Spermatozoa DNA and plasma membrane integrity after pellet optimized processing for cryopreservation in meat type chicken breeders

ABSTRACT

1. Aim of this study was the development of an optimised cryopreservation pellet procedure for chicken semen and the assessment of DNA and membrane integrity in frozen/thawed spermatozoa in a Hubbard F15 meat type selected strain.

2. The following semen processing conditions were studied: spermatozoa working concentration (SWC), 1.5 vs 2 × 10⁹ cells/ml in pre-freezing extender; equilibration of diluted semen at 5°C, 20 vs 40 min; dimethylacetamide concentration, 6% vs 9%; dimethylacetamide equilibration time at 5°C, 1 vs 30 min; thawing at 60°C for 10 vs 50°C for 30 sec. Spermatozoa viability (EtBr exclusion procedure – stress test), mobility (Accudenz® swim-down test) and subjective motility were assessed in fresh and frozen-thawed semen.

3. The lower SWC (1.5 × 10⁹ cells/ml) and the higher dimethylacetamide concentration (9%) had positive significant effects on the recovery rate of motile (22% vs 16%) and viable spermatozoa (39 vs 34%), respectively.

4.Membrane (SYBR14-PI staining) and DNA integrity (comet assay) were assessed before and after freezing/thawing according to the optimised protocol.

5. Recovery rates of spermatozoa with undamaged plasma membrane and DNA were 41% and 76%, respectively. The distribution of spermatozoa in classes of DNA damage was also analysed and discussed.

6. It was concluded that pellet cryopreservation was a damaging process mainly for plasma membrane rather than nuclear DNA in chicken spermatozoa.

KEYWORDS:

• Chickens
• DNA integrity
• pellet cryopreservation
• plasma membrane integrity
• spermatozoa

Acknowledgments

The study was funded by the Italian Council of Education, University and Research (PRIN project no. 20066072850). The authors would like to thank Grazia Mangiagalli (Department of Veterinary Medicine, Università degli Studi di Milano) for technical assistance in bird management and semen collection.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The study was funded by the Italian Council of Education, University and Research (PRIN project no. 20066072850).