136 EFFECTS OF INSULIN-LIKE GROWTH FACTOR I (IGF-1) ON THE DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION BUFFALO (BUBALUS BUBALIS) EMBRYOS

Abstract

The aim of this study was to explore the effects of insulin-like growth factor-I (IGF-1) on the development and apoptosis of preimplantation buffalo (Bubalus bubalis) embryos derived from IVF or somatic cell nuclear transfer (SCNT) in order to improve the quality of in vitro embryo culture (IVC). Buffalo oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L^–1 NaHCO₃ + 5 mmol L^–1 HEPES + 5% FBS) for 22–24 h, and fertilized in vitro, or enucleated and reconstructed for SCNT. Embryos were then cultured in the culture medium (CM: TCM-199 + 3% FBS) supplemented with different concentrations of IGF-1. Blastocyst development was evaluated after 7 days of culture. A total of 1566 oocytes were used in this study. The experimental data were analyzed using two-way ANOVA, P < 0.05 was considered to be statistically significant. The results showed that the cleavage rates of IVF or SCNT embryos cultured with 0, 10, 50, or 100 ng mL^–1 IGF-I, were not significantly different (P > 0.05). However, the blastocyst rate of IVF embryos cultured with 50 ng mL^–1 IGF-1 was significantly increased compared to the 0 ng mL^–1 group (35.1 v. 23.0%; P < 0.05), but not significantly different among the 0, 10, and 100 ng mL^–1 groups (23.0 v. 28.2 and 26.5%; P > 0.05). In the same line, more SCNT embryos could develop to the blastocyst stage when cultured in the CM supplemented with 50 ng mL^–1 IGF-I by comparison with the 0 ng mL^–1 group (32.3 v. 20.2%; P < 0.05), but the blastocyst development decreased with 100 ng mL^–1 (32.3 v. 21.4%; P < 0.05). Apoptosis and total cell number (TCN) of IVF/SCNT blastocysts were respectively detected by TUNEL or Hoechst 33342 staining. By comparison with the 0 ng mL^–1 group, the TCN of IVF/SCNT blastocysts was significantly increased (IVF: 91.7 ± 6.9 v. 108.7 ± 3.9, SCNT: 76.3 ± 5.6 v. 92.8 ± 3.9; P < 0.05) and the apoptotic index was obviously decreased (IVF: 3.9 ± 0.7 v. 2.5 ± 0.7; 7.2 ± 0.5 v. 2.9 ± 0.5; P < 0.05) when the embryos were cultured in the CM with 50 ng mL^–1 IGF-I. The result of RT-qPCR analysis showed that the mRNA expression level of the anti-apoptotic bcl-2 gene was distinctly enhanced, while the mRNA expression level of the pro-apoptotic bax gene was remarkably reduced in IVF/SCNT embryos cultured with 50 ng mL^–1 IGF-I by comparison with the 0 ng mL^–1 group (P < 0.05). These results demonstrated that supplementing CM with 50 ng mL^–1 IGF-1 could improve the developmental competence of buffalo embryos, increase the TCN of blastocysts and decrease their apoptotic index, probably by down-regulating the mRNA level of pro-apoptotic bax gene and up-regulating the mRNA level of anti-apoptotic bcl-2 gene.

This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).