In Experiment 2, 3 ejaculates per stallion ( nвЂ‰=вЂ‰6) were included. Semen motility and membrane integrity were completely absent, while DNA-methylation was similar in raw (0.4вЂ‰В±вЂ‰0.2%) and shock-frozen (0.3вЂ‰В±вЂ‰0.1%) semen (not significant). In Experiment 1, 1.5вЂ‰mL of raw semen ( nвЂ‰=вЂ‰6 stallions, 1 ejaculate each) was shock-frozen at вЂ“196вЊ for 15вЂ‰min and thawed at 38вЊ for 60вЂ‰s. Values are meansвЂ‰В±вЂ‰standard error of the mean.

Statistical analysis was done with the SPSS Statistics 21 software. The level of 5-methylcytosin in DNA is reported as the amount of methylated cytosine relative to the cytosine genomic content (%). Global DNA-methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) with a monoclonal antibody sensitive and specific for 5-methylcytosin after DNA extraction and denaturation (100вЂ‰ng of DNA per sample). For this purpose, semen was collected from fertile Shetland pony stallions. We hypothesised that the cryoprotectant may influence the DNA-methylation level of frozen-thawed semen. In the present study, we therefore addressed changes in sperm DNA-methylation induced by cryopreservation of stallion semen. However, semen processing may also contribute to epigenetic changes in spermatozoa. This has been mainly attributed to changes in sperm membrane function caused by the freezing-thawing procedure.

The use of cryopreserved semen for insemination of mares facilitates breeding management but often results in reduced conception rates.

Minitube 2.2