|COLOMBINI, STEFANIA - Universit Di Milano|
|CLAYTON, MURRAY - University Of Wisconsin|
Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/2010
Publication Date: 4/1/2011
Citation: Colombini, S., Broderick, G.A., Clayton, M.K. 2011. Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system. Journal of Dairy Science. 94:1967-1977.
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 breakdown by these same organisms. Rumen microbes often reduce feed protein value by degrading more protein than they resynthesize. As a result, dietary protein often is 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 feedstuffs. As yet, there are no reliable methods to determine protein escape that can be used by commercial feed testing labs. Earlier, we developed a technique to estimate dietary protein escape from the rumen. This method has proven to be reasonably reliable for evaluating rumen protein escape for a number of feeds. However, the technique sometimes gives variable results. Peptides, which are small fragments of proteins that are formed during the process of protein breakdown, were previously not measured in our assay or any of the other in vitro tests commonly used to evaluate protein degradation. The objective of this research was to develop a new chemical assay for protein degradation products that measured the peptides and to evaluate this assay in our rumen in vitro method to see if improved measurements were obtained. Overall, accounting for peptide formation 1) reduced variation, 2) increased the observed protein degradation rate by 28%, and 3) yielded estimated protein escape values that were similar to measurements made in living dairy cattle. These results were more reliable than those obtained using the most widely used test for evaluating proteins fed to lactating cows. This research indicates that the new in vitro method will give improved estimates of protein escaping the rumen for common feedstuffs. This research also indicates that the test most widely used to evaluate rumen protein escape is not as reliable as previously thought and consistently over-estimates protein escape. Dairy farmers will benefit because application of this new method will yield more reliable information on the true nutritional and economic value of feed proteins. This will help improve the economic sustainability of dairy farming.
Technical Abstract: The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.2 to 3 mg N/ml inoculum) and incubated with 10 ml of ruminal inoculum and 5 ml of buffer; fermentations were stopped after 2 h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer soluble N and buffer extracts were treated with TCA to determine N degraded at t = 0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA) (OPA-F) and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was calculated as the sum of 1) ammonia-N plus N in free TAA or 2) ammonia-N plus N in free TAA and peptides. Rate of degradation (kd) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters Km (Michaelis constant) and kd (Vmax/Km) were estimated directly; kd values were adjusted (Akd) for the fraction FD0 using the equation: Akd = kd - FD0/2. The OPA-C assay yielded faster degradation rates for all proteins due to the contribution of peptides to the fraction degraded (overall mean = 0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were 2.5 (SSBM 1), 1.9 (SSBM 2) and 3.6 (ESBM) times more rapid than rates reported by NRC. Values of rumen-undegraded protein (RUP) estimated with the OPA-C assay were lower than the OPA-F assay (overall means = 30.5% and 35.2% for, respectively, the OPA-C and OPA-F assay). Both assays indicated that the 2 SSBM differed in RUP content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value but similar to an RUP value previously observed in vivo. The OPA-C assay is a simple, rapid and reliable method to quantify formation of small peptides.