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FINES will be charged if book is returned after the date stamped below. J“, ._ ‘ G / / , '- ‘ ‘ ‘n/ (0 MILK PRODUCTION AND NITROGEN METABOLISM OF HIGH PRODUCING COWS EARLY IN LACTATION FED NON-PROTEIN NITROGEN AND RUMEN UNDEGRADABLE PROTEIN By Limin Kung, Jr. A DISSERTATION Submitted to Michigan State university in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1982 ABSTRACT MILK PRODUCTION AND NITROGEN METABOLISM OF HIGH PRODUCING COWS EARLY IN LACTATION FED NON-PROTEIN NITROGEN AND RUMEN UNDEGRADABLE PROTEIN By Limin Kung, Jr. The objectiVe of this research was to increase the efficiency of nitrogen utilization in high producing dairy cows early in lac- tation. Experiment 1 Data from these experiments showed that formaldehyde treat— ment autoclaving, and dry heat treatment of soybean meal (SBM) decreased nitrogen degradability in the rumen. Compared to un- treated SBM the treated exhibited less ig_yi££2_ammonia release, less nitrogen disappearance from rumen suspended nylon bags and less nitrogen disappearance during protease incubation. Experiment 2 Eighty four high producing multiparous cows were placed on pre- trial ration immediately after calving until day 21 postpartum. Cows were placed on one of the following rations from days 22 to 91 postpartum; l) 11% crude protein (CP), corn silage (CS) and soybean meal (SBM); 2) 14% CP ammonia-treated corn silage (AS) and Limin Kung, Jr. heated soybean meal (HS); 3) 14% CP, CS-HS; 4) 14% CP CS-SBM; 5) 17% CP AS-HS; 6) 17% CP, CS-HS; 7) 17% CP, CS-SBM. Dry matter intake and adjusted milk production increased with protein level. Milk production was greater for cows fed rations containing AS and/or HS when compared to SBM controls. Cows fed 17% CP AS-HS were most productive and profitable. Interpretation of results from this experiment suggests feeding for maximum peak milk production even though return over feed costs may be less for productive rations early in lactation, contradicting the well accepted idea to feed for maximum profit in early lactation. The substitution of natural protein with ammonia added to corn silage was compatable with high milk yields at both 14 and 17% dietary protein. Experiment 3 Four lactating cows fitted with rumen fistula, and duodenal and ilieal cannula were used to measure flow and digestion of nitrogen- ous compound in the digestive tract. Diets were 17% CP and similar to Experiment 2. Flow of dry matter to the duodenum appeared to be grossly overestimated when lanthanum was the marker, resulting in high estimates for microbial protein efficiencies at the duodenum. Estimates made with lignin were similar to accepted values and were deemed more appropriate. No significant differences in digesta flow or digestion were but trends between diets were apparent. Non ammonia nitrogen (NAN) flow was greatest for cows fed heated soybean meal CS-HS and AS-HS. Limin Kung, Jr. Digestion of NAN in the small intestine was equal for all treat- ments. This suggests that availbility of heated soybean meal was not different in the lower gut even though rumen degradability was decreased. This thesis is dedicated to my family, Limin Kung Myrtle Kung Lani Bushnell Mann-Chi Kung Linza Kung ii ACKNOWLEDGMENTS I would like to express my sincerest appreciation to my major professor Dr. J. T. Huber for providing me with the opportunity for pursuing my graduate degree. I would also like to thank all the members of my committee including Drs. J. T. Huber, W. G. Bergen, J. C. Waller, and J. W. Thomas for their support, help- ful suggestions, and guidance. I am extremely grateful to Drs. J. T. Huber, J. W. Thomas, and R. S. Emery for giving me the encouragement and opportunity in doing non thesis related research. Special thanks to Ellen Fitzgerald, Sue Brecht, Denise Snyder, and Denise Davis for helping in management of animals, sampling, and analyses. Barry Jessie, Amir Shanan, Doug Grieve, Ken King, and Stephanie Yang were also invaluable in giving assistance and suggestions in these experi- ments. A sincere thank you goes to all the student workers and full time workers at the MSU Dairy Barns. Special thanks to Jim Liesman for his suggestions and inter- est in my research and for assistance in computer programming. Thanks to Bill Rumpler and Gary Weber for some stimulating con- versations in ruminant nutrition. Sincere appreciation to Dr. L. D. Satter and the University of Wisconsin for use of animals and facilities in which a part of iii my thesis research was conducted. I would also like to thank Elaine Kibbey who was invaluable in many ways. Finally, I would like to thank Andrea Curato for her encour- agement, patience, and moral support throughout my Ph.D. program. To all those that I touched and who touched me, Mahalo and Aloha. iv TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . LISTOFFIGURES..................... INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . Protein Digestion in the Rumen. . . . . . . . . . . Factors Affecting Microbial Growth and Production . Nitrogen requirements for microbial growth . . Ammonia fixation in the rumen. . . . . . . . . Other essential growth factors . . . . . . . . Systems for Estimating Protein Requirements . . . . Efficiency of Amino Acid Use. . . . . . . . . . . . Amino Acid Requirements . . . . . . . . . . . . . . Amino Acids as Energy Sources . . . . . . . . . . . Protein Needs for Lactating Cows. . . . . . . . . . Amount of protein . . . . . . . . . . . . . . Protein quality. . . . . . . . . . . . . . . . Protein Degradation in the Rumen. . . . . . . . . . Form of nitrogen . . . . . . . . . . . . . . . Feed storage and processing. . . . . . . . . . Heat treatment . . . . . . . . . . . . . . . . Formaldehyde treatment . . . . . . . . . . . . Tannin treatment . . . . . . . . . . . . . . . Measurements of protein degradation and digestabilities . . . . . . . . . . . . . . . Amino Acid Supplementation. . . . . . . . . . . . . Nonprotein Nitrogen: Replacement of Dietary Protein Urea and ammonia treatment of silage . . . . . Problems with feeding NPN. . . . . . . . . . . Page viii xi ll 14 16 16 25 26 27 28 34 36 38 39 41 42 45 46 46 48 49 52 55 Page Rumen Thrnover and Marker Methodology. . . . . . . . 59 Significance of turnover. . . . . . . . . . . . 59 Marker methodology for determination of turnover and digestability. . . . . . . . . . . 60 Affect of intake level on turnover. . . . . . . 65 Affect of altering forage: concentrate ratio. . 67 Affect of turnover on rumen microbes. . . . . . 68 Turnover and protein metabolism . . . . . . . . 69 Affect of turnover on carbohydrate digestion. . 69 Affect of buffers on turnover . . . . . . . . . 71 OBJECTIVES. O O O O O O O O O O O O O I O O I O O O O O O 73 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . 74 Experiment 1 O C O O O C O O O O O O O O I O O C O O 74 General treatments. . . . . . . . . . . . . . . 74 Laboratory evaluations. . . . . . . . . . . . . 74 Experiment 2 . . . . . . . . . . . . . . . . . . . . 76 Animals, treatments, feeds. . . . . . . . . . . 76 Collection and preparation of samples . . . . . 77 Statistical analysis. . . . . . . . . . . . . . 78 Experiment 3 . . . . . . . . . . . . . . . . . . . . 78 Animals and treatments. . . . . . . . . . . . . 78 Marker preparation. . . . . . . . . . . . . . . 79 Collection and preparation of samples . . . . . 80 Statistical analysis. . . . . . . . . . . . . . 81 Calculations. . . . . . . . . . . . . . . . . . 82 RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . . . 85 Experiment 1 O O O C O O O O O O O O O O O O O O O O 85 Laboratory studies. . . . . . . . . . . . . . . 85 Field samples 0 O O O O O O O O O O O O O O O O 98 Experiment 2 . . . . . . . . . . . . . . . . . . . . 98 Ration composition. . . . . . . . . . . . . . . 98 Dry matter intake . . . . . . . . . . . . . . . 102 Milk production . . . . . . . . . . . . . . . . 104 Energy intake and body weights. . . . . . . . . 109 Production efficiencies . . . . . . . . . . . . 111 Rumen and blood parameters. . . . . . . . . . . 111 Economic evaluation . . . . . . . . . . . . . . 114 Reproductive efficiency . . . . . . . . . . . . 118 Experiment 3 . . . . . . . . . . . . . . . . . . . . 121 Feed composition and milk production. . . . . . 121 Rumen parameters. . . . . . . . . . . . . . . . 121 vi Page Neutron activation and marker analysis. . . . . 127 Sample collection . . . O O O O O C O O O O O O 131 Dry matter flow and digestion . . . . . . . . . 131 Nitrogen flow and digestion . . . . . . . . . . 134 NAN flow and digestion. C O O O O O O O O O O O 138 Dietary N and microbial N flow. . . . . . . . . 138 Microbial protein synthesis . . . . . . . . . . 140 Organic matter and acid fiber digestion . . . . Experiment 1 . . . . . . . . Experiment 2 . . . . . . . . Experiment 3 . . . . . . . . CONCLUSIONS . . . . . . . . . . . APPENDIX A : Additional tables. . APPENDIX B : Procedures . . . . . BIBLIOGRAPHY. O O O C O O C O O O Vii detergent O O O O O O O O O O O O 141 . . . . . . . . . . . . 144 . . . . . . . . . . . . 144 . . . . . . . . . . . . 144 . . . . . . . . . . . . 145 . . . . . . . . . . . . 147 . . . . . . . . . . . . 149 . . . . . . . . . . . . 160 O O O O O O O O O O O O 162 Table 1. 10. 11. 12. 13. LIST OF TABLES Km's for ammonia of some ammonia fixing enzymes. . . . Estimates for amino acid flow according to different mOdelS O I O O O O O O O O O O O O O O O O O O O O O 0 Recommended level of protein feeding for maXi-mum prOfit O I I O O O O I O O O O O O O O O O O O Marginal changes in milk production and dry matter intake as a result of changing rations protein percent Effect of level of milk production and extent of negative energy balance on the net AAN need in cows weighing 600 kg . . . . . . . . . . . . . . . . . Effect of protein level on reproductive per- formance of dairy cows . . . . . . . . . . . . . . . . The influence of casein infusion on the milk production and milk protein content of dairy cows. . . Summary of experiments feeding heat treated soybean products to lactating dairy cows . . . . . . . . . . . Effect of feeding nonprotein nitrogen on repro- ductive performance of dairy cows. . . . . . . . . . . In vitro ammonia release of formaldehyde (HCHO) and autoclaved soybean meal. Experiment 1. . . . . . . In vitro dry matter disappearance of treated soybean meals. Experiment 1 . . . . . . . . . . . . . . . . . Nitrogen solubility, in vitro dry matter disappear- ance, and detergent insoluble nitrogen and nitrogen disappearance from nylon bags suspended in the rumen of soybean meal subjected to 149°C for varying times. Experiment 1. . . . . . . . . . . . . . . . . . . . . Nitrogen degradation by a FICIN protease of soybean meal subjected to 149°C for varying times. Experimentl viii Page 15 24 31 32 34 36 37 44 58 86 96 97 99 Table Page 14. Nitrogen disappearance of soybean and soybean meal from nylon bags suspended in the rumen for various times. Experiment 1. . . . . . . . . . . . . . 100 15. Ingredient composition of rations fed to cows re- ceiving varying protein from different sources. Experiment 2. . . . . . . . . . . . . . . . . 101 16. Dry matter intake of cows fed varying protein amounts from different sources. Experiment 2. . . . . . . . . 103 17. Milk production, persistency and milk composition of cows fed varying amounts of protein from different sources. Experiment 2. . . . . . . . . . . . 105 18. Comparison of main effects of milk production results of cows fed varying protein from different sources. aperiment 2. I O O I O O O O O O O O I O O O O O O O O 106 19. Estimated net energy intake and requirement and body weight and efficiency of milk production of cows fed various protein from different sources. Experiment 2 . 110 20. Rumen ammonia-nitrogen of cows fed varying protein from different sources. Experiment 2 . . . . . . . . . 112 21. Rumen pH of cows fed varying protein from different sources. Experiment 2. . . . . . . . . . . . . . . . . 113 22. Plasma urea-nitrogen of cows fed varying protein from different sources. Experiment 2. . . . . . . . . . . . 115 23. Rumen volatile fatty acids of cows fed varying protein from different sources. Experiment 2.. . . . . . . . . 116 24. Economic evaluation of rations containing varying amounts of protein from different sources. Experiment12.117 25. Breeding data of cows fed varying protein from dif- ferent sources. Experiment 2.. . . . . . . . . . . . . 119 26. Breeding data of cows fed varying protein from different sources grouped by protein levels. Experiment 2 O O C O C O O O I O O O O O I O O O O O O O 120 27. Composition of feeds in experiment 3. . . . . . . . . . 122 28. Dry matter intake and milk production of cows in experiment 3. O O O C O O O O O O O C O O O O O O O O 123 ix Table Page 29. anen ammonia nitrogen of cows in experiment 3. . . . . 126 30. Rumen pH of cows in experiment 3. . . . . . . . . . . . 128 31. Rumen volatile fatty acids of cows in experiment 3. . . 129 32. Dry matter flow and digestion in various segments of the digestive tract of cows in experiment 3. . . . . 132 33. Nitrogen flow and digestion in various segments of the digestive tract of cows in experiment 3 . . . . . . . . 135 34. Flow and digestion of non-ammonia nitrogen (NAN) and efficiency of microbial protein production of cows in experiment 3 . . . . . . . . . . . . . . . . 139 35. Organic matter intake, flow and digestion in various segments of the digestive tract of cows in experiment 3. . . . . . . . . . . . . . . . . . . . . . 142 36. Acid detergent fiber (ADF) intake and digestion of cows in experiment 3 . . . . . . . . . . . . . . . . . . . . 143 A1. Dry matter and crude protein of feeds fed in EXPeriment 1. I O O O O O O O I O O O O C O O O O O O O 149 A2. Selected data for cows fed rations containing various anmnints of protein from different sources. 1 Experiment 2. O O C O O O O O O O O O O O O 0 O O O O O 151 A3. Bacterial composition from Experiment 3 . . . . . . . . 154 A4. Ileal comosition from Experiment 3. . . . . . . . . . . 155 A5. Duodenal composition from Experiment 3. . . . . . . . . 156 A6. Fecal composition from Experiment 3 . . . . . . . . . . 157 A7. Lignin (%DMB) content of feeds, digesta, and feces from Experiment 3. . . . . . . . . . . . . . . . . . . 158 A8. Chromium and lanthanum concentrations in feed, digesta, and feces from Experiment 3. . . . . . . . . . 159 Figure 1. 10. 11. LIST OF FIGURES Crude protein of dairy rations according to different models with N degradability 0.66 . . . . . . Crude protein (Z of dietary DM) requirement as a function of milk production (kg/day) for five European systems with undegradability at optimum . . . . . . . The relationship between milk yield and the propor- tion of dietary protein required as UDP and RDP as predicted by the ARC model. . . . . . . . . . . . . The dietary protein requirements over a complete lactation for a Friesan cow peaking at 30 kg milk per day as predicted by the ARC model. . . . . . . . . Undegradability (Z) of dietary crude protein as a function of milk production (kg/day) for five European systems with crude protein at optimum . . . . Simplified diagram of nitrogen pathways through the rumen. . . . . . . . . . . . . . . . . . . Dry matter disappearance of soybean meal (SBM) treated with formaldehyde (HCHO) from nylon bags suspended in the rumen. . . . . . . . . . . . . . Nitrogen disappearance of soybean meal (SBM) treated with formaldehyde (HCHO) from nylon bags suspended in the rumen . . . . . . . . . . . . . . . . Dry matter disappearance of soybean meal (SBM) auto- claved for various lengths of time from nylon bags suspended in the rumen. . . . . . . . . . . . . . Nitrogen disappearance of soybean meal (SBM) auto- claved for various lengths of time from nylon bags suspended in the rumen. . . . . . . . . . . . . . Nitrogen disappearance of normal soybean meal (SBM) and soybean meal heated for 2.5 hr at 140°C from nylon bags suspended in the rumen . . . . . . . . xi Page 19 21 22 22 23 4O 89 91 93 95 125 INTRODUCTION Valid estimates of protein digestion and amino acid absorption in the ruminant are complicated by rumen microbial fermentation. In monogastrics, feed and endogenous nitrogen are the two main sources of protein and amino acids reaching the small intestine for digestion and absorption. Microbial protein is a third and important contribution to this source in ruminants. Although energetically inefficient, mi- crobial fermentation does allow the cow to use fibrous feeds via the volatile fatty acids and 2) nonprotein nitrogen which is incorporated into microbial protein. Thus, ruminants have the ability of utilizing cellulose material which is not digestible by direct competition for the same feed sources by the two types of animals. There has recently been much attention given to refining our knowledge of protein metabolism in ruminants. This is especially true for factors which alter rumen fermentation and increase quantity and/ or quality of protein reaching the small intestine for digestion and absorption. Dairy cattle early in lactation are unable to consume suffi- cient dry matter to support maximal milk production (Bines, 1976; Clark and Davis, 1980; Kuber and Kung, 1981) and body fat is mobilized to provide significant amounts of energy for productive purposes during this period. However, mobilization of body protein is minimal and its rapid depletion occurs during periods of negative nitrogen balance (Botts et al., 1979; Paquay et al., 1972; Swick and Benevenga, 1977). Thus, quantity and/or quality of protein reaching the small intestine might reduce peak milk yields in early lactation. Peak milk and persistency of production are influenced by nutri- ent intake and body stores. If persistency is normal, peak milk produc- tion is the major determinant of total milk yield for the lactation. Broster and Strickland(1977) showed that every kg increase in peak milk production results in 200 kg increase in total milk yield during the entire lactation. Assuming cows calve in good body condition and a balanced ration is fed, protein becomes the most limiting nutrient in early lactation. Increasing protein early in.lactation has not always increased milk production, suggesting that amount and type of protein as well as genetic ability of cows and other factors might govern responsiveness. Systems have been identified by Huber and Kung (1981) which quantitatively and qualitatively define nutrients for lactating dairy cows. Amino acid requirements for ruminants are unknown due to extensive rumen degradation of dietary protein andtflmxcontri- bution of microbial protein at the duodenum. Oldham and Tamminga (1980) point out that increasing the supply of amino acids to the duodenum is only important if there is a con— comitant increase of amino acids at the tissue and a change in animal performance. Huber and Kung (1981) also stated that quality as well as quantity of amino acids reaching tissue of high yielding cows must be improved. The quantitative requirements of protein and amino acids for high milk production are unknown due to problems in sample collec- tion and partitioning of microbial and feed protein at the duodenum. Sutton and Oldham (1977) reported a 10% animal variation in duodenal digesta flow between animals. These workers suggested that in order to obtain a significant difference (P < 0.05) between animals in two out of three experiments, the following was required: four animals/ treatment (4 x 4 latin square) for a 20% difference and six animals/ treatment (6 x 6 latin square) for a 15% difference. Problems in partitioning microbial and feed proteins at the duodenum have been thoroughly reviewed elsewhere (Hume, 1976). Rumen bacteria can supply considerable quantities of microbial protein to meet the cows protein requirement. Since these bacteria cannot distinguish the source of ammonia nitrogen needed for their protein synthesis, it is logical to feed bacteria inexpensive sources of nitrogen (from nonprotein nitrogen) while maximizing the amount of plant protein reaching the small intestine. The results of this study describe: 1) methods to decrease rumen nitrogen degradation 2) milk production from high producing cows fed nonprotein nitrogen and rumen undegradable protein 3) the digestion and flow of nutrients in various segments of the digestive tract when cows are fed nonprotein nitrogen and rumen undegradable protein. LITERATURE REVIEW Protein Digestion in the Rumen Digestion of protein to peptides and amino acids is prerequi- site to utilization by animals and microorganisms. In the rumen, protein digestion occurs primarily via bacterial proteolysis and pro- tozoal engulfment. Proteolytic activity in the rumen was first described by Sym (1938). Casein was rapidly degraded although he found little protease in rumen fluid. Blackburn and colleagues (1965) have also studied rumen proteolytic activity under ig_!i££9- and in vivo conditions and reported rapid hydrolysis of casein and other proteins within 48 hr. These workers showed that B. amylo- philus discharged 20% of its total proteolytic activity into an in_ y_i_t_r_o_medium. Although extracellular proteases are able to hydrolyze protein, proteolytic activity in cell-free rumen fluid is low (Isaacson and Owens, 1971). Allison (1969) suggested that this was not surprising since extracellular enzymes are primarily associated with gram positive bacteria and rumen bacteria are predominantly gram positive. Bacterial proteases are cell bound, but are closely associated with the cell surface thus providing easy access to substrates. Since contact between enzyme and substrate is necessary, surface exposure may alter protein digestion. Thus, rupture of the herbage cell mem- brane or ingestion of large quantities of feed protein may limit protease activity for a few hours. It is generally agreed that rumen proteases are not subject to metabolic control, and are constitutive enzymes (Allison, 1970; Hogan and Hemsley, 1976). Proteins, peptides or amino acids have no effect on production of proteases. Proteo- lytic activity, however, is directly related to cell mass, making it difficult to distinguish effects on the microbes or on their pro— teases. Fbr example, Hume (1974) and Nugent and Mangan (1978) did show an increase in proteolytic in the rumen when protein in the diet increased. Nikolic et al. (1975) reported that urea spared protein from ruminal degradation, but others suggested that it did not (Orskov, 1979). Temperature and pH can alter protease activity, but tend to be relatively constant in the rumen. Blackburn and Hobson (1960) reported optimal proteolytic activity between pH 6 and 7. Hence, activity is primarily related to enzyme and substrate concentration, time of asso- ciation, and inherent differences in protein degradability. In terms of the latter, rate of protein digestion by rumen bacteria was positively correlated with protein solubility in salt solutions (Hendrickx, 1963). Peptides and amino acids are products of protease action. Rumen concentrations of these moieties tend to be low. Allison (1970) estimated the pool of free amino acids to be 2.6 to 65 xlo-SM. Bac- teria appear to preferentially take up peptides over free amino acids via a translocase or permease system. Di- and oligo- peptide translocases both require a free carboxyl group while the latter can transport peptides lacking carboxyl groups (Hogan and Hemsley, 1976). Prins et a1. (1979) reported that the net rate of amino acid disappearance from the rumen was increased when peptides rather than free amino acids were used. The translocase system also allows inexpensive nitrogen uptake compared to active transport re- quired for amino acid uptake. Although peptides and some amino acids are taken up by bacterial cells, most of the nitrogen is re- turned to the rumen fluid as ammonia nitrogen prior to incorporation into microbial protein (Nolan et al., 1973). The added cost of amino acid synthesis from ammonia and carbohydrates has been reported to be relatively small (Forest and Walker, 1971). Within the bacterial cells, peptides are extensively attacked by a wide range of peptidases, resulting in release of amino acids. Upon completion of peptidase reactions, amino acids may be subject to deamination. Extensive amino acid degradation may be due to the absence of a transport mechanism for amino acids from cytoplasm to the external media (Pittmanenzal., 1967). The optimalpH for deaminase activity appears to be between 4.5 (Lewis