal lids equipped with gas valves were used as experimental silos as shown in Figure 3. Fifteen jars were filled with 1.5 kg of untreated silage and another fifteen were filled with 1.5 kg of Pro-Sil treated silage. Pro-Sil was applied at a rate equivalent to 22.5 kg/MT of 35% dry matter silage and the silage was mixed in a small cement mixer. The silage was packed by hand in the jars, then a vacuum pump was used to exhaust all air (—.21 kg per cmz). The jars were then filled with carbon dioxide (.21 kg per cmz). The process was repeated and then the CO2 pressure was vented to zero kg per cmz, thus creating an anaerobic atmosphere. The experimental silos were placed in an incuba- tor at 400 C for the first three days, after which, they remain- ed at room temperature (220 C) for the duration of the exper- 2 daily during iment. Pressure was vented to zero kg per cm the fermentation. Silage from one jar in each treatment was removed and frozen on each of the following days for laboratory analysis: 1 - 10, 15, 20, 3o, 60 and 90. Figure 3. 63 Experimental Silo Unit. This one gallon glass jar with a rubber seal metal lid equipped with a gas valve was used as the experimental silo unit. 64 Silage Analysis Silage extracts were prepared by homogenizing a 25 g aliquot of the sample and 100 m1 of distilled water with a Sorvall Omni-Mixer for two minutes and then straining the homogenate through two layers of cheesecloth. A portion of the unstrained homogenate was used to determine total nitro- gen by micro-Kjeldahl procedures. After the first straining, the residue material was resuSpended in distilled water at 600 C and then restrained. The two extracts were finally combined and a 30 ml aliquot of the combiqed extract was used to determine pH and Soluble nitrogen. Another 27 m1 aliquot of extract was deproteinized using one ml of 50% sulfosali- cylic acid (SSA) per nine ml of extract. The sample was cen- trifuged at 18,000 rpm for 10 minutes and stored for nonpro- tein nitrogen, ammonia, volatile fatty acids and lactic acid determinations. Soluble nitrogen was determined by the micro- Kjeldahl method. Other silage parameters were determined as outlined in the procedures for Experiment I. Determinations of neutral detergent fiber (cell wall con- stituents) and acid detergent fiber were made using the met- hod of Goering and Van Soest (1970). Total nitrogen in the acid detergent fiber residue was determined by micro-Kjeldahl. Experiment VI - Intake, Metabolic and In Vitro Studies of Corn Silage Containing'VEry- ing Levels of Unidentified Soluble Nitrogen Design A 4 x 4 Latin Square design was utilized in this 'L4 .L IVY-.7 F 65 experiment. Four silages were fed to four mature crossbred wethers fitted with permanent rumen cannulas over four 22-day periods. Weights were taken on days 5, 15 and 22 of each period. Animals were randomized by time and treatment as shown in Table 11. Out of each 22-day period, 14 days were allowed for the wethers to adjust to the new ration before being placed in collection crates. Intake was measured the last 10 days of the l4-day acclimation period. After the adjustment period, wethers were placed in collection crates and feed intake, fecal output, and urine production were mea- sured and sampled for chemical analysis over a period of seven days. Water intake was also measured during the collec- tion period. On the day following the collection period, jugular blood was sampled immediately before feeding and four hours after feeding. Rumen samples were secured immediately before feeding, one hour after feeding, two hours after feed- ing and at two hour intervals thereafter up to 8 hours post- feeding. The experiment was initiated on November 9, 1971 and terminated February 4, 1972. Forage Preparation Corn silage was harvested on August 30 and 31, 1971 at approximately 32% dry matter. All treatments with the exception of three barrels of autoclaved silage were made from one load of silage chopped on the 30th of August. The following four silage treatments will be discussed: 1) air dried, 2) sun dried 50% DM, 3) autoclaved, and 4) control silage. 66 The air dried silage treatment consisted of 29 burlap sacks filled with approximately 27.2 kg of fresh silage each. The sacks were placed in a gas heated crop drier at 1180 F for four days. The DM was about 80% at the end of the drying process. Burlap sacks were then stored in steel barrels until fed. The sun dried treatment was prepared by unloading the fresh silage in rows onto a paved parking lot that had been swept clean. The silage was spread into a layer about 5 to 10 cm deep and was mixed several times during the day using a garden rake. Eleven experimental silos were filled late in the afternoon with the 50% DM material. Five barrels of autoclaved silage were prepared on Aug- ust 30 and three were prepared on August 31. Each barrel contained about 70 kg of silage. Two metal barrels, 88.9 cm high and 57.2 cm in diameter with 1.27 cm holes drilled approx- imately 45 cm apart in the sides and bottom, were used to autoclave the silage. A metal pipe 5.1 cm in diameter and containing holes 10 cm apart was placed in the center of each barrel to facilitate autoclaving. The silage was auto- claved for one hour at 2550 F and at a pressure of approxima- tely 6.7 kg per square centimeter. After autoclaving, the silage was dumped onto a clean cement slab and spread in a thin layer until it had cooled. It was then innoculated with 10% of a three day fermented silage, mixed, and placed in experimental silos. The control silage was placed in experimental silos 67 without any treatment at approximately 84 kg per silo. Experimental Silos Metal barrels 88.9 cm high and 57.2 cm in diameter were used as containers. Two vinyl bags 12 mm thick, 1.37 m long, and 50.8 cm in diameter were placed in each barrel. The bags were filled with the various treatments and tramped several times to insure maximum compaction. After each bag was filled to the capacity of the barrel, a vacuum hose attached to an industrial floor sweeper was used to remove as much of the remaining’air as possible. The bag was then sealed with tape. The above procedure does not apply to the air dried material which was stored in burlap sacks in the barrels. Feeding Regime The sheep were fed twice daily at 8:00 a.m. and 5:00 p.m. at N 15% excess of voluntary consumption, hence, insuring 33 libitum intakes during the intake trial. While the wethers were in collection crates, they were fed at approximately 90% of voluntary intake. The ration was composed of the respec- tive corn silage plus a mineral supplement (Table 12) added at 2% of the silage dry matter. The silage was weighed prior to each feeding and the mineral supplement was thoroughly mixed with the silage. Unconsumed feed residue was removed and weighed each morning prior to feeding. Water was available at all times. Sample Collection Intake Period: During the lO-day intake study, daily 68 Table 11 Experiment VI - Metabolic Study Design of Experiment Treatment Control Autoclaved Air Dried Sun Cured Period Silage Silage Silage Silage Sheep No. 1 2 4 3 1 2 4 3 1 2 3 3 l 2 4 4 l 2 4 3 Table 12 Experiment VI - Metabolic Study Formulation of Mineral and Vitamin Supplement Ingredient Percent Dicalcium phosphate (26.5% Ca — 20.5% P) 47.3 Trace mineral salt (high Zn) 47.3 Sodium Sulfate (22.5% S) Vitamin A (10,000 IU/g) Vitamin D (9,000 IU/g) TOTAL 100.00% 69 samples of the silage and unconsumed residue were taken and frozen. At the end of the intake trial, a composite sample of each silage treatment and unconsumed residue was oven dried at 600 C for 24 hoursto determine dry matter consump- tion. Collection Period: Daily samples of silages and uncon- sumed residue were frozen for laboratory analysis. Water intake was obtained by measuring the water presented to each sheep each morning and then measuring the unconsumed quantity 24 hours later. Evaporative loss was assumed to be negligible. Total fecal collection was made by fitting each sheep with a canvas zipper bag collection harness. After weighing, the feces were placed in a cooler until the end of the collec- tion period when they were thoroughly mixed and subsampled for laboratory analysis. Total urine was collected in a two litre glass bottle which contained 25 m1 of 20% sulfuric acid and 1 ml of 10% copper sulfate. The total urine volume was measured and then diluted with water to a volume of three litres. One-sixth of the diluted urine was saved from each of the seven days' collections and a composite sample was taken for later ana- lysis. The pH of the rumen samples was determined with a Corning Model 12 pH meter. A 50 gm aliquot was then dried at 60° C for 48 hours to determine rumen dry matter. The remainder of the rumen sample was strained through two layers of cheesecloth. One ml of saturated mecuric chloride was 70 added to 19 ml of the strained rumen fluid and restrained for ammonia analysis. Five ml of the above 20 ml mixture was added to 1 m1 of metaphosphoric acid and centrifuged at 10,000 rpm for 10 minutes. The supernatant was retained for volatile fatty acid analysis. The whole blood was centrifuged and a portion of the plasma was retained for plasma urea determinations. Two m1 of the plasma was processed for plasma amino acid analysis by adding 0.2 ml of norleucine (lum/ml) to 2 m1 of plasma and then deproteinizing the sample with 0.2 m1 of 50% sul- fosalicylic acid. After one hour on ice, the sample was centrifuged at 18,000 rpm for 15 minutes. The supernatant was kept in the freezer for further analysis. gaboratory Analysis Dry matter of the fecal samples was determined daily by oven-drying at 600 C for 24 hours. Total nitrogen contents of feed, feces and urine were Ianalyzed by macro-Kjeldahl procedures on wet samples. Rumen volatile fatty acid concentrations were determined by injecting samples into a Packard gas chromatograph, as described previously (page 51). Rumen ammonia and blood urea levels were determined by the micro-diffusion method of Conway (1950). Determination of amino acids was performed on a Technicon— TSM-l amino acid analyzer, according to Bergen and Potter (1971) and Makdani, Huber and Bergen (1971). Additional silage parameters were determined as in 71 Experiment V. Procedures for 13_Vitro Fermentation The basic system used in this series of trials was a modification of the Ohio System (Johnson, 1966). The sub- strate was prepared by taking each silage and lyophylizing it in a Stakes Model 21003F-2 Freeze Drying Unit. The freeze dried material was ground through a 40 mesh screen in a Wiley mill. The "Ohio" 13_31333 fermentation media (Johnson, 1966) was utilized. Rumen fluid inoculum was obtained from a donor sheep maintained on a Pro-Sil treated corn silage ration. On the day of the initiation of the trial, rumen con- tents were collected from the donor sheep in the morning prior to feeding and strained through cheesecloth. Fifty ml quantities of the strained material were used to inoculate each fermentation bottle. Each bottle contained 4.0 g of ground freeze-dried silage, 150 m1 of nutrient solution (Johnson, 1966) and 50 ml of