4 MATERIALS AND METHODS
6.7 Surgical transfer of fresh, frozen-thawed and vitrified-warmed embryos
A total of 89 kits were born to 28 recipient females. The overall survival rate of 30%
including fresh (42% survival, II), frozen-thawed (11% survival, III) and vitrified-warmed (16% survival, IV) embryos was similar to those reported earlier after transfer of fresh embryos in the domestic ferret (Chang 1968: survival rate 33%, surgical transfer; Kidder et al. 1999b: survival rate 26%, nonsurgical transfer) and in the American mink (Zhelezova and Golubitsa 1978: survival rate 26%, surgical transfer; Adams 1982: survival rate 25%, surgical transfer). The overall kit result (an average of 3 kits/recipient) after the transfer of an average of 10 embryos/recipient was higher or similar to those obtained in earlier studies in the ferret, except that Li et al. (2001) obtained an average of 9 kits/recipient after transferring an average of 18 embryos/recipient.
Reasons for not producing kits after embryo transfer may be related to several factors:
weight of the recipients, synchronization of donors and recipients, cryopreservation of embryos, number of transferred embryos, embryo processing before transfer or the transfer technique itself. During the first year of this study it was noticed that better kit results (6.4 versus 3 kits/recipient) and higher survival rates (52% versus 32%) were achieved when the recipient weighed >1 kg (12.4 embryos transferred per recipient) compared to <1 kg (9.4 embryos transferred per recipient). Females weighing under 1000 g were excluded from embryo transfer programmes in the subsequent years.
Induction of ovulation was used to synchronize donors and recipients. More kits were born (6.8 versus 3.1 kits/recipient) and higher survival rates achieved (52% versus 33%) after transfer of embryos from multiple donors (13.2 embryos transferred per recipient) than from a single donor (9.3 embryos transferred per recipient). Vicente and García-Ximénez (1993) transferred frozen-thawed rabbit embryos from a single donor versus two donors into recipients (9 embryos versus 16 embryos). The survival
rates were higher but not significantly higher for the group of two donors than for the group of single donors. Li et al. (2001) pooled the ferret embryos, and 50% continued embryonic development to full term kits. The use of multiple donors seemed to increase the number of transferred embryos per recipient as did synchrony between donors and recipients, which is required to obtain normal development as demonstrated by Chang (1969) in the domestic ferret. Additionally, altered embryo development conditions after embryo transfer may have a positive effect on survival as has been reported in the pig (Kvasnitski 2001).
In this study, all of the recipients which received ≥12 transferred embryos gave birth.
More embryos were required for birth of kits if the embryos had been frozen-thawed or vitrified-warmed (13 embryos on average) rather than if the embryos were fresh (11 embryos on average). Pope et al. (1993) reported more pregnancies in the domestic cat after transfer of ≥12 embryos than <12 embryos. However, live offspring have been born after transfer of 3 and 6 fresh embryos (McRae 1994, Li et al. 2001).
In our study, ≥12 embryos were transferred only to 36% (10/28) of the recipients. It seems that increasing the number of transferred embryos per recipient could well have increased the survival rate (= live kits/transferred embryos).
When transferring vitrified-warmed embryos, in vitro culture for a couple of hours did not affect the synchronization but culture periods longer than 24 hours seemed to compromise further development. The 7 embryos of the first OPS recipient were warmed too early by mistake and were cultured for 3 days. This may have accelerated development during these 3 days, and thus, the embryos were actually Day 11 embryos which were being transferred into a Day 8 uterus, or perhaps the culture period had compromised the developmental capacity of these embryos and they were no longer competent at transfer. An asynchrony of 2 days (but not 3 days) between donor and recipient has been reported to be acceptable for production of viable offspring (Chang 1969; own personal observations). Also, culture periods longer than 24-48 hours in TCM-199 medium in this study compromised the developmental capacity of the polecat in vivo embryos when compared to development in vivo. This indicates that extended in vitro culture of embryos after vitrification decreases the chances of survival. Therefore direct transfer without in vitro culture or a short culture
period <24 hours may rescue the transferred vitrified-warmed embryos and increase the kit yield.
In this study the technique of surgical embryo transfer was less traumatic to the females than the surgical embryo recovery. The recipients which did not whelp have been subsequently used by mating them during the second oestrus of the season. All that have conceived (75%, 9/12) have reproduced normally. This indicates that the technique of embryo transfer used throughout this study is not detrimental to further reproduction and can be applied in programmes designed to preserve endangered mustelids (Amstislavsky et al., in press).
