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

SIMPLE SUMMARY: Our previous study found that the addition of L-citrulline (L-Cit) to feed improved ram semen density as well as vitality and sperm mitochondrial membrane potential. In this experiment, after feeding 12 g/d of L-Cit to rams for 70 consecutive days, the contents of amino acids, sugars, and pyruvate in seminal plasma, in addition to the changes in metabolites in plasma, were analyzed by metabolomics, and the differentially expressed genes in the hypothalamus and testes were further studied through a transcriptome analysis. The results showed that L-Cit could increase the contents of pyruvate and most amino acids in seminal plasma, and that the differentially expressed genes in the hypothalamus and testes were significantly enriched in protein-metabolism-related pathways. This shows that L-Cit can improve the contents of amino acids in rams’ semen by promoting the protein metabolism of the hypothalamus–testes axis, thus improving semen quality. ABSTRACT: 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.