Effect of two extenders on the cryopreservation of Biyang donkey semen

Abstract

The Biyang donkey is a medium-sized Chinese donkey. Very little research has been done on semen cryopreservation in this donkey breed. Dimethylformamide (DMF) and ethylene glycol (EG) are small molecular weight osmotic cryoprotectants that enter equine spermatozoa more readily and effectively than glycerol, and spermatozoa have reduced swelling mortality when equilibrated in amide-containing extenders. In recent years, EG and DMF have been used in extenders to improve the quality of cryopreserved donkey semen. It is, however, unknown whether their combination with glycerol is more effective than glycerol alone in protecting the semen of Biyang donkeys. In this experiment, we aimed to compare the effects of two different semen extenders on semen cryopreservation in Biyang donkeys. Both extenders were based on the CAU1 extender (Feng Yang et.al. Equine Veterinary Journal 2021, 53: 1218-1226). The cryoprotectant 1 consisted of 2.5% egg yolk and 2.5% glycerol in CAU1 extender, cryoprotectant 2 consisted of 2.5% yolk, 1.5% DMF, 0.5% glycerin and 0.5% EG in CAU1 extender. In five donkeys, semen was collected three times to deplete epididymalsperm reserves, then one ejaculation of each jackass was collected for semen freezing was described (Feng Yang et.al. Equine Veterinary Journal 2021, 53: 1218-1226). Briefly, after the semen was collected, an equal volume of CAU1 dilution was added and left to stand at room temperature (22-25°C) for 30 min, thereafter semen was divided into two equal portions and centrifuged to remove the semen plasma, one was diluted with cryoprotectant 1 and the other with cryoprotectant 2. Diluted semen was filled into 0.5 mL straws with a filling machine and left to stand at 4°C for 2-3 hours. Cryopreservation was carried out at a height of 3-5 cm above liquid nitrogen for 15 min, straws were then stored in liquid nitrogen. Two days later, semen was thawed in a water bath at 37°C for 40 seconds. Sperm motility was examined by CASA (TYCASA1.0, Beijing Tianyuan Aorui Biotechnology Co., Ltd). Motility of the raw semen (n=5) was 89.6±4.0%. The frozen-thawed motility was 56.0±4.18% and 51.6±4.3% in cryoprotectant 1 and cryoprotectant 2, respectively (not significant). In conclusion, the two cryo-extenders were equally suitable for cryopreservation of Biyang donkey semen.