Synergistic Effects of Thyroxine and Nano‐Melatonin on Post‐Thaw Sperm Quality, Antioxidant Defence and Fertility of Cryopreserved Simmental Bull Semen

ABSTRACT Background Cryopreservation induces oxidative stress and functional damage in spermatozoa, negatively affecting sperm quality and fertility. Nano‐melatonin (NM) and thyroxine (T) have been suggested as potential cryoprotective agents because of their antioxidant and metabolic regulatory properties. Objectives This study aimed to evaluate the effects of thyroxine and nano‐melatonin supplementation in semen extenders on post‐thaw sperm quality, antioxidant status, apoptosis, and fertility of cryopreserved Simmental bull semen. Methods Ninety‐six ejaculates were collected from six healthy Simmental bulls over 8 weeks. Semen samples were pooled and diluted with extenders supplemented with T (0.5, 0.75 and 1 µg/dL), NM (30, 45 and 60 µg/dL), or their combinations. Post‐thaw sperm motility, kinematics, viability, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, DNA integrity, antioxidant parameters, lipid peroxidation, apoptosis‐like changes, and conception rates were evaluated. Results The combined thyroxine and nano‐melatonin treatment (T0.5 + NM30) significantly improved (p < 0.05) sperm motility and velocity parameters (VAP, VCL and VSL) compared with the other groups. This group also exhibited significantly higher viability, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, and DNA integrity. Antioxidant activity (GPx, SOD and TAC) was significantly enhanced, whereas MDA levels and late apoptotic sperm cells were reduced in the nano‐melatonin‐treated and combination groups, particularly T0.5 + NM30. Furthermore, conception rates were significantly increased in the NM30 (77.14%) and T0.5 + NM30 (82.86%) groups compared with the control group (48.57%). Conclusions Supplementation of semen extenders with thyroxine and nano‐melatonin, particularly the T0.5 + NM30 combination, improved post‐thaw sperm quality, antioxidant defence and fertility of cryopreserved Simmental bull semen, indicating a synergistic cryoprotective effect during semen cryopreservation.