Effects of cholesterol-loaded cyclodextrins on quality of equine epididymal sperm after freezing and thawing

Abstract

The objectives of this study were to examine the effects of cholesterol-loaded cyclodextrins (CLCs) on amount and distribution of cholesterol in equine epididymal sperm (EXP.1, EXP.2), and to study the effect of CLCs on quality of epididymal sperm during cryopreservation. EXP.1Sperm from Thai crossbreed stallions (n=3, aged 3-12 years old) were loaded with three concentrations of CLCs: 1) 1.5 mg, 2) 3 mg, and 3) 6 mg of CLCs per 120x10⁶ sperm. The CLCs loaded sperm were then analyzed for the amount of total cholesterol in the sperm plasma membrane at 15, 30 and 45 min of CLC incubation using gas chromatography. Sperm incubated in extender without CLCs served as a control.The cholesterol contents in epididymal sperm were increased in a manner of dose and time dependence. Incubating sperm with 1.5 mg CLCs/120 x 10⁶ sperm preserved sperm quality similar to non-CLC treated control (P>0.05). However, high CLC concentration, especially at 6 mg/ 120 x 10⁶, significantly decreased sperm motility and viability in all incubation times. EXP.2 Sperm from Thai crossbreed stallions (n=3, aged 3-12 years old) were treated with 1.5 mg CLCs/ 120x10⁶ sperm. After 15 min, the sperm were fixed and stained with filipin to examine the cholesterol distribution. Filipin-sterol complexes were distributed throughout the sperm head with a lower intensity at apical and pre-equatorial subdomains of non-CLC treated sperm. In CLC treated sperm, two patterns of filipin-sterol complexes were predominantly detected. A large proportion of CLCs treated sperm (64.3 ± 2.8%) had a marked increase of filipin-sterol complex intensity over the sperm head compared to non-CLC treated sperm. However, 27.7 ± 3.2% of sperm demonstrated an absent or patchy labeling of filipin at apical and pre-equatorial parts. Small number of sperm (8.0 ± 4.6%) had pattern of filipin-sterol complexes similar to that of non-treated control. EXP.3 Epididymal sperm from Thai crossbreed stallions (n=7, aged 3-12 years old) were treated with CLCs as similar to EXP.1 and then submitted to cryopreservation. The sperm quality was examined at before cryopreservation, post-equilibration and 10 min, 2 h and 4 h after freezing and thawing. CLCs at 1.5 mg/120 x 10⁶ sperm significantly improved sperm quality during sperm equilibration and cryopreservation compared to CLCs at 3 and 6 mg/120 x 10⁶ sperm and non-CLC treated sperm. It is concluded that cholesterol loading to the sperm plasma membrane by CLCs at optimal concentration decreases chilling sensitivity and also improves epididymal sperm cryopreservability.