2004 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The major issue addressed in this research is inefficient use of dietary crude protein (nitrogen) by lactating dairy cows. This matters because inefficient nitrogen utilization makes it necessary to feed large amounts of crude protein to dairy cows, thereby increasing feed costs and increasing nitrogen excretion in manure. When protein efficiency is depressed, or when dairy farmers overfeed protein, virtually all of the extra nitrogen is excreted as urinary nitrogen, the form that gives rise to the greatest pollution of both the air and surface and ground water. Dairy farms are major non-point sources of nitrogen pollution of the nation¿s streams and rivers and atmosphere. Specific ways in which this problem is being addressed include:. 1)developing effective ways to reduce silage nonprotein nitrogen (which will improve nitrogen efficiency in dairy cows);. 2)developing rapid and accurate tests for rumen protein breakdown (a major factor influencing nitrogen use by dairy cows;. 3)determining the factors that influence protein formation by the rumen microbes (which will minimize the nitrogen lost from dairy cows; and. 4)identifying management practices so farmers can reduce purchases of feed protein and nitrogen fertilizer (to reduce overall nitrogen pollution coming from dairy farms). 2.List the milestones (indicators of progress) from your Project Plan. Objective 1. Develop and test a series of methods for reducing excessive formation of NPN during ensiling of legumes to improve efficiency of utilization of forage protein in lactating dairy cows. i. Begin characterization of the mechanism of polyphenol oxidase (PPO) reduction of proteolysis in silages. ii. Evaluate methods for PPO activity for screening divergent genotypes of red clover germplasm. iii. Determine inhibition characteristics of quinones involved in PPO action on silage proteolysis. iv. Complete characterization of the recombinant PPO system in terms of proteolytic inhibition. v. Determine binding sites of quinones on proteolytic enzymes. vi. Select and intercross high and low PPO activity red clover genotypes to generate two divergent populations with high and low PPO activity. vii. Develop regression predictions of optimal nonprotein nitrogen (NPN) in alfalfa silage to maximize crude protein (CP) utilization. viii. Establish high and low PPO populations in field plots to generate materials for future research. ix. Test high and low PPO red clover populations in small-scale silos and ruminal in vitro studies. x. Evaluate high and low PPO red clover populations using in vivo feeding studies. xi. Use formic acid treatments to define "target" NPN levels in alfalfa silage fed to lactating cows (small scale trial 1). xii. Use formic acid treatments to define "target" NPN levels in alfalfa silage fed to lactating cows (large scale trial 2). Objective 2. Develop and evaluate rapid and accurate in vitro methods, based on ruminal inocula, blends of commercial enzymes with proteolytic activity mimicking ruminal microbes, and near infrared reflectance spectroscopy, for quantifying protein degradation in the rumen. xiii. Complete adaptation of fluorescent assays for NH3 and total amino acids. xiv. Confirm the value of the Michaelis-Menten inhibitor in vitro (IIV) system for reliably estimating first order degradation rates for protein mixtures. xv. Begin assessing protease-fingerprinting systems using the protein inhibitory effect. xvi. Complete a second study estimating in vivo rates and extents of ruminal degradation for four additional proteins to use as in vitro standards. xvii. Validate Michaelis-Menten IIV degradation rates using the standard proteins and the in vitro 15N method. xviii. Complete fingerprinting of proteolytic activity of mixed ruminal organisms and commercial proteases. xix. Begin testing of NIRS calibrations for estimating ruminal protein degradation. xx. Elaborate protease blends and test these against the other (standard) in vitro systems. xxi. Continue testing of NIRS calibrations for determining ruminal protein degradation. xxii. Begin NRC (2001) model validation of results on protein degradation. xxiii. Using protein standards from in vivo research, complete validation of methods based on Michaelis-Menton IIV, commercial proteases and NIRS for assaying protein degradation. xxiv. Complete NRC (2001) model validation of results on protein degradation. Objective 3. Quantify requirements of ruminal microbes for degraded protein and fermentable energy with the objective of developing practical strategies for optimizing the balance between protein degradation and ruminal protein escape to minimize losses of degraded N from the rumen. xxv. Milk production and omasal sampling to assess level of RDP required for ruminal