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Manipulating ration formulations to reduce nitrogen excretion from lactating cows, while maintaining… Dinn, Nelson Edward 1996

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MANIPULATING RATION FORMULATIONS TO REDUCE NITROGEN EXCRETION FROM LACTATING COWS, WHILE MAINTAINING MILK PRODUCTION by NELSON EDWARD DINN B.Sc. (Agr.), The University of British Columbia, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE We accept this thesis as conforming iOj the^required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1996 © Nelson Edward Dinn, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT Two studies were carried out to determine if nitrogen excretion could be reduced through ration formulation, while maintaining milk production in dairy cows. In each study, 18 lactating Holstein cows were randomly assigned to treatment sequences in a 3 x 3 Latin Square design, replicated 6 times. Milk, blood, and rumen fluid samples were taken during the third week of each 28 day experimental period in the first experiment, and during the second and third week of each 28 day period in the second experiment. Total collection of urine and feces occurred during the last 5 days of each experimental period. In the first experiment, total mixed rations were formulated to contain 15.3 (A), 16.4 (B), and 12.3% (C) crude protein. Ration A (control) contained a standard 18% dairy concentrate, ration B was balanced in terms of protein and carbohydrate degradation and rates of passage using the Cornell Net Carbohydrate and Protein System (CNCPS), and ration C was a low protein diet balanced to meet the amino acid requirements of the cows using the CNCPS model. There was a reduction (P<0.05) in dry matter intake among cows fed diets A, B, and C (22.8, 21.8, and 20.8 kg d"1, respectively). Milk production and milk protein output were lower (P<0.05) for the cows fed the low protein diet than for the other two diets, but N efficiency expressed as milk nitrogen as a % of intake nitrogen tended to be greater (P>0.05) for animals on the low protein diet. Blood urea nitrogen values were different (P<0.05) among treatment groups (14.0, 19.1 and 6.8 mg dl"1 for diets A, B, and C, respectively), but blood non-esterified fatty acid levels were unaffected by dietary treatment. Fecal nitrogen excretion was lower (P<0.05) for cows fed diet C than for those fed diet A, but was not different from those fed diet B. Daily urinary nitrogen Ill varied significantly (P<0.05) among the 3 dietary groups (0.154, 0.180 and 0.092 kg d"1, for diets A, B, and C, respectively). Nitrogen balance was not different (P<0.05) among treatments. In the second experiment, total mixed rations were balanced in terms of degradation, rates of passage, and amino acid requirements using the CNCPS model. Rumen-protected lysine and methionine were used to balance amino acid requirements in diets B and C. Crude protein levels in the diets were 18.3, 16.7 and 15.3% CP, for diets A, B, and C, respectively. Diet A resulted in higher (P<0.05) dry matter intakes than diets B and C. Milk production was lower (P<0.05) for cows fed diets B and C than for cows fed diet A (34.2, 32.8 and 32.8 kg/d for diets A, B, and C, respectively) but, milk protein output did not differ (P>0.05) among dietary groups. Nitrogen efficiency, expressed as milk N as a % of intake nitrogen, improved (P<0.05) for diet B compared to diet A, and for cows on diet C compared to cows on diets B or A (25.8, 29.3, and 33.1%, respectively). Blood urea nitrogen values were different (P<0.05) among groups (15.9, 12.9, and 10.0 mg/dl for diets A, B, and C, respectively). Treatment differences (P<0.05) were seen in plasma arginine, aspartate, isoleucine, methionine, threonine and valine concentrations. Plasma methionine levels were (P<0.05) higher for cows fed diets B and C compared to those fed diet A, but no differences (P>0.05) were seen in plasma lysine concentrations. Although there was a significant decrease (P<0.05) in apparent crude protein digestibility among cows fed diets A, B, and C, respectively, urinary nitrogen excretion was decreased (P<0.05) dramatically (0.264, 0.195, and 0.162 kg d"1, for diets A, B, and C, respectively). Fecal nitrogen excretion and nitrogen balance were not affected (P>0.05) by diet. iv The results of these studies indicate that it is possible to maintain milk protein production, make more efficient use of dietary protein, and to greatly reduce waste nitrogen excretion. The addition of rumen-protected amino acids to rations and the precision of the CNCPS model in terms of formulating dairy rations can aid in this regard. V TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS v LIST OF TABLES vii ACKNOWLEDGMENTS ix I. GENERAL INTRODUCTION 1 1.1 NITROGEN LOSSES 1 1.2 AMINO ACID SUPPLEMENTATION 4 1.3 CORNELL NET CARBOHYDRATE AND PROTEIN SYSTEM 6 1.4 SUMMARY 7 1.5 R E F E R E N C E S 8 II. THE EFFECT OF DIETARY NITROGEN LEVEL ON THE NITROGEN EXCRETION OF LACTATING DAIRY COWS 11 2.1 INTRODUCTION 11 2.2 MATERIALS AND METHODS 12 2.2.1 Forage composition and degradabilities 12 2.2.2 Experimental design 14 2.2.3 Diets 14 2.2.4 Sampling 15 2.2.4.1 Feed 15 2.2.4.2 Milk : 15 2.2.4.3 Rumen fluid 16 2.2.4.4 Blood 16 2.2.5 Total collections 16 2.2.6 Efficiency calculations 17 2.2.7 Statistical analysis 18 2.3 RESULTS 18 2.3.1 Feed composition 18 2.3.2 Dietary intakes and milk production 18 2.3.3 Blood composition 19 2.3.4 Rumen fluid 19 2.3.5 Water intake and waste excretion 19 2.3.6 Apparent digestibility of nutrients 19 2.3.7 Nitrogen balance 20 2.3.8 Nitrogen efficiency 20 2.3.9 Energy efficiency 21 2.4 DISCUSSION 21 2.5 R E F E R E N C E S 26 vi III. THE USE OF THE CORNELL NET CARBOHYDRATE AND PROTEIN SYSTEM AND RUMEN-PROTECTED LYSINE AND METHIONINE TO REDUCE NITROGEN EXCRETION FROM LACTATING DAIRY COWS 4 1 3.1 INTRODUCTION 41 3.2 MATERIALS AND METHODS 4 4 3.2.1 Forage composition and degradabilities 4 4 3.2.2 Experimental design 4 4 3.2.3 Diets 4 4 3.2.4 Rumen-protected methionine and rumen-protected lysine 4 5 3.2.5 Sampling 4 5 3.2.5.1 Feed 4 5 3.2.5.2 Milk 4 5 3.2.5.3 Rumen fluid 4 6 3.2.5.4 Blood . . . . . 46 3.2.6 Total collections 4 7 3.2.7 Efficiency calculations 4 7 3.2.8 Statistical analysis 4 7 3.3 RESULTS 4 7 3.3.1 Feed composition 4 7 3.3.2 Dietary intakes and milk production 4 8 3.3.3 Blood composition 4 8 3.3.4 Rumen fluid 4 9 3.3.5 Water intake and waste excretion 4 9 3.3.6 Apparent digestibility of nutrients 4 9 3.3.7 Nitrogen balance 5 0 3.3.8 Nitrogen efficiency 5 0 3.3.9 Energy efficiency 5 0 3.3.10 Fecal amino acid composition 51 3.4 DISCUSSION 51 3.5 R E F E R E N C E S 5 6 IV. GENERAL CONCLUSIONS 73 vii LIST OF TABLES Table 2.1. Nutrient composition (DM basis) of forages 30 Table 2.2. Amino acid composition of the forages (% UIP) 31 Table 2.3. Degradability of the nitrogen fractions of forages in situ (%) 32 Table 2.4. Composition of concentrate mixtures (kg t"1) 33 Table 2.5. Composition of total mixed rations (% DM) 34 Table 2.6. The influence of diet on dry matter intake, milk yield, and milk composition 35 Table 2.7. The effect of diet formulation on blood composition 36 Table 2.8. The effect of diet on rumen fluid pH and volatile fatty acid concentration (mg ml"1) 37 Table 2.9. The effect of diet on water intake, urine output, and fecal output 38 Table 2.10. The effect of diet formulation on apparent nutrient digestibility (%), N efficiency and N balance during week 4 of each experimental period 39 Table 2.11. The effect of diet on energy expenditure and efficiency 40 Table 3.1. Nutrient composition (DM basis) of forages 59 Table 3.2. Amino acid composition of the forages (% UIP) 60 Table 3.3. Degradability of the nitrogen fractions of forages in situ (%) 61 Table 3.4. Composition of concentrate mixtures (kg t"1) 62 viii Table 3.5. Composition of total mixed ration components (% DM) 63 Table 3.6. The influence of diet on dry matter intake, milk yield, and milk composition 64 Table 3.7. Milk composition and milk component yields during weeks 2 and 3 of each experimental period 65 Table 3.8. The effect of diet formulation on blood composition 66 Table 3.9. The effect of diet on plasma amino acid composition (nmolmr1) 67 Table 3.10. The effect of diet on rumen fluid pH and volatile fatty acid concentration (mg ml"1) 68 Table 3.11. The effect of diet on water intake, urine output, and fecal output 69 Table 3.12. The effect of diet formulation on apparent digestibility of nutrients (%), N efficiency and N balance during week 4 of each experimental period 70 Table 3.13. The effect of diet on energy expenditure and efficiency 71 Table 3.14. Diet effect on fecal amino acid levels 72 ix ACKNOWLEDGMENTS I would like to express my sincere thanks to my supervisors Dr. J. A. Shelford of the Animal Science Department at U. B. C. and Dr. L. J. Fisher at Agriculture Canada's Pacific Agricultural Research Center in Agassiz, B. C. for their dedication, encouragement, and assistance in carrying out this research and for their valued remarks on the thesis. Thanks are extended to Dr. R. M. Tait, Dr. R. M. Beames, and Dr. A. Bomke for their comments on the thesis. Appreciation is also extended to M. L. Swift of ProForm Feeds, Inc. for initiating the experiment, to Dr. Marina von Keyserlingk for her ever cheerful encouragement throughout the writing of the thesis, to my parents for their support, and to my colleagues for their valued input. The assistance of the dairy staff at the Pacific Agricultural Research Center in Agassiz, B. C. in the care and milking of the animals used in the studies was also appreciated. Gratitude is extended to the summer students who assisted with data collection at Agassiz, to R. Sun, M. Varenitch, and G. Wilson for assisting with some of the laboratory analysis, and to M. Fraser for preparing the concentrates used in the trials. A special thank you goes to the B. C. Canada Green Plan and ProForm Feeds Inc., Chilliwack, B. C. for providing the funding necessary to carry out this research; and to Dr. J. Kelly of Rhone-Poulenc Animal Nutrition, Mississauga, Ont. for supplying the Smartamine™. 1 I. GENERAL INTRODUCTION Nutritional management of dairy cattle has traditionally been used as a way to maximize milk production. Recent concerns about manure disposal, ammonia emissions to the environment, ground water quality, and excess nitrate in forage crops have increased interest in the effective and efficient use of nitrogen. Under current feeding practices, Dutch dairy cattle excrete 75-85% of dietary N in urine and feces (Tamminga, 1992). It therefore becomes apparent that work needs to be done to improve nitrogen utilization of dairy cattle rations in order to achieve minimum losses of nitrogen to the environment. 1.1 Nitrogen losses In order to realize improved nitrogen efficiency, it is important to understand where nitrogen losses originate. Urinary and fecal nitrogen losses result from different sources. Urinary nitrogen losses are the sum of rumen loss, replacement of metabolic losses in the gut, the formation of nucleic acids from dietary protein nitrogen, conversion losses in maintenance, and losses associated with milk production (Tamminga, 1992). Fecal losses of nitrogen include undigested feed nitrogen, undigested microbial nitrogen and endogenous nitrogen. Van Vuuren et al. (1993) concluded that the major source of urinary nitrogen loss from grass-based diets is rumen ammonia that is not used for microbial nitrogen. If more ammonia is produced than can be used by the microbes, the excess ammonia is absorbed from the rumen, or is later absorbed from the small intestine, and converted to urea by the liver (Satter and Roffler, 1975). A low percentage of urea is recycled, but the majority is excreted in urine. Van Vuuren et al. (1993) reported that 2 urinary nitrogen excretion was significantly related to the ratio between nitrogen and digestible organic matter in the diet. Urinary nitrogen losses were reduced by 30-40% when grass was partially replaced by concentrate (64% grass : 36% concentrate). Protein quality and degradability both play a role in determining urinary nitrogen loss (Wohlt et al., 1991). The authors reported that urinary nitrogen as a percentage of absorbed nitrogen was highest for cows supplemented with corn gluten meal (46%), intermediate for cows supplemented with soybean meal (42%), and lowest for cows supplemented with fish meal (38%). It was suggested that greater amino acid deamination of the poor quality protein in corn gluten meal was most likely responsible for the higher urinary nitrogen loss. The reduction of endogenous nitrogen losses has been suggested to be the most effective way to reduce fecal nitrogen. Tamminga (1992) noted that reducing the amount of undigested feed nitrogen in the feces has little impact on the level of nitrogen excretion from the body. It has also been suggested that a reduction in undigested microbial protein is unlikely to make a significant difference in fecal nitrogen losses, as about 85% of microbial protein is digested in the lower gut (Storm et al., 1983). Van Vuuren et al. (1993) indicated that due to a greater amount of indigestible crude protein in the small intestine, or an increase in hindgut fermentation, the feeding of concentrate mixtures high in starch or fiber or both resulted in higher fecal nitrogen excretion than when corn silage was fed as a supplement to grass diets. Lower rumen nitrogen losses can be achieved by reducing dietary crude protein, reducing dietary protein degradability, or by enhancing microbial protein synthesis by increasing capture of rumen degraded nitrogen (Tamminga, 1992). It 3 has been reported that little change in milk production or dry matter intake occurs when dietary protein exceeds 14 to 16% of dry matter in dairy rations, but that during early lactation, when dry matter intakes lag behind milk production, high producing cows may require up to 19% crude protein (Christensen, 1993). However, Tamminga (1992) noted that when dietary crude protein exceeds 15% on a dry matter basis, nitrogen losses from the rumen are high. If dietary crude protein levels are below 15%, digestion of other dietary components may also be reduced. Several studies have demonstrated a reduction in apparent crude protein digestibility when dietary crude protein levels were reduced from 19 to 13% (Ha and Kennelly, 1984) from 14.5 to 11.0% (Klusmeyer et al., 1990) and from 16 to 12% (Wohlt et al., 1991). Reduced microbial fermentation and reduced digestion in the gastrointestinal tract when lower dietary protein levels were fed may have been responsible for the reduced apparent dry matter and crude protein digestibilities (Ha and Kennelly, 1984). Others have reported that an improvement in nitrogen utilization was achieved when the ratio of dietary N to digestible organic matter was reduced, resulting in a reduction in the ratio of absorbable protein to dietary net energy for milk production and less nitrogen loss during milk protein synthesis (Van Vuuren et al., 1993). To improve utilization of dietary nitrogen and to lower nitrogen intakes, carbohydrate fermentation should be balanced with rate of crude protein breakdown (Tamminga, 1992). To benefit from this concept, it is necessary to provide a grain mixture with the appropriate level of undegradable protein to supplement the forage that is fed. However, little is known concerning the amount of forage proteins escaping degradation in the rumen and the relative amino acid content of those forage proteins. Observations of von Keyserlingk et al. (1996) indicated that the 4 variability in degradability of forage protein is high, ranging form 33-78% for grass silages and 63-80% for corn silages. Due to the unpredictability of the degradability of forage proteins from one sample to the next, there is a necessity to sample and analyze forages more often. Therefore, the use of book values has limited usefulness for balancing rations in terms of degradability. 1.2 Amino acid supplementation It has been suggested that the most meaningful time to determine amino acid requirements and responses to amino acid supplementation is during early lactation, when dry matter intake lags behind milk production (Patton, 1996). Patton (1996) concluded that it is difficult to use milk production or milk protein yields as a measure of amino acid response once the cow has maximized dry matter intake, weight gain has become evident, and milk production has begun to decrease, since these are indications that nutrient intakes are more than adequate. Therefore, comparison of studies undertaken to determine the effects of the supplementation of limiting amino acids to dairy cows is made difficult by the wide variation in stage of lactation, type of diet fed, and degree of amino acid supplementation in the various studies. A study by Fisher (1972), in which amino acids were infused intravenously into cows fed corn based diets, indicated that methionine was first limiting for milk production. Based on work in which amino acids were infused into the abomasum, Schwab et al. (1976) suggested that lysine and methionine were the first and second limiting amino acids for lactating cows fed low-protein, corn-based rations. In a similar study by Erfle and Fisher (1977), results indicated that methionine, lysine and threonine were co-limiting for dairy cows. 5 Work by Papas et al. (1984) demonstrated that encapsulating methionine with a pH sensitive coating is an effective way to deliver amino acids post-ruminally. However, studies on the effects of the addition of rumen-protected amino acids to dairy rations, which have concentrated on maximizing production of milk and milk component yields without monitoring nitrogen losses, have had varying success. Armentano et al. (1993) found no significant effects on dry matter intakes, milk or milk fat yields for cows in early or mid-lactation that were fed diets containing low or recommended levels of degradable intake protein, with or without supplementation with rumen-protected lysine and methionine. However, they achieved significant increases in milk protein %, milk protein yield and casein concentration for cows in early lactation that were supplemented with methionine and lysine. It was suggested that the increase in milk protein content was due to an increase in casein synthesis. Amino acid supplementation had no significant impact on milk protein yield for the cows in mid-lactation. Rogers et al. (1989) demonstrated that milk and milk protein yields could be increased when lactating cows were fed a corn based diet supplemented with corn gluten meal and rumen-protected lysine and methionine, but that no response was seen when soybean meal replaced the corn gluten meal. In a different trial, the feeding of 14.7 and 17.3% CP corn-based diets supplemented with rumen-protected lysine and methionine resulted in increases in milk true protein concentration and yield, but no differences were seen in milk production (Colin-Schoellen et al., 1995). Polan et al. (1991) suggested that the material entering the small intestine may be altered in terms of amino acid profile by feeding undegradable intake protein with different amino acid profiles than rumen microbial protein, but that the addition of 6 rumen protected limiting amino acids is a more effective way to improve amino acid availability in the small intestine. However, limited information is available regarding exact levels of individual amino acids that must bypass the rumen to maximize milk and milk protein production in high producing dairy cows (Rogers et al., 1989). Also, microbial protein availability is affected by differences in microbial turnover within the rumen and rumen out-flow rates (Leng and Nolan, 1984). 1.3 Cornell Net Carbohydrate and Protein System The Cornell Net Carbohydrate and Protein System is a ration modeling program that predicts the production of microbial protein and fermentation end products by comparing the rate of carbohydrate breakdown with the rate of protein breakdown (Russell et al., 1992; Sniffen et al., 1992). The rates of passage of the various microbial products and undigested feed out of the rumen are also predicted with this system. To achieve this, the model assumes feed intake is constant, rate of passage and extent of digestion are a function of intake, and that all dry matter intake is either digested or passed through the intestinal tract (Fox et al., 1990). An amino acid submodel, which allows prediction of absorbed amino acid supply based on ration formulation, and estimates requirements of these amino acids based on level of production, has been published by O'Connor et al. (1993). The assumed specificity and accuracy of the Cornell model in terms of ration composition and animal requirements should allow for a more efficient use of protein. However, the specificity of the model in terms of ration composition makes the practicality of this model on a farm level questionable. 7 1.4 Summary Improvements in nitrogen efficiency are limited by the amino acid that is most limiting in terms of the animal's required amount of that particular amino acid at the tissue level (Bergen, 1979). By using tools such as the Cornell Net Carbohydrate and Protein System and rumen-protected amino acids to balance dairy cattle rations for amino acid requirements at lower crude protein levels, an improvement in nitrogen efficiency should be possible, through a reduction in nitrogen excretion in urine and feces. Therefore, the objective of this study was to manipulate dairy ration formulations in order to reduce nitrogen excretion in urine and feces, without compromising milk production or composition or overall performance of the cows. 8 1.5 References Armentano, L. E., Swain, S. M., and Ducharme, G. A. 1993. Lactation response to ruminally protected methionine and lysine at two amounts of ruminally available nitrogen. J. Dairy Sci. 76: 2963-2969. Bergen, W. G. 1979. Free amino acids in blood of ruminants-physiological and nutritional regulation. J. Anim. Sci. 49: 1577-1589. Christensen, R. A., Lynch, G. L., Clark, J. H. and Yu, Y. 1993. Influence of amount and degradability of protein on production of milk and milk components by lactating Holstein cows. J. Dairy Sci. 76: 3490-3496. Colin-Schoellen, O. Laurent, F., Vignon, B., Robert, J. C , and Sloan, B. 1995. Interactions of ruminally protected methionine and lysine with protein source or energy level in the diets of cows. J. Dairy Sci. 78: 2807-2818. Erfle, J. D. and Fisher, L. J. 1977. The effects of intravenous infusion of lysine, lysine plus methionine or carnitine on plasma amino acids and milk production of dairy cows. Can. J. Anim. Sci. 57: 101-109. Fisher, L. J. 1972. Response of lactating cows to the intravenous infusion of amino acids. Can. J. Anim. Sci. 52:377-384. Fox, D. G., Sniffen, C. J., O'Connor, J. D., Russell, J. B., and Van Soest, P. J. 1990. The Cornell Net Carbohydrate and Protein System for Evaluating Cattle Diets. Part 1: A model for predicting cattle requirements and feedstuff utilization. Search:Agriculture, No. 34, pp7-83. Cornell Univ. Agr. Exp. Sta., Ithaca, NY. Ha., J. K. and Kennelly, J. J. 1984. Effect of protein on nutrient digestion and milk production by Holstein cows. J. Dairy Sci. 67: 2302-2307. Klusmeyer, T. H., McCarthy, R. D., Jr., Clark, J. H., and Nelson, D. R. 1990. Effects of source and amount of protein on ruminal fermentation and passage of nutrients to the small intestine of lactating cows. J. Dairy Sci. 73: 3526-3537. Leng, R. A., and Nolan, J. V. 1984. Symposium: protein nutrition of the dairy cow. Nitrogen metabolism in the rumen. J. Dairy Sci. 67: 1072-1089. O'Connor, J. D., Sniffen, C. J., Fox., D. G., and Chalupa, W. 1993. A net carbohydrate and protein system for evaluating cattle diets: IV. Predicting amino acid adequacy. J. Anim. Sci. 71: 1298-1311. Papas, A. M., Sniffen, C. J., and Muscato, T. V. 1984. Effectiveness of rumen-protected methionine for delivering methionine post-ruminally in dairy cows. J. Dairy Sci. 67: 545-552. 9 Patton. R. A. 1996. Methionine nutrition in dairy cows remains difficult. In Feedstuffs. March 11, 1996. Polan, C. E., Cummins, K. A., Sniffen, C. J., Muscato, T. V., Vincini, J. L, Crooker, B. A., Clark, J. H., Johnson, D. G., Otterby, D. E., Guillaume, B., Muller, L. D., Varga, G. A., Murray, R. A., and Peirce-Sandner, S. B. 1991. Responses of dairy cows to supplemental rumen-protected forms of methionine and lysine. J. Dairy Sci. 74: 2997-3013. Rogers, J. A., Peirce-Sandner, S. B., Papas, A. M., Polan, C. E., Sniffen, C. J., Muscato, T. V., Staples, C. R., and Clark, J. H. 1989. Production responses of dairy cows fed various amounts of rumen-protected methionine and lysine. J. Dairy Sci. 72: 1800-1817. Russell, J. B., O'Connor, J. D., Fox, D. G., Van Soest, P. J. and Sniffen, C. J. 1992. A net carbohydrate and protein system for evaluating cattle diets: 1. Ruminal fermentation. J. Anim. Sci. 70: 3551-3561. Satter, L. D., and Roffler, R. E. 1975. Nitrogen requirement and utilization in dairy cattle. J . Dairy Sci. 58: 1219-1237. Sniffen, C. J., O'Connor, J. D., Van Soest, P. J., Fox, D. G., and Russell, J. B. 1992. A net carbohydrate and protein system for evaluating cattle diets: II. carbohydrate and protein availability. J . Anim. Sci. 70: 3562-3577. Schwab, C. G., Satter, L. D., and Clay, A. B. 1976. Response of lactating dairy cows to abomasal infusion of amino acids. J. Dairy Sci. 59: 1254-1270. Storm, E., Brown, D. S., and 0rskov, E. R. 1983. The nutritive value of rumen-microorganisms in ruminants. 3. The digestion of microbial and nuclei acids in, and losses of endogenous nitrogen from, the small intestine of sheep. Br. J . Nutr. 50: 479. Tamminga, S. 1992. Nutrition management of dairy cows as a contribution to pollution control. J . Dairy Sci. 75: 345-357. Van Vuuren, A. M., Van Der Koelen, C. J., Valk, H., and De Visser, H. 1993. Effects of partial replacement of ryegrass by low protein feeds on rumen fermentation and nitrogen loss by dairy cows. J. Dairy Sci. 76: 2982-2993. von Keyserlingk, M. A. G., Swift, M. L, Puchala, R., and Shelford, J. A. 1996. Degradability characteristics of dry matter and crude protein of forages in ruminants. Anim. Feed Sci. Technol. 