198 BENEFICIAL EFFECT OF GHRELIN ON IN VITRO DEVELOPMENT OF PORCINE IN VITRO-FERTILIZED AND PARTHENOGENETIC EMBRYOS

Abstract

Accumulating evidence suggests that ghrelin plays an important role in female reproduction. The objective of the present study was to investigate the effect of ghrelin on pre-implantation development of porcine in vitro-fertilized (IVF) and parthenogenetic embryos. Cumulus–oocyte complexes were matured for 44 h in BSA-free NCSU23 supplemented with 10 ng mL^-1 epidermal growth factor, 10 ng mL^-1 leptin, 0.57 mM cysteine, 10 IU mL^-1 pregnant mare serum gonadotropin, and 10 IU mL^-1 hCG. After removal of the cumulus cells, some oocytes were fertilized with fresh boar semen (1 × 10⁵ sperm mL^-1) in modified Tween medium B with milk powder (Abeydeera and Day 1997 Theriogenology 48, 537–544) and some oocytes were activated by a single, 100-µs, direct current pulse of 1.4 kV cm^-1. Presumptive zygotes (Experiment 1) and parthenogenetic oocytes (Experiment 2) were subsequently cultured in PZM3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) supplemented with ghrelin at 0 (control), 0.5, 5, 50, and 500 ng mL^-1 (ghrelin 0.5, 5, 50, 500 groups, respectively) under 5% CO₂, 5% O₂, 90% N₂ and 100% humidity at 39.0°C. Cleavage and blastocyst rates were assessed on Days 2 and 6 (Day 0: the day IVF and activation were conducted). The total cell number in blastocysts was determined by Hoechst 33342 staining on Day 6. All data were analyzed by using SPSS (13.0) with one-way ANOVA. All experiments were done at least 4 times. In Experiment 1, the rate of blasotcyst formation in IVF embryos was significantly (P < 0.05) increased in the ghrelin 500 group compared with that in the control group (26.1 ± 1.8 vs. 12.4 ± 6.0%, mean ± SEM). Furthermore, increased total cell numbers (P < 0.05) were observed in the ghrelin 50 and 500 groups compared with that in the control group (63 ± 6.6 and 64 ± 5.5 vs. 42 ± 6.6). In Experiment 2, we found that the blastocyst rate of parthenogenetic embryos was significantly (P < 0.05) higher in the ghrelin 5 and 500 groups than in the others (24.6 ± 4.7 and 25.0 ± 3.3 vs. 13.3 ± 2.7, 14.9 ± 2.4, 18.1 ± 2.3% in the control and ghrelin 0.5 and 50 groups, respectively; P < 0.05). The total cell number per blastocyst was significantly increased in the ghrelin 50 group compared with that of the control group (85 ± 10.2 vs. 56 ± 8.0, P < 0.05). The maximum total cell number in the ghrelin treatment groups of parthenogenetic embryos was higher than in the control group (82, 93, 102, 100 in the ghrelin 0.5, 5, 50, 500 groups, respectively, vs. 69; P < 0.05). We also found that more embryos were developed to the morula stage and fewer embryos died early at the 2- to 4-cell stage in the ghrelin treatment groups than in the control group (data not shown) in both Experiments 1 and 2. The results suggest that supplementation with ghrelin in the embryo culture medium could enhance the pre-implantation development of porcine IVF and parthenogenetic embryos.

This study was funded by the Natural Scientific Foundation of Beijing (5030001).