Journal of Integrative Agriculture ›› 2015, Vol. 14 ›› Issue (10): 2034-2041.DOI: 10.1016/S2095-3119(14)60970-9

• 论文 • 上一篇    下一篇

Production of early monozygotic twin bovine embryos in vitro by the blastomere separation and coculture technique

 ZHAO  Shan-jiang, ZHAO  Xue-ming, DU  Wei-hua, HAO  Hai-sheng, LIU  Yan, QIN  Tong, WANG  Dong   

  1. Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences,Beijing 100193, P.R.China
  • 收稿日期:2014-12-11 出版日期:2015-10-09 发布日期:2015-10-12
  • 通讯作者: ZHU Hua-bin,Tel: +86-10-62815892, Fax: +86-10-62895971,E-mail: zhuhuabin@caas.cn
  • 作者简介:ZHAO Shan-jiang, Mobile: +86-18612246578,E-mail: shanjiangzhao@126.com;
  • 基金资助:

    The work was supported by the China Agriculture Research System (CARS-37) and the Agricultural Science and Technology Innovation Program, China (ASTIP-IAS06-2015).

Production of early monozygotic twin bovine embryos in vitro by the blastomere separation and coculture technique

 ZHAO  Shan-jiang, ZHAO  Xue-ming, DU  Wei-hua, HAO  Hai-sheng, LIU  Yan, QIN  Tong, WANG  Dong   

  1. Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
  • Received:2014-12-11 Online:2015-10-09 Published:2015-10-12
  • Contact: ZHU Hua-bin,Tel: +86-10-62815892, Fax: +86-10-62895971,E-mail: zhuhuabin@caas.cn
  • About author:ZHAO Shan-jiang, Mobile: +86-18612246578,E-mail: shanjiangzhao@126.com;
  • Supported by:

    The work was supported by the China Agriculture Research System (CARS-37) and the Agricultural Science and Technology Innovation Program, China (ASTIP-IAS06-2015).

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摘要： The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres (groups of at least 30 at the eight-cell stage) were separated into eight segments (to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments (blastomeres) were cultured respectively in microwells on the bottom of the four-well dish (Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics (cocultured with nothing, intact embryos, bovine cumulus cells (bCCs), intact embryos & bCCs) were applied to the group of four segments (blastomeres). Finally, diameter and inner cell mass (ICM):trophectoderm (TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group (P<0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & bCCs (P<0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo (P<0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group (P>0.05). Thus, these results suggest that combined with intact embryos & bCCs coculture system, culturing four isolated segments (blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.

关键词: cattle , in vitro embryo culture , blastomere separation , microwell , coculture

Abstract: The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres (groups of at least 30 at the eight-cell stage) were separated into eight segments (to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments (blastomeres) were cultured respectively in microwells on the bottom of the four-well dish (Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics (cocultured with nothing, intact embryos, bovine cumulus cells (bCCs), intact embryos & bCCs) were applied to the group of four segments (blastomeres). Finally, diameter and inner cell mass (ICM):trophectoderm (TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group (P<0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & bCCs (P<0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo (P<0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group (P>0.05). Thus, these results suggest that combined with intact embryos & bCCs coculture system, culturing four isolated segments (blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.

Key words: cattle , in vitro embryo culture , blastomere separation , microwell , coculture