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Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure.

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Equestrian sports require the cooperation of two species, horses and humans, but it is unknown to what extent stress responses in the rider affect the horse. In this study, the stress response of experienced and less-experienced horses and riders at showjumping was analysed. Sixteen sport horses were divided into two groups (n = 8 each) by experience and were ridden by highly experienced professionals (n = 8) and less-experienced riders (n = 8). Riders jumped a course of obstacles with an experienced and a less-experienced horse and horses took part with an experienced and less-experienced rider. Salivary cortisol, heart rate and heart-rate variability (HRV) variables, standard deviation of RR interval (SDRR) and root mean square of successive RR differences (RMSSD) were analysed. Cortisol and heart rate increased and HRV decreased in all riders and horses. In less-experienced riders, cortisol release was higher on a lessexperienced versus an experienced horse but the horses' cortisol release was not affected by experience of their riders. Heart rate did not differ between groups of horses and was not affected by experience of the rider but was higher in less-experienced versus experienced riders. The HRV decreased in horses and riders and SDRR was lower in less-experienced versus experienced riders. Thus, lower experience of riders appears not to affect physiological stress parameters in their horses during a showjumping course.