Estimation of oestrogenic activity in red clover (Trifolium pratense) using ovariectomized immature mice

Abstract

TWO experiments concerned with the testing of plant material for oestrogenic activity, utilizing vaginal reactions and uterine weight changes in immature ovariectomized mice are reported.In the first experiment the effects of varying the feeding period of an oestrogenic diet, containing 20 % of freeze-dried red clover, and of varying the interval between the last feeding of this diet and slaughter were investigated. In general, uterine weight increased linearly with increased period of feeding and decreased linearly with delay in slaughtering. A feeding period of 4 days was satisfactory for comparisons based on uterine weight. If vaginal reactions were also to be utilized a feeding period of not less than 6 days was desirable.In the second experiment the dose-response relationships of diethylstilboestrol, genistein and oestrogenic red clover were investigated. All materials were incorporated in the diet of mice, and the level of plant material was kept at 20 % of the mixed feed by adding inactive plant material where necessary. In general, either diethylstilboestrol or genistein could form a satisfactory standard for biological estimation of oestrogenic activity in red clover. Relative potencies based on uterine weight response and occurrence of vaginal opening and oestrus-type vaginal smears are given.The biological estimation of oestrogenic activity in plant material involves a number of problems. Among the most important of these are: the choice of an appropriate response in a given test animal, a suitable duration of treatment and a satisfactory reference standard. Biggers (1959), who has discussed the first of these problems, concluded that in mice vaginal cornification was more suitable than increase in uterine weight, because the former, unlike the latter, was a specific reaction to oestrogens. On the other hand, the uterine weight response has the advantage that it provides quantitative data. Flux, Munford & Barclay (1961) decided that, for initial screening purposes, uterine weight increase was more satisfactory because it was likely to show a response with low doses of oestrogens. Uterine weight increase has been commonly used to detect oestrogenic activity in plant materials (Cheng,

Bioassay of Oestrogen Using Sheep

“…Because of the uncertainty of the nature and actions of the oestrogenic substances, qualitative as well as quantitative assay methods must be developed to a high degree of reliability. To date these methods have been investigated in laboratory animals such as the guinea pig (Alexander and Watson 1951) and mouse (Robinson 1949;Biggers and Curnow 1954;Kitts et al 1959;Mumford and Flux 1961). The ewe has been used occasionally for qualitative assay (Sanger, Engle, and Bell 1958;Jennings and Dow 1959), but has not received as much attention as may be warranted, particularly in view of the fact that ingestion of pastures by the sheep is a mode of administration of which little is known.…”