Effects of Epidermal Growth Factor and Myo-Inositol on Nuclear Configuration and Subsequent Embryonic Development of Sheep Oocytes

Growth factors and vitamin-like substances have great positive importance in most biological interactions in the cellular level. The addition of these elements in the culture media will increase the yield of the resulting embryos and improve quality. We examined the effects of epidermal growth factor (EGF) and myo-inositol (MI) on meiotic maturation and yields of blastocyst of Awassi sheep oocyte across two experiments. The oocytes obtained were subjected into three treatments: A (without EGF nor MI), B (10 ng/ml EGF + 20 mmol/l MI) and C (50 ng/ml EGF +40 mmol/l MI). Oocytes were then cultured in Ham's F-10 medium supplemented with 5% (v: v) fetal calf serum and 40 ng/ml follicle - stimulating hormone. In the first experiment, during the 27-h culture period, the oocytes were assessed for germinal vesicle break down, metaphase-I and metaphase-II stages across three-time intervals (9, 21 and 27-h). Results of the experiment showed that EGF and MI enhanced the rates of germinal vesicle break down phase (1.53%; 27-h interval; lowest value), metaphase-I (33.87%; 21-h interval) and metaphase-II (89.23%; 27-h interval). In the second experiment, the oocytes incubated in treatment B achieved the highest rates of cleavage (81.96%), 2-8 cell (62.35%) and blastocyst (45.09%). It is concluded from the present study that incubating sheep oocytes in culture media containing a cocktail of EGF (10 ng/ml) and MI (20 mmol/l) significantly improves the rates of metaphase-II, fertilization and blastocyst rates.

Effect of Adding Insulin Transferrin Selenium (ITS) in the Medium on Maturation and Fertilization Rates of Bali Cattle Oocytes

The aim of this study was to determine the effect of adding Insulin Transferrin Selenium (ITS) in the medium on maturation and fertilization rates of Bali cattle oocytes. The ovarium of Bali cows were sliced to produce oocytes, then the collected oocytes were subsequently selected based on their quality. Those oocytes were maturated for 24 hours and then fertilized for 18 hours in the incubators of 5% CO2 with temperature of 38.5ºC. Oocytes were stained using 2% of aceto orcein, and observed under a microscope. This study was divided into four treatments at different addition of ITS in the medium and replicated four times, and analized using a Factorial Complete Randomized Design (CRD)at each treatment. The treatments consisted of P0 as control, P1 (5 ng/ml), P2 (10 ng/ml), and P3 (15 ng/ml). Parameters observed in this study were the stages of oocytes maturation rate that consisted of germinal vesicle (GV), germinal vesicle break down (GVBD), metaphase I (M-I), and metaphase II (M-II), and fertilization rate that consisted of absence of pronucleus (P0), 1 pronucleus (PN1), 2 pronucleus (PN2), and more than two pronucleus (>2PN). The results of this study showed that addition of ITS at 5-15 ng/ml did not effect maturation and fertilization rates of Bali cows oocytes.The conculision of the study is addition of ITS at 5 ng / ml produces the best level of maturation and for the best level of fertilization with addition ITS at 15 ng / ml.

Early germinal vesicle breakdown is a predictor of high preimplantation developmental competent oocytes in mice

SummaryThe preselection of highly developmentally competent oocytes for in vitro maturation (IVM) is crucial for improving assisted reproductive technology. Although several intrinsic markers of oocyte quality are known to be closely related to the onset of nuclear maturation (germinal vesicle break down, GVBD), a direct comparison between GVBD timing and oocyte quality has never been reported. In this study, we established a non-invasive oocyte evaluation method based on GVBD timing for preselecting more developmental competent oocytes in mice. Because the O2 concentration during IVM may affect the nuclear kinetics, all experiments were performed under two distinct O2 concentrations: 20% and 5% O2. First, we determined the time course of changes in nuclear maturation and preimplantation developmental competence of in vitro-matured oocytes to estimate GVBD timing in high developmental competent oocytes. Two-thirds of oocytes that underwent GVBD in early IVM seemed to mainly contribute to the blastocyst yield. To confirm this result, we compared the preimplantation developmental competence of the early and late GVBD oocytes. Cleavage and blastocyst formation rates of early GVBD oocytes (80.2% and 52.7% under 20% O2, respectively, and 67.6% and 47.3% under 5% O2, respectively) were almost double those of late GVBD oocytes (44.8% and 26.0% under 20% O2, respectively, and 40.4% and 17.9% under 5% O2, respectively). With no observable alterations by checking the timing of GVBD in preimplantation developmental competence, oocyte evaluation based on GVBD timing can be used as an efficient and non-invasive preselection method for high developmental competent oocytes.

In vitro culture of oocytes with surrounding cumulus complexes and granulosa cells (COCGs) from bovine early antral follicles

Cumulus-oocyte complexes with surrounding granulosa cells (COCGs) in early antral follicles (0·5 to 0·7 mm in diameter) were surgically collected from sections of bovine ovarian cortex under a dissection microscope and subsequently cultured in vitro using follicle stimulating hormone (FSH), epidermal growth factor (EGF), insulin-transferrin-selenium (ITS) and hypoxanthine, singly or in combination, to obtain fully grown matured oocytes. Oocytes cultured in the presence of FSH + hypoxanthine increased (P < 0·05) in diameter from 93 µm on the day of commencement of culture to 106·37±0·34 µm on day 5. Oocytes cultured in the presence of FSH, hypoxanthine or hypoxanthine + ITS + FSH increased (P < 0·05) to mean diameters of 105·40 (s.e. 0·47) µm, 105·50 (s.e. 0·39) µm and 105·35 (s.e. 0·55) µm, respectively. By day 11 of culture, oocyte diameters 110·50 (s.e. 0·35) µm, 110·13 (s.e. 0·39) µm, 109·49 (s.e. 0·46) µm, 109·53 (s.e. 0·58) µm and 109·16 (s.e. 0·43) µm were recorded for treatments FSH + hypoxanthine, hypoxanthine + ITS + FSH, FSH, hypoxanthine and FSH + EGF + hypoxanthine + ITS, respectively. The proportions with COCGs which formed an antrum while cultured in vitro; were categorized as morphologically normal following recovery from the gel; matured in vitro; showed germinal vesicle break down and reached metaphase II were highest (P < 0·05) for the FSH + hypoxanthine treatment (49/60 (81·7%), 48/60 (80·0%), 47/60 (78·3%), 45/60 (75·0%) and 15/60 (25·0%), respectively, followed by hypoxanthine + ITS + FSH (47/60 (78·3%), 44/60 (73·3%), 41/60 (68·3%), 41/60 (68·3%) and 12/60 (20%), respectively), FSH (43/60 (71·7%), 42/60 (70%), 40/60 (66·7%), 39/60 (65·0%) and 9/60 (15%), respectively) and hypoxanthine (41/60 (68·3%), 38/60 (63·3%), 36/60 (60%), 35/60 (58·3%) and 8/60 (13·3%), respectively). In experiment II, the in vitro fertilization and cleavage rates of COCGs were highest (P < 0·05) for FSH + hypoxanthine treatment (17/60; 28·3%) followed by hypoxanthine + ITS + FSH (13/60; 21·6%), FSH (12/60; 20%) and hypoxanthine (11/60; 18·3%) treatments. The results of this study show that COCGs from early antral follicles can be isolated, cultured and grown in vitro. Furthermore, supplements like FSH and hypoxanthine can be used singly or in combination(s) in culture medium to enhance the growth of COCGs.