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

Abstract

Abstract.We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 µl/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P<0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p<0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos. Key words: Bovine, Conditioned medium, Development, Embryo, Single embryo (J. Reprod. Dev. 52: [137][138][139][140][141][142] 2006) ecently, ultrasound-guided ovum pick-up (OPU) has enabled repeat collection of oocytes from live cattle for genetic improvement [1][2][3]. However, the efficiency of embryo production by OPU is still low because of the limited number of oocytes collected and used for in vitro fertilization (IVF) and culture (IVC). In our field data, the blastocyst formation rate decreased when the number of in vitro fertilized oocytes collected from individual heifers was less than 3. Therefore, it is important to increase the efficiency of blastocyst production from small numbers of in vitro fertilized oocytes collected by OPU.Previous studies in mice [4, 5], sheep [6], and cattle [7][8][9] have demonstrated that group cultures promote embryo development compared with single-culture or co-culture with small numbers of 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

Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality

“…Another possibility could be to modify directly the composition of culture media with the use of cell co-cultures (bovine oviduct epithelial cell, Lim and Hansel, 1996;granulosa cells, O'Doherty et al, 1997;cumulus cells, Goovaerts et al, 2009), conditioned media (Fujita et al, 2006) or addition of GF (Paria and Dey, 1990;Carolan et al, 1996;Lim and Hansel, 1996;Brison and Schultz, 1997). The review by Hardy and Spanos (2002) clearly reflects the importance of a number of GF affecting preimplantation embryo development.…”

“…In particular, when embryos were cultured individually or in reduced groups, in addition to a lower efficiency of embryo production, the obtained embryos usually presented a lower number of cells per blastocyst (Paria and Dey, 1990;O'Doherty et al, 1997; Nagao et al, 2008: 5 v. 25 embryos). Numerous authors have previously indicated, in several mammalian species, that IVEP efficiency is negatively affected when a single or a low number of oocytes/embryos were in vitro cultured in comparison with a higher number of the same (Paria and Dey, 1990;Lane and Gardner, 1992;Carolan et al, 1996;O'Doherty et al, 1997;Fujita et al, 2006). Moreover, we have observed that post-fertilisation culture, especially the last part of the culture period, seems to be the most important period for bovine embryo development when single and/or low numbers (5 to 10) of embryos were cultured (Salvador et al, 2011).…”

“…One of them would be the lack of growth factors (GFs) in the culture medium (CM), which could exert a beneficial effect stimulating embryo development, in a paracrine and/or autocrine manner, when embryos were in vitro cultured in groups (Paria and Dey, 1990;Lim and Hansel, 1996). Previously, Bavister (1995) had indicated that a rise in embryo number in culture could deplete embryotoxic substances or diminish the inhibitor concentration.To improve the production and quality of embryos cultured in low numbers or singly, several physical modifications to optimise embryo environment have been proposed, the most simple being reducing the ratio embryo/volume in microdrops Fujita et al, 2006;Nagao et al, 2008); other options are 'Well of Well' system (Vajta et al, 2000), glass oviduct (Thouas et al, 2003), woven polyester mesh (Booth et al, 2007), Cell-Tak grids (Gopichandran and Leese, 2006) or the use of microfluidics (Krisher and Wheeler, 2010). Another possibility could be to modify directly the composition of culture media with the use of cell co-cultures (bovine oviduct epithelial cell, Lim and Hansel, 1996; granulosa cells, O'Doherty et al, 1997; cumulus cells, Goovaerts et al, 2009), conditioned media (Fujita et al, 2006 or addition of GF (Paria and Dey, 1990;Carolan et al, 1996;Lim and Hansel, 1996;Brison and Schultz, 1997).…”

Efectos de los RNAm maternos sobre la maduración del ovocito y el desarrollo embrionario temprano en mamíferos

“…En los animales domésticos como el bovino, uno de los principales problemas a los que se enfrenta la fertilización in vitro es el bajo porcentaje de blastocistos viables obtenidos a partir de ovocitos madurados in vitro (5,6) . De acuerdo a la experiencia de diversos grupos de investigación (7)(8)(9), en los sistemas bovinos, del total de ovocitos que se someten a maduración in vitro (IVM) sólo se logra obtener aproximadamente un 40 % de blastocistos viables; lo que es muy diferente a lo observado a partir de ovocitos madurados in vivo, donde el porcentaje de blastocistos viables obtenidos es cercano al 80 % . Esto sugiere que la IVM modifica el microambiente del ovocito, al ocasionar un cambio celular y genético que impide que esta célula se desarrolle de manera normal (10) .…”

“…In domestic animals like cattle, one of the main problems faced by in vitro fertilization is the low percentage of viable blastocysts derived from in vitro matured oocytes (5,6) . According to the experience of different research groups (7)(8)(9) in cattle systems, the total number of oocytes undergoing in vitro maturation (IVM) only manages to get about 40 % of the viable blastocysts; versus in vivo matured oocytes, where the percentage of viable blastocysts obtained is close to 80 %. This suggests that the IVM modifies the microenvironment of the oocyte, to cause cellular and genetic change that prevents this cell to develop as normal (10) .…”