57: 291-311. 10 Wohlt, J. E., Chmiel, S. L, Zajak, P. K., Backer, L, Blethen, D. B., and Evans, J. L. 1991. Dry matter intake, milk yield and composition, and nitrogen use in Holstein cows fed soybean, fish, or corn gluten meals. J. Dairy Sci. 74: 1609-1622. 11 II. THE EFFECT OF DIETARY NITROGEN LEVEL ON THE NITROGEN EXCRETION OF LACTATING DAIRY COWS 2.1 Introduction Concerns about manure disposal, ammonia emissions to the atmosphere, and nitrate in drinking water justify the need for more efficient use of the dietary nitrogen in the diets of lactating cows, thereby reducing the amount of N excreted in urine and feces. Approximately 50% of nitrogen (N) required for lactating cow diets is imported as concentrates and protein supplements in the Lower Fraser Valley region of British Columbia, making the nutritional management of N an increasingly important tool to reduce N excretion from dairy cattle and to reduce the cost of diets. To improve utilization of dietary N, carbohydrate fermentation should be balanced with rate of crude protein breakdown. (Tamminga, 1992). Balancing rations in such a way should also reduce total nitrogen requirements and maximize the contribution of microbial populations, not only as a source of amino acids but to improve digestibility and intake of the total ration. However, little is known concerning the amount of forage proteins escaping degradation in the rumen and the respective amino acid contents of those forage proteins. The observations of von Keyserlingk et al. (1996) indicated that the variability in rumen degradability of forage protein is high, ranging from 33-78% for grass silages and 63-80% for corn silages. Therefore, in order to match the level of protein degradability of the concentrate with the degradability of the forage being fed, more information on forage quality is required to accomplish efficiencies of nitrogen utilization. It has also been noted that intestinal absorption of amino acids can be improved by increasing the proportion of rumen undegradable protein in the diet (Nocek and Russell, 1988). Microbial protein must be supplemented with rumen 12 undegradable intake protein in order to maximize production in high producing dairy cows (Leng and Nolan, 1984; Wohlt et al., 1991; Christensen et al., 1993). However, it is essential that microbial nutrient supply be maximized before rumen bypass nutrients are added to the diet (Stokes et al., 1991). In other words, dietary carbohydrate and protein fractions should be synchronized in terms of degradation and quality. The Cornell Net Carbohydrate and Protein System is a computer modeling program that assumes this achievement (Fox et al., 1990; O'Connor et al., 1990). Pell (1992) noted that due to the weak relationship between milk N and N excreted in urine and feces, N excretion could be substantially reduced without the impairment of milk production if rations were properly balanced. Recent concerns about manure disposal, ground water quality, and excess nitrate in forage crops have increased interest in the effective and efficient use of nitrogen. It has also been reported that under current feeding practices in The Netherlands, 75 - 85% of the N ingested by dairy cattle is excreted in urine and feces (Tamminga, 1992). The objective of the current study was to identify ration formulations to minimize the excretion of nitrogen from lactating dairy cows without impairing milk production. 2.2 Materials and Methods 2.2.1 Forage composition and degradabilities Forage composition and degradability data were determined for use in pre-trial ration formulations (Tables 2.1 and 2.3). Forage samples were dried in a forced air oven at 60°C for 48h and then ground through a 2 mm screen prior to chemical analysis (Table 2.1) or ruminal incubation for degradability determination (Table 2.3), using the nylon bag technique (0rskov and McDonald, 1979). Nylon bags (5x10 cm), 13 prepared in duplicate (two replicates per cow per time), were incubated in reverse order for 96, 72, 48, 24, 12, 8, 4, 2 and 0 hours. Sample quantities decreased with decreasing time of incubation, ranging from 3 g for 96 h to 1.5 g for 0 h. Each feed was incubated in two cows. The reverse order of incubation was employed in order to minimize the time that the bags already in the rumen were exposed to air and to enable the washing of all bags simultaneously. Nylon bags were sealed using rubber bands which were easily removed following rumen incubation. Bags were suspended in the rumen in a polyester mesh bag which was attached to the end of a 50 cm line weighted with a sand-filled bottle. Following ruminal incubations, the nylon bags were placed in a washing machine. The machine was allowed to fill with water and to agitate for 5 minutes prior to draining. This was repeated until the rinse water remained clear. Samples were dried in a forced air oven at 60°C until a constant weight was achieved. Replicates within cows were pooled and ground through a 0.5 mm screen prior to all laboratory analyses. Amino acid compositions were determined on the material from the 12 h bags by high performance liquid chromatography (HPLC), following derivation with o-phthaldialdehyde, using the method of Puchala et al. (1994), and expressed as a percentage of undegraded intake protein (Table 2.2). The HPLC system used included two Waters Model 510 HPLC Pumps (Waters Corp., Milford, MA), a Waters 717plus Autosampler (Waters Corp., Milford, MA), a Supelcosil LC-18 HPLC Column (7.5 cm x 4.6 mm I. D.) (Supelco Ltd., Bellefonte, PA) and a Waters 474 Scanning Fluorescence Detector (Waters Corp., Milford, MA) with wavelength settings at 340 nm for excitation and 450 nm for emission. Millennium Chromatography Manager software (Waters Corp., Milford, MA) was used for 14 automatically controlling the system as well as acquiring and treating chromatographic data. 2.2.2 Experimental design Eighteen lactating Holstein cows were utilized in a production trial. The animals were housed in a free stall barn located at the Pacific Agriculture Research Centre in Agassiz, B.C.. Cows were blocked according to age, milk production and weight, and randomly assigned to three treatments in a replicated 3 x 3 Latin square design. Experimental periods were 28 days in length. Individual feed intakes were monitored using Calan feeding doors (American Calan, Inc., Northwood, NH). The animals were cared for according to standards set by the Canadian Council on Animal Care (1993) and the experimental plan was approved by the Research Station Animal Care Committee. 2.2.3 Diets The animals were fed total mixed rations consisting of 25% tall fescue grass silage, 25% corn silage and 50% grain concentrate, on a dry matter basis (Table 2.4). Treatment A was a 15.3% CP diet formulated to meet NRC (1989) requirements, treatment B was a 16.4% CP diet balanced for rumen degradable and undegradable protein, as well as rate of passage using the Cornell (CNCPS ) computer model (O'Connor et al., 1990), and treatment C was a 12.3% CP diet balanced using the CNCPS computer model to meet amino acid requirements of cows weighing 615 kg, producing 39 kg d"1 of milk and consuming 21.9 kg of feed on a dry matter basis. Grain mixtures were ground and mixed with the silage to minimize sorting. A specific total mixed ration for each treatment group was prepared daily. Equal portions of the rations were fed at 8:30 A.M. and 3:30 P.M.. Total feed offered per day was equal to 15 110% of estimated intake, with weighbacks measured each morning. Feed intakes were calculated daily, averaged by week, and corrected for dry matter in order to calculate dry matter intake. 2.2.4 Sampling 2.2.4.1 Feed Total mixed rations were sampled three times per week, composited weekly, and frozen. Weighback samples were taken twice weekly from each cow and composited by week for each ration. Dry matter content was determined by drying samples in a forced air oven for 72 h at 60°C. Samples were then ground through a 1 mm screen and stored until subsequent chemical analysis. Ration samples were analyzed for acid detergent fiber (ADF) and neutral detergent fiber (NDF) using a modified method of Van Soest et al. (1991) called the filter bag technique (ANKOM Co., Fairport, NY), as well as N using a Le|o FP-428 N analyzer (Leco Corp., St. Joseph, Ml., USA) (Komareket al., 1994). 2.2.4.2 Milk Milk yield was recorded twice daily for all cows. Milk samples were collected from each cow in the third week of each treatment period, from four consecutive milkings, and analyzed for fat and protein by Infra Red Milk Analysis System (B. C. DHIS Lab, Chilliwack, B. C ) . Total daily yields of milk components were calculated using the A.M. and P.M. milk yield for the test date. Milk production values were used to calculate 4% fat corrected milk (FCM) using the equation: 4% FCM = (0.4 * kg milk) +(15 * (% fat /100) * kg milk). 16 2.2.4.3 Rumen fluid Rumen fluid samples (200 ml) were collected by vacuum stomach tube from each cow on day 21 of each period between 10:00 A.M. and 11:30 A.M. Rumen fluid pH was determined. Samples were acidified to pH 2 with 50% sulfuric acid, centrifuged and the supernatant frozen until volatile fatty acid (VFA) analysis by gas chromatography. The VFA were determined using a Shimadzu gas chromatograph equipped with a capillary column (30m x 0.25mm I.D. Stabilwax-DA). Injection port temperature was set at 170 °C. The column temperature was set at 120 to 180 °C at 10 °C min'1, with an initial time of one minute and a final time of two minutes. The internal standard used was isocaproic acid (0.70 g in 200 ml water). 2.2.4.4 Blood Samples of blood (20 ml) were taken in heparinized vacutainers by jugular venipuncture on day 21 of each period between 10:00 A.M. and 11:30 A.M.. Blood was centrifuged and plasma was frozen for later analysis of urea nitrogen and glucose using a Kodak Ektachem DT 60 Analyzer with Disc Two Module (Clinical Products Division, Eastman Kodak Co., Rochester, NY) and non-esterified fatty acid levels (NEFA kit, Wako Pure Chemical Industries, Ltd., Osaka, Japan). Red blood cell percentages were also determined. 2.2.5 Total collections For the last five days of each period, cows were confined to metabolism stalls for the total collection of urine and feces. Individual water intakes were also monitored and calculated daily for the cows in the metabolism stalls. External urine cups were used to collect urine (Fellner et al., 1988). A flexible vacuum hose connected each cup to a stainless steel container. The cups were fastened to the 17 cow with Velcro straps. Urine was sampled daily from each cow (250 ml), acidified (50% sulfuric acid), composited for each 5 day collection period and frozen at -4 °C until further analysis. Kjeldahl N content of the urine samples were determined using a Technicon Autoanalyzer Two system (Technicon Instruments Corp., Tarrytown, NY) after digestion on a block digester (Parkinson and Allen, 1975). Feces were collected in large pans placed in the gutter behind the cows. Fecal material for each cow was removed from the pans every four hours and placed in a covered garbage can. Daily fecal composites were mixed using a Hobart mixer, sampled (700 g) and frozen at -4 °C. Fecal samples were later dried in a forced air oven at 60 °C for 72 h for the determination of dry matter. Dried fecal samples were then ground through a 1 mm screen, composited for each cow-treatment period, and stored. Samples were later analyzed for ADF and NDF ) using a modified method of Van Soest et al. (1991) called the filter bag technique (ANKOM, Co., Fairport, NY) and for N using a Leco FP-428 nitrogen analyzer (Leco Corp., St. Joseph, ML, USA.) (Komarek et al., 1994). Cows were weighed on three consecutive days at the beginning of each period and at the end of the experiment to provide an estimate of changes in body weight. 2.2.6 Efficiency calculations The energy value of milk was calculated by the following equation: E (kcal lb"1) = 40.72 (% fat) + 22.65 (% protein) + 102.77 (Tyrrell and Reid, 1965). This value was then converted to metric units. The energy yield of milk was then added to the energy required for maintenance plus 10% allowance for activity (Moe et al., 1972; NRC, 1989) and a correction added for body weight change (NRC, 1989). Total energy yield (Meal NEL) was then divided by the total dry matter intake to provide an estimate of efficiency for utilization of dry matter. 18 2.2.7 Statistical analysis Statistical analysis was via least squares ANOVA, following the general linear models procedure of SAS (1985). The model statement used was: YiijP= p. + Tj + a p + Pi(p> + 8i(p) + Oj i + 6iji(p) H = overall mean Tj = effect of treatment, j = 1 ,...,3 a p = effect of square, p = 1,...,6 PKP) = effect of cow within square, i = 1.....3 8i(p) = effect of period within square, I = 1.....3 coji = effect of treatment by period interaction eiji(p) = experimental error 2.3 Results 2.3.1 Feed composition Feed composition data is given in Tables 2.1 to 2.5. Crude protein levels were 15.3%, 16.4%, and 12.3%, for diets A, B, and C, respectively. Acid detergent fiber percentages were different (P<0.05) among diets (23.5, 22.7, and 22.0%, for diets A, B, and C, respectively. The control ration (A) contained significantly higher (P<0.05) NDF than rations B and C (40.4% for diet A compared to 38.9 and 38.6% for diets B and C). 