EFECTO DE TRES DIFERENTES FRACCIONES DE PESO MOLECULARALTO DEL MEDIO CONDICIONADO POR CÉLULAS BRL EN ELDESARROLLO EMBRIONARIO Bovino In Vitro

To investigate the presence of embryotrophic factors in conditioned media from BRL cells (CMBRL), three high molecular weight fractions (>1, >5, and >10 kd) were added to TCM-199 culture media. Oocytes were obtained by cutting ovaries from cows at slaughter houses, then they were maturated under 5% CO2 at 39º C for 24 hours in TCM-199 media, supplemented with 10% of estrous cow serum. After maturation, oocytes were washed three times in TL-HEPES media, and transferred to fertilization drops. Oocytes were fertilized in 50ml drops of TL-FERT media under mineral oil, with frozen-thaw sperm, selected by Swim-up and capacitated with heparin. Eighteen to 20 hours after fertilization, zygotes were cultivated in CMBRL media alone (control) or with either >1, >5, and >10 KDa fractions. The different fractions incorporated in CMBRL increased the proportion of embryo that reach division (2-8 cells) compared to control after three culture days (61-75%). The higher proportion of divided embryos was obtained with the >10 Kda fraction. Embryo development to morula/blastocyst stage and nuclei count did not show significative differences under different culture conditions (P>0.05). The number of cells per embryo varied between 15 and 23 cells per embryo. In conclusion, these results showed that removal of low molecular weight factors from CMBRL improve embryo development to the division stage, however it is deficient to promote the morula/blastocyst stage