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The estrogen-like substances in various legumes and grasses, and the effect of these compounds on the… Swierstra, Ernest Emke 1958

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THE ESTROGEN-LIKE SUBSTANCES IN VARIOUS LEGUMES AND GRASSES, AND THE EFFECT OF THESE COMPOUNDS ON THE REPRODUCTION AND GROWTH OF CERTAIN LABORATORY ANIMALS by ERNEST EMKE SWIERSTRA B. S. A., The University of B r i t i s h Columbia, 1956. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the D i v i s i o n of Animal Science We accept t h i s thesis as conforming to the required standard Members of the D i v i s i o n THE UNIVERSITY OF BRITISH COLUMBIA May, 1958. THE ESTROGEN-LIKE SUBSTANCES IN VAftlOUS LEGUMES AND^GRASSES, AND THE EFFECT OF THESE COMPOUNDS ON THE REPRODUCTION AND GROWTH OF CERTAIN LABORATORY ANIMALS by ERNEST EMKE SWIESSTRA Abstract A three day mouse bio-assay procedure, using Immature female mice 20-21 days old and weighing 8-11 gm., was developed f o r the determination of the estrogen-like substances i n forages. With d i e t h y l s t i l b e s t r o l (D.E.S.) as reference compound a dose response curve was constructed. From t h i s curve the estrogenic a c t i v i t y of the samples of plant material was estimated and expressed i n terms of D.E.S. An extensive study was undertaken on the effects of stage of maturity and frequency of cutting of a l f a l f a , white clover, red clover, birdsfoot t r e f o i l and orchard grass, on the levels of estrogen-like substances i n these plant species. The data of these experiments revealed that there was a great difference i n estrogenic a c t i v i t y between samples taken from d i f f e r e n t plant species at the same time of the year. There also was a seasonal v a r i a t i o n between f i r s t cuttings of second year growth of the diff e r e n t plant species. A l f a l f a and white clover were high i n estrogenic a c t i v i t y i n the spring, showed a sharp decrease i n June and Jul y , and a f t e r August 1 possessed once again considerable potency. Birdsfoot t r e f o i l and orchard grass showed estrogenic a c t i v i t y only i n the spring, and t h i s was, r e l a t i v e l y small as compared to the a c t i v i t y of a l f a l f a , white clover and red clover. Red clover d i f f e r e d very much from the previously mentioned species. A l l samples studied showed considerable potency. There was no sharp decrease during June and July as was the case with a l f a l f a and white clover. Data obtained by' varying the number of hours of daylight (photoperiod) received by red clover seemed to indicate that estrogenic potency was decreased when the hours of daylight were reduced. Proximate analysis of the plant material revealed that estrogenic potency was not correlated with nitrogen content. Studies on the s t a b i l i t y of the estrogen-like compound i n a l f a l f a , white clover and red clover showed that the a c t i v i t y of dried ground a l f a l f a and white clover plant material was e a s i l y destroyed during storage, while the potency of dried ground red clover remained f a i r l y constant over a s i x month period. However, the estrogen-like compounds of these three species are much more stable a f t e r they had been extracted and the extract mixed with feed. Red clover and birdsfoot t r e f o i l extract interfered with the reproduction i n the mouse. Females were affected more severely than males, but both recovered t h e i r reproductive a b i l i t y quickly a f t e r feeding of the estrogen-like compounds ceased. The growth rate of the female guinea pig weighing between 500-600 gm. was not s i g n i f i c a n t l y influenced when red clover extract was added to t h e i r d i e t . - i i -I I . ABS'-. FACT A three day mouse bio-assay procedure, using immature female mice 20-21 days old and weighing 8-11 gm., was developed for the determination of the estrogen-like substances i n forages. With d i e t h y l s t i l b e s t r o l (D.E.S.) as reference compound a dose response curve was constructed. From t h i s curve the estrogenic a c t i v i t y of the samples of plant material was estimated and expressed i n terms of D.E.S. An extensive study was undertaken on the effects of stage of maturity and frequency of cutting of a l f a l f a , white clover, red clover, birdsfoot t r e f o i l and orchard grass, on the levels of estrogen-like substances i n these plant species. The data of these experiments revealed that there was a great difference i n estrogenic a c t i v i t y between samples taken from different plant species at the same time of the year. There also was a seasonal v a r i a t i o n between f i r s t cuttings of second year growth of the diff e r e n t plant species. A l f a l f a and white clover were high i n estrogenic a c t i v i t y i n the spring, showed a sharp decrease i n June and Ju l y , and af t e r August 1 possessed once again considerable potency. Birdsfoot t r e -f o i l and orchard grass showed estrogenic a c t i v i t y only i n the spring, and t h i s was r e l a t i v e l y small as compared to the a c t i v i t y of a l f a l f a , white clover and red clover. Red clover d i f f e r e d very much from the previously mentioned species. A l l samples studied showed considerable potency. There was - i i i -no sharp decrease during June and July as was the case with a l f a l f a and white clover. Data obtained by varying the number of hours of daylight (photoperiod) received by red clover seemed to indicate that estrogenic potency was decreased when the hours of daylight were reduced. Proximate analysis of the plant material revealed that estro-genic potency was not correlated with nitrogen content. Studies on the s t a b i l i t y of the estrogen-like compound i n a l f a l f a , white clover and red clover showed that the a c t i v i t y of dried ground a l f a l f a and white clover plant material was e a s i l y destroyed during storage, while the potency of dried ground red clover remained f a i r l y constant over a s i x month period. However, the estrogen-like compounds of these three species are much more stable after they had been extracted and the extract mixed with feed. Red clover and birdsfoot t r e f o i l extract interfered with the reproduction i n the mouse. Females were affected more severely than males, but both recovered t h e i r reproductive a b i l i t y quickly after feeding of the estrogen-like compounds ceased. The growth rate of the female guinea pig weighing between 500-600 gm. was not s i g n i f i c a n t l y influenced when red clover extract was added to t h e i r d i e t . In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of AfiJJ/lA L ±c/£A/ Cf The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8\ Canada. Date Zfi) APRIL /<gs~P - i -I. ACKNOWLEDGEMENT The w r