and Emery, 1962) and 7.2 (Chalmers, 1969). In ig_gi!g_and in_zi££g_studies by Chalupa (1976) free amino acids were utilized to establish apparent degradation rates of some essential amino acids. Prins et al. (1979) demonstrated that net rates of in_vi££g_disappearance of amino acids by mixed rumen micro- organisms was dependent on diet of the inoculum donor, form of amino acids (free or peptides), and presence or absence of energy sources in the incubation media. High rates of net disappearance of glycine, methionine, valine, and histidine from peptides were found, but vmax values were low for free amino acids, suggesting that pep- tides were preferred. Deamidases have also been found in rumen bac- teria and can liberate ammonia from amides (Warner, 1956). Like bacteria, protozoa do not excrete extracellular proteases. Protozoa can engulf protein and peptides and are able to degrade pro- tein to ammonia at a pH optimum of 6.5 to 7.0 (Hogan and Hemsley, 1976). Little if any amino acid uptake occurs in protozoa (Coleman, 1967). Warner (1956) suggested that protozoa could contribute 50% of the rumen proteolytic activity. Deamination of amino acids by protozoa is likely because rumen ammonia levels are higher in ani- mals containing protozoa, compared to defaunted controls (Purser and Moir, 1966). It is uncertain whether proteolysis or deamination is the rate— limiting step in rumen protein degradation. Data show an increase in free amino acids after a meal, which suggests that proteolysis occurs faster than use of free amino acids (Leibholz, 1969). Degradation of protein within the rumen is random, and reasons for extensive break- down are unknown (Tamminga, 1979). Prins (1977) described a strain of rumen bacteria requiring amino acids as a source of energy and suggested that ATP may be generated. However, under anaerobic con- ditions, proteolysis cannot yield ATP (Tamminga, 1979). Another degradation route which may be more important is the deamination of amino acids, followed by decarboxylation of the alpha— keto acid which yields one ATP per decarboxylation (Prins, 1977). Lack of an amino acid transport mechanism from cytoplasm to media has already been mentioned. Recent estimates of protein degradation in ruminants show con— siderable variation between feedstuffs. For example, the protein in soybean meal and haylage is 65-75% degraded in the rumen, but that from corn gluten meal is only 45%. Protein degradation will be dis- cussed in greater detail in a later section. Factors Affecting Microbial Growth and Production The full benefit of microbial fermentation can only be realized when growth and turnover of the microbial population are maximized. This can be achieved when requirements for carbon, energy, nitrogen, and other limiting elements are met. Energy and carbon are provided from fermentation of carbohydrates resulting in ATP, while ammonia, and to a lesser extent amino acids, provide nitrogen for protein syn- thesis. Energy may be the first limitation to microbial growth in the rumen since anaerobic fermentation limits ATP yield to 3.5-4.5 moles(m) per fermented hexose equivalent (Baldwin, 1970). Using batch cultures, Bauchop and Elsden (1960) related microbial ATP production and growth in defining YATP as the grams of microbial dry matter produced per mole of ATP. These workers suggested that YATP was a constant 10.8. However, Hespell and Bryant (1979) have calculated ATP expenditures for synthesis of microbial cells from preformed monomers and suggest theoretical values for YATP of 27 to 32. Indeed, Stouthamer and Bettenhauser (1973) showedthat the efficiency of ATP utilization for growth in continuous culture systems at steady state was determined by specific growth rate and maintenance requirements. Observed YATP values determined in pure bacteria and mixed cultures of rumen bac- teria range from 8 to 23 (Satter and Slyter, 1974; Isaacson and Owens, 1975; Hespell and Bryant, 1979). Hespell and Bryant (1979) suggest changes in cell composition, nutrient availability and transport cost account for the lower than theoretical values of YATP found in most studies. In order to maximize microbial synthesis, energy from rumen fer- mentable organic matter must be supplied at a rate which parallels the synthetic abilities of rumen microbes (Oldham et al., 1977). Readily available carbohydrates have been more effective than struc- tural carbohydrates in increasing uptake of degraded nitrogen, in_xivg (Offer et al., 1978) and in_zi££g_(8tern et al., 1978). Russell and Baldwin (1978)showedthat rumen bacteria have marked preferences for sugars as carbohydrate sources. McAllan and Smith (1976) found that starch supplied the greatest amount of energy for bacteria, and that high starch diets induced greater bacterial polysaccharide accumulation than soluble sugars (McAllan and Smith, 1974). Better conversion of urea to microbial protein with starch may then be ex— plained by a gradual release of polysaccharides (McAllan and Smith, 1974). The source of starch also appears to affect efficiency of microbial growth as Oldham et al. (1981) found 30% more bacterial nitrogen per kg organic matter digested when barley was substituted for corn as an energy source. McMeniman (1975) reported that efficiency of microbial protein synthesis was 33 g nitrogen/kg rumen digested organic matter (Rpom) in a forage-type diet vs 22 g N/kg RDOM for animals fed high grain. 10 These results differ from those discussed. However, rumen dilution rate tends to be faster on forage-type diets, which might parti- ally explain these findings. Microbial growth becomes more efficient as dilution (D) or turnover rate increases. The rumen half-life of a solid material may range from 30 minutes for casein to 48 hours for low quality forage (Sutherland, 1976). On the other hand, the half-life for liquid turnover may be 5-6 hours for sheep on pasture, or 18-20 hours for sheep fed all concentrate diets (Sutherland, 1976). As D increases, specific growth rate (U) also increases, resulting in less ATP used for maintenance and/or efficient microbial growth. Prins and Clarke (1980) reported maximum specific growth rates (umax) for various rumen bacteria ranging from 0.28 h"1 for 1 Lactobacilis _p, to 2.04 h- for S, Bovis. However, only 24% of YATP was used for maintenance when D was increased to 0.12 h-l. Using mixed rumen bacteria, Isaacson et al. (1975) altered D from 0.02, 0.06, and 0.12 h‘1 and found respective YATPs to be 7.5, 11.6, and 16.7. Harrison and McAllan (1980) recalculated data of Isaacson et al. (1975) and showed that when D was 0.02, 65% of YATP was used for maintenance. By infusing artificial saliva intrar ruminally in sheep, Harrison et a1. (1975) increased D from 0.38 to 0.98 h"1 with a 24% increase in YATP. Microbial ecology of the rumen is also drastically changed by D. Hobson and Summers (1972) studied growth rates of various bac- teria, and found that efficiency appeared to always be a function of D. Latham and Sharpe (1975) reported a predominant population 11 of selonnomads and bacterioides in sheep with a low D. An increase in D by addition of minerals, resulted in a decrease in bacterioides and increase in gram variable, chain cocci organisms. At high rates of D, decreases in protozoa and microbial cell lysis may also contribute to the increased microbial efficiency. The slower growth rate of protozoa would limit their numbers at high rates of D (Leng, 1976). Sutherland (1976) reported that when D rate was increased, microbial crude protein was increased 20 to 35% in defaunted sheep due to a reduction in cell autolysis and recycling within the rumen. Nitrogen Requirements for Microbial Growth Assuming sufficient energy and carbon, nitrogen becomes the next major limiting factor for microbial growth. Ammonia is the cen- tral and preferred N compound for synthesis of microbial protein (Bryant, 1970). Tracer studies using N15 suggest 50 to 80% of micro- bial N goes through an ammonia pool (Nolan and Leng, 1972; Mathison and Milligan, 1971). Pilgrim et al. (1970) reported that 63% of bacterial N and 37% of protozoal N was derived from an ammonia pool. The concentrations of ammonia needed for optimal microbial growth has been controversial. Allison (1970) reported maximal growth until ammonia was less than 4.6 mM. These findings are similar to those found ig_vi££g_in mixed cultures by Satter and Slyter (1974). Roffler and Satter (1975) presented in_vivo evidence that rumen 12 ammonia concentration of 3.6 mM was sufficient to obtain max— imum yields of cell protein. Orskbv (1973) could not increase the flow of protein from the rumen of sheep when ammonia was greater than 6.3mM, Hume et al. (1970) also found no increase in rumen protein concentration when rumen ammonia was in excess of 6.4 mM, al- though flow of protein from the rumen was not maximized until 9 mM , In rumen fermentors similar to those used by Slyter and Satter, Bull et al. (1975) showed an increase in microbial protein production until rumen ammonia reached 10 mM. Similarly, Kansas workers (Edwards et al., 1980) demonstrated increased microbial protein pro- duction with rumen ammonia as high as 52 mM. Using cannulated animals, Miller (1973) suggested maximum non ammonia nitrogen (NAN) flow at the duodenum when rumen ammonia was 17 mM . In an original approach, Mehrez et al. (1977) reported that maximum rates of fer- mentation did not occur until rumen ammonia reached 11.4 mM. However, Ortega et al. (1979) found no effect on fermentation rates above 4.0 mM ammonia. More recently, Tamminga (1979) reported that dietary protein of 13.4% was inadequate to sustain maximum microbial fer- mentation of crude fiber, however, no effect was observed on degrada- tion of the nitrogen free extract. In sheep fed semi-purified diets of concentrate, roughage and concentrate, or roughage, microbial protein production did not increase when rumen ammonia exceeded 2.8, 6.9, and 1.6 mM for the three rations (Pisulewski et al., 1981). Wallace (1979) reported similar findings to Mehrez when rumen ammonia was increased to levels considered by some (Satter and Slyter, 1974) as excess. As mean rumen ammonia 13 concentrations increased from 6.1 to 13.4 mM, a 90% increase in rate of degradation of rolled barley, and smaller increases in rates of protein and fiber degradation occurred. Despite low activity of ala- nine dehydrogenase and glutamine pyruvate-aminotransferase, alanine concentrations were increased with greater ammonia. Wallace suggested that bacteria which use ammonia by the alanine pathway require high ammonia for growth and these same bacteria may be responsible for plant degradation. In vitro, Illinois workers (Schaefer et al., 1980) showed ammonia saturation constants of less that 50 uM indicating that microbial growth was 95% maximal when ammonia was 1 mM. These data are much lower than data just discussed. Hespell and Bryant (1979) suggested that ammonia level in the rumen should seldom limit growth of ammonia requiring bacteria based on these constants. They believe that responses to greater levels of ammonia may be indirect such as forma- tion of ammonium bicarbonate causing a higher pH. In practice, rumen ammonia levels may be misleading as a guide for achieving maximal microbial growth, due to normal daily fluctua— tions (Coppock et al., 1976) and formation of microcolonies where ammonia concentrations may differ greatly from that in the surrounding media (Cheng and Costerton, 1980). Although ammonia is the predominant nitrogen source for rumen microbial protein synthesis, peptides and amino acids may also be stimulatory to microbial growth (Pittman and Bryant, 1964; Hungate, 1966), especially for cellulolytic bacteria (Hespell and Bryant, 1979). sauer et a1. (19 75) reported that all amino acid carbon could be synthesized 14 by ferredoxin dependent reductive carboxylation to appropriate keto acids. However, in y_i_v_o_ tracer studies with N15(Salter et al. 1979) indicated that rates of methionine and phenylalanine may limit micro— bial growth on protein-free diets. Isonitrogenous substitution of small amounts of amino acids for urea markedly stimulated rumen mi- crobial growth (Maeng and Baldwin, 1976). Pheng and Baldwin (1976) suggested ureazamino acid N