SOURCES OF VARIATION IN RATES OF IN VITRO RUMINAL PROTEIN DEGRADATION

Authors: Broderick, Glen; UDEN, PETER - KUNGSANGEN; MURPHY, MICHAEL - KUNGSANGEN; LAPINS, AIVARS - RIGA, LATIVA

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/26/2003
Publication Date: 5/1/2004
Citation: 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.

Interpretive Summary: Dairy cows obtain their protein needs both from synthesis by the microbes living in the rumen (the first compartment of the cow's stomach) and from dietary protein that escapes digestion by these same organisms. Rumen microbes often reduce the protein value of feeds by degrading more protein then they resynthesize. As a result, high quality feeds may be used inefficiently for milk production. The amount of protein escaping the rumen, referred to by farmers as 'bypass protein', determines much of the value of feeds. As yet, there are no reliable methods to determine bypass protein that can be used by commercial feed testing labs. We developed a technique to measure bypass protein involving collecting microbes from the cow's rumen and incubating them in vitro (that is, outside the live animal) with the feeds being tested. Metabolic inhibitors are added to these incubations so that the rate of release of protein fragments can be used to calculate the amount of protein bypass that would occur in the living cow. This method has proven to be accurate for predicting bypass protein for a number of feeds. However, the technique gives variable results because the activity of the rumen microbes fluctuates from day to day for unknown reasons. This means that the test must be repeated several times to get a reliable average result. The objective of this research was identify causes of this variation because, if it could be reduced, fewer assays would be needed and bypass protein value could be measured more rapidly. Dietary energy content and time of microbe collection relative to feeding were found to be important factors influencing variation and the bypass value obtained. Rumen microbes collected before feeding gave more reliable measurements of rumen bypass for three major feed proteins. Growing the microbes for 3 to 4 hours after removing them from the rumen was found to be useful for removing interferences in the determination. Also, results obtained with casein, a pure protein that is a commonly used as a standard by dairy scientists, proved to be an unreliable indicator of activity of the rumen microbes toward typical feed proteins. Although further work is required to perfect this method, this research shows how to reduce much of the variation in measuring bypass protein. This means that the bypass protein value of dairy feeds can be determined more rapidly and inexpensively by laboratories adopting this technique

Technical Abstract: Rates and extents of ruminal protein degradation for casein, solvent soybean meal (SSBM), expeller soybean meal (ESBM), and alfalfa hay were estimated from net appearance of NH3 and total amino acids in inhibited in vitro media containing 1 mM hydrazine (HS) and 30 mg/L of chloramphenicol (CAP). Protein was added at 0.13 mg N/ml of medium and incubations were conducted for 4 to 6 h. Inocula were obtained from ruminal cannulated donor cows fed diets of grass silage or legume silage plus concentrate mixtures. Pre-incubation or dialysis of inocula was used to suppress background NH3 and total amino acids; however, pre-incubation yielded more rapid degradation rates for casein and SSBM and was used in subsequent incubations. Pre incubation with added vitamins, VFA, hemin, or N did not alter protein degradation. Similar estimates of protein degradation rate were obtained for SSBM, ESBM, and alfalfa when computed from total N release or N release in NH3 plus total amino acids, regardless of whether amino acids were quantified using ninhydrin colorimetry or o-phthaladehyde fluorescence. Accounting for peptide N release did not affect estimated degradation. However, casein degradation rates were more rapid when using total N release or accounting for peptide N, indicating significant accumulation of small peptides during its breakdown. Rates also were more rapid using inocula from lactating cows versus non-lactating cows with lower feed intakes. Protein degradation rates were different due to time after feeding: casein rate was more rapid, but SSBM and ESBM rates slower, using inocula obtained after feeding. Several characteristics of ruminal inoculum that influenced breakdown of the rapidly degraded protein casein did not appear to have direct effects on degradation of protein in soybean meal.