i t e r wishes to take t h i s opportunity to thank Dr. B. A. Eagles, Dean of the Faculty of Agriculture and Chairman of the D i v i s i o n of Animal Science, for his permission to undertake t h i s project and for the use of the departmental f a c i l i t i e s . Sincere thanks are expressed to Dr. W. D. K i t t s , Assistant Professor i n the Division of Animal Science, for h i s d i r e c t i o n , assistance and c r i t i c i s m during the course of t h i s study. The writer also would l i k e to thank Dr. A. J . Wood for his many suggestions, and Dr. V. C. Brink and the s t a f f of the D i v i s i o n of Plant Science for t h e i r pleasant and h e l p f u l assistance i n supplying the plant materials used i n t h i s investigation. TABLE OF CONTENTS PAGE I. ACKNOWLEDGEMENT i I I . ABSTRACT i i I I I . INTRODUCTION 1 IV. HISTORICAL • 3 A. Physiological Functions of Estrogens i n the Animal Body 3 1. Control of Estrus Cycle 4 2. Control of Mammary Development 5 and Lactation 3. Estrogens i n the Milk * 6 4. Some Pathological Effects on the Female Reproductive System 6 5. Effects on the Male Reproductive System 7 6. Effects on the Skeleton 8 B. Estrogens i n Forage Plants 8 1. Subterranean Clover «... 9 2. Red Clover 11 3. A l f a l f a 11 4. White Clover 12 5. Birdsfoot T r e f o i l 12 6. Orchard grass 12 7. Other Forage Plants 12 8. Hay 13 9. Silage 13 C. Different Methods of Determining Estrogens 13 1. Chemical and Physical Methods 14 2. Bio-assay Methods 14 3. Units Used i n Expressing Estrogenic A c t i v i t y 17 D. Location, Is o l a t i o n and A c t i v i t y of the Estrogen-l i k e Substances i n Plants 19 1. Genistein 20 2. Genistin 21 3. Formononetin 21 4. Daidzein 22 5. Biochanin A 22 6. Coumestrol 23 TABLE OF CONTENTS - Continued PAGE E. Related Plant Products 23 V. MATERIALS AND METHODS A. Materials 26 1. Plant Materials 26 2. Experimental Animals 28 3. Bio-assay Control Diet 28 4. Control Guinea Pig Diet 29 5. Plant Material Used i n the Mouse Reproduction Experiment No.I 29 6. Plant M a t e r i a l i s e d i n the Guinea Pig and Mouse Reproduction Experiment No.II 30 B. Methods 1. Drying of Plant Material 30 2. Grinding of Dried Plant Material 31 3. Storage of Ground Samples 31 4. Extraction Procedure 31 5. Proximate Analysis of Samples 32 6. Housing and Care of Bio-assay Animals 32 7. Sacrifice,and Dissection of Bio-assay Mice 33 8. Housing and Feeding of the Mature Mice on Reproduction Experiments Nos. I and I I 33 9. Housing and Feeding of .Guinea Pigs 34 10. Mating Plan of Mice of Reproduction Experi-ment No. I 34 11. Mating Plan of Mice of Reproduction Experi- . ment No. I I 35 12. Mating Plan of Guinea Pigs 36 VI. RESULTS AND DISCUSSION A. Development of the Bio-assay Procedure 37 1. Determination of Feed Intake of Immature Female Mice over a Three Day Period 38 2. The Time of Feed Passage i n 21 Day Old Swiss Albino Mice 38 3. The Influence of Time of Fasting on Uterine Weight of Intact Immature Female Mice 39 4. The Length of the Bio-assay Period 41 5. The Effect of Dehydration of the U t e r i on the Accuracy of the Bio-assay 41 B. Construction of the Dose Response Curve 44 C. Plant Extract Bio-assays 45 TABLE OF CONTENTS - Continued PAGE 1. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of A l f a l f a 50 2. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of White Clover 62 3. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of Red Clover 70 4. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of Birdsfoot T r e f o i l 70 5. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of Orchard Grass 84 6. The Effect of Stage of Maturity on the Estrogenic A c t i v i t y of the Second Year Growth of Alsike Clover 84 7. The Influence of Length of Daylight on the Estrogenic A c t i v i t y of Red Clover 92 D. The S t a b i l i t y of the Estrogen-like Substances of Plant Origin .. 102 E. The Effect of Plant Estrogens on Certain Laboratory Animals 109 1. Mouse Reproduction Experiment No. I .110 2. Mouse Reproduction Experiment No. I I 114 3. Guinea Pig Experiment 115 VII. SUMMARY AND CONCLUSIONS 122 VI I I . BIBLIOGRAPHY 128 - 1 -I I I . INTRODUCTION The term estrogen has been defined as any pure chemical substance which has the a b i l i t y when injected into an adult ovariec-tomized mouse or r a t , to produce c o r n i f i c a t i o n of the vagina si m i l a r to that occurring during normal estrus. This d e f i n i t i o n has further been extended to apply i n a c o l l e c t i v e sense to mean an estrogenic substance of unspecified chemical i d e n t i t y . In 1940 Emmens (56, 57) c l a s s i f i e d the estrogens into two main groups. 1 - the true estrogens, as those compounds capable of i n -ducing the characteristic response r e l a t i v e to the A l l e n - Doisy test (3, 4) and, 2 - the pro-estrogens, as those compounds when applied to the vagina do not give the char a c t e r i s t i c response u n t i l they have been' absorbed into the c i r c u l a t o r y system and returned to the vagina. The presence of estrogen-like compounds i n plant material capable of inducing estrus i n animals was f i r s t reported i n 1926 by Loewe (85). Since that time approximately 50 plant species have been shown to possess these active compounds. The amounts and a c t i v i t y of the substances i n the d i f f e r e n t species vary considerably. In 1946 Bennetts et al.(18) observed that subterranean clover (Trifolium subterranean) of Western Au s t r a l i a contained suf-f i c i e n t amounts of estrogen-like substances to affect adversely the reproductive performance of gjtazing ewes and to cause g e n i t a l and mammary abnormalities i n wethers. Many other experiments have been - 2 -reported on the effect of natural and synthetic substances having estrogenic a c t i v i t y i n livestock production. These findings have led the author to investigate the levels of estrogenic compounds i n a l f a l f a (Medicago sativa'), a l s i k e clover (Trif o l i u m hybridum), red clover (Trifolium pratense), white clover (Trifolium repens), b i r d s -foot t r e f o i l (Lotus corniculatus), and orchard grass (Dactylis  glomerata), at di f f e r e n t stages of maturity,- and to study the extent to which these levels are influenced by frequency of cutting and length of daylight. A number of animal experiments were carried out to determine the influence of the estrogen-like substances of red clover and birdsfoot t r e f o i l on the growth and reproduction of the mouse and the guinea p i g . I t seemed advisable to use laboratory animals since more fundamental knowledge was necessary before an experiment with domestic animals could be designed i n order to obtain valuable r e s u l t s . - 3 -IV. HISTORICAL The early work of Loewe (85) i n 1926, where he reported that ether extract of willow catkins and ovaries of the water l i l y -(Nuphare