NUTRITIONAL STRATEGIES TO IMPROVE NITROGEN EFFICIENCY AND REDUCE NITROGEN EXCRETION OF LACTATING DAIRY COWS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Susan M. Noftsger, M.S. The Ohio State University 2003 Dissertation Committee: Dr. Normand R. St-Pierre, Advisor Approved by Dr. Jeffrey L. Firkins Dr. William P. Weiss ________________________ Dr. Mark Morrison Advisor Animal Science Graduate Program ABSTRACT Accurate prediction of amino acids (AA) reaching the intestine is imperative if reduction in N excretion by dairy cows is to be achieved through nutrition strategies. Provision of feed amino acids to the metabolizable supply is dependent on the rumen degradability of protein sources and their digestibility in the intestine. In the first experiment, we hypothesized that milk production and composition could be maintained and dietary crude protein (CP) decreased to improve efficiency of N utilization through selection of highly intestinally digestible rumen undegradable protein (RUP) and supplementation of Met. Sixty cows in their fourth week of lactation were assigned to four diets for 12 weeks in a balanced randomized block design. Diets differed in estimated digestibility RUP (high vs. low), CP concentration (17 vs. 18.3%), and supplementation of Met in rumen degradable and undegradable forms. Diets contained equal concentrations of net energy for lactation (NEL), acid detergent fiber (ADF), neutral detergent fiber (NDF), and ash. Maintaining the higher concentration of dietary RUP while increasing metabolizable protein (MP) concentration through higher intestinal digestibility of RUP (HiCP-HiDRUP), and decreasing dietary CP and supplementing Met (LoCP-HiDRUP + Met) increased milk yield and component production over the control (HiCP-LoDRUP). Both higher digestibility of the RUP source and supplemental Met were necessary to maintain milk production when reducing CP in the diet. Supplemental ii Met effects of rumen available 2-hydroxy-4-(methylthio) butanoic acid (HMB) and rumen bypass (polymer coated dl-Met; Smartamine) could not be differentiated in this trial. Lowering dietary RUP while maintaining MP concentration through higher intestinal digestibility of RUP (LoCP-HiDRUP) allowed some increases in milk, protein, and fat production, further indicating the nutritional limitations of the unselected protein source. Supplementing the highly digestible RUP source with rumen available and rumen escape sources of Met resulted in maximal milk and protein production and maximum N efficiency by cows, indicating that post-ruminal digestibility of RUP and AA balance can be more important than total RUP supplementation. In this study, the combined supplementation of Met in rumen available and rumen undegradable forms had a greater impact on milk protein yield than expected from prior research when both sources were not fed in combination. This points to an additive effect of the two sources, indicating that the HMB has a different mechanism of action, possibly through a stimulation of microbial growth. The hypotheses for the second experiment were that HMB supplementation increases microbial growth by either sparing Met precursors for more efficient protein synthesis or by shifting rumen bacterial species. These effects were quantified by measuring effluent flow of N; digestibilities of organic matter (OM), ADF, NDF and hemicellulose; and volatile fatty acid (VFA) concentrations in continuous culture. Four dietary treatments consisted of a control, two concentrations of HMB (0.055%, 0.110% of DM) and one concentration of dl-Met (0.097%). Digestibilities of OM, hemicellulose, and NDF were largely insensitive to treatment. Digestibility of ADF showed a quadratic iii effect to HMB in the diet, being highest at 0 and 0.11% HMB. There were trends (P < 0.15) for linear increases in production of isobutyrate and isovalerate with increasing concentration of HMB, whereas isobutyrate concentration increased linearly. Valerate concentration was affected quadratically by concentration of HMB, with estimated peak concentration at 0.055%. Propionate concentration decreased linearly with level of HMB supplementation, whereas a quadratic trend (P = 0.13) was noted for its production, which was estimated to peak at 0.047% HMB. There was a significant linear decrease (P = 0.032) in the amount of bacterial N obtained from NH3-N as concentration of HMB increased. The lack of major effects may indicate that a high rumen degradable protein (RDP) concentration in the diet provided enough excess AA so that Met was not limiting, that changes in bacterial populations were small and were concealed by the larger unaffected populations of common species of rumen bacteria, or that there may be a pronounced effect on protozoa in the rumen which are not maintained in continuous culture fermenters. The hypotheses for the third trial were: (1) that proportions of dl-Met and HMB escaping rumen metabolism through passage with the liquid fractions through the omasum are small, (2) that dl-Met and HMB