Quantitation of rat embryonic development in vitro: a morphological scoring system

Abstract

In order to realize the maximum potential of whole-embryo culture techniques, definitive parameters must be developed for the quantitative estimation of embryonic growth and development. An objective scoring system has been devised which provides a precise measure of morphological development. Examination of rat conceptuses from dams on pregnancy days 10, 11, 12, and 13 (sperm positive = day 1) led to the selection of 17 morphological features for use in the system. Up to six developmental stages of each feature were defined and assigned scores of 0 to 5. The numerical total of scores for an individual embryo is taken as the overall morphological score (MS). The system was applied prospectively to 103 Sprague-Dawley rat conceptuses aged 9 to 12.7 days (0 = time of copulation). The variation of score with embryonic age (EA, days) was highly linear: MS = 202.28 + 20.932 EA (R2 = 0.991). Thus, the morphological score can be used to compute apparent embryonic age, and the calculated standard error of prediction is +/- 2.2 hrs. The number of somites (SN) was also found to vary linearly with age over this period: SN = -126.23 + 13.217 EA (R2 = 0.982), and could be used to estimate development. However, the standard error for prediction of apparent embryonic age is greater (+/- 3.1 hours) than that for morphological score, and this single feature will not necessarily reflect overall development. Several parameters were examined as estimates of embryonic growth, as distinct from development. Yolk sac diameter, crown-rump length, and head length were found to vary with EA as quadratic functions over this time. Total embryonic protein increased logarithmically with EA and was considered to be the most suitable measure of embryonic growth. Use of the morphological scoring system in embryo culture experiments provides a precise index of embryonic development, aids the detection of retardation or dysmorphogenesis of specific primordia, and allows a quantitative comparison of development and growth.