2.3.2 Dietary intakes and milk production There was a significant (P<0.05) reduction in dry matter intake among cows fed diets A, B, and C (Table 2.6). The low protein diet resulted in a significant body weight loss (Table 2.6), as well as lower (P<0.05) milk production (Table 2.6), milk 19 protein percentage (Table 2.6) and milk N output (Table 2.10) when compared to cows fed the other two diets. Fat-corrected milk production was 33.7, 34.4, and 32.0 kg d"1 for diets A, B, and C, respectively. Milk fat percentage and milk fat output (Table 2.6) were not affected by dietary treatment (P>0.05). 2.3.3 Blood composition Hematocrit and blood glucose values were unaffected by treatment (Table 2.7). Blood non-esterified fatty acid concentrations were also not different (P>0.05) among diets. Blood urea nitrogen values were significantly different (P<0.05) among all three diets (14.0, 19.1, and 6.8 mg dl"1, for cows on diets A, B, and C, respectively). 2.3.4 Rumen fluid Rumen fluid pH was not affected (P>0.05) by dietary treatments (Table 2.8). No significant differences (P>0.05) were seen in rumen acetate, propionate, isobutyrate, butyrate, or valerate (Table 2.8). However, isovalerate concentrations were significantly higher (P<0.05) for cows fed diet B than those fed diet A, but were not different from those fed diet C. 2.3.5 Water intake and waste excretion Water intake and urine output (Table 2.9) reflected the level of dry matter consumed by the animals. The differences observed in kg of fecal dry matter (Table 2.9) were not unexpected since they reflected the differences in dry matter digestibility (Table 2.10). 2.3.6 Apparent digestibility of nutrients The effect of diet on apparent digestibility of nutrients is given in Table 2.10. The control and low protein diets resulted in significantly lower (P<0.05) dry matter digestibilities than for cows fed B diet. The apparent digestibility of crude protein was 20 significantly greater (P<0.05) for cows fed diet B compared to cows fed diets A or C. Cows fed the control diet A also had a significantly higher apparent CP digestibility than those fed the 12.3% CP diet, which again is an indication of the inadequacy of the third diet in terms of meeting the microbial N needs. Diet B, which was balanced using the CNCPS model, resulted in a significantly (P<0.05) higher apparent NDF digestibility than the low protein diet, but was not different (P>0.05) from cows fed the control ration. No differences (P>0.05) were seen among treatment groups in the apparent digestibility of ADF. 2.3.7 Nitrogen balance Nitrogen intakes were significantly (P<0.05) lower for those cows fed the 12.3% CP diet (Table 2.10), compared to the other two diets. The 25% reduction in N intake for cows on the low protein diet compared to the other two diets is a reflection of the lower dry matter intakes and protein percentage of that third diet. Fecal N (Table 2.10) excretion was significantly lower (P<0.05) for cows fed diet C when compared to cows fed diet A (control), but was not different from those fed diet B. The 12.3% CP diet resulted in nearly a 50% reduction in urinary nitrogen compared to diet B (P<0.05). Overall, N excretion in urine and feces was 29% lower for cows fed diet C compared to those fed diet A, but N balance (Table 2.10) was not affected (P>0.05) 2.3.8 Nitrogen efficiency The percentage of N intake (Table 2.10) excreted in milk tended to be greater (P>0.05) for cows fed diet C compared to those fed diets A or B (30, 29, and 32%, for diets A, B, and C, respectively). Urinary excretion represented 29, 35, and 23% of dietary N, for diets A, B, and C, respectively, with cows fed diet B excreting a 21 significantly (P<0.05) larger proportion of dietary N in urine than those fed diets A or C. Significant (P<0.05) differences were seen among all dietary groups (31, 27 and 34%, for A, B, and C, respectively) in percentage of dietary N excreted in feces. 2.3.9 Energy efficiency Cows fed diet C were losing weight (P<0.05) at a rate of 0.246 kg d' 1 compared to weight gains of 0.198 and 0.109 kg d'1, for diets A and B, respectively (Table 2.6). Daily energy expenditure for maintenance and milk production was significantly (P<0.05) reduced for cows on diet C, reflecting the lower milk yield of these cows (36.3, 37.2 and 32.5 Meal NEL for diets A, B and C, respectively). Energy efficiency of ration dry matter utilization was not different (P>0.05) among diets (Table 2.11). 2.4 Discussion It has been reported that feed intake is stimulated to a greater extent by natural protein supplements with low soluble N than by urea (or highly soluble N) supplemented diets (Nocek and Russell, 1988). The lower solubility of N sources in the concentrate portions of diets A and B may explain why cows fed diets A and B had significantly higher intakes than those fed diet C, which contained a concentrate that consisted mainly of barley. Crish et al. (1986) found reduced intakes when comparing 15 and 12% CP corn silage based diets and suggested that in order to maintain adequate rumen ammonia levels, sufficient soluble N must be fed. The low protein percentage of diet C may have been inadequate in terms of peptides and amino acids for microbial growth (Wohlt et al., 1991). This would suggest that perhaps inadequate protein intake was limiting energy intake and thus having a negative effect on milk production. Kung and Huber (1983) suggested that higher 22 milk production attained by cows on higher protein diets may reflect the higher overall nutrient intake of those cows. Ha and Kennelly (1984) noted that dietary CP levels did not affect rumen pH, which agrees with results of the present study. Although there were significant differences in fiber composition of the rations in the present study, the differences were small enough that nutrient digestion was not affected to any great extent, as indicated by the relatively small differences in rumen VFA concentrations. The differences in isovalerate concentrations, which were significantly higher for cows fed diet B than for cows fed diet A, but were not different from those fed diet C, may be a reflection of the carbohydrate composition of the rations (Stokes et al., 1991). The corn-gluten meal in diet B may have contributed to the increase in isovalerate, as Casper and Schingoethe (1989) and Aldrich et al. (1993) have reported an increase in isovalerate concentrations on corn-based diets when compared to barley-based diets. However, it should also be pointed out that in the present study both diets B and C contained considerably larger proportions of barley than diet A, which contained barley and corn. Sources of protein also varied widely among the three rations. Blood non-esterified fatty acid concentrations, which are often used as an indicator of the mobilization of fat during energy depletion (Christensen et al., 1993) were not different among dietary groups in the present study, indicating that type and level of dietary protein had no effect on body fat reserves. The excessive weight loss of cows on diet C suggests that cows were mobilizing protein tissue, although differences in rumen fill due to the lower dry matter intake and water intake for cows fed diet C as well as experimental error may have contributed to the differences in 23 weight change. Cows fed the 12.3% CP diet not only had reduced milk production and milk protein output when compared to the other two diets, but were also losing body weight at a rate of 0.246kg d"1. Longer experimental periods may have resulted in more severe production losses for cows on diet C since productivity in early lactation cows has been shown to suffer when low protein diets are fed, due to limited body reserves (Wohlt et al., 1991). Wohlt et al. (1991) found production differences when cows were fed corn silage based diets differing in crude protein level, and suggested that when cows were fed a low protein diet (12%), rumen microbial action may be limited due to an inadequate supply of peptides and amino acids. Performance on the 12.3% CP diet in the present study reflected this. However, differences in the degradabilities of the the nitrogen fractions among the rations may explain some of the production differences. In order to support high milk production, diets must contain bypass nutrients as the protein to energy ratio used for high milk production is greater than that produced by rumen fermentation (Leng and Nolan, 1984). Klusmeyer et al. (1990) reported that daily microbial N flow to the small intestine was not affected when comparing diets containing 14.5 and 11.0% CP. However, they reported that milk production was significantly reduced and may be attributed to differences in the flow of amino acids to the small intestine. The lower milk protein yield in the current study is a reflection of the lower milk production and lower milk protein percentage for cows on the 12.3% CP diet. Blood urea nitrogen is a reflection of protein metabolism in the cow (Baker et al., 1995). Blood urea nitrogen concentrations, which were significantly different among dietary groups, reflected dietary CP levels, as has been reported in previous studies (Wohlt et al., 1991; Christensen et al., 1993; Roseler et al., 1993). Excess 24 protein, whether degradable or undegradable intake protein, will elevate BUN since ruminal and post-ruminal N excesses are converted to urea by the liver (Roseler et al., 1993). This includes ammonia produced in the rumen that is not used for microbial protein production and that from the deamination of amino acids arising from protein escaping ruminal degradation (Butler et al., 1995). In the present study, the significantly greater dry matter and CP apparent digestibilities for cows fed diet B compared to those on diets A or C may be attributed to the improved balancing of ration ingredients in ration B. The higher N intake and higher subsequent rumen ammonia levels may have had a positive effect on dry matter digestibility (Kwan et al., 1977). Differences in the degradabilities of the protein fractions of the diets may also have contributed to the differences in apparent CP digestibility. Cows fed diet A also had significantly higher apparent CP digestibilities than those fed the 12.3% CP diet, which again is an indication of the inadequacy of the third diet in terms of meeting N needs. Others have reported improved apparent CP digestibility as dietary protein increased from 13 to 19% (Ha and Kennelly, 1984), from 12 to 16% (Wohlt et al., 1991) and from 11.0 to 14.5% (Klusmeyer et al., 1990). Ha and Kennelly (1984) suggested that higher apparent digestibilities of DM and CP at higher dietary protein levels were probably due to a combination of improved microbial fermentation and greater digestion in the lower gastrointestinal tract. A reduction in microbial protein synthesis and organic matter (OM) degradation has been noted when there is a deficiency of N for ruminal bacteria (Clark et al., 1992). In the present study, the extremely low BUN values (6.8 mg dl"1) and the significantly lower apparent CP digestibility for cows fed the 12.3% CP diet indicate that the cows receiving the low protein diet were receiving inadequate levels 2 5 of N. It has been reported that BUN levels should be above 8-10 mg dl"1 for maximum organic matter digestion (NRC, 1989). A decrease in the proportion of endogenous fecal N from cows on the higher CP diets may also have improved CP apparent digestibilities (Ha and Kennelly, 1984). This would explain the significantly higher proportion of dietary N excreted in feces at the low CP levels in the current study, as Tamminga (1992) noted that reducing the amount of undigested feed in the feces will not sufficiently influence the level of N excretion from the body. The differences in apparent NDF digestibility in the present study may reflect differences in diet formulations. Not only is excess dietary protein expensive, but there is also the energy cost to the animal associated with conversion of ammonia to urea. Although diet B, which was balanced in terms of degradable and undegradable protein as well as rate of passage, resulted in the highest apparent CP digestibility, N excretion in urine was also highest compared to the other two diets (35% of dietary N intake, compared to 29 and 23% for diets A and C, respectively). Wohlt et al. (1991) found that cows fed diets supplemented with corn gluten meal excreted a greater proportion of absorbed N in urine than soybean meal or fishmeal supplemented diets. Corn gluten meal, which is considered a poor quality protein, may result in greater amino acid deamination (Wohlt et al., 1991). The presence of corn gluten meal in diet B may partially explain the increased BUN values