Comparison of different cryoprotectants for freezing donkey jack (Equus asinus) semen

Abstract

The objective of this study was to extrapolate a rapid sperm freezing technique developed in horses to Remonta Argentino donkey semen, evaluating, in vitro and in vivo, the effect of freezing media that had two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) at two different concentrations (5 and 7%). Twenty-four ejaculates from 8 fertile jacks (n=8; r=3) were divided into 8 extenders: BotuSemen Gold with 5% or 7% MF and 5% or 7% DMF and EDTA-glucose with 5% or 7% MF and 5% or 7% DMF, all containing 11% lactose, 20% egg-yolk and Equex and frozen: to -15 oC (10-12 oC/min) then to -120 oC (25-40 oC/min.) then plunged in N2. Post-thaw evaluations included: sperm motility (Computer Assisted Semen Analysis; AndroVision, Minitüb GmbH, Tiefenbach, Germany), morphology (Diff Quick stain), membrane function and acrosome status (combined hypoosmotic test and Coomassie blue stain). Sperm data were analyzed using Kruskal Wallis and pregnancy data using Chi-Square. Significant differences were observed in the post-thaw progressive sperm motility between individuals (p< 0.05) creating three subgroups (low: 9.23  0.16; intermediate: 22.92  9.81 and high: 41.33  5.41. Values are mean  SD). Within each subgroup, in vitro the samples did not present statistically significant differences among the 8 media used, for any of the seminal parameters evaluated. However samples in BotuSemen Gold with 5% DMF showed the highest percentages (P>0.05) of sperm with acrosomes and functional membranes (DMF: 5%: 53.67  22.01; 7%: 33.92  23.4; MF: 5%: 44.5  20.46; 7%: 38.75  27.4. Values are mean  SD). In vivo, 300x106 PM sperm were deeply inseminated post-ovulation in 30 mares: 15 with BotuSemen Gold 5% DMF and 15 with BotuSemen Gold 7% DMF. A 46% (7/15) pregnancy rate was obtained using the extender with 5% DMF and no pregnancies (0%; 0/15) were obtained using the extender with 7% DMF (P=0.003). Perhaps uterine inflammatory response post-insemination was too high when using 7% DMF or sperm chromatin was damaged, affecting in vivo results. In conclusion, the rapid semen freezing technique was successfully applied in jacks. Regarding the extenders, in vitro no significant differences were observed using either the BotuSemen Gold or the EDTA-glucose media, whether adding MF or DMF at either 5% or 7%. However, when inseminating mares, pregnancies were only obtained using 5% DMF. It would be interesting to look at sperm DNA and peroxidation parameters, as well as uterine response to insemination to determine possible reasons for the failure in fertility and the variability observed between individuals.