Viability and Freezing Ability of Rabbit Collected in the Vagina after Prostaglandin Treatment

Transcription

1 Technical Note Japanese Journal of Physiology, 38, , 1988 Viability and Freezing Ability of Rabbit Collected in the Vagina after Prostaglandin Treatment Embryos Vlviane GARNIER, Jean Paul RENARD, * and Yves MENEZO* * INRA, Physiologie Animale, Jouy en Josas, France * Institut Pasteur, 25 rue Dr. Roux, Paris, France ** INSA, INRA LA 23203, Biologie 406, Villeurbanne Cedex, France Summary Morulae were collected from rabbit vaginas after prostaglandin treatment 65 h post coitum. The optimum embryo recovery was obtained when the flushings started around 12 h after the prostaglandin injection. The mean embryo collection was around 10 embryos per animal. These embryos had the same viability as those collected by the classical technique of uterine flushing (around 80%). The freezing ability of these embryos was also similar to that observed for uterus-collected embryos. Embryos not ejected after the treatment can develop in vivo and give birth to living progeny. The results obtained suggest that this technique can be used for egg transfer in rabbit genetic improvement programs. Key words prostaglandins, vaginal expulsion rabbit embryo. Embryo transfer is now a widely used large-scale procedure for genetic improvement, mainly in cattle but also in goats and sheep. In the rabbit, it is considered as a tool to further research in other animal species. However, this technology may also have a possible contribution of offer to genetic improvement programs. Sugical embryo collection (STAPLES, 1971) commonly leads to fertility losses (MAURER et al., 1968). TsUTSUMI et al. (1976) and TAKEDA et al. (1977, 1978) proposed vaginal collection following hormonal treatments (estrogens and prostaglandins) to accelerate embryo transport. Their data were preliminary, the number of embryos was small and the development rate after transfer was poor. Similarly TESTART (1969) claimed that a shortened period of oviductal transport has detrimental effects on embryo viability and ADAMS (1973) suspected that the vaginal environment was harmful for the embryo. Received for publication March 14, 1988 ** To whom all correspondence should be addressed. 585

2 586 V. GARNIER, J. P. RENARD, and Y. MENEZO In this study we modified the technology of TAKEDA et al. (1977) to control the viability and the freezing ability of these embryos ejected in the vagina after prostaglandin treatments. The fertility of the female donors after such treatments was also investigated. Materials and methods Embryo production. Six-month-old New Zealand female rabbits were treated for superovulation during spring and summer with pure FSH (Burns Biotech, Omaha, U.S.A.) (2 mg) and LH (0.33 mg) as described by TECHAKUMPHU et al. (1986, 1987). At the time of LH iv. injection, donor females were mated with entire or vasectomized bucks (for the recipients). Before embryo collection, the animals were anesthetized with diazepam (Valium, 5 mg/kg). Recipients were also anesthetized before transfer according to the same procedure. Vaginal collection. Fifty-four hours p.c., each donor female received a s.c. injection of the prostaglandin (Pg) F2 analog (Imperial Chemical Industries; estrumate: 500 µg/2 ml). Ten to 15 h following the Pg analog injection, female donors were submitted to 6 flushings at 2-h intervals. Each flushing was realized with 3 washings of 8 ml of PBS containing 0.4% of BSA. The washing device was derived from the one used by ROUSTAN (1982). It consisted of a Pyrex tube (o.d., 5 mm; id., 2.5 mm) slightly bent at one extremity, the other being connected to a collecting tube and a 20 ml syringe. The washings were collected in a final volume of 25 to 30 ml, and observed in a binocular microscope. The embryos were kept in B2 medium (API System, Montalieu Vercieu, France; MENEZO et al., 1984) for less than 5 h before either direct transfer (6 embryos per uterine horn) or immediate freezing. The freezing procedure was performed according to TECHAKUMPHU et al. (1987). Efficacy of collection. Six females (out of the 34) were sacrificed after collection. The number of corpora lutea was determined on the ovaries. In the remaining 28, the number of collected embryos was registered for each flushing. Pregnancies after Pg treatment. In all donors, the pregnancies, if any, were allowed to proceed after the end of the collection procedures. Embryo viability. The viability of the embryos was controlled after direct transfer or after freezing and compared to embryos collected, surgically, by uterine flushings. Results 1) Efficacy of embryo collection (Table 1). The mean collection rate was 11.4 embryos/animal. Of the 34 animals flushed, 30 were positive (88.2%). The number of embryos collected per female was variable. Nine females (26.5%) gave 20 or more embryos. The changes in time-schedule of flushing after Pg treatment modified the efficacy of collection. It increased when the begining of the flushing was slightly delayed around 12 h after the Pg injection. However, due to the wide variability in the samples, the difference was not significant at the 5 % level. Japanese Journal of Physiology