ratio of 3:1 for optimal microbial growth. In explaining these data, Hespell and Bryant (1979) suggested that amino acids may reduce the degree of energetic uncoupling. This is supported by an increase in the ratio of cell yielszFA and YATP with amino acid additions. Ammonia Fixation in the Rumen Ammonia fixation in the rumen has been studied by many workers (Allison, 1969; Erfle et al., 1977; Schaefer et al., 1980). The primary reaction involves the addition of ammonia to an alpha-keto acid. Glutamic acid is formed by reductive addition of ammonia to alpha keto glutarate. The former can then be converted to essential or non essential amino acids by transamination reactions. Addition of ammonia to amino acids to form amides can also occur. Amidic NH can then transform keto aCids to amino acids. Glutamine synthe- tase and glutamate dehydrogenase are the primary enzymes involved in rumen ammonia fixation. At low concentrations of ammonia, glu- tamine synthetase increases 10-fold and facilitates transfer of amide nitrogen from glutamine to alpha keto glutarate. It is of primary 15 importance when ammonia is low since its Km of 0.2 mM is relatively low, which suggests a high affinity for ammonia. Glutamate de- hydrogenase becomes more active as ammonia concentrations increase above 5 to 6 mM. One molecule of ATP is required for every molecule of ammonia fixed via the glutamine synthetase reaction. Schaefer et al. (1980) calculated that 14% of the ATP available for metabolism would be needed to fix all ammonia by this reaction and would therefore re- sult in low cell yields. Enzymes involved in ammonia fixation in the rumen are presented in Table 1. TABLE 1 . Km's FOR AMMONIA OF SOME AMMONIA-FIXING ENZYMESa Enzyme Source Km pH of Determination Alanine dehydrogenase B. subtilis 3.8x10'2M ‘ 8.0 Glutamate dehydrogenase Yeast 5.0x10’4M 7.6 (NADPH) Aspartase B. cadavaris 3.0x10’2M 6.8 Asparagine synthetase S. bovis 4.0x10-3M 7.2 Glutamine synthetase E. coli 1.8x10-3M 7.0 Carbamoyl phosphate synthetase E. coli 1.2x10-2 M 8.0 a Data taken from Barman (1969) 16 Other Essential Growth Factors Branched chain fatty acids of four and five carbons have been reported as essential nutrients for rumen microorganisms. These acids stimulate growth of cellulolytic organisms resulting in in- creased fiber digestion (Bryant, 1973; Dehority et al., 1967; Allison, 1970). Rumen microbes are able to carboxylate fatty acids to form alpha-keto acid analogs of amino acids (Allison and Robinson, 1970). A deficiency of branched-chain fatty acids could occur on low protein diets since they result from normal protein de- gradation. Sulfur has been suggested as an essential element for optimal fixation of ammonia by rumen microbes. The optimum nitrogen to sulfur ratio is between 12 and 15:1 (NRC, 1978). Other minerals have also been identified as essential elements (Durand and Kawashima, 1981). Systems for Estimating Protein Requirements Traditional methods to estimate protein requirements for cattle have used practical feeding trials in which response to increments of protein in the diet has been determined. This method provides direct answers applicable to particular conditions of the trial; however, variable responses occur due to varying management condi— tions, level of production and types of feed. Traditional systems for calculating nitrogen requirements have been based on digestible crude protein or available protein. Shortcomings of this approach 17 are many: 1) Extensive degradation of dietary protein in the rumen and incorporation into microbial protein invalidate employing classical protein systems used in non ruminants (e.g., Biological value); 2) Fecal nitrogen contains large quantities of undigested microbial protein from the rumen and hind gut thus complicating simple calculations of digestibility; 3) There is an inability to differentiate between absorption of various nitrogen sources, such as amino acid nitrogen, ammonia nitrogen or nucleic acid nitro- gen; 4) Relationships between energy intake, protein requirements and rumen fermentation are not considered; and 5) The value of non protein nitrogen (NPN) under various circumstances is not estimated. In ruminant animals, the protein value of the diet is reflected by the total amount and composition of amino acids absorbed at the small intestine. Hence, there have been many attempts to de- velop a reliable system for protein evaluation for ruminants (Waldo and Glenn, 1982; Huber and Kung, 1981). These systems equate protein not degraded in the rumen and microbial protein synthesis with flow of amino acid protein reaching the small intestine for absorption. By determining microbial protein and duodenal NAN, one can estimate undegraded dietary protein. One difficulty is that methods for estimating microbial protein are tentative (Smith, 1975; Theurer, 1982).. Thus, accuracy of estimating undegraded feed protein is also suspect. Despite variability in rumen undegraded protein and the varying contribution of endogenous l8 nitrogen to digesta flow, amino acid composition of duodenal diges— ta is relatively constant (Tamminga and Van Hellemond, 1977). This can be partly explained by the appreciable amount of microbial protein and its uniform composition. Waldo and Glenn(1982) have discussed the assumptions made with various protein systems. Microbial crude pro— tein produced per unit of fermented organic matter was similar for the four systems tested, however, digestibility of that protein was variable. The British and French systems assumed 0.56 digest- ibility,while the German and Danish systems were 0.68 and 0.63, respectively. Likewise, digestion of rumen undegraded dietary protein tended to be low for the British and French systems, com- pared to the German and Danish. Verite et al. (1979) compared a number of systems in formu- latzing diets for dairy cows. Crude protein content of a theoreti- cal diet was calculated for different levels of milk production in each system using a nitrogen degradability of 0.66 (see Figure 1). The Burrough's system gave unacceptably low values. The French system was similar to NRC recommendations, except at high levels of milk production where intake was low (early lactation). At low levels of production, the model of Satter and Roffler appeared to underestimate protein needs as rations with 10% protein would limit overall rumen fermentation (Huber and Kung, 1981). The British system generally called for less protein which increased 19 .Aaema ..He 00 mueem>v 00.0 >ufiHHnmomuwmo z :uH3 mHoooe udmumwmao ou wcaouooom mcowumu hufimv mo auououa mosuo .fi ouswfim 359.2558 3: m3. 8...; a 352.2... :5 8 98 e: 3: 3529. ma 8 mu m— ...llulm . a . a .. S . a. as. .3... 239:5 x as. 5:3: 9.22:.» o .. E 22.3.12. I .DQ ‘ I or .5: 6:: o 8:2...er o J or :20 .meo 20 to a lesser extent with increasing production. Requirements during high levels of production and energy deficits do not increase because they are dependent on microbial nitrogen requirements. Waldo and Glenn (1982) have simulated typical rations of thenvarioussystems when nitrogen undegradability was optimized ( Figure 2) . Differences between systems at low production levels were small, 9 to 12.5% protein, and were all apparently below opti- mum requirements for rumen fermentation (Huber and Kung, 1981). Differences at high production levels ranged from 12 to 17% pro- tein. The French system required the most protein because they assumed lowest dry matter intakes. On the other hand, British requirements were low because dxnrassumed lower metabolizable pro- tein requirements for maintenance and milk. In all systems, degradability of dietary protein should be such that little or no ammonia nitrogen is wasted. Treacher (1979) has presented two figures relating this concept to requirements for production. Figure 3 depicts the relationship between milk yield and the proportion of dietary protein required as degradable (RDP) or undegradable (UDP). As production increases, percent UDP in- creases. Figure 4 presents a similar relationship between RDP, UDP, and milk produced throughout the entire lactation. Similar estimates for UDP have been made by Waldo and Glenn (1982) in Figure 5. The metabolizable protein system of Satter and Roffler (1982) is not included because it uses the point of ruminal ammonia accumulation as a benchmark for protein degradability and ..-—— .- “-. UM a! lo 14 .a.—.—-. -_.. 12 Crude protein, 10 / 4P. - ’2/ 2% M /8 Figure 2. 10 20 30 40 Milk, kg/day Crude protein (% of dietary DM) requirement as a funCtion of milk production (kg/day) for five European systems with undegradability at opti- mum. B, British; D, Danish; F, French; G, German; and S, Swiss. (Waldo and Glenn, 1982). 22 50 ‘- -l 100 ' ~90 40 P .0 80 * _(,,170 30-' -60 9' 96 Pediau PROTEIN as .5 " I so RDP uop ------ 20 *- - 40 ‘ '30 10 '- - 20 " - 1O 1 3 1 a i 1 4 10 20 30 40 50 60 KG MILK/DAY Figure 3. The relationship between milk yield and the proportion of dietary protein required as UDP and RDP, as predicted by the ARC model. Vertical lines indicate the commercial range of milk yield (Treacher, 1979). 2000 30 25 1500 mm ":O/Tdil'yN 20 kn/day 1000 - - 15 ”“300 I” \s -I 10 I \ 500 ~ 1’ ~\~_ ’ .......... I, -------- -' 5 I, --- ...... ’ 1 1 1 1 1 1 1 1 s 10 1s 20 25 :0 35 40 STAGE ucnnou “Necks; Figure 4. The dietary protein requirements over a complete lactation for a Friesian cow peaking at 30 kg milk per day, as-pre- dicted by the ARC model. The diagram takes account of body-weight changes and of restricted appetite in early lactation (Treacher, 1979). 40 1: F/: ”E 6 °' 0 E /B/ .2 I G g’///,r”’ "3 I I. :0; /B 33 z s :3 I ': i E l 0 E 101 E? i B C . D ,... .b O 10 20 30 Milk, kg/day Figure 5. Undegradability (%) of dietary crude protein as a function of milk production (kg/day) for five European systems with crude protein at-optimum. B, British; D, Danish; F, French; G, German; and 8, Swiss (Waldo and Glenn, 1982). 24 microbial protein synthesis. Moreover, this system does not re- quire determination of protein degradability on most low pro- tein feeds and does not rely on an estimate of microbial protein synthesis. Whitlow and Satter (1979) recently compared the ability of various systems to predict the flow of amino acids to the intestine, including those of Burroughs et a1. (1975), Journet and Verite (1977), Kaufmann (1977), Roy (1977), and Satter and Roffler (1975). For most rations (low and high protein fed to sheep and cattle) Satter and Roffler best predicted actual flow of amino acid nitrogen to the small intestine. However, in rations fed only to cattle, the French system of Journet and Verite was most accurate (Thble2). Whitlow and Satter did point out that apparent over- TABLE 2 ESTIMATES FOR AMINO ACID FLOW ACCORDING TO DIFFERENT MODELS Rations Fed to Cattle (n = 26) AAFa= 1093 AAF = —144.09 + 1.50 AAF-Burroughs et al. 823 AAF = 28.24 + 0.97 AAF-Journet and Verite 1100 AAF = -33.97 + 1.16 AAF-Kaufmann 971 AAF = -194.77 + 1.42 AAF-Roy et al. 906 AAF = 26.45 + 0.96 AAF-Satter and Roffler 1108 , (Constant Degradation) AAF = 30.04 + 0.96 AAF-Satter and Roffler 1102 (Variable Degradation) AAF = 22.14 + 0.84 Crude protein intake 1280 a Amino acid flow to small intestine measured in g/day. or under-estimations of amino acid flow in certain systems were 25 probably compensated for by lower or higher efficiency of amino acid absorption and use by tissue. Tamminga and Van Hellemond (1977) suggested it was easier to relate total amount of amino acids reaching the small intestine to readily determined characteristics of feed such as crude protein, digestible protein, organic matter, of digestible organic matter. These workers studied relationships where organic matter intake ranged from 4.7 to 14.6 kg/day and nitrogen intake varied from 140 to 430 g/day. They concluded that amino acid flow to the small intestine was more dependent on dietary supply of digestible organic matter than nitrogen or digestible crude protein. The current status of most protein systems is under intensive scrutiny. Overall, these systems better predict nitrogen needs compared to the crude protein or digestible crude protein approaches, but more infor— mation and field testing are needed before accurate prediction of nitrogen requirements