. EFFECT OF SUPPLEMENTING CORN SiLAGE WITH lSOACiDS AND UREA ON PERFORMANCE OF HiGH PRODUCING COWS Dissertation fer the Degree of Ph. D. M§CHEGAN STATE UMVERSITY ARTHUR FELiX 1976. ’— nmomc av ' ' me & ms- anox mm mm, ' LIBRARY muons ABSTRACT EFFECT OF SUPPLEMENTING CORN SILAGE WITH ISOACIDS AND UREA ON PERFORMANCE OF HIGH PRODUCING COWS BY Arthur Felix The effect of supplementing high urea-corn silage- based rations for Holstein cows with isoacids on nitrogen utilization, growth, and milk production was studied over a period of five years. A total of 112 high producing lactat- ing cows and 20 growing heifers were used in four separate milk production trials and one growth study. One in vitrg experiment was also conducted. The addition of isoacids to urea rations increased rumen microbial activity in_zi§gg, improved rate of growth of young animals, increased persis- tency of lactation, and improved nitrogen retention in lactating cows. In the in gitgg study, five experiments were carried out using gas production as a parameter to estimate the effect of different concentrations of isoacids on rumen microbial activity. Concentrates or corn starch and filter paper were used as the source of energy. The addition of Arthur Felix isoacids to urea depressed the rate of gas production irres- pective of the source and the amount of substrate used in the medium. This study showed that the isoacids affected the fermentation process, and therefore, enhanced rumen microbial activity. In the growth study, isoacids and phenylacetate were added with urea to low quality grass hay fed to 20 dairy heifers averaging from 171 to 327 kg body weight. Isoacids and phenylacetate increased the growth rate of the younger animals but not of the older animals. In the first milk production trial the isoacids and phenylacetate were fed with urea to 24 lactating cows in a randomized block design using soy protein as a positive con- trol, urea control, and urea plus isoacids and phenylacetate treatment. Corn silage was used as the only roughage and concentrate was not fed. Soy protein was most effective in increasing milk production, persistency of lactation and body weight. Isoacids improved milk production, persistency of lactation and body weight over the urea alone. In the second trial, corn silage was supplemented with urea plus isoacids fed to 28 lactating cows in a randomized block design consisting of a positive control, a negative control, urea, and urea plus isoacids. Concentrate ‘mas fed according to the level of milk production. Isoacids improved corn silage dry matter intake and persistency of lactation over the urea control. Arthur Felix The third milk production trial used 30 lactating cows fed corn silage in a cross-over design consisting of a urea control and urea plus two different levels of isoacids. Concentrate was fed according to milk production. A nitrogen balance trial was also conducted at the end of each period. The addition of isoacids to urea improved fat corrected milk, persistency of lactation, decreased plasma urea nitrogen and rumen ammonia nitrogen, increased rumen acetate and nitrogen retention over urea fed alone. In the fourth trial, 30 lactating cows fed corn silage and concentrate and urea as supplemental crude pro- tein, were assigned to two treatment groups in a continuous feeding experiment. Isoacids increased the persistency of lactation and total feed intake. EFFECT OF SUPPLEMENTING CORN SILAGE WITH ISOACIDS AND UREA ON PERFORMANCE OF HIGH PRODUCING COWS BY Arthur Felix A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy Science 1976 DEDICATED TO The memory of my mother, Elucia; my father, Darius, whose many sacrifices and courage have enriched my life, and my wife, Jeanine, whose patience and encouragement have made this work possible. ii ACKNOWLEDGMENTS The author wishes to express his deepest and most sincere gratitude to his major professor, Dr. Robert M. Cook, for his continual counsel, his encouragement, enthusiasm and his high interest during the development of this work. Also his patience and moral support throughout the graduate program are highly appreciated. Sincerest gratitude and appreciation are extended to Dr. John T. Huber for his very generous assistance during the absence of Dr. Cook and during the nitrogen balance study, and his valuable suggestions and interest throughout the entire program. Dr. Huber's help in sampling and laboratory analysis is immensely appreciated. The author is also grateful to Drs. Clinton E. Meadows and Duane E. Ullrey for graciously consenting to serve on the guidance committee, and for their constructive criticism in the preparation of this dissertation. Special appreciation is due to Dr. Kim A. Wilson for his constructive reading and suggestions during the prepara- tions of this manuscript. iii The author acknowledges the generous contribution of Dr. John W. Thomas to this work by providing the fistulated cow used during the in zitrg study. Appreciation is extended to Dr. John Gill for statistical advice, and to Dr. Roger R. Neitzel for his invaluable assistance in the computer analysis. The generous assistance of Dr. Gustave Kulasek dur- ing the in 11259 study is gratefully acknowledged. Thanks are due to the Chairman of the Department of Dairy Science, Dr. Charles A. Lassiter for financial support in the form of Research Assistantship in the years 1972-1976. The author is grateful to Mrs. Catherine Ricks and Miss Mary Araiza for their efficient drawing of the figures. Special gratitude is due to Miss Becky Winters for her invaluable help during the nitrogen balance study and in laboratory analysis. Appreciation is extended to the fellow graduate students, the staff of the Dairy Research Center, laboratory personnel and Deparmental Secretaries for their assistances in one way or another during the author's stay at Michigan State. Above all the author is indebted to his wife, Jeanine, whose incomparable sacrifices, continual encourage- ment and patience have made these years of study tolerable and worthwhile. iv Q 1::v ‘w..‘ TABLE OF CONTENTS Page LIST OF TABLES C O O O O O O O O O O O O Vii LIST OF FIGURES . . . . . . . . . . . . . xi INTRODUCTION . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . 7 .Feeding Corn Silage and Urea to Dairy Cattle. . . 7 Some of the Dietary Factors that Affect Microbial Protein Synthesis. . . . . . . . 12 .Amino Acid Synthesis by Rumen Micoorganisns . . . 28 Supplemental Feeding of Isoacids and Urea to Ruminants . . . . . . . . . . . 34 mTERIALS AND “THODS O O I O O O O O O O O 39 In Vitro Experiment . . . . . . .. I . . . 39 Growth Study . . . . . . . . . . . . . 4 8 IEffects of Isoacids on Milk Production. . . . . SO Trial I O O O O O 0 O O O O O O O O 50 Trial II 0 O O O O O O O O O O O O O 5]- Trial III. . . . . . . . . . . . . . 55 Trial IV 0 O O O O O O O O O O O O O 61 RESULTS AND DISCUSSION 0 O O O O O O O O O O 6 6 In.vitro Experiment . . . . . . . . . . . 66 <3rowth Study . . . . . . . . . . . . . 91 Ififfects of Isoacids on Milk Production. . . . . 94 Trial I O O O O O O O O O O O O O O 9 4 Trial II C O O O O O O O O O O O O 0 Trial I I I O O O O O O O O O O O O O O l o 0 Page SUMMARY AND CONCLUSION. . . . . . . . . . . 114 LITERATURE CITED. . . . . . . . . . . . . 116 APPENDIX 0 . O O O O O O .0 O O O O O O 134 vi Ill. :12 LIST OF TABLES Composition of the in Vitro media . . . . Composition of the concentrate . . . . . Quantities of isoacids used in the in vitro experiment . . . . . . . . Composition of the concentrates for trial II. 0 C C O O O O O O O 0 Experimental design for trial III . . . . Composition of the concentrates for trial III C O O O O O O O O O 0 Composition of the diets for trial III . . Composition of the concentrates for trial IV. . . . . . . . . . . . Composition of the diets for trial IV. . . The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate on gas production in vitro. The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate on rumen ammonia production in vitro. . . . . . . . . . . . The effects of different concentrations of isovalerate, 2-methylbutyrate and n-valerate on rumen pH in vitro. . . . . . . . vii Page 41 43 44 54 56 58 59 63 65 67 69 72 Table 13. 14. 15. 16. 17. 18. 19. 20. 21” Analysis of variance for overall gas production, rumen ammonia-N and rumen pH for the in vitro experiment. . . . . Analysis of variance for overall VFA production for the in vitro experiment . . The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n—valerate on VFA production in vitro using 10 g of concentrate as a substrate (experiment 1) . . . . . . . . . . The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate on VFA production in vitro using 5 g of concentrate as a substrate (experiment 2) . . . . . . . . . . The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate on rumen VFA production in vitro, using corn starch and filter paper as substrates (experiment 3) . . . . . . The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate on rumen VFA production in vitro, using corn starch, filter paper and methionine as substrates (experiment 4). . The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate on rumen VFA production in vitro, using 20 g concentrates and methIEF _I—— 0 nine as substrates (experiment 5). . . . The effects of isobutyrate, isovalerate, 2- methylbutyrate, n-valerate and phenylacetate on growth of dairy heifers (growth study) . The effects of supplementing corn silage with soy protein, urea or urea plus isoacids and phenylacetate on milk yields, perisitency and body weight in Holstein cows (trial I). viii Page 73 74 75 76 77 78 79 92 95 Table Page 22. Milk yields, persistency of lactation, body weight changes, feed dry matter consumption and feed efficiency of cows fed corn silage supplemented with urea, urea plus isoacids, soybean meal and no nitrogen supplementation (trial II). . . . . . . . . . . . . 98 23. The effects of two different mixtures of isoacids on milk yields, persistency of lactation, body weight changes, feed dry matter intake and feed efficiency (trial III) . . . . . . . . . . . . 102 24. The effects of two different mixtures of isoacids on milk composition, plasma urea and rumen pH, ammonia nitrogen and VFA (trial III) . . . . . . . . . . . . 105 125. The effects of two different mixtures of isoacids on feed dry matter (DM) digesti- bility, nitrogen digestibility and nitrogen retention by Holstein cows (trial III) . . . 107 :26. Milk yields, persistency of lactation, body weight changes, feed dry matter intake of cows fed equal amounts of isoacids, (trial IV). . . . . . . . . . . . . 109 27. Milk composition, plasma urea nitrogen and VFA of cows fed isoacids (trial IV) . . . . 112 28. Analysis of variance for gas production 3:21—1- Vitro O O O O O O O O O I O O O 134 29. Analysis of variance for acetate production in vitro . . . . . . . . . . . . . 135 130. Analysis of variance for propionate produc- tion in vitro. . . . . . . . . . . . 136 31. Analysis of variance for isobutyrate pro- duction in vitro. . . . . . . . . . . 137 32 - Analysis of variance for butyrate production i—n Vitro C C C C C C C C C C C C C 138 33 - Analysis of variance for 2-methy1butyrate production in vitro. . . . . . . . . . 139 ix Table 34. 35. 36. 37. 38. 39. 40C 41. 42. 43. 44. Page Analysis of variance for isovalerate production in vitro. . . . . . . . . 140 Analysis of variance for valerate production in vitro. . . . . . . . . 141 Analysis of variance for overall N83 and pH in_vitro . . . . . . . . . . 142 Analysis of variance for trial I . . . . . 143 Analysis of variance for trial II. . . . . 144 Analysis of variance for milk yield, persis- tency of lactation and silage DM intake (trial III) 0 o o o c o o o o o o 145 Analysis of variance for nitrogen' balance (trial III). . . . . . . . . 147 Analysis of variance for rumen VFA (trial III) 0 o c c o o o c o o o 149 Analysis of variance for milk total solids, fat, protein and solids nonfat (trial III) . 150 Analysis of variance of total fat, protein and nonfat (trial III) . . . . . . . 151 Analysis of variance for blood urea nitrogen, rumen NHB-N and pH (trial III) . . . . . 152 Figure LIST OF FIGURES 1. Apparatus for measuring fermentation rates of rumen microorganisms. 2. Gas production experiment 1 3. Gas production experiment 2 4. Gas production experiment 3 5. Gas production experiment 4 6. Gas production experiment 5 and and and and and ammonia ammonia xi output output output output output for Page 47 81 83 85 87 89 INTRODUCTION The world population is still growing at a rate suf- ficient to double its current number every thirty years. The most important concern is how to properly feed the people in the world today. Of almost equal importance is how to provide improved diets for people in the future. The rate of population growth is faster than the rate of food produc- tion, at least in many countries of the world (Hodgson, 1971). Requirements to meet projected food needs are indeed alarming. In 1968, estimates indicated that to provide about 2,400 kcal of energy and 35.1 g of protein per capita per day to the world population, the supply will have to be increased by about 38 percent over the 1970 needs by 1985 (Agr. Statistics, 1968). Further estimates suggest that supplies of food crops, sugar, starchy food, vegetable and oil seed crops, as well as milk, meat, eggs, and fish all would need to be increased by 37 to 39 percent to meet human physiological caloric needs (Hodgson, 1971). The shortage of protein-rich foods to meet future world demands is con- stantly being stressed. Current estimates indicate that more than 50 percent of the world pOpulation is suffering from 5‘3?! mi: :‘ema Scie 9:3: dew fee: I f ’4‘ Kw from diet protein shortages. United Nations agencies recom- mend that priority be given to increasing world protein supplies. In more affluent countries such as the United States, animal products become more expensive to produce as world demands for cereals and protein supplements increase. Scientists all over the world are striving to increase the production of conventional protein of high quality and to develop procedures for preparing new proteins suitable for feeding both man and animals. Two foods, rich in protein of high biological value, are milk and meat, in the production of which the bovine holds the key position. Because of the microbial population inhabiting the rumen, the ruminant animal is unique in its ability to convert to milk or meat for human consumption those feeds which are metabolically less available to other species. For example, if ruminant animals are fed forages and other feeds inedible by man along with limited amounts of cereal grains the efficiency of producing proteins for humans in terms of total resource utilization can be enhanced (Moore 33 31., 1967). A report by Hardin and Rogers (1970) indicated that about 29 percent of total land area is forested, 38 percent is apparently unused, and 22 percent is in permanent meadow and pasture. The latter area is a large supplier of feed for the world's ruminant population. Only 11 percent of the total land is in permanent crop production and an undetermined amount of this is in rotation forage crops of various kinds. A (1.1 6' g ‘(1 5L La ‘5 i I t. f . “v. 7 qsn ”H considerable portion of the forested land does, or can, pro- vide grazing. Also, the forage and crop refuse from land under permanent crOps can be added to that forage supply. The grassland crops have a limited role in human food pro- duction except as feed for runinants, especially dairy cattle, which have the highest efficiency in converting feed protein to food protein. The case for the ruminant animal as a producer of protein is even stronger when their capacity to use non- protein nitrogen (NPN) is considered. Many feeding experi- ments with various non-protein nitrogen compounds have been performed. One of these compounds most widely used and most thoroughly investigated is urea. The use of the feed-grade urea in the United States in 1973 was approximately 800,000 tons (Allen, 1974). The use of this quantity of urea spares approximately 4.5 million tons of 50 percent protein supplement that can be used for feeding man and other mono- gastric animals. The real ability of ruminants to utilize urea as the sole source of nitrogen for protein synthesis is most clearly seen when urea is fed to milk-producing cows, which require protein in especially high amounts (Virtanen, 1966). Moore 23 31. (1967), calculating the protein input and output of a cow producing 5,295 kg of milk annually with usual feeding practices of the 0.8., found that the recovery of protein in the milk was about equal to the protein consumed as grain and oilseed concentrates. However, when urea was used to partially replace protein concentrates, the protein in the milk averaged about 21 kg more per cow per year than was consumed in cereals and legumes. Because of the increas- ing and severe competition for natural protein by human beings and non-ruminant animals, it becomes necessary that a portion of the natural protein in ruminant diets be replaced with non-protein nitrogen. But a most outstanding feature of urea feeding is the extent of its utilization with the basic diet especially by high-producing cows. Protein is the nutrient which often limits the per- formance of high-producing cows. Because it is quantita- tively the major component of animal tissue dry matter, because of various physiological and biochemical functions of proteins in the animal body, and because of high rates of milk production and the relatively high content of milk, high-producing lactating cows are more likely to encounter a shortage of protein than any other ruminants. An often forgotten feature of protein nutrition in ruminants is its dualistic nature: the microbial metabolism in the fore- stomachs on one hand, and the non-microbial metabolism in the remainder of the alimentary tract and the tissues of the animal on the other hand. Thus, the fate of the dietary protein fed will depend upon digestive processes as well as the physiological status of the animal. Furthermore, cows producing more than 35 kg of milk daily may fail to produce to their genetic capacity because of the lack of some key nutrients such as protein. In some cases, a cow's physical capacity limits ’9 (I '(I her feed intake so that nutrient supply to the mammary gland is not enough for maximum production of milk and milk protein. Thus, in many cases, the high producing lactating cow, at the peak of lactation is deficient in energy, protein and cal- cium. One of the most important goals for the animal nutritionist to pursue is to increase the efficiency of pro- tein utilization to fulfill the requirement of the high- producing cows. In cases where the digestive processes improve the quality of protein reaching the absorptive sites. of the small intestine, they would be classified as beneficial such as the case of urea utilization (Chalupa, 1972). However, a major problem in the efficient use of a high level of dietary urea is its rapid hydrolysis into ammonia and the subsequent inability of the rumen micro- organisms to utilize the ammonia at a rate comparable to its production. A considerable portion of the excess ammonia nitrogen is lost through metabolic excretions. Furthermore, the ability of the rumen bacteria to utilize ammonia depends on the availability of a suitable source of energy and carbon skeletons. If the nutritional status of high-producing cows is to be improved, it is imperative that the key nutrients be identify, nutrients which may be in short supply to the microbial population, and be provided in the time of greatest need. Several investigators have reported that supplement- ing urea-based rations with the isoacids* (n-valerate, isobutyrate, isovalerate and 2-methylbutyrate) improved the nitrogen economy of the animals. There is considerable evidence that rumen microorganisms utilize the carbon skele— tons of the isoacids in the synthesis of the corresponding branched-chain amino acids and for microbial protein forma- tion. But, to date, there exists no evidence of the beneficial effect of feeding a combination of urea and these isoacids on the performance of high-producing lactating cows. The objective of the studies reported herein was to investigate the influence of the direct supplementation of the isoacids to high urea-corn silage-based rations on nitrogen utilization and on the subsequent performance of high-producing lactating cows. *The term isoacids for the purposes of this disser- tation refers to a mixture of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate. LITERATURE REVIEW Feeding Corn Silage and Urea to Dairy Cattle The use of corn silage as a basic roughage consti- tuent for dairy cattle has increased rapidly because of its high energy yields ease of mechanization and storage, and uniform feeding value. The number of hectares of corn harvested as corn silage has nearly doubled in the United States during the past two decades. In Michigan, corn silage is the most important silage crop with over four million metric tons produced in 1973 (Mich. Agr. Stats., 1974). Cattle feeders have become increasingly aware of the excellent forage characteristics of corn silage and its value in the ruminant ration. The corn plant is similar to other forage grasses in that it contains most of the same components, although the concentrations are somewhat different from those in other forages. The high digestibility of its crude fiber by rumi- nants, and high concentrations of starch and other soluble carbohydrates are peculiar to corn silage and enhance its energy value compared to most forage crops. Research on corn silage diets for dairy cattle has been reviewed (Riley, 1967; Coppock and Stone, 1968; Huber, Polan and Hillman, 1968a). Several trials comparing corn silage to other for- ages have been reported (Holter 35 gig, 1973; Belyea gg_al., 1975a, 1975b; Thomas 33 31., 1970; Vandersall 35 31., 1970). These trials showed that corn silage or corn silage plus hay gave similar intake and production responses and that there appeared to be no advantage for including hay in corn silage rations. Because corn silage is widely recognized as a high quality roughage, it has been used as a standard of compari- son for many other silages. As a source of energy for milk production, corn silage has been shown to be equal or superior to dried-corn silage, alfalfa-bromegrass silage, corn-treated meadow-crop silage, broomcorn silage, beets and many other types of silages (Coppock and Stone, 1968). The voluntary consumption of corn silage dry matter «appears to increase as the dry matter content increases, at least within certain limits (Huber _e_1_:_ §__l_., 1965) . Corn Si;1age has a relatively low crude protein content of 8 to 9 Percent on a dry matter basis. Various approaches have been uBed to increase the protein content of corn silage or to supplement it economically with non-protein nitrogen (NPN) . (his: supplemental method, which is widely practiced, is to fSéeed corn silage in combination with high-protein legumes or 9136153 and legume mixture (Coppock and Stone, 1968). Another P“?c>cedure which is increasing rapidly in popularity is to Edit: urea or other non-protein nitrogen compounds to the whole corn plant at ensiling. Experiments conducted by Huber 95 §__1_. (1965, 1967, 1968b, 1971) and by Schingoethe and Beardsley (1975) have shown that urea added to corn silage at ensiling is efficiently utilized as a nitrogen source for milk production. However, Coppock and Stone (1968) reported that the higher protein content of the urea- corn silage is not due to the urea per se, but rather to its sparing action since it appears to reduce the degradation of protein during the silage fermentation. Corn silage is also a convenient carrier for urea. The feeding urea with corn silage masks the undesirable taste of urea, and also allows more intake of urea over the whole day and minimizes possible excesses in the blood compared to including the urea in concentrate which is consumed in relatively short periods (Huber £3 31., 1968a). Polan and co-workers (1968) reported that adding urea to silage at ensiling to supplement as much as 38 percent of the total Ilitrogen in the ration did not significantly reduce milk Prxoduction. A more uniform intake of NPN during the day may Partially explain those results (Huber _e_t 31., 1968) . Some lOsses of urea-nitrogen usually occur during storage and feeding. These losses may be even greater if corn is defi- cxieant in energy. Conrad and Hibbs (1968) point out that atbcnut 1 kg of rapidly fermentable carbohydrate is required P631: 100 g of urea for maximum utilization of urea in the adEipted dairy cow. It was later stipulated that at least t'VVCD-thirds of that fermentable carbohydrate should be in the ED rm of starch . 10 However, the problem of using urea to supplement corn silage rations lies not in the question of whether to use it, but rather under what condition and by what tech- niques and routes of administration of apprOpriate levels urea can be most effective. Huber and co-workers (1967) compared the utilization of nitrogen by lactating cows fed corn silage (ad libitum as the only forage) which had been ensiled with 0.0, 0.5, or 0.7 percent urea. The level of urea in the silage did not significantly influence the level of milk production, silage or total intake. But in a second trial it was discovered that cows fed a 0.85 percent urea-corn silage ration were in severe negative nitrogen balance because of lowered protein digestibility and large urinary nitrogen losses. This was substantiated by the work of Polan gt 31. (1968, 1970) Showed that adding urea to corn silage at ensiling at levels 015 0.5 to 0.85 percent resulted in increases in urinary nitrogen and decreases in nitrogen retention, although milk Production was not affected. It was then postulated that tunes cows receiving the high urea (0.85 percent) silage were drawing heavily on endogenous protein supplies. The benefits of using both urea and natural protein t<> supplement corn silage for balancing rations for high- pa?<>ducing cows have been studied (Huber and Thomas, 1971; C(311rad and Mugerwa, 1970; Van Horn and Jacobson, 1969). Ho\vever, divergent results have been observed concerning the optimum level of urea which can be successfully used for 11 milk production. Huber and associates (1967a) observed a depression in milk production when high corn silage rations were supplemented with urea in the concentrate to supply 21 percent or more of the total nitrogen in the diet. But when urea supplied only 11 percent of the nitrogen, no depression occurred. Later Van Horn and Jacobson (1971) found that the addition of urea beyond 11.4 percent dietary protein was of little benefit. In a earlier study the same investigators (Van Horn and Jacobson, 1967) observed a reduction in feed intake by cows consuming a dairy concentrate containing 2.2-2.7 percent urea. However, addition of 0.5 percent urea to corn silage and 1.0 percent to concentrate did not affect fleed.intake or milk production. Holter 23.21: (1968) found IND depresSing effect on feed intake and milk production, but decreases in nitrogen retention were found when high quality Huxlti-ingredient concentrate mixtures containing up to 2.5 Percent urea were fed with corn silage forage. Their highest level of urea provided about 300 g of urea per day. Urea iJ'lcreased rumen ammonia nitrogen from 15 mg to 30 mg/100 ml fJJJid during the first hour after feeding. The concentrate m«'i-thure contributed to the high feed intake and excellent Performance. Boman gt a1. (1969) studied restricted concentrate SuPplementation with corn silage ad libitum. Diets compared were : 12 1. Corn silage plus a 20 percent protein grain supple- ment; 2. Corn silage plus 45 percent cottonseed meal; and 3. Urea-corn silage (0.5 percent) urea plus cottonseed meal added to shelled corn. Differences among treatment groups were small in total dry matter intake, but there was a trend in favor of groups fed restricted concentrate and more urea with respect to milk production and weight gain. Consumption of the urea-corn silage was depressed during the first month of the study. This subject deserves more attention in high-producing cows early in lactation. Conrad and Hibbs (1967b) pointed out that ingestion 'the animal and upon intake are particularly relevant when low protein roughages, by-product feed and non-protein nitro- gen are fed. Thus, it is important to identify factors which infldaence rumen microbial protein synthesis. One of the important nutrients for rumen microbial growth is ammonia. The increasing substitution of urea and other non-protein nitrogen sources for plant protein in the 15 rations of dairy and beef cattle is economically important because worldwide demand for natural protein exceeds avail- able supplies. However, although cows have demonstrated an ability to lactate and reproduce on purified diets containing only NPN (99 percent) as a source of nitrogen (Oltjen and Bond, 1967; Virtanen, 1966, 1969), only partial replacement of dietary plant protein nitrogen with NPN has been recom- mended for lactating dairy cows. The variable factors involved in urea feeding have been well-reviewed (Reid, 1953; Freitag gt 31., 1966; Oltjen, 1969, Chalupa, 1968, 1970, 1972, 1973; Helmer and Bartley, 1971). Irrespective of the source of dietary nitrogen or non-protein nitrogen, ammonia is a central intermediate in the degradation and assimilation of nitrogen and it is the main nitrogenous nutrient for ruminal microbial growth (Bryant and Robinson, 1961, 1962; Bentley 3 21., 1955; Bryant, Robinson and Chu, 1959; Dehority, 1963; Hungate, 1966) . The relative importance of ammonia as compared to amino acids in the nutrition of rumen bacteria was discussed by Bryant and Robinson (1962) . They examined the nitrogen requirement of 44 strains of rumen bacteria and found that 32 percent could be grown with ammonia as the sole nitrogen Source, 30 percent would not grow unless ammonia was present, and 56 percent could use either ammonia or amino acid nitro- gen (Bryant and Robinson, 1962) . Information summarized by Bryant (1963) and Hungate (1966) indicates that ammonia is an essential nutrient for growth of Bacteroides succinogenes, 16 Ruminococcus flavefaciens, Ruminococcus albus, Bacteroides amylophylus, Methobacterium ruminantium and Eubacterium ruminantium, even when preformed organic nitrogen is present in the media (Warner, 1955; Abou Akkada and Blackburn, 1963). Under such conditions, ammonia is not essential, but may stimulate growth of some strains of Succinovibrio dextrino- solvens, Butyrivibrio fibrisolvens, Bacteroides ruminicola, and Streptococcus bovis (Bryant and Robinson, 1962; Gill and King, 1958; Shane EE.El-r 1969; Chalupa, 1972). The positive relationships between conversion of nitrogen sources to ammonia and the in_zi£rg_magnitude of .both cellulose and starch degradation indicate the importance (If ammonia as an essential nutrient for fiber--and starch-- digesting bacteria (Acord gt _a_l_., 1966; Chalupa gt 11., 1963) . Therefore, it is reasonable to assume that dietary NFTU will be of little benefit to the ruminant animal unless it: is first converted into ammonia and then utilized for microbial protein synthesis in the rumen. Slyter and CO-workers (1968) found that 7.4 percent of 403 strains of rumen bacteria possessed urease activity. Some strains which Possessed ureolytic activity were identified as Propinoi- b_¢'=1<=terium _s_p., Bacteroides EB” Ruminococcus g2” Strepto- EESEHEE.EQXi§r and an anaerobic Lactobacillus. Allison (1969, 1970) reported that ammonia nitrogen is primarily incorporated int£> bacterial cells and appears in protozoal cells as a °°n8equence of ingestion of bacteria by protozoa. Al Rabbat SE.§EL. (1971) observed that 61 percent of microbial nitrogen 17 was derived from ammonia and 39 percent was from ammino acid and peptide nitrogen. A similar observation has been reported by Pilgrim and associates (1970) indicating that 70 percent and 64 percent of bacterial nitrogen was derived from ammonia when animals were fed wheaten hay and lucerne hay, respec- tively. The percentage of microbial protein derived from urea was greatest with a diet containing low protein (Nikolic Anna, 1972). A more recent in vitro study by Maeng and co-workers (1976) indicated that the optimum ratio of non-protein nitro- gen for microbial growth was 75 percent urea nitrogen and 25 percent amino acid nitrogen. Allison (1969) stipulated that when microbial growth is limited by the availability of nitrogen, it is probable that ammonia concentration is criti- caJ.and supplementation or replacement of dietary protein vmith urea may, in certain instances, increase bacterial or protozoal concentrations. But when dietary protein is adequate, addition of urea to the ration may have little effect on microbial growth (Satter and Roffler, 1975) . The point at which the ammonia concentration becomes limiting for growth of ruminal bacteria has not been clearly defined. Neither has the optimum concentration of ruminal ammonia required for maximum cell yield been established. This is probably because the concentration depends upon such factors as level of feeding, solubility of dietary protein, availa- bility of carbohydrates and minerals to the microbes, fmeclllency of feeding, etc. Recent i_n vitro research by Satter . AI‘ ,‘ mu! AN- Vin '7”: an. PA 29-" M... p. M. (I C-< H '." fr) (II In I ‘\.i I .1: F of. 18 and Slyter (1974) indicated that increasing the ammonia concentrations above 5 mg NH3-N per 100 ml fluid was of little benefit to the ruminant animals. However, in 1139 results by Slyter gt 31. (1973) showed that nitrogen retention of steers was improved by maintaining ruminal ammonia concentrations above 8 mg/100 ml. Miller (1973) also studied this problem in 3139 and reported that the greatest microbial flow from the rumen was achieved with rumen ammonia concentrations of approximately 28 mg NH3-N/100 ml fluid. Probably the increased performance with the higher ammonia concentrations 12.2139 over that in zit£g_is due partially to the possible beneficial effects of ammonia outside the rumen, such as synthesis of non-essential amino acids in the liver (National Research Council, 1976). As indicated earlier, ammonia is the common denomi- ruitor in the utilization of NPN by ruminants (Hungate, 1966). Ii? the rumen microorganisms cannot degrade the compound in question to yield free ammonia, it is useless as a nitrogen scnirce to the microorganisms. The ability of rumen micro- organisms to utilize ammonia is dependent upon the presence Of a suitable source of energy (Lewis, 1961, 1962; Briggs, 1967: McDonald, 1948; Otagaki at 31., 1955). Briggs (1967) and McDonald (1948) demonstrated that available energy enhanced microbial protein synthesis. It is well established that readily fermentable carbohydrates, starches or grains, rat-her than roughages are required for optimum utilization °f urea (Conrad and Hibbs, 1968; Belasco, 1956). The W tn Cr; Uni 9 v in a 9;! inl“ 0L: '&A\ 19 availability of energy from different sources is the key to evaluating their dietary effects in the ruminant. Adequate available energy is essential for evaluating the utilization potential of any nutrient. Lewis (1961) demonstrated the effect of different carbohydrate sources and concluded that the type of carbohydrate present regulates ammonia concen- tration in the rumen and the extent of microbial protein synthesis. This conclusion is substantiated by the widely successful use of urea as the supplementary source of nitro- gen in high-grain diets. Chalupa (1968) reported that rumen microorganisms have a definite energy requirement and that the degree to which this requirement is met definitely influences the utilization of urea. In experiments in which animal performance appears to be superior on preformed protein-supplemented diets over performance on NPN-supple- mented diets and when protein needs were low, the differences were likely due to the energy contributions of the preformed Portein in low-protein diets. Burroughs gt §_l_. (1971a, 1971b, 1974) prOposed a system for the evaluation of feeds based on eStimated urea fermentation potential (UFP) . This system recognizes that microbial protein synthesis is primarily dependent upon energy availability and that the conversion of rumen degraded protein into microbial protein represents 311 energy cost. 599%. In addition to furnishing energy, dietary preformed I _‘. J") pli‘Oteins are sources of the branched-chain carbon skeletons (isobutyrate, isovalerate and 2-methy1butyrate). Rumen 20 microorganisms can use NPN for protein synthesis if the necessary carbon skeletons are present or if these can be synthesized fast enough from dietary carbohydrate or alter- nate carbon sources. Bryant (1972) reported that strains of cellulolytic species usually required some carbon sources other than those used as the energy source. Primary sources of carbon fragments that arise from carbohydrate fermentation are carbon dioxide and volatile fatty acids (VFA's). The use of carbon dioxide by the rumen microbes as the source of carboxyl carbon has been reported by Otagaki gt _a_1_. (1955) and Wright (1960), showing that 14c from 14CO2 was utilized in protein synthesis by mixed rumen bac- Other investigators have noted this requirement and The teria. most of the work was summarized by Dehority (1971). species fall roughly into two groups. The first group includes Bacteroides succinogenes and R. flavefaciens which have an absolute requirement and require large amounts of these compounds for optimal growth since they fix CO2 into PYruvate in the path of succinate (White, Bryant and Cald- Well, 1962) . Without carbon dioxide, these species are urtable to obtain energy for growth. They also use CO2 for biosynthetic purposes (Allison, 1969, 1970). The second group includes Butyrivibrio fibrisolvens and Ruminococcus albus which may or may not require a small amount of CO2 for .1._~__ itlitiation of growth but require a small amount for Optimal growth (Dehority, 1971) . These species probably require CO2 11Mainly for biosynthetic purposes. In their review, Kay and 21 Hobson (1963) noted that when rumen microorganisms were grown in a medium containing casein hydrolysate, isobutyrate, iso- valerate and 14CO2 (in carbonate form), 2 percent(§§)the cellular lipid carbon was from 14C of the 14C02, whereas 17 percent and 77 percent were in nucleic acid and protein respectively. The label in the protein was found in 15 amino acids including the three branched-chain amino acids, leucine, isoleucine and valine. All the radioactivity was observed in the carboxyl carbon of the amino acids. The investigators concluded that carbon dioxide is an important precursor of the carboxyl group of these amino acids. From this observa- tion the same workers listed the following sources of carbon: carbohydrates, carbon dioxide and volatile fatty acids (VFA). However, there are specific requirements for isobutyrate, isovalerate, 2-methylbutyrate, phenylacetate, and indole-3- acetate to provide for the synthesis of the specific amino acids (Allison, 1969) . There are potential sources of keto aCids in rumen fluid, but the branched-chain VFA's arise nlainly from the deamination of branched-chain amino acids Provided by dietary protein (El-Shazly, 1952a; Annison, 1954) . It: is significant that the feeding of protein-free diets causes a depression in the concentration of these acids (Clrskov and Oltjen, 1967; Oltjen, 1969; Chalupa gt_31., 1970), ‘Vjsth isovalerate and isobutyrate being greatly influenced (Itational Research Council, 1976). The high levels of the eIIZyme activity present in the rumen would indicate that jJlsufficient carbon skeletons could limit ammonia assimilation 22 (Hoshino gt gt., 1966; Chalupa, 1972). This observation is in agreement with the calculations of Balch (1967) showing that the level and type of dietary carbohydrate has a large influence on the efficiency of NPN utilization. (fr¢“wwri> Several investigations have been conducted, which mi ..\.--" H 173 showed that a number of rumen microorganisms require one or more of the volatile fatty acids, n-valeric, isovaleric, isobutyric and 2-methylbutyric acids for growth (Allison, Bryant and Doetsch, 1959, 1962a; Allison, 1969, 1970, 1965; Robinson and Allison, 1967; Bryant and Doetsch, 1955; Kuns- man, 1970; Slyter and Weaver, 1971). Bryant and Doetsch (1962a) observed that two fatty acid components are necessary for growth of Bacteroides succinogenes; one of these compo- nents is a branched-chain volatile fatty acid which may be either isobutyric, isovaleric or DL-a-methylbutyric acids; the other component is a straight-chain acid. Both straight- and branched-chain acids or any combination of them have been found to be growth stimulatory (Bryant and Robinson, 1962) . Bentley gt gt. (1954) , Dehority gt a_l_. (1957, 1958, 1967) and Allison and Bryant (1963') reported that four and five carbon branched- and straight-chain VFA are essential for the it v_i_tr_g growth of at least some of the rumen cellulolytic microorganisms. Allison e_t gt. (1958) reported that three cellulolytic strains of 5. flavefaciens and two Cellulolytic strains of 3. 11.2%?! which are among the most rUmmerous and most important of the cellulolytic organisms iSolated from the rumen, have nutritional requirements for l "\ a i) l K 23 isoacids. Recently Maluszynska and co-workers (1974) isolated a cellulolytic micromonospora which was found to be stimu- lated by valeric acid. Bryant and Robinson (1961) extended this finding to four additional strains of 3. gttgg, one of which was non-cellulolytic. Non-cellulolytic rumen bacteria with isoacid require- ments have been observed. Wegner and Foster (1960) isolated a bacterium and an unnamed gram-positive rod which require both branched- and straight-chain acids. Bryant (1959) noted that all of several strains of Eubacterium ruminantium required isoacids and that a number of other non-cellulolytic bacteria are greatly stimulated by these acids. It has also been reported that rumen bacteria that require 2-methylbutyric acid for growth include the Methanobacterium ruminantium strain M-1 and g. ruminicola strain H2b (Bryant, 1965; Dehority, 1966). 11 The requirement of these acids by the cellulolytic ’l /. bacteria emphasizes an interesting interaction among the rinnen bacterial species to obtain materials often essential/f ffxr their growth. When purified "poor quality" diets high 1J1 cellulose and lacking some of these essential materials are fed to ruminants, the cellulolytic bacteria grow and miiintain their functions, at least to a limited extent, Nébecause of production of these factors by other bacterigy/ l‘. ll- 1 . I . K . . ' .