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Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups. The loss of cells attributed to bisection was 25–30% in category 1 demi-embryos, and increased to 65% in category 3 demi-embryos. Demi-embryos in category 1 derived from early blastocysts and blastocysts experienced no loss of inner cell mass cells. It is concluded that (i) bisection of pig embryos can lead to a substantial proportion of demi-embryos lacking a functional inner cell mass, (ii) the proportion of total cells to inner cell mass cells is similar in demi-embryos and intact embryos, and (iii) the percentage of cell losses attributed to bisection increases with decreasing quality of demi-embryos. Collectively, these results indicate that the blastocyst is the optimal stage to obtain a maximum yield to viable pig demi-embryos.

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