bubble a mixture of 5% CO2, 5% O2 and 90% N2 through the medium prior
to use to establish the correct pH in the bicarbonate-based buffering
system (Carnevale et al. 1987). Recent studies have examined the use of
transport media that do not require gassing and are, therefore, easier to
use than Ham’s F-10 (Moussa et al. 2003). McCue et al. (2000) found no
difference in day 16 pregnancy rates between embryos stored in Ham’s F10 and those stored in the zwitterion buffered Emcare® holding medium
(ICP, Auckland, NZ). Subsequently, Moussa et al. (2004) reported that
while the cooled storage of day 7 horse embryos for 24 hours leads to an
increase in the percentage of dead cells (from 0 to 1.4%), there were no
differences between Hams F-10, Emcare or the hepes buffered holding
medium, Vigro Holding Plus (Bioniche Animal Health, Pullman, WA) in
their ability to prevent embryonic cell death. There are however, to date,
no published reports of pregnancy rates following large-scale use of
transport media other than Ham’s F-10; while it is, therefore, slightly
premature to advocate switching, initial anecdotal reports of the use of
both Emcare and Vigro in practice are promising (pers. comm. P. Daels,
Passendale, Belgium).
Embryo cryopreservation
Embryo cryopreservation has the potential to considerably simplify ET by
allowing flushing and transfer of embryos to be separate both in place and
time. It would, for example, no longer be necessary to synchronize
recipients for each and every flush, embryos could be frozen from young
mares and transferred only if and when the donor proved her worth in
competition, and possibilities for the international distribution of valuable
bloodlines could be extended via an international trade in frozen embryos,
as already exists for frozen stallion semen. Like superovulation, however,
the cryopreservation of horse embryos is a technique that has frustrated
researchers and practitioners since the infancy of equine ET. Although the
first foal born from a frozen horse embryo was reported in 1982 (Yanamoto
et al. 1982), this was the only foal resulting from 3 pregnancies generated
by the transfer of 11 embryos. Subsequent studies have established that,
irrespective of cryoprotectant used, acceptable pregnancy rates (50-60%)
can only be achieved using conventional slow-freezing techniques if the
embryos are frozen at an early developmental stage (morula to early
blastocyst) when they are less than 250mm in diameter (Czlonkowska et
al. 1985; Slade et al. 198 5; Skidmore et al. 1991; Squires et al. 2003).
Frustratingly, the embryo size criteria necessary to survive
cryopreservation are met only during a restricted time period shortly after
the arrival of the embryo in the uterus between 144 and 168 hours after
ovulation (Battut et al. 1997; 2001). Even a rigorous schedule of 4 daily
examinations to detect ovulation followed by embryo recovery 156 hours
post-ovulation was insufficient to ensure that all recovered embryos were
small enough to freeze (14% too large: Lascombes and Pashen 2001).
And while Eldridge-Panuska et al. (2005) described a much less labour
intensive system for recovering embryos of a size appropriate for freezing,
namely by flushing 8 days after the induction of ovulation with hCG (i.e.
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