The present experiment was conducted to investigate the effect of insulin-like growth factor-I on in vitro maturation, fertilization and early embryonic development of cattle oocytes. A total of 318 nos. bovine ovaries were collected from slaughter houses for one year and 1089 nos. culturable oocytes were collected. 357 out of 1089 nos. culturable oocytes, were subjected to IGF-I supplemented serum and serum free basic maturation media. There was no significant difference in respect of maturation, fertilization and embryonic development between IGF-I supplemented serum and serum free basic maturation as well as culture media. The mean percentage of in-vitro maturation of bovine oocytes (IVM) based on cumulus cell expansion was found to be significantly higher (P<0.05) in serum basic maturation media without IGF-I (75.43 ± 3.25) than the serum free basic maturation media with IGF-I (67.27 ± 6.33) respectively. The mean cleavage percentages at 4-cell, 8-cell, 16-cell, morula & blastocyst stages of embryos in serum culture (without IGF-I) and serum free culture media (with IGF-I) were recorded as 47.
Yak is one of the most important economically useful animals for highlanders. The decline in the yak population demands effective measures for conservation and multiplication of elite germplasm. In vitro production of embryos and their cryopreservation and transfer to suitable recipients for production of elite calves may contribute to fulfill the objectives. The work was conducted at the National Research Center on Yak over a period of 3 years. The ovaries of slaughtered animals were used for collecting oocytes through aspiration of follicles followed by slicing of ovaries in the conventional method. Trials were conducted using 7 cyclic parous yaks for ultrasound-guided ovum pickup (OPU) at Nyukmadung farm (2700 m above mean sea level). The technique followed was similar to that in buffaloes with slight modification. Categories of oocytes classified A (2–3 layers of cumulus) and B (at least one layer of cumulus) obtained through the processes were subjected to in vitro maturation using standardized maturation medium (TCM-199 + 10% follicular fluid + sodium pyruvate + l-glutamine + 10% heat inactivated oestrus cow serum + pFSH + 17β oestradiol). The frozen-thawed yak sperm were capacitated using the swing-up method before their incubation with matured oocytes using BO medium. Oocytes matured for 24 h were washed 5 to 6 times with BO medium and then co-incubated with in vitro capacitated spermatozoa (0.1 to 0.25 million) for fertilization (8–10 oocytes per group) in 100-µL droplets of BO medium under mineral oil in 35-mm Petri dishes and placed in a CO2 incubator (5% CO2, 90% RH) at 38.5°C for 16 to 18 h. The presumed zygotes were washed several times in mCR2aa (modified Charles Rosenkrans) washing medium and then cultured in culture medium for 7 days on original beds of granulosa cells. The rates of maturation and fertilization of oocytes collected by conventional and OPU technique were comparable (Table 1). This may be attributed to greater numbers of good quality oocytes recovered in the conventional method. Embryos developed up to the stage of compact morula and blastocysts (24.66% through conventional and 22.73% through OPU) were cryopreserved using the vitrification method for further study. Thirteen embryos were transferred non-surgically to one each of 13 yak recipients; 5 became pregnant and only 1 recipient transferred with a cryopreserved-thawed embryo, developed through OPU, delivered one male calf, leading to the first successful production of an IVF yak calf in the world. The present findings are suggestive of using the OPU technique for in vitro embryo production, though resulting in lower numbers of transferable embryos (Table 1), because availability of ovaries for conventional IVF is a major constraint in yak. Table 1.Comparative in vitro yak embryo production rate with recovery of oocytes by conventional or ovum pickup (OPU) method
The present experiment was conducted to investigate the effect of fetal bovine serum on in vitro maturation and fertilization of bovine oocytes. The mean percentages of in vitro maturation performance of bovine oocytes based on cumulus cells expansion in both serum and serum free basic maturation media in type-I & type-II ovaries were recorded as 76.52 ± 3.95 and 64.81+ 4.60 & 73.33 ± 5.71 and 62.96 ± 6.57 respectively. The mean percentages of in vitro maturation based on cumulus cells expansion were recorded as 75.43 ± 3.25 and 64.20 ± 3.77 respectively in serum and serum free culture media irrespective of ovarian types. So, the mean percentage of in vitro maturation performance of bovine oocytes based on cumulus cells expansion increased significantly (P< 0.05) in serum basic maturation media than in serum free media in both type I and type II ovaries and also irrespective of ovarian types. The mean fertilization rates (%) were found to significantly higher (P< 0.05) in serum culture media (70.33 ± 3.21) than the serum free media (55.81 ± 4.33). The mean cleavage percentages recorded from 4-cell to 16-cell stages of embryos in serum culture media were found to be significantly higher (P< 0.05) than the serum free media.
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