Germplasm cryopreservation in bulls: Effects of gonadal tissue type, cryoprotectant agent, and freezing-thawing rates on sperm quality parameters

Abstract

Germplasm preservation is crucial for reproductive programs involving farm and endangered species. This study describes the effects of slow-uncontrolled cryopreservation protocols on bovine sperm associated with testicular or epididymal tissues. Samples from the testis or epididymis (cauda) were cut into ∼0.5 or 1 cm3 fragments and cryopreserved using Me2SO (Dimethyl Sulfoxide) or glycerol-based cryoprotectants. Sperm were collected from testicular or epididymal tissue before and after freezing-thawing (38 °C or 40 °C) and kept at room temperature (RT) or 4 °C during handling. The parameters studied were viability, membrane integrity (HOS), motility, acrosome integrity, chromatin, and morphology. Pre-freezing parameters were lower in testicular sperm than epididymal: HOS+ and DNA integrity (P < 0.05). Normal-% pre-freezing testicular sperm morphology was lower than epididymal (43.3 ± 1.8% vs. 65.3 ± 14.8%). All testicular RT-kept sperm parameters decreased post-freezing, except for acrosome integrity, which remained constant (P > 0.05). There were no differences in Me2SO-frozen tissue sizes (P > 0.05). All epididymal RT-kept sperm parameters dropped post-freezing except for the constant DNA integrity (P > 0.05). 4oC-kept sperm were fitter than those at RT (P < 0.05). 4oC-kept testicular sperm viability, DNA, and membrane integrities declined after 38 °C or 40 °C thawing (P < 0.05). Acrosome integrity and motility remained unchanged after freezing (P > 0.05). 4oC-kept epididymal sperm acrosome integrity, motility, and HOS+% severely dropped post-thawing (P < 0.05). Viability and DNA integrity were unchanged (38 °C vs. 40 °C; P > 0.05). Overall, post-freezing sperm morphology was unaffected (P > 0.05), but Dag defect was significantly lower in testicular samples (P < 0.05). Whole-epididymis parameters were maintained up to 24h at 4 °C (P > 0.05). In conclusion, testis-epididymis freezing protocols should use small tissue pieces, Me2SO-based cryoprotectants, and 4°C-kept samples to reduce sperm damage.