The In Vitro Evaluation of Rooster Semen Pellets Frozen with Dimethylacetamide

Abstract

Sperm cryopreservation is an effective technique for conserving animal genetic diversity and transmitting superior genetic backgrounds, maintained via a non-invasive sampling and collection of huge quantities of sperm. Nevertheless, cryopreservation in avian species is not commercially viable because of the rooster sperm's susceptibility to damage. This study aims to estimate the impact of dimethylacetamide (DMA) as a cryoprotectant at different levels (3%, 6%, or 9%) on the post-thawed sperm quality, motility, antioxidant-biomarkers, and the expression of anti-freeze related genes. Semen samples were collected twice a week from twelve roosters aged 40 wk, weighing 3400 ± 70 g, and belonging to the Cairo-B2 chicken strain. Fresh semen samples were rapidly appraised, pooled, diluted with two volumes of a basic extender, and divided equally into three groups. The diluted groups were chilled at -20 °C for 7 min, then gently supplemented with 3, 6, or 9% pre-cooled DMA and equilibrated at 5 °C for a further 10 min. Semen pellets were formed by pipetting drops 7 cm above liquid nitrogen (LN₂), which were then kept inside cryovials in the LN₂. Thawing was performed 2 months later by taking 3-4 pellets of the frozen semen into a glass tube and warming it in a water bath for 8 s at 60 °C. The results showed that 3% DMA increased the proportion of total motile sperm, progressivity, viability, and plasma membrane integrity (%) compared to the 6% and 9% DMA groups. The lipid peroxidation and antioxidant enzyme activity were improved in the 3% group. At the same time, some anti-freeze-related genes' (including ras homolog family member A (RHOA), heat shock protein 70 (HSP70), and small nuclear ribonucleoprotein polypeptide A (SNRPA1)) expressions were upregulated within the 3% DMA group relative to other groups. In conclusion, the 3% DMA group maintained higher post-thawed sperm quality than the other tested groups.

Keywords: cryopreservation; dimethylacetamide; roosters; semen; sperm motility.

Figure 1

Impact of different DMA concentrations in freezing extender on the anti-freeze-related genes expression of post-thawed rooster sperm. Bars demonstrate the means of 3 samples (n = 3) as log₂ (fold change) ± standard error (SE). ^a,b Within the same gene, means denoted by various superscripts are significantly different (p < 0.05). HSP70: heat shock protein family A, RHOA: ras homolog family member A, SNRPA1: small nuclear ribonucleoprotein polypeptide A’, and RPL29: ribosomal protein L29.