Heat stress causes infertility in male rabbits in summer. This study was conducted to determine the effects of heat stress on semen quality and seminal plasma metabolites of male rabbits. To achieve these objectives, the temperature and humidity index (THI) was used to determine the stress state of male rabbits during different months, thereby the rabbits were divided into heat stress and no heat stress groups. The quality of the semen and the biochemical indices of seminal plasma were then analyzed. Next the plasma metabolites of rabbits in both groups were evaluated using the ultra-high performance liquid chromatography-mass spectroscopy (UPLC-MS)/MS technique. Our results showed that the THI value of the rabbit housing in May was 20.94 (no heat stress). The THI value of the housing in August was 29.10 (heat stress group, n = 10). Compared with the non-heat stress group, the sperm motility, density, and pH in the heat stress group (n = 10) were significantly decreased (P < 0.01); the semen volume decreased significantly (P < 0.05); and the sperm malformation rate increased significantly (P < 0.01). The number of grade A sperm significantly decreased, while the numbers of B and C grade sperm significantly increased (P < 0.01). The total sperm output (TSO), total motile sperm (TMS), and total functional sperm fraction (TFSF) decreased significantly (P < 0.01). Heat stress protein 70 (HSP70) and acid phosphatase (ACP) in the seminal plasma of rabbits in the heat stress group (n = 20) were significantly increased (P < 0.01). Seminal plasma testosterone (T), α-glucosidase (α-Glu), and fructose decreased significantly (P < 0.01). The concentrations of Mg²⁺ (P < 0.05), Na⁺ (P < 0.01), and K⁺ (P < 0.01) in metal ions were significantly decreased. These findings indicated that heat stress severely affected the quality of the male rabbit semen. Furthermore, UPLC-MS/MS technology was used to analyze the seminal plasma samples of rabbits in the heat stress group and non-heat stress group (n = 9 for each group). In total, 346 metabolites were identified, with variable importance in project (VIP) > 1.0, fold change (FC) > 1.5 or < 0.667, and P < 0.05 as the threshold. A total of 71 differential metabolites were matched, including stearic acid, betaine, arachidonic acid, L-malic acid, and indole. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential metabolites revealed 51 metabolic pathways, including synthesis and degradation of ketones, serine and threonine metabolism, tryptophan metabolism, and the citric acid cycle. Our study has shown that the sperm motility, sperm pH value, and sperm density of male rabbits decreased significantly under heat stress, and the sperm malformation rate increased significantly. Furthermore, the quality of semen was shown to deteriorate and the energy metabolism pathway was disturbed. These findings provide a theoretical reference for alleviating the adaptive heat stress in male rabbits.

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