Ruminal bacteria and protozoa composition, digestibility, and amino acid profile determined by multiple hydrolysis times

Abstract

Microbial samples from 4 independent experiments in lactating dairy cattle were obtained and analyzed for nutrient composition, AA digestibility, and AA profile after multiple hydrolysis times ranging from 2 to 168 h. Similar bacterial and protozoal isolation techniques were used for all isolations. Omasal bacteria and protozoa samples were analyzed for AA digestibility using a new in vitro technique. Multiple time point hydrolysis and least squares nonlinear regression were used to determine the AA content of omasal bacteria and protozoa, and equivalency comparisons were made against single time point hydrolysis. Formalin was used in 1 experiment, which negatively affected AA digestibility and likely limited the complete release of AA during acid hydrolysis. The mean AA digestibility was 87.8 and 81.6% for non-formalin-treated bacteria and protozoa, respectively. Preservation of microbe samples in formalin likely decreased recovery of several individual AA. Results from the multiple time point hydrolysis indicated that Ile, Val, and Met hydrolyzed at a slower rate compared with other essential AA. Singe time point hydrolysis was found to be nonequivalent to multiple time point hydrolysis when considering biologically important changes in estimated microbial AA profiles. Several AA, including Met, Ile, and Val, were underpredicted using AA determination after a single 24-h hydrolysis. Models for predicting postruminal supply of AA might need to consider potential bias present in postruminal AA flow literature when AA determinations are performed after single time point hydrolysis and when using formalin as a preservative for microbial samples.

Estimation of correction factors to determine the true amino acid concentration of protein after a 24-hour hydrolysis

“…These are the classical patterns reported, for example, in goat milk by Rutherfurd et al (2008) and in rumen microorganisms by Fessenden et al (2017). During examination of the results obtained from the equation proposed by Robel and Crane (1972) and used by others (e.g., Darragh et al, 1996;Rutherfurd et al, 2008; Fessenden et al, 2017), we observed that the loss rate from the hydrolysate was negative for the AA, still showing an increment of their concentration after 24 h of hydrolysis. Although this negative loss rate gives a good mathematical fit to the data, it does not apply to its initial representation in the model, which should be the proportion of state B (i.e., in the pool of AA in the hydrolysate) being degraded.…”

“…We excluded the max/24h ratio of Ser and Thr because, as discussed above, these 2 AA presented a significant loss before the 24-h measurements. The very low ratio of A 0 /24h compared with max/24h for the BCAA indicates that the current nonlinear model does not apply well to these AA Average of the current study, Darragh et al (1996), Rutherfurd et al (2008), and Fessenden et al (2017).…”

“…A third ratio reported in the literature was also used: the ratio of the true AA concentrations deter-mined based on the DNA sequence of a purified protein on the concentrations measured after a 24-h hydrolysis (gene/24h). In addition to the original work of Robel and Crane (1972) and the current study, the A 0 /24h ratios were estimated in egg white lysozyme (Darragh et al, 1996), goat milk formulation (Rutherfurd et al, 2008), and rumen microorganisms (Fessenden et al, 2017). The max/24h ratios were determined in wheat (Kohler and Palter, 1967), pig diet and digesta (Rowan et al, 1992), and different soybean products (Albin et al, 2000).…”

“…As previously mentioned, it has been known for a long time that acidlabile AA like Ser and Thr are partially destroyed after their release from the protein during a 24-h hydrolysis (Rees, 1946). Accordingly, when multiple time hydrolyses have been conducted, Ser and Thr are the AA with a positive and high loss rate (e.g., Darragh et al, 1996;Fessenden et al, 2017). Besides Ser and Thr, only Met had a positive loss rate across all the proteins with an average of 24 h to reach its maximal concentration.…”

“…Rumen fluid-associated bacteria (FAB) and particleassociated bacteria (PAB) were isolated according to Reynal and Broderick (2005), except that 2 layers of cheese cloth were used initially to separate the FAB and PAB and that the low speed centrifugations were at 500 × g rather than 1,000. Protozoa were extracted according to Fessenden et al (2017). Formalin was not used on bacteria or protozoa samples.…”