The Effect of Cholesterol-Loaded Cyclodextrin and Resveratrol Compounds on Post-Thawing Quality of Ram Semen

Ram sperm are more vulnerable to freezing than those of most other farm animals. During sperm freezing, the cell membrane loses some of its cholesterol, which regulates signalling mechanisms and prevents premature capacitation. Resveratrol (RES) increases the fluidity of the cell membrane, which becomes peroxidized during freezing and reduces free radicals. In this study, the effectiveness of RES, cholesterol-loaded cyclodextrin (CLC) and their combinations in ram sperm cryopreservation were investigated. The collected semen was divided into two equal volumes: One was diluted with tris-citric acid-glucose medium (TCG) containing CLC, whereas the other was diluted with a CLC-free TCG solution. After examining motility, both groups were further divided into two equal volumes, forming the following working groups: control (no RES, no CLC); RES (20 µg/mL); CLC (2 mg CLC/120 × 10⁶ sperm); and RES + CLC (RES 20 µg/mL + 2 mg CLC/120 × 10⁶ sperm). These groups were diluted with media containing their respective additives. Post-thawing, the samples were analysed for motility, acrosome and membrane integrity, membrane functionality, mitochondrial activity, capacitation status, oxidative stress and DNA integrity. CLC preserved sperm total motility, acrosome and plasma membrane integrity and decreased the rate of early capacitation (p < 0.05). RES had no significant effect on sperm quality before freezing and post-thawing (p > 0.05). However, RES + CLC increased mitochondrial activity post-thawing (p < 0.05). In conclusion, CLC minimized sperm membrane damage caused by cryopreservation in ram sperm. RES alone was ineffective, and the combination of RES and CLC did not yield a positive synergistic effect on ram spermatological parameters.