microbial growth. xxvi. Milk production and omasal sampling to identify optimal forage source (trial 1). xxvii. Milk production and omasal sampling to study dietary CP levels (trial 1). xxviii. Milk production and omasal sampling to identify optimum levels for sugar (molasses) feeding for utilizing alfalfa silage NPN (trial 1). xxix. Milk production and omasal sampling to study starch source and processing (trial 1). xxx. Milk production and omasal sampling to identify maxima for pectin feeding (trial 1). xxxi. Milk production and omasal sampling to assess source of RDP required for ruminal microbial growth. xxxii. Identify optima for nonfiber carbohydates (starch, sugars, pectins) for feeding with forages (long-term trials). xxxiii. Test CP-energy schemes for maximizing N efficiency (long-term trials). xxxiv. Evaluate accuracy of CNCPS and NRC models for predicting results from the lactation and omasal sampling studies. xxxv. Transfer information and technology. 3.Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004, and indicate, which ones were not fully or substantially met, briefly explain why not, and your plans to do so. iii. Determine inhibition characteristics of quinones involved in PPO action on silage proteolysis. iv. Complete characterization of recombinant PPO system in terms of proteolytic inhibition. v. Determine binding sites of quinones on proteolytic enzymes. xi. Use formic acid treatments to define "target" NPN levels in alfalfa silage fed to lactating cows (small scale trial 1). xii. Use formic acid treatments to define "target" NPN levels in alfalfa silage fed to lactating cows (large scale trial 2). xvi. Complete a second study estimating in vivo rates and extents of ruminal degradation for four additional proteins to use as in vitro standards. xvii. Validate Michaelis-Menten IIV degradation rates using the standard proteins and the in vitro 15N method. xviii. Complete fingerprinting of proteolytic activity of mixed ruminal organisms and commercial proteases. xix. Begin testing of NIRS calibrations for estimating ruminal protein degradation. xxviii. Milk production and omasal sampling to identify optimum levels for sugar (molasses) feeding for utilizing alfalfa silage NPN (trial 1). xxix. Milk production and omasal sampling to study starch source and processing (trial 1). xxx. Milk production and omasal sampling to identify maxima for pectin feeding (trial 1). xxxiv. Evaluate accuracy of CNCPS and NRC models for predicting results from the lactation and omasal sampling studies. Seven (7) of these milestones were substantially met during the past year. Three (3) of these milestones (items iii, iv, and v above), although involving research that was to begin under this CRIS, were transferred to another CRIS (3655-21000-034-00D, "Designing Forage Plants With Enhanced Value for Dairy Production, Profitability, and Sustainability"). Item xvii was not completed, pending installation of a new isotope-ratio mass spectrometer that will be used for 15-N determinations. This research will be addressed in the next year. The priority of item xviii was lowered due to preliminary evidence indicating that this approach may not be possible; priority of item xxx was lowered, pending availability of sufficient high-pectin forage for a trial with lactating dairy cows. B. List the milestones that are scheduled to be addressed over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? In FY 2005, we will accomplish the following milestones: xxi. Complete testing of NIRS calibrations for determining ruminal protein degradation. xi/xii. Additional studies will be conducted with condensed tannin legumes to determine an optimal levels needed to reduce silage NPN for feeding to lactating cows. xxiii. Using protein standards from in vivo research, complete validation of methods based on Michaelis-Menton IIV, commercial proteases and NIRS for assaying protein degradation. xxii. Begin NRC (2001) model validation of results on protein degradation. xxxiv. Evaluate accuracy of CNCPS and NRC models for predicting results from the lactation and omasal sampling studies. xxxv. Transfer information and technology. In FY 2006, we will accomplish the following milestones: xi/xii. An additional study will be conducted with formic acid applied at a range of levels to define optimal NPN levels in alfalfa silage fed to lactating cows. xxii. Complete NRC (2001) model validation of results on protein degradation. xxxi. Milk production and omasal sampling to assess source of RDP required for ruminal microbial growth. xxxv. Transfer information and technology. In FY 2007, we will accomplish the following milestones: xxxiii. Test CP-energy schemes for maximizing N efficiency (long-term trials). xxxii. Identify optima for nonfiber carbohydates (starch, sugars, pectins) for feeding with forages (long-term trials). xxxv. Transfer information and technology. 