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

Abstract

Sixteen embryos were recovered nonsurgically at day 6.5 after induced ovulation from Welsh pony mares and were evaluated for cellular changes that occur because of exposure to the cryoprotectant with or without the freeze and thaw process. Day 6.5 horse embryos were either (i) frozen and thawed using glycerol as cryoprotectant (n = 6), (ii) given only the glycerol treatment (n = 5), or (iii) washed in phosphate-buffered saline (PBS) the same number of times as in the glycerol treatment (n = 5). After treatments, embryos were incubated in Minimum Essential Medium (MEM), supplemented with BSA, glutamine, antibiotics and buffered with Hepes, for 1 h for one embryo per group and for 6 h for the others. After histological fixation, embryos were serially sectioned. On observation by light microscopy, the total numbers of interphasic, mitotic and pycnotic nuclei of each embryo were counted. Electron microscopy was used to evaluate the damage to the fine structure of intracellular organelles. The proportion of mitotic cells did not differ among groups (control 2.3%; glycerol-treated: 1.8%; frozen-thawed: 1.3%). There were significant differences in the proportion of pycnotic cells both between control (12.8% ± 5.6) and glycerol-treated embryos (39.4% ± 15.9) (P < 0.05) and between control and frozen-thawed embryos (42.2% ± 14.9) (P < 0.001), but no difference was found between treated embryos (glycerol-treated and frozen-thawed embryos). Degenerated cells were not localized in the same place in each embryo and no ultrastructural alteration was uniformly observed among every embryo of each group, but inner cell mass (ICM) cells were affected most by treatments (P < 0.001). These results indicate that cryopreservation induces considerable cellular damage. The deleterious effects of glycerol alone appear very important and further research is needed to find a suitable cryoprotectant for the horse embryo.