Search

Equine embryos were collected at exactly 156 +/- 0.5 (n=8) and 168 +/- 0.5 h (n=11) after ovulation. The embryos were fixed in glutaraldehyde, sectioned serially and observed using light microscopy. In the 156 h group, all embryos were early blastocysts except for one, which was a morula. The morula and one early blastocyst had no capsule. The capsules of the other embryos were thin. The mean +/- SD total number of cells was 275 +/- 105 (range 117-417). The mean +/- SD proportions of mitotic and pycnotic cells were 2.5 +/- 1.2 and 1.1 +/- 1.8%, respectively, and there were no differences between each inner cell mass (ICM) and trophoblast. The mean +/- SD proportion of ICM cells was 36.5 +/- 5.2%. In the 168 h group, there were early, mid- and expanded blastocysts, all of which had a capsule. The mean +/- SD total number of cells was 1093 +/- 666 (range 272-2217). The mean +/- SD proportions of mitotic and pycnotic cells were 3.5 +/- 1.4% and 0.1 +/- 0.03%, respectively, and there were no differences between each ICM and trophoblast. The mean +/- SD proportion of ICM cells was 21.1 +/- 9.7%. In the 168 h group, there was a significant correlation (P < 0.01) between the total number of cells and the diameters and proportions of ICM cells. The 168 h embryos were composed of significantly (P < 0.01) more cells (approximately four times) than were the 156 h embryos but had lower proportions of ICM cells. These results indicate that in equine embryos there is: (i) an individual (perhaps seasonal) variability in the rate of development; (ii) a doubling in the number of cells every 6 h between 156 h and 168 h after ovulation; and (iii) a decrease in the proportion of ICM cells between the early and expanded blastocyst stages of development.

Early (day 6) equine embryos (n=23) were assigned to four treatment groups to assess the cryoprotectant properties of glycerol and ethylene glycol and the effect of adding sucrose during removal of the cryoprotectant: (i) group GG (n=5) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as the cryoprotectant, which was added at 22 degrees C in four steps (0.375, 0.75, 1.125 and 1.5 mol glycerol l(-1)), and removed after thawing in five steps (1.5, 1.125, 0.75, 0.375 and 0.0 mol glycerol l(-1)); (ii) group GS (n=6) embryos were frozen and thawed using 1.5 mol glycerol l(-1) as for group GG, except that 0.25 mol sucrose l(-1) was added during removal of the glycerol; (iii) group EE (n = 6) embryos were frozen and thawed using 1.5 mol ethylene glycol l(-1) as the cryoprotectant, which was added in three steps (0.5, 1.0 and 1.5 mol ethylene glycol l(-1)) and removed after thawing in four steps (1.5, 1.0, 0.5, 0.0 mol ethylene glycol l(-1)); and (iv) group ES (n = 6) embryos were frozen and thawed using 1.5 mol ethylene glycol l(-1) as for group EE, except that 0.25 mol sucrose l(-1) was added during removal of the ethylene glycol. After thawing, the embryos were incubated at 37 degrees C in Ham's F10 medium supplemented with 10% (v/v) fetal calf serum and antibiotics, in 5% CO2 in air for 6 h. The embryos were fixed in glutaraldehyde, serially sectioned and observed using light microscopy. None of the frozen-thawed embryos treated with ethylene glycol (groups EE and ES) had any viable cells. There were no lysed cells in the frozen-thawed embryos treated with glycerol (groups GG and GS) and the proportion of cells with pyknotic nuclei was low (group GG = 1.1 +/- 0.8% and group GS = 2.5 +/- 1.5%). There were no differences between embryos treated with cyroprotectant diluted with or without sucrose. The embryos were morulae or early blastocysts and either did not have a capsule or had a very thin capsule. The results of the present study confirm that ethylene glycol is a poor cryoprotectant for early equine embryos and that the use of sucrose during dilution of the cryoprotectant after thawing does not improve the morphology of the embryos. The results of this study also indicate that glycerol is an effective cryoprotectant for freezing equine embryos with intact zonae pellucidae and with either very thin or no capsules.

A culture for equine oviduct epithelial cells is described. Primary cultures reached confluence in 5-8 days, forming a monolayer of polygonal cells and remaining morphologically intact for about 20 days. Subcultures were obtained by collecting cells detached spontaneously from the monolayers, and confluence was reached again after 5-7 days. Cells frozen before primary culture were confluent 10-15 days after thawing. Dishes containing confluent cells also were frozen, and some cohesive monolayers formed after thawing. Equine embryos, collected 2 days after ovulation, were cultured alone or with a monolayer of equine oviduct epithelial cells. Of 5 embryos cultured alone, 3 contained 12-20 cells, 1 was at the morula stage and 1 reached the blastocyst stage after 4 days. Of 5 embryos co-cultured with oviduct cells, 2 contained 12-16 cells, 1 was at the morula stage and 2 reached the blastocyst stage after 4 days. After 2 more days, the blastocysts showed only delayed development; there was no capsule, and limited increase in size. Equine embryos can develop in vitro from 4-8 cells to the blastocyst stage, in co-culture with equine oviductal monolayers and also without cellular support. The number of embryos studied was too small for us to draw conclusions about the benefits of co-culture.