the transfer of vitrified-warmed embryos to recipient mares. However, as
for controlled-rate freezing, only small embryos (<300 mm in diameter)
survived the vitrification process. Nevertheless, as a result of the very
promising results, a commercial equine embryo vitrification kit was recently
launched (Bioniche Animal Health, Pullman, WA) and, while initial
reactions in the field have not been uniformly positive, it should be borne in
mind that over-exposure to vitrification solutions is toxic to the embryo and
strict adherence to durations of immersion during both vitrification and
warming are likely to be critical to success.
In vitro produced horse embryos
In mares unable to produce embryos for ET, because for example of
ovulatory failure, occluded oviducts or recalcitrant uterine infection
(Carnevale et al. 2004), the two most obvious ways of salvaging that
mare’s fertility are oocyte transfer and in vitro fertilization. The former
involves surgical transfer of the oocyte to the oviduct of a recipient mare,
and is therefore forbidden in some European countries (e.g. the
Netherlands) on welfare grounds. Of course, conventional in vitro
fertilization, i.e. the co-incubation of oocyte with sperm, has proven poorly
successful in the horse, with only 2 foals produced (Palmer et al. 1991)
despite many years of research, primarily because of difficulties in
stimulating stallion sperm to undergo capacitation and subsequently bind
to and penetrate an oocyte in vitro (Palmer et al. 1991; Alm et al. 2001).
This obstacle has, however, been circumvented by the development of
intracytoplasmic sperm injection (ICSI) in which the sperm is injected
directly into the cytoplasm of a mature oocyte; during the last 10 years a
number of foals have been produced using this procedure (for reviews see
Squires et al. 2003; Allen 2005). Indeed, in vitro production (IVP) of equine
embryos via ICSI is in increasing demand as a last resort for producing
foals from chronically infertile mares, or when too few viable sperm are
available for intra-uterine insemination. That equine IVP is only now takingoff commercially is due largely to previously poor success rates for nearly
all components of the IVP process (e.g. ovarian stimulation, oocyte
recovery, fertilization, embryo culture). In particular, conventional in vitro
fertilization (IVF), i.e. incubating oocyte and sperm together, yielded
extremely poor results; sperm bound to but did not penetrate equine
oocytes in vitro, presumably because treatments for inducing capacitation
of stallion sperm were inadequate. However, intracytoplasmic sperm
injection (ICSI) proved a successful means of fertilizing equine oocytes,
and many foals have been produced via ICSI. Initially, cleaved oocytes
were transferred surgically to the oviducts of recipient mares; extended in
vitro culture was avoided, in part because too few oocytes per mare were
available (oocyte recovery from immature follicles was around 20%) but
also because blastocyst formation rates (<5%) and blastocyst quality were
low.
Equine IVP has finally become commercially viable because oocyte
recovery from immature follicles has improved markedly (50%), oocyte
maturation rates (60%) and post-ICSI cleavage rates (70%) are
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