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.

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.

Apoptosis in ovarian granulosa cells of cattle: morphological features and clearance by homologous phagocytosis.

Apoptosis is involved in many physiological processes of the ovary, such as recruitment of prenatal germ cells, follicular atresia, ovulation, and luteolysis. Based on the need for the involvement of phagocytic cells to achieve apoptosis clearance and that follicular atresia is triggered by weak apoptotic stimuli, we postulate that granulosa cells engullng apoptotic corpses (ACs) must carry out this macrophagic process. Since apoptosis was early defined in terms of morphological aspects, here we describe apoptosis induced by a GnRH analog (leuprolide acetate, LA) at histological level on bovine granulosa cells (primary culture, CPGB, and an established cell line, BGC-1). We observed two main types of apoptosis. In type A, the whole cell or most of it is compacted into a single large AC that is then engulfed by neighboring cells or simply detached. In type B, small portions of cells, either with or without nuclear material, become ACs that are also phagocytosed. Apoptosis and homologous phagocytosis were confirmed by TUNEL and immunocytochemistry for Bax and active caspase 3. Induction of apoptosis was significant in BGC-1 cells treated for 24 h with 100 nM LA. CPGB cells showed two types of response with different doses of LA. Fetal calf serum was necessary to find apoptosis induced by LA.