269 IN VITRO FERTILITY OF BOAR SPERMATOZOA PRESERVED AT 10^°C FOR 22 DAYS

Abstract

Fertility of boar spermatozoa as determined following artificial insemination seems to be maintained during liquid preservation at 10–15°C for several days, although prolonged liquid preservations reduce the pregnancy rate rapidly. However, it is not clear if spermatozoa can penetrate into oocytes in an IVF system even after a prolonged liquid preservation. Oxidative stress could also be one of the possible detrimental factors in liquid preservation of spermatozoa. In the present study, fertility of liquid-preserved spermatozoa was examined using an IVM-IVF system. Whether cysteine can improve the fertility was also determined. Spermatozoa (from four Berkshires) was resuspended at 1 × 10⁸ cells mL⁻¹ in Modena solution containing 15% (v/v) boar seminal plasma and 0 or 5 mM cysteine after washing 3 times. Sperm suspensions (1 mL) were then preserved at 10°C for 22 days following a program for cooling down (to 15°C for 4 h, keeping at 15°C for 12 h and then to 10°C for 6 h). At Days 1, 8, 15 and 22 after the start of preservation, spermatozoa (5 × 10⁵ cells mL⁻¹) were co-cultured with IVM oocytes in an IVM/IVF system (Funahashi et al., 1997 Biol Reprod 57, 49–53). Viability and functional status of spermatozoa were also examined at Days 8 and 15 of preservation by using LIVE/DEAD sperm viability kit and CTC fluorescence assay. Data (mean ± SEM) from 4–6 replicates were analyzed by ANOVA and Fisher’s protected LSD test. When spermatozoa that had been preserved without cysteine (Cys−) were used, penetration rates were not different (P > 0.05) from those with cysteine (Cys+) at Day 8 of preservation (91.4 ± 3.4% in Cys− and 99.3 ± 0.7% in Cys+), but lower (P < 0.02) at Days 15 and 22 (72.6 ± 13.6% and 33.8 ± 8.4% in Cys−; 94.8 ± 2.1% and 71.1 ± 10.8% in Cys+, respectively). Both viability and proportion of uncapacitated live cells were higher (P < 0.05) in Cys+ than Cys− at Days 8 and 15. These results demonstrate that boar spermatozoa can penetrate into oocytes in vitro even after a liquid preservation at 10°C for 22 days and that cysteine can improve the viability and penetrability in vitro of spermatozoa during liquid preservation. Supported by the Ito Foundation.