029 Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein organization of rooster sperm during cooling and cryopreservation using the fluorescent stains Merocyanine 540 (phospholipid order) and Rhodamine 640 (protein order). Rooster semen samples (3 roosters and breeds per pool; 4 pools total) were diluted in Lakes Low Temperature (LLT) and Sasaki diluent and cooled to 5°C over 30min (Step 1). The samples were then diluted with the penetrating cryoprotectants (CPA) glycerol (LLT) or methylacetamide (Sasaki), equilibrated for 30min (Step 2), loaded into straws and cryopreserved. The cryopreservation and thawing (Step 3) was performed using the optimized procedures for each diluent. Computer automated semen analysis (CASA) was used to analyze motility characteristics and flow cytometry was used to analyze plasma membrane phospholipid, protein organization and plasma membrane integrity (PMI) at each step. Percent values from CASA and flow cytometry fluorescent medians (FM) were transformed using arcsine and Box–Cox, respectively. The GLM ANOVA was used to detect differences in motility characteristics and membrane organization and included the effects of cryopreservation step and rooster semen pool. In the total motility analyses cryopreservation diluent/CPA was a significant (P