In vitro development of bovine embryos in conditioned media from bovine granulosa cells and vero cells cultured in exogenous protein- and amino acid-free chemically defined human tubal fluid medium

Abstract

We have investigated the effect of protein- and amino acid-free simple human tubal fluid (HTF) medium conditioned with bovine granulosa cells (BGC) or Vero cells on the development of early bovine embryos to the blastocyst stage. Serum-containing medium (SCM) and serum-free medium (CM) conditioned by BGC (BGC-SCM, BGC-CM) and by Vero cells (VC-SCM, VC-CM) were prepared. Early embryos (1-cell stage and 5- to 8-cell stage) were obtained by in vitro maturation and fertilization of oocytes from slaughtered cows. Embryos were randomly divided into 7 culture groups as follows: culture with BGC-SCM, BGC-CM, VC-SCM, or VC-CM; coculture with BGC or Vero cells; or culture with fresh HTF medium without serum. The proportion of 5- to 8-cell embryos developing to the blastocyst stage in BGC-CM (16%) and VC-CM (12%) was significantly lower (p < 0.05) than in BGC-SCM (41%) and in VC-SCM (29%) and after coculture with BGC (48%) and Vero cells (30%). Similarly, the percentages of 1-cell embryos developing to blastocyst in BGC-CM and VC-CM were significantly lower than in BGC-SCM and VC-SCM and after coculture. Cell numbers per blastocyst developed from 5- to 8-cell embryos in BGC-CM (96.8 cells) and in VC-CM (94.0 cells) were somewhat lower than those in BGC-SCM (128.5 cells) and VC-SCM (117.1 cells) and after coculture with BGC (124.2 cells) and Vero cells (115.3 cells). These results suggest that BGC and Vero cells cultured in a protein- and amino acid-free simple HTF medium synthesize and secrete factor(s) promoting blastocyst formation in vitro. Physiochemical analysis indicated that the embryotrophic substances in BGC-CM were distributed in two molecular size ranges, one between 10 kDa and 30 kDa and another greater than 30 kDa.

Effect of bovine cumulus–oocyte complexes‐conditioned medium on in‐vitro maturation of canine oocytes

“…In addition, during conditioning the culture secreted embryotrophic components including proteins into the media that support blastocyst formation in early bovine embryo fertilized in vitro [46]. Therefore, the ability of bovine COCs-conditioned medium to support nuclear maturation of canine oocytes recovered from domestic dog ovaries in random reproductive states was examined.…”

Effect of Group Culture and Embryo-culture Conditioned Medium on Development of Bovine Embryos

“…In murine embryos, the blastocyst formation rate is promoted by an increase of embryo density in culture drops [10], suggesting that specific factor(s) are secreted from the embryos to influence embryo growth and development in an autocrine or paracrine manner. In the non-coculture system, embryo development is promoted by adding conditioned medium derived from bovine oviductal epithelial cells [11,12], buffalo/rat hepatocytes [13], bovine granulosa cells, or Vero cells [14]. Therefore, some growth factors secreted from co-culture cells should be contained in the conditioned media and should promote growth of preimplantation embryos.…”

Beneficial effects of Vero cells for developing IVF bovine eggs in two different coculture systems

“…These authors reported a 30% blastocyst rate, but their rate is expressed as a ratio to cleaved eggs (2-4 cell) and not to inseminated oocytes as in our study. Recently, Maeda et al (1996) also used this cell line for coculture and they reported only I 1 % blastocyst formation. However, they used a protein and amino acid free simple human tubal fluid (HTF) medium instead of B 2 medium, which is considered to be more appropriate for coculture of bovine embryos than other media (Hasler et al, 1996).…”