Chronology of equine fertilisation and embryonic development in vivo and in vitro

Abstract

SummaryFertilisation and early embryonic development were studied in the following sequence: (1) detection of the pre‐ovulatory follicle by daily ultrasonography; (2) induction of ovulation by injection of pituitary extract: (3) artificial insemination 19 to 25 h after injection; (4) precise detection of ovulation by hourly ultrasonography from 34 or 35 h post injection (pi); (5) surgical collection of the ovary and oviduct at different intervals from ovulation (6, 12, 24, 48, 72, 96 h); (6) flushing of the embryo and immediate evaluation by microscopy; (7) either fixation for later morphological study or culture for 12 to 84 h. Induction of ovulation was attempted in 44 mares by intravenous injection of 25 mg crude horse pituitary extract when a growing follicle over 33 mm was detected. Twenty four mares ovulated between 34 and 40 h pi. Five ovulated before 35 h but had not been scanned at 34 h. Five ovulated before 34 h, three between 40 and 47 h and seven did not ovulate. Recovery of ova by flushing the oviduct with PBS containing antibiotics and 100 iu/ml heparin was successful in 24 of 32 attempts. In vivo development of the embryos was estimated at collection. At 6 h after ovulation (n=6), ova were in cumulus and fertilisation could not be determined. At 12 h after ovulation (n=4), ova had two polar bodies and had not yet cleaved. At 24 h (n=4), three had two cells but one had not cleaved. At 48 h (n=3), embryos had four to six cells. At 72 h (n=5), four embryos had seven to twelve cells and one had not divided. At 96 h (n=2), embryos had eight and twelve cells. From a total of 18 ova or embryos collected between 12 and 96 h after ovulation, 16 (89 per cent) were fertilised and close synchronisation of cleavage divisions was observed. In vitro culture of the embryos was performed at 38.5°C in tubes containing 0.5 ml of B2 medium with 15 per cent heat‐inactivated foetal calf serum under air and 5 per cent CO2. Of nine ova collected at 6 or at 12 h and cultured, six had cleaved at 24 h after ovulation; the other three did not divide and were not considered further. From 24 to 48 h, all seven embryos divided from two cells to four to six cells. From 48 to 72 h, three of four embryos divided from four to six to seven to eight cells and one did not develop. The two embryos cultured from 72 to 96 h divided from eight to twelve cells. In vitro cell divisions in embryos less than 96 h seemed to occur at a similar schedule as those in vivo. Six ova which divided following an attempt at in vitro fertilisation of 20 oocytes were compared for their division rate and aspect to the in vivo controls. Division rate seemed advanced in at least three of them but two had normal aspects and stages of division (two cells at 48 h and seven to eight cells at 70 h). All ova had been penetrated by spermatozoa which were found in the zona pellucida, under it or between blastomeres. A mid‐piece of a flagellum was found in the ooplasm providing proof of fertilisation rather than parthenogenesis. The reason for the abnormal development is suspected to be polyspermy.