luteum) induced estrus i n ovariectomized mice, has led to extensive investigations of estrogenic substances i n plant and animal materials. Positive r e s u l t s were obtained with a group of plant materials including beet seeds, potatoes (49), cooked potatoes (72), male and female alder catkins, alder leaves, sprouted oats, rhubarb leaves (109) and soybean meal (111). In 1941 Bennetts, (17) reported the sudden appearance of a serious breeding disorder i n sheep i n large regions of Western A u s t r a l i a . The flocks were grazing on pas-tures which were dominated by a l o c a l l y developed s t r a i n of subter-ranean clover (Trifolium subterranean L. var. dwalganup). Since the economy of the country depended to a large extent on the sheep industry, intensive investigations were carried out with sheep grazing on these "affected" clover pastures. In 1946 strong evidence was presented that the breeding disorder was due to an exces-sive stimulation of the reproductive organs of the animals by estrogens derived from the pasture (19). This led to subsequent research programs i n connection with these active compounds i n d i f f e r e n t species of plants. A. Physiological Functions of Estrogens i n the Animal Body; Since the plant estrogens appear to have sim i l a r physiologi-c a l properties as the natural estrogens i n the animal body, i t was f e l t that a short discussion of t h e i r functions would be appropriate. - 4 -A more extensive review on t h i s subject has been published by-Burrows (32) while the part played by estrogens i n l a c t a t i o n i s re-viewed by Folley (70). In . ivertebrates, estrogens,take part i n the control of the estrus cycle as the name suggests. Furthermore, the growth and functions of the female accessory reproductive organs, the secondary sex characteristics and the female mating behavior are controlled by these hormones. I t i s interesting t o note that i n the female mouse a l l tissues and organs with the exception of the brain and striped muscle respond to estrogens by c e l l d i v i s i o n . However, the i n t e n s i t y of the reaction depends on the organ concerned (31). 1. Control of Estrus Cycle. In mammals the anterior p i t u i t a r y secretes three hormones that d i r e c t l y influence reproduction. They are the f o l l i c l e - stimu-l a t i n g hormone, l u t e i n i z i n g hormone and pro l a c t i n or luteotrophin. The f o l l i c l e stimulating hormone stimulates the growth of the ovisacs to the point of rupture, while the actual rupturing of the ovisacs i s aided by the l u t e i n i z i n g hormone. After rupturing,the l u t e i n i z i n g hormone influences the formation of the corpus luteum or yellow body. The t h i r d hormone - p r o l a c t i n - causes the corpus luteum to secrete progesterone which i s essential f o r implantation of the f e r t i l i z e d egg within the placental t i s s u e s . The theca interna of the ovisacs secrete three estrogens, e s t r a d i o l , estrone and e s t r i o l . The most active of these three hormones i s e s t r a d i o l and the least a c t i v e , e s t r i o l (89, 6). - 5 -2 Control of Mammary Development and Lactation: Mammary development i s mainly i f not completely, controlled by the endocrine system. At least three hormones are concerned i n the growth and functioning of the mammary gland (86). Each one has i t s own function and they act i n a sp e c i a l sequence. E s t r a d i o l , secreted by the Graafian f o l l i c l e acts i n i t i a l l y and causes duct development i n the mammary gland. This i s followed by growth of the a l v e o l i under influence of progesterone from the corpus luteum. The t h i r d hormone, prolactin from the p i t u i t a r y , i n i t i a t e s milk secretion. The r e l a t i v e amount of these three hormones may d i f f e r between species and perhaps also i n t h e i r mode of action (86). A r t i f i c i a l mammary development and copious l a c t a t i o n has been induced i n intact and ovariectomized v i r g i n goats by use of estrogens and mixtures of estrogens and progesterone. Microscopic examination of the mammary glands of these goats revealed that less abnormalities were present i n animals receiving both estrogens and progesterone than i n those receiving estrogens alone. Administration of synthetic estrogens t o milking cows has produced controversial r e s u l t s . F o l l e y (66) and Folley et a l . (68) studied the influence of synthetic estrone, d i h y d r o - f o l l i c u l a r hormone and d i e t h y l s t i l b e s t r o l (D.E.S.) i n l a c t a t i n g cows. In some cases there was an increase i n milk s o l i d s , which was not due to a secretion of less water by the mammary gland. The nitrogen p a r t i t i o n i n these cows with increased milk production was normal indicating that the experimental treatment had not caused the secretion of colostrum. This galactopoietic effect was confirmed by Spielman et a l . (101) who showed that estrogens increased the lactose content of the milk. In other cases estrogens caused a considerable - 6 -i n h i b i t i o n of l a c t a t i o n i n the sense of a decreased milk yield(66). Hawkins et a l . (78) fed levels of D.E.S. to l a c t a t i n g cows within the range of the levels of estrogen-like compounds found i n forages (0.068-3.40 mcgm. per l b . of feed) and observed no s i g n i f i c a n t effects on the production of fat corrected milk, the gross e f f i c i e n c y of milk production, changes i n body weight and ef f i c i e n c y of feed u t i l i z a t i o n . Folley et a l . (68) also noticed that synthetic estrogen administration resulted often i n d i f f i c u l t i e s i n conception, while i n advanced pregnancy large doses of D. E. S. resulted i n abortion. 3. Estrogens i n the Milk In normal cows and goats small amounts of estrogens occur i n the milk during the l a s t month of pregnancy (87), immediately after p a r t u r i t i o n (67, 108), and during estrus (87). Human milk taken during the f i r s t 9 days of l a c t a t i o n was shown t o induce estrus i n ovariectomized mice (30), suggesting that estrogens were present. This was also recognized by Myers (88) who noticed that small amounts of milk were present i n the mammary glands of a young rat 12 hours after b i r t h suggesting that minimal amounts of hormones necessary f o r mammary secretion passed t o the suckling young from the mother. When D..E.S. became available f o r experimental use i t was found that most of the administered hormone was excreted i n the urine and that only very small amounts occurred i n the milk (74, 81, 104). 