Evaluation of equine semen frozen in extenders free of animal proteins

Various countries are reticent regarding importation of semen that contains egg-yolk, limiting its commercialization. Egg-yolk composition is complex, varying with the diet of the chicken and egg-yolk granules are similar in size to sperm, interfering with analysis. Therefore, development of a chemically defined cryopreservation extender that maintains seminal parameters is relevant. Twelve ejaculates from 4 stallions of proven fertility (n= 4; r=3) were diluted in one of 5 extenders: 1) EDTA-glucose with 11% lactose, Equex, 20% egg-yolk and 5% dimethylformamide (EY); 2) casein salt and cyclodextrin cholesterol extender (CE); 3) CE with 3% dimethylformamide (CE-3); 4) OptiXcell (OP); 5) BioXcell (BIO). Cryopreservation was carried out in a Cryogen, Neovet automatic freezing machine: -0.5°C/minute to 5°C, 180 minutes equilibration, then -5°C/minute to -20°C and -20°C/minute to -100°C, then plunging in N2. Thawing was carried out at 37°C for 1 minute and sperm kinematic parameters (CASA, AndroVision, Minitube), viability and acrosome status (FITC-PNA-PI stain), membrane lipoperoxidation (BODIPY stain) and DNA fragmentation (SCD) were evaluated. Data were analyzed using ANOVA or Friedman test and results are reported as mean ± SD. Higher (P