Can adding cholesterol help boar sperm survive freezing and retain their ability to bind to an egg's outer covering?

Details

Changing the lipid composition of sperm plasma membranes affects their ability to undergo capacitation and the acrosome reaction, as well as their response to cryopreservation. In bull sperm, incubating cells with cholesterol-loaded cyclodextrin (CLC) before freezing yields higher percentages of motile and viable cells after thawing than in controls. Membrane cholesterol is important for maintaining membrane integrity during cryopreservation; CLC changes sperm lipid composition and affects cryosurvival.

This controlled laboratory experiment evaluated whether adding cholesterol to boar sperm affected cryosurvival and the ability of cryopreserved sperm to bind to the zona pellucida.

To prepare CLC, 0.45 mL of cholesterol solution (200 mg mL–1 in chloroform) was added to 1 g of methyl-β-cyclodextrin dissolved in 2 mL of methanol, then stirred until clear. The mixture was poured into a glass dish, and the solvents were removed with a stream of nitrogen gas. The resulting crystals were dried for another 24 h, removed from the dish, and stored in a glass container at 22°C. A working solution was made by adding 50 mg of CLC to 1 mL of TALP at 37°C and briefly mixing with a vortex mixer.

Ejaculates from each of 8 boars were collected, diluted 1:1 in BTS® (Minitub, Brazil), and kept at room temperature for 2 h. They were cooled to 15°C over 60 min. They 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 containing 80 mL of 11% lactose solution and 20 mL of egg yolk.

The sperm were divided into two treatments: T1, the control, and T2, 1.5 mg CLC mL–1. Samples were incubated for 15 min at 15°C, cooled to 5°C over 90 min, and diluted 1:1 (v:v) with Freeze diluent containing 2.5 mL of 11% lactose solution, 6 mL of glycerol, and 1.5 mL of Orvus-es-Paste. Sperm were packaged in 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.

After thawing, sperm binding to chicken egg perivitelline membrane (EPM) and porcine zona pellucida (PZP) was assessed using epifluorescence microscopy. Post-thaw motility and binding efficiency to salt-stored EPM and PZP were analyzed by analysis of variance.

CLC-treated sperm had higher post-thaw motility than controls: 47% versus 34% (P < 0.05). Binding counts were also higher with CLC: 101 sperm/EPM and 166 sperm/PZP, compared with 77 sperm/EPM and 65 sperm/PZP in controls (P < 0.05). Sperm were easier to visualize on EPM than on porcine zona pellucida. The study concluded that adding CLC before cryopreservation increased the number of boar sperm surviving cryopreservation.

The abstract names Fapemig, CNPq, and CAPES from Brazil.