Effects of the addition of insulin-transferrin-selenium (ITS) and/or metformin to the in vitro maturation of porcine oocytes on cytoplasmic maturation and embryo development.

CONTEXT: One of the main problems of porcine in vitro maturation (IVM) is incomplete cytoplasmatic maturation. Nuclear and cytoplasmic maturation will determine the future success of fertilisation and embryo development. Insulin-transferrin-selenium (ITS) has insulin-like and antioxidant effects, and metformin (M) is an insulin-sensitiser and antioxidant drug. AIMS: To assess the effects of adding ITS and/or M in porcine IVM media on cytoplasmic maturation and early embryo development. METHODS: Cumulus -oocyte complexes (COC) were IVM with M (10-4 M), ITS (0.1% v/v), M+ITS or no adding (Control). KEY RESULTS: ITS increased glucose consumption compared to Control and M (P <0.01), and M+ITS did not differ from ITS or Control. Redox balance: M, ITS and M+ITS increased glutathione (P <0.01) and decreased lipid peroxidation (P <0.005). The viability of cumulus cells by flow cytometry increased with M (P <0.005) and decreased with ITS (P <0.001); M+ITS did not differ from Control. After IVF, M increased penetration and decreased male pronucleus (P <0.05). Embryo development: cleavage increased with M (P <0.05), and blastocysts increased with ITS and M+ITS (P <0.05). The number of blastocyst cells increased with ITS (P <0.05). CONCLUSIONS: Adding ITS and M+ITS to porcine IVM media benefits embryo development to blastocysts, but ITS alone has better effects than M+ITS. IMPLICATIONS: ITS is an excellent tool to improve IVM and embryo development after IVF in pigs.

The coculture of in vitro produced porcine embryos and oviductal epithelial cells improves blastocyst formation and modify embryo quality.

The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF.

Embryotrophic effect of a short-term embryo coculture with bovine luteal cells.

The coculture with somatic cells is an alternative to improve suboptimal in vitro culture (IVC) conditions and promote embryo development. Several cell types have been used for this purpose, but there is no information about using luteal cells in short-term coculture with embryos. Consequently, this study aimed to assess the effect of a short-term coculture of early bovine embryos-luteal cells on the in vitro development and embryo quality. Presumptive embryos were cultured from day 0 to day 2 in medium alone (control) or cocultured with bovine luteal cells (BLC-1). Then, embryos from both groups were cultured in medium alone from day 2 to day 8. The development rates on day 8 were compared between groups. The level of reactive oxygen species (ROS) and proliferation rates were evaluated in day 2 embryos and late apoptosis and proliferation rates were determined in day 7 blastocysts. Our results showed that the coculture with bovine luteal cells increased the blastocyst rate compared to the control (50.4% vs. 29.8%; P < 0.01), but there were no differences in the cleavage rates on day 2. The rate of stage 6 blastocysts was higher in the coculture (37.3% vs. 23.8% control; P < 0.01), without differences in the expansion and hatching rates compared to the control. The ROS level in day 2 embryos was higher in the coculture than the control (82 vs. 57.1; P < 0.05), and the cell proliferation rate was higher in the coculture (48% vs. 13% control; P < 0.01), without differences in the mean number of cells between groups. In day 7 blastocysts, the apoptosis rate decreased in the coculture with bovine luteal cells from day 0 to day 2 (4.1% vs. 10.9% control; P < 0.01), whereas the cell proliferation rate and the mean number of cells did not differ between groups. This is the first report of a short-term coculture of in vitro produced embryos and bovine luteal cells. Our model could be an alternative to increase the efficiency of the in vitro production of embryos in cattle.