The characteristics of frozen-thawed rooster sperm using various intracellular cryoprotectants

Abstract

Cryopreservation of rooster semen is essential for conserving genetic resources, genetic improvement, and increasing productivity. However, the nature of avian sperm presents a global issue in ensuring superior frozen semen for artificial insemination. Thus, the present study aimed to evaluate the impact of using dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) as cryoprotectants on post-thawed sperm motility, quality, antioxidant indicators, and fertilizing capacity. Twice a week, fresh semen ejaculates were collected from 15 adult roosters and immediately evaluated to constitute a pool from clean and qualified samples. The pooled semen was further diluted at a ratio of 1:2 (v/v) with an extender and then subjected to a freezing protocol in a liquid nitrogen vapor after adding a cryoprotectant solution containing 6% of either DMA, DMSO, or EG, respectively. After thawing, characteristics of sperm motion, quality, antioxidants, and fertilizing ability were evaluated and compared to fresh and cooled semen as controls. The results demonstrated that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. In comparison to the DMSO and EG groups, employing DMA considerably (P < 0.05) raised the percentages of sperm progressive motility, viability, plasma membrane intactness, and DNA integrity. The DMA group showed a significant increase in the catalase and glutathione reduced antioxidant enzyme activity and a reduction in nitric oxide and lipid peroxidation. After artificial insemination, the DMA and DMSO groups exhibited considerably (P < 0.05) better rates of hatchability and fertility than the EG group. It is concluded that freezing extenders containing 6% DMA is better than DMSO or EG to improve the post thaw semen quality and fertility in chickens.

Keywords: Rooster; cryoprotectant; fertility; semen freezing; sperm quality.

Figure 1

Effect of semen cooling and freezing with DMA, DMSO, and EG as cryoprotectants (CPA) on the quality parameters of rooster sperm. Viability (panel A), Plasma membrane integrity (panel B), and DNA fragmentation (panel C). Bars express means ± standard error (SE). Bars with different superscripts represent significant differences (P < 0.05). Fresh: fresh semen as control; Cooled: semen after cooling without CPAs: DMA: semen frozen with dimethylacetamide; DMSO: semen frozen with dimethyl sulfoxide; EG: semen frozen with ethylene glycol.

Figure 2

Effect of semen cooling and freezing with DMA, DMSO, and EG as cryoprotectants (CPA) on the antioxidant biomarkers of rooster sperm. TAC: total antioxidant capacity (panel A), SOD: superoxide dismutase (panel B), CAT: catalase (panel C), GR: glutathione reduced (panel D), LPO: lipid peroxidation (panel E), and NO: nitric oxide (panel F). Bars express means normalized per mg protein ± standard error (SE). Bars with different superscripts represent significant differences (P < 0.05). Fresh: fresh semen as control; Cooled: semen after cooling without CPAs: DMA: semen frozen with dimethylacetamide; DMSO: semen frozen with dimethyl sulfoxide; EG: semen frozen with ethylene glycol.