Effect of cholesterol-loaded cyclodextrin enriched extenders on the quality of prefrozen and frozen buffalo semen

Objective: To investigate the effects of non-permeable cryoprotectant, cholesterol-loaded cyclodextrin, when added at different concentrations into cooled and frozen-thawed semen extended with Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders. Methods: A total of 40 ejaculates from four buffalo bulls were collected using artificial vagina. Ejaculates were extended with one of Tris-citrate-fructose egg yolk glycerol and lecithin-based extenders which contained different concentrations [0 (control), 0.75, 1.50, 2.25 and 3.00 mg/mL] of cholesterol-loaded cyclodextrin. The extended semen samples were cooled to 5 °C and then frozen slowly to -196 °C in 0.25 mL ministraws before being stored in liquid nitrogen pending its evaluation. Sperm motility, live sperm, normal sperm morphology, sperm membrane integrity and acrosome morphology were measured. Results: Supplementation of cholesterol-loaded cyclodextrin improved progressive motility, viability, morphology and acrosome as well as plasma membrane integrities at 1.50-2.25 mg/mL depending upon types of used extenders and stages of pre- and post-freezing process (P<0.01). The best concentration was 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post- freezing. However, greater concentration (3.00 mg/mL) of cholesterol-loaded cyclodextrin had a detrimental effect compared to the control group with the two evaluated extenders (P<0.01). Conclusions: Cholesterol-loaded cyclodextrin supplementation at 1.50-2.25 mg/mL concentration could improve pre-frozen and post-thawed buffalo sperm quality. The most suitable concentration is 1.50 mg/mL at pre-freeze stage and 2.25 mg/mL at post-freezing.