Coculture of porcine luteal cells and cumulus-oocyte complexes during in vitro maturation: effect on the mature-oocyte lipid content, mitochondrial activity, and embryo development

TEPLITZ, G.; LORENZO, MS.; BASTIEN,A.; ROBERT,C.; SIRARD, MA.; LOMBARDO, DM.

Bologna

Congreso; 19th International Congress on Animal Reproduction; 2020

ICAR Committee

The coculture with somatic cells is an alternative to improve suboptimal in vitro culture conditions. In the pig, the in vitro embryo production is still inefficient biotechnology. This study aimed to evaluate the coculture of porcine luteal cells (PLC) on the in vitro maturation (IVM) and its effect on the oocyte lipid content, mitochondrial activity, and embryo development. Slaughterhouse ovaries were used to obtain PLC and cumulus-oocyte complexes (COC). COC were matured in vitro for 44 h in supplemented TCM-199 with human menopausal gonadotrophin (control) and in coculture with PLC. After maturation, oocytes were stripped and stained with 300nM MitoTracker Orange (CMTMRos) for 30 min; then, oocytes were fixed with paraformaldehyde 4% and immersed in 3 µg/mL Bodipy and 1mg/mL Hoechst blue dye 33342 for 10 min. Confocal and epifluorescence images were acquired using a Nikon TE2000 confocal microscope, and the intensity of fluorescence was measured using the mean greyscale in ImageJ software. IVF was performed for 4 h with frozen-thawed boar semen in 100 µL-drop of modified Tris-buffered medium (20 denuded oocytes per drop, 1.5x106 spermatozoa/mL). Presumptive zygotes were washed and cultured in porcine zygote medium at 39°C, 7 % O2, 5 % CO2, and humidity. The cleavage rate was registered on day 2 and the blastocyst rate on day 7. In the control and PLC group the MitoTracker fluorescence intensity was similar. The number of lipid droplets (LD) per embryo was similar in both treatments, but the mean volume of LD was higher in control. Likewise, the Bodipy fluorescence intensity was higher in control (p < 0.05, Two tail unpaired t-test). No significant differences were observed in the cleavage rate (control: 43.2%; PLC: 37.5%; p < 0.05, Chi-square test). The coculture with PLC significantly increased the blastocyst rate (blastocyst/cleavage) (control: 14.4%; PLC 21.2%; Chi-square test). The coculture with PLC during IVM decreased the amount of LD in the mature-oocyte and enhanced embryo development. Other parameters of embryo quality should be evaluated in the future. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in the pig.