189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

Abstract

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL⁻¹; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL⁻¹ in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL⁻¹; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL⁻¹ in ethanol) + EGF (25 ng mL⁻¹; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO₂ in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P < 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P > 0.05), whereas the combination P4 + EGF was better than P4 alone (P < 0.05). Blastocyst rate was not different (P > 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P < 0.05) blastocyst rate than control but it was lower (P < 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts.

Table 1.  Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS