Effect of an Improved Mechanical Method for Assisted Hatching on the in vitro Development of Mouse Embryos

Objective To evaluate the safety and efficiency of an improved shape of opening for mechanical assisted hatching （AH） on the in virto development of mouse embryos. Methods A total of 622 KM BAI mouse embryos in 2-cell-4-cell stage were randomly divided into group A, group B and control group. A new mechanical AH method by improving the shape of opening in the ZP was used in group A, and a ＂-/ ＂-shaped opening was created. A ＂＋ ＂ -shaped opening was made in group B, while no opening was made in control group. Comparisons have been made among the three groups with regard to the duration of AH, the blastocyst formation and complete hatching rate, etc. Results The duration of AH in group A （43.25 ±3.46 s） was significantly shorter than that in group B （52.81 ±4.32 s, P 〈0.05）. The blastocyst formation rate on d 5 was not significantly different among the three groups （92.27%, 93.66% and 94.92% respectively, P 〉0.05）. The complete hatching rate of blastocysts on d 6 between group A and group B was no statistical difference （94.09% vs 92.71%, P 〉0.05）, but significantly higher than that in control group （43.32%, P 〈0.001）. No significant difference in the percentage of grade 1 blastocysts was found among the three groups on d 5 （85.22%, 82.81% and 86.63% respectively, P 〉0.05）. Conclusion R could enhance the process of embryo hatching and facilitate the hatching rate of blastocysts by using the improved mechanical AH method, which is of safety and efficiency to mouse embryo in the in vitro development.

Effect of Different Antioxidants on in vitro Development of Mammalian Oocytes

In vitro maturation of mammalian oocytes is a key step during in vitro production of mammalian embryos. Studies have shown that the level of oocyte maturation is positively correlated with embryo cleavage rate and blastocyst rate. However,reactive oxygen species（ ROS） in vitro is one of the main influencing factors of oocyte maturation. The results indicate that the addition of antioxidants can effectively improve in vitro oocyte maturation level and reduce the adverse effects of reactive oxygen species,which contribute to oocyte development. This paper summarizes research progresses on the effects of different antioxidants on in vitro development of mammalian oocytes,which is conducive to further analysis of the mechanism of action.

Effects of Medium on in Vitro Maturation of Pig Oocytes and in Vitro Early Development of Parthenogenetic Embryos

[ Objective] To improve quality of oocytes maturing in vitro and to optimize in vitro culture system of porcine early embryos. [ Method ] Oocytes were cultured in the improved TCM199 and NCSU-23 basic media, which were added pig follicular fluid （PFF） or high-quality fetal bovine serum （FBS） both at a proportion of 10% （V/V）. After in vitro maturation and development, effects of medium on maturation of pig oocytes and development of eady parthenogenetic embryos were investigated using maturing rate of pig oocytes and development rate of parthenogenetic embryos as indicators. [ Result] After 42 h culture, the maturing rates of pig oocytes respectively cultured in the TCM199, TCM199 added FBS, TCM199 added PFF, NCSU-23, NCSU-23 added FBS and NCSU-23 added PFF were （54.2 ±3.5）%, （68.5 ±3.2）%, （69.3 ±3.7）%, （51.6 ±3.3）%, （63.2 ±3.1 ）% and （65.5 ±3.5）%, respectively. The pig oocytes cultured in the TCM199 or NCSU-23 that was added FBS or PFF had significantly higher maturing rate （P 〈 0.05）. The development rates of parthenogenetic embryos were not significantly different between these six experimental media. However, the parthenogenetic embryos which developed in the TCM199 added PFF （36.5 ±4.8） had significantly more blastomeres than those developed in the TCM199 or NCSU-23 （ 18.7 ± 3.2 and 15.5 ± 2.4, respectively） （ P 〈 0.05）. [ Conclusion ] The improved TCM199 and NCSU-23 added PFF or FBS can largely promote in vitro maturation of pig oocytes and in vitro development of early parthenogenetic embryos.

Studies on the in vitro fertilizaion in cattle

The in vitro maturation, fertilization and development of bovine ovary oocytes in two differentcultural systems A and B were studied under the conditions of 38.5℃,5%CO2,95%air and 100%humidity.The maturation rates were 94.5%and 91.3%,respectively,and the difference wasextremely significant.Frozen semen were thawed and sperm were capacitated with three kinds ofcapacitation agents for fertilization.The pronucleus rates were 76%,65%-68%and 62%respectively.The rates of embryos developed to morula and blastocyst were 19%,16% and 17%respectively.The developmental rates of embryos cocultured with bovine oviductal epithelium cellsand bovine granulosa cells were 25% and 23.4% respectively,with no significant difference. Freshembryos were transplanted into 15 recipicns,and three of them were pregnant and calves were bornin 1990 and 1991.The pregnant rate was 20%.The emryos developed faster before 8-cell stage andslower after 8-cell stage,in vitro than in vivo.

A Preliminary Observation on the Development of Mouse Embryos Co－cultured with Human Oviductal Tissue or Conditioned Medium i

The present investigation has been carried out to examine the effect of human oviductaltissue co-culture system on the development of mouse embryos in vitro. Two-cell embryos collected from superovulated mouse were co-cultured with human oviductal tissue suspended inHam 's F10+10% Fetal Calf Serum(F10 FCS),or,in oviductal tissue conditioned medium andF10 FCS as control.The results showed that the proportion developed into blastocyst,proportion of hatchedand the velocity of embryo development were higher in both tissue co-culture and conditionedmedium as compared with F10 FCS control. Furthermore,the velocity and percentage ofembryomic development were higher in co-culture with ampullary tissue or its conditioned medium than that of isthmus.The effects of co-culture and conditioned medium on embryo development had no significant difference. All the embryos obtained from two co-culture systemscould cleave normally.This experimental observation indicated that human oviductalepithelium might secrete some factors to promote the embryonic development in vitro.

Effect of Insulin on Development of ICR Mouse Embryos in Vitro

Objective To study the effects and the appropriate concentration of insulin on the development of mouse embryos in vitro, and the effects of insulin on the development of different stages of embryos.Methods Mouse embryos were cultured in vitro in the mKSOM media supplemented with insulin at different concentrations and with insulin during different stages of embryos. The blastoeyst rates and the cell numbers were counted.Results Additions of insulin significantly increased the rates of blastocyst and the total cell numbers. The concentrations of 0.005 μg/ml and 0.05 μg/ml insulin caused a significant increase in the total cell numbers compared with the control and experimental groups. Addition of insulin from 2-cell to 4-cell stage or from the 4-cell to morula stage, significantly increased the blastocyst rates compared with control and experi- mental groups.Conclusion Insulin can promote the development of mouse embryos in vitro. The appropriate concentration of insulin added in mKSOM was 0.005 μg/ml and 0.05μg/ml. Exposure of embryos to insulin, beginning at the 2-cell and extending to the 4-cell stage or beginning at the 4-cell and extending to the morula stage, is important for the development of lCR mouse embryos in vitro.