Bulls carrying the myostatin genotype show impaired sperm cryotolerance

While the myostatin (MSTN) gene is responsible for the double-muscled phenotype in cattle and has been associated with alterations in metabolism and energy homeostasis. its potential effects on sperm function and fertility remain poorly understood. This study evaluated whether the MSTN genotype affects post-thaw sperm cryotolerance and its relationship with field fertility in Asturiana de los Valles bulls. Frozen-thawed semen from 15 homozygous MSTN-mutated (mh/mh) and 15 wild-type (+/+) bulls were assessed after 30 and 240 min of incubation at 37 °C. Sperm motility and kinematic parameters were analyzed by computer-assisted sperm analysis (CASA), whereas sperm functionality variables were evaluated by flow cytometry. Fertility was assessed using 90-day non-return rates (NRR) from 10,416 artificial inseminations. Post-thaw incubation significantly reduced sperm motility and altered all CASA kinematic parameters. Although total motility declined more markedly over time in MSTN carriers, most kinematic descriptors did not differ between genotypes. In contrast, flow cytometry revealed clear genotype-dependent differences. Sperm from MSTN carrier bulls showed lower acrosome integrity, increased membrane lipid disorder, reduced intracellular calcium levels, and altered oxidative status, together with significant Genotype × Time interactions for reactive oxygen species, superoxide production, and calcium dynamics. These findings indicate reduced functional stability and lower cryotolerance in sperm from MSTN carriers. Fertility analyses further showed that post-thaw sperm viability after prolonged incubation was positively associated with fertility at first service in MSTN carriers, whereas CASA-derived kinematic traits exhibited weaker and non-genotype-specific associations. In conclusion, the MSTN genotype negatively affects post-thaw sperm cryotolerance and functional stability. Reduced fertility in double-muscled bulls appears to be more closely related to impaired resilience to cryopreservation-induced stress than to alterations in conventional sperm kinematic parameters.