This study aimed to evaluate the antioxidant properties of coenzyme Q10 (CoQ10) during cryopreservation of semen obtained from stallions having good and bad semen freezing ability (GFA vs. BFA, respectively). Forty ejaculates (n = 20 stallions) were split into five centrifugation and five freezing extenders containing different concentrations of CoQ10 (0, 25, 50, 75 and 100 μmols/L). If CoQ10 was added to the centrifugation extender, the freezing extender had no CoQ10 added; similarly, if CoQ10 was added to the freezing extender, the centrifugation extender had no CoQ10. Semen cryopreserved on extenders containing no CoQ10 served as the control. After post-thaw total sperm motility (TM) assessments, the stallions were classified as GFA (i.e., decrease of ≤25% in TM, n = 7) or BFA (i.e., decrease of ≥40% in TM, n = 5). Stallions not fitting (n = 8) this enrollment criteria had samples discarded. After that, two straws for each extender were thawed at 37 °C for 30 s; one straw was immediately used for evaluation of sperm kinetics, plasma membrane integrity, non-capacitated spermatozoa, reactive oxygen species production, mitochondrial activity and lipid peroxidation. The second straw was kept at 37 °C for 30 min and subjected to the same assessments. Expectedly, sperm motility parameters were significantly lower for stallions with BFA. There were no effects of CoQ10 concentration or time for all parameters evaluated in the group with GFA when compared with the control extender (p > 0.05), except lipid peroxidation (p < 0.05). However, stallions with BFA had improved sperm parameters for samples processed with extenders containing CoQ10 (particularly 75 μmols/L) (p < 0.05), except for the reactive oxygen species production and mitochondrial potential (T0) in which there were no differences between the groups (p > 0.05). In summary, 75 μmols/L appears to be the optimal dose of Co-Q10, particularly, when added to the centrifugation extender.

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Artificial insemination using cooled-transported semen has marked importance in equine breeding programs around the world, and the high value of mules has generated avid interest in donkey semen biotechnology. However, donkey semen cools poorly in commercially available equine extenders. Therefore, this study aimed to develop approaches to improve the ability of donkey semen to tolerate cooling. Ejaculates of seven donkeys (n = 21) were cooled at 5°C for 48 h in three different extenders (milk-based, SM; sodium caseinate-based, SC; or egg yolk-based, EY) in the presence or absence of seminal plasma (centrifugation, C). Sperm motility, plasma membrane integrity (PMI), plasma membrane stability (PMS), mitochondrial membrane potential (HMMP), intracellular hydrogen peroxide (H2O2), and intracellular superoxide (O2−) were assessed before, 24 h, and 48 h post-cooling. In addition, 15 mares (163 estrous cycles) were randomly inseminated with semen from two jacks (Jack 1, n = 90; Jack 2, n = 73) previously cooled for 24 h under one of the treatments (SM, SC, EY, SM-C, SC-C, or EY-C). Groups EY, SC-C, and EY-C (P &lt; 0.05) demonstrated superior sperm analytical parameters to SM at 24 and 48 h. Centrifugation positively affected sperm analytical parameters in cooled donkey semen extended in SM and SC (P &lt; 0.05). Mares bred with semen extended in SC (67%, 18/27), SC-C (89%, 24/27), EY (89%, 25/28), or EY-C (74%, 20/27) had significantly greater conception rates than mares bred with SM (33%, 9/27; P &lt; 0.05). Mares bred with SM-C had intermediate conception rates (59%, 16/27). In conclusion, SC and EY improved the cooling ability and fertility of donkey semen in horse mares, and centrifugation positively affected donkey semen extended in SM.

This study investigated the binding capacity of equine spermatozoa (both ejaculated and from different epididymal regions) to the oviductal epithelial cells (OECs) culture, before and after cryopreservation, using an oviduct explant assay. Ejaculated and epididymal sperm from caput, corpus, and cauda of 10 stallions were diluted and submitted to freezing process. Fresh and frozen-thawed sperm were evaluated for sperm kinematics, plasma membrane integrity (PMI) and incubated with oviduct explants. The cryopreservation process decreased significantly the sperm motility parameters of ejaculated sperm, and corpus and cauda epididymal sperm (P < .05). The percentage of PMI was significant higher in fresh samples versus frozen-thawed samples, in all analyzed groups (P < .05). Binding of ejaculated spermatozoa to oviduct epithelium was significantly higher than caput, corpus, or cauda epididymal sperm (P < .05). The caput epididymal sperm showed no binding capacity to oviduct explants; thus, significantly more sperm recovered from the corpus and cauda epididymis were bound to OEC compared to caput epididymal sperm (P <.05). No differences were observed in ejaculated and epididymal sperm before and after cryopreservation (P > .05). In conclusion, the ejaculated sperm has higher binding capacity than epididymal sperm, suggesting that the seminal plasma plays an important role in the establishment of the oviductal sperm reservoir. The cryopreservation process did not affect the binding capacity of ejaculated or epididymal spermatozoa to oviductal epithelium.