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

Ciudad Autónoma de Buenos Aires

Jornada; XX Jornadas Anuales de la Sociedad Argentina de Biología 2018; 2019

Sociedad Argentina de Biología

The coculture of gametes and embryos with somatic cells has been an attempt to improve embryo production. The use of oviductalepithelial cell could mimic the in vivo conditions. This study aimed to evaluate if the coculture of porcine embryos and oviductalepithelial cells increase embryo production. Oviducts from slaughtered sows with corpus luteum on their ovaries were dissected, andporcine epithelial oviductal cells (POEC) were obtained by pressing the isthmus surface with a slide. Individual cells were separated bycycles of aspiration-ejection with 21G-needle in a 1 mL-syringe and vortex. After 3 cycles of decanting in a 15-mL centrifuge tube, theywere seeded using DMEM F12 with 10% SFB. Culture medium was changed every 48 h until confluence. POEC were trypsinized andcryopreserved until use. For the passage 1 (P1) culture, 2 days before in vitro fertilization (IVF) the POEC were warmed and seeded at aconcentration of 50000 cells/mL in 50 uL-drops of NCSU 23 supplemented with 0,5 mM sodium pyruvate, 5 mM sodium lactate and2,5% SFB. They were cultured in 7% O2, 5% CO2 humidified air at 39°C. Cumulus-oocyte complexes were obtained by follicularaspiration of ovaries from slaughtered sows. They were matured in vitro in groups of 50 per well, in 5% CO2 humidified air at 39°, inmodified Medium 199 containing 1 mM AMPc and 1,5 UI hMG for the first 22 h and without them for the last 22 h. For IVF, 17°Crefrigeratedsperm 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 werecoincubated in 100 uL-drops of IVF media, for 4 h in 7% O2, 5% CO2 humidified air, at 39°C. Presumptive zygotes were washed threetimes 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 5 mM glucose) and without POEC-P1. They were cultured up to day 7 in the conditions previously described. Cleavage rate wasregistered 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-P12,5% 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 didnot differ between the coculture groups (p>0,05). We did not obtain blastocyst in control. The coculture of porcine embryos and POECP1during the first 2 days of culture appears to be a model which could improve pig embryo production. The addition of 2,5% SFBduring coculture would not affect embryo production, although embryo quality needs to be evaluated