102 Effect of conditioned media of oviductal epithelial cells on in vitro embryo development in pigs

We have previously reported that the co-culture of porcine in vitro-produced embryos with a monolayer of porcine oviducal epithelial cells (POEC) enhances blastocyst formation and lowers the level of ROS in cleaved embryos. This effect could be due to cell contact or the epithelial cell secretions. Adding media obtained from cell culture to embryo culture is a more straightforward and repeatable technique than co-culturing. Thus, this study aimed to evaluate the effect of POEC-conditioned media supplementation during embryo culture in porcine embryo development and quality. POEC from passage 1 were seeded in NCSU 23 and cultured at 39°C, 7% O₂, and 5% CO₂ in a humidity-saturated atmosphere for 48 h. Then, the media were centrifuged at 1,500 rpm for 10 min, and the supernatant was cryopreserved at −20°C until use. The oocytes were obtained by follicular aspiration from slaughterhouse ovaries. In vitro maturation was performed in wells containing medium 199 supplemented for the first 22 h with cAMP and hMG. At 44 h, the oocytes were denuded and co-incubated with semen refrigerated at 17°C from pigs of proven fertility for 4 h in medium 199 supplemented for IVF, in drops covered with mineral oil. Presumptive zygotes were pipetting, rinsed in TALP-H, and assigned to one of the following groups: CM (drops with 50% conditioned media in NCSU 23; n = 350) or control (drops of NCSU 23; n = 373). Other percentages of CM were discarded as they do not influence or negatively affect embryo development. Groups of 25 embryos per drop were cultured at 39°C, 7% O₂, and 5% CO₂ in a humidity-saturated atmosphere. On Day 2, the cleavage rate was determined, and the embryos of both groups were changed to drops containing NCSU 23. On Day 7, the percentage of blastocysts was determined by observation with a phase-contrast microscope. Blastocysts were fixed and stained with TUNEL-Hoechst to determine the number of cells per blastocyst and apoptosis index (TUNEL positive cells/total cells). The embryonic development indices were evaluated by the Fisher test and the number of cells, the number of TUNEL cells, and apoptosis index by ANOVA, considering significant P ≤ 0.05. CM allowed embryo hatching (12%), whereas no blastocyst hatched in the control group, but no other embryo-development parameters were affected. CM neither modified the number of cells (control 41.2 ± 3.3 vs CM 41.9 ± 4) nor the apoptotic index in blastocyst (control 0.085 vs CM 0.076). Taking into account our previous results and considering those obtained in this study, the cell-cell contact is the key to explaining the effect of the oviducal epithelial cell on in vitro-produced porcine embryos.

The authors thank Agroceres PIC for semen samples.