‘Y ' Bentley gt gt. (1954, 1955) reported that n-valeric, 1Sovaleric, isobutyric and n-caproic acids or their amino aCtid precursors stimulate cellulose digestion and the 24 conversion of urea nitrogen into protein by rumen microorga- nisms as measured by artificial rumen techniques. Burroughs and co-workers (1951) working with mixed cultures of bacteria from the rumen have shown that cellulose digestion occurs with ammonia or urea as the sole source of nitrogen. Cellu- lose digestion was stimulated if protein was included, but ammonia or urea stimulated that digestion above that observed with protein as nitrogen source. McLeod and Murray (1956) also showed that certain amino acids can replace protein in stimulating cellulose digestion. These included valine, leucine, isoleucine and proline (Dehority, 1958). It is generally accepted that nitrogen from catabo- 1ized amino acids enters an ammonia pool. Probably there are extracellular and intracellular pools. However, the pool size of free, extracellular amino acids in the rumen is usually quite low (Allison, 1970). Extracellular concentra- ‘tioms of a-amino nitrogen, measured, after dialysis or liltra-filtration, are usually less than half the values for t£rta1 free amino acids measured from acidified samples. It is! suggested that the differences may be due to release of 'amnino acids from microbial cells in acidified samples (Wright and Hungate, 1967a) . Qualitative and quantitative estimates of the free amino acids in rumen protozoa have been made. A small proportion of the free, extracellular glycine, gluta- mai‘te, or aspartate was incorporated intact into microbial cell substances, but a much larger proportion of labelled carbon from these amino acids was found in volatile fatty 25 acids and CO2 (Wright and Hungate, 1967b). While some species of rumen bacteria use exogenous amino acids (Bryant and Rob- inson, 1963), amino acids in peptides are more efficiently utilized than are free amino acids by certain species (Pitt- man and Bryant, 1964; Wright, 1967). Other species are less able to use preformed amino acids and assimilate amonia nitrogen in quantities approaching or equivalent to the amount of nitrogen incorporated into the cells. This occurs even when these organisms are grown in a medium containing a complete mixture of amino acids (Allison, 1969; Bryant and Robinson, 1961, 1963). The most probable explanation for the apparent failure of amino acids to complete with amonia is the low activity of, or absence of systems for transport of amino acids into the cells (Allison, 1969; Bryant, 1973). _ it The ability of many rumen bacteria to assimilate ammonia nitrogen implies also that they have the ability to Construct the carbon skeletons of the amino acids that con- stitute the protoplasm of the cells. In effect, the microflora of the rumen has been shown to be capable of both Producing and utilizing isoacids. El-Shazly (1952a) was the first investigator who reported the presence of isoacids in 3rtunen fluid. He indicated a positive correlation between the level of protein in the diet and the level of the iso- acBids in the rumen fluid. Later, Annison (1954) and El~Shazly (1952b) observed that the branched-chain VFA, iSobutyrate, isovalerate and 2-methylbutyrate arose from the degradation of dietary protein and the subsequent 26 deamination of the corresponding branched-chain amino acids. They then concluded that the probable origins of isobutyrate, isovalerate and 2-methylbutyrate are valine, leucine and isoleucine, respectively. Slyter and Weaver (1969) reported that branched-chain VFA may be synthesized by ruminal bac- teria when a substrate devoid of protein is supplied to a mixed rumen microbial population of cellulolytic bacteria which require them for growth. The same workers noted that even when a diet was fed which contained no branched-chain amino acids, the carbon skeleton precursors of branched- chain fatty acids, the cattle were still able to maintain a large population of cellulolytic bacteria that require fatty acids for growth. Other investigators have reported pro- nounced depressions in the ruminal concentrations of isobutyric and isovaleric acids (Orskov and Oltjen, 1967; Cline gt gt., 1966; Freitag gt gt., 1966), somewhat depressed levels of these acids (Clifford and Tillman, 1968) or lowered quantities of isobutyric acid (Matrone gt gt., 1966) when ruminants were fed a protein-free diet. Replacing isolated soy protein by urea has also resulted in lower acetic acid proportions and increased proportions of butyric acid (Orskov and Oltjen, 1967). In other studies Oltjen and associates (1969) observed less ruminal branched-chain fatty acids from cattle fed a purified diet containing urea than in cattle fed purified diets which contained soy-protein. Of interest is the observation by Annison (1954) that the branched-chain 27 VFA concentration in the rumen almost certainly depends not only on the rate and extent of degradation of dietary and microbial protein but also on the rate of absorption of these acids. Conversion of leucine into isovaleric acid by Bacteroides ruminicola has been reported by Bladen, Bryant and Doetsch (1961). Dehority gt gt. (1967) noted that tg gtttg digestion of cellulose by a mixed culture of microorganisms is stimu- lated by leucine, valine and isoleucine as well as by corresponding VFA's produced by anaerobic catabolism of these amino acids. The branched-chain amino acids, phenylalanine and tryptophan are deaminated and decarboxylated in the rumen and the acids produced accumulate in appreciable concentra- tion (El-Shazly, 1952b; Menahan and Schultz, 1964; Allison, 1970). Allison (1970) stipulated that the volatile products from alanine are acetate and formate, and that phenylacetate is the major catabolic product of phenylalanine metabolism. This was confirmed by Martin (1973) showing the conversion of phenylalanine to phenylacetate excreted in the urine of sheep. He then suggested that the amount of phenylacetate excreted in the urine is a measure of the equilibrium occurring in the rumen between catabolism of phenylalanine and reutilization of products of catabolism for phenylalanine synthesis. Yokoyama and Carlson (1974) reported that incubation of tryptophan with ruminal microorganisms resulted in for- mation of indole acetic acid (Lacoste, 1961). N-valerate was 28 found to be produced from either carbohydrate or from proline (Dehority gt gt., 1958; Elsden gt gt., 1956; Pottele gt gt., 1966). ' Although catabolism of dietary branched-chain amino acids is a major source of branched-chain VFA, small amounts of these acids were present in the rumen when purified diets containing no protein were fed (Clifford and Tillman, 1968). This suggests that the acids were produced from amino acids synthesized by microbial cells and may indicate turn over of these cells (Bryant, 1973; Allison, 1969). Amino Acid Synthesis by Rumen Microorganisms Information on free amino acid pool size, turnover, and metabolic fate and on nitrogen sources for pure cultures of rumen bacteria (Wright and Hungate, 1967a, 1967b; Bryant and Robinson, 1963) suggests that a relatively small portion of the amino acids that pass through the extracellular pool are incorporated intact into microbial protein. Furthermore, evidence has already been given that amino acids in peptides are incorporated by certain rumen microbes more efficiently than are free amino acids. Nevertheless, even in animals fed on ration contain- ing appreciable quantities of protein, it is likely that a large portion of the amino acids in microbial proteins are synthesized gg|gggg from intermediates or end-products of carbohydrate fermentation, or end-products of amino acid metabolism (Allison, 1970). Microbial biosynthetic 29 capacities are shown when ruminants grow and produce milk while fed on purified diets with NPN as the only source of nitrogen (Virtanen, 1966). Even on such diets, there are detectable quantities of branched-chain VFA in the rumen and also organisms that require them (Slyter and Weaver, 1969). Therefore, the metabolism of the branched-chain amino acids indicates a turnover of microbial protein or a metabolism of endogenous animal protein (Phillipson, 1964). Bacteriophages (Adams gt gt., 1966; Paynter gt gt., 1969) or unidentified factors (Jarvis, 1968) are potential sources, but branched- chain VFA are present in the rumen mainly as the result of degradation of dietary protein and deamination of branched— chain amino acids (Chalupa, 1972). Rumen bactgria are able “Fun-W WW... ___..—.___,n_._- to synthesize the.carbon_ skeletons of 19991091 isoleucine, valine, phenylalanine,tryptophan, alanine and glutamateabyfl _ fl...- 7 _. - .M- ”M ; ‘reductive carboxylationireactions not likely to function in N‘s-q... W v-u ""‘"’ ‘9‘5 .._,.— aerobic organisms (Allison, 1970). The use of carbon skele- tons of the branched-chain VFA for rumen microbial protein synthesis has been largely discussed (Allison gt gt., 1962a; Allison and Bryant, 1962, 1963; Allison and Peel, 1971; Allison, 1969; Cline gt gt., 1966;01tjengt gt., 1971; Hume, (”1970; Allison, Bryant and Doetsch, 1962). The branched-chain E VFA (Allison, Bryant and Doetsch, 1962; Allison and Bryant, 1 1963), phenylacetic acid (Allison, 1965b), and indole acetic acid (Allison and Robinson, 1967) are carboxylated and I aminated by mixed cultures of ruminal microorganisms incu- i l i hated tg vitro and by a number of pure cultures of important l 30 ruminal bacteria to resynthesize the original amino acid which is then incorporated into microbial protein. The path- ways are different from any that have been described for biosynthesis of these amino acids (Allison, 1969). Carbon skeletons of branched-chain fatty acids were incorporated mainly into the lipid and protein portion of the cells. Higher branched-chain fatty acids and fatty aldehydes were synthesized from isovalerate and isobutyrate (Allison, Bryant, Katz and Keeny, 1962; Wagner and Foster, 1963). In the protein fraction leucine was synthesized from isovalerate (Allison, Bryant and Doetsch, 1959, 1962; Hoover gt gt., 1963; Singer and Doolittle, 1975), valine from isobutyrate (Allison and Bryant, 1963; Allison and Peel, 1971; Quay 22.21:: 1975) and isoleucine from 2-methylbutyrate (Robinson and Allison, 1969; Hungate, 1966). The tracer evidence also indicated that carboxyl groups of these amino (acids were formed by carboxylation. Hoover and co-workers (1963) have observed that carbon from each of these sources (:2, C3, C4, CS was incorporated into the amino acids and 1:hat the rate of their utilization was proportional to their m3nmmurunuo mucmucoo smash mo HE OOH msficflmusoo huwommmo HE mnv mo Hmnllm .mEmwcmmnoouoflE owes“ mo mmumu coaumucoEHmm mcwusmmme “Om moumummmdlu.a mmDon 47 I WI L. 4 __ t; firflpr_ _ FL ____.__~_L o ,_L 48 Ammonia nitrogen was determined according to the method of Fawcett and Scott (1960) as modified by Kulasek (1972). Volatile fatty acids (VFA) were determined by the method of Ottenstein and Bartley (1971a, 1971b) with minor modifications. Three microliter aliquots were injected into a Hewlett-Packard gas chromatograph*, model 5730A, equipped with a model 7671A automatic sampler and an Integrator- Recorder, model 3880A. The all-glass column (6ft X 2mm i.d.) was packed with graphited carbon, CarbOpak A**. Nitrogen was used as the carrier gas. The acid standards were Eastman compounds***, containing 0.01N each of acetic, propionic, butyric, valeric, isobutyric, isovaleric and 2-methylbutric acids in deionized water. The column was conditioned at 200°C (overnight) and the running temperature was maintained at 175°C. The column was cleaned three times after condition- ing, with a 3 ul injection of deionized water. Carrier gas flow rate was 60 ml/min. Growth Study In many instances dairy herd replacements are wintered on high fiber-low protein roughages. Improved digestibility and, consequently, increased growth of heifers *Hewlett-Packard. ROute 41, Avondale, Pennsylvania. **Supe1 Co., Inc., Supel Co. Park, Bellafonte, Pa. ***Eastman Organic Chemicals, Eastman Kodak Company, Rochester, New York. 49 may be achieved if rumen microorganisms are supplemented with certain key volatile fatty acids. The objective of this study was to investigate the effects of feeding urea plus isoacids and phenylacetate on growth rate of dairy heifers fed on low quality roughages. Twenty yearling dairy heifers weighing from 121 to 327 kg were paired on the basis of body weight and used in a 90—day feeding trial. Heifers from each pair were randomly assigned to one of two treatments. All heifers were fed timothy hay gg libitum as the sole roughage. In addition, the control group received per head day 450 g of a pelleted supplement which consisted of 80 g of urea, 50 g of molasses and 270 g of ground hay. The other group was supplemented with 454 g of the pelleted diet containing the same ingredients and in equal amount as in the control plus 50 g of the acid mixture. The acids were added to the urea-treated ground hay prior to pelleting. Both groups of animals were fed free choice a salt-mineral mixture consisting ot 50 percent trace minera- lized salt and 50 percent dicalcium phosphate. All animals were weighed for two consecutive days at the beginning, twice during the trial and at the end of the experiment. The average body weights were 245 and 246 kg for the control and treatment groups, respectively. Differences in body weight changes between the two groups were tested by the student "t"-test technique. 50 Effects of Isoacids on Milk Production Urea has long been used as a source of supplemental nitrogen for cattle and sheep. But it has been recognized that carbon skeletons from branched-chain and some straight- chain volatile fatty acids (isoacids) were needed with urea for microbial protein synthesis. In a series of four feeding trials high-producing lactating Holstein cows were fed urea- based diets plus isoacids. Trial I.--This trial was designed to evaluate the effects of feeding a mixture of isoacids and phenylacetate with urea on milk production when corn silage was fed as the sole roughage, and without concentrates, on the assumption that high-producing cows in the peak of lactation are in high energy demand. A randomized block design of 3 treat- ment groups and 8 animals per group was used in this trial. Blocks were based on previous milk yields. Treatment groups were balanced for age, stage of lactation and producing ability. The treatment period was for 60 days Twenty-four lactating Holstein cows within 30 days postpartum and producing over 25 kg of milk per day were placed on a two-week preliminary period. All cows were fed control corn silage free choice and the regular herd corn grain-based concentrates at 1 kg per 3 kg of milk. The preliminary period was followed by a two-week transition period for adjustment to the experimental rations, to which the cows were randomly assigned. Cows were then gradually 51 converted to the experimental treatments in S-day intervals. During the treatment period cows were fed control corn silage gg libitum and the experimental rations. The three experi- mental diets consisted of the following ingredients: a. 300 g soy protein + 1200 g ground grass hay (premix); b. 300 g urea + 454 g dry molasses + 300 g grass hay; and c. 300 g urea + 454 g dry molasses + 300 g grass hay + 100 g isoacid mixture. The acid mixture consisted of 20 g each of isobutyrate, isovalerate, 2-methylbuty— rate, n-valerate and phenylacetate. The treatments were administered once a day and were well mixed with the top third portion of the silage. A salt mineral mixture containing NaCl, K, MgO, Ca and P04 was also fed free choice. Daily milk yields were recorded for each milking. Body weights were taken for two consecutive days at the beginning and at the end of the experimental period. Trial II.