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High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

Journal of Animal Science and Biotechnology 2020年第1期11卷 71-80页

作者： Sergio Ledda Jen M.Kelly Stefano Nieddu Daniela Bebbere Federica Ariu Luisa Bogliolo Dity Natan Amir Arav Department of Veterinary Medicine University of SassariSassariItaly South Australian Research and Development Institute Turretfield Research Centre129 Holland RoadRosedaleSA 5350Australia FertilSafe Ltd 11 Haharash st7403118 Ness ZionaIsrael.

Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a *** in vitro produced embryos(IVEP)were vitrified at either early(EBs:n=74)or fully expanded blastocyst stage(FEBs:n=195),using a new device named"***",composed by a 0.25-m L straw with a 50-μm pore polycarbonate grid at one *** at each stage(EBs and FEBs)were vitrified by either Two-step(TS)or Multi-step(MS;6 different concentrations of vitrification solutions)***-vitrified embryos(n=102)were maintained in in vitro culture as a *** consisted of placing the straws directly into 1.5 m L tubes containing a TCM-199 solution with three decreasing concentrations of *** re-expansion,embryo survival and hatching rate were evaluated at2,24 and 48 h post *** number of apoptotic cells was determined by TUNEL ***:Blastocyst re-expansion(2 h)after warming was higher(P<0.05)in FEBs group,vitrified with the MS and TS methods(77.90%and 71.25%,respectively)compared with the EBs group(MS:59.38%and TS:48.50%,respectively).Survival rates of vitrified FEBs after 24 h IVC were higher(P<0.001)in both methods(MS and TS)than vitrified EBs(MS:56.25%;TS:42.42%)and was higher(P<0.05)in the MS method(94.19%)compared with those in TS(83.75%).After 48 h of culture the hatching rate for FEBs vitrified in MS system(91.86%)was similar to control(91.89%),but higher than FEB TS(77.5%)and EBs vitrified in MS(37.5%)and TS(33.33%).Number of apoptotic cells were higher in EBs,irrespective of the system used,compared to *** number of apoptotic cells in FEBs vitrified with MS was comparable to the ***:A high survival rate of IVP embryos can be achieved by the new"***"device with hatching rates in vitro comparable with control fresh *** method has the potential for use in direct embryo transfer in field conditions.

关键词： blastocysts High survival In-straw In vitro embryo produced Ovine

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Relationships Between Icariin and Anti-Apoptotic miRNA-21 in Mouse Blastocyst Development In vitro

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Journal of Integrative Agriculture 2013年第4期12卷 663-669页

作者： SHI Ya-ran WANG Zhan-he CAO Yong-chun LU Yan TIAN Jin-ling ZhANG Chao JIA Zi-ye CHEN Wu GAO Jian-ming Animal Science and Technology College Beijing University of Agriculture

In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium （mCZB） as control group. The experimental group （Ica group） was supplemented with 0.6 μg mL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21： pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group （（85.14±6.57）% vs. （72.04±11.58）%; （82.50±7.11）% vs. （66.80±11.70）%, respectively, P〈0.01）. The total cell number ofblastocysts had extreme difference between Ica group and control group （（61.40±9.64） vs. （46.23±4.50）, P〈0.01）. The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group （（1.47±0.51） vs. （2.94±0.66）; （2.40±0.27）% vs. （6.25±0.62）%, respectively, P〈0.01）. Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 （P〈0.01）, down-regulated pro-apoptotic Caspase3 （P〈0.05） and PTEN （P〈0.01）, up-regulated anti-apoptotic Bcl-2 （P〈0.01）. In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro- apoptotic genes

关键词： blastocysts icariin miRNA-21 anti-apoptosis mice

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Effect of PMSG/hCG Superovulation on Mouse Embryonic Development

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Journal of Integrative Agriculture 2013年第6期12卷 1066-1072页

作者： WU Bao-jiang XUE Hong-yan CHEN Li-ping DAI Yan-feng GUO Ji-tong LI Xi-he Inner Mongolia Saikexing Reproductive Biotechnology Co. Ltd. Research Center for Animal Genetic Resources of Mongolia Plateau Inner Mongolia University

Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mice and embryo quality derived from the superovulated mice were assessed. Female mice at 6-8-wk old were superovulated with 0, 5, 7.5 and 10 IU PMSG/hCG and mated with male mice. The embryos were retrieved at 2.5 d post coitum. No statistic difference was observed for the number of 2-cell embryos collected per mouse between control and 5 IU PMSG/hCG treatment group, but the number significantly increased for 7.5 and 10 IU PMSG/hCG treatment group （P〈0.05）. The average number of 4- cell and 8-cell embryos collected from each mouse significantly differed between control and 5, 7.5, 10 IU PMSG/hCG treatment groups （P〈0.05）. When 8-cell embryos derived from mice administrated with 0, 5, 7.5 and 10 IU PMSG/hCG were cultured in KSOM, the blastocyst development rates were 88.1, 94.7, 96.1 and 94.3%, respectively, which were similar to control （P〉0.05）. This indicated that exogenous gonadotropins have no effects on development of Kunming mouse embryos. The quality of blastocyst was assessed by labelling with Hoechst and propidium iodide for inner cell mass and trophectoderm cells, the result showed that ICM/TE ratio significantly decreased for 10 IU PMSG/hCG treatment group compared with control, 5 and 7.5 IU PMSG/hCG treatment group （P〈0.05）. This suggested that the embryo quality of Kunming mouse has been affected by high dose of gonadotropins.

关键词： PMSG mouse 8-cell embryos in vitro culture blastocysts

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