4. Some Pathological Effects on the Female Reproductive System Administration of estrogens to animals leads to changes i n - 7 -the reproductive system. The severity of these changes depends on sex, age, species, kind of hormone used, the method and duration o f administration. Eromens (59) injected male and female mice with e s t r a d i o l benzoate and rendered the males s t e r i l e within s i x weeks i n 90 per cent of the cases by in j e c t i n g 10 mcgm. of the compound twice weekly. The females were more susceptible and became s t e r i l e when injected with 0.5 mcgm. twice weekly. Both groups recovered normal or nearly normal reproductive power when injections of the hormone ceased. I n f e r t i l i t y i n mice could also be produced by administering t h i o u r a c i l but again the animals recovered quickly after the treatment was stopped (9). H i s t o l o g i c a l examination revealed that estrogen administration resulted f i r s t i n p r o l i f e r a t i o n of the epithelium followed by extension of the uterine glands which penetrated into the submucosa and became distended with f l u i d (32). In immature mice estrogen administration also resulted i n an e a r l i e r opening of the vaginal t r a c t . Different species react d i f f e r e n t l y to estrogens. Injections of two to f i v e mcgm. of estrone per day f o r 8 to 15 days i n spayed guinea pigs and r a t s resulted i n the development of cystic glandular hyperplasia i n the guinea pig uterus, while the r a t s were affected to a much lesser extent (113). Simultaneous administration of estrogens and androgens causes synergystic and antagonistic reactions depending on the proportions i n which the hormone was supplied (32). 5. Effects on the Male Reproductive System. I t would be expected that males are more resistant to exo-genous estrogens, since the androgen complement of the male exerts some - 8 -protective action. Results confirming t h i s hypothesis were obtained by Emmens (59) with e s t r a d i o l benzoate injections i n mice, by East et a l . (52) with entire and castrated guinea pigs receiving subterranean clover and by Bennetts (13) with intact rams grazing on subterranean clover. However, East (54) showed that the "plant estrogen", genistein, affected the male mice to a greater extent than the female. Certain parts of the reproductive system are more susceptible to estrogens than others. In the guinea p i g , for example, the seminal vesicles are more susceptible than the other accessory sex organs (52). Prolonged estrogen treatment of the male mouse results i n metaplastic changes i n uterus masculinus, seminal v e s i c l e s , vas deferens, prostate, bulbo-urethral glands and urethra. 6. Effects on the Skeleton. In mammals estrogens aid i n the development of the female pelvis and relaxation of the ligaments during pregnancy. Implan-t a t i o n of synthetic estrogens into heifers or cows resulted i n loosening of the sacro-sciatic and s a c r o - i l i a c ligaments (42, 69). Furthermore, estrogen administration causes a f a l l i n serum calcium followed by transient increase i n serum phosphatase, in d i c a t i n g that the hormones are capable of exercising general effects on the s k e l e t a l system (66). B. Estrogens i n Forage Plants: Recently Bradbury and White (29) reviewed the complete subject of estrogens and related substances i n plants. Since t h i s and other reviews are a v a i l a b l e i t i s not proposed to give here a complete review of the l i t e r a t u r e , but t o point out some of the most in t e r e s t i n g facts r e l a t i n g to certain forages that have been found to possess - 9 -estrogenic a c t i v i t y . 1. Subterranean Clover (Trifol i u m subterranean). In 1941 Bennetts (17) reported the sudden appearance of a serious breeding disorder i n sheep i n large areas of Western A u s t r a l i a . The affected flocks were grazing on pastures dominated by the Dwalganup s t r a i n of subterranean clover. Three main c l i n i c a l symptoms of sheep grazing on t h i s clover were observed; dystocia, uterine prolapse, and female i n f e r t i l i t y . The i n f e r t i l i t y had no char a c t e r i s t i c c l i n i c a l features; there was no f a i l u r e of estrus. In many cases i n f e r t i l e sheep were served by the male, but did not conceive (10b). When the animals were s a c r i f i c e d post coitus unsegmented ova were recovered i n the f a l l o p i a n tubes or uterus, indicating that i n f e r t i l i t y was a result of f a i l u r e of f e r t i l i z a t i o n . Frequently, ewes grazing on subterranean clover showed cystic glandular hyperplasia of the endometrium, and i n many cases a number of cysts dist r i b u t e d throughout the fundus and horns of the uterus could be located by macroscopical examination (107). These </ cysts contained a colorless,serous f l u i d and varied i n size from 0.1 to 1.4 cm. i n diameter. Similar changes i n the endometrium of guinea pigs were produced when these animals were fed fresh, a i r dried or a r t i f i c i a l l y dried subterranean clover (18). When ovariectomized ewes were grazed on f i e l d s of subter-ranean clover atrophy of the uterus did not occur (22). The same happened when ovariectomized ewes were treated with adequate estrogens, the uterus was restored almost to i t s normal state; thus the estrogen-l i k e compounds i n subterranean clover function as natural estrogens i n preventing atrophy of the uterus of ovariectomized ewes (35). However, - 10 -when the clover reached the dry stage, atrophy of the u t e r i occurred. This meant that the forage had no longer any estrogenic a c t i v i t y (22). A high incidence of milk secretion i n v i r g i n ewes p a r t i c u l a r l y during the spring and early summer occurred. Microscopy showed normal alveolar development of the udder,and the gland yielded a secretion sim i l a r to colostrum (18). Bennetts et a l . (18) noted marked mammary development i n castrated rams when grazed on certain f i e l d s of sub-terranean clover. In some cases the nipples developed to the same size as those of ewes i n f u l l l a c t a t i o n . A f l u i d s i m i l a r to sheep's milk was obtained from these extended teats. During the same period Bennetts (19, 20) demonstrated the presence of squamous metaplasia i n the sex glands of wethers grazing on subterranean clover. In most cases there was an enlarged bulbo-urethral gland and i n some cases metaplasia was also observed i n the prostate gland and uterus masculinus, but i n these l a s t two cases to a r e l a t i v e l y small extent. In a few wethers a sac had developed on the dorsal w a l l of the pelvic urethra and was f i l l e d with urine. External rupture of the sac occurred sometimes and a permanent f i s t u l a was formed. Rams grazing on subterranean clover were unaffected and remained f e r t i l e (18, 52). Strong presumptive evidence was given by Bennetts et a l . (18) that there was an estrogen or a pro-estrogen present i n subterranean clover. Curnow et a l . (45) confirmed