Characterization of the embryotrophic activity of exogenous protein-free oviduct-conditioned medium used in culture of cattle embryos.

Bovine zygotes, obtained after in vitro maturation and fertilization of oocytes from slaughtered cow ovaries, were cultured in droplets of nonconditioned or conditioned medium on bovine oviduct cell monolayers. The media tested were Medium 199 alone and Medium 199 supplemented with 10% fetal calf serum (FCS). Oviduct conditioning increased both early cleavage and development to blastocysts. Only the effect on early cleavage was mimicked by FCS. The blastocysts obtained in serum-free conditioned medium (SFCM) appeared morphologically normal and had the same cell number as those produced in conditioned medium containing serum. Their hatching rates did not differ. Transfer of 16 blastocysts developed in SFCM to 16 synchronized recipients resulted in five pregnancies (31%), indicating good embryonal viability. Boiling of SFCM resulted in a total loss of activity, while heating at 56 degrees C for 30 min had no deleterious effect. A 10-kDa ultrafiltration of SFCM removed the blastocyst development-supporting activity from the filtrate but not the early cleavage-favoring activity. This allows us to conclude that at least two different factors are present in SFCM: one of low molecular mass (< approximately 10 kDa), needed to obtain the 5-8 cell stage and mimicked by FCS, and another of higher molecular mass allowing embryos to develop from the 8-cell to blastocyst stage.

Effects of dilution procedure and culture conditions after thawing on survival of frozen bovine blastocysts produced in vitro.

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.

Collection of oocytes and production of blastocysts in vitro from individual, slaughtered cows.

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 microliters droplets and those from pooled ovaries were cultured in groups of 50 in 50 microliters 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.(ABSTRACT TRUNCATED AT 250 WORDS)