149 INTRAUTERINE CULTURE OF IN VITRO PRODUCED BOVINE EMBRYOS AND RECOVERY OF THE EMBRYOS AT DAYS 12–14

Abstract

For stem cell production and detailed morphological analysis 12–14-day-old bovine embryos are suitable. However, it has been proven to be difficult to extend the in vitro culture period beyond Days 8–9, and it was the aim of the present experiment to examine whether it might be possible to culture 6–7-day-old in vitro-produced (IVP) embryos for a period of 5–7 days in the uterine horns of heifers. The IVP embryos were produced by standard procedures. Briefly, IVM took place in DMEM medium supplemented with 5% serum, EGF, and eCG/hCG, and IVF was carried out in TALP medium under 5% CO₂ in humidified air and at 38.5°C. IVC took place in SOFaaci supplemented with 10% serum under 5% CO₂, 5% O₂ and 90% N₂ at 38.5°C .The embryos were cultured in vitro to Days 6–7 post insemination, when morulas and blastocysts of excellent quality were placed in HEPES-buffered TCM199 with 10% serum, loaded in numbers of 10–30 into 0.25 mL straws, and then transported to the place of transfer in a portable incubator at 38.5°C. The embryos were transferred nonsurgically to the mid or distal part of the uterine horns of 28 dairy heifers which were heat synchronized with injections of cloprostenol (Estrumat Vet, Schering-Plough, Farum, Denmark) to a cycle stage of embryo age +1 day. In 16 heifers, embryos were transferred into both sides and for the remaining ones only into the horn ipsilateral to the ovary bearing the corpus luteum. After 5–7 days, the heifers were flushed nonsurgically by standard method, using a flushing catheter of large caliber (Minitab^® 18 G) and slow infusion and evacuation of the fluid. The differences in recovering rate among horns were identified by Fisher's Exact test. Data are given as LS means ± SEM values and statistical differences assigned at the P < 0.05 level. In 6 of the 28 heifers no embryos were obtained; in these 6 cases, the quality of the transferred embryos, the transfer procedure, the heifers, and the flushing procedures did not differ in any obvious way from those of the successful flushings, which numbered 22 (79%). The mean embryo recovery rate was 40 ± 3% with a variation from 7% to 93%. There was a minor but not statistically significant difference between the overall recovery rate of embryos from the ipsi- versus contralateral horn, respectively (44 ± 5% vs. 38 ± 6%). In only 4 of the 16 heifers where transfer occurred to both horns was the recovery rate higher in contralateral side, compared to 9 heifers where the highest recovery rate was seen in the ipsilateral side. The oldest elongated embryos were in one occasion damaged and in another tangled, making it difficult to isolate the individual embryo; apart from that, all of the embryos seemed of excellent quality making it possible to isolate the embryonic discs. It can be concluded that it is possible to culture in vitro produced Day 6–7 bovine blastocysts in the uterus of synchronized heifers and to achieve an acceptable recovery of Day 12–14 embryos.