Effect of different anti- freeze (glyco)proteins on cryotolerance of rooster sperm

Antifreeze- proteins (AFP) and antifreeze- glycoproteins (AFGP) are known to control the size and shape of ice crystals in freeze- tolerant organisms. The present work focused on studying the effect of AFPI from Winter Flounder, AFPIII from Ocean Pout and a synthetic AFGP (supplied by Ilja Voets ' group), on the cryotolerance of rooster sperm. Nine pools of 5 randomly selected roosters (45 animals) were diluted 1:1 (v:v) with poultry extender and cooled to 5°C. Each pool was divided into 3 portions [one per AF(G)P]; and each portion was subdivided into 5 aliquots to be supplemented with dimethylacetamide (DMA, 0.6 M) and 5 concentrations of AFP: 0, 0.3, 1, 3, 30 μg/mL of AFPI; 0, 0.01, 0.1, 1, 10 μg/mL of AFPIII; or, 0, 0.01, 0.1, 1, 10 nM of AFGP. After one hour of chilling, the semen was packed in 0.5 mL straws and frozen to −140°C at −50°C/min. Thawing was done in water at 5°C for 30 sec. Viability (DAPI) and motility parameters (CASA system) were determined. The results were analysed by ANOVA and Tukey ' s post- hoc tests. Concentrations of 0.3 and 3 μg/mL of AFPI increased ( p < 0.05) the viability (56.1 ± 1.5 and 56.0 ± 1.3%), compared with the control (50.2 ± 1.1%) while 1 μg/mL of AFPI improved total motility (29.7 ± 1.0 vs. 25.9 ± 1.0%), progressive motility (PM, 24.1 ± 1.1 vs. 20.6 ± 1.0%) VCL (60.2 ± 1.8 vs. 56.3 ± 1.3 μm/s) and ALH (0.44 ± 0.02 vs. 0.39 ± 0.01 μm) compared with the control. Regarding AFGP, 1 nM of the protein improved ( p < 0.05) PM (22.5 ± 1.4 vs. 19.9 ± 1.3%) compared with the control. In conclusion, AFP or AFGP at proper concentration can increase the cryotolerance of rooster sperm. Funding: European Union, HORIZON- MSCA- 2022- PF- 01- 01, Project 101,109,983 CRYOCHICK/Ministry of LNV, project KB- 34- 013- 002.