FUNCTIONAL COMPARISON OF BOVINE TROPHOBLASTIC
　　VESICLES DERIVED FROM FRESHLY COLLECTED
　　CONCEPTUSES AND SERIALLY-PASSAGED TROPHOBLAST CELLS
　　　　　　　　

　　　　Y. Hashiyada^A, H. Takahashi^B, M. Asada^A,C, S. Yamaya^A, N. Sakuta^A, S. Furuyashiki^A, T. Oike^A,
　　　　　　　　　　　　　　　　　　　　　　　　K. Konishi^A,C, and M. Takahashi^{D
　　　　A}Ohu station, National Livestock Breeding Center (NLBC), Aomori, Japan, ^BNational Institute of
　　　　Livestock and Grassland Science, Ibaraki, Japan, ^CTottori Station, NLBC, Tottori, Japan, ^DNational
　　　　Agricultural Research Center for the Kyushu Okinawa Region, Kumamoto, Japan

　　　　　　The co-transfer of trophoblastic vesicles (TVs), derived from in vivo recovered conceptuses, is a
　　　　　well-known method for promoting the successful implantation of embryos through the action of
　　　　　bovine interferon-tau (bIFN-τ) on maternal-fetal recognition. However, the preparation of these TVs
　　　　　is a tedious process. Techniques have progressed for obtaining large numbers of TVs, consistently,
　　　　　from serially-passaged trophoblast cells. The aim of the present study was to compare the function
　　　　　of TVs for co-transfer derived from in vitro production with those derived from in vivo flushed embryos
　　　　　and without co-transfer.
　　　　　　Production of TVs from serially-passaged trophoblast cells was carried out according to the method
　　　　　previously described by Takahashi M et al. (Cloned animals and placentation, Yokendo.2000 147-151).
　　　　　The in vitro TVs (IVP-TVs), 1-2mm in diameter, were taken from cells passaged 48 to 52th and 96th.
　　　　　In vivo-derived TVs (vivo-TVs) were prepared from recovered elongating blastocysts 7 days after transfer
　　　　　of frozen-thawed embryos on Day 8 of estrous cycle. Demi-embryos were produced from fresh embryos
　　　　　of flashed Japanese Black by bisection using a micromanipulator. Pairs of demi-embryos, with or without
　　　　　(control) 2 to 4 TVs, were transferred into the uterus ipsilateral of 65 Japanese Short Horn recipients to
　　　　　the functional corpus luteum. Pregnancy diagnoses were performed twice, from Day 30 to 70, by ultrasound
　　　　　scanning. BIFN-τ content of culture media was measured by RIA (Takahashi H et al. 2005 Theriogenology
　　　　　63,1050-1060). Data were analyzed by Chi-square test and Fisher's exact test.
　　　　　　The pregnancy rate in the IVP-TVs group (20.0％, 5/25) had a tendency to be lower than in the vivo-TVs
　　　　　(46.2%, 6/13) although this didn’t reach statistical significance (P=0.09). These pregnant animals were
　　　　　transferred TVs from passaged 48-52th (5/19). In the control group 33.3% (9/27) of recipients conceived.
　　　　　Twin pregnancy rate was also lower in the IVP-TVs (0/5) than in the other two groups (3/6 and 5/9,
　　　　　P=0.09 and P=0.04, respectively). Fetal losses occurred only in the single pregnancies of IVP-TVs
　　　　　(60.0％, 3/5) and vivo-TVs (16.7％, 1/6) up until around Day 70. Reproductive efficiency, based on numbers
　　　　　of delivered offspring, was significantly lower in the IVP-TVs (8％, 2/25) compared with the vivo-TVs
　　　　　(61.5％, 8/13) and control (51.9％,14/27) groups, respectively (Ｐ<0.01). BIFN-τ levels secreted from
　　　　　the TVs derived from passages 46 and 48th were 0.157ng/ml and 0.113ng/ml, respectively. In conclusion,
　　　　　compared with the in vivo TVs, those from erially-passaged trophoblast cells had a negative effect on pregnancy,
　　　　　although the morphology of the two differently-derived TVs was similar.

　


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