Effects of cholesterol supplementation on the cryosurvival of equine spermatozoa

Abstract

Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion spermatozoa to determine which concentration of CLC optimizes cryosurvival. Maximum percentages of motile spermatozoa were maintained after thawing when 1.5 mg CLC was added to sperm. Addition of CLC's also increased the percentages of viable spermatozoa surviving cryopreservation compared to non-treated spermatozoa (P<0.05). The amount of cholesterol that incorporated into the membranes of spermatozoa increased in a polynomial fashion (R2=0.9978) and incorporated into all spermatozoal membranes. In addition, there was significant cholesterol loss from spermatozoal membranes after cryopreservation, however, CLC's addition to spermatozoa prior to cryopreservation resulted in higher cholesterol levels in the spermatozoa after cryopreservation than untreated spermatozoa (P<0.05). Addition of CLC's also resulted in more spermatozoa binding to bovine zona pellucida after cryopreservation than control spermatozoa (48 vs. 15; P<0.05). In addition, when stallion spermatozoa were subjected to anisosmotic solutions, or after spermatozoa were returned to isotonic conditions, CLC treatment increased the osmotic tolerance limits as measured by spermatozoal motility (P<0.05). Additional experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. Stallion spermatozoa were determined to have a volume of 18.61 ± 0.51 μm3, and behaved as linear osmometers when incubated in anisosmotic conditions and exhibited an osmotically inactive volume of 83%. When spermatozoa were treated with CLC's and incubated with one of three cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and volume excursions measured during cryoprotectant removal at 5° and 22°C, stallion spermatozoa were less permeable to the cryoprotectants at 5°C than 22°C and glycerol was the least permeable cryoprotectant in control cells (P<0.05). The addition of CLC's to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants (P<0.05). A final study was conducted to determine the optimal cooling rate for stallion spermatozoa frozen in the presence of one of three cryoprotectants. Spermatozoa were frozen in a diluent containing 4% glycerol, ethylene glycol or dimethyl formamide at 10 cooling rates ranging from -5°C/min to -50°C/min. The percentages of viable spermatozoa were higher for spermatozoa cooled at -10°C/min compared to spermatozoa cooled at -50°C/min (P<0.05). Spermatozoa frozen using glycerol as the cryoprotectant had higher percentages of motile and progressively motile spermatozoa compared to those spermatozoa frozen using the other two cryoprotectants (P<0.05). In conclusion, the cryosurvival of stallion spermatozoa was similar when cooling rates of -5°C/min to -45°C/min were used and when 4% cryoprotectant was used. Glycerol was a more effective cryoprotectant, than ethylene glycol or dimethyl formamide at this concentration.