3 VAGINAL COLLECTION OF RABBIT EMBRYOS 587 Table 1. Efficacy of collection (embryos recovered) according to the time of the 1st flushing and the order of the flushing. Table 2. Flushing efficiency assessed by the number of corpora lutea (CL) and the number of embryos remaining in the uterus. 2) Flushing efficiency (Table 2). Out of the 6 sacrificed females, we detected 140 corpora lutea after Pg treatment. Fifty-six embryos had been collected from the vagina (40%), another 32 remained in the uterus (32%). There was a net loss of 52 embryos (37%). 3) Pregnancy in the female donors after treatments. Of the 34 superovulated, Pg-treated females, 12 remained pregnant giving birth to 73 offspring (6.1 per pregnant female or 2.6 per female submitted to treatment). 4) Embryo viability after treatment. a) Transfer of fresh embryos (Table 3): A total of 83 embryos collected from the vagina were transferred into the utreri of recipients, and compared with 92 embryo collected by uterine flushing. In both cases, the development rate (78.3 and 79.3, respectively) was around 80%. b) Transfer of vagina-collected frozen embryos (Table 4): Of the 171 embryos submitted to freezing, 125 looked normal after thawing (73%). After transfer these 125 embryos gave birth to 64 rabbits (51.2%). If we consider the embryo loss during the freezing procedure, the ratio was 37.4%. Vol. 38, No. 4, 1988

4 588 V. GARNIER, J. P. RENARD, and Y. MENEZO Table 3. Viability after transfer of the vagina-collected embryos. Table 4. Freezing ability of embryos collected from the vagina. Discussion and conclusion The present technique of superovulation associated with Pg treatment permits a vaginal, non-surgical collection of viable embryos. As most of the embryos were ejected within 18 h after the prostaglandin treatment irrespective of the time of the first flushing, it seems better to start collection 12 to 15 h after the Pg treatment. There is a wide variability in the number of embryos collected per animal. The highly variable responses may be due to the different schemes obtained after superovulation (7 to 31 corpora lutei per female). Variability is a common feature observed after superovulation. Prostaglandings, which increase the muscular contractions, should have more or less efficient action depending on the hormonal background: estrogen and progesterone (NoYES et al., 1959; KENNELLY and FOOTE, 1965). If we consider the viability, our data demonstrate a close similarity with uterine collection; however, our technical procedure avoids prolonged embryo exposure to the vaginal environment. The freezing ability is similar to the uterus-collected embryos. If we consider the net viability, around 80% of freshly collected embryos and around 40% of the frozen embryos give normal young. Moreover, the embryos and around 40% of the frozen embryos give normal yound. Moreover, the fertility of the female donors submitted to the Pg treatment is preserved. This technique is a useful tool for further genetic improvement, based on embryo transfer in the female rabbit. Japanese Journal of Physiology

5 VAGINAL COLLECTION OF RABBIT EMBRYOS 589 REFERENCES ADAMS, C. E. (1973) The development of rabbit eggs in the ligated oviduct and their viability after re-transfer to recipient rabbits. J. Embryol. Exp. Morphol., 29: KENNELLY, J. J. and FOOTE, R. H. (1965) Superovulatory response of pre- and postpubertal rabbits to commercially available gonadotrophins. J. Reprod. Fertil, 9: MAURER, R. R., HUNT, W. L., VAN VLECK, L. D., and FoOTE, R. H. (1968) Developmental potential of superovulated rabbit ova. J. Reprod. Fertil., 15: MENEZO, Y., TESTART, J., and PERRONE, M. D. (1984) Serum is not necessary for human in vitro fertilization, early embryo culture, and transfer. Fertil. Steril., 42: NOYES, R. W., ADAMS, C. E., and WALTON, A. J. (1959) The transport of ova in relation to the dosage of oestrogen in ovariectomized rabbits. J. Endocrinol., 18: ROUSTAN, A. (1982) L'insemination artificielle chez la lapine. Cuniculture, 9: STAPLES, R. E. (1971) Blastocyst transplantation in the rabbit. In: Methods in Mammalian Embryology, ed. by DANIEL, J. R., 290 pp. TAKEDA, T., TSUTSUMI, Y., TANABE, Y., and YAMAMOTO, K. (1977) Administration of prostaglandin F2 for the recovery of fertilized eggs from the vaginas of rabbits. Fertil. Steril., 28: TAKEDA, T., TSUTSUMI, Y., HARA, S., and IDA, M. (1978) Effects of prostaglandin F2 on egg transport and in vivo egg recovery from the vaginas of rabbits. Fertil. Steril., 30: TECHAKUMPHU, M., TORRES, S., HEYMAN, Y., and MENEZO, Y. (1986) Effect of culture, freezing and culture associated with freezing on the viability of rabbit embryos controlled by synchronous and asynchronous transfer. In: Workshop on Embryos and Oocytes Freezing. ed. by MENEZO, Y. and MERIEUX, Ch., Collection Fondation Marcel Merieux, pp TECHAKUMPHU, M., WINTENBERGER-TORRES, S., SEVELEC, C., and MENEZO, Y. (1987) Survival of rabbit embryos after culture or culture/freezing. Anim. Reprod. Sci., 13: TESTART, J. (1969) Comparison de differentes techniques de transplantation des blastocystes chez la lapine. Ann. Biol. Anim. Biochem. Biophys., 9: TSUTSUMI, Y., TAKEDA, T., YAMAMOTO, K., and TANABE, Y. (1976) Nonsurgical recovery of fertilized eggs from the vagina of oestrogen treated rabbits. J. Reprod. Fertil., 48: Vol. 38, No. 4, 1988