Triclosan (TCS), a widely used environmental antimicrobial agent, poses potential risks to female reproductive health, yet its toxic effects on oocyte maturation remain inadequately characterized. In this study, we established an in vitro maturation (IVM) model of porcine oocytes to investigate TCS-induced meiotic impairment and to evaluate the rescuing effects of melatonin (MT), an endogenous indoleamine with potent antioxidant and anti-apoptotic activities. Our results demonstrated that TCS exposure significantly disrupted oocyte maturation, as evidenced by suppressed polar body extrusion and compromised cumulus expansion. Furthermore, TCS triggered early apoptosis. Proteomic analysis revealed that the p53 signaling pathway was significantly dysregulated by TCS exposure. Notably, co-treatment with MT during IVM effectively restored meiotic progression, attenuated apoptosis, and rebalanced the disrupted proteomic profile. Mechanistic investigation, validated by Western blotting, confirmed that TCS upregulated p53 and downregulated its downstream cell cycle effector CCNB1 while concurrently altering the ratio of apoptosis-related proteins BAX/BCL-2. Melatonin treatment effectively normalized the expression of these key proteins (p53, CCNB1, BAX, and BCL-2). These findings illustrate that MT rescues TCS-impaired oocyte quality through p53-dependent suppression of apoptosis and restoration of meiotic progression, providing new insights into potential strategies for mitigating environmental pollutant-induced reproductive damage.

Sperm cryo-tolerance resulted in significant variations in post-thaw semen quality among breeds and individual boars. In the present study, semen samples from thirty-seven large white boars were cryopreserved to select individuals with strong and weak freezing tolerance according to their post-thaw sperm quality. Comparative TMT-based quantitative proteomic analysis between the two groups identified 22 significantly differentially expressed proteins. NT5C1B and ADA, the significantly downregulated proteins in the semen of the low cryo-tolerance group, were supplemented in the semen samples with lower cryo-tolerance. Supplementation with 1 µg/mL of NT5C1B dramatically (p < 0.05) improved kinematic parameters and structural integrity. In comparison with the control group, mitochondrial activity and antioxidant capacity were significantly enhanced in post-thaw sperm. In vitro fertilization assays revealed that the NT5C1B-treated group also has notably (p < 0.05) high sperm penetration and embryonic cleavage rates. ADA supplementation did not exhibit obvious freezing tolerance effects. NT5C1B can be a potential key functional protein to enhance the quality and cryo-tolerance during cryopreservation. Specifically, supplementation with 1 µg/mL NT5C1B significantly improved post-thaw motility, structural integrity, mitochondrial activity, and antioxidant capacity and ultimately enhanced the sperm penetration rate and embryonic cleavage rate in cryo-sensitive sperm, confirming its role as a functional protector during cryopreservation.