Assay of embryo-derived platelet activating factor (EDPAF) by an equine platelet aggregation assay: Preliminary data concerning its presence in bovine embryo culture media

Abstract

This study was designed to establish a sensitive bioassay for bovine platelet-activating factor (PAF), to determine if the bovine embryo secretes PAF in vitro and if PAF release is correlated with the embryo's potential to establish a pregnancy. Using an equine platelet aggregation assay, lipid extracted culture media from 33 Day-7 embryos (individually cultured for 18 hours in 1 ml of Ham's F10 containing 0.4% BSA at 37°C in an air: CO 2 mixture of 95:5 prior to their transfer to recipient heifers) and from control media (n=15, Ham's F10 + 0.4% BSA incubated simultaneously without embryos) were investigated. In addition, culture media from Day-6 (n=6) and Day-1 (2-cell, n=12) bovine embryos that were cultured for 4 hours but not transferred were examined. The aggregation assay proved to be sensitive to 5 pg of PAF. The assay proved to be specific, since the PAF receptor antagonist SRI 63–441 inhibited platelet aggregation induced by culture media in dosages comparable to aggregation induced by synthetic PAF 18. From the 15 Day-7 embryos that established a pregnancy 2 contained measurable amounts of PAF in their culture media. No PAF was detected in the culture media from 13 embryos that succeeded, in the 18 embryos that failed to establish a pregnancy, or in the control media. One of 6 Day-6 embryos and 3 of 12 Day-1 (2-cell) embryos secreted detectable amounts of PAF into the culture media. Although the results indicate that some bovine embryos release PAF or a PAF-like substance in vitro, PAF measurements in the culture medium seem not to be a suitable method for the evaluation of bovine embryos prior to transfer.