103 Co-culture of porcine in vitro-produced embryos and porcine luteal cells

The co-culture of somatic cells with gametes and embryos is used to improve culture conditions and thus favour embryonic development. Porcine luteal cells (PLC) used during in vitro maturation (IVM) promote oocyte maturation and blastocyst formation and modify the gene profile of porcine embryos. These cells produce progesterone, and adding it during embryo culture in pigs promotes development. This work aimed to evaluate the effect of the co-culture of porcine embryos with a monolayer of PLC during the first two days of embryo culture on the development and blastocyst quality. PLC were obtained by mechanical and collagenase chemical disintegration of corpora lutea from the ovaries of dioestrus slaughtered females. The expression of 3β-HSD by immunocytochemistry verified its luteal character, and passage 1 (PLC-1) was used for this study. The PLC-1 were thawed 48 h before the co-culture and seeded at 10,000 cell/well in medium NCSU 23 + 5% fetal bovine serum (FBS). On the day of IVF, the medium was changed to NSCU 23 without adding FBS. The oocytes were obtained by follicular aspiration from slaughterhouse ovaries. IVM was performed in wells containing medium 199 supplemented 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 and rinsed in TALP-H and assigned to one of the following groups: 10,000 PLC-1 (n = 185) or control (containing only NCSU 23; n = 143). Groups of 50 embryos were cultured in wells at 39°C, 7% O₂, 5% CO₂ in a humidity-saturated atmosphere. On Day 2, the cleavage rate was determined, and embryos of both groups were changed to wells 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, number of TUNEL cells, and apoptosis index by ANOVA, considering significant P ≤ 0.05. Three replicates were performed. Treatment with PLC-1 significantly improved cleavage (27.7% vs 43.7%) but not the blastocyst rate. The number of blastomeres, although not significant, was higher in the PLC-1 group in all replicates (35.5 ± 3.3 vs 46 ± 4.1), and it significantly lowered the apoptosis index (0.144 vs 0.082). The presence of PLC-1 during the first two days of embryo culture promotes cleavage and improves embryo quality. The effect may be due to the luteal cells per se or the presence of feeder cells.

The authors thank Agroceres PIC for semen samples.