Quantitative analysis of morphological modifications of day 6.5 horse embryos after cryopreservation: differential effects on inner cell mass and trophoblast cells.

This research examines the changes in cell structure in early-stage horse embryos, when exposed to cryopreservation processes. Significant differences in the proportion of dying cells were found between treated and non-treated embryos, with the inner cell mass cells of the embryo most affected by the treatments.

Objective and Method

• The researchers aimed to assess cellular modifications in day 6.5 horse embryos post cryopreservation, by examining the differential effects of the process on the inner cell mass and trophoblastic cells. These embryos were obtained non-surgically from Welsh pony mares following induced ovulation.
• The study involved 16 embryos that were all treated differently: Some underwent freezing and thawing using glycerol as a cryoprotectant, some were only exposed to glycerol treatment, and others were cleansed in phosphate-buffered saline (PBS) the same number of times as those receiving the glycerol treatment.
• Post treatment, the embryos were kept in Minimum Essential Medium (MEM), enhanced with bovine serum albumin (BSA), antibiotics, glutamine, and buffered with Hepes. Depending on the group, they were incubated for either 1 hour or 6 hours.
• Embryos were then sectioned serially post histological fixation.

Observation and Results

• Observations were made and numerical values obtained on the total count of interphasic, mitotic (cell division) and pycnotic (cell death) nuclei in each embryo. This observation was aided by deploying light microscopy techniques.
• Additionally, electron microscopy was utilized to evaluate possible damage to intracellular organelles’ fine structure.
• Results showed that the proportion of mitotic cells was almost consistent across all groups. However, notable differences were noted in the number of pycnotic cells between the control group and the treated groups.
• More specifically, there was a significant increase in the proportion of pycnotic cells in glycerol-treated (39.4% +/- 15.9%) and frozen-thawed embryos (42.2% +/- 14.9%) compared to the control group (12.8% +/- 5.6).

Conclusion

• The researchers found that the glycerol treatment and the freeze-thaw process induced cell death in day 6.5 horse embryos.
• The study also revealed that the impact of these treatments wasn’t uniform across the embryo. Notably, the inner cell mass (ICM) cells, a crucial part of the pre-embryo that forms the fetus, were most affected.