Establishment of a field-adapted biotechnological protocol for Duck semen cryopreservation

Cryopreservation of duck semen is a strategic tool for safeguarding genetic resources and maintaining breeding continuity, particularly in the context of sanitary crises such as highly pathogenic avian influenza outbreaks. However, progress has been limited by the intrinsic low cryotolerance of avian spermatozoa and the practical constraints of industrial artificial insemination. The study aimed to develop and validate cryopreservation protocols for Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) duck semen using permeating cryoprotectants compatible with direct insemination without post-thaw centrifugation. In Pekin ducks, four protocols combining different extenders (BHSV, FEB) and cryoprotectants (DMF, DMA) were evaluated by computer-assisted sperm analysis (CASA) and in vivo fertility trials. The combination of FEB extender with 6% DMA produced the best pos-thaw motility parameters and achieved the highest fertility rate 87.5%, comparable to fresh semen. Insemination volume (200 µL vs. 100 µL) significantly influenced hatchability rates (93.2% vs. 89.9%), whereas final dilution ratio (1/2 vs. 1/3) had no significant effect on reproductive performances. In Muscovy duck semen, direct application of the optimized Pekin protocol resulted in lower fertility (19%), highlighting interspecific differences in cryotolerance. Optimization of extender composition, including trehalose supplementation, identified BHSV+DMA 6% and LakeC+DMA 6% as the most effective formulations for intraspecific insemination, achieving fertility rates of 77.4% and 66.0%, respectively. However, insemination of Pekin females with frozen Muscovy semen for Mule production yielded low fertility (31.9%), suggesting a strong female-dependent effect to reproductive incompatibility. Taken together, these results demonstrate that robust, industry-compatible cryopreservation protocols can be successfully implemented for Pekin duck semen, and that progress has been made for Muscovy semen. Residual limitations for Mule duck production underscore the need for further protocol optimization tailored to interspecific hybridization contexts.