This study was designed to test if treating chicken sperm with i) the cyclodextrins 2-hydroxypropyl-β-cyclodextrin (HBCD) and methyl-β-cyclodextrin (MBCD) alone improve fresh, liquid-stored and cryopreserved semen quality, or ii) cholesterol-loaded cyclodextrins (CLCs): 2-hydroxypropyl-β-cyclodextrin loaded with cholesterol (HCLC) and methyl-β-cyclodextrin loaded with cholesterol (MCLC) enhance chicken semen quality for application to assisted reproductive technologies. Three consecutive experiments were performed with different concentrations of additives: Exp. 1: 1, 2, 4 mg of HBCD and MBCD in fresh and liquid stored semen; Exp. 2: 1, 2, 4 mg of HCLC and MCLC in fresh and liquid stored semen; and Exp. 3: 1, 2 mg of HBCD, HCLC, 1 mg MBCD, MCLC in cryopreserved and post-thaw storage semen. Sperm motility parameters were assessed by CASA system and comprehensive sperm characteristics were evaluated by flow cytometry. Supplementation with 4 mg HBCD, MBCD, HCLC and MCLC resulted in the lowest motility and functional parameters of fresh and stored spermatozoa for 24 h at 5 °C. After cryopreservation, spermatozoa stored with CLCs showed significantly lower progressive motility and velocity of movement, and exhibited the lowest percentage of cells with intact plasma membranes and acrosomes, mitochondrial activity and cells without apoptosis. These results indicated that CLCs did not improve chicken sperm viability after cryopreservation. However, spermatozoa treated with 2 mg HBCD showed higher proportion of motile sperm (28%; P < 0.01) together with higher proportion of sperm cells with high mitochondrial potential (25%; P < 0.05) compared to the control (18%; 21%, respectively) after 3 h of post-thaw storage. CLC, especially with methyl-β-cyclodextrin, appeared to be detrimental to chicken spermatozoa, causing apoptosis, impairment of mitochondrial potential, and damaged acrosomes and sperm plasma membranes.
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