| In vitro embryo production plays an important role on stockbreeding and research. It is more important in protecting and utilizing the local cattle by in vitro fertilization (IVF). In order to optimize IVF system in beef cattle, effects of different supplements (17-βestradiol, Caffeine and Cysteamine), dose of supplements and different treatment time on in vitro maturation (IVM) and IVF were investigated.Results are as following:1. The optimization of in vitro maturation techniques in beef cattle oocytesThe cumulus-oocyte complexes (COCs) were collected and cultured in TCM-199 supplemented with hormones (FSH,LH,17-βestradiol) for different times of 10h,12h,and 22h (control group). The maturation rates of IVM were 61.20%, 77.23%, 80.87%, respectively. The maturation rate of 10h treated group was significant (p<0.05)lower than those of 12h group and 22h group. However, no statistical differences were observed between 12h group and 22h group. The results indicated that oocyte begin to mature by interrupting the relationship between oocyte and cumulus cell after 12h hormon treatment when they were cultured in vitro.The COCs were cultured in four groups and supplemented with 0μg/ml, 1μg/ml, 2μg/ml, 4μg/ml 17-βestradiol (E2), respectively. Maturation rates of four groups were 60.52%, 74.91%, 77.25% and 62.20%. No statistical differences were observed (p> 0.05) between 1μg/ml of E2 (74.91%) and 2μg/ml of E2 (77.25%) group, same situation was seen between 0μg/ml of E2 (60.52%)and 4μg/ml of E2 (62.20%). However, maturation rates (74.91%-77.25%) of groups of 1μg/ml of E2and 2μg/ml of E2 were significant higher (P<0.05) than those (60.52%-62.20%) of rest two groups. The results showed the appropriate supplement of E2 (1μg/ml-2μg/ml)could improve in vitro maturation of oocytes. And, the excessive supplement of E2 (>4μg/ml)could inhibit the maturation.When IVM medium was supplemented with 0μg/ml, 50μg/ml and 100μg/ml of cysteamine, maturation rates were 75.25 %, 80.56 % and 76.83 %, respectively. And no significant difference (p>0.05) was observed among them. However there were approximately five pencents higher in supplement group with 50μg/ml of cysteamine than those of groups of 0μg/ml and 100μg/ml of cysteamine. Results showed that appropriate supplement of cysteamine (50μg/ml) is benefit to the IVM.2. The optimization of in vitro fertilization techniques in beef cattle oocytes Compared with the control group (0mM of caffeine), the supplements of caffeine (2.5mM and 5mM) could improve rates of cleavage and blastocyst. Rates of cleavage and blastocyst (64.95% and 25.56%) in 5mM of caffeine group were higher than those of 2.5mM of caffeine group (61.50% and 22.00%), but no significant difference (p>0.05) was observed between them. Results showed that 5mM of caffeine could maintain longer time for higher sperm viability and benefit to the development of zygotes.3. The optimization of culture techniques of in vitro fertilized egg in beef cattle Zygotes were cultured in SOF and TCM-199 two different medium added with 10%FCS and cocultured with GCM. There was no significant difference(p>0.05) between blastocyst rates in the two medium (21.01% vs 26.67%). However, there was 5 percents of blastocyst rate higher in TCM-199 than that of in SOF. The results showed that both SOF and TCM-199 could be used as developmental medium, but the TCM-199 is better than SOF.Zygotes were cultured in TCM-199 medium supplemented with 0μg/ml, 50μg/ml and 100μg/ml of cysteamine, respectively. The blastocyst rates in supplemented groups (26.14% and 22.56%)were higher than that of non-supplemented group (20.87%), but there was no significant difference (p>0.05) between them. However, there was 5 percents higher in 50μg/ml of cysteamine group(26.14%) than that of non-cysteamine group (20.87%). The results showed appropriate supplement of cysteamine (50μg/ml) is benefit to zygote development.
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