of ruminants can be made. Efficiency of Amino Acid Use The absorption efficiency of amino acids from the small in— testine is about 0.6 to 0.8 (Oldham, 1980 ). On the average a greater proportion of essential amino acids are absorbed. Values for early lactation are lacking but are probably closer to 0.8. Although it is difficult to quantitate amino acid absorp- tion, Cripps and Williams (1975) and Weston (1979) have shown greater absorption in early lactation in rats and ewes. Oldham 26 (1980) speculated that absorption efficiency is 0.8 in early and 0.7 during the remainder of lactation. With an estimated trans- fer of absorbed amino acids to product of 0.60 to 0.85, he suggested the net transfer of duodenal amino acid to product was 0.60 for early and 0.46 during the remainder of lactation. Amino Acid Requirements In lactating animals the mammary gland extracts large amounts of amino acids for synthesis of milk protein. However, the animal still has a maintenance requirement that must be met which includes amino acids as precursors for gluconeogenesis. Estimates of nitrogen requirements for maintenance are con— flicting. When Verite et al. (1979) summarized maintenance re- quirements of the various systems, values ranged from 100 to 395 g of alpha-amino nitrogen x 6.25 needed to be absorbed daily from the intestine. Tamminga and Oldham (1980) stated these differ- ences were due to differences in definition of "alpha-amino nitro- gen absorbed" from the small intestine and much of the disparity was due to inclusion of metabolic fecal nitrogen and its defini- tion. A substantial proportion of amino acids required for main- tenance are metabolized in the gut and the liver. There appears to be no estimates for dairy cows of amino acids metabolized in the gut wall. Tagari and Bergmann (1978) reported that a substantial part of most amino acids absorbed from the 27 small intestine of sheep were metabolized in the gut wall, but the former study showed no preference for either essential or non- essential amino acids. When a 15.6% protein diet was fed, 67 and 71% of the essential and non-essential amino acids were meta- bolized in the intestinal wall. Tamminga and Oldham (1980) cal- culated that increasing the amino acids absorbed from the small intestine of sheep from 2.8 to 4.5/kg w0.75 per day caused a de- crease in gut wall metabolism from 0.71 to 0.52. If dairy cattle absorbed 2 to 3 times this amount of amino acids, then a much smaller proportion would be metabolized in the gut wall. Amino Acids as Energy Sources Amino acids may be converted to glucose during either a shortage of glycogenic precursors, or a surplus of absorbed amino acids. A discussion of amino acids as energy sources was reported by Lindsay (1980). Clark (1975) showed preferential use of NEAA for gluconeo- genesis, although essential amino acids could be used. Of the total amino acids present in the duodenal digesta, 16% are essential and glucogenic while 15% are essential and partially glucogenic (Tamminga, 1975). Since differences in amino acid absorption are relatively small, no more than 25% of amino acids are used for gluconeogenesis could be essential. Branched chain amino acids and lysine are not available for gluconeogenesis because they are ketogenic or partially ketogenic. During early lactation when glucose is limiting, amino acids 28 may be used to meet glucose requirements. Bruckental (1980) suggested that in cows producing over 30 kg milk/day less than 5% of the glucose supply was from amino acids. Oldham (1978) pre- sented evidence from dairy cows that protein conversion to end pro- duct was done 0.65 to 0.85 efficient; thus 0.15 to 0.35 of absorbed protein might be available for gluconeogenesis. Assuming a major part of surplus protein was oxidized and that 55 g of glucose was synthesized from 100 g of protein, high yielding cows would obtain less than 2% of their required glucose from protein. After maintenance needs are met, protein is used for milk protein production with an efficiency of 0.60 to 0.75 (Tamminga and Oldham, 1980). Oldham (1980) reported that increased energy supply increased efficiency of protein utilization. Clark et al. (1978) calculated that amino acid nitrogen was utilized by the mammary gland with an efficiency of 0.91. Part of the NEAA excreted in milk protein are synthesized de novo in the mammary gland. A major portion of the NEAA nitro- gen is from essential amino acids, primarily arginine. Estimates for EAA nitrogen in milk range from 0.6 to 0.71. This would suggest that for a given output of EAA in milk, a surplus of about 50% must be extracted from the blood. Pfgtein Needs for Lactating Cows Revisions in suggested protein allowances for lactation have recommended higher protein intake per unit of milk (NRC, 1971; NRC, 29 1978). These increases generally were supported by research re- sults showing greater milk yields at higher protein percentages (Clay et al., 1978; Edwards et al., 1980; Gardner and Park, 1973; Grieve et al., 1974; Murdock and Hodgson, 1979; Sparrow et al., 1973; Van Horn and Zometa, 1978) but not always (Chandler et al., 1976; Foldager and Huber, 1979; Patton et al., 1970, Van Horn et al., 1976). Where milk yields were increased by increasing crude protein concentrations above 13 to 14% of the ration dry matter, total energy (Clay and Satter, 1979; Claypool et al., 1980; Cressman et al., 1980; Davis, 1978; Grieve et al., 1974; Murdock and Hogdson, 1979; Sparrow et al., 1973; Van Horn and Zometa, 1978) or concentrate (Edwards et al., 1980) intakes usually were improved. Van Horn and Zometa (1978) summarized 13 experiments where soybean meal was used to increase protein in rations for lactating cows and concluded that higher protein had much of its effect through stimulation of energy intake. Foldager and Huber (1979) demon- strated that when high protein did not increase feed intakes, milk yields of early lactation cows (3 to 20 wks postpartum) fed 13% crude protein were equal to those fed 16%. Others have found little response in milk yields to dietary protein in excess of 13% when dry matter intakes were similar to lower protein control diets (Chandler et al., 1976; Clay et al., 1978; Van Horn et al., 1976). Satter and Roffler (1975) recommended that average ability cows during the first 4 months after calving receive 16% protein of only 30 plant origin. A decrease to 12.5% was proposed for the remainder of the lactation durihg‘which NPN might be included. As protein content of the ration increases, response in milk yield to higher protein diminishes (Claypool et al., 1980; Edwards et al., 1980; Satter et al., 1979). Satter et al. (1979) suggested that the amount of dietary protein for lactating cows should depend upon protein price relative to profit from the in- creased milk resulting from a given increment in ration protein. When the cost of soybean meal was 36¢, milk 26¢, and shelled corn 11¢ per kg, recommended crude protein for high producing cows early in lactation did not exceed 16%. Changes in prices of feed protein, feed energy, or milk will alter the percent of dietary pro- tein that is most profitable, but several recent studies showed that income over feed costs was maximal for high yielding cows at 14 to 16% (Claypool et al., 1980; Satter et al., 1979). Satter's (1982) recommended protein requirements relative to feed costs are presented in Table 3. Higher levels of protein were not profit— able due to the diminishing increase in milk yield with increase in ration protein. Similarly, Van Horn (1982) has suggested that 14% CP is optimum in dairy rations and that the like- lihood of higher protein being needed was remote. As mentioned earlier, Broster (1977) has estimated a 200 kg increase in total milk yield for every -1kg increase in peak lactation. The typical response to increasing protein in the diet (Table 4) would be drastically different in mid to late lactation where nutrient needs 31 TABLE 3 RECOMMENDED LEVELS OF PROTEIN FEEDING FOR.MAXIMUM PROFIT Level of Milk Peak Daily Days of Lactation Production for Milk Total Lactation Production 0-100 100-200 200-305 (kg) (kg) (% of total ration dry matter) Relatively low protein prices2 <5,450 <27 14.53 12.51 12.5 5,450-6,800 31 16.03 13.0 12.5 6,800-8,18O 38 17.53 14.53 13.0 >8,180 >41 19.03 16.03 14.53 Relatively moderate protein prices <5,450 <27 13.0 12.5 12.5 5,450-6,800 31 14.53 12.5 12.5 6,800-8,180 38 16.03 13.0 12.5 >8,18O >41 17.53 14.53 13.0 Relatively high protein prices <5,450 <27 13.0 12.5 12.5 5,450-6,800 31 13.0 12.5 12.5 6,800-8,l80 38 14.53 12.5. 12.5 >8,180 >41 16.03 13.0 12.5 TUnderlined values mean that nonprotein nitrogen can betised to supply all or nearly all of the supplemental protein. 2A5 a crude guide to determine whether protein prices are relatively low, moderate, or high, use the following: Take the difference between the price of 1 lb of soybean and 1 lb of shelled corn and subtract it from the price of 1 1b of milk: If you obtain 6¢ or more, protein is relatively low in price; If you obtain 2 and 6¢, protein is relatively moderate in price; If you obtain less than 2¢, protein is relatively high in price. Example: When milk is 14¢, soybean mean 15¢, and shelled corn 6¢ per lb, then subtracting the difference between soybean mean and shelled corn (15-6, or 9¢) from milk gives 5¢ (14-9 or 50). This is a moderate protein price. 3Situations where relatively resistant protein sources might be advantageous. Feeding resistant proteins would tend to lower the suggested requirement, assuming the resistant protein sources had comparable essential amino acid content. (from Satter, 1982). 32 TABLE 4 MARGINAL CHANGES IN MILK PRODUCTION AND DRY MATTER INTAKE AS A RESULT OF CHANGING RATION PROTEIN PERCENT1 Change in Protein % of kg/Day Increase In} Ration Dry Matter From: Milk DM Intake 10 to 11 1.9 1.4 11 to 12 1.5 0.7 12 to 13 1.0 0.4 13 to 14 0.8 0.3 14 to 15 0.6 0.2 15 to 16 0.5 0.1 16 to 17 0.4 0.1 17 to 18 0.3 0.1 ISoybean mean was the dominant protein supplement, and corn grain and corn silage were the major ration ingredients. Cows were in early lactation, and were capable of producing about 7000 kg of milk per lactation. (from Satter, 1982). 33 would be low. Indeed, Barney et a1. (1981) showed no response to protein level. Switching cows from a basal ration of 16% CP producing 28 kg of milk 19 wks postpartum to either 12, 14, 16, or 18% CP had no effect on milk yields or dry matter intake. In light of these findings and those of Broster (1977), it is the opinion of this author that cows early in lactation should be fed for maximal milk production regardless of feed prices. A differential response to protein was observed for multi- parous cows and first calf cows (Cressman et al., 1980; Roffler et al., 1978). Roffler et al. (1978) reported an increase in milk yields of about 6 kg/day when dietary protein was raised from 12.2 to 16.2%, but no response was noted on first calf cows. Sig- nificantly greater dry matter intakes on high protein were noted for cows but not heifers. Second-calf cows and older responded sim- ilarly. Cressman et al. (1980) confirmed that mature cows and first calf heifers respond differently in milk yields when dietary protein increased above 12%, but age did not affect nitrogen utili- zation, digestabilities, or rumen or blood measurements. The effect of level of milk production and extent of negative energy balance on net amino acid nitrogen need in cows is presented in Table 5. As milk production increases, microbial amino acid ni- trogen cannot meet demands for high levels of production. Thus it is apparent that undegraded dietary protein must be increased as production increases. Systems for establishing. protein requirements have been 34 discussed earlier. TABLE 5 EFFECT OF LEVEL OF MILK PRODUCTION AND EXTENT OF NEGATIVE ENERGY BALANCE ON THE NET AAN NEED IN COWS WEIGHING 600 kg3 Level of ME ME Net AANb Microbial Net AAN’Defi- Production Requirement Intake Required Contribution (g/MJ cit (kg Fan/d) 00/0) (MJ/d) (g/d) (Net AAN g/d) of ME) (2) 0 61.9 61.9 9.7 32.8 0.16 —- 10 110.5 110.5 65.7 58.6 0.59 11 20 159.1 159.1 121.7 84.3 0.76 31 40 256.3 256.3 232.7 135.8 0.91 42 40 256.3 207.8 232.7 110.1 1.12 53 60 353.5 353.5 346.7 187.4 0.98 46 60 