The recipients gave birth to more male than female offspring after embryo transfer.
During the corresponding years 1998-99 and 2001 on the research farm, there were no significant differences between the sexes of the kits of the breeding females and the embryo transfer kits. However, when combining the three years it seems that the treatment of embryos significantly favors male offspring (P=0.040, nonparametric binomial test). Also, several reports have described male-biased sex ratio of cattle and mouse preimplantation embryos among blastocyst stage after in vitro culture (Valdivia et al. 1993, Sato et al. 1995, Gutiérrez-Adán et al. 1996, Carvalho et al.
1996, Pegoraro et al. 1998, Peippo 2001). For conservation purposes, excess production of male offspring by assisted reproductive technology is undesirable, as increased efforts are required to produce sufficient number of breeding females.
7 CONCLUSIONS
Early embryonic development in the farmed European polecat is a process that is typical of all carnivores: oviductal passage is slow, the embryos enter the uterus on Day 6 after the first mating and implantation occurs late, between Days 11 and 14 after the first mating. The embryos enter the uterus mainly as morulae, some as early morulae and some as early blastocysts. On Days 6 and 7, nearly 80% of embryos can be flushed from the uterus and on Day 8 almost all embryos have arrived in the uterus.
It can be concluded that for the best recovery results, surgical embryo recovery ought to be performed on Day 8. However, 50% of polecat embryos have already expanded to a large size on Day 8 and become difficult to handle and cryopreserve using the methods described in this study. Embryo recovery on Day 7 results in smaller blastocysts which are more suitable for embryo transfer but 20% of the recovered blastocysts have already expanded too much to stand cryopreservation. Embryo recovery on Day 6 would be the best solution for cryopreservation because blastocysts have not yet expanded, but the recovery rate on Day 6 is only around 30%. In conclusion, in the present situation, the best compromise seems to be the selection of Day 7 after the first mating as the optimal day to harvest embryos for cryopreservation in the farmed European polecat using surgical embryo recovery method and cryopreservation methods described in this study. However, after development of a reliable method for cryopreservation of expanded blastocyst stage embryos in the polecat, embryo recoveries can be performed on Day 8 which results in good embryo recovery rates.
Earliest embryonic stages that developed to term after transfer into the uterus consisted of 8- to 16-cell stages. Kits were produced also from fresh, frozen-thawed and vitrified-warmed morulae and blastocysts and both fresh and frozen-thawed expanded blastocysts. All recipients receiving ≥12 embryos produced kits.
An asynchrony of ±1 day between a donor and a recipient did not affect the birth outcome. A reasonable survival rate (= live kits/transferred embryos) of 52% was achieved when fresh embryos were transferred to recipients weighing more than 1 kg.
Polecat recipients weighing more than 1 kg gave birth to an average of 6 kits
compared to 3 kits for recipients weighing less than 1 kg suggesting that light recipients (< 1 kg) should not be used in embryo transfer programs.
Cryopreservation seemed particularly harmful for expanded blastocysts as the majority of these embryos did not develop to term after freezing, thawing and transfer and most vitrified-warmed expanded blastocysts were totally destroyed. The compromised results of OPS vitrified-warmed expanded blastocyst stage embryos may have been a consequence of a minor technical problem (incubation time, temperature, concentration of cryoprotectans) that will undoubtedly be solved in future studies.
Surgical flushing is more sensitive to complications than surgical transfer, and further developmental work needs to be done to improve the flushing and transfer methods.
The surgical flank method results in high embryo yields from Day 7 and Day 8 donors, does not cause adhesions or jeopardize further reproduction of the donors and is undoubtedly a good option for embryo recovery.
Timing of blastocyst formation in vitro did not differ from that in vivo. Expansion of blastocysts in vitro was comparable to expansion in vivo during the first 24-h period for embryos placed in culture as morulae, early blastocysts or as expanded blastocysts which were considered to expand further. Embryos placed in culture at earlier stages of development failed to expand at all after reaching the blastocyst stage in vitro.
These results indicate that polecat in vivo embryos can be maintained in vitro, in culture medium designated for cattle, for a period of 24-h.
If one is to use large-scale embryo transfer programs effectively in a conservation programme, the kit yield should be increased. Our results are encouraging and the techniques may be applicable in experimental populations or in the management of small populations of endangered European mink in individual cases. Reliable methods of embryo collection and transfer already exist for immediate embryo transfer, and methods of semen collection, cryopreservation and transcervical artificial insemination are being developed.
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