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embryos and 26.5 43r162 for fresh embryos P - 0.05 . Of the 12 live calves born from the Ž
. frozenrthawed embryos, two males 18.3 were transgenic. None of the live-born calves derived
from fresh embryos exhibited the transgene. One of transgenic bulls did not produce transgenic Ž
. sperm. Three out of 23 calves 13.0 produced from cows inseminated with semen of the other
bull were transgenic, suggesting that this animal was a germ-line mosaic. These studies indicated that the viability of in vitro produced, DNA-injected bovine blastocysts was affected by freezing
and by both the quality and stage of development of the embryo prior to freezing. The generation of transgenic cattle demonstrates that it is feasible to freeze DNA-injected, in vitro produced
embryos. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Cattle; Embryology; DNA injection; Cryopreservation; Transgenic; Transmission
1. Introduction
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In vitro maturation IVM , fertilization IVF and culture IVC , DNA injection and embryo transfer are complex techniques that have been used to produce transgenic cattle
Ž in recent years Krimpenfort et al., 1991; Bowen et al., 1994; Hyttinen et al., 1994;
. Eyestone et al., 1998 . Transgenic animals that produce human pharmaceutical proteins
have many benefits compared to conventional production systems using microorganisms Ž
. or animal cells
Janne et al., 1992; Bremel, 1996 . The advantages include high productivity, low operating costs, appropriate post-translational modification of proteins,
and the production of transgenic progeny by most transgenic animals. Problems associ- ated with production of transgenic cattle are the supply of in vivo fertilized zygotes, the
low developmental potential of injected embryos and their high costs of production. Production of transgenic livestock by pronuclear injection is inefficient, with a low
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integration frequency around 1 of injected embryos Wall, 1996 . In vitro embryo production procedures have been used to provide a large number of synchronous zygotes
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for pronuclear microinjection Krimpenfort et al., 1991; Janne et al., 1992 . Embryos that develop successfully in culture can be biopsied or bisected to detect the transgene
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. by DNA polymerase chain reaction PCR
Janne et al., 1992 . Only those embryos containing the transgene are then transferred into recipients, thus increasing the probabil-
ity of transgenic calves being born. Even with these advances, the process of producing transgenic cattle remains inefficient. Recently, revolutionary techniques giving rise to
100 transgenesis in mammals, the so-called nuclear transfer using transformed somatic
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cells, have been achieved Schnieke et al., 1997; Cibelli et al., 1998; Brink et al., 2000 . Alternatively, cryopreservation may increase utilization of DNA-injected bovine em-
bryos. Studies on cryopreservation of IVF-derived bovine embryos have been reported Ž
. Suzuki et al., 1993; Han et al., 1994 . Transfer of the frozen IVF embryos results in a
Ž lower pregnancy rate than that of fresh IVF embryos Hasler et al., 1995; Agca et al.,
. 1998 . Factors affecting the in vivo viability after transfer of DNA-injected bovine
Ž .
blastocysts produced in vitro have been investigated Han et al., 1996 . However, little information has been reported on the in vitro and in vivo viabilities of DNA-injected
bovine embryos after freezing and thawing. The present study investigated the factors affecting the survival of in vitro produced,
DNA-injected bovine blastocysts after freezing and thawing. In addition, it was exam-
ined whether transgenic bulls derived from frozenrthawed embryos pass the transgenes to their progeny through the germ-line.
2. Materials and methods
