In vitro culture and interferon-tau secretion by ovine blastocysts

Embryos were collected surgically from superovulated ewes on days 7, 8, 9 and 10 (oestrus=day 0) to evaluate the long-term culture and interferon-tau (IFN-tau) secretion of ovine blastocysts. Embryos were cultured in 2 ml Dulbecco's modification of Eagle's medium (DMEM) supplemented with 15 mg/ml BSA in 5% CO(2) in air or DMEM without BSA in 5% CO(2), 7% O(2), and 88% N(2) at 39 degrees C, examined daily for morphological features and diameter and each day placed into fresh culture medium to enable daily measurement of IFN-tau secretion. Nine day-7 and two day-9 embryos were cultured in DMEM with BSA and nine continued to develop. The day-7 embryos reached a mean maximum diameter of 370.0+/-50.25 microm after 4 days in culture. Nineteen day-7, 12 day-8 and five day-10 embryos were cultured in DMEM without BSA but only six of the day-7 and one day-8 embryos survived for at least 7 days with the former reaching a mean maximum diameter on day 7 of 357+/-43.75 microm whereas all five day-10 embryos survived for at least 7 days reaching a mean maximum diameter on day 6 of 1038+/-155.8 microm. An anti-viral assay and a ELISA for IFN-tau were developed. There was a considerable variation in the time of onset and amount of IFN-tau secreted that did not seem to be related to embryo morphology. Of 28 day-7 embryos cultured, 60.7% were secreting IFN-tau after 1 day of culture whereas 87.5% of day-8 embryos were secreting IFN-tau after 1 day in culture. The mean concentration of IFN-tau secreted by day-8 embryos after 1 day in culture (10.99+/-2.55 ng/ml) was not significantly different to day-7 embryos after 2 days in culture (8.8+/-1.75 ng/ml).