304 SHORT-PERIOD SPERM–OOCYTE INCUBATION AND FERTILIZATION MEDIA EFFECTS ON THE IN VITRO PRODUCTION OF SWINE EMBRYOS

Abstract

The aim of this study was to evaluate the effect of 2 sperm–oocyte incubation periods (30 min and 6 h) and 3 IVF media (TCM-199, TCM-199 with Ca-lactate, or mTBM). Cumulus–oocyte complexes (COCs) from abattoir-derived swine ovaries were matured in TCM-199 supplemented with 3.05 mM glucose, 50µg mL⁻¹ gentamycin, 0.91 mM sodium pyruvate, 10% swine follicular fluid, 0.57 mM cysteine, 10 ng mL⁻¹ of epidermal growth factor, 10 IU mL⁻¹ eCG, and 10 IU mL⁻¹ of hCG for 22 h, followed by a 22-h incubation without hormones. After in vitro maturation, oocyteswere allocated into 3 IVF media: TCM-199 (supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 50µg mL⁻¹ gentamycin, 1 mg mL⁻¹ BSA, and 3.06 mM mL⁻¹ caffeine-standard medium);TCM-199 Ca (standard medium with 2.92 mM Ca lactate); or mTBM (supplemented with 5 mM mL⁻¹ sodium pyruvate, 0.57 mM cysteine, 50µg mL⁻¹ entamycin, 1 mg mL⁻¹ BSA, and 2 mM mL⁻¹ caffeine). The refrigerated semen at 15°C was centrifuged and capacitated for 2 h at 38.5°C in an atmosphere of 5% CO₂ in air and under high humidity conditions in the respective IVF media. Oocytes were denuded, washed with the respective IVF media, and inseminated with the capacitated spermatozoa at a concentration of 6 × 10⁵ sperm mL⁻¹ in 90-µL microdroplets. After 30 min, a group of oocytes from each fertilization medium was gently washed to remove nonbound spermatozoa and returned to a new medium droplet for another 5.5 h (30-min group). The other group of oocytes remained in the same droplet with spermatozoa for 6 h (6-h group). After IVF, oocytes were cultured in porcine zygote medium-3 for 7 days. Data were analyzed by chi-squared test (P < 0.05).With regard to cleavage rates on the third day of embryo development (Day 3), no significant difference was observed among sperm–oocyte incubation periods; however, there was a difference in the TCM-199 group, compared with the mTBM and TCM-199 Ca groups, at the 30-min incubation period. With 6 h of incubation, no differences were observed among groups. When blastocyst (Day 7) rates were evaluated, no significant differences were observed between the 2 TCM-199 media groups; however, the mTBM groups showed higher blastocyst rates. The sperm–oocyte incubation period of 30 min or 6 h of IVF did not interfere with the blastocyst rate. In conclusion, the IVF medium mTBM was more efficient than the TCM-199 media when considering embryo production. Moreover, 30 min of IVF was sufficient for the spermatozoa responsible for oocyte fertilization to bind to the zona pellucida.

Table 1.  Cleavage (Day 3) and blastocyst (Day 7) rates of embryos incubated in two sperm–oocyte periods (30 min or 6 h) and cultivated in three IVF media

This work was supported by the FAPESP 05/01420-7.