Ice nucleating agents in rooster sperm cryopreservation

Previous studies by our group indicated that sub- zero temperatures in supercooled boar semen are a serious detrimental factor during cryopreservation. Like boar sperm, chicken semen is quite sensitive to the cryopreservation processes. The present work focused on reducing supercooling during rooster sperm cyopreservation by supplementing the freezing medium with ice nucleating proteins (Snowmax®). These proteins reduce supercooling by inducing freezing at high subzero temperatures. Nine pools of 5 randomly selected roosters (45 animals) were diluted with ASG extender 1:1 (v:v) and chilled to 5°C. Each pool was divided into 4 aliquots which were supplemented with dimethylacetamide (DMA, 0.6 M) and Snowmax to final concentrations of 0 (control), 0.01, 0.1 and 1 mg/mL. Snowmax did not dissolve completely in the freezing medium, observing particles suspended in it. After 1 h of refrigeration 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 s. Viability (DAPI) and motility parameters (CASA system) were determined. The results were analysed by ANOVA and Tukey ' s post- hoc tests. Snowmax did not affect viability but had a negative effect ( p < 0.05) on total motility (1 mg/ mL concentration) and progressive motility (0.01 and 1 mg/mL concentrations). VCL and VAP, decreased ( p < 0.05) as Snowmax concentrations increased. In conclusion, Snowmax affected negatively the post- thaw motility of chicken sperm. However, it is not clear if the effect was due to reducing supercooling or to the presence of high number of particles in the medium. Additional experiments with completely soluble ice nucleators are needed. Funding: European Union HORIZONMSCA-2022- PF- 01, Project 101109983 CRYOCHICK/Ministry of LNV, Project KB- 34- 013- 002.