--Trial I indicated that isoacids and phenylacetate fed with urea to high producing cows improve milk production as compared to urea alone. These sources of supplemental nitrogen are more economical than soybean meal. Furthermore, according to the National Research Council report (1976), an experimental diet should contain both a "negative" and a "positive control" in order to test both the animal protein needs and the efficiency of the source of nitrogen in fulfilling those needs. The "negative control" is used as a test diet known to be protein deficient. 52 The "positive control" is a test diet with preformed protein furnishing the supplemental protein and eliciting a positive response relative to the negative control. Trial II was then conducted to compare the effects of soybean meal (positive control), urea, urea plus isoacids and no nitrogen supplementation (negative control) on milk production by cows fed grain along with corn silage. Twenty-eight Holstein cows in early lactation and milking over 20 kg per day were alloted to 4 treatments in a randomized block design. Blocks were based on milk yields during a two-week preliminary period. Treatment groups were balanced for age, stage of lactation and breeding groups. Treatment comparisons were established as follows: a. Negative control b. Positive control c. Urea d. Urea + isoacids The negative control consisted of corn silage, 1.4 kg of hay and corn grain. The positive control included the same ingredients as for (a) plus soybean oil meal. The urea treatment was similar to (a) plus urea as supplemental nitro- gen. The urea—isoacid treatment included the same components as (c) plus 20 g of each acid. Total crude protein concen- tration of each diet was 14%. During the preliminary period cows were fed control corn silage gg libitum and the regular herd concentrate at 1 kg per 2.5 kg of milk. The concentrate ration contained 53 2.5 percent crude protein equivalent as urea and was well- mixed with the silage prior to feeding. Cows were gradually converted to the experimental concentrates over a 4-day period. During treatment, the control corn silage was fed and experimental concentrates at 1 kg per 2.5 kg of milk. Silage and concentrate were well-mixed before feeding. During the first 4 weeks of treatment, the amount of concentrate offered was based on milk production during the preliminary period; this concentrate was reduced 5 percent at 28 days and 5 percent more at 56 days. Sufficient corn silage was fed and regularly adjusted to provide a 10 percent weighback of total feed. The ingredient composition of the concentrates is indicated in Table 4. Daily milk yield was recorded five days a week from both the morning and evening milkings. Milk samples were collected from two consecutive milkings during the prelim- inary period, and at 2-week intervals during treatment, composite (AM and PM) samples were taken. Corn silage was sampled three times a week for a weekly composite and frozen for dry matter determination. Concentrate samples were taken at each feed preparation and kept in the cooler for further analysis. Feed was weighed in and weighed back from each cow on a daily basis. Cows were weighed as previously described. Milk fat was 54 Table 4.--Composition of the concentrates for trial II. Treatment Ingredient a b c d Ground shelled corn, % 55.5 44.5 56.2 54.0 Oats, % 27.5 22.0 23.5 25.5 Soybean meal, % .... 16.5 .... .... Urea, % .... .... 2.6 2.5 Beet pulp, % 10.0 10.0 10.4 10.0 Molasses, % 4.0 4.0 4.2 4.0 Dical phosphate, % 1.0 1.0 1.0 1.0 Ground limestone, % 1.0 1.0 1.0 1.0 TM salt, % 1.0 1.0 1.0 1.0 Isoacids, % .... .... .... 1.0 Vitamin A, IU/kg 4400.0 4400.0 4400.0 4400.0 Vitamin D, IU/kg 1100.0 1100.0 1100.0 1100.0 55 determined according to the method of Babcock*. Milk protein determination was performed by the Kjeldahl N procedures (N x 6.38 = CP in milk). Total solids in milk were deter— mined in duplicates by oven drying at 100°C for three hours. Two milliliters of milk were pipetted for weighing in aluminum pans of 3 cm diameter which were used for drying. The content of solids non fat of milk was estimated as total solids % minus fat%. Feed dry matter was determined by drying in a forced air oven for 24 hours at 100°C. Trial III.--In trial II, the use of a randomized block design showed that an equal weight of isoacids improved persist way of lactation. But trial II failed to show significant difference in milk yield between treatments. It has been reported that, using as few as eight Holstein cows per treatment in a 30-day feeding period, the probability of detecting a true mean difference in milk yield of 2 kg per day is much higher with a cross-over design as compared to a randomized block design (Gill, 1969). Therefore, in trial III a special Latin square cross-over design was used to evaluate the effects of two different blends of isoacids for milk production by Holstein cows. Thirty lactating Holstein cows producing more than 23 kg of milk per day were randomly assigned to a 3 x 3 cross-over design following a three-week *The fat test was performed at the center for the Dairy Herd Improvement Association, Forest Road, East Lans- ing, Michigan. 56 prelimanary period. Treatment allocation was based on milk production during the preliminary period, age, and breeding groups. The experimental design is shown in Table 5. Table 5.--Experimental design for trial III. Treatment periods (days) Groups Prelim. Period (days) I II III 0-21 22-49 50—78 79-107 I .... a c b II .... b a c III .... c b a Each treatment period was of 28 days duration. During the preliminary period the cows were fed control corn silage gg libitum as the sole roughage. In addition, the regular herd corn grain-based concentrate was fed at the rate of 1 kg per 3 kg of milk. Cows were gradually converted to experimental concentrates throughout the preliminary period. . During treatment cows were fed control corn silage free choice. In addition, cows on control (a) received per head per day 1 kg of concentrate per 3 kg of milk. The experimental concentrates contained 3.0 percent crude pro- tein equivalent as urea. Cows in treatment b were fed similar concentrate to (a) plus 80 g of the acid mixture 1, containing on a molar basis 28, 24, 24, and 24 percent of 57 isobutyrate, isovalerate, 2-methylbutyrate and n-valerate, respectively. Cows in treatment c were offered the same treatment as (b), but the corresponding values for the acid mixture 2 were 36, l7, l7 and 30 percent. Table 6 shows the ingredient composition of the concentrates. Corn silage was fed in the morning. Urea and acid mixtures were premixed with part of the concentrates and fed well-mixed with the top third portion of the silage during the morning feeding. The remaining amount of concentrate was fed in the afternoon. Feed amounts were adjusted to average a 10 percent refusal. The procedures used for sampling silages and concentrates and for weighback recording were similar to those previously described. Daily milk yield recording and milk sampling were similar to that described in trial II. Blood samples were collected bi-weekly from tail. veins of each cow three hours after the morning feeding. The blood was drawn in a 15 ml vacuum tube containing 30 mg potassium oxalate and 33 mg sodium fluoride. Plasma was prepared by centrifuging at 2000 x g for 15 minutes. The plasma was then stored for further urea analysis. Plasma urea--N determination was performed by the colorimetric method of Fawcett and Scott (1960) as modified by Kulasek (1972). At the end of each treatment period (28 days) rumen fluid was collected from all cows by stomach tube at 3 hours post—feeding. The rumen fluid was then strained through four layers of cheesecloth, and the pH was measured with a 58 Table 6.--Composition of the concentrates for trial III. A B C Ground shelled corn, % 54.0 53.5 53.5 Oats, % 26.0 25.8 25.8 Beet pulp, % 10.0 9.9 9.9 Dry molasses, % 4.0 3.9 3.9 Urea, % 3.0 3.0 3.0 Isoacids, % ..... 0.9 0.9 Defluorinated P04, % , 1.0 1.0 1.0 Ground limestone, % 1.0 1.0 1.0 TM salt, % 1.0 1.0 1.0 Vitamin A, IU/kg 4400.0 4400.0 4400.0 Vitamin D, IU/kg 1100.0 1100.0 1100.0 59 Beckman pH meter*, model G. A sample was prepared for ammonia and VFA determinations, using the same procedures as described in the in XEEEQ study. Body weights of each cow were taken at the beginning and the end of each treat- ment period as previously described. Proximate analysis including dry matter, crude fiber, crude protein, and ash was conducted for silages and concentrate samples according to the standard method of the A.0.A.C.** Table 7 shows the chemical composition of the diets. Table 7.--Composition of the diets for trial III. Corn Silage Concentrates Dry matter, % 38.47 88.45 Crude protein, % 8.42 . 18.47 Organic matter, % 35.31 83.16 Ash, % 3.16 5.29 Crude fiber, % 23.84 7.41 A digestion trial was conducted at the end of each experimental period, using three groups of three cows each and in the same experimental sequence. Since the cows were on the same diet for three weeks, no adaptation period was *Beckman Industries, Inc., South Pasadena, California. **Proximate analysis was performed at the analytical lab of the Biochemistry Department, Michigan State Univer- sity, East Lansing. 60 needed. However, the cows were equipped with urinary collection devices five days prior to the collection period. The collection period was of five days length. The urinary collection devices were made from light transparent plastic material at the Michigan State Univer- sity Veterinary Research Center. Cows were fitted with these apparati at the MSU Dairy Research Center. The devices were sutured along with a l-foot rubber tubing at six points surrounding the cow's vagina, following local anesthesia (Rapicaine*); the devices were then well-glued to the skin with branding cement to prevent leaks during collection time. One day prior to collection, cows were transferred to the digestion stall where they were fed and milked as usual. Samples from feed and orts were collected and analyzed as previously described. Milk yields were recorded as usual. The urine was collected in a 5 gallon plastic carboy containing 30 ml of 50 percent hydrochloric acid to acidify the urine and to prevent loss of nitrogen. The plastic carboy was connected with the collection device through a 5-foot plastic tube. Daily urine weight was recorded, and the carboy emptied following removal of a 10 percent aliquot, which was stored in the cooler. Upon termination of each collection period, samples from each cow were pooled. A 10 percent *Haver-Lockhart Laboratories, Shawnee, Kansas. 61 subsample was then taken for nitrogen determination using the Kjeldahl procedure as previously indicated. Total feces output was collected from each cow in a galvanized metal container placed immediately behind the cow. The weight of the feces was recorded once a day and a representative sample (15 percent) was taken after thourough mixing. At the end of each period a composite sample was prepared for each cow, and a 10 percent subsample was used for dry matter, crude protein, crude fiber and ash determinations. Proximate analyses for feed, orts, and feces were performed as pre- viously described. Trial IV.--The Latin Square cross-over design experiment was selected because it had been reported that in dairy animal feeding this type of design is more appro* priate to detect differences between treatments over a relatively short period of time and with much smaller numbers of animals per treatment than the randomized block design. However, results from trial III showed that, in the particular case of the isoacids, the cross-over design cannot show differences between treatments in milk production on a 28-day period because milk production declines when the cows are changed from one ration to another. Twelve cows showed significant reduction in feed intake and, consequently, reduction in milk production when their diets were changed from control to the acid mixtures. It took them more than 2 weeks to recover. Because of the initial effect Of changing 62 treatment on appetite, and because of the relatively short treatment period, the cows did not exhibit any positive response to the acid treatment in trial III. Thus, trial IV utilized two treatment groups in a continuous feeding randomized block design. This trial con- sisted of two treatments, acid mixture and control. The treatment period was 90 days. Thirty lactating Holstein cows averaging 40 days post partum and producing over 23 kg of milk per day were paired on age, stage of lactation, milk production, body weights and breeding groups. One cow from each pair was randomly assigned to each of the two treatments for a three-week preliminary period. Control corn silage was fed free choice as the sole roughage from the start of the preliminary period throughout the entire trial. Silage rations were supplemented with a corn grain-based concentrate at 1 kg per 3 kg of milk. The grain ration was initially the same as the regular herd rations. This ration was then gradually converted to the experimental concentrate over a series of 7-day intervals throughout the preliminary period. The composition of the concentrates is shown in Table 8. Total crude protein of the rations was 14 percent. During the treatment period the experimental concentrates were fed at 1 kg per 3 kg of milk for cows producing over 25 kg of milk per day.‘ The ratio was reduced by 10 percent for every 5 kg below that initial level. Urea and acid treatments were premixed with the concentrates and fed with silage as previously indicated. Each cow on the control diet received 63 Table 8.--Composition of the concentrates for trial IV. A* 3* Ground shelled corn, % 57.0 56.3 Oats, % i 20.0 19.6 Urea, % 3.0 3.0 I Beet pulp, 8 9.0 8.9 Molasses, % 8.0 7.9 Isoacids, % .... 1.1 Dical phosphate, % 1.0 1.0 TM salt, % 1.0 1.0 Ground limestone, % 1.0 1.0 Vitamin A, IU/kg 4400.0 4400.0 Vitamin D, IU/kg 1100.0 1100.0 *A = Control; B = Acid treatment. 