and urinary N output, compared to the other two diets. When data from several studies were compiled, regression analysis showed a poor relationship (R2=.05) between milk N and urinary or total N excretion (Pell, 1992). It was therefore suggested that nitrogen excretion could be lowered without impairing milk production. In the present study, the 12.3% CP diet did result 26 in decreased N excretion in urine and feces but milk N efficiency expressed as a percentage of N intake was not significantly different among dietary groups. The Cornell Net Carbohydrate and Protein System has been suggested to estimate dietary protein degradability and to predict accurately that rumen microorganisms have access to adequate levels of ammonia and peptides (Chalupa and Sniffen, 1993). Matching the degradation of dietary protein and carbohydrates should result in an improved capture of microbial N (Tamminga, 1992), but high blood urea N and urinary N excretion from diet B (16.4% CP) would indicate otherwise. Although cows fed the 12.3% CP diet had lower milk and milk protein outputs, waste N excretion was dramatically reduced without influencing energy efficiency, when compared to cows fed the other two diets. With greater N utilization and lower N intake, cows fed low protein diets have less of an impact on the environment. This could have important implications if government regulations are implemented to limit the amount of manure N that can be applied per area of land. If rations can be formulated that allow a more efficient use of N, and therefore result in less N excreted in urine and feces, more animals could be farmed per area of land. Producers feeding low protein diets, similar to diet C, could house approximately one-third more animals than those feeding diets containing over 15% CP, such as diets A or B, due to the differences in N excretion. In order to formulate rations that improve N efficiency while maintaining milk and milk protein production, a subsequent study was undertaken in which rumen-protected lysine and methionine were added to low protein diets. 27 2.5 References Aldrich, J. M., Muller, L. D., Varga, G. A., and Griel, L.C., Jr. 1993. Non-structural carbohydrate and protein effect on rumen fermentation, nutrient flow, and performance of dairy cows. J. Dairy Sci. 76: 1091-1105. Baker, L. D., Ferguson, J. D. and Chalupa, W. 1995. Responses in urea and true protein of milk to different protein feeding schemes for dairy cows. J. Dairy Sci. 78: 2424-2434. Butler, W. R., Cherney, D. J. R. and El rod, C. C. 1995. Milk urea nitrogen (MUN) analysis: field trial results on conception rates and dietary inputs. Proc. Cornell Nutrition Conference, pp. 89-95. Canadian Council on Animal Care. 1993. Guide to the care and use of experimental animals. Volume 1. E. D. Olfert, B. M. Cross, and A. A. McWilliam (Editors). 2nd Edition, Ottawa, Ontario. Casper, D. P., and Schingoethe, D. J. 1989. Lactational response of dairy cows to diets varying in ruminal solubilities of carbohydrate and crude protein. J. Dairy Sci. 72: 928-941. Chalupa, W., and Sniffen, C. J. 1993. Protein and amino acid nutrition of lactating dairy cattle - today and tomorrow. 1993 Western Nutrition Conference, pp. 87-114. Christensen, R. A., Lynch, G. L. and Clark, J. H. 1993. Influence of amount and degradability of protein on production of milk and milk components by lactating Holstein cows. J. Dairy Sci. 76: 3490-3496. Clark, J. H., Klusmeyer, T. H., and Cameron, M. R. 1992. Microbial protein synthesis and flows of nitrogen fractions to the duodenum of dairy cows. J. Dairy Sci. 75: 2304-2323. Crish, E. M., Wohlt, J. E. and Evans, J. L. 1986. Insoluble nitrogen for milk production in Holstein cows via increases in voluntary intake and nitrogen utilization. J. Dairy Sci. 69: 1576-1586. Fellner, V., Weiss, M. F., Belo, A. T., Belyea, R. L., Martz, F. A., and Orma, A. H. 1988. Urine cup for collection of urine from cows. J. Dairy Sci. 71: 2250-2255. Fox, D. G., Sniffen, C. J., O'Connor, J. D., Russell, J. B., and Van Soest, P. J. 1990. The Cornell Net Carbohydrate and Protein System for Evaluating Cattle Diets. Part 1: A model for predicting cattle requirements and feedstuff utilization. Search:Agriculture, No. 34, pp7-83. Cornell Univ. Agr. Exp. Sta., Ithaca, NY. 28 Ha, J. K. and Kennelly, J. J. 1984. Effect of protein on nutrient digestion and milk production by Holstein cows. J. Dairy Sci. 67: 2302-2307. Klusmeyer, T. H., McCarthy, R. D., Jr., Clark, J. H., and Nelson, D. R. 1990. Effects of source and amount of protein on ruminal fermentation and passage of nutrients to the small intestine of lactating cows. J. Dairy Sci. 73: 3526-3537. Komarek, A. R., Robertson, J. B., and Van Soest, P. J. 1994. A comparison of methods for determining ADF using the Filter Bag Technique versus conventional filtration. J. Dairy Sci. 77 (Suppl. 1): 114. (Abstr.) Kung, L., Jr., and Huber, J. T. 1983. Performance of high producing dairy cows in early lactation fed protein of varying amounts, sources, and degradability. J. Dairy Sci. 66: 227-234. Kwan, K., Coppock, C. E., Lake, G. B., Fettman, M. J., Chase, L. E., and McDowell, R. E. 1977. Use of urea by early postpartum Holstein cows. J. Dairy Sci. 60: 1706-1724. Leng, R. A., and Nolan, J. V. 1984. Symposium: protein nutrition of the dairy cow. Nitrogen metabolism in the rumen. J. Dairy Sci. 67: 1072-1089. Moe, P. W., Flatt, W. P., and Tyrrell, H. F. 1972. Net energy value of feeds for lactation. J. Dairy Sci. 55: 945-958. National Research Council (NRC), 1989. Nutrient requirements of dairy cattle. Seventh Revised Edition. National Academy Press, Washington, D. C. Nocek, J. E., and Russell, J. B. 1988. Protein and energy as an integrated system. Relationship of ruminal protein and carbohydrate availability to microbial synthesis and milk production. J. Dairy Sci. 71: 2070-2107. O'Connor, J. D., Sniffen, C. J., and Fox, D. G. 1990. The Cornell Net Carbohydrate and Protein System for Evaluating Cattle Diets. Part 2: A computer spreadsheet for diet evaluation. Search:Agriculture, No. 34, pp4-40. Cornell Univ. Agr. Exp. Sta., Ithaca, NY. 0rskov, E. R. and McDonald, I. 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92: 499-503. Parkinson, J. A. and Allen, S. E. 1975. A wet oxidation procedure suitable for the determination of nitrogen and mineral nutrients in biological material. Commun. Soil Sci. Plant Anal. 6: 1-11. 29 Pell, A. N. 1992. Does ration balancing affect nutrient management? Cornell Nutrition Conference, pp. 23-31. Puchala, P., Pior, H., von Keyseriingk, M. A. G., Shelford, J. A., and Barej, W. 1994. Determination of methionine sulfoxide in biological materials using HPLC and its degradability in the rumen of cattle. Anim. Feed. Sci. Tech. 48: 121-130. Roseler, D. K., Fergusen, J. D., Sniffen, C. J., and Herrema, J. 1993. Dietary protein degradability effects on plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J. Dairy Sci. 76: 525-534. Statistical Analysis System (SAS). 1985. SAS User's Guide: Statistics. SAS Institute Inc., Cary, N.C. Stokes, S. R., Hoover, W. H., Miller, T. K., and Blauweikel, R. 1991. Ruminal digestion and microbial utilization of diets varying in type of carbohydrate and protein. J. Dairy Sci. 74: 871-881. Tamminga, S. 1992. Nutrition management of dairy cows as a contribution to pollution control. J . Dairy Sci. 75: 345-357. Tyrrell, H. F. and Reid, J. T. 1965. Prediction of the energy value of cow's milk. J . Dairy Sci. 48: 1215-1223. Van Soest, P. J., Robertson, J. B., and Lewis, B. A. 1991. Methods for dietary fiber, NDF and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3583-3597. von Keyseriingk, M. A. G., Swift, M. L, Puchala, R., and Shelford, J. A. 1996. Degradability characteristics of dry matter and crude protein of forages in ruminants. Anim. Feed Sci. Technol. 57: 291-311. Wohlt, J. E., Chmiel, S. L., Zajak, P. K., Backer, L., Blethen, D. B., and Evans, J. L. 1991. Dry matter intake, milk yield and composition, and nitrogen use in Holstein cows fed soybean, fish, or corn gluten meals. J. Dairy Sci. 74: 1609-1622. 30 Table 2.1. Nu trient composition (DM basis) of forages nutrient1 corn silage grass silage % DM 28.71 33.23 % CP 8.26 17.51 % ADF 26.33 33.29 % NDF 43.85 50.20 % ADIP 4.95 5.87 % NDIP 4.03 8.48 % lignin 2.32 4.24 % ash 4.02 12.04 % starch 16.31 13.65 DM, CP, ADF, NDF, ADIP, and NDIP stand for dry matter, crude protein, acid detergent fiber, neutral detergent fiber, acid detergent insoluble protein, and neutral detergent insoluble protein, respectively. 31 amino acid corn silage grass silage methionine 1.15 1.01 lysine 2.07 3.44 arginine 2.81 3.55 threonine 3.60 3.87 leucine 11.57 6.99 isoleucine 3.8 3.90 valine 4.72 4.92 hisitidine 1.80 1.62 phenylalanine 5.18 4.85 UIP refers to undegraded intake protein after 12 h incubation in the rumen. Table 2.3. Degradability of the nitrogen fractions of forages in situ (%) corn silage grass silage soluble 41.6 57.55 potentially degradable 53.16 33.23 Kd 1 (% h"1) 0.01 0.03 lag 2.68 3.44 ED 2 (0.06) 47.76 67.71 ED 2 (0.08) 45.84 65.78 Rate of degradation in the rumen. Effective degradability of nitrogen (0.06 and 0.08 refer to hypothetical rates of passage used in the degradability calculations). 33 Table 2.4. Composition of concentrate mixtures (kg t"1) diet A B C barley 445.1 651.5 895.8 canolameal 121.7 0 15.3 distillers mash 258.3 0 0 soybean meal 0 131.3 12.8 cracked corn 115.6 0 0 corn gluten meal 34.7 153.2 12.8 tallow 0 39.6 38.8 multifos 15 15 15 cobalt iodized salt 5 5 5 vit-min premix 5 5 5 Table 2.5. Composition of total mixed rations (% n diet A B C S. E. crude protein 159 15.3b 16.4a 12.3c ±0.09 acid detergent fiber 178 23.5a 22.7b 22.0c ±0.3 neutral detergent fiber 178 40.4a 38.9b 38.6b ±0.5 a, b, c Means in the same row with different letters differ significantly (P<0.05) UJ CM CO o CN o CN CO CD o o CO d +1 d -H +1 d -H d -H d -H d +i d +i o oo d CN o CO CN CO CD CO CD CD X i X> X i T— T— CN LO o T— CO d CN CN 1 CO CO T J i T J ICQ CO X ) o CO XI CO CO CN O) CO o CD CO LO d LO CO CN CN CO CO CO CD CD CO "co E T J C o c o co o E o o CD T J o o c iE 5 CD c\i 0) X i (0 h-(0 CO 00 CO CO CD CD CO CO CN oo LO T— CN CD o CN CO h - d Tf' co CO CO CN CO CO CO CD CD •^ r O o o o LO CD co CD 1 T J CD ' . CD (0 c cd l _ 4— 0) o CO E T J Q O) CO CD CD C CO x: o c o CO ^ ^ K o g> CD T J O X) D ) CD >» T J O X ) O •D T J O i _ C L 5^ 1 C O l _ CL Table 21.The effect of d i ^ n diet A B C S. E. hematocrit (%) 41 29.3 29.1 28.6 ±0.4 glucose (mg dl"1) 41 63 63.8 62.7 ±0.6 urea nitrogen (mg dl"1) 41 14.0b 19.1a 6.8c ±0.4 NEFA (mEq I"1) 41 0.14 0.16 0.16 ±0.02 a, b, c Means in the same row with different letters differ significantly (PO.05). Table 2.8. The effect of diet on rumen fluid pH and volatile fatty acid concentration (mg ml"1) n diet A B C S. E. pH 41 6.85 6.87 6.85 ±0.07 acetate 41 2.2 2.5 2.4 ±0.2 propionate 41 0.85 0.96 0.89 ±0.07 isobutyrate 41 0.047 0.058 0.051 ±0.004 butyrate 41 0.75 0.82 0.77 ±0.07 isovalerate 39 0.071b 0.097a 0.086ab ±0.007 valerate 41 0.100 0.103 0.100 ±0.008 a, b Means in the same row with different letters differ significantly (P<0.05). Table 2.9. The effect of diet on water intake, urine output, and fecal output n diet A B C S. E. water intake (Id"1) 37 74.0a 69.7ab 66.2b ±2.3 urine output (I d"1) 41 17.4 15.4 14.0 ±1.4 feces output (kg d'1) 41 40.6a 35.9b 37.5ab ±1.2 feces output (kg DM d"1) 41 6.0a 5.3b 5.6ab ±0.2 feces dry matter (%) 41 14.9 14.2 15.0 ±0.3 a, b Means in the same row with different letters differ significantly (P<0.05). CO *- Z c C T J 0) o c E g o o . § 0) § * s z t _ o =s Co j^-E b o >» CDO TJ € c 0 CD CNJ -Q CO H CD £ D) C ' l . D . TJ CD " -o ro -2 CO CD _Q Q J 0) TJ LU CO O CD CD CD 00 CD d o d o •H -H -H -H X i o X i o CN CN i n c\i d CD CD CD CO CO CD X i CO CD O) TJ CO T O t co m CM I— I— CD CD X i X i CO 9> >> 3n T - l + ± — > CD O -•—« CO E "O to CD O) Jc CD  i _ Q . CD TJ CO CD O) TJ E Q CO CN CD CO i -CD CD CD ^ ^ '^ r <o CD D) TJ Li. Q < CN O o o d o o d -H o o d -H CN TJ ~ .i= CD _ CO ^ TJ CD ro c 1 E O) CO c CD Jul CO O 4^ TJ CO CO CO CO CO o X— T— LO oo LO CO o d d CD d LO d CM CO CN o -H X i o X i 8 X i LO CN CO 1 oo CN CD CN N" CO o -Q x— CO jo d d CN d CO d Id CO X I CO CO o CD X— LO IO LO LO LO X i _Q o d d o d CD d x— d CO CN CO CO CO CO CD CD CD CO CD CN CN CN CN CN CN CN CN o CD CO 0s CO c z z 8 .CD _ V U TJ O) CD O c ro co x i UJ ^ CO LO o •<- c i -H +1 CO T3 O 00 X ) LO CM CO CO CM CO CO CO CD CO o UJ z s CO l _ D T3 c CD Q . X CD >* O) CD c CD CO LO CD CD LO o D ) UJ 8 c CD O 5t= CD 41 III. THE USE OF THE CORNELL NET CARBOHYDRATE AND PROTEIN SYSTEM AND RUMEN-PROTECTED LYSINE AND METHIONINE TO REDUCE NITROGEN EXCRETION FROM LACTATING DAIRY COWS 3.1 INTRODUCTION Concerns about manure disposal, nitrates in ground water and high ammonia losses to the environment have hastened the need to utilize feed N more efficiently in dairy cattle rations, through the reduction of N excretion in urine and feces. Results described in chapter II showed that N excretion in urine and feces can be dramatically reduced when lower crude protein levels are fed. Supplementing