the inoculum. During the course of the fermentation, carbon dioxide was continuously bubbled through the closed flask system. Duplicate samples of 10 ml were removed at 0 and 12 hours after the initiation of the trial and 20 ml duplicate samples were removed at 48 hours. These samples were centrifuged at 7,000) cums can mum :tmaamcm: .wcHHHHm oHHm wsfiHSt cmxmu mmaeSmm mufimoesoo w mo mmsam> same we» can :smmum: "muoz .tmuUStsoo uoc mwmeamcm HmoflumHuMumH .Amo. v mv u m .Aao. v mV n < .uSmanMAt hausmoflmwcmfim no: can uefiuumquSm mama can wcfi>c£ no uafiuomquSm os Sues mcsHm> o o o o o o 0 mg mm m «am q me m mummno unmaummue oz Hz\wx e.mH Exxwx m.m unusummue oz suoo manumfioz_£wfim acoummsfiq twu< oaShom Houusou tum choc mHSumHoS swam tam mommafim mo mammm Hmuumz sun so mwmeamc< HmUHawcu owwum>< ma manna 77 buffering effect of the limestone. Elevated lactic acid levels in the limestone treated silage are in agreement with results received by Klosterman 33 31. (1961b), Klosterman 33 31. (1963b) and Nicholson and Cunningham (1964). The limestone addition may have altered the activity of plant enzymes that degrade protein thus accounting for the decrease in proteolysis previously discussed. Although the pH of the three silages was significantly different (P < .01), they were all within the range described by Barnett (1954) for maintaining desirable silage quality. Effect 33 Maximizing and Minimizing Silage Fermentation on Performance 33 Feedlot Cattle —_ Complete performance of all lots of cattle is shown in Table 14. The means for all performance traits for cattle fed silage treated with additives not containing nitrogen are shown in Table 15. Average daily gain was not affected by silage treatment and there were only small differences in feed efficiency. Daily feed consumption values were slightly reduced for the two treated silages (control - 7.88 kg, formic acid treatment - 7.78 kg, and limestone treatment - 7.58 kg). The reduced intake of the limestone treated silage fed cattle may be due to significantly (P < .01) higher lactic acid levels in the limestone treated silage; however, there are conflicting Opinions concerning the effect of lac- tic acid on voluntary dry matter consumption (Emery 33 31., 1961; Senel and Owen, 1966; and Allen 313 31., 1971). If lactic acid content of the silage did depress the intake of 78 steers fed the limestone treated silage, then the dry mat- ter intake of the steers fed the formic acid treated silage should have been the largest since it contained significantly (P < .01) less lactate than either of the other treatments. Amines, which are possible products of proteolysis, have been reported to depress intake (Neumark, 1964 and Dain 33 31., 1955) and most proteolytic products of silage fermentation are contained in the unidentified nitrogen fraction. Uniden- tified nitrogen levels as a percent of total nitrogen were similar for all three treatments (34% to 41%), which may explain the resulting small differences in dry matter intake received on the three rations. Greater differences in the unidentified nitrogen levels fed are needed before definite conclusions can be made regarding their effect on voluntary dry matter consumption. The absence of an effect on average daily gain, feed consumption and feed efficiency by cattle fed the formic acid treated silage is in disagreement with results reported by Waldo 33 31. (1969) and Waldo 33 31. (1971). Improvement in animal performance has been shown for formic acid treatment of low dry matter grass and legume silage (mzos DM) (Saue and Breirem, 1969a; Waldo 33 31., 1970; and Waldo 33 31., 1969) or high dry matter corn silage (44% DM) (Huber, 1970). All silages in this experiment were in the dry matter range of 30.98% to 35.66% which was sug- gested by Geasler (1970) as the most effective stage of maturity to harvest corn silage to Optimize all factors. Feed cost per 100 kg gain was elevated for the formic 79 Table 14 Corn Silage Additives Compared (October 22, 1970 to June 17, 1971) Type of Silage Treatment and Supplement Control Formic Acid Limestone Soy-mineral Soy-mineral Soy-mineral Supplement Supplement Supplement 402 Sh. 40% Sh. 407 Sh. All Corn-60% All Corn-60% A11 Corn-60% 161 - 238 Day Test Silage Silage Silage Silage Silage Silage Lot No. 38 40 37 35 39 36 No. of steer calves 10 10 10 10 10 10 Av. initial wt., kg 245 245 244 244 243 247 Av. final wt., kg 403 420 397 414 405 414 Av. daily gain, kg .71 .89 .70 .87 .73 .85 Daily Feed, kg 85% DM: Corn silage 6.64 4.30 6.71 4.11 6.77 3.91 Gr. sh. corn 3.42 3.32 3.10 Soy—mineral supplement . 84 . 55 . 88 . 53 . 85 . 47 TOTAL 7.48 8.27 7.59 7.96 7.62 7.48 Feed Efficiency: Feed per kg gain, kg 10.58 9.26 10.86 9.15 10.36 8.78 Feed cost per 100 kg gainl $ 40.74 $ 39.51 $ 47.54 $ 41.47 $ 41.03 $ 38.43 Carcass Evaluation: Carcass gradez 12.98 14.28 14.12 14.72 13.18 13.68 Marbling score3 15.51 19.01 18.09 20.89 15.19 17.11 Fat thickness, cm 1.54 1.77 1.62 1.51 1.64 1.67 Ribeye area, cm 69.94 64.88 66.63 66.44 70.50 69.13 Percent K.H.P. fat“ 3.62 3.77 3.38 3.58 3.33 3.47 Percent B.T.R. cuts5 49.36 47.87 49.09 48.88 49.27 48.88 Dressing percent6 60.52 61.78 59.30 60.98 60.95 61.31 Carcass price/100 kg $115.70 $114.82 $116.18 $116.18 $115.52 1Feed costs based on 30% DM corn silage $9.37/MT, urea—mineral $88.18/MT, Pro-Sil $71.65/MT, shelled corn $49.60/MT, soy-mineral supplement $110.23/MT. 2Good = 9, 10, 11; Choice = 12, 13, 14. 3Small = 10, 11, 12; Modest = 13, 14, 15; Moderate = l6, 17, 18. 1+Percent of carcass weight in kidney, heart and pelvic fat. 5Percent of carcass weight in boneless, trimmed retail cuts. 6Cold carcass weight over off experiment weight. Values with no superscript or having the same superscript are not signifi- cantly different, A = (P < .01), a = (P < .05). 80 Table 15 Corn Silage Additives Compared (October 22, 1970 to June 17, 1971) Type of Silage Treatment and Supplement Control Formic Acid Limestone Soy—mineral Soy-mineral Soy—mineral 161 - 238 Day Test Supplement Supplement Supplement No. of steer calves 20 20 20 Av. initial wt., kg 246 245 245 Av. final wt., kg 412 406 410 Av. daily gain, kg .80 .79 .80 Daily Feed, kg 85% DM: Corn silage 5.47 5.41 5.34 Gr. sh. corn 1.71 1.66 1.55 Soy-mineral supplement .70 .71 .69 TOTAL 7.88 7.78 7.58 Feed Efficiency: Feed per kg gain, kg 9.88 9.87 9.50 Feed cost per 100 kg gain1 3 40.08 $ 44.20 $ 39.82 Carcass Evaluation: Carcass grade2 13.631?b 14.42Aa 13.40:b Marbling score3 17.263 19.498 16.15 Fat thickness, cm 1.67 1.56 1.64 Ribeye area, cm2 67.38 66.50 69.75 Percent K.H.P. fat“ 3.70 3.48 3.40 Percent B. T. R. cuts5 48.61 48.99 49.07 Dressing percent6 61.15 60.14 61.13 Carcass price/100 kg $115.26 $116.18 $115.61 1Feed costs based on 30% DM corn silage $9.37/MT, urea-mineral $88.18/MT, Pro-Sil $71.65/MT, shelled corn $49.60/MT, soy—mineral supplement $110.23/MT. 2Good = 9, 10, 11; Choice . 12, 13, 14. 3Sma11 = 10, 11, 12; Modest = 13, 14, 15; Moderate = 16, 17, 18. ”Percent of carcass weight in kidney, heart and pelvic fat. 5Percent of carcass weight in boneless, trimmed retail cuts. 6Cold carcass weight over off experiment weight. Values with no superscript or having the same superscript are not signifi- cantly different, A = (P < .01), a = (P < .05). 81 acid treated silage fed group. This was due to the added cost of formic acid which resulted in no improvement in feed efficiency. The slight improvement in feed efficiency for the lime- stone treated silage fed group offset the cost of treating. This improvement in feed efficiency is in agreement with previous work by Klosterman (1963b) and the summary by Essig (1968). One possible explanation for the increased feed efficiency of the limestone treated silage fed cattle may be a reflection of the significantly (P < .01) higher lac- tic acid content of this silage. Increased feed efficiency when lactate was added to the ration was reported by Emery 35 31. (1961), Senel and Owen (1966), and Allen 33 a1. (1971). All groups had an average carcass grade of middle to high Choice; however, small differences were significant (P < .01). Marbling scores ranged from moderate - to slightly abundant - and again the small differences were significant (P < .05). Differences in all of the other car- cass traits were small and insignificant. Experiment II - Feeding Trial 2 Chemical Analyses 9f Silage Containing NPN Additions Results of 18 different composite analyses of each silage and the high moisture corn used during the experi- ment are shown in Table 16. Percent dry matter of the fresh and ensiled material varied little and all silages were within the dry matter range for excellent quality (Geasler, 1970). 82 Nitrogen Fractions Total nitrogen values of the three NPN treated silages compared to the control untreated silage were increased 44% by Pro-Sil treatment, 53% by urea-mineral treatment, and 57% by the urea-mineral plus formic acid treatment. In all cases, increases in total nitrogen accounted for essentially 100% of the Pro-Sil and urea applied. The apparent increase in total nitrogen of the control silage is attributed to samp— ling errors since no nitrogen was added at time of ensiling. The water insoluble nitrogen content of the control silage decreased approximately 41% (fresh vs. adjusted ensiled). The control ensiled values were adjusted downward to compensate for the apparent increase in crude protein. The decrease in water insoluble nitrogen content of the silage during fermentation was 12% for the Pro-Sil treated silage, 24% for the urea-mineral treated silage and 18% for the urea- mineral plus formic acid treated silage. These data indicate that Pro-Sil and urea-mineral addi- tions had a sparing effect on water insoluble nitrogen and are in agreement with work by Beattie (1970), Huber and Hillman (1970) and Henderson, 23 al.(l97l). This appears to be due to an increase in bacterial protein and/or a decrease in proteolysis during fermentation (according to Modyanov, 35 31., 1960 and Rayetskaya,‘gtfal., 1954)]. Table 16 shows that approximately 36% of the nitrogen 1As reported by Owens (1968). 83 in Pro—Sil was recovered as insoluble protein in silage, 63% was recovered as ammonium salts (assuming all ammonia present was linked to organic acids to form ammonium salts)1 and 1% as urea. The results agree with previous data of Henderson (1969) and (1970b). When silage was treated with urea-mineral, 13% of the added nitrogen was recovered as water insoluble nitrogen, 63% as ammonium salts, 14% as urea and 10% in the unidenti- fied fraction. The high ammonium salts value and low level of water insoluble nitrogen and urea in urea-mineral treated silage are not readily explainable. Apparently, a high level- of urease was present in the fresh corn plant material at ensiling time (as suggested by Karr, 33 31., 1955) which reduced the added urea to ammonia. The ammonia was then combined with organic acids during fermentation. This is suggested by the high level of lactic acid produced in this silage. When silage was treated with urea-minerals plus formic acid, 27% of the added nitrogen was recovered as water insol- uble nitrogen, 7% as ammonium salts, 54% as urea and 12% remained in the unidentified fraction. Actual levels of unidentified NPN in the four silages did not differ substantially; therefore, the elevated water insoluble nitrogen levels for the Pro-Sil, urea-mineral, and urea-mineral plus formic acid treated silages, as 1As suggested by Bentley, et a1. (1955); Klosterman, et al. (1961); Johnson, gt_al. (1967T? Huber and Hillman (1970)?— and Henderson, 35 31. (1970b). 84 compared to the control silage, are probably due to the pro- duction of microbial protein during fermentation rather than to a reduction in proteolysis. In can be concluded that corn silage treated with either Pro-Sil or urea-mineral will have a higher water insoluble nitrogen content at feeding time than untreated silage. Organic Acids Total organic acid (as percent of dry matter) was cal- culated by combining lactic, acetic and butyric acids. Other organic acids such as valeric and isovaleric were too low for accurate determination. Pro-Sil and urea-mineral treated silages had similar total organic acid contents which averaged 33% higher than the control. Urea-mineral plus formic acid treatment reduced the total acid content by 43%. Lactic acid levels in the silage were significantly (P < .01) increased by 39% for both the urea-mineral and Pro- Sil treated silages, when compared to the control silage. Acetic acid levels were unaffected by these two NPN treatments. Treatment of silage with urea-mineral plus formic acid significantly (P < .05) reduced both lactic and acetic acid levels by 43% and 46%, respectively, when compared to the control silage. This reduction is attributed to the formic acid addition discussed in Experiment I. Thus, the neutralizing effects of Pro-Sil and urea mineral when added without formic acid resulted in an increa— sed fermentation and bacterial activity, yielding signifi- cantly (P < .01) higher amounts of lactic acid. This is in .msfipoom wcfiunp coxmu moaaemm mufiwoaaoo wH mo some oSu mum mosam> :vmafimam: .wowaawm oawm wcfiusw aoxmu mmadsmm ouwmomaoo m mo some onu mum mo=Hm> :nmoum: "ouoz .pmuoavcoo uo: mwmhamcm Hmoaumfiumumfl .Amo. v mv n m .Aao. v mv u 4 .usouomwflp haucmoHMficme uoa mum unfinomummsm mean who wofl>m£ no unauowummam on cows mm=Hm> o o o o o o o o 0 mm. mm m m<~H 3 mm m 3mm 3 Nu m muomno ooosooooe oz Hzst m.mnoao< Hz\ws o.o~ Hz\ms m.- successes oz auoo musu ofishom .Hz\mx Hmuoaflzlmoun Hamloum Houunoo uoaoz swam o.o~uaoeoanuooee pom snow onnumaoz swam can mommaam mo mammm Houumz ham :0 mwmhamn< Hmowswso owmuo>< ca oases 86 agreement with previous work by Huber, et 31. (1968); Klosterman, §t_al. (1963); and Henderson, gt'al. (1970b, 1971). The pH of the control silage (3.89) was significantly (P < .05) lower than all other treatments; however, all silages were within the pH range suggested by Barnett (1954) as being required for maintaining excellent preservation of the silage during storage and feeding. Effect g£.Additives on Feedlot Performance Individual lot means for performance and carcass traits are shown in Table 17. The means for performance and car— cass traits for cattle fed NPN treated silages are shown in Table 18. Steers receiving either Pro-Sil, urea-mineral or urea—mineral plus formic acid treated silage gained sig- nificantly faster than steers receiving the control silage supplemented at feeding time with soy-mineral (P < .05) or Pro-Sil (P < .01). The difference in gain between Pro-Sil supplemented and soy-mineral supplemented groups (.75 kg vs. .80 kg) was not significant. Although Pro-Sil has a strong ammonia odor, and the nitrogen content is made up entirely of anhydrous ammonia; the ammonia readily combined with the organic acids con- tained in the silage. After approximately one minute of mixing in a horizontal mixer, the silage was free of ammonia odor and was quite similar to silage treated with Pro—Sil at time of ensiling with respect to physical characteristics and odor. 87 Previous work (Geasler and Henderson, 1970) showed a high correlation between feed efficiency and lactic acid con- tent of the silage fed. The superiority of the NPN treated silages may be attributed to their higher lactic acid levels. Differences in feed consumption were small; however, feed efficiency favored the NPN treated silages. Control silage supplemented with Pro-Sil at feeding time resulted in the poorest feed efficiency. Lower levels of organic acids in the control silage supplemented with Pro-Sil compared to Pro-Sil treated and urea-mineral treated silages may explain the reduced performance for this group. Another possible explanation of the lower daily gain and feed efficiency may be the formation of poorly utilizable NPN compounds other than ammonium salts and thus less available protein. Perhaps the latter explanation is more accurate since the urea-mineral plus formic acid treated silage had significantly (P < .01) lower lactate and acetate than the control, but resulted in performance superior to the controls. Feed cost per hundred kilograms of weight gain was lower for the NPN treated and supplemented silages. The increased feed cost for the urea—mineral plus formic acid treated silage fed group was due to the cost of the formic acid additon. Compared to the soybean supplemented silage, urea or Pro+Sil treament alone lowered feed cost approximate- ly 25%. Feed cost for Pro-Sil supplementation at ensiling time was 17% less than when added at feeding time. Carcass grade for all groups of cattle averaged between 88 .Amo. v mV n m .Aao. v mv u < .usoumMMfip mauamoflwwsmfim uoc mum umanomummsm 08mm may maw>m£ no unauomummsm on saws monam> .uanoB unoSHuoaxo «no uo>o unmaos wmmoumo waoUm .wuso meuou wosafiuu .mmmaodop nH uanoB mmmoumo mo unmouomm .umm oH>HoQ was unmoz .%oswfix ca uswwo3 mmmoumo mo undoumm: .mH .es .oH u ooosoooz ”ma .es .ms u osmooz ”NH .HH .oa n sesame .es .ma .NH u ooaoso was .oH .o u ooooN .Hz\mm.0HHw uGoEoHQQSm Hmuocaslhom .HZ\00.m3w oooo seasons .ez\mo.aem Hamuoea .Hz\wa.wmw Hooooasaooeo .Hz\em.om owoaaa osoo 2o Nom so oooso soooo oases om.mHHw ao.mHHm oo.sHHw we.mHHw Nw.aHHw oe.mH3m (ms ooa\ooaoo aeaooao mo.om He.em ma.om om.om w~.Ho ~m.oo anaconda masseuse mo.om 33.oa H~.oe «a.ma em.e3 om.oa moose .m.e.m ooooeom mm.~ em.m oo.m mm.m ek.m No.m some .m.e.e oooooom ma.mo Hm.mo Hm.mo om.3o mm.3o 3a.mo Nae .oous oaooam HN.H mm.H 3m.H so.s an.H 3m.a ao .ooooxoaro use. oH.eH Ho.3H 3H.oa mm.mH Ho.oH Hm.mH mooooo masseuse «a.ms eo.~H oa.mH em.~H mm.3s mo.NH meadow amoosso "GOHHNDHN>M mmmohmo oa.cm w Hm.mm m on.Hm m Nw.m~ m Hm.om 35.03 a Hoasm we OOH poo eaoo some 03.0 m0.HH 3H.w 0m.m 0N.m wm.0H wx .swmw m3 Hod pooh “Nusowofimmm pooh No.e No.e o~.m sa.o e~.w m3.e asaoa ma. mm. mm. 30. uaoaoaaasm mm.m mn.m N3.m shoe .sm .um oH.a am.e sa.a 3a.o om.e ao.o masses coco "so Nam we .ooom meson om. do. Ho.H me. am. an. we .oaom seams .es 003 003 033 M03 0N3 m03 wx 9u3 Hmawm .>< 33m n3~ 03m 33m m3~ m3~ wx ..u3 Hwaufiafi .>< 0H 0H m m 0H 0H mo>amo Hooum mo .oz me as me we o3 mm .oz coo seesaw Noo owosam omoaam moo owoflam owsaam Noo seesaw some moo wmm : Hoe Chou pmaaosm N03 HH< usmEmHQQSm Hamloum :uoo onHonm N03 HH< uaoaumoua Hamloum usoswwmmmw wsm uaoEumoHH owmafim mo ommH Aaeoa .es ooze oo odds .NN soooooov pmumafiou mo>Hufipp« zmz ommafim suoo choc voaaonm N03 HH< uaosoaamsm Hmumnwsl%om Houuaoo NH UHQNH 89 00.3HHO 00.0HHO 00.3HHO m0.mHHO 0x 00H\ooaua mmmonmu 3~.oo c0.0m mo.H0 H3.0m ouamouma woflmmoum 00.03 00.Hm 0N.03 00.03 mmuso .m.H.0 unwoumm No.0 00.0 03.0 03.0 summ .m.m.x unmouom 00.00 m0.Hn 0m.0o mm.0o So .moum ozobam 00.H 0H.H 30.H 00.H aw .mmocxoasu umm H~.0H 00.0H H0.3H 00.0H mouoom wafiHmez 0m.mH ~3.NH 00.NH mm.mH Nowmuw mmmoumu "coaumsam>m mmmoumo mm.0m m 00.~m m N0.Nm 0 00.0w 0 Hsflmw mx 00H you umoo comm 00.0 33.0 03.0 03.0 0x .swmw 03 non woom "Nucmwofimmm woom 00.0 3N.n N3.0 0H.“ A< m33 3H3 333 ma3 0x ..u3 Hanan .>< 03m 03m 03m m3~ 0x ..u3 HmHuHoH .>< 0H 0H 0H 0H wm>Hmo nomum 00 .oz 03 03 03 H3 .02 ac; owssam No3 owosam owosam Noo «wosam some zoo mmw 1 sea suoo coHHozm N03 HH< suoo voaaozm N03 HH< ucoaummue 0304 oHEpom unmaummufi Hmuoaazlmoup Hmumafizlmmun ucosmaamam wow ucmEummuH owmawm mo mama Assad .es more oo odds .NN soooooov ooeooaoo oo>aoaoo< zmz owosam osoo wussfiuaoo RH pomH 90 .000. v 00 .000. v .00 n 0 < .080000000 0008000008000 won 000 00000000080 080m 000 080>00 Ho 00000000080 on 0003 00:00> .00 .00 .00 mumsoooz 000 .30 .00 .000003 0808000000 000 H0>o 000003 0000000 00000 .0080 000000 0088000 .00000co0 80 000003 0000000 00 08000000 .000 00>00m 080 00000 .008000 80 000003 0000000 mo 08000003 ummaoz “00 .00 .00 u 0025m .30 .00 .00 n oo0o00 000 .00 .0 u ooo0N .9z\00.0000 0808000080 00008081000 .9z\00.03m cuoo 0o00oem .ez\m0.000 00muosm .02\00.000 0muos0aumouo .02\00.00 000000 osoo :0 000 so 0oome aumoo 0mo00 30.0000 30.0000 00.0000 00.0000 00.0000 00 000\oo0sa 0006000 03.00 00.00 00.00 00.00 00.00 00800000 00000000 00.00 00.03 00.03 00.03 00.03 00000 .m.0.m 0800000 0000.0 0<03.0 0m<00.0 0<03.0 0<00.0 300m .m.m.M 0800000 00.00 00.00 00.30 00.00 00.00 80 .0000 000000 0000.0 00000.0 000.0 0000.0 000.0 8w .000800000 000 0000.30 0000.00 003.30 0000.30 000.00 mosoom 00000002 0000.00 0000.00 000.00 0000.00 000.00 000000 0000000 "8o000800>m 0000000 00.30 0 00.00 0 03.00 0 00.00 00.03 0 08000 00 000 000 0000 0000 00.0 00.0 00.00 00.0 00.0 00 .o0mw ms 000 0000 ”0080000000 0000 00.0 00.0 00.0 00.0 00.0 03909 00. 00. 0808000080 00.0 30.0 00.0 00.0 00.0 nuoo .00 .00 00.0 00.0 00.0 00.0 03.0 000000 auoo ":0 000,00 .0000 00000 m 00. 00 00. a 00. 00. ms .o0am 00000 .>3 3 003 m3 003 00 003 3 003 003 N03 00 ..ea 0ao00 .>3 030 030 030 030 030 00 ..03 0w0o0o0 .>3 00 00 00 00 00 00>000 00000 00 .02 0000009 0000009 0808000080 0000009 0808000080 0009 000 000 I 000 0000 0088om 000080: 00mloum 00mloum 00008081000 00008021000: 10000 080800009 oz 0o008o0 0008000080 080 000800009 000000 «o 0009 A0000 .00 made ou 0000 .00 oosoooov omumdaoo mo>0u000< zmz 000000 :uou 00 00009 91 low and middle Choice; however, the group fed Pro—Sil supple- mented silage was significantly (P < .05) lower in carcass grade and marbling score than the control group. The Pro-Sil supplemented silage fed group had significantly (P < .05) less fat thickness than the groups fed control and Pro-Sil treated silage. The steers fed urea-mineral plus formic acid treated silage also had significantly (P < .05) less fat thickness than those fed control silage. Percent kid- ney, heart and pelvic fat significantly favored the Pro-Sil supplemented (P < .01) and urea-mineral plus formic acid treated silage (P < .05) fed groups. Other carcass traits were small and insignificant. All Silage Ration vs. 40% Shelled Corn and 60% Silage Ration .Table 19 shows mean values for performance and carcass traits summarized across level of silage in the ration for Experiments I and II combined. Average daily gain was significantly (P < .01) higher for the cattle fed concentrate (.92 kg vs. .73 kg). Higher dry matter intake (8.13 kg vs. 7.40 kg), greater feed effi— ciency (8.84 vs. 10.14) and higher energy content of the ration containing added shelled corn accounted for the increase in daily gain. This is in agreement with many pre— vious experiments conducted at this station. Cattle fed silage required a 12% longer feeding period to reach slaughter weight and Choice carcass grade (195 days vs. 220 days). Feed cost per hundred kilograms was lower for the all 92 silage ration ($36.06 vs. $36.76). Using a standard yield of 35 metric tons of 35% DM silage or 5 metric tons of shelled corn per hectare, beef produced per hectare increased from 1149 kg for the 40% shelled corn group to 1730 kg for the all silage group. This represents a 51% increase in beef produced per hectare with an all silage ration. Using the actual selling price of the cattle, gross returns per hectare of corn fed was increased (48%) from $803 for the 40% shelled corn group to $1191 for the all silage group. Cattle fed the all silage had significantly (P < .01) greater percent of boneless, trimmed retail cuts and signi- ficantly (P < .05) lower dressing percent. This was attri- buted to less fat thickness on the cattle fed all silage. The carcass grade of both groups of cattle averaged middle to high Choice; however, small differences significantly (P < .01) favored the 40% shelled corn group. 'Therefore, Choice carcasses with a higher cutability can be produced on an all silage ration. No significant differences were found in other carcass traits. Experiment III — Metabolism Study with Corn Silage Varied in Extent of Fermentation Chemical Analysis of Silage The three all silage rations used in Experiment I were compared in this metabolic study. The silages were not sup- plemented with additional nitrogen, but a mineral supplement was fed (Table 8). The chemical analysis of the silages is shown in Table 13 and discussed on pages 73 - 77. 93 Table 19 All Silage vs. 40% Shelled Corn and 60% Silage Ration (Dry.Matter Basis) (October 22, 1970 to June 17, 1971) 40% Shelled Corn 161 - 238 Day Test All Silage 60% Silage No. of steer calves 69 69 Av. initial wt., kg 245 246 Av. final wt., kg 406 A 426 Av. daily gain .73 .92 Daily Feed, kg 85% DM: Corn silage 7.00 4.38 Gr. sh. corn 3.51 Supplement .40 .24 TOTAL 7.40 8.13 Feed Efficiency: Feed per kg gain, kg 10.14 8.84 Feed cost per 100 kg gain1 $ 36.06 $ 36-76 Kg beef produced per hectare corn fed7 1730 1149 Grass returns per hectare corn fed8 $1191 $803 Carcass Evaluation: A Carcass grade2 13.033 13.61 Marbling score3 15.29 16.79 Fat thickness, cm 1.46 1.56 Ribeye area, cm 67.94 67.44 Percent K.H.P. fat“ 3.37A 3.33 Percent B.T.R. cuts5 49.498 48.87 Dressing percent6 59.44 60.62 Carcass price/100 kg $115.83 $115.17 1Feed costs based on 30% DM corn silage $9.37/MT, urea-mineral $88.18/MT, Pro-Sil $71.65/MT, shelled corn $49.60/MT, soy-mineral supplement $110.23/MT. 2Good = 9, 10, 11; Choice = 12, 13, 14. 3Small = 10, 11, 12; Modest = 13, 14, 15; Moderate = 16, 17, 18. ”Percent of carcass weight in kidney, heart and pelvic fat. 5Percent of carcass weight in boneless, trimmed retail cuts. 6Cold carcass WEight over off experiment weight. 7Based on corn yields of 35 MT of 35% DM silage or 5 MT of shelled corn er hectare. Based on selling price of cattle. Values with no superscript or having the same superscript are not signifi- cantly different, A = (P < .01), a = (P < .05). 94 Rumen Ammonia and Blood Urea Concentrations Mean values for rumen ammonia and blood urea are shown in Tables 20 and 21. The rumen ammonia concentration was highest at T (two hours post feeding) for the groups fed 2 limestone treated and formic acid treated silages. The con- trol silage produced a maximum concentration of ammonia at T4 and was higher at T4, T6 and T8 than the other two treat— ments. The limestone treated and formic acid treated silages had higher initial (To) and final (T10) rumen ammonia con— centrations than the control silage. Blood urea concentra- tions were highest during the period from four to eight hours post feeding for all three silages. The control silage produced blood urea levels that were higher at all times than the other two silage treatments and the limestone treated group had the lowest blood urea at all times. None of the rumen ammonia or blood urea values were significantly different. Rumen VFA Concentrations Tables 22, 23 and 24 show mean values for acetate, pro- pionate and butyrate concentrations expressed as mg of VFA per 100 ml of rumen fluid. There were no significant dif- ferences between treatments. However, the limestone treated silage had significantly (P < .01) higher lactic acid con— centration than either of the other two silage treatments and resulted in higher rumen acetate levels and more stable propionate levels than the control silage. The fate of lac- tate in the rumen in unclear. Baldwin at 31. (1962) and 95 Table 20 Mean1 Rumen Ammonia Values (mg/100 ml) Silage Treatment Formic Acid Limestone Time Control Treated Treated SE2 TO 1.17 2.44 4.10 1.76 T2 1.94 3.16 4.26 .77 T4 9.24 3.10 1.34 3.53 T6 5.44 1.50 2.03 2.69 T8 1.80 .73 .86 .64 T10 .58 1.20 2.30 .56 1Three observations per mean. 2SE = Standard error of means. No significant differences between means. Table 21 Mean1 Blood Urea Values (mg/100 ml) Silage Treatment Formic Acid Limestone Time Control Treated Treated SE2 TO 5.60 4.30 3.30 .90 T2 6.11 4.73 3.83 1.05 T4 7.01 5.43 4.33 1.22 T6 7.56 5.63 4.30 1.87 T8' 7.76 5.00 4.16 1.78 T10 6.93 4.76 4.00 1.35 1Three observations per mean. 2SE = Standard error of means. No significant differences between means. 96 Bruno §E_a1. (1962) used 14 C-lactate in an in vitrg ferment- ation and found acetate to be the main product of lactate fermentation. Others (Waldo gt al., 1956; Waldo at al., 1960 and Wallnofer et al., 1966) have found an increase in the proportion of propionate in the rumen contents when rumen lactate concentration is elevated. Satter (1968) reported that both acetate and propionate are important metabolites of ruminal lactate, with propionate increasing in importance as the amount of dietary starch or lactate increases. Ace- tate is not a terminal end product of lactate metabolism but is used in the synthesis of butyrate. Drpratter Intake and Dry Matter Digestibility As shown in Table 25, small differences in intake favored the control silage. These differences approached significance. The dry matter digestibility was not signi- ficantly different for the three silage treatments. Lower dry matter intake and digestibility were shown for the formic acid treated than the control silage. This is in disagree- ment with work done by Waldo et_al. (1969) and Thomas 33 31. (1969) using formic acid treated grass and legume silages. Huber also (mimeo D-235) reported increased dry matter intake of 44% dry matter silage comparing formic acid and control treatments. The nonsignificant differences in dry matter digesti- bility of the limestone treated and control silage fed groups was in agreement with previous results summarized by Essig (1968). 97 Table 22 Mean1 Rumen Acetate Concentration (mg/100 ml) Silage Treatment Formic Acid Limestone Time Control Treated Treated SE2 TO 289.0 353.0 425.0 33.56 T2 246.3 371.0 401.3 39.08 T4 374.0 387.7 418.3 31.00 T6 391.0 446.3 419.0 14.34 T8 385.0 407.3 389.0 12.54 T10 400.0 389.7 431.7 49.44 1Three observations per mean. 2SE = Standard error of means. No significant differences between means. Table 23 Mean1 Rumen Propionate Concentrations (mg/100 ml) Silage Treatment Formic Acid Limestone Time Control Treated Treated SE2 To 71.3 81.0 110.34 15.90 T2 67.0 102.0 109.0 8.35 T4 149.7 117.3 139.3 33.84 T6 147.7 149.3 138.7 49.97 T8 125.7 111.0 107.0 22.61 T10 102.7 139.3 126.7 8.04 1Three observations per mean. 2SE = Standard error of means. No significant differences between means. v r z t 1" 98 Table 24 Mean1 Rumen Butyrate Concentration (mg/100 ml) Silage Treatment Formic Acid Limestone Time Control Treated Treated SE2 T0 50. 60. 68.0 15.27 T2 44. 60. 59.0 16.73 T4 58. 68. 62.0 15.21 T6 58. 81. 65.7 13.48 T8 63. 80. 63.0 7.42 T10 60. 83. 71.0 11.79 1Three observations per mean. 2SE = Standard error of means. No significant differences between means. 99._ Nitrogen Balance The mean values for all nitrogen balance parameters are shown in Table 25. The nitrogen intake varied considerably for the three silages (control - 141.0 g/day, formic acid treatment — 132.8 g/day and limestone treatment — 116.1 g/day), but differences were not significant. The increase in nitro- gen intake for the control parallels the increase in dry mat- ter intake and the difference in the latter two treatments was probably due to the elevated crude protein content of the formic acid treated silage (see Table 13). The differences in nitrogen digestibility of the three silages (control - 70.6%, formic acid treatment - 47.3% and limestone treatment - 54.5%) were not significantly different. Nitrogen digested (g/day), nitrogen retained (g/day) and nitrogen retained as a percent of nitrogen intake were significantly higher (P < .01) for the control silage, com- pared to the two treated silages. Other nitrogen balance parameters were not significantly different. Experiment IV - Nitrogen Balance Study with NPN Silage Additives Chemical Analysis 9: Silage The four all silage rations utilized in this experiment were identical to the all silage rations utilized in Experi- ment II. The chemical analysis of the silages is shown in Table 16 and discussed on pages 81 - 86. mm Uessb 100 .Amo.v .3 u m .Aao. v mv u < .ucmumMMHt kfiucmofimecwfiw uoc mum umfluomuodsm mean gnu wcfi>m£ no unauomquSm oc nufis mosam> .fimma HGQ wGOHUMNrHOmn—O 00H£H "MUOZ .mcmma mo uouuo tuthMum u mmH m.Hq ¢.¢q «.mo N .vmummmwt 2 mo N mm tocfimumu z mm.H H.~N o.HN wuatt< mwmafim mo muommmm nu maan "v .1. L“ U) 101 Dry Matter Intake and Dry Matter Digestibility Mean values for dry matter intake and digestibility parameters are shown in Table 26. Dry matter intake and dry matter digestibility were lowest for the silage treated with urea-mineral plus formic acid. The other three rations had similar dry matter intakes and higher dry matter digesti- bilities. The dry matter digestibility of the control, Pro- Sil treated and urea-mineral treated silages are similar to those obtained by Beattie (1970). Nitrogen Balance Results of all nitrogen balance parameters are shown in Table 26. Daily nitrogen intake was similar for all three NPN treated silage rations (Pro-Sil - 87.0 g/day, urea-mineral - 93.3 g/day and urea-mineral plus formic acid — 86.5 g/day) and higher for the control soy-mineral supple- mented silage ration (104.4 g/day). Nitrogen digestibility was not significantly different for the four treatments (control - 70.7, Pro-Sil - 71.8, urea-mineral - 63.5 and urea—mineral plus formic acid — 55.3). All silage treatments produced a positive nitrogen balance. Although differences in nitrogen digestion para- meters did exist, they were not significant. Experiment V — Silage Fermentation Study Analysis of silage samples taken fresh and on days 1 through 10, 15, 20, 30, 60 and 90 after ensiling for the 102 .mcmme ammsumn mmodmumwmat unwowmwcwwm oz .GNOE HGQ mGOHUNPHmeO Mach "On—OZ .mcmms mo uouum wumchum n mmfi m.- «.mm N.om «.mm N .emummwfie 2 mo N we emafimumu z ©©.w w.w I o.e w.o~ o.o~ N .oxmuSN 2 mo N mm wmcfimumu z o¢.m w.NH o.¢H m.mH 0.0m kmt\w .wmcflmumu smwouuwz ww.m «.mq w.om m.mq o.n¢ hmv\m .cowouuwa %Hms«uD 0H.¢ m.mm m.mo w.Hn n.0n N .toumwwfiv smwouufic unmouwm mm.o N.om m.me a.me o.mm hmt\w .tmumowfiv cowouufiz mo.H m.om o.wN H.¢N N.w~ hmv\m .cmwouufis Hmoom Hq.N m.©w m.m¢ 0.5m q.qoa 5mb\m .mxmucH cowouuwz qo.~ o.Ho N.~N H.HN a.mo N .emummwfiw 2m unwoumm ~.Nme momm memm SHNM Nmmm see\m .eeeeewee ea N.NNN seNm case case mess sme\w .exeeea ea q q q q magnum mo .02 Hmm wmummuH pmumoua wmummue ucoamamdsm wHo< ofisuom Hmumafiz Hamloum Hmnmsaalhom Hmumcfialmmua Icon: Houucoo usmsummue mmeHm mumuosmumm coaummwwm so mm>wuatt< zmz mwmafim mo muoommm 0N mHQmH 103 control and Pro-Sil treated silages are shown in Tables 27 and 28, reSpectively. Experimental silos were used and the results may not fully apply to farm size silos. Control Silage Fermentation Parameters As shown in Table 27, pH decreased rapidly from 5.72 in fresh material to 4.65 on the first day of fermentation. The reduction in pH was variable from day one through day twenty and then appeared to level off at approximately 4.00. This is in agreement with data reported by Geasler (1970). There was a large increase in lactic acid production during the first day of fermentation (Figure 4). It appears that there was a trend for lactic acid production to continue to increase but at a much slower rate through day ten. Geasler (1970) reported similar trends. The rate of lactate production increased again between day ten and fifteen and remained at a level of approximately 4.00% of silage dry matter. Barnett (1954) concluded that lactic acid increased at a slow rate during phase one and two (day one to day two) but at an accelerated rate during phase three and four of fermentation. The high level of acetic acid in the fresh material is not readily explainable. The fresh sample was taken approx- imately one hour after field chopping. The acetic acid pro— duction was variable up to about day nine and then appeared to remain steady at approximately 1.50% of silage dry mat— ter rather than decrease as reported by Geasler (1970). Barnett (1954) reported acetic acid production was rapid 104 during phases one and two of fermentation and continued at a slower rate thereafter. Soluble nitrogen and soluble NPN, expressed as a per- cent of dry matter, had a rapid initial increase (fresh - day one) and then leveled off until about day fifteen, when another increase occurred which held fairly constant through day ninety. The ammonia concentration followed the trend of soluble NPN except that it did not increase until day three. All of the initial increase in soluble NPN appears to be due to increases in the unidentified nitrogen compounds. Geasler (1970) reported rapid increases in water soluble nitrogen and water soluble NPN initially. A more accurate comparison of the nitrogen fractions is to express soluble nitrogen as a percent of total nitrogen as shown in Figure 4. It is apparent that the majority of the proteolytic action occurs early in the fermentation process (fresh — day two) and then tends to continue at a slow rate through day ninety. The variability of the results may be due to individual experimental silo variation. Van Soest (1965) reported that when the cell wall con- stituents (CWC) made up 50 to 60% of the forage dry matter, they appeared to limit intake. 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N .z eNseNomeH N mm.H 44.4 44.4 NH.H NN.N 44.4 04.4 4N.H N .eewoeeez N em.Hm NN.mm H4.om 4N.4N mm.~m 4N.Nm 4~.~m NH.Hm N .eeueez see N oo.4 NH.4 mm.4 «4.4 No.4 m~.4 no.4 «N.m me N o m w m N H :moum Amanda uwuums sue a co emwwmunxm mosam> HHummbo m mo smmz4 coaumusmakmm mo hon 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 4444 444444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 4444 444444 N 44. 44. 44. 44. 44. 44. 44. 44. N .z 444444444444 N 444. 444. 444. 444. 444. 444. 444. 444. N .z-4mz N 44. 44. 44. 44. 44. 44. 44. 44. N .242 4444444 444 N 44. 44. 44. 44. 44. 44. 44. 44. N .2 4444444 444 N 44. 44. 44. 44. 44. 44. 44. 44. N .2 444444444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 N .44444442 N 44.44 44.44 44.44 44.44 44.44 44.44 44.44 44.44 N .444442 444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44 44 44 44 44 44 44 4 4 mhmn he muoumamuwm coauMusmsbmm omeHm Houucou mo 4mcmmz Amanda umuuma hut m so vmmmmumxo moDHm> HHummbo m mo :4424 sowumucmshmm MO 449 44. 44. 44.4 44. 44. 44. 44. 44. N .4444 444444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 4 N .4444 444444 N 444. 444. 444. 444. 444. 444. 444. 44. N .z-4mz N 44. 44. 44. 44. 44. 44. 44. 44. N .z 444444444444 N 44. 44. 44. 44. 44. 44.4 44.4 44.4 N .zmz 4444444 444 N 44. 44. 44. 44. 44. 44.4 44.4 44.4 N .2 4444444 444 N 44.4 44.4 44.4 44.4 44.4 44. 44. 44. N .2 444444444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 N .44444442 N 44.44 44.44 44.44 44.44 44.44 44.44 44.44 44.44 N .444442 444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 --- 44 4 4 4 4 4 4 4 44444 wN oHan Amfimmn Hmuuma sue a no tomwmumxm mMSHm> HHuwmno m 40 smwz4 44.4 44.4 44.4 44.4 44.4 44. 44.4 44.4 N .4444 444444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 N .4444 444444 N 444. 444. 444. 444. 444. 444. 444. 444. N .z-4ez N 44. 44. 44. 44. 44. 44. 44. 44. N .z 444444444444 N 44. 44.4 44. 44.4 44.4 44. 44. 44. N .242 4444444 444 N 44. 44.4 44. 44.4 44.4 44. 44. 44. N .2 4444444 444 N 44. 44.4 44. 44. 44.4 44. 44. 44.4 N .2 444444444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 N .44444442 N 44.44 44.44 44.44 44.44 44.44 44.44 44.44 44.44 N .444442 444 N 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44.4 44 44 44 44 44 44 44 4 4 sowumusmahom mo ken cmssfiucoo mu manna AmNmMQ umuuma 44p 4 so vommmumxm wmaam> HH ammouufiz mn< pom muowSuHumsoo Hamz Hamu mN mHQmH 114 harming plant enzymes (IAEA,_197Q)- The AI and II silages were inoculated with 10% by weight of a silage in the third day of fermentation. The A and I silage treatments that were not inoculated produced no bacterial growth in fluid thioglycollate medium. Bacterial growth was produced in fluid thioglycollate medium in a 24 hour incubation period with the following silage treatments: C, AI and II. The fermentation data (Table 30) and bacterial growth indicate that the attempted steriliza- tion and reinoculation were successful. The AI silage and the II silage did undergo fermenta- tion as revealed by the lactic and acetic acid concentrations, which were essentially equal to the C silage. The A and I silages (not inoculated) contained no lactate and low levels of acetate indicating the absence of fermentation. This was expected since autoclaving and irradiating are bacteri- cidal processes. The inclusion of bacteria in the II silage resulted in no increase in water soluble NPN as a percent of total nitro- gen compared to the I silage. The two irradiated silages and the C silage produced similar amounts of water soluble NPN as a percent of total nitrogen (N27%). Therefore, it appears that plant enzymes were reSponsible for the proteolysis. Further evidence is suggested by the absence of proteolysis in the A and AI silages. The level of water soluble NPN as a percent of total nitrogen was essentially the same for the fresh and two autoclaved silages (mll%). 115 .mmHBHonuo wmumum 4444c: uwuuma map mo ucooumd mm pomwmuexw mum mmsam> .mmsam> m mo cam: 44. 44. 44. 44. 44. 4 4444 444444 44.4 4 44.4 4 44.4 4 4444 444444 "mpHo< owomwuo 44. 44. m4. 44. 44. 44. N .44444444 444444444m4: 444. 444. 444. 444. 444. 444. N z- 44 4.44 4.44 4.4 4.44 4.44 4.44 N .44444444 44444 44 N 444 4444444 44. 44. 44. 44. 44. 44. N .444 4444444 44. 44. 44. 44. 44. 44. N .44444444 4444444 44. 44. 44.4 44. 44. 44. N .44444444 444444444 44.4 44.4 44.4 44.4 44.4 44.4 N .44444444 44444 "moofiuomum ammouuwz 44.4 44.4 44.4 44.4 44.4 44.4 44 4.44 4.44 4.44 4.44 4.44 4.44 N .444444 444 @wumHn—U OfiH Umumflwmh—HH fiwumHH—UOGH ©¢>flHUOufi< HOHUGOU fimmhm 444 444 wmuwfipmuuH po>mHooud< uomEumonH omda4m mHo>mg smwouufiz pmHMHuompHaD wownmufl< unmaauoexm uoH4m om wamH 116 Russell (19081 and Kirsch (1930) demonstrated that auto- claving silage. a process that denatures enzymes and steri— lizes the silage, prevented proteolysis even if inoculated with bacteria. Hunter (1921); Kirsch (1930), and Mabbit (1951) also reported that plant enzymes were responsible for proteolysis in silages. Russell (1908) suggested that plant enzymes degrade pro- tein to amino acids and that microorganisms may attack the nitrogenous products carrying them beyond the stage reached by plant enzymes. The amino acid content in the two irrad- iated silages was not evaluated; however, Bergen and Hender- son (unpublished) have shown that amino acids compose less than .1% of corn silage dry matter. Hughes (1970a) reported ’that amino acids composed 63% of the NPN in grass silage ensiled for two months. Since the AI silage resulted in fermentation essentially equal to the C silage in the absence of proteolysis, it was decided to use this silage treatment in Experiment VI. Other treatments altering the unidentified nitrogen content of silage were also compared in Experiment VI. Qhemical Analysis of Silage Results of four composite analyses of the silages are shown in Table 31. Dry Matter The dry matter means were significantly different (P < .01) and this difference was expected due to intended drying. 117 The dry matter levels for the four silage treatments were: control - 31.68%, autoclaved — 32.85%, sun—dried — 52.13% and air-dried - 84.51%. Nitrogen Fractions The silages were chopped at the same time and allowed to dry on a paved surface (52% DM) or in a crop drier (85% DM); therefore, stage of maturity was not a factor and dif- ferences in nitrogen content of the silages were not antici- pated. The means for total nitrogen did not differ signi- ficantly. Geasler (1970) and Byers and Ormiston (1966) reported significant decreases in total nitrogen as stage of maturity and consequently dry matter increased. The total nitrogen content expressed as a percent of dry matter (% DM) ranged from 1.34% to 1.43% and is within the range reported by Geasler (1970) and Gorb and Lebedinski (1960) for corn silage. Water insoluble nitrogen values (% DM) approached sig- nificance and when expressed as a percent of total nitrogen (% TN) the differences became significant (P < .01). The correlation between silage dry matter and water insoluble nitrogen was significant (r =_.50, P < .05). Hawkins (1969) using alfalfa silage and Geasler (1970) working with corn silage reported similar trends. The percent of water insol- uble nitrogen (% TN) in the autoclaved silage was signifi- cantly (P < .01) higher than the control silage and signi- ficantly (P < .05) lower than the air dried silage. 