64 275 g of urea per day. Cows on the acid treatment were fed per head per day an amount of urea similar to that fed the control group plus 80 g of the acid mixture. Feeding and sampling procedures were similar to those described in the previous trial except that corn silage and concentrates were fed in the afternoon (4 to 6 pm). Daily milk yields, body weights and orts were recorded; feed, milk, blood and rumen fluid samples were collected and analyzed similarly to the previous trial. The chemical composition of the rations is shown in Table 9. All data were statistically analyzed by analyses of various techniques, using a CDC 6500 computer at the Michigan State University computer laboratory. When necessary, differences between means were tested by ortho- gonal, Tukey's or "t"-test comparisons. Details of statis- tical analysis are reported in the appendix. 65 Table 9.--Composition of the diets for trial IV Corn Silage Concentrates Dry matter, % 30.70 88.11 Crude protein, % 7.95 18.72 Organic matter, % 26.66 82.15 Ash, % 4.04 5.96 Crude fiber, % 24.04 7.35 NFE*, % 61.92 64.12 Ether extract, % 2.25 3.92 *NFE = Nitrogen-free extract. RESULTS AND DISCUSSION In vitro Experiment The effects of different concentrations of the iso- acids on gas production for the five experiments are summa- rized in Table 10. The values represent an average of seven observations per treatment. In general, isoacids depressed (P<.01) the rate of gas production irrespective of the source and the amount of substrate used in the culture medium. However, the rate of gas production was different between experiments (P<.01; Table 13). Also the rate of gas production was higher for the experiments which contained higher levels of concentrates in the media. Thus, an excess of substrate is necessary for maximum gas production (El- Shazly and Hungate, 1965). When food is in excess and other conditions are favorable, fermentation is maximal and is a linear function of population size. The rate of gas produc- tion in this present study is in agreement with that observed by El-Shazly and Hungate (1965) and more recently by Naga and Harmeyer (1975). However, whatever level of gas produc- tion obtained is consistently depressed by the increasing concentrations of the isoacids in the media. Gas production 66 67 Table 10.--The effects of different concentrations of iso- butyrate, isovalerate, 2-methy1butyrate and n-valerate on gas production i§_vitro* Experiment** 1 2 3 4 5 No urea ' 22.90” 13.87” 12.25” 16.65” 26.90” Urea 21.92” 11.18” 13.60” 18.17” 33.23” Urea + 2u1*** - - - 17.82” - Urea + 401 21.28”” 11.05” 13.27” 18.33” 25.70” Urea + 8ul 20.30” 11.75” 12.15”” - 22.77” Urea + 1201 21.02”” 10.17”” - 15.33” 19.10” Urea + 16ul 16.65” 12.02”” 10.35” 10.13” - Urea + 2001 19.42” - 9.65” - - Urea + 24u1 16.42” - - 6.26” - Urea + 2301 16.96” - - - 15.33f Urea + 3201 14.15” - - 5.26f - sn**** .99 .25 .42 .41 .52 *Acid mixture was an equal weight of each one of the four acids used. **Gas production measured in ul/ml rumen fluid/ minute. ***Amounts of acid mixtures are expressed in micro- liters/100m1 fermentor. adeefValues in same column with different super- scripts are significantly different (P<.05), using Tukey's Test comparison. ****Standard deviation. 68 was lower for corn starch or cellulose than for concentrates (experiments 1 and 5). One would have expected higher rates on gas produc- tion from those two experiments which contained corn starch as a more readily fermentable carbohydrate. Yet such was not the case. The probable explanation for this is the , presence of cellulose in combination with corn starch. The 90 minute incubation time may have been insufficient to allow a full degradation of cellulose by the microbes. Umunna (1975) reported that the source of energy affected the extent of microbial utilization of isoacids. For example, the rumen microbes were more active with corn starch than with solka floc as a substrate. The difference in rate of gas production between concentrates and corn starch plus cellulose as source of energy could be explained in the light of a recent report by Maeng et 31. (1976). These workers observed that the optimum ratio of non-protein nitro- gen to amino acid nitrogen for microbial growth was 75 per- cent urea nitrogen and 25 percent amino acid nitrogen. The concentrate contained protein and probably many other chemicals that would stimulate microbial activity. The effects of isoacid concentrations on ammonia nitrogen output are presented in Table 11. The statistical analysis is reported in Table 13. Contrary to the gas production pattern, the ammonia nitrogen levels tended to increase with increasing concentrations of isoacids. This tendency was more marked in experiments 3 and 4 where corn Table ll.--The effects of different concentrations of isobutyrate, isovalerate, 2-methylbutyrate and n-valerate on rumen ammonia production in vitro* 69 Experiment** 1 2 3 4 5 No additions 21.6 12.2 11.5 10.6 11.2 No urea 46.1 24.1 5.5 6.2 29.1 Urea 48.7 38.3 21.7 22.3 34.7 Urea + 2u1*** - - — 22.8 - Urea 4u1 47.6 36.8 22.7 21.1 32.9 Urea 8u1 51.1 39.7 23.1 - 36.0 Urea 12U1 46.9 38.5 - 23.5 31.0 Urea 16u1 48.4 37.8 23.9 28.9 - Urea 20ul 49.6 - 24.3 - - Urea 24ul 49.5 - - 28.1 - Urea 28ul 49.1 - - - 33.4 Urea 32ml 49.2 - — 28.0 - SD**** 8.26 10.4 7.40 9.12 8.49 each one of the four acids used. **Ammonia levels are expressed as mg/100ml of incubation media. *Acid mixture was composed of an equal weight of ***Acid mixtures are expressed as microliters/lOOml fermentor. ****Standard deviation. 70 starch plus cellulose were used as sources of energy. In experiment 5 which contained the highest level of concentrate NH3-N decreased slightly with increasing concentrations of isoacids. The levels of NHB-N in the rumen fluid alone varied from 21 to 11 mg/100 ml from experiment 1 to experi- ment 5. Thus, there was some variation in the NH3-N content of the rumen fluid from sampling to sampling. The additions of concentrates (experiments 1, 2 and 5) to the rumen fluid increased by more than 100 percent the levels of ammonia as compared to the negative control (no urea). It is obvious from the NH3-N level of the negative controls of experiments 3 and 4 that NH3 had been utilized during the fermentation process. In the first two experiments the amount of nitrogen equivalent from urea additions was double that in experiment 3, and 4 times that in the last two experiments (Table l). NH3-N was utilized by the microbes throughout the incubation process, but it is difficult to estimate the magnitude of NH3-N used throughout the different concentrations of the isoacids in the media. The level of ammonia supplied from urea was probably in excess of that required for maximal microbial growth and, therefore, a major disappearance of NH3-N could not be detected. Helmsley and Moir (1963) noted a slight reduction in ruminal ammonia concentration due to the addition of isoacids to a urea roughage diet i§_yiyg. Using an isolated soy protein diet, ammonia levels did not appear to be affected. Umunna and associates (1975) did not show consistent differences in ammonia concentrations between 71 urea and urea-isoacids or casein treatments during an in ‘yitrg trial. Even though the present work differed from experiments of those workers because of many variations in the in 3139 system, the similarity between results appears obvious. However, the data in Table 11 show that NH3-N was used by the microbes. The effects of isoacids on pH variations are reported in Table 12. The final pH for each experiment was different from the initial ph (P<.05). The greatest pH decline appeared in experiments 3 and 5, with a final pH of 5.2 and 5.3, respectively. This is about the pH at which microbial activity begins to cease (Brown and Tucker, 1962; Slyter 25 31., 1966). The higher levels of isoacids tended to decrease the initial pH measurements. Volatile fatty acid production for the five experi- ments is presented in Tables 15 through 19. The statistical analysis is presented in Table 14. There were no significant differences in the concentrations of acetate, propionate or butyrate within experiments. The VFA values were slightly lower when compared to those reported by Naga and Harmeyer (1965) and Slyter 33 El- (1966). However, the low level of VFA in the fermentors is not necessarily an indication of low fermentation. A negative correlation coefficient was reported by El-Shazly gt 31. (1969) and by Valthauer 33 31. (1970) between @dggnlentration and microbial growth. Recently Naga and Harmeyer (1975) indicated that no constant relationship exists in vitro between microbial protein 72 HOUflQEHmM HEOOH\mumUHHOHUflE mm OGmWGHme ”HM mmHDUXHE Ufl0¢t¥ 0866I060~ um 06:60 c0856 8060 mm« Nv. ma. mm. mm. 00. vN. 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I I OH. no. «a. pm. vm. mm. ~.H ¢«««am I I I I I I I I I damn + mmua v>.m vh.m wn.o om.o mm.o mo.H no.0 m~.H mh.v Hamm + «was I I I I I I I I I Havm + «was I I I I I I I I I anon + ammo I I I I I I I I I .33 + «85 hh.m mw.oa m>.o h¢.o vm.o eo.~ mm.o o¢.H m~.m Hana + mmua mm.o mm.~a om.o om.o mm.o vH.H mm.o hN.H vm.m Ham + «mus oh.m Hm.m mm.o om.o H~.o vo.a mm.o Hm.H mm.¢ «:«H1v + nous mm.m mb.h I no.0 ma.o mo.H mm.o vm.H hh.v «who oh.m Hm.> I I I mo.H mm.o mm.a oo.m awn: uoz a¢.v m~.h I I no.0 mo.H ~H.0 MH.H mm.¢ *«mco«uwnum oz 0\ 0 dance mumumam> mumumam> mammauzn mumuhuzm mumumuan mumco «mumumoc m N IOmH stumelu IOmH Iwmoum Am ucmawuwmxov mmvmuumnum mm mcwcownums can mmumuuamocoo mo m ow Guam: ouuw>.mw cowuoaooum ¢m> swank co oumumam>Ic new mumuwvanauzuoaIm .oumumam>omfl .mumuhuanOmw mo mnowumuuamocoo unmummmflv mo muowmmm anaII.mH manna 80 FIGURE 2.--Gas production and ammonia output for experi- ment 1. 10 g concentrate ( O~———O ). gas; ( O-———O ), NH3. pl GAS/MLIMIN 81 aoq 1'60 \o \O 20. \ '40 /\ 09 z I n I z 10. L20 6 i 5 1'2 1‘6 2'0 2'4 2'8 35 VFA plIML 82 .52 .A TIIoc 3mm i Toy oumuucmocoo m m .m ucmEHumaxo How usmuao mwcoeam can cowuoscoum mmoII.m mmame 83 O -8 g9“ N Hg 3 9: 9 I? 5? I ° I \ /. /O a“! C 0 "D g 'i E C 0 w I O r0 J.“ J. V ' a, N umnmsvo Irf 84 .mmz Q olIIov $8 .5 ollov omoHsHHwo me and confirm cuoo m mé .m ucoEwummxm How unmuoo mwcoaam can GOwuosooum mmoII.v mmoon 85 0109'! N-cHN O 2 3 a, 9 L l l l ' a JD ‘ v- .N P J 2 2 ; ‘ Lou. CD > /o . b' o 0 _o a f X, N P P NIwnmsvo II{ 86 .nououm :uoo w H .32 .A Iv “mam 2 oIIIoV .mcwcowcuoe 06 H uncaoaaoo w a .v ucmswummxm Mom usmuoo mwcoafim can cowuooooum mmoII.m mmame 87 0,0 9“ N—ci-IN o O o ‘1 ‘1’ ‘=‘ " . O ’3 T 0 ha “.1 .. z 1 If > no 3’ .0 \9 \H IIIIIIIII1WIsvo Ir! / . \ I 6 N 88 FIGURE 6.--Gas production and ammonia output for experi- ment 5. 20 g concentrate 5 mg methionine ( O————O )I gas; ( O——-—O ), NH3. 89 ,40 401 -30 Eco: zInxz 0 2 p D (V W O 2 2.23553 i #10 m 1 1'2 1'6 23 2'4 2'8 VFA yum. f 8 I4 90 synthesis and end product formation. These observations might explain the negative relation observed in this study between gas production and the addition of isoacids. That is, isoacids stimulate cell growth, so that more C02 and carbon were incorporated into cells. Since the carbon skeletons of the isoacids have been shown to stimulate cellulolytic activity of rumen microorganisms, one would have expected an increase in the rate of gas production as the concentration of the isoacids increased up to a point where maximum microbial activity would be reached. The rate of gas production would then decrease for any further addi- tion of isoacids in the fermentors. However, the results of all five experiments showed the opposite. Gas production decreased as the concentration of the isoacids increased. The reasons for the depressing effect of the isoacids on gas “a... production are not known. However, the general outcome of this in yitgg study clearly showed that the isoacids plus urea stimulated the fermentation process. This is evidenced by the reduction of gas production, decrease of pH, reduction of ammonia level and increased molar concentration of VFA. The reduction of gas production may be a consequence of rapid microbial growth. CO2 which is a major component of gas produced is used for the synthesis of cellular consti- tuents. Therefore a rapid microbial growth would reduce the concentration of C62 in the fermentor. This is also eVidenced by the reduction of ammonia nitrogen observed throughout the five experiments. Contrary to the suggestions 91 of some workers that isoacids may not show an effect on microbial growth when the concentration of the rumen fluid in the fermentor exceeds 20 percent, data from this study showed that even with a nearly 100 percent rumen fluid concentration in the media the isoacids stimulated fermen- tation. Also the utilization of the carbon skeletons from the isoacids may decrease the fermentation of natural protein resulting in less gas production. Growth Study This study was an evaluation of the growth rate of dairy heifers as a response to supplementing grass hay with urea plus isoacids and phenylacetate. The isoacids increased the growth rate of the younger animals but not of the older animals. There are two explanations for this. First, iso- acids increase nitrogen retention in cattle. Second, younger animals are laying down more muscle than are older animals. Therefore, the nitrogen needs of younger animals are greater (Lassiter 22.2l-r 1958a). In Table 20 the first group included the five pairs of smaller animals having an average initial weight of approximately 200 kg per animal. The second group consisted of seven pairs of heifers with an average initial weight of about 225 kg. The third group included all nine pairs of animals averaging an initial weight of about 240 kg. The difference between treatment and control group was close to significant (P<.20) for the first two groups of heifers (200 and 225 kg). However, only 92 .cOmaummEoo ummuI:u= unocsum mcwms .Ao~.vmv ucwsummuu can Houucoo cmosuon mocmuoumac unmoamwcmfiw no m.MH H.mH mImH & .ucmfiummua m.oa h.~H m.HH a .Houucou unmwmz HowUficH 0:0 Ho>o ommouosu unmouom 0mm. Mbm HVN Damn. mmN ¢NN Ahmm. ova how ucmsummua 0mm. how HVN mmom. mvN ANN thN. mNN vow HOHUGOU chow amcwh Hmwuwcu came Hmswm Hmwuficu Gama Honda HmwuwcH wHMEHcd Ham madfiwfld h mafififlcm m pcosumwua mom mamsacd ox .Hmswcc Mom unmflos upon ommum>¢ Ahosum susoumv muomwoa mufimv mo cpsoum so mumumomaacmnm can oumuoHM>Ic .oumumusnamnumEIN .oumuoam>omw .oumuhusQOmH mo nooommo mnvII.om manna 93 a slight difference was observed between the isoacid-treated and the urea-treated groups when the average initial weight was around 240 kg. An examination of the isoacid-treated groups shows that the younger (200 kg) animals averaged an increase of 15.9 percent over the initial weight, whereas the second group gained slightly less weight (15.1 percent). When the entire treatment group was considered (240 kg) the percentage gain was even less than that of the second group. Lassiter gt El° (1958a) fed to dairy heifers corn cobs supplemented with valerate and isovalerate in a lSO-day feed- ing trial. They observed a significant increase in growth rate by the group which was fed the acid supplement. However, the greatest effect of these acids was manifested during the first 30-day-period when the animals gained about 1.5 times the weight they gained during the remaining 120 days of the experiment. Previous trials conducted by Beeson 25 al. (1964) have indicated that a combination of rumen factors including valeric and isovaleric acids improved cellulose digestion in an artificial rumen, and the growth rate of beef cattle. The pmesent study showed that feeding a combination of isoacids and phenylacetate with urea to dairy heifers increases the growth rate. The mechanism through which the isoacid addi- tion improves growth rate is probably by supplying their carbon skeletons to the ammonia released from urea hydrolysis. 94 Effects of Isoacids on Milk Production Trial I--This feeding trial was conducted to test the effects of isoacid—urea-supplemented corn silage without any other source of energy on milk production by high- producing cows in early lactation. For this feeding trial using lactating cows the rationale was that the cows would be in negative energy balance without a concentrate supple- ment and any improvement caused by isoacids in extracting more energy from the roughage by the rumen microbes would be easier to detect when measuring milk production. The parameters considered in this trial were milk yields, per- sistency of lactation and body weight changes as summarized in Table 21. The soy protein and the iSoacid treatments resulted in significantly-higher (P<.05) milk yields as compared_to the control. However, soy protein supplementa- tion showed better performance as compared to the isoacids (P<.05) when milk yields are considered. The persistency with which cows maintained their milk yields was higher (P<.05) among cows fed soy protein supplement as compared to the control. In turn, cows fed with the isoacids maintained their milk production at a higher level than those fed with the control, but the difference between both groups was not statistically significant. Cows fed the urea treatment alone lost 486 g of body weight per day; whereas the groups on isoacids and soy protein treatments gained 94 g and 422 9 weight per animal per day, respectively. The difference in 95 Table 21.--The effects of supplementing corn silage with soy protein, urea or urea plus isoacids and phenyl- acetate* on milk yields, persistency** and body weight in Holstein cows*** (trial I) Urea + isoacids Soy Protein Urea + phenylacetate Milk yields, 24.03 d 19.86 e 22.13 f kg/cow/day (6.67) (3.45) (2.43) Persistency, % 93.45 d 82.31 e 86.99 de (6.67) (7.75) (9.77) Body weight .143 d -.486 e .094 d changes**** (.422) (.845) (.372) kg/cow/day *Equal amounts of isobutyrate + isovalerate + 2-methylbutyrate + phenylacetate + n-valerate **Persistency of lactation = 100 x (treatment/ ~standardization) ***All values are means with standard deviation in parentheses defValues in same rows with different superscripts are significantly different (P<.05) ****Differences between control and treatments; P<.lO 96 body weight changes was statistically significant between the soy protein group and urea (P<.01), and between isoacids and urea (P<.05). But no significant difference was observed in body weight changes between soy protein- and isoacid-fed animals. The results of this trial show that soy protein was most effective in producing milk and maintaining milk production and body weight when corn silage was fed as the sole source of energy for high-producing cows. Data from this trial also indicated that isoacid addition to an urea- corn silage diet improved milk production and animal body weight over urea alone. This work was a preliminary feeding trial to evaluate the feeding value of isoacids as a comple- mentary source of protein in a urea diet fed to dairy cows. The trial was designed so that the cows would be at the highest protein and energy demand. The positive response of cows fed a soy protein diet, which is a high source of natural protein, was expected. However, we are not aware of any studies concerning the use of isoacids with urea for milk production. Lassiter gt 31. (1958b) failed to show any difference in milk production when they supplemented corn silage and alfalfa hay with isovalerate and valerate in a complete ration for lactating cows. The results of this trial show the potential value of isoacids as a part of the supplemental crude protein for lactating cows. From litera- ture on the nutrition of rumen bacteria, it is known that fiber-digesting bacteria require isoacids and ammonia for microbial protein synthesis. Without the carbon skeletons 97 from isoacids, urea or ammonia nitrogen cannot be used by rumen microbes. The major sources of carbon skeletons for biosynthesis of branched-chain amino acids (valine, leucine, isoleucine) are from degraded protein or exogenous branched- chain amino acids. With urea feeding as the sole source of supplemental nitrogen the rumen bacteria are not able to synthesize enough protein for maintainance of animal body tissues and for productive purposes. Because of lack of dietary preformed proteins, which are sources of the carbon skeletons, some exogenous branched-chain acids are needed when urea or other non-protein nitrogen sources are used. Therefore, the importance of supplying the isoacids in this particular situation of high protein demand is evidenced by the results of this trial. It is obvious from the results of this trial that the isoacids would be most effective when dietary protein is low and the protein requirement for ani- mal performance is high. Trial II--Since a positive response to isoacids was observed in trial I, it was decided to test the effects of addition of isoacids to a nutritionally complete ration. The ration consisted of corn silage and a supplemental concentrate. The results of this work are summarized in Table 22. Milk yields were not significantly different between animals in all four treatments. Addition to isoacids to urea improved, but not significantly, the persistency of lactation over both positive control and urea rations. Also 98 m~.vm unconnecmwm coHumw>mp cumusmum mum mononucmumm cw mosam?’4 an msomwumgsoo Hmcomoauuo mcwms Amo.vmv ucoummmwp haucmoamwcmwm mum mumfluomummsm acouommwc sues 30H 088m ca mosam> m n mumuoam>Ic can muwumusnaacuofiIm .oumuoam>0mw .oumuausnomw mo mucsosm Hmswma 1m.mov Am.moae AH.o~He Ah.msac mas. mmm. mmm. use. menus“ 2a mx\xafle as Amm.v Ams.e .mm.c “om.v saunas soon so m an m~.m m~.m no.m so.m mmxmucn cwmm sauce Halam.e mlm~.e mlom.v alom.c unmwmz soon w an ma.H om.H H>.H mo.H menace «madam Amm.v .oe.e Ame.c AH¢.S mmc\3oo\mx emu. m~o. ohm. o~m. momcmeo unmfims soon n1m~.HH. almm.¢c also.oac almm.mc aa.nm no.4m H5.mm en.m> m .socmumnmuwm Ama.ee Ahm.mv Aho.mv «Ixem.mc smo\3ou\mx 5H.H~ -.H~ m~.H~ so.ma moamnm sans mowomomw mono Houucou Houusoo + mono m>wuwmom m>wummmz AHH Hmfiuuv cOAumucmEmHmmsm comouuwc on can Home :wmnaOm .«mpflomomfl msam nous .mmus spas woucmamammsm monawm suoo mom msoo mo woodwoammm comm can GOwumfismcoo umuume who comm .mmmsmso unmwm3 anon .GOflumuoma mo hocmumfiuumm .mvamwh waZII.NN manna 99 the isoacid treatment tended to maintain body weight at a higher level when compared to the three other groups. However, the difference was not statistically significant. Corn silage dry matter intake was slightly higher (P<.25) among cows fed the isoacid diet as compared to the other treatment groups. However, total feed dry matter intake per 100 kg body weight was only slightly higher for the isoacid ration over the remaining groups. The efficiency with which feed was converted to milk was essentially the same for all treatments. Therefore, the slight increase in milk yields between the negative control and the other treatment groups may not be explained on the basis of higher feed efficiency per se. In general this trial shows that the addition of isoacids to urea was most effective in maintaining body weight and the level of milk production, and in increasing corn silage dry matter intake, as compared to the other' treatments. The positive effect of isoacids on increasing roughage intake is consistent with the works of Hensley and Moir (1963), and Van Gylswyk (1970). Both groups of investi- gators reported significant increases in voluntary hay intake when they fed isoacids with urea to sheep. The increase in hay intake was accompanied by an increase in the number of cellulolytic bacteria (Gylswyk, 1970) and an increase in the level of microbial protein (Hensley and Moir, 1963). Among several possible reasons why isoacids did not show any increase in milk production in comparison to urea alone, three explanations are notable: (1) the cows may have 100 been able to mobilize body tissue to meet their energy needs and the effects of the isoacids could not be detected by measuring milk production; (2) the level of protein equiva- lent supplied by the urea ration may have been sufficient to fulfill the protein requirements for milk production so that the addition of the isoacids could not contribute any further increment. The level of milk yields shown in Table 22 required only a ration with about 14 percent crude protein (National Research Council, 1971). This amount of protein had been reported in the current trial; and, (3) as pre- viously suggested, isoacid addition to urea may be more effective when the level of natural protein in the diet is low and when the protein demand is high. It was reported (National Research Council, 1976) that the basal diet must be deficient in nitrogen or natural protein if supplementary urea is to be beneficial to the animal. However, even though significant differences were not uniformly observed for all parameters, the isoacid addition to urea reveals that better performance is obtained when compared even to the positive control. Trial III--Since trial II did not result in signifi- cant differences between treatments using a randomized block design it was decided to conduct another trial using a Latin square cross-over type design which is much more sensitive than the former design (Gill, 1969). Using this design, the theoretical probability of showing a 2 kg difference in milk 101 production using 8 cows per treatment is about 80 percent. However it was found that when the cows were changed from one treatment to another milk production declined for about the first two weeks, recovering at the end of the fourth week, which was the duration of the treatment period. Thus, it was found that this design is not satisfactory for test— ing the parameters of interest. Thirty cows were used in this experiment. The milk production of 12 of them decreased more than 2 kg per day when the ration was changed. There— fore, the data from these cows were not included in the statistical analysis. The effects of the two different mixtures of isoacids on milk yields, persistency of lactation, body weight changes, feed intake and feed efficiency are shown in Table 23. Data in this table are means of eighteen repetitions per treatment from the 18 cows which were less affected by the effect of changing diet in the cross-over experiment. Milk yields per cow per day were slightly, but not significantly, higher in both isoacid mixtures than in the control. However, fat-corrected milk (FCM) was significantly higher (P<.05) among cows fed the isoacid mixture 2 when compared to either the control or isoacid mixture 1, indicating that isoacid mixture 2 increased the fat content of the milk over that of the control and even over that of mixture 1. Cows fed the acid mixture 2 maintained their milk production at a higher level (P<.18) than those fed urea alone or the acid mixture 1. The group fed mixture 1 were slightly more persistent in 102 Table 23.--The effects of two different mixtures of isoacids on milk yields, persistency of lactation, body weight changes, feed dry matter intake and feed efficiency (trial III) Control Acid Mixture Acid Mixture 1* 2** Milk yields, kg/cow/day 28.1 28.5 29.3 FCM*** b kg/cow/day 25.6 (4.13)a 25.9 (3.24)a 27.1 (3.59) Persistency, 5 89.3 (14.7)c 90.3 (9.06)° 94.6 (11.28)d Body weight changes, kg/cow/day - .11 (5.5) - .27 (5.8) - .01 (6.1) Silage intake, kg % body weight 1.67 (.15) 1.67 (.15) 1.70 (.16) Total feed intake, kg % - body weight 3.12 (.20) 3.17 (.4) 3.18 (.22) Kg milk/kg feed DM 1.4 (.24) 1.4 (.25) 1.5 (.4) *Twenty-eight percent isobutyrate, plus 24 percent of isovalerate, 2-methy1butyrate and n-valerate. abValues in same row with different superscripts are significantly different (P<.05), using orthogonal comparisons. CdSignificant difference at P<.10 **Thirty-six percent isobutyrate, 30 percent n-vale- rate, and 17 percent each of isovalerate and 2-methy1butyrate. ***Fat corrected milk 4 percent at 305 days (.4 x milk + 15 x fat). 103 lactation than the group receiving the control ration. Cows fed isoacids 2 lost less body weight than those on acids 1 or on control. However, the difference was not statistically significant. Corn silage dry matter intake, total feed intake and feed efficiency were essentially unaffected by the acid treatments. Generally the group on isoacids 2 reveals a trend towards better performance as compared to control or isoacids 1. There is no apparent explanation for the differ- ence between the responses resulting from isoacid mixtures l and 2. The difference between the composition of the two isoacids is that the molar proportions of isobutyrate and n-valerate were higher (36 and 30 percent) in mixture 2 than in mixture 1 (28 and 24 percent). This may suggest that these two acids were the most limiting. The work of Lassiter et al. (1958b) showed that cows fed with n-valerate gave better performance than when n-valerate was associated with isovalerate. However, Umunna and co-workers (1975) found much higher incorporation of isovalerate into the microbial cells than isobutyrate. In this trial II, the addition of the isoacids did not show any improvement in milk production. However, acid mixture 2 improved the per— sistency of lactation over the control. Lassiter's group (1958b) could not improve milk production by feeding iso- valerate and n-valerate to lactating cows fed corn silage and hay. But they did not feed urea. In the current trial the isoacids probably improved utilization of nitrogen from urea. There were some similarities between these two trials. 