Studies On The In Vitro Fertilization And Effect Of Igf-i On Bovine Oocytes Under Heat Shock

With the further studies on embryo engineering technique, the technique of bovine oocyte fertilization in vitro has become more and more popular in many areas. This technique not only provides cheaper embryo sources for commercial application of bovine embryo transfer, but builds technology platform for research on animal reproductive function, developmental mechanism of germ cell in vivo and influence mechanism of environmental stress factors in animal production on reproductive mechanism. Heat stress is an enviromental factor which is popular in the world and affects dairy cow fertility. It is well known that IGF-I improves embryo development in the early stage, while it is unkown about how IGF-I affects in vitro maturation of bovine oocytes. In this study, experiments of factors affecting oocyte in vitro maturation, fertilization and embryo culture were performed (Expriment 1). In addition, in cellular and molecular level, we examined effect of heat shock and also of IGF-I addition before and after in vitro maturation on developmental competence of bovine oocyte by several experimental groups (Expriment 2).Results are as following.Eexperiment 1, studies on bovine oocytes in vitro maturation, fertilization and preimplantation embryo culture.1) There's no difference between maturation rate within 3h and that of 3-6h, while maturation rate decreased significantly when ovary holding time was more than 6h. Maturation rate of oocytes in follicles with diameter less than 2mm was low (51.66±3.15), maturation rate increased significantly when follicles diameter were more than 2mm. However, there were no difference among those with follicle diameter among 2-6mm,7-8mm and 9-10mm (80.73±2.81,84.32±1.85, 87.27±6.23, P>0.05). Maturation rate was higher when the oocytes were from COCs of grade A and B (85.92±3.35 vs 82.47±3.24, P>0.05). While it significantly decreased compared with results of grade A and B (60.26±5.46, P<0.05) and the lowest result was from grade D COCs (18.23±4.15, P<0.05).2) The cleavage rate of oocytes was greatly affected by follicle diameter. It increased significantly with increasing of follicle diameter (P<0.05). As to the blastocyst rate, it was only 6.37±2.58 when diameter is less than 2mm and increased significantly when diameters were from 2 to 8mm (24.73±1.63 vs 28.21±1.89, P>0.05). While the blastocyst rate developed from oocytes in 8-10mm follicles was significantly higher (31.43±7.49, P<0.05) accept for 6-8mm group. The cleavage rate and blastocyst rate were not affected when cumulus cells were kept completely or removed partly before in vitro fertilization, but significantly decreased when cumulus cells were completely removed. Oocyte developmental competence was not affected when sperm-egg incubation time was during 8-18h.3) The cleavage rate and blastocyst rate were not affected when half of the embryo culture medium was renewed every 24h or 48h compared with control group with culture medium completely unchanged (P>0.05). While development of blastocsyt was greatly improved when the culture medium was fortified with 10% FCS on day 3 of in vitro culture.Experiment 2, Effects of IGF-I on developmental competence of bovine oocytes exposed to heat shock.1) Effects of IGF-I on developmental competence of GV-stage oocytes exposed to heat shockThere's no any effect of HS and IGF-I on distribution of cortical granules in oocyte cytoplasm, nuclear maturation and nuclear apoptosis after in vitro maturation.2) Effects of IGF-I on developmental competence of oocytes exposed to heat shock during in vitro maturationThe cleavage rate was decreased in HS group compared with C group (C vs HS,75.60±3.54 vs 56.55±5.24, P<0.05) and the percentage of embryos with more than 2 cells 42h after in vitro fertilization (C vs HS,39.11±5.17 vs 22.66±4.99, P<0.05) was significantly decreased by heat shock. However, only in group LCR, IGF-I showed its beneficial effect by decreasing ratio between 2-cell embryos and embryos with more than 2 cells (3.42±0.53% vs 1.8±0.53%; P<0.1). There's no any effect of HS and IGF-I on distribution of cortical granules in oocyte cytoplasm after in vitro maturation. HS increased percentage of oocytes in MⅠstage (C vs HS,1.75±1.75 vs 39.34±5.39, P <0.05) and decreased percentage of oocytes in MⅡstage (C vs HS,57.9±13.27 vs 13.91±4.16, P <0.05). Percentage of oocytes in late nuclear stage (TeloⅠand MⅡ) was lower in HS than that in C (P< 0.01). IGF-I increased slightly percentage of oocytes in late nuclear stage (P>0.05). The proportion of TUNEL-negative oocytes tended to be higher in the C group relative to the HS group (47.94±14.24 vs 27.98±5.95; P≤0.09). However, under HS, IGF-I decreased the proportion of TUNEL-positive oocytes to a level similar to that in the C group (P>0.05). HS and IGF-I didn't have any effect on expression of GAPDH and GDF9 in 4-cell embryos.