| In this article we tested the effect of cryoprotectant on in vitro development of embryo derived by in vivo fertilization (1-cell embryo or pronuclear embryo), and the experimental animal was Kunming mouse. The purpose was to seek for an effective vitrification method and optimize culture system, which hopefully could be used as a model for other animals.(1) we tested the effect of body weight and the dosage of hormone treatment on mouse superovulation. The results showed that:â‘ As for superovulation rate, the mouse body weight (BW) <20g group was significantly lower than that group when the BW ranged from 20 to 31g (P<0.05).â‘¡the superovulation result was worse in 5IU PMSG/hCG group than that in the 10IU PMSG/hCG treatment group, so it's better to choose 10 IU PMSG/hCG as the dosage to superovulate Kunming mouse.(2) we investigated the effect of different loading carriers:(open pulled straws, OPS)and Cryotop on the in vitro development of pronuclear embryos after vitrification/warming, The results shows that the rate of 2-cell embryo, 4-cell embryo, and blastocyst was 84.28% vs 74.10%, 57.35% vs 65.87%, 28.34% vs 43.57% respectively in OPS and Cryotop group, respectively. No significant difference among groups (81.19%, 64.25%, 40.04%; (P>0.05), but the results form the Cryotop group were slightly closer to the control.(3)We examined the effect of Cryotop and self-made Cryotip on in vitro developmental capacity of the pronuclear embryos after vitrification/warming. We found that the rate of 2-cell embryo,4 -cell embryo, and blastocyst was similar between the two group, and no significant difference was observed (P>0.05). Therefore, our results indicate that Cryotip could be an alternate loading device for vitrification with less cost.(4)The third, we tested the effect of cryoprotectant recipe (15%EG+15%DMSO+sucrose, EDS vs. 15%EG+15%PROH+sucrose, EPS) on in vitro developmental ability of pronuclear embryos after vitrification/warming, and the results were:â‘ the rate of embryo recovery (96.35% vs 94.95%) and survival (84.83% vs 95.83%) after vitrification/thawing was similar in both group , and no significant difference (p>0.05) was observed.â‘¡the rate of 2-cell embryo was 60%, 70% for EDS and EPS group,respectively, and significantly lower than that in the control (92.5%, p<0.05), but there were no obvious difference between the vitrification groups (p>0.05).â‘¢there were significantly difference between EPS group and the control (33.84% vs 76.90%), and the both vitrification groups (33.84% vs 56.35%) in the rate of blastocyst (p<0.05), but there were no significant difference between the EDS group and the control (p>0.05).â‘£The total cell number per-blastocyst was a little higher in EDS group than the EPS and the control (p<0.05). Our data imply that the EDS group is more suitable for Kunming mouse pro-nuclear embryo vitrification.(5) Permeable cryoprotectants play important roles in vitrification, and so as the nonpenetrated ones, and in this article we tested the effect of sucrose and trehalose on mouse pro-nuclear embryo vitrification, and at the same time we choosed the different embryo in vitro culture density:1:2, 1:4, 1:8. The results showed that no significant difference regarding to the rate of 2-cell, 4-cell, morula and the blastocyst between sucrose and trehalose group (70% vs 67.5%; 69.04% vs 72.44%; 57.22% vs 58.75%; 54.05% vs 53.83%; p>0.05). All current data demonstrate that both sucrose and trehalose can be used in Kunming mouse pro-nuclear embryo vitrification; and there were no significantly difference among the different embryo culture density(p>0.05).In conclusion, in the process of pro-nuclear embryo vitrification and thawing in mouse, Cryotop as the vitrification carrier, and EG+DMSO+sucrose or trehalose as the cryoprotectant are feasible to vitrify embryos prodced from the mouse whose BW is ranged from 20g~30g, and treated with 10 IU PMSG/hCG hormone. |
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