title: Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 degrees 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 degrees 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 degrees 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.
authors.affiliation: Physiology of Reproduction and Behavior, INRA, Nouzilly, France. moussa@tours.inra.fr
authors.forename: M
authors.initials: M
authors.lastname: Moussa
authors.forename: J L
authors.initials: JL
authors.lastname: Tremoleda
authors.forename: G
authors.initials: G
authors.lastname: Duchamp
authors.forename: J F
authors.initials: JF
authors.lastname: Bruyas
authors.forename: B
authors.initials: B
authors.lastname: Colenbrander
authors.forename: M M
authors.initials: MM
authors.lastname: Bevers
authors.forename: P F
authors.initials: PF
authors.lastname: Daels
mesh_terms: D000818:Animals; D017209:Apoptosis; D003080:Cold Temperature; D053938:DNA Fragmentation; D004622:Embryo, Mammalian / Q000166:cytology* / Q000502:physiology; D005260:Female; D005456:Fluorescent Dyes; D006736:Horses / Q000196:embryology*; D020287:In Situ Nick-End Labeling; D007211:Indoles; D007315:Insemination, Artificial / Q000662:veterinary; D010062:Ovulation Induction / Q000662:veterinary; D013997:Time Factors; D014021:Tissue Preservation / Q000662:veterinary; D020858:Tissue and Organ Harvesting / Q000662:veterinary
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI