103 CHOLESTEROL-LOADED CYCLODEXTRIN ADDED TO FRESH BOAR EJACULATES AND SPERM CRYOSURVIVAL

Sperm cryosurvival is affected by altering the lipid composition of sperm plasma membranes and causes damage to spermatozoa during the cryopreservation process as loss of motile cells and functionality, compared with fresh sperm. Our objective was to compare the effect of adding cholesterol-loaded cyclodextrin (CLC) on sperm quality after freezing boar sperm. The CLC was prepared as described: 200 mg of cholesterol was dissolved in 1 mL of chloroform, and 1 g of methyl-β-cyclodextrin was dissolved in 2 mL of methanol. A 0.45-mL aliquot of the cholesterol solution was added to the cyclodextrin solution, after which the mixture was poured into a glass dish and the solvents removed using a hot plate for 24 h. The crystals were removed from the dish and stored at 22°C. A working solution of the CLC was prepared by adding 50 mg of CLC to 1 mL of BTS at 37°C. Thirty-five ejaculates from 5 boars were collected, diluted 1:1 in Beltsville thawing solution, and kept to 2 h at 22°C. The ejaculates were held at 15°C for 60 min and centrifuged at 15°C for 400g for 10 min; the pellet was suspended to 120 million cells in cooled diluent (80 mL of lactose solution 11%, 20 mL of egg yolk) and divided in 2 treatments: control and 1.5 mg of CLC/mL. The samples were incubated for 15 min at 15°C, cooled to 5°C over a 90-min period, and diluted 1:1 with freeze diluent (72.5 mL of lactose solution 11%, 6 mL of glycerol, 1.5 mL of Equex). Sperm were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen. Straws were thawed in a water bath at 37°C for 30 sec, extended in Beltsville thawing solution, and analyzed by optic microscopy. Sperm were stained with 35 μg mL-1 of Hoechst 33342 and incubated for 15 min at 37°C, centrifuged at 400g for 5 min, and suspended in BTALP to a final concentration of 2 million spermatozoa/mL. A total of 10 000 spermatozoa (5 μL) from each sample were added to droplets containing 10 porcine oocytes. Porcine cumulus oocyte complexes were aspirated and placed in BTALP. The cumulus cells of the oocytes were removed by vortexing for 2 min at maximum speed. Denuded oocytes were washed 4 times in BTALP and incubated for 1 h at 38.5°C in an atmosphere of 5% CO2 in air, following which 10 oocytes per treatment were randomly placed into 45 μL droplets of BTALP, using a small bore fire polished glass pipette to remove loosely bound spermatozoa. Five oocytes were placed onto glass slides and covered with a cover slip supported by a mix of paraffin wax and petroleum jelly. Oocytes were viewed using an epifluorescence microscope, and the total number of spermatozoa bound to each zona pellucida (ZP) was determined at 400× magnification. Treatment differences for sperm motility and zona binding were determined using ANOVA. The addition of CLC to boar sperm before cryopreservation resulted in higher percentages of motile sperm and higher numbers bound to the ZP (35% and 67 sperm/ZP) compared with control cells (26% and 36 sperm/ZP; P < 0.01). In summary, adding CLC to boar sperm before cryopreservation improved cells. FAPEMIG, Piglandia, CNPq, FACEPE.