4.What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: Feeding crude protein in excess of the amount needed by the cow is wasted and will be excreted largely as urinary nitrogen, the form that contributes most to nitrogen pollution of water and air. Much of the loss in protein efficiency occurs in the rumen because the microbes there degrade (to ammonia) more protein than they resynthesize. A trial was conducted with dairy cows that had cannulas into their rumens to study the effects of level of dietary crude protein on the amount of usable protein that is available to the animal. Diets contained normal amounts of alfalfa silage, corn silage, and high-moisture corn. High-moisture corn was replaced with soybean meal to increase crude protein in 5 steps from 13.5% to 19.4%. Increasing dietary crude protein did not affect energy or fiber digestion in the rumen. However, level of crude protein did influence the amount of useful protein available to the cow from both the rumen microbes (the major source) plus the diet ("bypass protein"), but the supply of useful protein was not increased by feeding more than 16.5% crude protein. This finding supports results from earlier studies that showed that about 16.5-16.7% crude protein was adequate to support milk yield. These results indicate that, if farmers reduce dietary crude protein to about 16.5%, nitrogen pollution can be substantially reduced without loss of milk production. B. Other Significant Accomplishments: Optimizing the amount of protein formed by the rumen microbes is important because this represents more than half of the protein actually used by the cow. An in vitro trial, where rumen microbes were incubated in the laboratory, was conducted using one of 3 common forage sources (alfalfa silage only, alfalfa silage plus corn silage, or corn silage only) at the 5 levels of crude protein ranging from 13.5 to 19.5% (same as used in the experiment described above with live cows). Overall, there was little effect of any of the 15 diets on rumen fermentation. Amounts of protein produced by the rumen microbes in these laboratory incubations were not affected by the level of crude protein in the diet. However, efficiency of protein formation of was greater when corn silage only was the forage versus when forage came from alfalfa silage or alfalfa plus corn silages. Research is continuing to determine how the energy in alfalfa silage can be made more available to support microbial protein formation in the rumen. Lactating dairy cows, including several with cannulas in their rumens, were used to investigate the effect of protein source on milk production and rumen metabolism. Diets were composed of alfalfa silage, corn silage, and high moisture corn, typical feeds used for dairy cows in the U.S., plus equal crude protein from urea (a source of nonprotein nitrogen) or from one of three protein meals (solvent soybean meal, cottonseed meal, or canola meal). All diets contained 16.6% crude protein. Feed intake and yields of milk, fat, and protein were greater when cows were fed a protein meal rather than urea. Rumen pH and concentrations of volatile fatty acids (the major energy sources used by the cow) were similar on all diets. However, rumen ammonia concentration, an indicator that protein is being wasted, was greater on urea, and protein formation by the rumen microbes was greater when one of the protein meals was fed. The protein meals did not give equal production. Milk fat yield were greatest on canola meal, intermediate on soybean meal, and lowest on cottonseed meal. Nitrogen efficiency was greatest on soybean meal, intermediate on canola meal, and lowest on cottonseed meal. Overall, feeding a protein meal improved nitrogen utilization versus nonprotein nitrogen. However, greater nitrogen efficiency and production on soybean meal and canola meal indicated that feeding these supplements would reduce nitrogen pollution compared to feeding cottonseed meal. Condensed tannins are compounds present in certain forages that may reduce protein losses were these forages are fed to dairy cows. Silages made from 3 different sources of birdsfoot trefoil (containing low, medium, or high levels of condensed tannins) were compared to standard silages made from alfalfa or red clover as the sole forage source for lactating cows. Diets also contained high moisture corn and soybean meal was adjusted to equalize crude protein. There were no differences in feed intake or in milk composition due to feeding the different silages. However, milk and fat yields were greater on the trefoils containing medium or high tannin than on low tannin trefoil; milk yield on any of the 3 trefoils was greater than that on either alfalfa or red clover