Redox homeostasis modulation promotes capacitation-associated events in equine sperm

Sperm capacitation, a crucial process for successful fertilization, is modulated by reactive oxygen species (ROS) and involves molecular changes including increased membrane fluidity, cholesterol depletion, and protein tyrosine phosphorylation. Although an effective equine in vitro fertilization protocol has recently been described, its requirement for prolonged capacitation limits applicability to high-quality ejaculates. Because stallion spermatozoa possess endogenous antioxidant systems that may mitigate ROS accumulation and delay ROS-mediated capacitation, this study investigated whether inhibition of catalase with 3-amino-1,2,4-triazole (3-AT), superoxide dismutase 1 with LCS1, γ-glutamylcysteine synthetase with buthionine sulfoximine (BSO), or their combination (INH), could enhance endogenous ROS and promote capacitation. Sperm were incubated for 24 h under control, capacitating, or inhibitor-treated capacitating conditions (3-AT, LCS1, BSO, or their combination [INH]). Samples were evaluated using CASA and flow cytometry. Incubation increased intracellular calcium in all samples, but sperm under capacitating conditions showed lower calcium levels than control after 22 h (100.8 ± 2.4 vs 112.7 ± 4.81 AU respectively; p ≤ 0.01), an effect further enhanced by 3-AT (p ≤ 0.05) and BSO (p ≤ 0.01). After 22 h, BSO concentrations above 500 μM increased membrane lipid disorder (CTR: 70.81 ± 5.2 vs 500 μM BSO: 104.85 ± 5.1 AU, p ≤ 0.01), and hyperactivation-associated motility kinematics (TM, VCL, ALH, BCF; all p ≤ 0.05). When data were pooled across time points, INH increased tyrosine phosphorylation compared with control (31.55 ± 2.1% vs 25.24 ± 1.8% respectively; p ≤ 0.01). In conclusion, inhibition of antioxidant systems, predominantly the glutathione system, may drive stallion sperm capacitation and reduce the time required for IVF protocols, although further studies are required to determine its impact on in vitro fertility