Season affects sperm quality, seminal extracellular vesicles, and antioxidant-related genes in Egyptian Buffalo bulls.

Abstract

Global climate change threatens livestock production, especially in regions where heat stress diminishes fertility. Extracellular vesicles (EVs), key players in biological processes like intercellular communication and oxidative stress regulation, are crucial in reproductive biology. The objective was to determine seasonal impacts on semen quality, oxidative stress, and antioxidant enzyme activity in Egyptian buffalo bulls, focusing on differences in EV content between bulls having high- or low-quality sperm (HQS and LQS, respectively), based on sperm motility and morphology. Semen samples were collected by artificial vagina in summer and winter. Oxidative stress markers, such as malondialdehyde (MDA), and antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPx]) were measured in seminal plasma. Seminal plasma EVs (SP-EVs) were isolated and characterized by size, concentration, and surface markers (CD9 and CD63). Additionally, gene expression of antioxidant and apoptotic markers was analyzed using qRT-PCR. Semen quality peaked in the winter, with HQS exhibiting superior total motility (winter total motility: 79.4 ± 0.65% vs. summer: 69.9 ± 0.65%) and morphology (winter normal morphology: 75.5 ± 0.87% vs. summer: 71.3 ± 0.87%). Furthermore, LQS semen had higher MDA concentrations (summer: 1.31 ± 1.67 nmol/ml; winter: 2.62 ± 1.21 nmol/ml), indicating greater oxidative stress, whereas HQS exhibited elevated antioxidant enzyme activity, especially in summer (SOD: 292.0 ± 3.93 U/ml; CAT: 949.7 ± 15.23 U/ml; GPx: 77.7 ± 2.15 mU/ml; TAC: 9.83 ± 4.75 mM/L). Notably, SP-EVs from LQS were larger and had increased CD9 and CD63 expression, suggesting their association with oxidative damage. Conversely, SP-EVs from HQS were smaller, had lower CD9 and CD63 expression, and had upregulated (p ≤ 0.001) antioxidant genes (SOD, NFE2L2) and downregulated (p ≤ 0.001) apoptotic markers (CASP3). Expression of DNMT1 and ATF6 mRNA were elevated (p ≤ 0.001) in HQS sperm and SP-EVs, especially in summer, indicating a seasonal effect on epigenetic regulation and stress response in reproductive health. These findings highlighted the potential of SP-EVs as biomarkers for semen quality, their role in protecting sperm functions under seasonal stress, and epigenetic mechanisms, providing critical insights for improving the reproductive performance of buffalo bulls under climate-induced stress.