118 The water soluble nitrogen levels (% DM) ranged from _28% to .55% and were significantly lower in the air dried and autoclaved silages than in the control silage (P < .01) and the sun dried silage (P < .05). The air dried silage contained only 50% of the water soluble nitrogen that was contained in the control silage. Water soluble nitrogen was reduced 33% in the autoclaved silage compared to the control silage. The difference in water soluble nitrogen between the autoclaved (.37%) and the air dried silage (.28%) was significant (P < .05). Water soluble nitrogen and silage dry matter had a significant negative correlation (r = -.64, P < .01). Since total nitrogen of all four silages was essentially the same, the water soluble nitrogen expressed as a percent of total nitrogen followed the changes of water soluble nitrogen expressed as a percent of dry matter. The water soluble nitrogen represented 27.61% and 19.58% of total nitrogen in the autoclaved and air dried silage, respectively; the difference was significant (P < .05). Both the auto- claved and air dried silages had significantly (P < .01) less water soluble nitrogen (% TN) than the control silage. Hawkins (1969) and Geasler (1970) reported that protein degradation during ensiling measured by increased water soluble nitrogen (% TN) decreased as dry matter increased. Brody (1965) also reported higher protein hydrolysis in moist silage than in drier silage. Autoclaving reduced but did not inhibit proteolysis as 119 was the case in the pilot experiment. There are two pos- sible explanations: either the silage was not autoclaved properly or proteolysis occurred prior to autoclaving. The later possibility seems most likely since data reported in Experiment V and by Geasler (1970) indicated that proteolysis is rapid during the first day of fermentation. There were small but significant differences between silages in the ammonia levels (% DM); however, ammonia (% TN) was so small (range 1.50% to 4.00% of total nitrogen) that it appeared unimportant. The ammonia values observed for the control, autoclaved and sun dried silages are simi- lar to those found by Henderson (1971c) and (197le). The unidentified nitrogen levels of the four treatments are shown in Table 31 and follow the trend of water soluble nitrogen levels. The air dried silage resulted in a signi- ficantly (P < .01) lower level of unidentified nitrogen com- pounds than all other treatments. The autoclaved silage had significantly lower unidentified nitrogen content than the control silage (P < .01) and the sun dried silage (P < .05). The latter two treatments were not significantly dif- ferent. The unidentified nitrogen expressed as a percent of soluble nitrogen did not vary greatly (80% to 85%) because changes in soluble nitrogen and unidentified nitrogen para- lleled each other. A more meaningful expression is the per- cent of total nitrogen contained in the unidentified nitro- .gen fraction and these values were: control - 33.89, auto- claved - 22.81, sun dried - 29.55 and air dried - 15.95. 120 It is apparent that increasing the dry matter content of silage decreased the unidentified nitrogen (% TN) and these two silage parameters were significantly and negatively correlated (r = -.60, P < .05). The air dried silage had approximately 50% less unidentified nitrogen (% TN) than the control and sun dried silages. The unidentified nitrogen (% TN) in the control silage was approximately 34% and this is in agreement with values reported by Henderson 33 31. (1971c) of 30%. Analyses of cell wall constituents and acid detergent fiber (ADF) in the four silages are shown in Table 32. The results did not vary greatly. The percent of the total ni- trogen contained in the ADF fraction was reduced approxima- tely 60% in the control and sun dried silages and 37% in the air dried silage compared to the autoclaved silage. The autoclaved silage contained 13.81% of the total nitrogen in ADF fraction and the air dried silage contained 8.53%. Ni- trogen contained in the ADF is essentially indigestible as reported by Van Soest (1962). Qrganic Acids Total organic acid content of the silage (% DM) and silage dry matter had a significant negative correlation (r = -.87, P < .01). The range in total acid content was from..03% to 12.16% of silage dry matter. It is apparent that the air dried silage did not undergo fermentation as revealed by virtually no organic acids and consequently a higher pH. The other three silages did undergo fermentation 121 with all three having higher lactic acid levels than reported by Barnett (1954) and Watson and Nash (1960). The acetic acid concentrations are near expected values reported by the above researchers. The pH paralleled the acid con- tent of the silages. The acid concentrations illustrate again that there is less fermentation with increasing levels of dry matter as reported by Hawkins (1969) and Geasler (1970). In this experiment the acid content of the control, sun dried and air dried silages were all significantly dif- ferent either at (P < .01) or (P < .05). It is important to note that the autoclaved silage did have less lactic and total acid content than the control; however, the difference was not significant. The acetic acid in the autoclaved silage in this experiment was significantly (P < .05) lower than in the control silage. Studies In Vivo Water Balance Trial Water balance data are shown in Table 33. Free water intake by the sheep differed significantly (P < .01) for the four silage treatments. Silage dry matter and free water intake were significantly correlated (r = .86, P < .01). The sheep fed the air dried silage drank 90% of their total water intake and the sheep on the control and auto- Claved silages drank approximately 14% of their total water intake. The silage water intake of the sheep fed the con- trol and autoclaved silages exceeded the total water intake 0f the sheep fed the air dried silage by an average of 14%. 122 .444. v 44 n 4 .440. v 4V 4 .ucmummm4w kHuSMUHMHQme uoc mum uQHHUmHmmBm 4844 4:4 wc4>mn no uewuomumdom on 5443 mmoam> .mcmms mo uouum vumtoMum u Mmm .ms0444>ummno 4 mo ammz4 44.4 44.4 44.4 44.4 44.4 44 NH.o 4.mo.o nom.o pr.o 404.4 4404 UHumo< 4m.o MHUOus< Houusoo s044m>ummno uaosummufi mwmafim 44 44444 mmwmafim mo 44mmm 444442 hum no mwmhamn< HmUHEwSU Hmwwum>< 123 44.4 44.4 44.44 44.4 N .2 44444 44 N 44 z 4444 m4.4 4m.4 4m.4 N4.4 N .cmwou44a 44404 444. 444. 444. 444. N .44444444 444 44.44 40.44 44.44 O4.mm N .44444 444444444 4444 44.44 om.mm 4m.4m m4.mq N .444404444400 4443 4440 @444Q 44< @444Q cam vm>44004=< 4044400 4444544448 0w444m 44:44> cmm0444z mm< cam m4dmnu4umaou 4443 4440 Nm M434H 124 Sheep fed the 52% dry matter sun dried silage had a total water intake that was less than the silage water intake of the control silage fed sheep and equal to the silage water intake of the autoclaved silage fed sheep. Therefore, sheep fed the control and autoclaved silages were at the point of being forced to increase their water intake by eating. Water intake (ml per g of dry matter intake) was signi- ficantly different (P < .01) and decreased with increasing dry matter content of the silage fed. The values ranged between 2.51 for the control and 1.89 for the air dried silages. Hawkins using alfalfa reported values of 3.52 for 22% DM silage, 2.42 for 40% DM silage and 2.23 for 80% DM alfalfa. Calder et_al. (1964) reported water to dry matter consumption ratio of 2.5 for hay and 3.3 for silage. Fecal water did not differ significantly; however, total water output was significantly (P < .05) higher for the control and autoclaved silages than the other two treat- ments. Total water balance varied between 788 ml and 458 ml but was not significantly different. Therefore, the pri- mary method of regulating water balance was by urinary excre- tion which did differ significantly (P < .01). Urinary water was significantly correlated to total water intake (r =_.85, P < .01). Dry Matter Intake Silage dry matter intake measured when the sheep were fed ad libitum is shown in Table 34. Values are expressed 125 as dry matter intake g per kg body weight'75. Differences in dry matter intake were not significant and were not cor- related with silage acid content or water soluble nitrogen parameters. Geasler (1970) reported a negative correlation between water soluble nitrogen and dry matter intake (r = -.84, P < .01). Senel and Owen (1967) and Allen at El. (1971) observed no reduction in dry matter intake when acetic acid was added to the ration. Allen at El. (1971) reported no reduction in dry matter intake when lactic acid was added to the silage. However, King (1943), Emery 33 31. (1961), Harris at El. (1966) and McCarrick at 31. (1966) attributed reduced dry matter intake to the content of organic acids. Dry matter intake did not increase as silage dry matter increased. This is in disagreement with results reported by Klosterman et_al. (1963a), Huber 35 El. (1965), Johnson and McClure (1968) and Henderson et_al. (1971a). One ex- planation may be due to observed differences in eating habits of the sheep during the course of the experiment. Dry_Matter Digestibility Dry matter digestibility differences (Table 34) were small and nonsignificant. The range in digestibility was from 66.93% to 71.60% and these values are within the range of those reported by Geasler (1970). The digestibil— ity values were all above the 52% to 66% range where rumen fill may limit intake as suggested by Conrad et_§l, (1964). Campling (1966c) found no relationship between digestibility EIE as To 126 Table 33 Means1 for Water Balance Study Treatments Control Autoclaved Sun Dried Air Dried 882 ml m1 ml ml Free water 385 372 1304B 1838A 111.7 Silage water 2390A 2239A 938B 196 99.0 Total intake 2775a 26113 2242b 20342: 148.5 Fecal water 834 710A 649 777 47.0 Urinary water 1154a 1208 964AB 800B 76.4 Total output 19883 1918a 1613 1577 95.2 Water balance 788 693 630 458 76.9 Water intake, ml/g 2.51Aa 2.36Aab 2.16ABb 1.89B 0.09 dry matter intake 1Mean of 4 observations. 2SE = Standard error of means. Values with no superscript or having the same superscript are not signi- ficantly different, A = (P < .01), a = (P < .05). Table 34 Means1 for Dry Matter Intake-and Digestibility Treatments ”CEnETOI’ Autoclaved’ Sun Dried’ Air Dried SE2 Dry matter intake, 1092 1085 1038 1073 30.67 g/day Dry matter digested, 738 724 735 778 41.37 g/day Dry Inatter digest- 67.65 66.93 70.58 71.60 2.03 ibility, ‘7. Dry Inatter intake7 65.81 68.84 63.47 68.48 4.10 8/kgg body weight' 5 1 Means of 4 observations. SE == Standard error of means. N0 Significant differences between means. 127 and intakes of silage which agrees with data in this exper- iment. NitrOgen Balance There were no significant differences in the nitrogen balance values shown in Table 35. Since dry matter intake and silage nitrogen content were not significantly differ- ent for the four silage treatments, significant differences in nitrogen intake were not expected. The percent nitrogen digested for the autoclaved silage was lower than the other three treatments and dif- ferences approached significance. Apparently, autoclaving denatured some protein rendering it indigestible and it contained more nitrogen in the ADF which is indigestible (Van Soest, 1962). It is impossible to determine the fate of the unidentified nitrogen compounds based on this trial due to lack of trends between nitrogen balance parameters and unidentified silage nitrogen compounds. All sheep were in a positive nitrogen balance. If the unidentified nitrogen compounds were entirely unavailable (% of total nitrogen) then some of the sheep should have been in a negative nitrogen balance because the rations were just over the 8% crude protein level required for main- tenance. None of the nitrogen balance parameters were sig- nificantly correlated to silage fermentation parameters. Rumen Ammonia and Blood Urea Rumen ammonia values are shown in Table 36. Rumen 128 .30. v my .Amo. v .3 u m < .ucoumMMfip hausmofiMfiswfim uoc mum umwuomHmQSm memo man wsH>ms no udwuomumdsm o: nufiz mmSHm> .mdmoa mo Hound unochum u mmm .msoaum>uomno q no coo:H wm.q HH.Nq mw.m¢ oo.cm cm.~q N .mxmuaH 2 mo N mm 2 humswua om.~ em.oe ma.me Hm.Nm aa.~a N .mxnuae z «6 N an z aroma m~.a wo.- um.- mm.m~ mm.o~ N .umumowfiv 2 mo N we wmaemumu z om.m mo.NH mm.~a mq.HH mq.ma N .poummmaH 2 mo N mm pmofimumu z mm.o mw.~ mw.H om.H om.N ch\w .pocfimuou omwouuaz mq.o «m.c mH.o 0H.m no.0 mmp\w .smwouufic humoaub ow.~ oa.mm Nw.cm mq.nq Hw.nm N apoummwfip smwonuas usmoumm mw.o wH.a was. oo.n oo.m hmp\w apoummwwp cowouufiz mq.o 00.0 ma.o wo.n Ho.o . hmp\w .cmwouuas Hooch ww.o mm.mH HH.¢H mo.qa No.mH mmp\w .mxmuafi smonUHz Nmm twang ufi< poaun cam po>mH00us< .Houuaou muowaummua kpaum monmamm sowouufiz How Hummus mm mHan 129 ammonia levels were significantly (P < .01) higher at feed- ing time (To) on the air dried and sun dried silage rations than on the control silage ration. This could be due to a longer or more extensive fermentation period in the rumen for the higher dry matter silages. Significantly (P < .05) higher rumen ammonia levels were produced by the air dried silage ration than all other rations at T Ammonia con- 0. centrations at other times were not significantly different for the four silage treatments. The autoclaved silage pro- duced lower rumen ammonia levels than the other treatments at T1 - T This could be attributed to autoclaving since 6' the digestibility of the nitrogen was lower than the other treatments. The air dried silage had significantly (P < .01) less unidentified nitrogen as a percent of total nitrogen and the rumen ammonia levels did not peak as high or drOp as fast as they did in the control and sun dried silage. The unidentified compounds may have been (same for control and sun dried silage) converted to ammonia faster than the. water insoluble protein which was higher in the air dried silage. £2 yitrg studies (discussed later) indicate that most of the unidentified nitrogen compounds are converted to ammonia. The relationship of low unidentified nitrogen compounds in the silage to more stable rumen ammonia levels did not hold true for the autoclaved silage. However, some of the protein was apparently altered in the autoclaved 130 Table 36 Mean1 Rumen Ammonia Values (mg/100 ml) _‘ Treatments Auto- Sun Air Time Control claved Dried Dried SE2 AB To 5.67C 6.95BC 8.62 10.68Aa 0.59 T1 13.25 10.62 14.62 12.37 1.86 T2 12.18 8.23 12.15 10.48 2.14 T4 5.70 4.90 5.15 6.39 0.90 T6 3.35 3.55 4.07 5.66 0.79 T8 3.67 3.80 4.02 5.35 0.74 1Mean of 4 observations. 2SE = Standard error of means. Values with no superscript or having the same superscript are not significantly different, A = (P < .01), a = (P < .05). Table 37 Mean1 Blood Urea Values (mg/100 ml) Treatments Auto- Sun Air 2 Time Control claved Dried Dried SE TO 8.10 8.77 9.07 9.78 0.54 T4 9.28 9.53 10.13 11.30 0.68 1Mean of 4 observations. 2SE = Standard error of means. 131 silage and thus responsible for the change in the relation- ship deScribed above. Although there were no significant differences in blood urea levels (Table 37), they followed rumen ammonia concentrations. There was an increase in blood urea with the higher dry matter silages. The autoclaved and control silages produced similar blood urea concentrations. Rumen pH and VFA Concentrations None of the silage treatments utilized in this experi- ment had a significant influence on rumen pH (Table 41). Rumen pH values exhibited a normal pattern (Fenner at al., 1967) of decrease in pH during active fermentation after feeding and then increased as fermentation declined. The drop in pH at T in the autoclaved and air dried silages 8 can be ascribed to increased fermentation due to the con- sumption of silage over a longer period of time. Mean rumen volatile fatty acid concentrations for the various silages fed are shown in Tables 38, 39 and 40. The small differences in VFA concentrations may be due to the eating habits of sheep on the different rations. The low level of propionate at all times after feeding (significant (P < .01) at T and T2) is not readily explainable. The l significantly low level of rumen butyrate in sheep fed the control silage compared to the sun dried silage (P < .05) and the air dried silage (P < .01) at T0 was not observed after feeding. 132 Table 38 Mean1 Rumen Acetate Concentrations (mg/100 ml) Treatments Auto- Sun Air Time Control claved Dried Dried SE2 T0 340.0 352.5 355.0 382.5 17.20 Tl 385.0 417.5 385.0 397.5 17.14 T2 377.5 397.5 395.0 360.0 21.60 T4 360.0 402.5 362.5 380.0 14.58 T6 365.0 367.5 327.5 402.5a 17.19 T8 332.5 382.5a 330.0 372.5 11.70 1Mean of 4 observations. 2SE = Standard error of means. Values with no superscript or having the same superscript are not significantly different, a = (P < .05). Table 39 Mean1 Rumen PrOpionate Concentrations (mg/100 ml) Treatments Auto- Sun Air Time Control claved Dried Dried SE2 To 97.5 95.5 102.5 92.5 7.77 T1 174.6 165.0 162.5A 105.0% 11.06 T2 185.0Aa 142.5 162.5 a107.5 11.79 T4 140.0 142.5 127.5 112.5 5.91 T6 125.0 120.0 105.0 97.5 7.60 T8 115.0 ‘125.0 100.0 87.5 10.18 1Mean of 4 observations. ZSE = Standard error of means. Values with no superscript or having the same superscript are not significantly different, A = (P < .01), a = (P < .05). 133 Table 40 1 Mean Rumen Butyrate Concentrations (mg/100 ml) Treatments Auto- Sun Air Time Control claved Dried Dried SE2 To 65.0Bb 72.5ABab77.5ABa 82.5A 3.15 Tl 70.0 65.0 90.0 80.0 9.47 T2 85.0 60.0 105.0 75.0 12.99 T4 70.0 65.0 87.5 90.0 6.42 T6 72.5 60.0 67.5 90.0 11.27 1Mean of 4 observations. 2SE = Standard error of means. Values with no superscript or having the same superscript are not significantly different, A = (P < .01), a = (P < .05). Table 41 Mean1 Rumen pH Treatments Auto- Sun Air Time Control claved Dried Dried SE2 To 6.59 6.58 6.57 6.50 0.046 T1 6.44 6.49 6.39 6.48 0.056 T2 6.43 6.47 6.33 6.45 0.064 T4 6.51 6.44 6.42 6.43 0.027 T6 6.51 6.51 6.52 6.46 0.037 T8 6.51 6.47 6.54 6.44 0.022 1Mean of 4 observations. 2SE = Standard error of means. No significant difference between means. 134 Rumen Dry Matter Rumen dry matter percentages are shown in Table 42. The significant differences at T1 may be a reflection of silage dry matter, since the control silage (31.68% DM) pro- duced a rumen dry matter that was significantly lower than the sun dried silage (P < .05) and the air dried silage (P < .01). The rumen dry matters did not follow the expected pattern of decreasing with time after feeding and then increasing by eight hours post-feeding reported by Hawkins (1969). This may have been due to sheep eating throughout the day. Plasma Amino Acid Analyses The average values for total essential, total non-essen- tial, and sulfur containing amino acids are shown in Table 43. There were no significant differences in any of the values; this may have been due to the large variation as indicated by the standard error. The reasons for the varia- bility are not readily explainable. Only the prefeeding values (T0) are shown because the values for four hours post-feeding (T4) were essentially the same. Fenderson and Bergen (1972) using four rations consisting of varying levels of roughage and/or grain (varied in protein from 6.9 to 20.2%) demonstrated that plasma amino acid concentrations were not affected by the diets used or time after feeding in sheep. Purser 35 31. (1966) administered a starcheglucose mixture intraruminally and observed a decline in plasma amino acid levels in sheep. However, the starch-glucose Table 42 Means1 for Rumen Dry Matter Determinations Treatments , Time Control Autoclaved Sun Dried Air Dried .SE2 To 8.25 9.00 9.13 9.50 0.34 Tl 8.00Bb 8.43Bab 8.95ABa 9.66A 0.22 T2 7.80 8.42 9.00 9.02 0.28 14 7.48 8.34Ab 8.83Aa 9.02Aa 0.16 T6 8.00 8.42 8.37 9.53A 0.19 T8 8.24 8.32 8.92 9.75 0.32 1Mean of 4 observations. 2SE = Standard error of means. Values with no superscript or having the same superscript are not signi- ficantly different, A = (P < .01), a = (P < .05). Table 43 Means1 Plasma Amino Acid Concentrations (um/100 m1) Treatments Control Autoclaved Sun Dried Air Dried SE2 Total EAA T0 90.9 92.7 95.9 99.3 28.3 Total NEAA TO 198.6 192.8 235.0 246.1 65.2 NEAA/EAA TO 2.48 2.09 2.41 2.39 0.18 Lysine 9.75 9.63 9.2 9.2 3.9 Sulfur con— 7.58 7.78 8.1 7.6 2.2 taining AA 1Mean of 4 observations. 2SE = Standard error of means. 136 mixture was a more readily available source of energy than found in most conventional rations. Salas (1971), using soybean supplemented corn silage rations fed to steers, reported NEAA/EAA ratios of 1.00. In this experiment, using unsupplemented corn silage, NEAA/EAA ratios of 2.09 to 2.48 were observed. The increased ratio was due to increased NEAA levels. Studies £2_Vitro The rate and extent of cellulose digestion, using silage alone and silage plus urea as the substrates, are shown in Table 44 for the four silage treatments. The digestibility of 12 hour samples estimated the rate of cell- ulose digestion while the 48 hour sample estimated the ex- tent of digestion. Cellulose digestibilities were about 50% less for the control and autoclaved silages without added nitrogen than the sun dried and air dried silages. This may suggest that nitrogen is limiting in the control and autoclaved silages; however, the water soluble nitrogen compounds in all silages were converted to volatile base (NH3) after 12 hours as illustrated in Figure 5. At 48 hours, the two drier silages had greater cellu- lose digestibilities; however, the difference was not as great as at 12 hours. When urea (108 mg ml) was added to the silage, cellu- lose digestibility was increased at 12 hours to the level attained at 48 hours with silage alone. Under these 137 conditions, nitrogen was not limiting. A higher cellulose digestibility was observed for the autoclaved silage (61.4%) than all other treatments which averaged approximately 54%. All of the water soluble non-ammonia nitrogen was converted to volatile base (NH3). The urea elevated the levels of water soluble nitrogen initially (Figure 6). Based on these results, it appears that the water solu- ble unidentified nitrogen compounds are converted to ammonia and if they are converted to ammonia, they should not reduce voluntary dry matter intake. However, the unidentified nitro- gen compounds may escape the rumen before being degraded to ammonia and thus could interfere with intake. Correlation Coefficients Correlation coefficients are shown in Appendix I. 138 Table 44 Means of Cellulose Digestibility for 13_Vitro Studies of Corn Silages1 Silage Treatment Sampling Time Control Autoclaved Sun Dried Air Dried Z Z Z Z No N added in the_in vitro system 12 hr. 11.2 10.7 20.9 20.5 48 hr. 28.2 32.6 35.9 36.9 40 mg Z Urea - N/100 ml in the_in vitro system 12 hr. 31.7 37.2 29.0 27.0 48 hr. 55.0 61.4 53.0 54.4 1Two observations per mean. 139 10 — Solub 1e NPN - - Volatile Bases -4 Meg. N Base x 10 U! 12 48 Time (hours) Figure 5. Changes in water soluble nitrogen and volatile bases during in vitro digestion. 