Effect of Honey, Coenzyme Q10, and β-Carotene/α-Tocopherol as Novel Additives in Rabbit-Sperm Cryopreservation Extender

SIMPLE SUMMARY: Rabbit sperm cryopreservation efficiency is still suboptimal. Post-thawing qualities, such as motility and membrane integrity, are severely impaired after the cryopreservation procedure. One strategy to increase cryopreservation efficiency is the use of novel extender components with cryoprotective effects. In the current study, we aim to test the use of honey, coenzyme Q10, and β-carotene/α-tocopherol as novel additives for rabbit-sperm cryopreservation. Additionally, we used, for the first time, proAKAP4 as a molecular marker candidate for sperm quality in rabbits. Our results demonstrated that adding 2.5% honey to the semen extender improved the post-thawing rabbit-sperm motility compared to higher concentrations of honey. In contrast, coenzyme Q10 was harmful to sperm motility after cryopreservation. Furthermore, the β-carotene/α-tocopherol neither improved nor worsened the post-thawing sperm motility compared to the base extender media. In conclusion, the cryopreservation protocols used in this study failed to preserve the fresh-sperm parameters, paving the way for additional studies to improve rabbit sperm cryopreservation protocols. ABSTRACT: The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 μM), and β-carotene/α-tocopherol (500 μM/620 μM and 250 μM/310 μM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the β-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation.