65 Co-culture of porcine epithelial oviductal cells and in vitro-produced embryos: Effect on embryo development and quality

Abstract

The oviduct is involved in many reproductive functions, including early embryo development. The epithelial cells that cover the oviduct produce oviducal fluid and could be used to recreate the in vivo environment into which embryo development takes place. This study aimed to evaluate the co-culture of porcine embryos with a monolayer of porcine oviducal epithelial cells (POEC) and its effect on embryo development and quality. The POEC were obtained by pressing the isthmus (from diestrus sow oviducts) using slides and performing 3 cycles of vortexing and decanting in DMEM-F12 medium. Passage 1 cells were used for these experiments (POEC-1). Oocytes were obtained from follicular aspiration of slaughterhouse ovaries. Oocytes were in vitro matured for 44 h in TCM-199 supplemented with human menopausal gonadotrophin and cyclic AMP during the first 22 h. In vitro fertilization was performed with 17°C-refrigerated boar semen for 4 h in 100-µL drops of TCM-199 with caffeine, bovine serum albumin, sodium lactate, and sodium pyruvate (20 denuded oocytes per drop, 1 × 10⁶ spermatozoa mL⁻¹). Presumptive zygotes were washed and randomly assigned to one of the following groups for in vitro culture: control (50-µL drop of NCSU-23 with sodium pyruvate and lactate), POEC-1 (same as the control + POEC-1 50 000 cells mL⁻¹), POEC-1 + FBS (same as the control + POEC-1 50 000 cells mL⁻¹ and 2.5% of fetal bovine serum). Culture conditions were 7% O₂, 5% CO₂, 39°C, and humidity. On Day 2, the cleavage rate was recorded, and embryos were transferred to NCSU-23 drops with glucose and without cells. The blastocyst rate was recorded on Day 7. Embryo quality was assessed by counting the number of cells per blastocyst (Hoechst) and the apoptosis index (TUNEL-positive cells/total cells). Co-culture with POEC-1 significantly increased the blastocyst rate (control: 14%; POEC-1 + FBS: 10%; POEC-1: 28%; P < 0.05 Chi-squared test) and allowed embryo hatching (control: 0; POEC-1 + FBS: 22.2%; POEC-1: 7; P < 0.05 Chi-squared test). However, there was no significant difference in the number of cells per blastocyst (control: 58.6 ± 6; POEC-1 + FBS: 50.3 ± 3.7; POEC-1: 50.6 ± 4.8; nonparametric ANOVA) or in the apoptosis index (control: 8.1; POEC + FBS 8.3; POEC: 7.4; nonparametric ANOVA). The use of POEC-1 during the first 2 days of embryo culture enhanced embryo development and improved culture conditions, allowing embryo hatching. The effect on embryo development could be due to an effect of POEC itself or the effect of feeder cells. Other parameters of embryo quality should be evaluated in the future.