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The aim of this study was to examine ovine sperm cryoresistance during the rutting season (RS) and its association with sperm head area and seminiferous epithelium proliferation. Small ruminants show fluctuating testosterone levels throughout the year, which could interfere with spermatogenesis and sperm cryopreservation. Ejaculates, testicular biopsies and blood were collected during the middle and at the end of the RS (Middle-RS vs End-RS) during periods of high and low testosterone levels in Merino and Mouflon rams. Fresh and frozen–thawed sperm quality, sperm morphometry, seminiferous tubule morphometry and testicular proliferation markers (proliferating cell nuclear antigen, proliferation marker protein Ki-67 and transcription factor GATA-4) were evaluated. Post-thaw sperm viability was higher in the End-RS group in both Merino (69.9±8.2 vs 41.6±7.3%; P=0.020) and Mouflon rams (40.9±3.3 vs 24.2±5.0%; P=0.008). Mouflons had larger sperm head area at the End-RS (38.3±0.2 vs 34.3±0.1µm2; P=0.029), whereas there was no difference between Merino groups (35.7±0.5 vs 34.8±1.0µm2). Seminiferous tubule morphometry and proliferation markers showed higher levels of germinal epithelium proliferation in the Middle-RS of both species. In conclusion, sperm freezability is affected during the RS in domestic and wild rams, which could be correlated with changes that occur during spermatogenesis, since there is an effect of season on cell proliferation in the testis.

Spermatozoa must undergo the process of capacitation to fertilize the egg which involves a cell destabilizing process. Capacitation-like changes such as protein tyrosine phosphorylation (PTP) are associated with cryopreservation. The aim of this study was to compare the cryoresistance and capacitation response of epididymal and ejaculated sperm of European mouflon (Ovis musimon). Post-thaw sperm parameters were analysed from epididymal and ejaculated samples cryopreserved by slow-freezing or ultrarapid-freezing for comparison. Sperm capacitation status was assessed by the semiquantification of PTP levels, cell localization of PTP and kinematic clustering. Epididymal sperm had higher cryoresistance than ejaculated sperm in both freezing techniques, and slow-freezing rendered better results than ultrarapid-freezing in both sperm samples. Ejaculated sperm had higher PTP levels than epididymal sperm and, additionally, ejaculated sperm showed higher phosphorylation in capacitating (CA) than in non-capacitating (NCA) conditions while there was no effect of medium in epididymal sperm. There was a higher tail PTP in CA than in NCA conditions in both types of sperm. Kinematic analysis revealed that the cluster associated with hyperactivated movement increased in ejaculated sperm incubated in CA whereas no effect of medium was observed in epididymal sperm clusters. In conclusion, epididymal sperm showed better freezability and lower capacitation status compared to ejaculated sperm.

The journey of spermatozoa through the female genital tract is facilitated by rheotaxis, or the cell’s preference to swim against a flow, as well as thigmotaxis, the wall tracking behaviour, which guides them to the site of fertilisation. The aim of this study was to characterise the rheotactic and thigmotactic response of stallion sperm within a microfluidic channel. Stallion sperm rheotaxis was assessed within the microfluidic channel with regard to: (i) A range of flow velocities, (ii) Varying media viscosity and (iii) Sperm hyperactivation. Sperm distribution across the microfluidic channel was also studied and compared to human and ram sperm. Stallion sperm progressed furthest at a velocity range of 10–30 µm/s, with an optimum velocity of 20 µm/s. A flow viscosity of 2.5cP or greater reduced sperm rheotaxis (P < 0.05). Stallion sperm that were hyperactivated were unable to exhibit rheotaxis within the microfluidic channel, whereas, both hyperactivated human and ram sperm did exhibit positive rheotaxis under the same conditions. The number of sperm swimming near the microfluidic channel walls was higher than in the microfluidic channel centre (P < 0.05). This is the first study to illustrate that stallion sperm are rheotactically responsive and increasing viscosity reduces this response. We also demonstrated that sperm are predominantly inclined to swim along a surface and uniquely, hyperactivated stallion sperm are non-progressive and do not exhibit a rheotactic response unlike other species.

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Sperm cryopreservation protocols are not well defined in many wild species such as the mouflon. The aim was to study the effect of different concentrations of EY on mouflon epididymal sperm cryosurvival. Samples were collected by the flushing method and cryopreserved by the conventional freezing technique in straws using a TEST (TES, Tris, glucose) 5% v/v glycerol medium containing either 6% v/v (n=16) or 12% v/v (n=13) clarified EY. The membrane integrity, acrosome integrity, motility, and morphological abnormalities were assessed in fresh and frozen/thawed samples. Fresh sperm quality parameters did not differ between groups except for the acrosome integrity that was lower in the TEST-6% EY than in the TEST-12% EY group (88.9 ± 2.1% vs 94.7 ± 0.8%). Membrane integrity (31.6 ± 4.6% vs 11.6 ± 4.5%), total motility (32.8 ± 4.6% vs 17.2 ± 5.6%), progressive motility (13.3 ± 2.7% vs 6.1 ± 2.9) were higher in frozen-thawed sperm with TEST-6% EY than with TEST-12% EY (p<0.05). Other motility parameters such as curvilinear velocity, straight-line velocity, average path velocity and amplitude of lateral head displacement were also higher (p<0.05) in frozen-thawed sperm with TEST-6% EY. Frozen-thawed acrosome integrity (85.1 ± 3.3% vs 91.9 ± 2.3) and morphological abnormalities (34.0 ± 3.7% vs 29.1 ± 3.6%) did not differ between extenders. In conclusion, high EY concentration had detrimental effects on post-thaw quality parameters, therefore the use of TEST based extender containing 6% EY is recommended for the cryopreservation of mouflon epididymal sperm.