295 INFLUENCE OF FERTILIZATION MEDIUM ON THE INCIDENCE OF POLYSPERMY IN THE PIG

Abstract

The in vitro production of embryos is well established in most domestic species including cattle, pigs, sheep, and goats. However, a major problem of IVF in the pig is the high incidence of polyspermy. In our laboratory, we investigated the effect of 2 different media, TALP and SOFaa, on the rate of fertilization and polyspermy of pig oocytes. Preliminary experiments indicated that TALP provided the highest fertilization but also the highest polyspermy rates, as reported in the literature (Coy et al. 2002 Reproduction 124, 279–288). By contrast, much lower polyspermy rates but also much lower fertilization rates were obtained in SOFaa. Therefore, we made a direct comparison between the 2 media and a third medium prepared by mixing TALP and SOFaa equally (1 : 1 TALP–SOF) and using 2 different boars for IVF. Porcine oocytes were recovered from slaughtered donors and matured in vitro for 40 to 44 h in DMEM-F12 supplemented with 10% FCS, 0.05 IU of LH and FSH (Menogon; Ferring, Milan, Italy), 0.3 mM cystine, 0.5 mM cysteamine, 50 ng mL⁻¹ of long-EGF, 100 ng mL⁻¹ of long-IGF1, and 5 ng mL⁻¹ of bFGF (Sigma-Aldrich, Milan, Italy) in 5% CO₂ at 38.5°C. Boar frozen–thawed semen was separated on a Percoll gradient (45–90%) and diluted in TALP or in SOFaa with PHE (penicillamine, hypotaurine, epinefrine) and heparin (1 µg mL⁻¹) to concentrations ranging from 0.01 to 0.15 million sperm/mL. The concentration was optimized for each boar and medium: For boar A, the concentration was 0.015 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF; for boar B, the concentration was 0.1 million sperm/mL for medium TALP and TALP–SOF and 0.15 million sperm/mL for medium SOF. The oocytes were co-incubated with the sperm suspension for 18 h and then were denuded of the surrounding cumulus and fixed in acetic acid–ethanol (1 : 3) for 48 h. Finally, they were stained with lacmoid and observed under phase-contrast microscopy. The data are shown in Table 1 and were compared by a chi-squared test. Our results indicated that TALP was the most efficient medium for pig IVF but over 50% of the oocytes were polyspermic. By contrast, very low polyspermy, but also very low fertilization, was observed in SOF medium for both boars A and B. Interestingly, the empirical approach of mixing the 2 media 50% each provided a dramatic reduction of the polyspermy rate while maintaining the fertilization rate at over 60% in both boars. At present, experiments are ongoing to clarify the role of specific components of the 2 media on the fertilization and polyspermy rates of pig oocytes.

Table 1.  Effect of different media on fertilization and polyspermy rates with 2 different boars

This work was supported by grants from EUROSTELLS-ESF (ERAS-CT-2003-980409).