these findings and showed that a r t i f i c i a l l y dried clover and the ether extracts of these samples when given per os produced changes i n ovariectomized mice s i m i l a r to the changes that occurred when the natural estrogen, e s t r a d i o l , was fed. These physiological changes were: - 11 -a) early vaginal opening i n immature mice. b) vaginal c o r n i f i c a t i o n i n mice. c) uterine hypertrophy i n mice and guinea pigs. d) c y s t i c endometrium i n guinea pigs when administered over longer periods. Further studies showed that estrogens were not only present i n the Dwalganup s t r a i n of subterranean clover (97, 13) but also i n the Mount Baker, the Burnerany (46), the Red Leaf (46), the Tallarook (46), and at least f i f t e e n other strains (21). Bradbury and White (28) showed i n 1951 that the estrogenic a c t i v i t y of subterranean clover was mainly due to genistein ( 5 s 7: 4' trihydroxyisoflavone) and f o r lesser part to formononetin (7-hydroxy-4' methoxyisoflavone). The estrogenic a c t i v i t y of genistein has been confirmed by Carter et a l . (34). 2. Red Clover (Trifolium pratense L.) Red clover has been shown, by bio-assay procedures, to possess estrogen-like substances (10, 82, 48, 93, 94, 92). However, the l e v e l s of these substances reported i n the l i t e r a t u r e vary con-siderably. To date,three compounds showing estrogenic a c t i v i t y have been iso l a t e d from red clover. In 1953, Pope et a l . (93) i s o l a t e d the i s o -flavone, biochanin A, and i n 1954 the isoflavone, genistein (94). The t h i r d isoflavone, formononetin, having very l i t t l e estrogenic a c t i v i t y was i s o l a t e d i n 1953 by Bate-Smith et a l . (12). 3. A l f a l f a (Medicago s a t i v a ) . There are c o n f l i c t i n g reports i n the l i t e r a t u r e i n connection - 12 -with the estrogen-like substances i n a l f a l f a . Dohan et a l . (48) and Legg et a l . (82) reported negative r e s u l t s , while other (91, 92, 24) demonstrated d e f i n i t e estrogenic a c t i v i t y . In 1957 Bickoff et a l . (24) i s o l a t e d a new estrogen-like substance, coumestrol, from a l f a l f a and showed that i t was ten times more active than genistein. 4. White Clover (Trifolium repens). Many reports (97, 82, 38) state that white clover does not contain any estrogen-like substances, while others (91) state the opposite. Curnow (46) studied white clover f o r the presence of genis-t e i n with s i l i c a g e l chromatography and spectophotometry and was unable to detect the presence of t h i s active compound. 5. Birdsfoot T r e f o i l (Lotus corniculatus). Very l i t t l e l i t e r a t u r e i s available i n connection with estrogen-like substances i n t h i s species. However, a few reports (92, 6l) state that estrogenic a c t i v i t y has been demonstrated i n cer-t a i n samples by the mouse uterine weight technique. 6. Orchard Grass (Dactylis glomerata). Estrogenic a c t i v i t y of orchard grass has been demonstrated i n very few samples (82). In most cases (82, 48) no estrogen-like com-pounds could be detected. 7. Other Forage Plants. Other forage plants which have been reported to contain estrogen-like compounds include - strawberry clover ( T r i f o l i u m  frageferum) (97, 24), ladino clover (Trifolium repens, Ladino) (92, 61, 24), rye grass (Lolium perenne L.) (82, 48), oats (Aoena sativa L.) (77, 92), and wheat (Triticum aestioum) (92). - 13 -No estrogen-like substances have been detected i n brome grass (Bromus inermis) (92), fescue (Festuca e l a t i o r ) (92), sweet clover (Melilotus alba) (92), timothy grass (Phleum pratense) (82), veldt grass (Erharta calycina) (82), and blue grass (Poa pratense)(48, 37, 61). . 8. Hay. Hay samples of the following forages have been shown to possess estrogenic a c t i v i t y : a l f a l f a (38 , 92) , a l s i k e clover (38), red clover (38), and white clover (38). Pieterse and Andrew (92) noticed that a l f a l f a hay had a r e l a t i v e l y high estrogenic a c t i v i t y . In one case a hay sample was found to be more active than any of the a l f a l f a pasture samples. They suggest that there may be a compound present other than those i n sub-terranean clover which increases i n a c t i v i t y during the curing period. 9, Silage. Pieterse et a l . (91) state that the estrogenic a c t i v i t y of a l f a l f a silage was s i g n i f i c a n t l y greater than that of fresh a l f a l f a . Corn or brome silage did not contain any estrogenic a c t i v i t y . When molasses or sodium metabisulfite was added to a mixture of a l f a l f a , ladino clover and brome grass the estrogenic a c t i v i t y increased during fermentation. C. Different Methods of Determining Estrogens: Quantitative and q u a l i t a t i v e determination of estrogens i n plant and animal material can be accomplished by chemical and physical methods and bio-assay procedures. - 14 -1. Chemical and Physical Methods. Chemical procedures are very tedious, especially when plant materials are involved (79). I t i s es s e n t i a l to have an accurate a n a l y t i c a l method i n order t o f ollow the presence of the active com-pounds during separation of the mixtures. In many cases better results are obtained with chromatography combined with absorption measurements and spectophotometry. Chromatography enables the inves-t i g a t o r to separate a mixture of compounds quickly so that i d e n t i f i -cation of the pure substances can proceed. When the chemical and physical properties of estrogens are unknown, a quantitative estimate of the estrogenic a c t i v i t y can be obtained by the use of bio-assay techniques. Since t h i s i s the case with plant estrogens, bio-assay procedures have been used extensively. 2. Bio-assay Methods. The f i r s t bio-assay method f o r estrogens was developed i n 1923 by A l l e n and Doisy (3, 4) and has been used quite extensively. In t h i s method the estrogens are injected subcutaneously i n t o spayed rats or mice. I f the sample being assayed has estrogenic a c t i v i t y t y p i c a l e s t r a l hyperemia, growth and hypersection i n the g e n i t a l t r a c t and growth of the mammary glands occur. These changes include thickening and c o r n i f i c a t i o n i n the vaginal w a l l which can be followed very e a s i l y i n the l i v i n g animals. Additional proof that the injected hormones are estrogenic can be obtained by mating the ovariectomized females. Copulation i s normal, and t h i s i s followed by the formation o f a vaginal plug, which c l e a r l y demonstrates that the animals were i n estrus as a result of the injected material. - 15 -Emmens (55, 56, 58, 60) presented several modifications of the A l l e n - Doisy technique. The substances i n t h i s case are not injected subcutaneous l y but administered per vaginum into ovariecto-mized female mice. There i s a fundamental difference between the intravaginal method and the A l l e n - Doisy method. In the f i r s t method the hormone action i s e s s e n t i a l l y l o c a l i n character and has l i t t l e opportunity to e l i c i t systemic.'