162 Effects of methionine supplementation on seminal plasma miRNAs and proteome in sheep

Seminal plasma (SP) primarily serves as the transport medium for sperm; however, protein and miRNA expression in SP have been found to be indicative of reproductive function and have been used to identify changes in spermatogenesis. It is also postulated that SP proteins and miRNAs play a role in direct communication with the female reproductive tract to alter uterine function and drive physiological changes that increase pregnancy success. The lack of SP during conception in mice has been shown to promote the development of obesity and glucose intolerance in the adult offspring, demonstrating a role for SP in fetal programming. Importantly, SP has been shown to be susceptible to paternal diet, indicating another way in which altered fetal programming may occur. Most work regarding the role of SP in pregnancy establishment as well as dietary contributions to SP composition has been done in mice and humans; however, the effects of diet on SP composition in livestock species warrants further investigation. Therefore, this study aims to investigate how paternal diet can alter the proteome and miRNA composition in SP from sheep to better understand its potential roles in male fertility and fetal programming. Here, we examined how a prepubertal methionine-enriched diet affected the miRNA and protein compositions of SP in Polypay rams. Nine male twin pairs (18 rams in total) were randomly divided into two groups, in which one twin from each pair was fed a general basal control diet consisting of corn, oats, molasses, ammonium chloride, calcium carbonate, and a commercial lamb supplement, and the other twin was fed the control diet plus a 0.44% (3 g) added top-dress of rumen-protected methionine. Rams were individually fed 0.45 kg of their respective diet once daily from weaning until the time of semen collection, for a total duration of 105 days. Between feedings, the rams were housed in groups of nine with free access to forage, water, and a total of 1 kg of the basal diet. Semen was collected from all rams and centrifuged to remove sperm cells. Micro RNAs were then extracted from the SP using the Zymo Quick-cfRNA Serum and Plasma Kit, and proteins were lysed using RIPA buffer. These samples were respectively submitted for miRNA sequencing and liquid chromatography with tandem mass spectrometry (LC-MS/MS) to evaluate changes influenced by the methionine-enriched diet. In total, 85 significant miRNAs were identified in the SP from treatment versus control rams, with 69 upregulated and 16 downregulated (P < 0.05; fold change > 2). Additionally, 38 proteins were upregulated and 26 were downregulated (FDR < 0.01; P < 0.05; fold change > 1.5) from the SP of treatment versus control rams. Overall, these results demonstrate that the SP composition of rams is susceptible to a methionine-enriched diet, which may have an impact on fertility and future offspring. Work is currently being conducted to evaluate the miRNA targets and better understand the pathways that the miRNAs and proteins are involved in. Future work should investigate the role that diet may play in pregnancy establishment through alterations of the SP proteome and miRNA landscape.