353.5 256.3 346.7 135.8 1.35 61 a Orskor, 1980. b Amino Acid Nitrogen. Amount of Protein At excessive protein intakes, efficiency of utilization is re- duced because more protein is available than the host can process physiologically (Broster, 1972). Inefficiencies arise from elimina- tion of surplus urea that results from protein catabolism (Tyrell, 1970). Metabolic abnormalities also have been reported in cows fed more protein than needed (Gould, 1969; Jordan and Swanson, 1979; Julien et al., 1977). Julien et a1. (1977) reported a high inci— dence of downer cow syndrome when dairy cows were fed excessive pro— tein (15 vs 8% CP). These problems were compounded by more abortions, displaced abomasums, and milk fever. Gould (1969) suggested consumption of too much protein tended to increase anestrus, decrease conception rate, and lower peak milk 35 production. Jordan and Swanson (1979) fed isocaloric rations of 12.7, 16.3, and 19.3% CF from 0 to 95 days postpartum. Highest pro- tein produced fewest days to first observed estrus and most services per conception. Days open increased from 69 to 96 to 106 for low, medium, and high protein. Treacher et a1. (1976) reported in- creased liver glutamic dehydrogenase and ornithine carbamyl trans- ferase in plasma of cows fed excessive protein. Huber (unpub- 1ished data) recently summarized breeding data from 11 studies in- volving 1,109 cows fed varying protein levels in early lactation. The data in Table 6 would suggest no relationship between protein level and reproductive performance up to 18% CP. Cows fed 19 to 20% CP tended to have more services per conception than when less protein was fed, but also tended to have less days open. Recommen- dations never exceed 18% CP in dairy rations, which was reflected in only 3% of the animals fed in excess of this amount in the 11 studies. Caution should be taken because diets can contain exten- sive amounts of rumen degradable protein leading to high levels of plasma urea and ammonia which may be deleterious. Examples of such diets would be: 1) NPN-treated corn silage and soybean meal or 2) wet haylage and high moisture corn diets. In ruminants, low protein intakes are utilized at relatively high efficiencies because of nitrogen recycling to the rumen as urea and reduced losses through the kidneys (Mercer and Annison, 1976). At the rumen. level, increased retention time of nutrients, depressed intakes, and a lowered capacity to digest organic matter (Huber, 1978; Huber and Thomas, 1971) result from too low protein, 36 probably because microbial fermentation is curtailed (Orskov, 1976). TABLE 6 EFFECT OF PROTEIN LEVEL 0N REPRODUCTIVE PERFORMANCE OF DAIRY COWSc % Protein Number of Services/ a Days Settled Firfit Sold a b Cowsa Conception Open Service (%) Open (%) 9 7 2.10 114.0 ---- ---- 11-13 306 2.14 123.2 35.4 16.7 14-15 259 2.09 121.9 38.8 13.9 16-18 328 1.92 119.3 41.3 13.7 19-20 35 2.34 103.8 ---- ---- a These data only include cows which settled in the trials. b Number of cows averaged for protein levels (ll-13, 14-15, 16—18) was 192, 188, and 256, respectively. c From Huber (unpublished data). Low protein also depresses milk production through decreased lactose synthesis and reduced mobilization of body fat (Orskov et al., 1977). Protein Quality It was thought that quality of protein was not critical in ruminant diets because of extensive modification and synthesis by rumen microbes (Brady, 1976). However, certain protein supplements such as distillers dried grains and soybean meal resulted in higher milk yields than others (Lossli et al., 1958; Lossli et al., 1961; Lossli et al., 1960; Warner et al., 1957), which authors attributed 37 to differences in energy and not protein. Delivery of protein or amino acids directly to the postruminal digestive tract to escape rumen breakdown enhanced milk and milk protein production (Clark, 1975; Schwab et al., 1976; Vik—Mo etal., 1974). Magnitude of re- sponse to abomasal infusion of casein varied with type of ration, protein content of the ration, and production by cows. In early lactation, cows producing over 25 kg/day of milk and fed adequate ration according to NRC (1971) increased milk protein production from infused casein 10 to 15%. Approximately 50% of the increase TABLE 7 THE INFLUENCE OF CASEIN INFUSION ON THE MILK PRODUCTION AND MILK PROTEIN CONTENT OF DAIRY COWSa Author Daily In- Milk Yield (kg/day) Protein Content fusion (g) control infusion control infusion Orskov 300 14.4 17.0 3.07 3.51 et al. (1977) 250 16.8b 19.8 2.52 2.84 500 16.8b 21.6 2.52 2.96 750 16.8b 21.4 2.52 3.15 Broderick 800 30.4 32.0 3.14 3.34 et al. (1970) ' Spires 32.6 34.1 2.86 3.11 et al. (1973) Derrig 400 23.3 24.6 3.08 3.24 et a1. (1974) Tyrell 860 224.0 227.0 --- ---- et al. (1972) Vik-Mo 285 17.0 17.9 3.00 3.09 et al. (1974) Schwab 425 28.5 30.1 2.88 3.04 et a1. (1976) a Kaufmann, 1979. Glucose infusion. 38 was attributable to more milk and the remainder to higher percent protein in milk (Vik-Mo et al., 1974). Infusion of casein re— sulted in greater increases in milk protein production than iso- caloric amounts of glucose, suggesting that milk protein synthesis was limited by both energy and amino acid shortage (Vik-Mo et al., 1974). The influence of casein infusion on milk production and milk protein content of dairy cows is presented in Table 7. Specific amino acids identified in the lowest supply rela- tive output in milk were methionine, lysine, threonine, and phenyl- alanine (Clark, 1975; Schwab et al., 1976; Vik-Mo et al., 1974). The former three (in that order) were first limiting amino acids of microbial protein to support nitrogen balance in prowing lambs (Hatfield, 1971). Wisconsin workers (Schwab et al., 1976) infused free amino acids in various combinations into the abomassum of lac- rating cows and showed that the combination of methionine and lysine gave the greatest response of production. In contrast to plant proteins, the an no acid composition of rumen microbial protein is constant unde. a variety of nutri- tional regimes (Bergen et al., 1967,1968; Hungate, 1966; Purser and Buechler, 1966; Syvaoja and Kreula, 1979). Protein Degradation in the Rumen There has been a concerted effort to develop methods for minimizing degradation of protein in the *umen through a selection 39 of feedstuffs. Variation in distribution of amino acids in in- soluble and soluble fractions of plant protein supplements (Macgregor et al., 1978) is an important finding because response to undegraded protein depends on the pattern of amino acids supplied for post ruminal absorption. Hence a protein source may contain an abundance of needed amino acid, but if that amino acid is de- graded disproportionately compared to others in the rumen, post- ruminal absorption may be minimal. A simplified diagram of nitro- gen metabolism is presented in Figure 6. Form of Nitrogen Dietary nitrogen often is divided into its protein and NPN components. The latter includes free amino acids, peptides, nu- cleic acids, free ammonia, ammonium salts, urea, biruet, and ni- trates. Also, dietary nitrogen is separated into insoluble and soluble fractions. Soluble nitrogen is that extracted by a neu- tral buffer with low ionic strength (Wohlt et al., 1973) and the insoluble nitrogen often is calculated by subtraction of soluble from total nitrogen. Estimates of nitrogen solubility for various feeds have been reported (Crawford et al., 1978; Crooker et al., 1978; Wohlt et al., 1973). Even though nitrogen solubility of a protein and its degradability in the rumen are related (Hendrickx, 1975), they do not always equate to one another. For example, nitrogen solubility of soybean meal is about 15 to 20%. However, the nitrogen in soybean meal is probably 65 to 80% degraded in the 40 Excess Excreled ' DIETARY . ' PROTEIN - Amino Acods Absorbed q—___—., RUMEN LOWER GUT Figure 6. Simplified diagram of nitrogen pathways through the rumen. RDP = rumen degradable protein; UDP = undegraded dietary protein; NPN = non-protein nitrogen (Treacher, 1979). 41 rumen. Thus, it is apparent that solubility may not be the best way to estimate undegradable protein in dairy rations. Regardless of the shortcomings of a nitrogen solubility in— dex, its usefulness was demonstrated by significant increases in milk production when diets were formulated for lower protein solu- bility (Braund et al., 1978; Aitichison et al., 1976; Majdoub et al., 1978). Majdoub et al. (1978) fed two levels of protein (13 and 15%) and two levels of soluble protein (22 and 42%) to lactating cows. Milk, milk fat, milk protein, and solids not fat were great- est when cows were fed a diet that contained 15% CP with 22% sol- uble nitrogen. Of interest was the fact that cows fed 13% protein and 22% soluble nitrogen produced more milk than cows fed 15% pro- tein but with a solubility of 42%. After correlating several laboratory methods for predicting degradation of protein for nine classes of feeds with beef cattle grains, Poos et al. (1980) concluded that nitrogen solubility esti— mates were of questionable validity, that sampling time was criti- cal for predicting from rumen dacron bag measurements, and that assay with fungal protease was the most accurate technique. The latter gave correlations with grain which exceeded 0.92 for all the times determined. Feed Storage and Processing Storage and processing of feeds greatly affects nitrogen utilization by ruminants. Ensiling increases soluble nitrogen to 42 40 to 60% of the total nitrogen in corn silage (Huber et al., 1973), to similar concentrations in low moisture haylage (Sutton and Vetter, 1971) and to over 70% in high moisture haylage. These sol- uble nitrogens are 2 to 3 times greater than in the fresh crop. Ensiled grains also increase in soluble nitrogen. Jones (1973) reported that 42% of the total nitrogen was soluble in high moist- ured shelled corn compared with 25% in dried corn. Additions of ammonia (Bergen et al., 1974; Buchanan-Smith, 1980; Huber et al., 1979; Huber et al., 1973; Waldo et al., 1980) and formaldehyde (Waldo et al., 1973) reduce proteolysis of ensiled forages. Har- vesting too dry for good compaction, filling silos too slowly, or inadequate structures often causes excessive heating of forages (Thomas, 1982). Heat damage in forages measured by increased acid detergent insoluble nitrogen, results in less protein and energy for microbial and host needs. In excess of 40% of the nitrogen in haylage can be made unavailable due to excessive heating during storage. Grinding, pelleting, rolling, cracking, and micronizing of feemsnmy affect protein utilization through altering rates of pass- age, degree of rumen degradation, and microbial protein synthesis (Osbourn et al., 1976; Thompson, 1972). Heat Treatment Controlled heating might decrease nitrogen solubility of for- ages (Beever et al., 1971, 1975) and grains (Glimp et al., 1967, 43 Nishimuta et al., 1972, 1974; Sherrod and Tillman, 1962; Tagari et al., 1962) without substantially diminishing overall protein a- variability. The heat causes carbonyl groups of surgars to com- bine with free amino groups of proteins in the Maillard reaction (Bjarnason and Carpenter, 1969). These linkages are more resistant than normal peptides to enzymatic hydrolysis. Even in the ab- sence of ssugars and carbohydrates, extensive heating causes un- natural amide bonds to form between the free amino groups of lysine and carbonyl groups of proteins (Bjarnason and Carpenter, 1969). Rakes et al. (1972) roasted soybeans at 118°C for 3.5 minutes and noted slightly greater milk production for cows fed the roasted than raw beans, but the difference was not significant. Block et al. (1980) also reported that cows fed heated soybeans produced slightly more milk than those fed unheated beans. There was, however, a significant depression in milk fat for the group fed heated (2.52%) compared to unheated beans (3.50%). A summary of trials feeding heat treated soybean or soybean products is presented in Table:8. Soybean meal (Ahrar and Schingoethe, 1979) or whole soybeans (Kenna and Schwab, 1979; Mielke and Schingoethe, 1980; Schwab et al., 1980; Smith et al., 1980) were heat processed in a cooker-extruder and fed as the main pro- tein supplement to cows. For two early lactation studies (Mielke and Schingoethe, 1980; Smith et al., 1980), milk yields on extruded soy significantly exceeded controls (means of 32.9 vs 29.5 kg/day); but for the other trials, advantage for heat treated protein was 44 .coaumuummmwo mafia Scum muHSmmm n .oouumum ucoafiuooxo soLB msoo pom coaumuoma :H mxwoB mmmuwe< m 2mm mamonzom 2mm osmm mammnmom me mamonhom mqmonhom mamonhom mwxmaw 2mm mammnkom 8.0m ~.6~ meaHme aa< m.e~ o.6~ m A w.~m e.om n v mu< meme 666666x6 6.6N 6.6m a .6eu6omeee6m e 66eu< Heme 666:06x6 H.6N m.m~ a .6e66omeee6m a 6xa6ez 66666; £6656 e.m~ o.m~ wens Name ..H6 66 6656a6u6z 66666: has e.mm N.em m Name .66eem 6 wees 6666660 o.me 6.m2 e Name ..H6 66 6636a meme 666806x6 w.om m.6~ m .6e66omaesem e 6xe6ez UNMXHfiE fiwmmmuowfi .6666e 666=eex6 o.mm 6.mm m owed ..H6 66 emseem ummMHHE wwwmwhuww .6866e 666:66x6 m.em e.em m mama .eeseum a 6:86x UNMXHHE vwmmmhuwv .6e66e 666:66x6 6.em m.mm m Emma ..H6 66 xeoem 66x6H6 66666e66 w.om 6.~m N owed .xeeeo 8 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5.655 6.665 6.655 6.665 6.66 65 .56666656666 6. 5.66 6.66 5.66 5.66 6.66 5.66 6.56 566565 .555: 6555 66566666 111 6.66 6.66 6.56 6.66 5.66 6.66 5.66 566565 0055: u808u0055 111 6.56 6.56 5.56 6.56 6.56 6.56 6.66 566565 65552 555656 66 266166 66166 66166 266166 65166 66166 566166 6666666665 55 65 55 5 6566666 .N 58085500x0 .0005800 u80500050 8050 8505050 00 058808< 085050> 000 0300 no 805u5000800 055: 080 00805050500 .8655080050 0552 .05 0508008= 5:00:00 n 5050 50805580>800 w .5008 8005000 005000100 .000550 8500I0580880I0< .5008 8000000I200 .000550 8500I000 .000050 00 N05 080 05 50 8002 0 .5 6566666 6666 655653 666666 266u6< 666 66-60 .266u60 66 66626 555050 555 x 0050 .05005080 050550>00 05 8055000050 0558 5808500551050 505 005005000 a w.N55 0.mm m.mm w.5m mmlm< N.w05 N.0m m.¢m 0.5m Emmlmo 505.v0v 5005050 50805580>80080 .0> 50805580>800 w.N55 0.mm m.mm w.5m mmlm< w.555 «.mm «.mm 0.5m mmlmo 565.A60 66666556 66 6505 w.555 «.mm «.mm 0.5m mmlmo 5.665 5.66 6.66 6.56 266-60 Amo.v0v 005002 8000000 50 000W 5.M55 0.mm m.mm w.5m 005 0.w05 0.0m n.0m 0.5m 005 0.00 0.5m 0.0m 0.0m 55 6566. 6 .55 .6> 65 556. 6 .55 666 65 .6> 556 5 .6566666 00805050500 0580850055 0050050< 580850055 58085005H|050 8055 56665656 6656666666 555: .N 5808550050 .0005800 580500050 8055 8505050 085050> 000 0300 50 0550000 8055000050 055: 50 0500550 8502 00 8005500800 .05 0500.15) affect milk yields (35.4 vs 35.9 kg/day; Table 13). In comparison, cows fed CS-HS at 14% CP had milk yields (35.1 kg/day) equal to those fed the conventional <0 .005500 580850055 085500 550503 0005 080 00000050 0558 00050>0 80 00000 .500050 002 8050 00505005000 .005080 508 u 025 .5008 8005000 005005100 .000550 85001058088010< .5008 00050001200 .000550 85001000 66.5 65.5 66.5 65.5 56.5 65.5 66.5 65 565500 050565550 61 65- 55- 55- 65- 6- 66- 65 .66665 656563 6666 656 666 666 666 666 566 566 65 6666562 6666 6.+ 6.51 6.5- 5.5- 6.6- 6.1 5.6- 6666566 62 5.66 6.66 5.56 6.66 6.66 6.66 6.56 6665566: 058080550000 02 6.66 6.66 6.66 6.66 6.56 5.66 5.65 66655660 656665 62 5 566166 66166 66166 066166 66160 66-66 266166 6666666665 55 65 55 5 6566666 .0 5808550050 .0005000 580500050 8050 8505050 000550> 000 0300 00 8055000050 0552 00 0080505000 080 550503 0000 080 50080550000 080 000585 005080 502 005085500 .05 05008 111 consumed close to NE requirements and tended to lose least weight during treatment. Body weight losses were greatest for 11 CS-SBM and least for 17 CS-SBM. Cows fed 17 AS-HS produced the most milk and lost the most weight of groups fed 17% protein. Orskov et al. (1977) have observed similar findings, and Robinson et al. (1974) suggested that at high energy intakes, increased protein may favor the partitioning of energy towards milk rather than tissue stores. Production Efficiency Because of low DM intakes for cows fed 11 CS-SBM ration, they were efficient in conversion of feed to milk; however, the 14 CS-HS group was most efficient, and 17 CS-SBM was the least. Rumen and Blood Parameters There were no significant (p>0.10) differences due to hour of sampling for rumen ammonia-nitrogen (RAN; Table 20). RAN increased (p<0.05) as protein percent increased. Cows fed 11 percent pro- tein had RAN less than estimates needed for optimal microbial pro- tein synthesis (Satter and Slyter, 1974). In general, cows fed 14 and 17% protein had RAN's comparable to suggested optimal con- centrations (5 to 25 mg/dl). Within protein levels, cows fed CS-HS tended to have lowest levels of RAN, suggesting lower protein degrad— ability in the rumen. Rumen pH values were not significantly (p>0.10) affected by hour of sampling or treatment (Table 21). They were higher than 112 .Amc.v0v 0558005058050 500050 050550050000 055580 5553 0800200 .08055085850500 00 00 8008 055 05 0050> 50000 .0508580 05 8050 8008 055 05 0050> 50005 .5008 8005000 005005100 .000550 85001058088010< .5008 80050001200 .000550 85001000 O O O O O O O 0 mwmum> m 5 00 55 00 0 0m 05 00 0 00 0 0o 0 00 m 0 0 111 0.0 0.0 0.0 0.0 0.0 0.0 0.0 50 111 0.05 0.0 0.05 0.0 0.0 0.0 0.0 50 111 0.55 «.0 0.05 0.0 0.5 5.0 0.0 50 085000015000 05000 111111 565 6a 00 200100 00100 0010< 200100 00100 00104 200100 0580850058 05 c5 55 N 8505050 .0 5808550050 .0005000 580500050 8050 8505050 085050> 000 0300 50 800055521058088< 80800 .ON 050 50000 .0508580 N5 00 8008 055 05 0050> 50005 .005.A0v 580500050 0558005058050 508 0503 000805000500 55. 00.0 50.5 00.5 50.5 00.5 50.0 50.5 000050>< 111 00.5 00.0 00.5 00.5 55.0 00.5 00.5 50 111 00.5 00.5 50.5 00.5 00.5 00.5 00.5 55 111 00.0 55.0 00.0 50.0 00.5 05.0 50.0 50 005000015000 05000 00 200100 00100 0010< 200100 00100 00100 200100 580850059 55 65 55 5 6566656 0.0 58085500x0 .0005000 580500050 8050 8505050 085050> 000 0300 00 00 80800 .5N 0500B 114 expected and near neutral, probably due to salivary contamination during sampling. Plasma urea nitrogen was also not affected (p>0.10) by hour of sampling but increased significantly (p<0.05) with increasing protein (Table 22). Molar concentrations of volatile fatty acids were not different (p>0.10) between hour of sampling, thus, values presented in Table 23 are means of O, 4, and 8 hrs. These data suggest that rumen fermentation was similar for all diets. ~ Molar percentages of butyrate, iso-butyrate, and valerate tended to be slightly greater when cows were fed 17% protein and supports findings of others (Folman et al., 1981). Economic Evaluation Increased milk production when feeding greater amounts of pro- tein is generally beneficial if income over feed costs is increased (Satter et al. , 1979). Table 24 shows an economic evaluation of' treatments based on treatment means. Cows fed 17 AS—HS were most productive and gave the greatest profits. The 14% protein groups ranked second, third, and fourth in profitability. Note that cows fed 17 CS—HS and CS-SBM produced more milk than 11 CS-SBM, but returned less income due to greater feed intakes and cost of additional protein. How- ever, it is still more economical to feed greater amounts of pro- tein if one considers projected milk production and differences in income returned over feed costs. For example, cows fed 17 CS-SBM 115 .500.v0v 0558005058050 500050 050550050000 005580 0553 08002 000 .08055085850500 00 8050 8008 005 05 00500 00000 .0508580 05 8050 8008 005 05 0050> 00000 .5008 8000000 005000100 .000550 850010580880104 .5008 80000001200 .000550 85001000 00. 050.55 050.55 000.05 000.55 000.55 000.55 050.5 000050>< 111 00.05 00.05 00.05 00.05 05.05 00.05 50.0 0.0 111 00.05 00.55 50.05 00.05 00.55 00.05 50.5 00 111 05.05 00.55 00.05 00.05 50.55 05.05 00.5 00 085000015000 05000 1 1 5205 111 1 00 200100 00100 0010< 200100 00100 00100 200100 0580850059 55 05 55 N 8505050 .0 5808550000 .0005000 580500050 8050 8505050 085050> 000 0300 00 8000555010050 080050 .00 050 00 00005085 085000015000 0 0 080 0 .0 00 8002 0 .5008 8000000 005000100 .000550 850010580880104 .5008 80000001200 .000550 85001000 0. 0.0 0.0 0.5 5.0 5.5 0.5 0.5 0505050> 0. 5.5 5.5 0.5 0.5 5.5 5.5 5.5 0505050>005 0.5 0.05 0.0 0.05 0.0 0.0 0.05 0.05 05050500 5. 0.5 5.5 5.5 5.5 5.5 5.5 5.5 mum555=0o05 «.5 5.55 5.05 5.05 m.55 «.05 0.05 5.05 mumao5aoum 0.5 5.00 0.00 0.00 0.50 0.00 5.00 0.00 050500< N 50502 .000> 00 200100 00100 00100 200100 00100 0010< 200100 0580850058 55 05 55 N 8505050 .0 58085500x0 8050 8505050 085050> 000 0300 00 0050< 05500 0555050> 80800 .0005000 580500050 .00 0500 080085 0552 5050 05000 555: 508 non cmuum5o55 5 gang m>5um5mm 05.0 5500500 5000 0000 50>0 080085 05.0 u5508500 maoua5 5550 No.0 05000\00 5000 0000 200100 00100 0010< 200100 mmlmo 0010< 55 05 000100 0580850055 55 N 8505050 .0 5808550000 00 058008< 085050> 0858505800 0805500 00 80550050>0 05808000 .qm 000umm umH coauumm ma\q NH\~ NH\m ~H\m NH\N ma\¢ MH\m ammo vaom w m o w HH w HH mBoo ow mm Hm OOH mm mm on ammo mhmo mn.H NN.N mw.H oo.m wH.N mN.N oq.m aowunmodou \mmua>umm smuH 2mmlmu mmlmo mmlm< 2mmlmu mmlmo mmlm¢ 2mmlmo wunmaummua NH «H HH N camuoum .N ucmEHumaxm .moounom ucmumwmfio Eoum aHmuoum wafihum> pom m3ou mo Mama wcfiwmwum .mN m4m0.10) different between treatments although cows fed diets with ammonia-treated silage (AS) tended to be more productive. There were no significant differences (p>0.10) in milk composition between treatments. Rumen Parameters Nitrogen disappearance of soybean meals used in this study from nylon bags suspended in the rumen are presented in Figure 11. Estimated nitrogen degradability based on 12 hr incubation was 75% for SBM and 40% for HS. Rumen ammonia nitrogen measured at O, 2, 4, and 6 lurs after feeding is presented in Table 29. Peak RAN was observed 2 122 TABLE 27. Composition of Feeds in Experiment 3. a Item DM, Z CP, Z ADF, Z Grain Mixb AS-HS 90.7 21.1 6. AS-SBM 88.9 21.7 6. CS-HS 90.2 23.9 6. CS-SBM 88.7 24.4 5. Corn Silage 35.1 8.1 29. Ammonia-Corn Silage 34.2 12.5 26. Alfalfa Hay 85.0 17.1 36. Complete Ration AS-HS 55.0 17.7 18. AS-SBM 54.4 17.8 18. CS-HS 54.6 17.0 18. CS-SBM 55.0 17.2 17. aAS-ammonia-corn silage, HS-heated soybean meal, SBM-soybean meal, CS-corn silage. bIncludes ground corn, limestone, dical, calcium sulfate, vitamins A--30-40,000 I.U./day, D--3,000—5,000 I.U.lday, E--500 I.U.lday. 123 TABLE 28. Dry Matter Intake and Milk Production of Cows in Experiment 3.8 Treatmentb AS-HS AS-SBM CH-HS CS-SBM SE Dry Matter Intake kb/day 17.6 17.5 17.9 16.9 1.2 Milk, kg/dayc 24.1 24.2 23.6 22.6 1.0 2 BF 3.03 3.47 3.41 3.33 .24 2 cp 2.69 2.91 2.81 3.08 .09 % Solids 11.00 11.21 11.41 11.48 .28 8None of the differences were significant (P>.10). b AS-ammonia-corn silage, HS-heated soybean meal, SBM-soybean meal, CS-corn silage. cMean of days 8 through 14 averaged for 4 periods. 124 Figure 11. Nitrogen disappearance of normal soybean meal (SBM) and soybean meal heated for 2.5 hr at 1400 C from nylon bags suspended in the rumen and fed in Experiment 3. Legend, 1, SBM, normal; 2, SBM, heated. 125 42¢ n=ewom .fifi muswfim VN In- 3 q. zoapmmzozm mo manor mu q b d e P H J 1:- s .- .ON 1 Lac“ BONUHUBAAUSIU NHOOHlIN 126 TABLE 29. Rumen Ammonia Nitrogen (ng/dl) of Cows in Experiment 3.3 Hours After FeedingC Treatmentb 0 2 4 6 Rd SE AS-HS 10.8 19.0 10.9 12.5 13.3ef 4.4 AS-SBM 14.9 25.1 13.1 18.7 17.9e 6.4 CS-HS 9.4 12.7 7.8 9.7 9.9f 3.9 CS—SBM 19.7 18.7 13.7 11.2 15.8e 5.7 aRumen fluid collected via rumen fistula. bAS-ammonia-corn silage, HS-heated soybean meal, SBM-soybean meal, CS-corn silage. cValues are averages from 4 determinations. dValues are averages from 16 determinations. efMeans with unlike superscripts differ significantly (P<.05). 127 hr post feeding for all treatments. As expected, RAN was greatest for cows fed AS-SBM, intermediate for AS—HS and CS-SBM, and least for CS-HS. Rumen pH (Table 30) was not different (p>0.10) between treatments, but lower than in Experiment 2 since rumen fluid collection was made via a fistula in Experiment 3. Molar percents of rumen volatile fatty acids (VFA) were not significantly (p>0.10) affected by treatments (Table 31). Pro- pionate tended to be greatest for cows fed ammoniated silage (AS) while butyrate tended to be greatest for cows fed normal silage (CS). Neutron Activation and Marker Analysis Neutron activation was performed on triplicate samples at the University of Wisconsin Nuclear Reactor Laboratory (UWNRL). The marker ratios of chromium (Cr) and La in duodenal ileal, and fecal contents as percent of their ratios in feed ranged from 74 to 107. Calculated rumen digest- ibility of dry matter, nitrogen, and efficiencies of microbial pro- tein synthesis were questionable when La was used to determine di- gestibility. Dry matter digestibility in the rumen was much lower than expected, and in some instances, thesexvalues were negative suggesting a net gain in dry matter at the duodenum. On the aver- age, nitrogen and non ammonia nitrogen flow to the duodenum were 110 to 130% of nitrogen intake. Microbial efficiencies were 128 TABLE 30. Rumen pH of Cows in Experiment 3.ab Hours After FeedingC Treatment 0 2 4 6 id SE AS-HS 6.39 6.20 6.24 6.38 6.30 .25 AS-SBM 6.49 6.34 6.30 6.54 6.42 .17 CS-HS 6.35 6.43 6.30 6.54 6.41 .23 CS-SBM 6.41 6.33 6.26 6.38 6.35 .17 aRumen fluid collected via rumen fistula. bDifferences were not significant for treatment or hr after feeding. cValues are averages from 4 determinatins. dValues are averages from 16 determinations. eValues were not significantly different (P>.10). 129 TABLE 31. Rumen Volatile Fatty Acids of Cows in Experiment 3.3 Treatmentb AS-HS AS-SBM CS-HS CS-SBM SE Molar, Z Acetate 67.6 66.3 67.8 66.4 1.7 Propionate 21.1 20.1 17.4 18.9 1.2 Butyrate 9.8 9.9 11.3 10.6 .9 Total mmoles/dl 8.6 8.7 9.1 9.3 .7 aAverage of 0, 2, 4 and 6 h post—feeding. Differences were not significantly different (P<.10). 130 also extremely high with some individual values over 100 g N/kg rumen fermented organic matter. These findings are disturbingbut are similar to those found in a number of recent experiments also conducted at the University of Wisconsin using La as a marker. Close inspection of the data show no detectable error in analysis or computation of data. Wisconsin workers (L. D. Satter, person- al communication) have suggested discrepancies in counting of rare earth elements at the UWNRL. Coefficient of variation (CV) for counting of La were larger (8 tolO% CV)than from other rare earth ele- ments (less than 5% CV). However, samples submitted over time show no statistical difference between activation runs. In one experiment, inspection of individual samples collected throughout the four-day collection periods showed marked variation of La attjm: duodenum but not at the ileum or feces. Statistical calculation resulted in theoretical rumen dry matter digestibilities of approximately -10 to 40% (D.K. Combs, personal communication). Possibilities which might explain these findings are: 1) La {is not behaving ideally as an inert marker at the duodenum, 2) digesta flow is not in a steady state because of large dry mat- ter intakes, 3) sampling of liquids and solids from the t-cannulae is not proportional to amounts present, 4) La is absorbed prior to the duodenum and is re—secreted into the lower tract. Total tract dry matter digestibility appears normal when La was used as a marker. These and other data suggest that fecal La was accurately estimated; digestibility. Personal communication with Wisconsin workers (L. Rode) indicate that use of lignin 131 results in rumen digestibility similar to accepted literature values with total tract digestibilities similar to those calcu— 1ated with La. In the present study, lignin was used as a digestibility mar- ker after obtaining abnormal rumen digestion values with La. Co- efficients of variation (CV) for lignin determination in hay and corn silage were less than 5%, while a CV for a grain sample of less than 20% was deemed acceptable since their lignin content of grain was less than 1%. Lignin was determined by the acid deter- gent 1ignin method (Goering and Van Soest, 1970). Sample Collection Visual observations of digesta flow from the duodenal cannula revealed marked variation in apparent liquid to dry matter content. To obtain unbiased samples, sample collection was as follows: 1) all solid material from the cannula was removed, 2) the first 100 ml. of digesta were discarded, 3) complete collection of the next 400 m1 of digesta were saved for compositing. Ileal digesta flows were not always sufficient to obtain a representa- tive sample for compositing. Ileal flows were poorest during the early morn- ing hours (12 AM to 6 AM) suggesting the possibility of some diurnal variation in digesta flow. Dry Matter Flow and Digestibility When La was used as a flow marker, DM entering the duodenum was approximately 16 kg/day which resulted in low average Inunen dry matter digestion (RDMD) (Table 32). Estimates for 132 .wcflumucm wummwfic wo hufiawnaummwfin v .sbamcuamH wafims pmumasoamuo .aficwwa wswma vmumasoamo A .mwmawm ducalmu .Hmme amonhomlzmm .Hmoe amonhom wmummnlmm .mwmafim cMOUIMHcoEEMImHum0wHQ use mo mucmewmm m=OHum> :H cowummwwo cam 30Hm Hmuumz >ua 133 DM flow were reduced 24% to 11 kg/day when lignin was used to calculate digesta flow. Average RDMD was 32.72% when estimated by lignin ratios and was much closer to literature values. Rumen dry matter digestion estimates with La suggest an inhibition of fermentation which was not corraborated by other data in this trial. While some (Muntifering et al., 1981; Fahey et al., 1979) have cautioned against the use of lignin, it is apparent that in our study lignin resulted in more accurate estimates of RDMD than La. Muntifering et al. (1981) reported that lignin was digested in the rumen and intestine and that extent of digestion varied greatly with diet and method of lignin determination. Lambs fed Kenhy tall fescue had 35.2% of ingested lignin digested in the to- tal tract. Of this, 97.2% and 7.3% were digested in the rumen and:hm— testines, respectively, if the permanganate (1011104.) assay was used. By the acetyl bromide solubilization assay (ABSL),tota1 tract digestion of lignin was 29.7% but, only 13.5% was digested ruminally and 86.5% in the small intestine. Lignin digestibility for the same diet was only -0.6 using acid detergent lignin procedure (ADL). Across three methods of determination and three diets, lignin di- gestibility in the rumen, intestine, and total tract were 13.1, 11.7, and 26.2%. Fahey et a1. (1979) also fed various diets to lambs and compared lignin digestibility using the ADL and ABSL methods. They reported a negative correlation between the two methods of -— 0.60 and suggested that the latter method was more 134 sensitive in detecting soluble lignin, which would be degraded by the ADL method. Negative lignin digestibility may reflect artifact lignin formation due to heat damage (Morrison, 1972), incomplete removal of interfering materials during lignin analysis (Van Soest and Wine, 1968), or artifact lignin formed by postruminal formation of non—conjugated phenols that analyze as lignin (Allinson and Osbourn, 1970). In the present study, heat was not used in preparation or analyses of ruminal, duodenal , or ileal contents so that artifact lignin formation due to heat damage did not exist. Moderate heat (55°C for 48hr) was applied to feces and would only.moderate1y increase heat damage. Although Muntifering et al. (1981) showed lignin digestion in the intestine, ou1' estimates of dry matter flow to the ileum and feces using lignin averaged within 10% of flows calculated with La. Because lignin gave lower estimates of DM flow to the duodenum but similar ileal flows, apparent DM digestibility in the small intestine was approximately 31% less with lignin than with La. Dry matter digestibilities in the total tract were similar for lignin and La. Nitrogen Flow and Digestibility Nitrogen intake and a comparison of N flow and digestibili- ty calculated with lignin or La is presented in Table 33. As ex- pected, N flow to the duodenum was 29% less with lignin than with 135 .wcfiumuam wummwfiu mo uuwawnfiummwfiov .EbamnuamH magma pmumadoamou .awcwfia magma woumasoamun .mwmawm cuoolmu .Hmwa :mwnaomlzmm .Hmms Gwmnhom pmummnlmm .mwmafim shoalmfiaoaam1mHummeQ onu mo munmawom m30Num> aw coaummwfin paw 30am cowouufiz .mm mqmufiafinwumowwam .20 Hmwumuomp mopsaoaHo p .awawfifi mafia: poumasuamon .mmeHm cuoolmo .Hmms somehomIEMm .Hmms cmwnhow vmummnlm: .mmeHm cuoolmficosamamooa=v Ebmmson<\amE:m new coaummwfim umuumz oanmwuo 88. 34.8 33.m mm.m w3.m N66\mx .mmumm om. 88.m m~.8 no.6 8N.6 N66NNN .8563H so. 6m.w os.w m3.m om.a sme\mx wfiwmuomuuoUV abamvoan 3N. 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Iuwmua Iumm m HZMZHmmmxm 20mm mmumm Qz< 53-4. Solution should have no precipitate if pH is correct. Add 80 mlll M CaClz. pH can be adjusted with NaOH pellets. Add slowly! This addition causes violent boiling for a few seconds. 8. Precipitate will form if pH is too high. Use HCl to lower. Lanthanum 1. Requires about 0.5 g La intake/cow/day. Calculate requirement. Use Lanthanum Oxide (89.67 Z La). 2. Dissolve 27 g LaO (gives about 24 g La) in 96 ml conc. HCl. Boils violently. 3. Dilute with dist. H20 to 624 m1 H20. 10. 11. 161 PROCEDURE Processing Rumen Fluid For Bacterial Isolate A total of 800 to 1000 ml rumen fluid is needed to obtain 3 to 6 g of dry bacteria. Collect rumen fluid on last two days of collection period at 0, 2, 4, and 6 hr after morning feeding; strain through cheesecloth. Save 50 ml from each sample, acidify and freeze for analysis of VFA and ammonia. Take remaining sample and add 1 m1 37Z HCHO (N0.9Z NaCl) to every 4 ml of strained rumen fluid. Pool samples at end of trial and make 1 composite per animal. Centrifuge at 2,500 - 3,000 rpm (500 x g) for 5 minutes and discard pellet. Repeat. Centrifuge at 20,000 x g for 20 minutes and discard supernatant. Wash pellet with N15-20 ml of 0.9Z NaCl. Centrifuge at 20,000 x g for 20 minutes. Resuspend pellet in distilled water. Freeze until sample can be lyophilized. BIBLIOGRAPHY BIBLIOGRAPHY Abgarowicz, F., N. Grzeszezak-Swietlikowska, and T. Truszynski. 1963. An attempt at evaluating silages of corn with an addition of nonprotein nitrogenous compounds: urea, ammonium sulfate, and ammonia water. (Transl. from Polish.) Agric. Sci. Ann. 81:695. Ahrar, M., and D. J. Schingoethe. 1979. Heat-treated soybean meal as a protein supplement for lactating cows. J. Dairy Sci. 62:932. Aitchison, T. E., D. R. Mertens, A. D. McGilliard, and N. L. Jacobson. 1976. Effect of nitrogen solubility on nitrogen utilization in lactating dairy cattle. J. Dairy Sci. 57:2056. Allinson, D. W., and D. F. Osbourn. 1970. The cellulose-lignin complex in forages and its relationship to forage nutritive value. J. Agr. Sci., Camb. 74:23. Allison, M. J. 1969. Biosynthesis of amino acids by ruminal microorganisms. J. Anim. Sci. 29:797. Allison, M. J. 1970. Nitrogen metabolism in ruminal microorganisms. ln_Phillipson, A. T., ed., Physiology of Digestion in the Ruminant. Oriel Press Ltd., Newcastle upon Tyne, England p. 456. Armstrong, D. C., and R. R. Smithard. 1979. The fate of carbohydrates in the small and large intestines of the ruminant. 38:283. Association of Official Analytical Chemists. 1975. Official methods of analyses 12th ed. AOAC, Washington, D.C. Balch, C. C., and R. C. Campling. 1965. Rate of passage of digesta through the ruminant digestive tract. IN: Physiology of Digestion in the Ruminant. Doughtery, R. W., R. S. Allen, W. Burroughs, N. L. Jacobson, and A. D. McGilliard, eds., Butlerworths, Washington, p. 108. Baldwin, R. L. 1970. Energy metabolism in anaerobes. Amer. J. Clin. Nutr. 23:1508. Barney, D. J., D. G. Grieve, G. K. MacLeod, and L. G. Young. 1981. Response of cows to dietary crude protein during midlactation. J. Dairy Sci. 64:655. 162 163 Barry, T. N. 1976b. Evaluation of formaldehyde treated lucerne hay for protecting protein from ruminal degradation, and for increasing nitrogen retention, wool growth, liveweight gain and voluntary intake when fed to young sheep. J. Agr. Sci. (Camb.) 86:379. Bartley, E. E., A. Davidovich. G. W. Barr, G. W. Griffel, A. D. Dayton, C. W. Deyoe, and R. M. Bechtel. 1976. Ammonia toxicity in cattle. 1. Rumen and blood change associated with toxicity and treatment methods. J. Anim. Sci. 43:835. Bauchop, T., and S. R. Elsden. 1960. The growth of micro-organisms in relation to their energy supply. J. Gen. Microbiol. 23:457. Beardsley, G. L., L. W. Whitlow, R. E. Roffler, and L. D. Satter. 1977. Ruminal degradation of soybean meal protein in sheep fitted with duodenal re-entrant cannulae. J. Dairy Sci. Suppl. 60:66. 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