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

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7%) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n=8; r=3) were divided into 4 extenders: BotuSemen Gold with 5% or 7% MF and 5% or 7% DMF, all containing 11% lactose, 20% egg-yolk and 0.5% Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P>0.05). However, samples with 5% DMF showed the highest percentages 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) (Data: mean ± SD; P>0.05). Hence, thirty mares were inseminated: 15 with 5% DMF and 15 with 7% DMF. The pregnancy rate was 46% (7/15) and 0% (0/15) using the extender with 5% or 7% DMF, respectively (P=0.003). To conclude, the use of 5% or 7% of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5% DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates.