Identification of pronuclei inin vitrofertilized cow embryos

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Abstract
Experiments were conducted to determine the efficiency of visualizing pronuclei in living zygotes. Oocytes aspirated from ovaries of slaughtered cows were matured for 24 hr, fertilized, and examined by differential interference contrast microscopy (DICM) to identify pronuclei, then fixed, stained and examined by phase contrast microscopy (PCM) for stage of development. In the first experiment, 1028 oocytes were examined at 3‐hr intervals between 10 and 22 hr after exposure to sperm. In the second experiment, 759 oocytes were classified by type of cumulus investment (complete, incomplete, and all others, such as loose, expanded, or no investment) and examined at 13 or 19 hours. The proportion of oocytes and embryos in which two pronuclei were seen by DICM increased from 5% at 10 hr to 38% at 19 and 22 hr, with greatest increase between 10 and 13 hours. In exp. 2, oocytes with incomplete investments had the highest proportion of zygotes with two pronuclei. Over both experiments, the proportion of embryos in which one pronucleus was seen by DICM ranged from 14 to 18%. Examination of stained oocytes and embryos indicated that, over both experiments, the fertilization rate was 68%, and the proportion of fertilized oocytes with 0, 1 or 2 pronuclei was 16%, 11% and 57%; 15% of fertilized oocytes were polyspermic. The proportion of embryos with two pronuclei increased over time. Examination of stained ova agreed with DICM of live ova in assessing the number of pronuclei in 67% of oocytes and embryos. Evaluation of ova by PCM, which was considered to provide the more accurate assessment of the state of embryonic development, agreed most frequently with DICM evaluation in embryos with two pronuclei (85%) and ova with no pronuclei (70%) and least frequently in ova with one pronucleus (30%). Of ova in which DICM and PCM disagreed, 55% of those in which no pronucleus was found by DICM showed two after being stained, and 73% of those in which one pronucleus was found by DICM showed two after being stained. Results suggest that all embryos in which at least one pronucleus is seen by DICM will often contain two pronuclei and should be treated accordingly.