Evaluation of equine semen frozen in extenders free of animal proteins

Abstract

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<0.05) percentages of total and progressively motile sperm were observed in samplescryopreserved in EY (29.25 ± 11 and 21.17 ± 10), CE-3 (30.4 ± 15 and 22 ± 12) and OP (23.1 ± 14 and 15.5 ± 10) compared to those in CE (4.75 ± 3 and 1.58 ± 2) and BIO (8 ± 4 and 3.25 ± 3). Straight-line, curvilinear and average-path velocity parameters, amplitude of lateral head and beat-cross frequency were significantly higher in samples frozen with EY, CE-3 and OP compared to CE and BIO media. Straight-line and average-path velocities were also significantly higher in OP compared to EY medium. Live acrosome-intact sperm were significantly higher in EY (45 ± 14) compared to the rest of the extenders; with CE-3 (25 ± 10) being significantly higher than CE (8 ± 5), OP (9 ± 5) and BIO (13 ± 5). Live, non-peroxidized sperm were significantly higher in EY (47 ± 15) compared to the other extenders; with CE-3 (29 ± 15) being significantly higher than CE (14±10), OP (21 ± 15) and BIO (23 ± 14). No significant differences were observed in the percentages of sperm with intact DNA in any of the extenders: EY (95 ± 4); CE (95 ± 2); CE-3 (93 ± 8); OP (94 ± 7) and BIO (94 ± 5). To conclude, the casein salt and cyclodextrin cholesterol extender with 3% dimethylformamide, without egg-yolk could be a suitable alternative for extenders with animal proteins. Further studies are ongoing to evaluate in vivo fertility of ejaculates cryopreserved in this extender.