assuming that ovulation will occur approximately 36 hours after hCG
administration), it is not clear how well such a protocol will stand up to the
vagaries of clinical practice and variations in the timing of ovulation after
hCG and in the rate of embryo development due to factors like mare age,
season and semen type (Stout 2003). An alternative approach to
recovering equine embryos at a stage when they are amenable to freezing
was described by Robinson et al. (2000). Based on earlier reports by
Weber et al. (1991a,b) that, from approximately day 5 after ovulation,
equine embryos secrete PGE2 which relaxes the smooth muscle of the
oviduct and thereby allows the embryo to pass into the uterus, Robinson et
al. applied PGE2 gel to the ipsilateral oviduct 4 days after ovulation and
were able to recover early-stage embryos from the uterus one day later.
This technique has not, however, been adopted in clinical practice largely
because the PGE2 has to be applied via a laparoscope, and the need to
starve the mare, surgically prepare its flank and enter the abdominal cavity
greatly reduces its appeal to the owners of valuable Sporthorse mares
(Allen 2005).
Other studies have investigated why larger embryos do not tolerate
freezing and thawing. In general, reduced post-thaw viability is
undoubtedly a product of the higher levels of cell death and organelle
disruption sustained by larger embryos during freezing (Bruyas et al. 1993,
1995, 2000; Tharasanit et al. 2005). Attempts to identify why large
embryos should be more prone to such damage have focused on the
acellular glycoprotein capsule that forms around the embryo during
blastulation (Betteridge et al. 1982; Flood et al. 1982). A negative
correlation between capsule thickness and freezability led to the
suggestion that the capsule may impede the access of cryoprotectants to
the embryo proper (Legrand et al. 1999; Bruyas et al. 2000). However,
experiments to test this hypothesis by immersing the embryo in a trypsin
solution to partially digest the capsule prior to freezing have produced
conflicting results; improved pregnancy rates in one report (6/8: Legrand et
al. 2001) but not in others (3/11: Legrand et al. 2002; 0/14: Maclellan et al.
2002). The reason for this discrepancy may reside in conflicting effects of
trypsin treatment; while it reduces disruption to the embryo’s cytoskeleton
during freezing, it also makes the capsule sticky and more prone to loss
during subsequent embryo handling (Tharasanit et al. 2005), where
absence of the capsule is detrimental to embryonic survival in vivo (Stout
et al. 2005a). Clearly, further research is needed to determine whether the
capsule really is the reason for the reduced freezability of expanded
blastocysts and how adequate cryoprotectant penetration can be achieved
without removing this vital structure.
A small number of recent studies have examined the possibility of
cryopreserving horse embryos by vitrification (i.e. ultra-fast freezing; Vatja
et al. 1998), because it is simpler, faster and cheaper than conventional
controlled-rate freezing (e.g. it does not require an expensive
programmable freezing machine). Post-thaw embryo quality after
vitrification appears to be similar to that after controlled rate freezing
(Oberstein et al. 2001; Moussa et al. 2005) and, recently, EldridgePanuska et al. (2005) reported exceptional pregnancy rates (>60%) after
15-‐18
February
2016
East
London
Convention
Centre,
East
London,
South
Africa
141