Studies on the developmental potential and the survival after the deep freezing of microsurgically dichotomized morula embryos in rats and rabbits

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  • NAGASHIMA Hiroshi
    Faculty of Agriculture, Meiji University Stock Farm, Faculty of Agriculture, University of Tokyo
  • OGAWA Shyoso
    Faculty of Agriculture, Meiji University

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Other Title
  • ラットとウサギにおける切断二分離桑実期はいの発達および凍結生存性に関する研究
  • ラット ト カト ニ オケル セツダン ニブンリ ソウジツキ ハイ ノ ネイタ

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Halved morula embryos of rats and rabbits were obtained by a new practical method for the dichotomy and their developmental ability in vitro and in vivo was investigated as well as the viability of the halved embryos after deep freezing. The results were statistically analyzed after arc sin transformation.<BR>In our new method, a morula embryo was dichotomized by using fine glass needle and a simple micromanipulator, without using any other device such as a "suction pipette" for holding the embryo. The morula, of which the zona had been softened by pronase pre-treatment, was placed in a droplet of culture medium on a glass slide. The tip portion of the needle (10-15 μm in diameter) was touched exactly on the top of the zona and then pressed gently down as the needle axis to pass through the center of the embryo. By this means the squashed embryo was readily divided into two equal pieces. The successful dichotomies, which gave two equal half-embryos, were achieved in 277 out of 365 morulae in the rat (75. 9%) and 50 out of 63 in the rabbit (79. 4%).<BR>These halved morula embryos were cultured in vitro. Fifty-seven out of 73 halved embryos in the rat (78. 1%) and 37 out of 39 in the rabbit (94. 9%) developed into morphologically normal blastocysts, though their sizes were significantly smaller than the respective control blastocysts derived from the intact whole morulae.<BR>To the uteri of 33 pseudopregnant rats, 274 halved morula embryos of rats were transferred. Seventeen recipients showed placental sign. Of these recipients three were laparotomized between 8 and 11 days after the transplantation. They had 4, 2 and 6 implantation sites, respectively. Two other recipients delivered two live pups each (3 females and 1 male) 20 and 23 days after the transplantation.<BR>The birth rates based on the number of embryos transferred were 25. 0 and 28. 0% in these rats, and all offspring were proved to possess normal reproductive ability.<BR>Survival of halved morula embryos in the rat and the rabbit were found after deep freezing at -80C, though in a low percentages. Thirty out of 135 in the rat (22. 2%) and 26 out of 45 in the rabbit (57. 8%) maintained normal morphology after freezing and thawing. Of these halved embroys recovered in normal appearance, eight embryos in the rat (26. 7%) developed in vitro into morpho-logically normal blastocysts, although none of the halved embros showed normal development in the rabbit.

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