effects are ruminal, and (3) that HMBi (the isopropyl ester of HMB) does supply metabolizable Met, resulting in milk composition effects characteristic of an increased Met supply. These effects were quantified using eight ruminally-cannulated cows. Effects on milk production, N utilization, ruminal VFA, and protozoa were determined. Samples of omasal fluid were used to determine the amount of Met supplements passing out of the rumen. Treatments were: (1) no iv methionine (Control); (2) 2-hydroxy-4-methylthiobutanoic acid (HMB) at 0.10% of DM; (3) isopropyl HMB (HMBi) at 0.13 % of DM; and (4) dl-methionine (dl-Met) at 0.088% of DM. Dry matter intakes were not different and averaged 20 kg/d. Milk yields averaged 37.7 kg and were not different among treatments. Milk protein concentration was significantly affected by treatment (2.91, 2.95, 3.02, 2.96%) for control, HMB, HMBi, and dl-Met, respectively, but milk fat concentration was not affected. Short-term increase in milk true protein content is characteristic of an increase in metabolizable Met supply. Rumen VFA profile and NH3 concentrations were similar among treatments. Total tract digestibilities of OM, NDF, and N were not different. In situ rate of digestibility of NDF in TMR was increased with HMBi, whereas rate of in situ digestibility of alfalfa hay CP was increased by HMB and dl-Met when compared with control or HMBi. Passage rates of small particles (0.071/h) and fluid (0.157/h) were not affected by treatment. Protozoa were increased numerically, but not statistically, in the omasum by HMB and HMBi treatments. The percentage of HMB ingested that passed into the omasum was 5.3% (± 1.5%). This, along with numerical protozoal increases and digestibility changes with HMB and HMBi, indicated that most of the activity of HMB was in the rumen. Only a small amount of HMBi was found as HMB in the omasum (2.3%). If HMBi remaining in the rumen is assumed to break down to HMB and isopropanol and act similarly to unmodified HMB, this number would be consistent with prior research claiming that 50% of the HMBi is absorbed through the rumen wall, while the remaining 50% is converted to HMB in the rumen and is rapidly used by the microbial population. v ACKNOWLEGMENTS I would like to thank my advisor, Normand St-Pierre, for intellectual support, encouragement, and enthusiasm, which made this thesis possible. I would also like to thank Dr. Jeff Firkins for taking the time to help me out. I thank my fellow graduate students, both for helping me with the animal experiments and in the lab, and allowing me to bounce ideas off of them, especially John Sylvester, Dana Harvatine, and Carine Reveneau. I also wish to thank the undergraduate students who have helped me at the farm and the lab, and without whom I would never have finished. Last, I would like to express my thanks to Dr. Brian Sloan and his colleagues from Adisseo, Aventis, and Rhone-Poulenc for their intellectual as well as financial support of my research. The financial support of Venture Milling, a division of Perdue Farm must also be acknowledged and was greatly appreciated. vi VITA August 24, 1971…………………………………………………….Born-Shelby, NC 1992-1997…………………………………………………….……Research Assistant North Carolina State University Dairy Research and Teaching Unit 1997-1999…………………………………………………………Graduate Teaching and Research Assistant, North Carolina State University 1999-Present………………………………………………………Graduate Research Assistant The Ohio State University PUBLICATIONS Peer-Reviewed Journal Articles Noftsger, S. M., N. R. St-Pierre, S. K. R. Karnati, and J. L. Firkins. 2003. Effects of 2- Hydroxy-4-(methylthio) butanoic acid (HMB) on microbial growth in continuous culture. J. Dairy Sci. J. Dairy Sci. 86:2629-2636. Noftsger, S., and N. R. St-Pierre. 2003. Supplementation of methionine and selection of highly digestible rumen undegradable protein to improve nitrogen efficiency for milk production. J. Dairy Sci. 86:958-969. Firkins, J. L., M. L. Eastridge, N. R. St-Pierre, and S. M. Noftsger. 2001. Effects of grain variability and processing on starch utilization by lactating dairy cattle. J. Anim. Sci. 79(E. Suppl.):E218-E238. Noftsger, S. M., B. A. Hopkins, D. E. Diaz, C. Brownie, and L. W. Whitlow. 2000. Effect of whole and expanded-expelled cottonseed on milk yield and blood gossypol. J. Dairy Sci. 83:2539-2547. vii Abstracts J. T. Sylvester, N. R. St-Pierre, B. K. Sloan, J. L. Beckman, and S. M. Noftsger. 2003. Effect of HMB and HMBi on milk production, composition, and N efficiency of Holstein cows in early and mid-lactation. J. Dairy Sci. 86(Suppl. 1):60. Noftsger, S.M., J. Firkins, and N. St-Pierre. 2002. Effects of 2-hydroxy-4-(methylthio) butanoic acid (HMB) and dl-methionine on microbial growth, VFA production and nutrient digestion in continuous culture. J. Dairy Sci. 85(Suppl. 1):240. Noftsger, S. and N. St-Pierre. 2001. Effects of rumen undegradable protein digestibility and supplemental methionine on production parameters and nitrogen efficiency of Holstein cows in early lactation.