Advances in Horse Nutrition and Muscle Protein Synthesis: Grazing Warm Season Annual Forages and Evaluating Muscle Protein Synthesis in Response to Leucine

Abstract

Warm-season grasses have the potential to provide forage in Upper Midwest pastures. The objectives of this research were to determine yield, forage nutritive value and preference of annual warm-season grasses under horse grazing. Four adult horses (Equus caballus) grazed Japanese millet (Echinochloa esculenta (A. Braun) H. Sholz) Siberian millet (Setaria italica (L.) P. Beauv.), teff (Eragrostis tef (Zucc.) Trotter) sorghum sudan BMR (Sorghum bicolor (L.) Moench), sudangrass (Sorghum sudanense (Piper) Stapf.), and annual ryegrass (Lolium multiflorum L.) at mature and vegetative stages. Forages were seeded in two separate plots utilizing a randomized complete block design with three replicates per plot. Each plot was grazed at a distinct target maturity and each maturity was grazed a minimum of three times each grazing season over a two-year period. Sudangrass was consistently the highest yielding grass (P ≤ 0.0002), producing ≥ 5.5 MT ha-1 at the vegetative stage and ≥ 9.7 MT ha-1 at the mature stage while Japanese and Siberian millet exhibit the lowest yields at ≤ 5.5 MT ha-1 at the vegetative stage and ≤ 6.3 MT ha-1 at the mature stage. Annual ryegrass was the most preferred grass (P ≤ 0.0057) with ≥ 60% removal at the vegetative stage and ≥ 40% removal at the mature stage. Siberian millet was the least preferred grass with ≤ 40% removal at the vegetative stage and ≤ 5% removal at the mature stage. While warm-season grasses meet the nutritional requirements of many classes of horses, Ca:P under 1:1 was observed as well as high NO3-N levels. These conditions could lead to limited mineral availability and possible nitrate toxicity. While teff and sudangrass have potential to be grazed by horses, Ca:P and NO3-N levels should be determined before grazing. Limited research is available regarding the role of leucine in regulating equine skeletal muscle protein synthesis. The objective of this study was to evaluate the effect of leucine on protein synthesis in cultured equine satellite cells by evaluating translation initiation factors in a western blot, the incorporation of puromycin using a nonradioactive surface sensing of translation (SUnSET) method, and measuring the incorporation of [3H]Phenylalanine (3HPhe) in a protein synthesis assay. Satellite cells from equine semimembranosus muscle were grown in cell culture until they developed into myotubes. Treatments used in the western blot consisted of a no leucine control (CON), leucine (LEU), control plus rapamycin (CR), and leucine plus rapamycin (LR). LEU exhibited higher 4E-BP1 and rpS6 phosphorylation when compared to CON with no change observed in mTOR phosphorylation. No increase in phosphorylation was observed in CR or LR treatments. Puromycin incorporation was measured on treatments including a no puromycin control, a no leucine control (CON), and a leucine treatment (LEU). These results showed a 1.6-fold increase in puromycin incorporation between CON and LEU (P = 0.0004). Treatments used in the protein synthesis assay consisted of a leucine titration ranging from 0-nm to 408-nm. These results showed muscle protein synthesis rates increased as a result of leucine treatments with significant differences observed at 204-nM and 408-nM leucine compared to the untreated cells. These treatments exhibited a 1.6-fold increase in protein synthesis rates when compared to the untreated control (P ≤ 0.02). This study demonstrated the phosphorylation of translation initiation factors downstream of mTOR as well as increased 3HPhe and puromycin with leucine treatment. These results suggest leucine can trigger muscle protein synthesis in horses through activation of the mTOR pathway.