College of Agriculture & Natural Resources

Satellite cells (SC) are a population of muscle resident stem cells that are responsible for postnatal muscle growth and repair. With investigation into the genomic regulation of SC fate, the role of the epigenome in governing SC myogenesis is becoming clearer. Histone deacetylase (HDAC) inhibitors have been demonstrated to be effective at enhancing the myogenic program of SC, but their role in altering the epigenetic landscape of SC remains undetermined. Our objective was to determine how an HDAC inhibitor, butyrate, promotes myogenic differentiation. SC from tributyrin treated neonatal piglets showed a decrease relative to SC from control animals in the expression of enhance of zeste homologue-2 (EZH2), a chromatin modifier, ex vivo. Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) analysis of SC isolated from tributyrin treated pigs showed a global reduction of the tri-methylation of lysine 27 of histone H3 (H3K27me3) repressive chromatin mark. To determine if reductions in EZH2 was the primary mechanism through which butyrate affects SC behavior, SC were transfected with siRNA targeting EZH2, treated with 0.5 mM butyrate, or both. Treatment with butyrate reduced paired-box-7 (Pax7) and myogenic differentiation-1 (MyoD) gene expression, while siRNA caused reductions in EZH2 had no effect on their expression. EZH2 depletion did result in an increase in differentiating SC, but not in myotube hypertrophy. These results indicate that while EZH2 reduction may force myogenic differentiation, butyrate may operate through a parallel mechanism to enhance the myogenic program.

Mechanistic knowledge of urea-N partitioning has the potential to reveal targets that can be manipulated to improve protein efficiency of ruminants, and hence, reduce N excretion to the environment. The objective of this research was to establish the role of rumen volatile fatty acids (VFA), particularly propionate and butyrate, in regulation of N utilization, urea-N recycling and gluconeogenesis in growing lambs. For these studies, sheep were fitted with a rumen cannula and fed a pelleted ration to ≥ 1.5 × maintenance energy intake. Total urine and feces were collected for determination of N balances. In addition, [15N₂]urea was infused to determine urea-N kinetics, [13C₆]glucose was infused to estimate gluconeogenesis and [ring-D₅]phenylalanine was infused to estimate protein fractional synthesis rate (FSR) of rumen tissue. The first study was conducted to evaluate the perturbations in rumen VFA profiles as a result of rumen starch infusion and the association of these perturbations to changes in urea-N kinetics and gluconeogenesis. Sheep (n=4) were infused into the rumen with either water (control) or gelatinized starch (100 g/d) for 9-d periods in a balanced crossover design. The rumen VFA profile was not affected by starch infusion. Fecal N output tended to increase with starch infusion; however, there were no effects on N retention and urinary N excretion. In addition, starch infusion did not alter urea-N entry rate (UER, i.e. synthesis) nor urea-N recycled to the gut (GER); however, starch infusion increased urea-N excreted in feces (UFE). Glucose entry, gluconeogenesis and Cori cycling were increased by starch infusion. The results suggest that under the feeding conditions of this study, starch infusion shifted the elimination of urea-N from urine to feces but this did not lead to an increase in N retention. Two companion studies were conducted to determine the role of rumen butyrate in urea-N recycling and rumen FSR. In Exp 1, sheep (n=4) were given intra-ruminal infusions of either an electrolyte buffer solution (Con-Buf; control) or butyrate dissolved in the buffer solution (But-Buf). In Exp 2, sheep (n=4) were infused into the rumen with iso-energetic (1 MJ/d) solutions of either sodium acetate (Na-Ac; control) or sodium butyrate (Na-But). Butyrate infusion treatments increased the proportion of rumen butyrate whereas acetate infusion increased rumen acetate. No difference in N retention was observed between treatments in either experiment. In Exp 2, UER was reduced by Na-But compared to the Na-Ac control, thus, a higher proportion of urea-N entering the rumen was utilized for microbial protein synthesis. In Exp 1, although But-Buf infusion increased the FSR of rumen papillae, urea kinetics were not altered. This study is the first to directly assess the role of butyrate in urea-N recycling and effects on rumen papillae protein turnover in growing lambs. Under the conditions in the present studies, butyrate did not affect overall N retention in growing sheep; however, butyrate reduced urea synthesis and altered the distribution of urea-N fluxes. Lastly, two companion studies were conducted to determine the role of rumen propionate in urea-N recycling and gluconeogenesis. In Exp 1, sheep (n=6) were continuously infused into the rumen with iso-energetic (1 MJ/d) solutions of either Na-Acetate (control) or Na-Propionate for 9-d periods in a balanced crossover design. In Exp 2, a different group of wether sheep (n=5) were fed on an equivalent protein intake basis either a control or Na-propionate supplemented ration. Propionate treatments increased the proportion of rumen propionate in both experiments. In Exp 1, urea kinetics and N retention were not affected by propionate infusion compared to iso-energetic acetate infusion. However, in Exp 2, the propionate diet increased N retention by ∼50%, which resulted from reductions in UER (−2.1 g urea-N/d) and UUE (−0.8 g urea-N/d). Glucose entry and gluconeogenesis were increased by propionate treatments. Under the conditions of these studies, higher ruminal propionate did not affect urea-N fluxes to the rumen. The results from this research provide an understanding of the role of individual rumen VFA in N retention and urea-N recycling in ruminants.

College of Agriculture & Natural Resources

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