silage. Protein yield also was greater when the forage was from a trefoil silage, regardless of tannin level, than on either alfalfa or red clover silage. Concentration of milk urea (an indicator of nitrogen wastage) was lower on diets with trefoil containing medium or high tannin than on diets with low tannin trefoil, alfalfa or red clover. These results suggest that feeding forages containing condensed tannins will improve nitrogen efficiency and reduce nitrogen pollution from dairy production. Research is continuing to determine the optimal level of condensed tannins. C. Significant Activities that Support Special Target Populations: None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Major accomplishments thus far in this project have been:. 1)Finding that the amount of degraded protein required for maximal protein formation by the microbes in the rumen was about 15% less than indicated in NRC feeding standards. This means that less degraded protein can be fed, which would reduce nitrogen pollution from dairy production.. 2)Showing that reducing rumen degraded protein from about 14 to 10% of the diet did not affect milk or protein yield but caused a 12% reduction in urinary nitrogen excretion. However, a further reduction in rumen-degraded protein to about 8% of the diet reduced urinary nitrogen a further 19% but also depressed milk protein yield.. 3)Showing that reducing nonprotein nitrogen in alfalfa silage improved efficiency protein utilization, thus reducing the need to add more protein to the dairy cow's diet.. 4)Showing that bottom loading of dairy manure slurry into manure storage was effective in reducing loss of volatile nitrogen. This will reduce nitrogen pollution of the atmosphere. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Presentations were made at conferences in North America (Dia Internacional del Ganadero Lechero, World Dairy Expo, California ARPAS, Wisconsin, upstate New York, ADSA) to producers, consultants, and other scientists on ways of improving use of feed protein by dairy cows and on application of the newer rationing systems for precision feeding to maintain production while reducing nutrient excretion. Other significant technologies transferred to dairy farmers and consultants were the relative rumen bypass and digestibility of several commonly fed protein supplements, and on the effect of dietary protein and energy levels on nitrogen excretion in dairy manure. This information can now be used in ration formulation and to aid in pollution abatement by both farmers and feeding consultants. Within the next year, a new NIRS calibration for rumen bypass protein will be released via two commercial feed testing labs for use by North American producers and consultants. The major constraints to acceptance and application of much of this information is convincing farmers, but especially feeding consultants, that reducing total crude protein in the diet will not depress production, but will both reduce nitrogen pollution and lower feed costs. Feeding consultants are particularly reluctant to make this type of ration change because of what they perceive as increased risk of underfeeding crude protein (that might reduce production and result in loss of clients). 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Broderick, G.A., Muck, R.E., Shaver, R. 2003. Sound management practices for ensiling whole-crop corn and alfalfa. Delicias Internacional Ganadero Lechero Conference. September 11-12, 2003. Broderick, G.A. 2003. Optimal utilization of forage protein by high producing cows. Delicias Internacional Ganadero Lechero Conference. September 11-12, 2003. Broderick, G.A. 2003. Protein precision feeding in the dairy cow. Proceedings of the ARPAS, California Chapter, Continuing Education Conference. p. 17. Broderick, G.A., Olmos, J., Adams, L.N. 2004. Are we feeding our cows too much protein?. Hoard's Dairyman. 149(6):214. Review Publications Broderick, G.A., Uden, P., Murphy, M., Lapins, A. 2004. Sources of variation in rates of in vitro ruminal protein degradation. Journal of Dairy Science. 87:1345-1359. Broderick, G.A., Murphy, M., Uden, P. 2004. Effect of inhibitor concentration and end-product accumulation on estimates of ruminal in vitro protein degradation. Journal of Dairy Science. 87:1360-1371. Broderick, G.A. 2004. Effect of low level monensin supplementation on the production of dairy cows fed alfalfa silage. Journal of Dairy Science. 87:359-368. Moreira, V.R., Satter, L.D., Harding, B. 2004. Comparison of conventional linted cottonseed and mechanically delinted cottonseed in diets for dairy cows. Journal of Dairy Science. 87:131-138. Broderick, G.A., Albrecht, K.A., Owens, V.N., Smith, R.R. 2004. Genetic variation in red clover for rumen protein degradability. Animal Feed Science And Technology. 113:157-167.