140 10 III-I Soluble NPN a? - - Volatile Bases O H x m m m m z 5 8 ‘~-- 2 12 48 Time (hours) Figure 6. Changes in water soluble nitrogen and volatile bases during in_vitro digestion (urea added). V SUMMARY The results of six experiments are presented and dis- cussed. A common objective of all six experiments was to vary and evaluate the unidentified water soluble nitrogen frac- tion in corn silage, which represents 1/3 to 1/2 of the total silage nitrogen. The literature suggests that the unidentified nitrogen fraction may contain compounds respon- sible for depressed dry matter consumption by ruminants fed silage vs. hay or green chop. Perhaps the unidentified nitrogen fraction is unavailable for rumen microorganisms, thus reducing dry matter intake of silage as a result of less utilizable nitrogen. Attempts to link other products of silage fermentation (organic acids) to reduced silage dry matter intake have produced inconsistent results; therefore, other silage fermentation products such as the unidentified nitrogen fraction must be investigated. Experiment I was designed to measure the effects of maximizing fermentation of corn silage with limestone treat- ment and minimizing fermentation with formic acid treatment without the confounding effects of added nitrogen on steer performance, silage nitrogen and acid fractions, and steer metabolic parameters, when compared to control silage 141 142 receiving no treatment. All three silages were fed to steer calves in a 161 — 238 day experiment and were compared on both an all silage ration and a 60% corn silage and 40% high moisture shelled corn ration on a dry matter basis. Results of the metabolic study, using the three all silage rations without protein supplementation, are presented in Experiment III. Organic acid levels were significantly (P < .01) increased and decreased with limestone and formic acid treatments, respectively. The unidentified nitrogen coumpounds made up 41% of the total nitrogen in the control silage and 34% of the total nitrogen in the formic acid and limestone treated silages; therefore, the content of unidentified water soluble nitrogen compounds was not greatly affected by the extent of fermentation. Average daily gain was essentially identical for all treatments. Dry matter consumption was decreased slightly for the lime- stone treated silage; however, this was offset by an improve- ment in feed efficiency. Dry matter intake was not increased for the formic acid treated silage, which contained signi- ficantly (P < .01) less lactate than the control or lime- stone treated silages. The nitrogen retained (g/day) was sig- nificantly (P < .01) higher for the control silage and blood and rumen parameters were not significantly different. Experiment II was designed to measure the effects of stimulating fermentation of corn silage with NPN additions on steer performance, nitrogen balance parameters and Silage nitrogen and organic acid fractions. Five silage 143 treatments were studied: .control untreated silage, Pro—Sil supplemented and treated silages, urea-mineral treated silage and urea-mineral plus formic acid treated silage. All silage treatments were compared on an all silage ration and a 60% corn silage and 40% high moisture shelled corn ration on a dry matter basis. The results of the nitrogen balance study, using the control soy-supplemented and the NPN treated all silage rations, are presented in Experiment IV. The neutralizing effect of Pro-Sil and urea-mineral addition without formic acid resulted in stimulated fer- mentation and bacterial activity, yielding significantly (P < .01) greater lactate levels. Lactate production was reduced when formate was added to the urea-mineral treated silage. Total nitrogen was increased approximately 50% by NPN additions. Water insoluble nitrogen levels were signi- ficantly (P < .01) elevated for all NPN treated silages compared to the control silage; however, unidentified nitro— gen levels were not significantly altered by stimulating fermentation with NPN additions. The increase in water insoluble nitrogen content appears to be due to the pro- duction of microbial protein during fermentation rather than a reduction in proteolysis. Average daily gains were significantly higher for the NPN treated silages than for the control silage supplemented at feeding with soy-mineral (P < .05) or Pro-Sil (P < .01). Differences in feed con- sumption were small; however, feed efficiency and cost favored the NPN treated silages. Differences in nitrogen 144, balance parameters were not significantly different. Experiment V was designed to monitor changes occurring during fermentation in untreated control and Pro—Sil treated corn silages. Samples of each silage treatment were taken when fresh and on days 1 through 10, 15, 20, 30, 60 and 90 after ensiling. Water insoluble nitrogen level in Pro-Sil treated silage was increased to a maximum at day five when the soluble nitrogen as a percent of total nitrogen had decreased to a low level (Figure 4). The unidentified nitrogen level was slightly higher in the Pro-Sil treated silage than in the control silage. These results indicate that water soluble nitrogen compounds are incorporated into microbial protein, consequently increasing the water insol- uble nitrOgen. Experiment VI was designed to vary the resulting level of water soluble nitrogen compounds occurring in fermented silage and determine the effects of these compounds on silage dry matter intake and metabolic and blood parameters. .32 yitrg studies were also conducted to examine the avail- ability of the nitrogen contained in the unidentified com- pounds for rumen microorganisms. Dry matter intake was not affected by experimentally altered unidentified nitro- gen levels in corn silage and was not significantly cor- related with acid levels, nitrogen fractions or dry matter content of the silages. The unidentified nitrogen ranged from 15.95% to 33.89% of total nitrogen for the air dried and control silages, reSpectively. Silage dry matter 145 content and unidentified nitrogen levels had a significant negative correlation (r = —.60, P < .05). Results of the nitrogen balance study were not significantly different. The nitrogen balance data indicate that the unidentified nitrogen compounds are at least partially available for rumen microorganisms, if not entirely, otherwise some sheep would have been in a negative nitrogen balance. £2.213E2 studies showed that all of the unidentified nitrogen compounds were converted to volatile base (NH3) within a 12 hour period. Therefore, it is unlikely that the unidentified nitrogen compounds are responsible for reduced dry matter intake unless they escape the rumen before being degraded to ammonia. It appears that plant enzymes are responsible for protein hydrolysis (increased levels of water soluble nitro- gen) in silage, based on the literature and indirect evi- dence from the pilot experiment in Experiment VI. Plant enzyme levels were not measured in the pilot study, how— ever. The plant enzymes may degrade protein to amino acids and either plant enzymes or microorganisms may carry the amino acids to other nitrogenous products as indicated in the literature. Perhaps bacterial activity is greater in corn silage than in grass and legume silages, since amino acid levels were never greater than .1% of the silage dry matter in Experiment V and levels of .56% of silage dry matter were reported by Hawkins (1969) using alfalfa silage. 146 The unidentified nitrogen compounds appear to be con— verted to ammonia, which is utilizable by rumen microorganisms. Therefore, unless they escape the rumen before being degra— ded they would not be expected to reduce silage dry matter intake. More research is needed before unidentified water soluble nitrogen compounds can be eliminated as depressors of silage dry matter intake. Once the compounds are identi— fied in corn silage, it will be possible to follow their metabolism in vivo. BIBLIOGRAPHY BIBLIOGRAPHY Allen, C. K., H. E. Henderson and W. G. Bergen. 1971Aa. Ammonium salts as a source of crude protein for feedlot cattle. Research Report 143:16. Mich. Agr. Expt. Sta. Allen, C. K., H. E. Henderson and W. 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APPENDIX 166 m Hmm.ou z mz mamaam “muses sue manaummmee a ~mm.o mxmuae umuuns sun saunas sue maneunmmee a mew.o omgwmumn ammonuwz Hmuuma who manfiummmflo w rmom.on somouues Hmomm Hmuums map manwpmmmeo w «vmh.o nouns Hmomm mxmusw Hmuums mun roam.o Hmum3 mumsflna oxmucfl Hmuumfi mum r.mne.o emcenuwn ammouuez minute “whens sea mmH.o cmmonufls whmsfluo wxwpcfl Hmuvmfi who «mnm.o somehow: Hooch mxmusw Hmuumfi mun «ramm.o excuse cwmouuwz mxmuga Hmupme who ewo.ou z maasaon earns emaeeeameaca manaem manure sweets sum mmo.on z oHQsHOm kum3 mmmaflm mxmuow Hmuums who th.oI pflom ospmom momaem excuse Hmuums who Hmo.ou whom oeuoma mmmHem menace ampere sun rmmm.o: nouns mumsflub Hmuume who mmmaflm «rmmm.o mxwugfl Hmumz mosh Hmupme who mmmaflm .reme.o mm mmnaem ampere sec mamaem «rwmh.ol pflom oeumom momaflm Hmuumfi mum mmwaflm rrmvw.ot Uflom owuoma mmmaflm Hmuume who momaflm rmom.o z wHQsHomsw Hmuw3 mmmaflw Humane who mmmawm rammm.01 ZMmZ mmmawm Hmupmfi hut momawm «rmvw.o1 z mHaDHom Hmumz mmmaflm kuumfi hut mmmaflm .moe.ou z manaaon amen; ememeenmaeas monaem smears sue manaem mmv.o umuume who manflpmwmflo w Hmuume who womaam mvo.o mxmucfl Hopumfi who Hmupmfi hum mmmHflm H HH mabmwum> H manmwum> Hfi magma monum OAHOQmumz I v unmawnmmxm I musmwoflmmmoo coanmamuhoo mameflm 167 mvma .HoomomsmH AHo. v my *4 Amo. v my « mmm. hmv. amaH6> Hmoaueao «room.ou wxmucfi nouns mosh axons“ nouns mmmaflm «renm.o wxmusfi Hmum3 Hmuoe orange Hopm3 mmmaflm «rmmw.o mxmpcfl Hmum3 momaflm umum3 wumcwub. «rmvm.o mxmucw kum3 Hmuoa Hmum3 masses: «thah.o pwom oaumom mmmaflm z wansaom “mums womaflm «omm.o pHom oauwma momawm z mansaom Hmum3 mmmafim «omm.o z mz mosaflm z mansaom Hmum3 pmHMHusmoHcs mmwaflm rtvmm.o z mansHOm Hmum3 ommaflm z manoaom H0u03 omflmwusmoacs womawm «rmhm.o meow owumom monaflm z mHnSHom Hmumz cosmeusmpflcs momawm «ovm.o oflom oauoma mmmafim z mannaom Hmumz Umwmwuompflos mmmaflm oom.o: z mesHom Hmumz pmflmwpsmcwss momawm pmswmpmn somouuwz mam.01 z mHnDHOm umum3 mmmHflm Uwswmumu smmouuwz ¢m¢.o Hmum3 Hwomm somouufls anodes: mom.o z mahaaon “mum; ememeuamcaaa mmmaem ammonuea senses: mmH.o| omcwmuwu ommouuwz cmmouufi: mumswuo He~.o whom oeuoma mmmaem ammouues Hmomm vma.o omgflmumu omoouuwz cmmoupflg Hooch mmm.o z Hmuou momaflm mxwusw cmmonnflz «.mee.o emcemumu ammouuez manure ammouuez mm¢.o cmmonuflc mamas“: expose cmmouuez ramee.o ammoauec aroma manage ammouuez nae.ou neon oeuvre manaam Hugues was maneunmmee a mea.ou z annoy monaem ampere sue maneunmmec a vvm.ol z mansHOm kums mmmaam Houumfi who manwummmwo w H HH manmflum> H mabmwum> A.ucoov H4 magma . .l. ii.1|1.4 WIN 0 3 9 2 1 uufimwu