Blastocysts derived from bovine zygotes fertilized and matured in vitro and cultured for 7 days in conditioned medium were frozen in 1.36 mol glycerol l−1 and 0.25 mol sucrose l−1. In vitro survival after thawing was unaffected by dilution rate in 0.25 mol sucrose l−1. The proportion of blastocysts that re-expanded after 24 h was 81% (70 of 86) and 47% (33 of 70) hatched. Seven pregnancies beyond 2 months resulted from transfer of 21 blastocysts to 19 recipients. Total embryonic loss was 46.2%, of which 31% occurred between days 21 and 35. In vitro survival after thawing was influenced by culture conditions, the best being culture with oviduct epithelial cells, where 55–82% of blastocysts re-expanded, of which 41–54% hatched. Conditioned medium also supported re-expansion, but low hatching (6%), whereas M199 plus fetal calf serum allowed only limited re-expansion (19–40%). This behaviour was not a consequence of freezing. It is suggested that blastocysts produced in vitro have reduced metabolic activity leading to high embryonic loss before or just at the time of implantation and that oviduct cells create a favourable environment after thawing, allowing hatching in vitro.
A. Massip, P. Mermillod, C. Wils and F. Dessy
P. Mermillod, C. Wils, A. Massip and F. Dessy
Summary. On four occasions ovaries from a total of 35 cows were collected separately at the abattoir where they had been killed. The age of 20 of these cows was recorded. Oocytes from these ovaries were collected separately and were submitted to in vitro maturation, in vitro fertilization and in vitro culture procedures. Ovaries of 34 randomly chosen cows were pooled and treated as the control. Ova from individual cows were cultured in 10 μl droplets and those from pooled ovaries were cultured in groups of 50 in 50 μl droplets of oviductal cell-conditioned medium. The 35 cows treated individually supplied 493 oocytes (mean 14·1 oocytes per cow) with high individual variation (sd = 10·0; range = 0–38) and 47 expanded blastocysts (9·5% of oocytes; mean 1·3 blastocysts per cow; range = 0–6). Among these cows, 16 produced one or more blastocysts. Considerable variation in average development rates was detected over the four replicate experiments (11·3, 4·0, 9·0 and 13·5%). The 34 cows treated as the control supplied 397 oocytes (mean 11·7 oocytes per cow) and 44 expanded blastocysts (11·1% of oocytes; mean 1·3 blastocysts per cow) with high variations between replicates (11·1, 4·0 and 18·1%). No difference was observed between individual and pooled ovaries regarding either the number of oocytes, the rate of blastocyst formation, or the number of blastocysts per cow. No effect of age was detected.
The conclusion was that culture of zygotes in small groups does not impair bovine embryo development in vitro but that high individual variation in oocyte number and the rate of embryonic development may explain the variable results observed in this study between replicate experiments and in general in bovine IVF. These variations will impair the prediction of blastocyst production from individual cows of high genetic value.
Keywords: in vitro fertilization; cow; embryo; blastocyst; breeding; oviduct; conditioned medium