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The aim of this study was to determine if the conditions of glycerol or ethylene glycol incorporation could improve the viability of equine embryos frozen with a conventional slow cooling method.Embryos were recovered 6.75 days after ovulation.After cooling, freezing, and storage, straws were thawed and transferred. The in vitro viability of embryos was appreciated by DAPI staining to count dead cells. In the Control group, embryos were placed successively in 4 baths containing glycerol (2.5%, 5%, 7.5%, 10% (v/v)). In the Long incubation group (n=11), the process was similar except for the duration of incubation in the 4th bath (20 min instead of 5 min). The Exp II and III compared different concentrations of CPA (glycerol in Exp II, and ethylene glycol in Exp III) 1), and sequential versus one step CPA incorporation. In Exp II, in the High concentration group (n=10 embryos), glycerol was incorporated in 5 baths (5 min each): 2.5%, 5%, 7.5%, 10% ; 15% (2M) (v/v). In the Low concentration group (n=10), glycerol was incorporated in 3 baths, 2.5%, 5% for 5 min each and in 7.5% (1M) for 15 min. In the Control group (n=10), glycerol was incorporated in 4 baths, 2.5%, 5%, 7.5% for 5 min each and in 10% (1.3 M) for 10 min. In the One step group (n=10), embryos were directly incubated for 25 minutes in 10% glycerol. In Exp III, groups were the same as in Exp II except that glycerol was replaced by ethylene glycol and the concentrations used in the 4 groups were changed: 1) High: 2.1%, 4.2%, 6.3%, 8.4% ; 11.2% (2M). 2) Low: 2.1%, 4.2%, then 6.3% (1 M). 3) Control: 2.1%, 4.2%, 6.3% then 8.4%, (1.5M) and 4) One step: 8.4%. The Exp IV tested the in vivo embryonic survival after freezing and thawing according to 2 procedures described in Exp II: One step (n=19) versus Control (n=19). A pair of thawed embryos frozen with each procedure was transferred surgically in the same recipient mare (age: 3-5 years). Embryos were collected 5 days later and classified as well developed (normal size at ten days, spherical and translucent aspect) or degenerated. In case of a single recovered embryo, its experimental group was determined by parentage testing. In all experiments and in all groups, the morphologic score decreased strongly just after CPA addition (2.9-3.3 with glycerol and 2.4-2.6 with ethylene glycol) and then remained low after the other steps. In ExpIII, this score decreased also between ethylene glycol addition and thawing. There was no significant difference among groups in each experiment. In all experiments and in all groups, the inner relative volume of embryos (in comparison with their respective inner volume at collection (100%)) was significantly decreased just after CPA addition (47-67% with glycerol and 68-81% with ethylene glycol) and never re-expanded to collection values excepted after thawing in Control group in Exp I. There was no significant difference among groups in each experiment. For instance in ExpI, inner relative volume after glycerol addition and before freezing was 67 and 64% in Control and Long incubation groups, respectively. In ExpI, the mean rate (%) of dead cells did not differ between the two groups: 10± 5 (sem) (Control) and 7± 1 (Long incubation). In Exp II, this rate was 40± 13, 40± 13, 41± 13 and 18± 9 in High, Low, Control, and One step groups, respectively. There were no significant differences among the first 3 groups but the rate was lower in One step vs Control group (P=0.04). The number of embryos with a percentage of dead cells > 18% was significantly lower in the One step group (1/10) than in the other groups (7/10 in High, 5/10 in Low, 6/10 in Control) (P= 0.04). In Exp III, the rate of dead cells was: 32± 11, 29± 12, 27± 10 and 63± 14 in High, Low, Control and One step groups, respectively. The rate tended to be higher in the One step than in Control group (P=0.07). In Exp IV, the in vivo development was similar in the 2 groups: 9/19 embryos were recovered in Control group (5 well developed and 4 degenerated) and 7/19 in One step group (4 well developed and 3 degenerated). of the incubation in glycerol was neither improving nor decreasing embryo survival. It demonstrates that glycerol is certainly not very toxic for equine embryos. When CPAs were progressively incorporated, no effect of the concentrations used (1 to 2M) on the embryo viability was observed. However, the one step incorporation tended to have a slight positive effect on dead cells ratio with glycerol, and a negative effect with ethylene glycol. It could be due to the higher permeability and/or higher toxicity of ethylene glycol for equine embryos (Pfaff et al., 1993, Bruyas et al, 2000). In all experiments, the morphologic score decreased very rapidly after the CPA addition. It seems that this score is more influenced by the shrinkage of embryos than by the intrinsic quality of embryos. Osmotic effects of addition of CPA with no total re-expansion of the embryos have been observed previously (Pfaff et al, 1993, Hochi et al., 1994). This phenomenon is different from what is observed in ruminant embryos where a complete re-expansion of embryos was obtained at the end of the incorporation of cryoprotectant suggesting that there was an equilibration with a similar concentration of cryoprotectant inside and outside the embryos. In vivo, the low survival rates in Exp IV were disappointing but consistent with literature. The incorporation of glycerol in one step did not improve the embryo development, but did not decrease it. Further studies with more embryos will be necessary to conclude definitively. However, the one step technique is more rapid to perform and appeared as efficient as sequential one as far as glycerol was used as CPA.

In equine species, embryo cryopreservation is not as widely developed as in some other species. Slow freezing has been applied to equine embryos but with relatively low success rates. This higher sensitivity to conventional freezing procedures may be explained by the presence of a capsule surrounding the equine embryo that may impair cryoprotectant penetration. Recently, good in vitro embryo survival rate was obtained after OPS vitrification (Moussa et al, Theriogenology, 2005, in press). The aim of the present study was to evaluate in vivo survival of vitrified embryos five days after surgical transfer into Welsh pony mares. Morulae (M), early blastocysts (EB) and blastocysts (B) ranging from 140 to 320 µ ; m in diameter were collected (n=20) in a Ringer lactate solution on Day 6.75 after ovulation. Before vitrification, embryos were assessed morphologically and their size was measured (McKinnon AO and Squires EL, J Am Vet Med Assoc, 1988, 192:401-406). Then, embryos were vitrified using the OPS method described by Berthelot et al. (Reprod Nutr Dev 2001, 41:267-72). Briefly, embryos were twice washed in HepesTCM199 + 20% newborn calf serum(NBCS) for 1 min, equilibrated in Hepes TCM199 + 20% NBCS with 7.5% DMSO + 7.5% EG for 3 min and with 18% DMSO + 18% EG + 0.4 M sucrose for 45 sec. One embryo was then loaded per straw. For transfer, four straws were quickly thawed (5 sec in air) and the narrow end of the straw containing the embryo was immersed in Hepes TCM199 + 20% NBCS + PBS + 0.2 M sucrose. Five to 8 min after thawing, four embryos were surgically transferred into the cranial portion of the uterine horn in each of five pony mare recipients. Five days after transfer, embryos recovered by transcervical flushing of the uterus were classified as viable if morphology was normal, no dark inner cells were present, the capsule was intact and diameter was at least 1000µ ; m. The results are shown in the table. One recipient of vitrified embryos had an endometritis and no embryo was recovered. From the four other recipients, nine embryos were recovered out of 16 (56%) embryos transferred, seven of which were viable (44%). The results of the present preliminary study demonstrating survival of equine embryos transferred after OPS vitrification is very encouraging. However, the results should be confirmed by birth of foals after transfer of OPS vitrified embryos to recipients.