Effects of cholesterol-loaded cyclodextrins on the quality of frozen–thawed equine epididymal sperm

Abstract

Equine epididymal sperm are known to be severely sensitive to cryopreservation, in terms of sperm quality and pregnancy rate. The objective of this study was to examine the effects of cholesterol loaded cyclodextrins (CLCs) on the quality of stallion epididymal sperm during cryopreservation.In experiment I, sperm were treated with different concentrations of CLCs: (1) 0mg (control), (2) 1.5mg, (3) 3mg, and (4) 6mg per 120×106 sperm. The sperm viability and amount of cholesterol were determined at 15, 30 and 45min after CLC treatment using viability markers (Ethidium homodimer-1 and Calcein AM) and gas chromatography, respectively. In experiment II, CLC treated sperm (1.5mg CLC per 120×106 sperm) were fixed and stained with filipin to examine the cholesterol distribution. In experiment III, sperm were treated with CLCs at concentrations of 1.5, 3.0, 6.0mg per 120×106 sperm for 15min, then equilibrated with freezing extender at 4°C for 1h prior to cryopreservation. Epididymal sperm without CLC loading (0mg) were used as the control group. The sperm quality was examined at post-equilibration and 10min, 2h and 4h after freezing and thawing.The cholesterol was successfully loaded into the plasma membrane of stallion epididymal sperm. The amount of cholesterol was increased in a manner of dose and time dependence, and the filipin–sterol complexes were increasingly labeled over the sperm head. CLCs at 1.5mg/120×106 sperm significantly improved sperm quality during sperm equilibration and cryopreservation compared to other doses of CLCs and non-CLC control. An increasing concentration and incubation time of CLCs was detrimental to sperm quality.It is concluded that cholesterol loading to the sperm plasma membrane via CLCs decreases chilling sensitivity and also improves epididymal sperm cryopreservability.