Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage

Immature bovine oocytes were matured and fertilized in vitro, and the resulting zygotes were cultured to the blastocyst stage in droplets of tissue culture medium 199 (TCM 199) conditioned by oviduct cells in the absence of serum. In Experiment 1, the effect of the number of zygotes in a constant culture volume was investigated by culturing 1, 4 or 40 zygotes in 40 microl of culture medium. The cleavage rate was low with a single embryo (36%) but increased with the number of embryos, to reach 50% with 4 embryos/40 microl and 59% with 40 embryos/40 microl. Blastocyst formation was nil with 1 embryo per 40 microl, reaching 2.5% with 4 embryos/40 microl and 18% with 40 embryos/40 microl. The effect of the size of the drop was assessed in Experiment 2, the concentration of embryos remaining constant (1 embryo/1 microl). The volumes tested were 10, 20, 30 and 40 microl. Development into blastocysts increased gradually from 12% in the 10 10 group to 20% in the 40 40 group. Experiment 3 was designed to find a minimal droplet volume able to support the development of a single embryo to the blastocyst stage. The minimum tested volume was 5 microl and was not successful. These results show that bovine embryos cultured in oviduct-conditioned TCM 199 need to cooperate to reach the blastocyst stage. The mechanism of this cooperation is not known, but some autocrine/paracrine factors, probably growth factors, could promote embryo development as was demonstrated in mice. From Experiment 2 we can hypothesize that the surface volume ratio of the droplets could play a role in the culture conditions by interfering with the exchanges between the culture medium and the surrounding environment.