Improving drone sperm cryopreservation: Investigating cryoprotectant combinations and PVP supplementation.

Cryopreservation of drone sperm is a widely studied method for long-term genetic preservation and offers key advantages. However, despite successful cryopreservation, fertilization rates remain unsatisfactory. Artificial insemination with cryopreserved sperm reduces spermatozoa concentration in the queen's spermatheca, highlighting the need for alternative cryoprotectants and extenders. Research on other species suggests that combining cryoprotectants may enhance preservation outcomes compared to using them individually. This study evaluated the effects of various cryoprotectant combinations on drone sperm quality after cryopreservation. Phase I assessed the individual and combined effects of dimethyl sulfoxide (DMSO), glycerol (GLY), dimethylacetamide (DMA), and ethylene glycol (EG). In Phase II, different concentrations of polyvinylpyrrolidone (PVP) were added to DMSO. Finally, the three best combinations were tested in vivo for sperm concentration and plasma membrane integrity (PMI) in queen spermathecae following artificial insemination. DMSO-based cryoprotectants, particularly combinations with EG, GLY, and PVP, significantly enhanced post-thaw sperm quality (P ≤ 0.001). In Phase I, DMSO, EG, and DMSO + EG yielded the highest motility and PMI values. In Phase II, DMSO+6 % PVP achieved optimal results in all parameters. In Phase III, although all cryopreserved groups showed lower spermathecal sperm concentration and PMI compared to fresh semen, the DMSO + PVP group performed best compared to the other cryopreserved treatments. In conclusion, the combined use of cryoprotectants yielded superior cryopreservation outcomes compared to their individual utilization. DMSO showed greater efficacy than GLY, DMA, and EG, while PVP supplementation showed promise in improving drone sperm cryopreservation, warranting further investigation with expanded parameters.