. • responses, while i n the A l l e n - Doisy test the hormone i s absorbed i n the blood stream and carried to the g e n i t a l t r a c t . Systemic responses then occur. Furthermore, Biggers (25) showed that the vehicle used i n intra-vaginal application also influences the res-ponses. Estrone dissolved i n one per cent aqueous albumin had 2.5 to 4.9 times the a c t i v i t y as shown i n 50 per cent aqueous g l y c e r o l when administered by the intra-vaginal route. One per cent egg albumin alone does not produce c o m i f i c a t i o n of the vagina. These differences may account f o r the dif f e r e n t results obtained by the two methods f o r the same hormone. In 1939 Fierz and coworkers (65) developed the "LOKALEN NIPPLE TEST," (L.N.T.), involving cutaneous application of the te s t solution to the nipple of intact male guinea pigs. Extreme small quan-t i t i e s of estrogens can be tested by t h i s method since the nipple i s very sensitive to these hormones. East (51) confirmed the fact that estrogens influence teat length i n guinea pigs by using castrated animals. The "sexual skin" and nipples of female monkeys provide other areas which are sensitive to estrogenic substances (36). In the f i r s t - 16 -case there i s a reddening and swelling of the skin and i n the second an increase i n nipple s i z e . Evans et a l . (64) developed the uterine weight method by using immature female mice and i n j e c t i n g the estrogens subcutaneously. The amount of increase of the uterus was used as a measure of potency of the estrogen tested. The a c t i v i t y i s generally expressed i n terms of estrone, e s t r a d i o l or D.E.S. This increase i n uterine weight has been shown t o be an increase i n both t o t a l water and t o t a l s o l i d s (7, 33). Alexander et a l . (1, 2) determined the estrogenic a c t i v i t y of clover samples by o r a l administration to spayed guinea pigs. The re s u l t i n g increase i n uterine weight was used as a measure of estro-genic a c t i v i t y of the clover sample. The above investigators also demonstrated that the weight of the uterus of spayed guinea pigs i s unaffected by injections of as much as 20 mgm. of testosterone or proges-terone (1). This would indicate that these compounds do not affect the results obtained using the guinea pig as the bio-assay animal. However, i t was shown (64) that testosterone and progesterone cause hypertrophy of the uterus of mice. Alexander et a l . ( l ) further showed that much i f not a l l of the increase i n uterine weight occurs during the f i r s t two days when "estrogenic" clover was fed t o guinea pigs. Recently o r a l administration of the sample to be tested t o int a c t or ovariectomized mice, and measuring uterine weight increase, has been employed frequently by United States workers. The mice are fed ad l i b i t u m (102) or ad li b i t u m and food consumption recorded (39) or controlled feed intake (91, 92, 105), while the assay time varies between ten (102, 91, 92, 96, 105) and three days (103) which has been - 17 -shown to be s u f f i c i e n t for a l l p r a c t i c a l purposes as far as ovariecto-mized mice are concerned (103). After terminating the assay, the mice are s a c r i f i c e d and the u t e r i removed. These are weighed d i r e c t l y a f t e r surplus moisture i s removed with f i l t e r paper (102, 10-5, 91, 92) or after having been fix e d i n Bedouin solution for 24 hours (96). The uterine weight i s d i r e c t l y used as an index of estrogenic a c t i v i t y (102, 105, 91, 92), or the weight of the uterus i s expressed as a percentage of body weight and t h i s i s used as the index of hormone a c t i v i t y (102). Few rats are employed i n determining estrogenic a c t i v i t y of plant and animal material, since immature albino mice are more sensitive to gonadotrophins (75, #4, 62, 63). 3. Units Used i n Expressing Estrogenic A c t i v i t y . Early workers i n t h i s f i e l d expressed the estrogenic a c t i v i t y of a substance i n terms of the natural hormones, v i z . , e s t r a d i o l , estrone, e s t r i o l (100). However, when synthetic hormones became a v a i l -able the estrogenic a c t i v i t y of b i o l o g i c a l material was often expressed i n terms of D.E.S. (19, 92). This l a t t e r compound i s r e l a t i v e l y stable and can be obtained i n pure c r y s t a l l i n e form. In 1941 Evans et a l . (64) gave the potencies f o r a number of natural and synthetic hormones as obtained by the mouse uterine weight technique (TABLE I ) . This i s based on a 6 mgm. increase i n uterine weight and estrone having 100 per cent a c t i v i t y . - 18 -TABLE I Potencies of diff e r e n t n a t u r a l and synthetic estrogens i n terms of estrone (64) Compound Potency Estrone 100 E s t r i o l 40 oC e s t r a d i o l 300 S t i l b e s t r o l 250 ©^estradiol acetate 110 ©^estradiol benzoate 220 oCes t r a d i o l (dipropionate) 70 est r a d i o l (from estrone) 7.5 /tf e s t r a d i o l (from mare urine) 5.0 Eq u i l i n 110 Af c E q u i l i n 7.5 oC dihydroquilin 200 In 1948 Pearlraan (89) recorded the physiological potencies of the estrogens (TABLE I I ) . TABLE I I Comparative physiological potency of the estrogens Potency Conroound Effect i v e dose levels for vagi-nal response i n rats ^ig per Relative a c t i v i t y Subcut., Oral, Rat unit Mouse unit Spayed rat method Immature mouse -uterine-weight method Estrone e s t r a d i o l °^estradiol E s t r i o l 0.7 0.3-0.4 20-30 20-30 1.0 0.08-0.125 3.2-12.5 1.0 0.05 1.25 100 1,000 10 20 100 1,000 7.5 40 - 19 -From these two tables one notices that d i f f e r e n t i n v e s t i -gators report d i f f e r e n t potencies f o r the same hormone. D. Location. I s o l a t i o n and A c t i v i t y of the Estrogen-like Substances  i n Plants; Legg et a l . (82) showed that i n perennial rye grass (Lolium  perenne L.), cocksfoot (Dactylis glomerata L.) and i n red clover (Trifolium pratense) estrogen-like compounds occur i n a l l sections of the plant, i . e . , l e a f , p e t i o l e , stem and inflorescence, with the highest concentration i n the leaves (2). Maximal concentration did not occur at the same time i n a l l organs of the plant and considerable seasonal v a r i a t i o n was noticed (82). This l a s t phenomenum has been postulated to be due t o a change i n the r a t i o of lamina and stems (2). The chloroplasts have been suggested as the s i t e of produc-t i o n , since these are present i n high concentration i n the leaves. I t i s i n these parts of the plant that estrogen-like compounds occur early (82). Curnow i n 1954 (46) i s o l a t e d 12 mgm. of genistein per 100 gm. of dry matter from the "chloropLast" f r a c t i o n , while from fresh clover 369 mg. of genistein per 100 gm. of dry matter were obtained. He stated, therefore, that only three per cent of the estrogenic com-pounds occur i n the cloroplast f r a c t i o n of the plant. Walker (109) suggests that estrogenic a c t i v i t y i s related to the phase of rapid growth of the plant i n the spring, while others (82) state that i t i s associated with the reproductive growth. Spring growth precedes flowering and shows high estrogenic a c t i v i t y , while f a l l growth, when the plant i s not i n i t s reproductive state, i s low i n a c t i v i t y . A decrease i n the estrogenic a c t i v i t y i n subterranean clover, - 20 -when i t reached the dry state, was also observed by Australian workers (22). Red clover on the other hand w i l l produce flowers throughout the summer and f a l l and as a r e s u l t i s continuously high i n estrogen-like substances(82). There i s considerable v a r i a t i o n between the potency figures f o r the same plant estrogen as obtained b y 7 d i f f e r e n t workers. This may be partly due to the fact that, investigators used different bio-assay techniques. Furthermore, the vehicles by which the estrogens are ad-ministered have been shown to influence the a c t i v i t y of the compound (52, 97, 25). 1. Genistein The estrogenic isoflavone, genistein, was isola t e d i n 1951 from subterranean clover ( T r i f o l i u m subterranean) (28) and i n 1954 from red clover (Trifolium pratense) (94). The compound was completely charac-t e r i z e d by Bradbury and White (28), and has the following chemical configuration. OH O Genistein (5: 7: 4 1 - trihydroxyisoflavone) Baker et a l . (8) i n 1928 and Cheng et a l . (39) i n 1954 synthesized t h i s active compound. Bradbury et a l . (28) state that natural and synthetic genistein have a potency 10"^ times that of estrone. Biggers —5 et a l . (26) revised t h i s and showed i t to be either 1.25 x 10 times that of e s t r a d i o l when injected with propylene g l y c o l as solvent, or - 21 --5 4.53 x 10 times that of e s t r a d i o l when peanut o i l was used as vehicle -5 of administration. Pope et a l . (94) estimated the potency to be 10 times that of e s t r a d i o l , while Cheng and coworkers stated that i t had - 5 an a c t i v i t y of approximately 2 x 10 ^ that of D.E.S. 2. Genistin The glucoside of genistein, genistin, i s present i n substan-t i a l amounts (0.1 per cent) i n soybean o i l meal (100) and was is o l a t e d i n 1953 from t h i s source by Cheng et a l . (37). These workers reported that both genistein and genistin are estrogenic. However, the glucoside was shown t o be less active than the aglucone. The estro-genic a c t i v i t y of genistin was confirmed by Carter et a l . (34). 3. Formononet i n In 1951 Bradbury and White (28), and i n 1953 Bate - Smith et a l . (12) isolated a second isoflavone, formononetin, from subter-ranean clover (Trifolium subterranean) and red clover (Trifolium  pratense) respectively. Formononetin (7 - hydroxy - 4' - methoxyisoflavone) This compound had previously been obtained from Ononis spinosa L. where i t occurs as the glucoside of the.isoflavone ononin (112). The synthetic form of formononetin has been shown to exhibit low estrogenic - 22 -a c t i v i t y (39), while the natural compound has been reported to be inactive (94). 4. Daidzein. A fourth compound which has been shown to possess estrogenic a c t i v i t y i s the glucoside daidzein. This compound has been is o l a t e d from soybean meal ( i l l ) . The aglucone was synthesized by Cheng et a l . (39) but has not been reported to be present i n nature (39). HO OH Daidzein (41, 7 dihydroxyisoflavone) Cheng et a l . (39) also reported that the synthetic aglucone was more estrogenic than genistein and biochanin A. 5. Biochanin A. Pope et a l . (93, 94) i s o l a t e d another estrogenic isoflavone, biochanin A, from red clover (Trifolium pratense) and from subterranean clover (Trifolium subterranean'). This compound had previously been isolated from germinated chana grain ("27, 99) and from the heartwood of Ferreirea sp e c t a b i l i s (80). OH O Biochanin A (5: 7 dihydroxy -4 1 - methoxyisoflavone) - 23 -Pope et al .(93, 94) i n 1953 showed that biochanin A was estrogenic and reported i n 1954 that the estrogenic a c t i v i t y of the synthetic form was equal t o that of genistein, while the natural compound had a potency of O.63 times that of genistein. 6. Coumestrol. In 1957 Bickoff et \ § 1 . (24) isolated an estrogenic coumarin derivative, coumestrol, from ladino clover (Trifolium repens L.). Proposed structure of coumestrol (24). This compound i s the predominant estrogen-like substance i n ladino clover (Trifolium repens, ladino), strawberry clover (Trifolium  fragiferum) and a l f a l f a (Medicago s a t i v a ) . The estrogenic a c t i v i t y of coumestrol was shown to be higher than that of genistein but less than D. E.S. as measured by the mouse uterine weight technique. E. Related Plant Products: The estrogenic compounds previously mentioned belong to two groups of compounds known as isoflavones and coumarins. Genistein, genistin, diadzein and formononetin are isoflavones and have a l l the same basic carbon skeleton. ' o 6' - 24 -They d i f f e r i n the number and location of hydroxyl groups and methoxyl groups. In many synthetic compounds the estrogenic a c t i v i t y i s related t o the number and arrangement of the hydroxyl groups. This would also explain why the synthetic isoflavone daidzein has more a c t i v i t y than genistein and biochanin A, and that formononetin i s least active of the four (39). To t h i s same group of compounds belong p r u n i t i n (4', 5-dihyd-roxy-7-methoxy- ? -glucoside) which has been shown to be s l i g h t l y estrogenic, ononin, i s o g e n i s t i n , methylisogenistin and methylgenistein. Closely related t o the isoflavones are the flavones and flavonones. Coumestrol belongs to the group of compounds known as coumarins. These compounds are widely d i s t r i b u t e d i n nature, and are p a r t i c u l a r l y abundant i n the Umbelliferae and Rutaceae (73)• I t has been noted that numerous st r u c t u r a l l y - r e l a t e d coumarins may occur i n the same species, genus or family (73)• This may mean that i n the future more estrogenic coumarins w i l l be isolated from related species or fa m i l i e s . Closely related t o isoflavones are the stilbenes, which have a basic carbon skeleton. The nat u r a l l y occurring stilbene derivatives vary only s l i g h t l y with respect t o t h e i r hydroxylation pattern (73) and are isolat e d from Pinaceae and L i l i c e a e . Synthetic stilbene derivatives