The Effects of Antifreeze Protein III Supplementation on the Cryosurvival of Goat Spermatozoa During Cryopreservation

Antifreeze protein (AFP) has been shown to have beneficial effects on frozen mammalian spermatozoa. However, rare reports have been published regarding the use of AFPs in storage of goat spermatozoa. The aim of this study was to investigate the effects of AFPIII on the quality of goat semen during cryopreservation. Ejaculates were collected from six Yunshang black goats through an artificial vagina. The collected semen was pooled, divided into five aliquots, and diluted with the commercial bull semen extender containing: no AFPIII (AFP-0, control), 1 mu g/mL AFPIII (AFP-1), 10 mu g/mL AFPIII (AFP-10), 50 mu g/mL AFPIII (AFP-50), and 100 mu g/mL AFPIII (AFP-100), respectively. Spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial function, distribution of phosphatidylserine, and formation of reactive oxygen species (ROS) were measured after the freezing and thawing process. The results showed that the spermatozoa motility, membrane integrity, acrosome integrity, and mitochondrial function were significantly higher in frozen spermatozoa using the extender containing 1 mu g/mL AFPIII as compared with the other groups (p < 0.05). Furthermore, the extender supplemented with 1 mu g/mL of AFPIII resulted in higher viable and lower nonviable spermatozoa compared with the other treated groups (p < 0.05), after staining using Annexin V-fluoresceine isothiocyanate (Annexin V-FITC) and Propidium Iodide. No significant differences were found between these groups in relation to viable cells with lower ROS production. In conclusion, the addition of AFPIII to the freezing extender improved the post-thaw quality of goat semen. The optimal concentration used in this study was 1 mu g/mL. However, excessively high concentrations of AFPIII were unable to exhibit their cryoprotective effects on goat spermatozoa. However, the presence of AFPIII cannot mitigate oxidative stress caused by the freezing and thawing process. In addition, in vitro fertilization or artificial insemination can further evaluate the effects of AFPIII on frozen-thawed goat spermatozoa.