104 Lassiter's trial used a Latin square cross-over design in a 28-day period. It would appear that the effect of changing diet on the appetite of the animals and the duration of the treatment period are the main explanation for results obtained in both trials. Milk composition, plasma urea nitrogen, rumen pH, ammonia-N and VFA production are presented in Table 24. There were no significant difference among the three treat- ment groups as far as milk composition is concerned. However, for the isoacid groups milk protein, fat and solids nonfat were slightly higher. Rumen.ammonia-N was lower in the isoacid treatments than in the control, although this dif- ference was not significant. The same trend was observed~ for plasma urea-N.r This suggests that there was higher utilization of ammonia by the cows on the acid treatments as compared to those on control. It is possible that carbon skeletons from isoacids have been associated with ammonia in the process of microbial synthesis. This is supported by the increase in acetate in both acid treatments over the control, indicating that fermentation is stimulated by the addition of the acids to urea. However, the addition of the isoacids did not appear to influence the molar proportions of propionate and butyrate. Isobutyrate levels increased slightly in the rumen fluid with the addition of the iso— iacids. But a close examination of Table 24 indicates that 'the isoacids were utilized since isobutyrate was the only one among the four acids added that appeared in the rumen fluid. 105 mo.vm um uGMOHMHsmHm co “Ho.vmv ucouwMMHo hHucmoHMHcmHm mum mumHuomummsm ucmquMHo nuHs sou meow CH mosHm>no w. m :.m _m w MdNN5 Amo.v mo. Amo.v mo. AHo.V mo. oumuausnomH xaH.e mm. Ao~.v mm. AN~.V em. mumumunm Hmm.v h¢.H Amm.v mv.H Ace.v o¢.H oumconoum oAmm.Hv mo.¢ oGHHV Hm.¢ oA>>.v HH.¢ mumuood HsoOmeHoes .¢m> amass pamm.ev n~.m came.mv «o.OH oxmn.ov mm.HH HEo0H\mE .ZIMHcoEEm coasm Ahm.v mm.m Avu.v Hm.o Amm.v.mm.m mm coesm clom.~c mv.a elem.~e mm.a olvm.~e ~G.¢H Hsooa\ms .zImmus mammam amo\300\m Av.bHHv ~m.mvm Am.mmv me.mom am.vHHv Hm.mwm coHuosooum sHououm xHHz moo\3oo\m Av.mmmv mh.mmm~ Am.m~mv mv.mmH~ A~.vmmv ms.mmv~ cOHuosooum mzm xHHz hm0\3oo\m nAH.meV mH.ovoH on.omHv mm.mnm mA~.m>Hv mm.vom cOHuoooonm you xHHz Amm.v vm.m Amm.v 5H.m Aom.v HH.m m .cHououm xHHz Amh.v m>.m Ahm.v hm.m Amm.v m>.m a .mZm xHHz Amm.v mm.m .mm.v m¢.m Amm.v m¢.m w .umu xHHz hm.v om.~H Anm.v mv.NH Hom.v «H.~H w .moHHOm Hmuou xHHz m ousutz oHom H musutz oHoa Houucou AHHH HMHuuv can comouuHc MHcoEEm .mo smash can mono QEmMHm .GOHuHuooEoo xHHE co moHUMOmH mo mousuxHE ucoumMMHo 03» mo muoommm onaII.¢~ mHnma 106 The effects of isoacids on dry matter digestibility, nitrogen digestibility, nitrogen retention and nitrogen balance are summarized in Table 25. Feed dry matter digest- ibility was essentially the same among the three treatment groups. .However, nitrogen intake and fecal nitrogen were higher in both isoacid treatments than in the control. Nitrogen digestibility appears to be higher in the acid treatments as compared to the control. However, urinary-N was significantly higher (P<.025) in the control than in both acid treatments. Nitrogen retained was significantly higher (P<.01) for the acid treatments compared to the con- trol. The addition of both isoacid mixtures improved (P<.10) the nitrogen balance of the lactating cows. The effect of isoacids on nitrogen retention is the most obvious beneficial effect showed by the use of isoacids in cattle and sheep diets (Cline et_al., 1966; Lassiter EEH2£" 1958b; Moore, 1964; Oltjen 22 21., 1971; Umunna st 21., 1975). A11 pre- vious work in which nitrogen balance studies were_conducted shows that more of the available ammonia is being converted into microbial protein. In the present trial the addition of the acids reduced the urinary nitrogen loss and therefore increased the utilization of the absorbed nitrogen as was indicated by an improved nitrogen retention. Kay and Phillipson (1964) have reported an increase in nitrogen flow rate to the duodenum of sheep fed poor quality hay diets by infusing isobutyrate, 2-methylbutyrate and isovalerate; whereas similar infusion of the straight-chain acids had 107 OH.vm um oocmuomuHooo .ummuucoo Hmcomonuuo mchs .Ammo.vmv usoquMHo hHucmoHMHcmHm mum mumHuomHmmsm ucoummuHo suHs sou meow sH mosHm>nm .mmmonucoumo cH GOHuMH>mo oumocmum :qu .hmo moo 300 you momma mum nosHm> HH4¢ elm.oe m.mHI ulh.mv h.mHI olm.mae p.ovI m .oocmamn z n.4ma 5.H~H m.-H m .z I xHHz conuonnm mo om.¢m am.mm oH.m¢ w an nochuou z olv.qc a.¢oa 615.41 o.ooH oxo.mc 5.Hm m .nmcnmumu z m.me~ o.me~ m.om~ m .cmumuoxo z Hmuoa alm.mv q.e~ n.~.me e.e~ m.>.pe m.m~ menses s .zIsumawuo alm.mv e.ow alp.me m.mm «15.5. m.oa m .zIsumcfiua 1m.me o.em .m.¢v m.qm Amadoc e.mm a .muaafinwummmwu z 1m.me a.~oH Am.qv ~.omH 15H.mc m.om~ m .nmmouufic swoon Amo.v «.emm Ama.v o.~mm lm~.e «.mmm m .msau:H cmmouufiz n.5m m.nm 4.5m a .sunaflnflummmno an N musuxflz unom H musuxnz cane Houucou AHHH HMHHHV smzoo :HoumHom an coHucmuou cmmouuHc can huHHHnHumomHo comouuHc .muHHHnHumomHo Azov Houume who comm so moHomomH mo monouxHE ucmHoMMHo 039 no muoommo onaII.mN oHoma 108 little effect on the nitrogen flow rate. This indicates an increase in microbial growth rates due to the addition of the isoacids. Although the addition of isoacids in the cur- rent trial did not improve milk yields or feed intake, the fact remains that all of these parameters showed a positive influence of these isoacids on nitrogen utilization as evidenced by the nitrogen retention. The maintenance of the level of milk production by these acids over the urea diet is additional evidence of the beneficial effect of isoacids on the nitrogen economy of ruminants fed urea. It is also possible that a greater response could have been obtained if greater quantities of the acids were fed. Trial IV--From the results of trial III it was felt necessary to conduct another continous feeding trial using pairs of cows in a randomized block design for 90 days. Thirty animals were paired, adapted to the feeding of iso- acids and high level of urea for a period of 21 days prior to the treatment period. The results of this trial are reported in Tables 26 and 27 and are the summaries of observations for 14 pairs of cows. Half of the animals were heifers in the first or second lactation. Milk yields, persistency of lactation, body weight changes and feed dry matter intake are shown in Table 26. The addition of iso- acids to urea did not appear to improve significantly milk yields as compared to the control diet. However, the acid treatment showed a slight increase over the control. Fat 109 Table 26.--Milk yields, persistency of lactation, body weight changes, feed dry matter intake of cows fed equal amounts of isoacids*, (trial IV) Control Treatment Milk yields, kg Fat corrected milk, k9 Persistency, % Body weight changes, k9 Silage intake (DM % body weight) Total feed intake (kg % body weight) 17.36 (3.09) 16.32 (3.89) 76.87 (9.56)a .029 (.17) 2.03 (.34) 2.61 (.04)° 18.69 (3.42) 17.56 (4.35) 81.79 (10.20)b .027 (.19) 2.18 (.28) 2.83 (.06)d *Data are means per cow with standard deviation in parentheses. ab Values in same row with different superscripts are significantly different (P<.001), using "t"-test comparisons. cd Significant difference at P<.05. 110 corrected milk showed the same trend in favor of the acids, but without any significant difference between treatment groups. However, the persistency of lactation was signi- ficantly higher (P<.001) among cows fed the isoacid treat- ment than among those fed urea alone. This is consistently observed throughout all four trials, indicating a beneficial influence of the isoacids on urea feeding. Persistency of lactation expresses the level at which a cow maintains her production throughout the entire lactation. A cow with low persistency would indicate that the rate of milk production has been markedly reduced from one month to another, which would affect the total milk produced during the entire lactation compared with a higher persistency. Therefore information about persistency of lactation may be a more accurate parameter to estimate the effect of a diet than the actual milk production which is subjected to many variations from one animal to another. The gain in body weight was essentially the same between the two groups of animals. The isoacids increased slightly the corn silage dry matter intake over the control. But the total feed dry matter intake expressed as a percentage of body weight was significantly increased (P<.05) by the acid treatment over the control. This is another major benefit of the isoacid feeding as substantiated by Van Gylswyk (1970) and Hensley and Moir (1963). Although no significant difference was observed in daily milk yields, the beneficial effect of the isoacid 111 feeding is manifested by the significantly high persistency of lactation and the significant increase in feed dry matter consumption. Milk composition, plasma urea nitrogen, rumen pH, rumen NH3-N and VFA are summarized in Table 27. The addition of the isoacids to the diet did not significantly affect the milk composition, rumen pH, total rumen VFA, rumen ammonia- nitrogen or plasma urea-nitrogen. The only significant difference observed in VFA concentrations was for isobutyrate (P<.01), which reflects the addition of the isoacid to the ration. The molar concentrations of acetate and propionate increased slightly for the isoacid group over the control group. The level of butyrate remained essentially the same in both the control and treatment groups. It is important to note that isovalerate, n-valerate and 2-methylbutyrate were not found in detectable amounts in rumen fluid of trials III and IV. This is in contrast to the observations by Oltjen gt 21° (1971), Umunna et 31. (1975) and Hume (1970). These workers reported significant increases in the rumen fluid of the animals to which these acids were administered. However, the significant increase in isobutyrate level observed in the current trial is in agreement with those findings. Probably the dilution factor used in our study did not permit the detection of the other acids. A close examination of the results of the present trial reveals that the addition of isoacids to urea resulted in significantly higher levels of milk production, higher feed dry matter ‘_._. ("r0 3‘. r . 112 Table 27.--Milk composition, plasma urea nitrogen and VFA of cows fed isoacids* (trial IV) Control Treatment Milk composition total solids, % 12.18 (.46) 12.09 (.65) Fat, % 3.60 (.36) 3.57 (.46) Solid non fat, % 8.57 (.35) 8.52 (.42) Protein, % 3.11 (.41) 3.11 (.38) Fat production g/cow/day 645.25 (146.8) 650.84 (145.32) SFN production g/cow/day 1544.77 (396.45) 1591.22 (462.54) Protein production g/cow/day 540.01 (389.6) 581.21 (416.8) Plasma urea-N mg/100m1 11.55 (3.47) 10.95 (3.22) Rumen pH 7.05 (.20) 7.05 (.24) Rumen NH -N mg/10031 9.64 (5.63) 10.40 (4.28) Rumen VFA, mmole/ 100ml Acetate 3.96 (.59) 4.10 (.57) Propionate 1.06 (.17) 1.11 (.23) Butyrate .69 (.10) .69 (.17) Isobutyrate .04 (.01)a .12 (.03)b 133 367 *Data are means per cow with standard deviation in parentheses ab Significant difference at P<.Ol 7? - .- 5" 5‘1w 5’ 7.3; 1"." . __ “-1"- x.— 113 intake, lower plasma urea nitrogen and higher rumen acetate, propionate and isobutyrate concentrations. These data indi- cate a beneficial effect of isoacids on urea nitrogen utili- zation by dairy cows. Although statistical differences were not always observed for all parameters considered in this trial, information from persistency of lactation and feed intake are sufficient to support the evidence of beneficial effects of isoacids on the performance of the dairy animals. I (1‘ SUMMARY AND CONCLUS ION One in yitgg experiment, one growth trial and four separate milk production trials were conducted to investi- gate the nutritional response of dairy cattle to high-urea ration supplemented with isoacids. The results of the in gitrg study showed that isoacids enhanced rumen microbial activity. The growth study was conducted to investigate the effect of supplementing grass hay with isoacids and urea on growth rate of dairy heifers. This study showed that addition of isoacids to urea increased the growth rate in young animals but not in older animals. The four milk productionsfiwere carried out to deter- mine the performance of high—producing cows fed corn silage as the sole roughages supplemented with isoacids and urea. Those four trials showed that isoacids had a positive effect on milk production, persistency of lactation, body weight, feed intake and nitrogen balance, when added to a urea-based diet for lactating cows. It is well known that the utiliza- 'tion of low protein roughage by ruminants is influenced by several factors. One of these factors is unquestionally an 114 -u,' . .I‘Ah-leh ‘ OW ' I' 115 insufficiency of nitrogen to satisfy the growth requirements of an adequate microbial population in the rumen. The addition of urea alone to improve the nitrogen level of such dietary material has produced variable responses. This is because after the deficit in nitrogen has been removed by (feeding urea, the next factor limiting the microbial growth is the lack of carbon skeletons from isoacids. One or more of these acids is required for the growth of several species of cellulose-digesting bacteria (Allison, 1965). These isoacids are normally derived from protein in the diet. The addition of the isoacids to urea in the present studies no doubt resulted in increasing the rate of proliferation of cellulose-digesting bacteria in the rumen. These studies have shown that isoacids: a. increased rumen microbial activity in gitgg b. improved rate of growth of young animals c. increased persistency of lactation d. improved nitrogen retention in lactatingirbws :It is apparent that feed supplements must be considered not nuerely in terms of their gross nitrogen, mineral and energy FF.“ " _\ - - __._...—...._._k , supplements, but also in terms of their ability to satisfy the full nutrient requirements of the rumen microbiota. J '4” ‘-.‘l~v‘- _2____ LITERATURE CITED LITERATURE CITED Abou Akkada, A. R. and T. H. Blackburn. 1963. 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