low protein diets, balanced for microbial nutrient supply and requirements for milk production, with rumen-protected lysine and methionine, may make it possible to improve the proportion of dietary N found in milk (Robinson et al., 1995). Many of the previous studies that have used protected amino acids have concentrated on trying to maximize production of milk and milk protein, but without monitoring amount of nitrogen lost in urine and feces (Rogers et al., 1989; Armentano et al., 1993; Colin-Schoellen et al., 1995). Methionine and lysine have been found to be limiting for milk production when many different dairy cattle rations have been fed (Schwab et al., 1976; Erfle and Fisher, 1977; Schwab et al., 1992a). The dietary addition of rumen stable methionine and lysine allows for the effective delivery of these amino acids to the small intestine and hence should provide a more favorable amino acid profile for absorption and utilization. In doing so, there will be a reduction in nitrogen losses and therefore the subsequent impact of the dairy industry on the surrounding environment will be lower. The Cornell Net Carbohydrate and Protein System (CNCPS) allows rations to be balanced in terms of protein and carbohydrate degradation and rates of passage 42 (Fox et al., 1990; O'Connor et al., 1990) and thus could also aid in improving feed efficiency. The model predicts, based on a minimum of actual measurements, the rates of protein and carbohydrate degradation from the degradation and flow rates of the various fractions and subfractions of these components (Sniffen et al., 1992). Crude protein is divided into nonprotein nitrogen, true protein, and unavailable nitrogen. The true protein is subsequently fractionated into a further three portions based on degradation rate. Carbohydrate content, which is estimated by difference, after determination of crude protein, fat, and ash levels in the ration, is also fractionated on the basis of degradation rates into sugars, starch, available cell wall, and unavailable cell wall. It is assumed that dietary protein and carbohydrate degradations follow first order kinetics and that all material disappears through passage or digestion (Russell et al., 1992; Sniffen et al., 1992). The production of microbial protein and fermentation end products is predicted by comparing the rate of carbohydrate breakdown with the rate of protein breakdown (Russell et al., 1992; Sniffen et al., 1992). The rates of passage of the various microbial products and undigested feed out of the rumen are also predicted with this system. To achieve this, the model assumes feed intake is constant, rate of passage and extent of digestion are a function of intake, and that all dry matter intake is either digested or passed through the intestinal tract (Fox et al., 1990). Dietary amino acid sufficiency has been predicted in a CNCPS submodel (O'Connor et al., 1993). The submodel estimates absorbed amino acid supply from predicted ruminal microbial growth and composition as well as level and composition of undegraded intake protein. Amino acid requirements are based on level of production. Meeting the amino acid requirements of lactating dairy cows on low protein diets should improve N efficiency 43 and result in less wastage of dietary N. The CNCPS computer model should aid in this regard (O'Connor et al., 1993). Improved N utilization by dairy cattle is necessary if government regulations are enforced to restrict the amount of manure nitrogen that can be applied per area of land. The objectives of this study were 1) to use rumen-protected methionine and lysine and the Net Carbohydrate and Protein System to improve the efficiency of N utilization in lactating dairy cows by minimizing N excretion in urine and feces, without impairing milk production and 2) to evaluate the Net Carbohydrate and Protein System as a means of improving the overall dietary efficiency of dairy cattle. 44 3.2 Materials and Methods 3.2.1 Forage composition and degradabilities Forage composition and degradability data were determined for use in pre-trial ration formulations (Tables 3.1, 3.2, and 3.3). The in situ incubation of the forage samples and the estimation of rumen degradation parameters are described in Chapter II, section 2.2.1. 3.2.2 Experimental design Animals were cared for and the experiment was designed as described in Chapter II, section 2.2.2. 3.2.3 Diets The animals were fed total mixed rations consisting of 30% grass silage, 20% corn silage and 50% grain concentrate (Table 3.4). All rations were formulated in terms of protein and carbohydrate degradabilities and passage rates using the Cornell (CNCPS) model (O'Connor et al., 1990). Crude protein levels for the three treatments (Table 3.5) were 18.3, 16.7 and 15.3%, for diets A (control), B, and C, respectively. Formulations for diets B and C (Table 3.4) incorporated rumen-protected lysine and methionine (Smartamine™, Rhone-Poulenc Animal Nutrition, Atlanta, GA) to ensure optimal dietary amino acid profiles at lower dietary crude protein levels. The rumen-protected amino acids were top-dressed and mixed by hand into the total mixed ration for each cow twice daily to ensure that each cow was receiving adequate quantities of the material and to ensure that the protective coats of the amino acids were not destroyed by mechanical mixing or by the acidity of the silage. Grain mixtures were ground and mixed with the silage to minimize sorting. A specific total mixed ration for each treatment group was prepared daily. Equal 45 portions of the rations were fed at 8:30 A.M. and 3:30 P.M.. Total feed offered per day was equal to 110% of estimated intake, with weighbacks measured each morning. Feed intakes were calculated daily, averaged by week, and corrected for dry matter content in order to calculate daily dry matter intake. 3.2.4 Rumen-protected methionine and rumen-protected lysine A pH-sensitive poly (2-vinylpyridine-co-styrene) coating is used in the production of Smartamine™ M and Smartamine™ ML (Rhone-Poulenc Animal Nutrition, Atlanta, GA). The coating is resistant to rumen microbes and is reported to be stable at ruminal pH. In the acidic abomasal environment, the coating breaks down and releases the amino acids for absorption in the small intestine. Smartamine™ M contains 70% DL-methionine and Smartamine™ ML contains 15% DL-methionine and 50% L-Lysine monohydrochloride (Rhone-Poulenc Animal Nutrition, Atlanta, GA). The rumen-protected amino acids were mixed with ground barley ( 1 : 3 ratio) with a Hobart mixer to ensure more even distribution in the TMR. Samples of the Smartamine™ :barley mixes were later tested for stability (Rhone-Poulenc Animal Nutrition, Atlanta, GA). One-hundred percent of the lysine was found to be protected and 94% of the methionine was found to be protected. 3.2.5 Sampling 3.2.5.1 Feed. Feed was sampled and analyzed as described in Chapter II, section 2.2.4.1. 3.2.5.2 Milk. Milk yield was recorded twice daily for all cows. Milk samples were collected and analyzed as described in Chapter II, section 2,2.4.2, except that samples were 46 collected from each cow in the second and third weeks of each treatment period, from four consecutive milkings. 3.2.5.3 Rumen fluid Rumen fluid samples were collected and analyzed as described in Chapter II, section 2.2.4.3, except that samples were taken from each cow on days 14 and 21 of each period. 3.2.5.4 Blood. Samples of blood (20 ml) were taken in both heparinized and EDTA-containing vacutainers by jugular venipuncture on days 14 and 21 of each period between 10:00 A.M. and 11:30 A M . . Blood was centrifuged and plasma was frozen for later analysis of urea nitrogen and glucose using a Kodak Ektachem DT 60 Analyzer with Disc Two Module (Clinical Products Division, Eastman Kodak Co., Rochester, NY) and non-esterified fatty acid levels (NEFA kit, Wako Pure Chemical Industries, Ltd., Osaka, Japan). Red blood cell percentages were also determined. Free amino acid profiles were determined on the plasma samples by high performance liquid chromatography (HPLC), following derivitation with o-phthaldialdehyde, using the method of Puchala et al. (1994), after deproteinization with 5% trichloroacetic acid. The HPLC system used included two Waters Model 510 HPLC Pumps (Waters Corp., Milford, MA), a Waters 717plus Autosampler (Waters Corp., Milford, MA), a Supelcosil LC-18 HPLC Column (7.5 cm x 4.6 mm I.D.) (Supelco Ltd., Bellefonte, PA) and a Waters 474 Scanning Fluorescence Detector (Waters Corp., Milford, MA) with wavelength settings at 340 nm for excitation and 450 nm for emission. Millennium Chromatography Manager software (Waters Corp., Milford, MA) was used for automatically controlling the system as well as acquiring and treating chromatographic data. 47 3.2.6 Total collections For the last five days of each period, cows were confined to metabolism stalls for the total collection of urine and feces, as described in Chapter II, section 2.2.5 except that Foley bladder catheters (French 75, Rusch of Canada, Scarborough, ON) were used to facilitate collection of urine in this trial. Flexible rubber tubing connected the catheter to a stainless steel container. Cows were weighed on three consecutive days at the beginning of each period and at the end of the experiment to provide an estimate of changes in body weight. 3.2.7 Efficiency calculations Energetic efficiency values were calculated as described in Chapter II, section 2.2.6. 3.2.8 Statistical analysis Statistical analysis was via least squares ANOVA, following the general linear models procedure of SAS (1985). The model statement used is described in Chapter II, section 2.2.7. 3.3 Results 3.3.1 Feed composition Feed composition data is given in Tables 3.1 to 3.5. Crude protein levels were 18.3%, 16.7%, and 15.3%, for diets A, B, and C, respectively. Acid detergent fiber percentage was lower (P<0.05) for diet B than diets A or C (19.4,18.5, and 19.1%, for diets A, B and C, respectively). Ration A contained significantly higher (P<0.05) NDF than rations B and C (34.3, 32.5, and 32.8%, for diets A, B, and C, respectively). 48 3.3.2 Dietary intakes and milk production Dry matter intake, in kg d"1 or as a % of body weight, and milk production were significantly (P<0.05) higher for cows fed diet A than those fed diets B or C (Table 3.6). Fat-corrected milk production was also higher (P<0.05) for cows on diet A than those on diets B and C (32.8, 31.4, and 30.7 kg d'1, for diets A, B, and C, respectively). Body weight change was positive for all three diets. Milk fat % was not different (P>0.05) among treatments during week 2 (Table 3.7). During week 3, milk fat % was higher (P<0.05) for cows fed diet B when compared to those fed diet C, but was not different (P>0.05) from those on diet A (Table 3.7). No differences (P>0.05) were seen in milk fat output, milk protein % or milk protein output during week 2 or 3 of each experimental period. However, if data from week 3 is compared with results from week 2, milk fat %, milk fat output and protein % were significantly (PO.05) greater during week 3 than during week 2. 3.3.3 Blood composition Hematocrit and blood glucose values were unaffected by treatment (Table 3.8). Blood non-esterified fatty acid concentrations were also not different (P>0.05) among dietary groups. Blood urea nitrogen values were significantly different (P<0.05) among all dietary groups (Table 3.8). There were a number of differences in plasma amino acid levels related to the diet fed (Table 3.9). Plasma arginine and aspartate concentrations were significantly lower (P<0.05) for cows fed diet C than for cows fed diet A, but were not different (P>0.05) from those fed B. Isoleucine levels in plasma were lower (P<0.05) for cows fed diet C than for cows fed either ration A or B. Methionine concentrations in the plasma were higher (P<0.05) for cows fed diets supplemented with rumen-protected methionine (17.9, 25.1, and 23.8 nmol ml"1, for 49 diets A, B, and C, respectively). Plasma lysine levels were not influenced (P<0.05) by diet (Table 3.9). Free plasma threonine levels were higher (P<0.05) for cows fed ration B when compared to those fed diet C, but were not different (P>0.05) from cows fed diet A. Plasma valine concentrations of the cows decreased (P<0.05) significantly across diets A, B, and C, respectively. 3.3.4 Rumen fluid Rumen fluid pH was not affected (P>0.05) by dietary treatments (Table 3.10), although there was a trend to lower pH for cows fed diet C compared to those on diet A. No significant differences (P>0.05) were seen in rumen fluid volatile fatty acid concentrations (Table 3.10). 3.3.5 Water intake and waste excretion Water intakes (Table 3.11) were significantly lower (P<0.05) for cows fed diet C when compared to those on diet A, but were not different (P>0.05) from those fed diet B. Urine output decreased (P<0.05) across diets A, B and C. The differences observed in kg of fecal dry matter (Table 3.11) reflected the level of dry matter intake consumed by the animals as there were no differences (P>0.05) in dry matter digestibility (Table 3.12). 3.3.6 Apparent digestibility of nutrients The effect of diet on apparent digestibility of nutrients is given in Table 3.12. The apparent digestibility of nitrogen was significantly lower (P<0.05) for cows fed diet B compared to those on diet A, and for cows diet C compared to those fed diets B and A (76.4, 73.1, and 71.0%, for diets A, B, and C, respectively). No differences (P>0.05) were seen among dietary groups in the apparent digestibility of NDF, but 50 feeding diets B and C resulted in significantly (PO.05) higher apparent ADF digestibility than the control diet (A). 3.3.7 Nitrogen balance Nitrogen intakes were significantly (P<0.05) lower across three treatment groups (0.68, 0.58, and 0.53 kg d"1, for diets A, B, and C, respectively). Milk N output and fecal N excretion were not different (P>0.05) among dietary groups (Table 3.12). There was a dramatic reduction (P<0.05) in urinary N excretion when comparing cows fed diets B or C to those fed diet A, but N balance (Table 3.12) was not affected (P>0.05). 3.3.8 Nitrogen efficiency The percentage of N intake (Table 3.12) excreted in milk was greater (P>0.05) for cows fed diet B or C compared to those fed diet A (25.8, 29.3, and 33.1 %, for diets A, B, and C, respectively). Significant differences (P<0.05) were seen in percentage of dietary N excreted in urine among all dietary groups (38.9, 34.0, and 31.0% of dietary N, for diets A, B, and C, respectively). Significant (P<0.05) differences were also seen in percentage of dietary N excreted in feces among all dietary groups (23.5, 26.8, and 29.0%, for A, B, and C, respectively). 3.3.9 Energy efficiency Cows fed diet B appeared to gain weight at a faster rate than those fed diets A or C (Table 3.6), but the differences were not significant (P<0.05). Daily energy expenditure, for maintenance and milk production, was also not different (P>0.05) among dietary groups. Energy efficiency of ration dry matter utilization was significantly (P<0.05) greater for cows fed diet B than those fed ration A, but was not different from cows fed diet C (Table 3.13). 51 3.3.10 Fecal amino acid composition Data in Table 3.14 indicates that all fecal amino acid concentrations were greater (P<0.05) for cows on diet A, when compared to those fed diets B or C, except methionine which was not different (P<0.05) among diets. 3.4 Discussion Reports have indicated that since histidine, lysine, methionine, phenylalanine, and threonine are the most important essential amino acids for milk protein synthesis, methionine and lysine are most limiting for milk synthesis since they are both heavily utilized by the mammary gland and are present in low concentrations in plasma (Clark, 1975). Others have also noted that lysine and methionine were limiting for milk production (Schwab et al., 1976; Erfle and Fisher, 1977; Schwab et al, 1992a). Amino acid requirements depend on stage of lactation and level of production, making it difficult to balance dairy cattle rations to meet amino acid demands (Rode and Kung, 1996). The use of the Cornell Net Carbohydrate and Protein System as a basis of formulating diets and the inclusion of rumen-protected lysine and methionine in the rations seemed to aid in this regard, as was indicated by the maintenance of milk protein percentage and milk protein yield at the lower dietary crude protein levels, but dry matter intake and milk production were lower for cows on the low protein diets. Although milk production was not maintained for all three treatment groups, the higher milk production for cows fed diet A may be partially explained by a higher nutrient intake of those cows, as suggested by Kung and Huber (1983). Dry matter intake and milk production were both reduced for cows fed lower levels of crude protein so it is difficult to determine if the decrease in crude protein or energy intake was having more of an impact (Roffler et al., 1978). 52 Since all rations were, in theory, formulated to meet amino acid requirements of the cows, and the grain concentrate portions of the rations differed from each other in terms of composition, it is difficult to interpret the plasma amino acid concentrations. The CNCPS predicted absorbed essential amino acids, as a percentage of requirement, for the rations formulated were based on cows producing 39 kg of milk containing 3.5% fat and 3.2% protein. Lysine concentrations available at the small intestine would not appear to be limiting for cows fed any of the diets when comparing plasma lysine levels. However, the availability of lysine is difficult to predict because of its vulnerability to heat-damage and hence the variations in heat exposure of the various protein fractions fed (Schwab et al., 1992b). Methionine was not limiting in the low protein diets, as plasma amino acid concentrations increase only when supply is greater than demand (Clark, 1975). At higher protein intakes, increased activity of enzymes that degrade amino acids has been reported to be responsible for maintaining relatively constant amino acid levels in the plasma (Broderick et al., 1974). This may explain some of the other changes in plasma concentrations of the amino acids on the lower protein diets, but it is difficult to determine which of the amino acid differences are due to dietary differences and which are due to other factors. Comparison of the patterns of amino acid concentrations in the feces would indicate that the rumen-protected lysine and methionine were not over-protected. The improvement in nitrogen efficiency has important implications for both dairy producers and the environment. Not only was waste nitrogen excretion reduced by as much as 26% in the present study, but this also translated into an improvement in the proportion of dietary N excreted in milk. Improved N efficiency resulted in lower 53 blood urea nitrogen (BUN) values, which could also result in improved cow fertility as high BUN levels have been shown to result in reduced conception rates in dairy herds (Ferguson etal., 1993). Concentrations of BUN reflected dietary CP levels, as has been reported in previous studies (Wohlt et al., 1991; Christensen et al., 1993; Roseler et al., 1993). Blood urea nitrogen levels have been reported to reflect protein metabolism in the cow (Baker et al., 1995). Excess protein, whether degradable or undegradable intake protein, will elevate BUN since ruminal and post-ruminal N excesses are converted to urea by the liver (Roseler et al., 1993). This includes ammonia produced in the rumen that is not used for microbial protein production and that from the deamination of amino acids arising from protein escaping ruminal degradation (Butler et al., 1995). Not only is excess dietary protein expensive, but there is also the energy cost to the animal associated with conversion of ammonia to urea. Nitrogen excesses were much lower for the low protein diets, as indicated by the linear decreases in both BUN and urinary N excretion. A decrease in the proportion of endogenous fecal N from cows on the higher CP diets may have improved CP apparent digestibilities (Ha and Kennelly, 1984). This would explain the significantly higher proportion of dietary N excreted in feces at the low CP levels in the current study, as Tamminga (1992) previously noted that reducing the amount of undigested feed in the feces will not sufficiently influence the level of N excretion from the body. Although there were significant differences in fiber composition of the rations in the present study, the differences were small enough that nutrient digestion was not affected to any great extent as indicated by the similarities in rumen VFA levels. The 54 lower apparent digestibility of ADF for diet A may have been due to an increased flow of digesta resulting from the higher dry matter intakes of those cows. Ha and Kennelly (1984) noted that dietary CP concentrations did not affect rumen pH, which agrees with results of the present study. Increased water intakes and subsequent urine output on the higher protein diets may have been due to the diuretic effect of increased protein intake (Broderick et al., 1974). The improved energetic efficiency of cows fed diet B compared to diet A was due to a combination of lower dry matter intakes for cows fed B and a tendency for those cows to be gaining more weight. This trend toward increased body weight gains may have been due to the high levels of available methionine, as indicated in the plasma, since methionine is involved in fat metabolism (Rode and Kung, 1996). Improved apparent CP digestibilities have been reported when dietary protein increased (Ha and Kennelly, 1984; Klusmeyer et al., 1990; Wohlt et al., 1991). This agrees with the present results. It has been suggested that higher DM and CP apparent digestibilities at higher dietary protein intakes result from a combination of improved microbial fermentation and greater digestion in the gastrointestinal tract. (Ha and Kennelly, 1984). The reduced CP digestibilities on the lower protein diets may also be due, in part, to differences in degradation of dietary CP fractions for the three diets. Although apparent CP digestibilities improved as dietary CP levels increased, apparent DM digestibilities remained constant, indicating that N levels were adequate for ruminal microorganisms. A reduction in microbial protein synthesis and organic matter (OM) degradation has been noted when there is a deficiency of N for ruminal bacteria (Clark et al., 1992). 55 The Net Carbohydrate and Protein System has been suggested to estimate dietary protein degradability and to predict accurately if rumen microorganisms have access to adequate levels of ammonia and peptides (Chalupa and Sniffen, 1993). Although the CNCPS model can be used to balance rations that improve nitrogen utilization, the cost and time associated with the detailed chemical analysis makes the widespread adoption of this model unlikely. The difficulty and time involved in becoming familiar with the program also makes its usefulness limited. Results of this study indicate that cows should not be fed to maximize milk production, when efficient utilization of dietary nitrogen is the major objective. The supplementation of low CP diets with rumen-protected lysine and methionine can improve N utilization of dairy rations. With a greater N utilization and lower N intake, cows fed low protein diets have less of an impact on the environment. If government regulations are implemented that limit N application per area of land, diets being fed on farms could dictate the number of animals available for production. Rations formulated to improve N efficiency result in less N excreted in urine and feces, and therefore more animals could be farmed per area of land. 56 3 . 5 R e f e r e n c e s A r m e n t a n o , L . E . , S w a i n , S . M . , a n d D u c h a r m e , G . A . 1 9 9 3 . Lactation response to ruminally protected methionine and lysine at two amounts of ruminally available nitrogen. J. Dairy Sci. 7 6 : 2963-2969. B a k e r , L . D . , F e r g u s o n , J . D . a n d C h a l u p a , W . 1 9 9 5 . Responses in urea and true protein of milk to different protein feeding schemes for dairy cows. J. Dairy Sci. 7 8 : 2424-2434. B r o d e r i c k , G . A . , S a t t e r , L . D . , a n d H a r p e r , A . E . 1 9 7 4 . Use of plasma amino acid concentration to identify limiting amino acids for milk production. J. Dairy Sci. 5 7 : 1015-1023. B u t l e r , W . R . , C h e r n e y , D . J . R . a n d E l r o d , C . C . 1 9 9 5 . Milk urea nitrogen (MUN) analysis: field trial results on conception rates and dietary inputs. Proc. Cornell Nutrition Conference, pp. 89-95. C h a l u p a , W . , a n d S n i f f e n , C . J . 1 9 9 3 . Protein and amino acid nutrition of lactating dairy cattle - today and tomorrow. 1993 Western Nutrition Conference, pp. 87-114. C h r i s t e n s e n , R . A . , L y n c h , G . L . a n d C l a r k , J . H . 1 9 9 3 . Influence of amount and degradability of protein on production of milk and milk components by lactating Holstein cows. J. Dairy Sci. 7 6 : 3490-3496. C l a r k , J . H . 1 9 7 5 . Lactational responses to postruminal administration of proteins and amino acids. J. Dairy Sci. 5 8 : 1178-1197. C l a r k , J . H . , K l u s m e y e r , T . H . , a n d C a m e r o n , M . R . 1 9 9 2 . Microbial protein synthesis and flows of nitrogen fractions to the duodenum of dairy cows. J. Dairy Sci. 7 5 : 2304-2323. C o l i n - S c h o e l l e n , O . L a u r e n t , F . , V i g n o n , B . , R o b e r t , J . C , a n d S l o a n , B . 1 9 9 5 . Interactions of ruminally protected methionine and lysine with protein source or energy level in the diets of cows. J. Dairy Sci. 7 8 : 2807-2818. E r f l e , J . D . a n d F i s h e r , L . J . 1 9 7 7 . The effects of intravenous infusion of lysine, lysine plus methionine or carnitine on plasma amino acids and milk production of dairy cows. Can. J. Anim. Sci. 5 7 : 101-109. F e r g u s o n , J . D . , G a l l i g a n , D . T . , B l a n c h a r d , T . , a n d R e e v e s , M . 1 9 9 3 . Serum urea nitrogen and conception rate: the usefulness of test information. J. Dairy Sci. 7 6 : 3742-3746. F o x , D . G . , S n i f f e n , C . J . , O ' C o n n o r , J . D . , R u s s e l l , J . B . , a n d V a n S o e s t , P. J . 1 9 9 0 . The Cornell Net Carbohydrate and Protein System for Evaluating Cattle Diets. 57 Part 1: A model for predicting cattle requirements and feedstuff utilization. Search:Agriculture, No. 34, pp7-83. Cornell Univ. Agr. Exp. Sta., Ithaca, NY. Ha, J. K. and Kennelly, J. J. 1984. Effect of protein on nutrient digestion and milk production by Holstein cows. J. Dairy Sci. 67: 2302-2307. Klusmeyer, T. H., McCarthy, R. D., Jr., Clark, J. H., and Nelson, D. R. 1990. Effects of source and amount of protein on ruminal fermentation and passage of nutrients to the small intestine of lactating cows. J. Dairy Sci. 73: 3526-3537. Kung, L., Jr., and Huber, J. T. 1983. Performance of high producing dairy cows in early lactation fed protein of varying amounts, sources, and degradability. J. Dairy Sci. 66: 227-234. O'Connor, J. D., Sniffen, C. J., and Fox, D. G. 1990. The Cornell Net Carbohydrate and Protein System for Evaluating Cattle Diets. Part 2: A computer spreadsheet for diet evaluation. Search:Agriculture, No. 34, pp4-40. Cornell Univ. Agr. Exp. Sta., Ithaca, NY. O'Connor, J. D., Sniffen, C. J., Fox, D. G., and Chalupa, W. 1993. A net carbohydrate and protein system for evaluating cattle diets: IV. Predicting amino acid adequacy. J. Anim. Sci. 71:1298-1311. Puchala, P., Pior, H., von Keyserlingk, M. A. G., Shelford, J. A., and Barej, W. 1994. Determination of methionine sulfoxide in biological materials using HPLC and its degradability in the rumen of cattle. Anim. Feed. Sci. Tech. 48: 121-130. Robinson, P. H., Fredeen, A. H., Chalupa, W., Julien, W. E., Sato, H., Fujieda, T., Suzuki, H. 1995. Ruminally protected lysine and methionine for lactating dairy cows fed a diet designed to meet requirements for microbial and postruminal protein. J. Dairy Sci. 78: 582-594. Rode, L. M. and Kung, L, Jr. 1996. Rumen-protected amino acids improve milk production and milk protein yield, in Advances in Dairy Technology Proceedings of the Western Canadian Dairy Seminar, ed. Kennelly, J . J . University of Alberta, Edmonton, Canada. Roffler, R. E., Satter, L. D., Hardie, A. R., and Tyler, W. J. 1978. Influence of dietary protein concentration on milk production during early lactation. J. Dairy Sci. 61: 1422-1428. Rogers, J. A., Peirce-Sandner, S. B., Papas, A. M., Polan, C. E., Sniffen, C. J., Muscato, T. V., Staples, C. R., and Clark, J. H. 1989. Production responses of dairy cows fed various amounts of rumen-protected methionine and lysine. J. Dairy Sci. 72: 1800-1817. 58 Roseler, D. K., Fergusen, J. D., Sniffen, C. J., and Herrema, J. 1993. Dietary protein degradability effects on plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein cows. J. Dairy Sci. 76: 525-534. Russell, J. B., O'Connor, J. D., Fox, D. G., Van Soest, P. J. and Sniffen, C. J. 1992. A net carbohydrate and protein system for evaluating cattle diets: I. ruminal fermentation. J. Anim. Sci. 70: 3551-3561. Schwab, C. G., Satter, L. D., and Clay, A. B. 1976. Response of lactating dairy cows to abomasal infusion of amino acids. J. Dairy Sci. 59: 1254-1270. Schwab, C. G., Bozak, C. K., Whitehouse, N. L, and Mesbah, M. M. A. 1992a. Amino acid limitation and flow to duodenum at four stages of lactation. 1. Sequence of lysine and methionine limitation. J. Dairy Sci. 75: 3486-3502. Schwab, C. G., Bozak, C. K., Whitehouse, N. L, and Olson, V. M. 1992b. Amino acid limitation and flow to the duodenum at four stages of lactation. 2. Extent of lysine limitation. J. Dairy Sci. 75: 3503-3518. Sniffen, C. J., O'Connor, J. D., Van Soest, P. J., Fox, D. G., and Russell, J. B. 1992. A net carbohydrate and protein system for evaluating cattle diets: II. carbohydrate and protein availability. J. Anim Sci. 70: 3562-3577. Statistical Analysis System (SAS). 1985. SAS User's Guide: Statistics. SAS Institute Inc., Cary, N.C. Tamminga, S. 1992. Nutrition management of dairy cows as a contribution to pollution control. J. Dairy Sci. 75: 345-357. Wohlt, J. E., Chmiel, S. L., Zajak, P. K., Backer, L, Blethen, D. B., and Evans, J. L. 1991. Dry matter intake, milk yield and composition, and nitrogen use in Holstein cows fed soybean, fish, or corn gluten meals. J. Dairy Sci. 74: 1609-1622. 59 Table 3.1. Nutrient composition (DM basis) of forages nutrient corn silage grass silage % DM 30.00 27.10 % C P 8.45 13.62 % ADF 24.21 31.44 % NDF 42.65 52.48 % AD IP 3.09 3.33 % NDIP 3.04 5.99 % lignin 3.80 4.85 % ash 4.48 9.43 % fat 3.06 4.00 1DM, CP, ADF, NDF, ADIP, and NDIP stand for dry matter, crude protein, acid detergent fiber, neutral detergent fiber, acid detergent insoluble protein, and neutral detergent insoluble protein, respectively. 60 Table 3.2. Amino acid composition of the forages (% UIP1) amino acid corn silage grass silage methionine 0.89 0.95 lysine 4.36 4.24 arginine 3.64 3.18 threonine 3.52 3.64 leucine 6.94 6.96 isoleucine 3.45 3.77 valine 4.20 4.39 hisitidine 1.26 1.39 phenylalanine 3.97 4.53 UIP refers to undegraded intake protein after 12 h incubation in the rumen. 61 Table 3.3. Degradability of the nitrogen fractions of forages in situ (%) corn silage grass silage soluble 43.54 51.90 potentially degradable 44.29 40.19 Kd 1 (% IV1) 0.068 0.060 lag 2.0 0.63 ED 2 (0.04) 69.65 74.88 ED 2 (0.06) 64.95 70.54 Rate of degradation in the rumen. Effective degradability of nitrogen (0.04 and 0.06 refer to hypothetical rates of passage used in the degradability calculations). 62 Table 3.4. Composition of concentrate mixtures (kg t"1) diet A B C ' barley 625.6 463.5 497.6 canola meal 187.7 0 0 soybean meal 153.8 235.3 130.6 cracked corn 0 267.4 337.7 urea 0 0 0.783 multifos 15 15 15 lime 10 10 10 cobalt iodized salt 5 5 5 vit-min premix 5 5 5 1Smartamine M and ML top-dressed manually on each cow's TMR each feeding (animals on diet B received 13g Smartamine™ M and 12g Smartamine™ ML daily; animals fed diet C received 7g Smartamine™ M and 15g Smartamine™ ML daily). Table 3 .5 . C n diet A B C S T E crude protein 201 18.3a 16.7b 15.3c ±0.1 acid detergent fiber 198 19.4a 18.5b 19.1a ±0.1 neutral detergent fiber 198 34.3a 32.5b 32.8b ±0.2 a, b, c Means in the same row with different letters differ significantly (P<0.05) TJ CD J* CD ±£ CO CD iTJ 0) CO E TJ C o TJ CD -21 O CD 0 TJ H C0 | CD 00 0) Xi CO I-LU CN oo O o CO T— 00 CO d d •H oo -H d d -H d -H Xi Xi LO o oo Xi oo Xi o CN CN cd CD d CN 00 d oo Xi CO Xi CO LO Xi oo Xi m CN CN oo LO CD d CN OO CO CO CO CD CD 00 CO CO CN CO oo < CO CN CO 00 co d CO CN CO CN O CN CN O) oo oo CN o CN £ T— CD ^ 2 5 .£ m i — u -0) O ro s£ • E r TJ Q O) CD O) ^ "5 x: CD >% TJ O Xi x: O) CD TJ O Xi TJ CD O) ; * 0 2 1 a TJ vg 2 § Q . 65 CD c T J •a T J CD •~ a c _ CD ro 11 E .§. E " -D O C CO co a> § ° 2 0 0 CO TJl O C CL (0 E CM 8 j» ^ CD I I co a) jQ co | T J UJ CD CD o LO o CO o CO o ai O +1 c i +1 d +1 d +1 d +1 X) CM LO CM CD CD o O co CO CO ^— CO T— ,9ab CN LO LO CM o CQ CO CO CO CO CO LO CO CD CD o CM o < LO CO CO CO CD CD CD CD CD T J CD T J CM CD j*: CD CD E CD CD T J CD >> C 0) o CL LO CO o o CM O CM O d +i d +i d +i d +1 d +1 2ab LO CD T-CO CM h-o CO CO co CO T— .a CO o o NT CM LO CM LO q CO CO CO CO co CO CO O CO CO CM CM CO CO CO CD 1 T J CO T J CO CD J £ >. CD CD E o LO o LO o LO CO o LO = ^ o o m >- <-a ex CD T J CD c CD  I— C L Table 3.8. The effect of diet formulation on blood composition diet n S. E. A B C hematocrit (%) 101 30.3 30.7 30.7 ±0.3 glucose (mg dl"1) 101 65.5 65.8 67.3 ±0.7 urea nitrogen (mg dl"1) 101 15.9a 12.9b 10.0c ±0.3 NEFA (mEq r1) 101 0.10 0.12 0.09 ±0.01 a, b, c Means in the same row with different letters differ significantly (P<0.05). Table 3.9. The effect of diet on plasma amino acid composition (nmol ml"1) n diet A B C S. E. alanine 99 183 176 175 ±5 arginine 99 81a 78ab 73b ±2 aspartate 99 20a 18ab 17b ±1 glutamate 99 104 107 112 ±3 glycine 97 175 184 197 ±8 isoleucine 99 102a 93a 80b ±3 leucine 99 95 92 87 ±3 lysine 99 74 72 67 ±3 methionine 99 17.9b 25.1a 23.8a ±0.8 phenylalanine 99 48 41 46 ±5 serine 99 77 71 73 ±3 threonine 97 146ab 149a 134b ±5 tyrosine 99 44 43 39 ±2 valine 99 220a 195b 163c ±8 a, b Means in the same row with different letters differ significantly (P<0.05) Table 3.10. The effect of diet on rumen fluid pH and volatile fatty acid concentration (mg ml"1) n diet A B C S. E. PH 101 6.85 6.79 6.75 ±0.04 acetate 98 2.55 ' 2.49 2.60 ±0.07 propionate 98 1.30 1.25 1.32 ±0.05 isobutyrate 98 0.060 0.062 0.055 ±0.003 butyrate 98 0.72 0.73 0.76 ±0.03 isovalerate 98 0.120 0.127 0.116 ±0.005 valerate 98 0.130 0.121 0.114 ±0.006 Table 3.11. The effect of diet on water intake, urine output, and fecal output n diet A B C S. E. water intake (I d'1) 43 75.3a 72.3ab 68.5b ±2.0 urine output (I d"1) 48 23.3a 20.6b 17.7c ±0.6 feces output (kg d"1) 48 38.8a 35.9b 35.4b ±0.8 feces output (kg DM d'1) 48 5.8a 5.4b 5.4b ±0.1 feces dry matter (%) 48 15.2 15.0 15.1 ±0.1 a, b, c Means in the same row with different letters differ significantly (P<0.05). 70 CD [TJ — D) i _ CO u CD "O M i— CO CO X i Q . . CL ' CO i _ C 8 o co £ > , c o O O i s I CD X CD TJ O p ® .9 — x: CO ° w ©I o _ £ . g * CD ~ C CD CD CD I- o CN CO 0) X i CO H | L U !co ICQ oo LO CN o o O T— LO CD CD o o o O o c i c i d d d d d d d -H -H -H -H -H •H -H -H -H o o V CN x— O CO oo CD CO CO o LO co LO o O oo CO LO T— T— o X— o O LO CN CD d d 00 d d CD d CO LO 00 00 CN Si Si LO LO X i CO CO Si O) LO LO o T— LO CD LO 00 T— o T— oo o LO CO 00 CO d d CD d d CD d CD LO CN oo CM CO CO CD 00 C0 X i oo o CD CO LO O 00 CO CD CD T— oo CN CD t — LO o CD CN LO d d LO d CO d 00 d h~ CO LO CN oo CN 00 00 OO OO oo 00 OO CO 00 CD CD 5 " 8 to CD D) TJ t_ 0) "co E TJ 2> >» >, TJ C O Q . CD TJ i _ O CD O) E 9 to CD O) TJ Ll_ Q < CD CO CD CO c o TJ ~ -9 TJ 2 rj) o v x CD z z 1 E O) CD CO O) CO c CD O 1— *- +2 CD CO C o O) CD o X CD CO c s s - , <D <D iLO I O i d v i o- . _>» -I—* C 8 > ^ ' c to [I : TJ to c CD I— E£ JTJ ix: o CD E CO CO CO c CO CD o i x f co" CD T J LU CO CQ O +1 LO CO CO CO CO CD CO CO LU CO o (D T J c CD CL X 0 > » CD c CD LO O O -H . O CO CO CO CO LO CD LO CO O) LU 3 8 c CD o it CD n diet A B C S. E. alanine 49 1.32a 1.05b 1.15b 0.05 arginine 49 0.69a 0.54b 0.56b 0.03 aspartine 49 1.99a 1.60b 1.68b 0.07 glutamine 49 2.37a 1.89b 2.02b 0.09 glycine 49 0.93a 0.75b 0.81b 0.04 histidine 49 0.42a 0.30b 0.32b 0.02 isoleucine 49 0.87a 0.68b 0.72b 0.03 leucine 49 1.38a 1.06b 1.14b 0.06 lysine 49 1.22a 0.88b 0.98b 0.04 methionine 49 0.20 0.17 0.20 0.01 phenylalanine 49 0.90a 0.72b 0.77b 0.03 serine 49 0.80a 0.61b 0.65b 0.03 threonine 49 1.09a 0.79b 0.84b 0.03 tyrosine 49 0.74a 0.60b 0.63b 0.03 valine 49 1.05a 0.83b 0.88b 0.04 a, b Means in the same row with different letters differ significantly (P<0.05). 73 IV. GENERAL CONCLUSIONS Results of these studies indicate that low protein diets can result in dramatic reductions in the excretion of nitrogen in urine and feces from lactating dairy cows. However, it is essential to ensure that the rations are properly balanced to maintain milk protein yields. In doing so, not only are nitrogen intakes reduced, but nitrogen efficiency expressed as milk nitrogen as a percentage of nitrogen intake can also be improved. The incorporation of rumen-protected lysine and methionine into low protein rations is an option that can be used to aid in this regard. In these trials, the low protein diets supplemented with rumen-protected lysine and methionine did, however, result in lower dry matter intakes, and thus milk production was not maintained due to reduced energy or crude protein intake. Therefore, dairy producers should reduce dietary crude protein levels and strive for an optimum, rather than maximum, milk production if nitrogen efficiency is to be improved through the reduction of nitrogen excretion in urine and feces. By reducing urea excretion the resulting manure nitrogen will be more stable, resulting in lower ammonia emissions from the manure and therefore less pollution to the environment. The assumed specificity and accuracy of the Cornell Net Carbohydrate and Protein System in terms of ration composition and animal requirements can assist in improving the efficiency of use of protein, but the time and expense involved in determining the detailed chemical analysis of the ration components makes its usefulness limited and its widespread application unlikely. As Tamminga (1992) indicated is the case in The Netherlands, there is increasing pressure from the government and the general public for dairy producers 74 to reduce environmental pollution. This is very evident in the Lower Fraser Valley of British Columbia, where there is a large concentration of animal production occurring on a shrinking land base. Therefore, it is not unlikely that government regulations will be put in place to limit the amount of manure nitrogen that can be applied per area of land. Thus, the adoption of such measures to reduce the excretion of waste nitrogen will be essential if farmers want to maintain animal numbers and economic viability. 

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