. Ž
. respectively; P - 0.05 . DNA-injected embryos co-cultured with MEF 13.7, 28r205 showed a
Ž higher developmental rate than that of the embryos cultured without MEF 6.7, 13r193;
. P - 0.05 in Experiment 2. Following the transfer to recipients of one or two DNA-injected
Ž blastocysts, the pregnancy rates for two culture systems were similar MEF co-culture 27.4,
. 23r84; CR1aa culture 24.2, 16r66 . However, the numbers of calves born alive from these
Ž .
pregnancies were higher on the MEF co-culture group 82.6, 19r23 than the CR1aa culture Ž
. group 56.2, 9r16 . It was concluded that in vitro embryo development to the blastocyst stage
and subsequent in vivo development to term of DNA-injected bovine embryos was improved in comparison to culture in CR1aa alone when the last 5 days of in vitro culture were in a MEF
co-culture system. q 2000 Elsevier Science B.V. All rights reserved.
Ž .
Keywords: Cattle-reproductive technology; Mouse embryonic fibroblasts MEF ; DNA injection; Embryo co-culture
1. Introduction
In the production of transgenic animals, especially transgenic cattle, it is important to establish an in vitro culture system for mass production of transferrable embryos.
Several studies have shown that the development of microinjected, in vitro-produced bovine embryos to the blastocyst stage was considerably lower than that of non-injected
Ž .
control embryos Gagne and Sirard, 1990; Krimpenfort et al., 1991; Peura et al., 1994 . The reduced development of DNA-injected, in vitro-produced bovine zygotes may be
due to the pronucleus-injection itself rather than injection-related handling or the overall Ž
. Ž
damage caused by zygote piercing Peura et al., 1994 . In our previous report Han et .
al., 1996 , the developmental rate of DNA-injected bovine embryos to blastocysts was low, approximately 5 when the embryos were cultured in CR1aa medium alone. Thus,
a better culture system needs to be developed in order to improve subsequent develop- ment of the DNA-injected bovine embryos.
Ž .
When bovine embryos derived from in vitro fertilization IVF of in vitro matured follicular oocytes were cultured, they were delayed or arrested at the 8- to 16-cell stage
Ž .
Camous et al., 1984; Heyman et al., 1987; Eyestone and First, 1989 . A breakthrough which overcame this problem was co-culture of the early bovine embryos fertilized in
Ž .
vitro. We have used mouse embryonic fibroblasts MEF for co-culture of bovine Ž
. embryos. MEF or STO cells irradiated mouse fibroblasts have been generally used for
Ž .
Ž .
the development and maintenance of mouse embryonic stem ES cells Joyner, 1993 . Ž
. Primary MEF can be easily isolated from fetuses in the third trimester 14 to16 days of
pregnancy, and 1- and 2-cell ovine embryos subjected to 5 days of co-culture showed Ž
. significantly better development on STO than EF Rexroad and Powell, 1993 . However,
little information is available on the effects of MEF on the in vitro development of IVF-derived, especially DNA-injected, bovine embryos and on the viability after transfer
of DNA-injected bovine embryos co-cultured with MEF.
The purpose of this study is to examine if MEF affected the in vitro development of IVF-derived or DNA-injected bovine embryos. In addition, in vivo viability after
transfer of DNA-injected bovine embryos co-cultured with MEF was compared with the embryos cultured without MEF.
2. Materials and methods
