Abstract
Problem statement: Development of efficient culture system to support embryonic development would be valuable when quality of produced embryos was important. However, the rate of bovine embryo production in vitro was still lower than expected. Present study, incl uding of three experiments, was carried out to investigate the eff ect of insulin on nuclear maturation and subsequent development of immature bovine oocytes and in vitro fertilized embryos. Approach: Grade one cumulus-oocyte-complexes harvested from slaughterhouse ovaries were selected and randomly allocated in each treatment groups. In experiment 1 , in vitro maturation medium (Hepes-buffered medium 199 + fetal calf serum + gonadotrophins + antibiotics) supplemented with 0 (control), 1, 10, 20 and 100 µg mL -1 of insulin. In experiment 2, to eliminate the effe ct of serum and hormones, Hepes- buffered medium 199 was supplemented with 1 mg mL -1 polyvinyl alcohols (PVA) and same levels of insulin. In experiment 3, the effect of insulin on bovine in vitro embryo development was assessed. Presumptive zygotes were randomly cultured in synthetic oviductal fluid added with 0 (control), 1, 10, 20 and 100 µg mL -1 of insulin. Results: In experiment 1, nuclear maturation and embryo development rates were significantly higher in 1 and 10 µg mL -1 compared with other groups (P<0.05). In experiment 2, both maturation and cleavage rate sig nificantly increased in 1 and 10 µg mL -1 insulin. The only treatment resulted in higher hatchability was 10 µg mL -1 insulin (17.1±2.34%) compared with control (11.34±3.94). In experiment 3, cleavag e and morula rates were significantly greater in 1 and 10 µg mL -1 insulin compared with other groups; although the h ighest rates resulted by using 10 µg mL -1 . Conclusion: Obtained results show that inclusion of 10 µg mL -1 insulin in maturation and culture medium exerted beneficial effects on nuclea r maturation of bovine oocytes and in vitro embryo development till morula stage.
Highlights
The effect of insulin on in vitro maturation of bovine immature oocytes and subsequent embryo development was evaluated using 1081 COCs. Data obtained in this experiment (Table 1) indicated that, after 24 h of culture, the proportion of immature bovine oocytes reaching metaphase II (MII) stage significantly increased when 1 and 10 μg mL−1 insulin (92.70±1.83% and 93.19±1.82%, respectively) were included in the In Vitro Maturation (IVM) medium compared with the control group (85.80±3.40%; p
Maturation rate was slightly increased at 20 μg mL−1 insulin (86.37±2.63%), compared with the control but not significantly (p>0.05)
The results demonstrated that presence of 1 or 10 μg mL−1 insulin in the maturation media showed a positive effect on maturation and cleavage rates of bovine immature oocytes in vitro
Summary
Approximately 60-80% of in vivo embryo production system includes three consequent matured bovine oocytes are competent to reach to the steps: in vitro oocyte maturation (IVM), in vitro metaphase II stage while only 25-40% of in vitro. Animal & Vet. Sci., 5 (4): 258-265, 2010 matured oocytes reach to this stage It is necessary to modify the culture medium and conditions to support higher percentage of embryo development in vitro. Several factors such as, hormones, proteins and growth factors supplemented to the culture medium may have crucial roles on the outcome of IVEP. Several studies have indicated that insulin increases the in vitro oocyte maturation and development of human (Dashtizad et al, 2003), mice (Demeestere et al, 2004) and porcine. Thereafter, groups of 7 -10 COCs were randomly distributed in each 50 μL pre-equilibrated
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