80 TREATING BOAR SPERM WITH CHOLESTEROL IMPROVES CRYOSURVIVAL

Abstract

Altering the lipid composition of plasma membranes not only affects the ability of sperm to capacitate and acrosome react, but also affects the way sperm respond to cryopreservation. When cyclodextrins are preloaded with cholesterol to form cholesterol-loaded-cyclodextrin (CLC) and then incubated with bull sperm before cryopreservation, higher percentages of motile and viable cells are recovered after freezing and thawing compared with control sperm. The amount of cholesterol in a membrane is important for maintaining its integrity during cryopreservation, and CLC alters the lipid composition of sperm, affecting their cryosurvival. This study evaluated the effect of adding cholesterol to boar sperm on cryosurvival rates and the ability of cryopreserved sperm to bind to the zona pellucida. Methyl-β-cyclodextrin was loaded with cholesterol as follows: 0.45 mL of cholesterol (200 mg mL^–1 in chloroform) was added to 1 g of methyl-β-cyclodextrin dissolved in 2 mL of methanol, and the solution was stirred until clear. The mixture was poured into a glass dish and the solvents removed using a stream of nitrogen gas. The resulting crystals were allowed to dry for an additional 24 h, at which time they were removed from the dish and stored in a glass container at 22°C. A working solution of the cholesterol-loaded cyclodextrin was prepared by adding 50 mg of CLC to 1 mL TALP at 37°C and mixing the solution briefly using a vortex mixer. Ejaculates from each of 8 boars were collected, diluted 1:1 in BTS^® (Minitub, Brazil), and maintained for 2 h at room temperature. The ejaculates were then cooled to 15°C over 60 min. The ejaculates were then centrifuged at 400 × g for 10 min (at 15°C), the supernatant was discarded, and the sperm were suspended to 120 × 10⁶ cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk). The sperm were divided into 2 treatments (T): T1 = control and T2 = 1.5 mg of CLC mL^–1. The samples incubated for 15 min at 15°C, after which they were cooled to 5°C over 90 min and diluted 1:1 (v:v) with Freeze diluent (2.5 mL of lactose solution 11%, 6 mL of glycerol, and 1.5 mL of Orvus-es-Paste). The sperm were then packaged into 0.5-mL French straws and frozen in static liquid nitrogen vapor (4.5 cm above the liquid nitrogen) for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the efficiency of the sperm to bind to both the chicken egg perivitelline membrane (EPM) and porcine zona pellucida (PZP) were determined using epifluorescence microscopy. The post-thaw motility and binding efficiency of sperm to salt-stored EPM and PZP were analysed by analysis of variance. Boar sperm treated with CLC maintained higher post-thaw motility than control sperm (47 and 34%, respectively; P < 0.05) and had higher numbers of sperm binding to the PZP and EPM (101 sperm/EPM and 166 sperm/PZP) than control samples (77 sperm/EPM and 65 sperm/PZP; P < 0.05). In addition, sperm were easier to visualise on the EPM than the porcine zona pellucida. Adding CLC to boar sperm before cryopreservation increased the number of sperm surviving cryopreservation.