Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 °C

Abstract

The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 °C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution ® for 6 and 24 h, in Hams’F10 or Vigro Holding Plus ® for 24 h at 5 °C ( n=9–10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fragmentation (TUNEL). Finally, all the fixed embryos were re-stained with DAPI to determine the total number of cells. The percentage of cells stained with both TUNEL and DAPI or TUNEL-only or DAPI-only were determined. The percent of dead cells (DAPI-labelled) per embryo increased with duration of storage, but no differences were detected between the storage media. The percentage of early apoptotic cells (TUNEL+/DAPI−) in fresh and stored embryo for 6 h or 24 h did not differ significantly ( P>0.05). There was a significant correlation between the percentage of cells labelled by TUNEL and DAPI ( R=0.87) ( P<0.001). These results suggest that cooled storage increases cell death but this does not appear to occur by induction of apoptosis and that DAPI staining proves to be a quick and reliable method for assessing embryo viability.