In vitro culture of sheep zygotes following pronuclear microinjection

THERIOGENOLOGY In Vitro Culture of Sheep Zygotes Following Pronuclear Microinjection. SK Walkerl, T.M. Heard1,P.J. Verma2, C.S. Bawden2, A.V. Sivaprasad2, G.E. Rogers2 and R.F. Seamarks. 1Department of Agriculture, Turretfield Research Centre, Rosedale, South Australia 5350 and Departments of 2Biochemistry and 3Obstetrics and Gynaecology, University of Adelaide, Adelaide, South Australia 500 1. Cloned DNA can be routinely transferred by pronuclear microinjection to However, the mammalian embryos for the production of uansgenic offspring. procedure results in a substantial reduction in the proportion of viable embryos. The availability of an in vitro culture system to assess embryo viability with thus enhance the efficiency of handling microinjected embryos. ln this study microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM; Proc. 1lth Int. Cong. Anim. Reprod. A.I. 4,483-485) for either one or three days and an assessment made of the effect of culture on subsequent viability. Zygotes were collected from FSH-treated ewes by mid-ventral laparotomy approximately 12-17 h after the expected median time of ovulation. Microinjection occurred immediately after collection using a Nikon Diaphot inverted microscope incorporating Nomarski differential interference contrast optics. Pronuclei were injected with DNA (5 ng/ul) under a pulse pressure of 220 kPa for 0.1-0.3 seconds. One of two cysteine gene constructs were microinjected; each contained a 6.4 kb fragment containing the Cys E and Cys M genes from Salmonella tvohimurium (J. Cell. B&hem. 13B, 183). Zygotes were either transferred to synchronized recipient ewes within 4 h of microinjection (connol) or cultured for one or three days before transfer. Zygotes were cultured in 20 ul microdrops of medium under paraffin oil in an atmosphere of 5% C02, 5% 02 and 90% N2; temperature was 38.5oC and humidity 93%. Between 3-5 zygotes/embryos were @ansferred per ewe. Ewes were scanned by ultrasonography 50 days after transfer to determine the number of fetuses per ewe. There were no significant differences between the two constructs in the proportion of zygotes/embryos that developed to day 50. Overall, 21.6 (40/185), 21.1 (32/152) and 26.0% (401154) of the control zygotes and those cultured for either one or three days respectively developed. Of the cultured embryos, 20.6% (63~06) failed to divide and 23.0% (56~43) were classified as grade 2 embryos on the basis of fragmentation and the development 01 large irregular blasromeres. Significaurly iewer grade 2 embryos developed into fetuses compared with grade 1 embryos (14.3 v 34.8%, P&01). The ability to identify embryos less likely to develop was not influenced by the time of culture. It is concluded that microinjected sheep zygotes may be cultured for up to three days without loss of viability compared with zygotes transferred shortly after microinjection. The ability to identify zygotes that fail to divide as well as embryos less likely to develop will enhance the efficiency of embryo transfer programs following microinjection. Supponed by the Australian Meat and Livestock Research and Development Corporation and the Australian Wool Research and Development Fund.

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JANUARY

1990 VOL. 33 NO. 1