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

The research aimed to evaluate the survivability and DNA fragmentation of horse embryos stored in different solutions at a temperature of 5 degrees Celsius. The study found that while cell death increased over time, this did not appear to be due to induced apoptosis. Additionally, staining with a compound called DAPI was found to be an effective method of assessing embryo viability.

Study Design and Methodology

• The primary goals of this study were to examine the proportion of dead cells (referred to as viability) and the occurrence of DNA fragmentation in horse embryos stored in three distinct media at 5 degrees Celsius for time durations of 6 and 24 hours.
• A total of 40 embryos were used in the study. They were preserved in Emcare Holding Solution for 6 and 24 hours, in Hams’F10, or Vigro Holding Plus for 24 hours with 9-10 embryos per group. An additional set of 10 embryos were assessed shortly after collection as a sort of ‘control group’.

Assessment Procedures

• Firstly, the embryos were stained right after collection or after being stored. This staining was conducted for the detection of dead cells using a compound called DAPI.
• Next, the DAPI-stained embryos were fixed and then stained once more to detect DNA fragmentation. This was accomplished through a process known as TUNEL.
• Lastly, all of the fixed embryos were re-stained with DAPI in order to calculate the total cell number.
• The researchers determined the percentage of cells stained with both TUNEL and DAPI, TUNEL-only, or DAPI-only.

Findings and Conclusions

• The results indicated that the percentage of dead (or DAPI-labelled) cells per embryo increased as the duration of storage extended. However, there were no detectable differences between the various storage media used.
• The percentage of early apoptotic cells (labelled as TUNEL+/DAPI-) in both fresh as well as those embryos stored for either 6 or 24 hours did not show significant differences.
• A strong correlation was observed between the percentages of cells labelled by TUNEL and DAPI, demonstrating the consistency in both measurements of cell death.
• The findings imply that while cooled storage raises the rate of cell death, this does not seem to directly result from induced apoptosis – a process where cells naturally die as part of their lifecycle.
• DAPI staining was found to be a robust and quick method for evaluating the viability of the embryos, making it a useful tool for similar future studies.