QUANTITATIVE-ANALYSIS OF MORPHOLOGICAL MODIFICATIONS OF DAY 6.5 HORSEEMBRYOS AFTER CRYOPRESERVATION - DIFFERENTIAL-EFFECTS ON INNER CELL MASS AND TROPHOBLAST CELLS

Sixteen embryos were recovered nonsurgically at day 6.5 after induced ovulation from Welsh pony mares and were evaluated for cellular change s that occur because of exposure to the cryoprotectant with or without the freeze and thaw process. Day 6.5 horse embryos were either (i) fr ozen and thawed using glycerol as cryoprotectant (n = 6), (ii) given o nly the glycerol treatment (n = 5), or (iii) washed in phosphate-buffe red saline (PBS) the same number of times as in the glycerol treatment (n = 5). After treatments, embryos were incubated in Minimum Essentia l Medium (MEM), supplemented with BSA, glutamine, antibiotics and buff ered 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 micr oscopy was used to evaluate the damage to the fine structure of intrac ellular organelles. The proportion of mitotic cells did not differ amo ng groups (control: 2.3%; glycerol-treated: 1.8%; frozen-thawed: 1.3%) . There were significant differences in the proportion of pycnotic cel ls both between control (12.8% +/- 5.6) and glycerol-treated embryos ( 39.4% +/- 15.9) (P < 0.05) and between control and frozen-thawed embry os (42.2% +/- 14.9) (P < 0.001), but no difference was found between t reated embryos (glycerol-treated and frozen-thawed embryos). Degenerat ed cells were not localized in the same place in each embryo and no ul trastructural alteration was uniformly observed among every embryo of each group, but inner cell mass (ICM) cells were affected most by trea tments (P < 0.001). These results indicate that cryopreservation induc es considerable cellular damage. The deleterious effects of glycerol a lone appear very important and further research is needed to find a su itable cryoprotectant for the horse embryo.