Metabolomic and Transcriptomic Changes Underlying the Effects of L-Citrulline Supplementation on Ram Semen Quality

This study examined the effects of L-Cit supplementation on ram semen quality through metabolomics and transcriptomics. A total of 16 rams were randomly categorized into two groups. The control group was fed a basic diet, whereas the experimental group received feed supplemented with 12 g/d of L-Cit. Semen and blood were collected from the rams on days 0 and 72 to measure sugar, pyruvate, amino acid, and nontargeted metabolite contents. Additionally, hypothalamic and testicular tissues were collected for a transcriptomic analysis. We found 27 differential metabolites between the control and experimental groups, of which 21 were downregulated (p < 0.05) and 6 were upregulated (p < 0.05). Compared with the control group, xylose and pyruvate contents in seminal plasma increased by 43.86% and 162.71%, respectively (p < 0.01). Additionally, the levels of 11 amino acids showed a significant increase in seminal plasma (p < 0.01). Furthermore, 961 and 715 differentially expressed genes were detected in the hypothalamic and testicular tissues, respectively. The pathways of significant enrichment in the hypothalamus and testes were protein digestion, absorption, glycolysis/gluconeogenesis, and amino as well as nucleotide sugar metabolisms. In the present study, L-Cit improved protein synthesis and blood metabolism, consequently increasing the contents of most amino acids in ram seminal plasma. Specifically, the hypothalamus controlled the expression of glycolysis/gluconeogenesis-related genes in the testes through its metabolites released into the serum, thereby providing energy for sperm production, which led to a decrease in the sugar content of seminal plasma.