124 SUPPLEMENTATION OF IN VITRO MATURATION MEDIUM WITH GROWTH HORMONE DID NOT AFFECT THE TIMING OF THE FIRST ZYGOTIC CLEAVAGE BUT SIGNIFICANTLY IMPROVED THE QUALITY OF RESULTING BOVINE BLASTOCYSTS

Abstract

It is well documented that growth hormone (GH) supplemented to maturation medium positively affects in vitro oocyte maturation and embryonic development in cattle. Conditions of in vitro maturation significantly influence oocyte competence for successful fertilization and preimplantation embryo development. It has been shown that bovine zygotes that cleave early are characterized by better quality than their later cleaving counterparts. In this context, the aim of this study was to determine whether supplementation of maturation media with GH affects the timing of the first zygotic cleavage and quality of produced blastocysts. Oocytes were matured in TCM-199 supplemented with fatty acid free BSA and hormones (FSH and GH) and then fertilized (Parrish et al. 1998 Biol. Reprod. 38, 1171–1180), whereas embryos were cultured in sequential media (Lane et al. 2005 Theriogenology 60, 407–419). All embryos that cleaved by 30hpi (early cleavers, EC) were selected and cultured separately. The remaining embryos cleaved by 48hpi (non early cleavers, NEC) were also incubated in separate drops. Blastocysts of proper morphology, collected at 176hpi, were subjected to TUNEL assay and differential staining (TE:ICM ratio). Due to technical problems only expanded blastocysts were analyzed by differential staining. The significance of GH supplementation in relation to apoptotic index and blastomere number (total, TE, ICM) was analyzed by ANOVA. This experiment included 250 blastocysts obtained from 1092 oocytes. The presence of GH did not influence the basic developmental parameters: cleavage rate, percentage of EC embryos and blastocysts (P > 0.05). Blastocysts derived from oocytes matured with GH more often hatched regardless of the timing of the first zygotic cleavage when compared with blastocysts that developed from the control group (P < 0.05). GH supplementation to IVM medium significantly reduced apoptotic index in EC blastocysts (2.9 and 4.8% for control, P < 0.05). Interestingly, blastocysts containing none or one apoptotic blastomere were observed only among EC embryos (20.6%). Although exogenous GH did not affect the cell count in TE and ICM of expanded EC blastocysts, it increased total cell number and reduced the TE:ICM ratio in embryos derived from oocytes culture with GH. In summary, although exogenous GH was previously shown to accelerate the kinetics of oocyte maturation, it has no effect on the timing of the first zygotic cleavage in cattle. The results of the present work support previously published data on GH as growth stimulating and antiapoptotic factor. Its stimulatory effect was especially evident in NEC blastocysts. Those embryos exhibited even better quality (higher cell count, lower apoptotic index) than control EC blastocysts.

This work was supported by the project No. 3780/P01/2006/31 of the Ministry of Science and Higher Education, Poland.