COCULTURE OF PORCINE EMBRYOS AND OVIDUCTAL EPITHELIAL CELLS: A MODEL TO IMPROVE EMBRYO PRODUCTION

LORENZO, MS.; TEPLITZ, G.; CRUZANS, PR.; LUCHETTI, CG.; MARURI, A.; TELLO, MF.; LOMBARDO, DM.

Buenos Aires

Encuentro; XX ANNUAL MEETING OF THE ARGENTINEAN BIOLOGY SOCIETY (SAB); 2018

Argentinean Biology Society

The coculture of gametes and embryos with somatic cells has been an attempt to improve embryo production. The use of oviductal epithelial cell could mimic the in vivo conditions. This study aimed to evaluate if the coculture of porcine embryos and oviductal epithelial cells increase embryo production. Oviducts from slaughteredsows with corpus luteum on their ovaries were dissected, and porcine epithelial oviductal cells (POEC) were obtained by pressing the isthmus surface with a slide. Individual cells were separated by cycles of aspiration-ejection with 21G-needle in a1 mL-syringe and vortex. After 3cycles of decanting in a 15-mL centrifuge tube, they were seeded using DMEM F12 with 10% SFB. Culture medium was changed every 48 h until confluence. POEC were trypsinized and cryopreserved until use. For the passage 1 (P1) culture, 2 days before in vitro fertilization (IVF) the POEC were warmed and seeded at a concentration of 50000 cells/mL in 50 uL-drops of NCSU 23 supplemented with 0,5 mM sodium pyruvate, 5 mM sodium lactate and 2,5% SFB. They were cultured in 7% O2, 5% CO2 humidified air at 39°C. Cumulus-oocyte complexes were obtainedby follicular aspiration of ovaries from slaughtered sows. They were matured in vitro in groups of 50 per well, in 5% CO2 humidified air at 39°, in modified Medium 199 containing 1mMAMPc and 1,5 UI hMG for the first 22 h and without them for the last 22 h. For IVF, 17°C-refrigerated sperm of proven fertility was centrifuge at 490 x g for 5 minutes and resuspended in Medium 199 supplemented with 0,4% BSA, 5 mM caffeine, 2,9 mM sodium lactate and 1,25 mM sodium pyruvate (IVF media). Denuded oocytes and 1x106 sperm/mL were coincubated in 100 uL-drops of IVF media, for 4 h in 7% O2, 5% CO2 humidified air, at 39°C. Presumptive zygotes were washed three times and randomly placed in drops of 50 uL NCSU 23 (with sodium pyruvate and lactate) alone (control, n=97), with POEC-P1 (n= 99) or with POEC-P1 and 2,5% SFB (n= 107). After two days of culture, they were washed and changed to drops containing fresh NCSU 23 (with 5mM glucose) and without POEC-P1. They were cultured up to day 7 in the conditions previously described. Cleavage rate was registered at day 2 and blastocysts rate at day 7. The rate of cleavage was 35,05% (control) 36,36% (POEC-P1) and 38,32% (POEC-P1 2,5% SFB) and it did not differ between categories. The rate of blastocyst was 3% (POEC-P1) and 2% (POEC-P1 2,5% SFB) and it did not differ between the coculture groups (p>0,05). We did not obtain blastocyst in control. The coculture of porcine embryos and POEC-P1 during the first 2 days of culture appears to be a model which could improve pig embryo production. The addition of 2,5% SFB during coculture would not affect embryo production, although embryo quality needs to be evaluated.