Effect of Dimethylacetamide Concentration on Motility, Quality, Antioxidant Biomarkers, Anti-Freeze Gene Expression, and Fertilizing Ability of Frozen/Thawed Rooster Sperm

SIMPLE SUMMARY: Although sperm cryopreservation has a great potential for application in the poultry industry, it is still very limited at the economical scale due to the low fertility of frozen/thawed avian spermatozoa. Dimethylacetamide (DMA) has been widely employed as a cryoprotectant for the cryopreservation of chicken spermatozoa, but with variable results in motility and fertility. To determine the appropriate concentration of DMA in the chicken semen freezing extender, in vitro sperm quality and motility analyses and in vivo field trials based on the fertility of the frozen/thawed rooster sperm were conducted in the present study. The antioxidant biomarkers and novel anti-freeze-associated genes were also analyzed in the post-thawed sperm. Results conclude that DMA is preferably used at a low concentration to obtain applicable results in sperm motility and fertility after thawing without harming the quality and antioxidant system defense. The current study could highlight the possible molecular mechanisms behind the obtained fertility results of DMA-frozen sperm, especially when artificial insemination is implemented in the poultry industry. ABSTRACT: Sperm cryopreservation is of great importance for the poultry industry but still needs to be optimized. The high susceptibility of poultry sperm to cryodamage leads to low fertility rates after cryopreservation. Therefore, the present study aimed at evaluating the effect of including a cryoprotectant, dimethylacetamide (DMA), in the chicken semen freezing extenders at a final concentration of 3%, 6%, or 9% on the post-thawed sperm motility, quality, antioxidant biomarkers, anti-freeze gene expression, and fertilizing ability. Results showed that the total motile sperm, progressivity, and viability were quadratically increased (p < 0.05) in the 6% DMA group. The antioxidant enzyme activity and lipid peroxidation were negatively (p < 0.05) affected by the increase in DMA concentration. Furthermore, some anti-freeze-associated genes such as heat shock protein 70 (HSP70) and ras homolog family member A (RHOA) were linearly and quadratically down-regulated (p < 0.05) with the high concentration of DMA. Finally, the fertility and hatchability rates did not indicate statistical differences between DMA groups. It can be concluded that using the low concentration of 3–6% DMA in the freezing semen extender is preferable to obtain acceptable results in the post-thawed sperm quality and fertility.