Influence of protein supplements on the secretion of leukaemia inhibitory factor by mitomycin-pretreated Vero cells: possible application to the in vitro production of bovine blastocysts with high cryotolerance

A co-culture system for bovine embryos using mitomycin-treated Vero cells a nd serum-supplemented modified synthetic oviduct fluid (mSOF) supports the development of in vitro maturation and fertilization-derived oocytes to hat ched blastocysts. In this system, it has been suggested that one contributi on made by the co-culture cells to embryo development is production of the cytokine leukaemia inhibitory factor (LIF). However, there are concerns abo ut exposure of early embryos to serum due to its incompatibility with embry o cryosurvival. Ln this study, the influence of two protein supplements (sy nthetic serum substitute (SSS), a lipid-free human serum-derived product) a nd oestrous cow serum (ECS)) on Vero cell LIF secretion was compared, with the aim of designing a co-culture system that is supportive of bovine embry o cryopreservation. Vero cells cultured for 72 h in medium 199 + 5% fetal b ovine serum (FBS) (recommended maintenance medium for this cell line) secre ted detectable amounts of LIF (13.1 +/- 0.9 pg LIF per 10(5) cells). Cultur e in mSOF, the medium routinely used in this laboratory for embryo culture, also supported LIF secretion in Vero cells. However, the amount of LIF was tenfold higher (24.7 +/- 6.2 pg LIF per 105 cells; P < 0.05) when mSOF was supplemented with 10% (v/v) ECS compared with supplementation with 2% (v/v ) SSS. Results of a second series of experiments in which supplementation w ith each protein was normalized to 10% revealed similar differences in LIF secretion, indicating that LIF secretion was affected by the type, not the amount, of protein. Time course analysis revealed stepwise increases (P < 0 .05) in cumulative LIF secretion with every 24 h of culture in mSOF + eithe r SSS or ECS. Ln terms of embryo development and post-cryopreservation viab ility, medium supplementation with 2% (v/v) SSS alone versus the two-step s ystem of 2% (v/v) SSS (days 1-4) + 10% (v/v) ECS (days 4-10) had no influen ce (P > 0.05) on the ability of bovine blastocysts to hatch, with or withou t intervening cryostorage. However, the rate of blastocyst formation (expre ssed as the percentage of cleaved embryos) was only 27% in the presence of 2% (v/v) SSS, and increased almost twofold (P < 0.05) when ECS was added be ginning on day 4 of coculture. Ln summary, Vero cell LIF secretion was incr eased markedly by ECS. A two-step system of medium supplementation, in whic h embryos are exposed to ECS beginning on day 4 of in vitro development com bined high rates of blastocyst formation with cryotolerance. This effect ma y be a result of limiting embryo exposure to serum-derived lipid until afte r the eight-cell stage and providing an increase in LIF during the critical developmental stages of compaction and cavitation.