have gained much attention - 25 -since 1938 when Dodds et a l . (47) found that D.E.S. had sim i l a r action t o estrone on the u t e r i of intact immature r a t s , rabbits and ovariec-tomized r a t s , on the vagina and the mating reaction of immature r a t s , and on the teat length i n guinea pigs. D i e t h y l s t i l b e s t r o l and the cl o s e l y resembling hexotrol have also been found to have growth promoting properties i n c a t t l e (5, 40) and sheep (15, 16), but not i n swine (90, 14). - 26 -V. MATERIALS AND METHODS A. Materials; 1. Plant Materials. The samples of the second year growth of a l f a l f a (Medicago sativa var. "Rhizoma"), birdsfoot t r e f o i l (Lotus corniculatus var. "Cascade"), orchard grass (Dactylis glomerata). a l s i k e clover (Trifolium hybridum) and white clover (Trifolium repens) were taken from a s p l i t plot design. This s p l i t plot design was sown i n the spring of 1956 and located on the agronomy f i e l d s of The University of B r i t i s h Columbia. These f i e l d s have an Alderwood sandy loam s o i l type. Each major plot was 6' x 12' and replicated four times. These 6» x 12' major plots were divided into f i v e ultimate plots of equal s i z e , which were assigned l e t t e r s A to E at random. The plant material of the four ultimate A p l o t s , one i n each major p l o t , was pooled a f t e r eliminating border material. The same was done f o r the four B p l o t s , the four C p l o t s , etc. In 1956 a l l plots received 200 l b s . of f e r t i l i z e r (10-20-10) per acre on May 11. The samples of second year growth of red clover (Trif o l i u m  pratense, var. "La Salle") studied to determine the effect of stage of maturity and frequency of cutting on the estrogen-like a c t i v i t y were obtained from, f i e l d s located at the R. Reynold's farm near Ladner, B. C. The s o i l type was Ladner clay. The samples of f i r s t year growth of orchard grass and red - 27 -clover were obtained from a s p l i t plot design s i m i l a r to that previously-described for second year growth of a l f a l f a , birdsfoot t r e f o i l , etc. This second s p l i t plot design was sown i n June 1957. The red clover plots received 400 l b s . of 4-10-10 per acre on J u l y 26, 1957 and the orchard grass plots 400 l b s . of 10-20-10 per acre on the same date. Plant material from the f i r s t year growth of a l f a l f a , birdsfoot t r e f o i l and white Dutch clover was obtained from plots measuring 12.5 by 45 feet. These plots were sown at. the end of May 1957 and received 300 l b s . of f e r t i l i z e r (10-20-10) per acre before seeding. The second year growth of red clover used to study the effect of length of day on the a c t i v i t y of the estrogen-like substances i n plants was also part of a s p l i t plot design. Each 6' x 12' major plot was replicated four times. These major plots were divided i n t o three 3'7" x 6"6" p l o t s , which were surrounded by a wooden frame. These three plots A, B and C were again subdivided i n t o two ultimate p l o t s , i d e n t i f i e d as k± and A 2, B-L a n d B 2 , C X and G 2 respectively, as given i n the diagrams below. A l B l c i C2 The number of hours of daylight was controlled by covering them with plywood boxes measuring 6'6" x 3'7" x 1'6". A l l these plots received 300 l b s . of f e r t i l i z e r (4-10-10) per acre at the beginning of May immediately after the f i r s t cuttings were removed. - 28 -2. Experimental Animals. Immature female mice of the U.B.C. Swiss albino s t r a i n , 20-21 days old and weighing between 8 and 11 gm.,. were used i n a l l of the bio-assays. Mice of the same s t r a i n , 60 days old and weighing from 22-30 gm., and guinea pigs weighing between 500 and 600 gm., were used to study the effect of the estrogen-like substances on t h e i r growth and reproduction. 3. Bio-assay Control Diet. This bio-assay diet was composed of ingredients which were free from estrogen-like substances.' The composition of t h i s control d i e t i s given i n TABLE I I I . TABLE I I I Composition of the control diet Ingredient s Lbs. Rolled oats 52.50 Ground wheat 26.25 Fishmeal (70$) 8.75 Meat scraps 3*75 Skim milk powder 7.50 Steamed bone meal 1.00 Iodized s a l t 0.25 100.00 lbs. During the experimental period t h i s diet was stored at 6°C. i n the dark. The proximate analysis of t h i s control d i e t i s given i n TABLE IV. TABLE IV The proximate analysis of the control diet Moisture 17.91 per cent Protein 19.00 Fat 4.65 Crude f i b e r 1.80 Ash 6.86 Nitrogen free extract 49.78 100.00 per cent - 29 -4. Control Guinea Pig Diet. The control diet used f o r the guinea pig growth and reproduc-t i o n experiment had the following composition. TABLE V Composition of the control guinea pig diet Ingredients Lbs. Ground wheat 30 Ground barley 30 Ground oats 12.5 Wheat bran 10 Fishmeal (70%) 5 Meat scraps 5 Molasses 5 Steamed bone meal 1 Iodized s a l t 1 Dried yeast .5 100 l b s . 5. Plant Material Used i n the Mouse Reproduction Experiment  No. I. The red clover used f o r t h i s experiment was a f i r s t cutting of second year growth. I t was cut on May 3, 1957, i n the vegetative stage and after drying at 150°F. for 24 hours had the composition as given i n TABLE VI. TABLE VI Composition of the red clover used for  the mouse reproductive experiment No. I . Total dry matter 13.2 per cent Protein 23.56 " Fat 5.92 » Fiber 12.55 " Ash 8.54 " Nitrogen free extract 49.43 11 The birdsfoot t r e f o i l used was also a f i r s t cutting of second year growth. I t was cut on July 2, 1957, i n the l a t e bloom stage, and - 30 -contained 26.4 per cent dry matter and 18.45 per cent protein a f t e r drying at 150°F. f o r 24. hours. The method used i n extracting the estrogen-like substances from the plant material i s given elsewhere under the section headed ''Methods." The red clover extract was mixed with mouse control diet so that one gram of the experimental d i e t corresponded with one gram of dried plant material, and the same procedure was followed f o r the birds -foot t r e f o i l extract. The D.E.S. diet used i n the mouse reproduction experiments contained 0.068 mcgm. of D.E.S. per gram of mouse control d i e t . The estrogenic potency of t h i s r a t i o n corresponded to that of the f i r s t cutting of red clover previously described. The feed-extract mixtures and the D.E.S. rati o n were stored during the experimental period i n "seal-tight" containers at 6°C. 6. Plant Material f o r the Guinea Pig and Mouse Reproduction Experiment No. I I . ' A second cutting of second year growth of red clover was used for t h i s experiment. The clover was harvested i n the early bloom stage from the Agronomy f i e l d on July 12, 1957. I t contained 13.2 per cent dry matter, 12.51 per cent protein (dry weight) and had an estrogenic a c t i v i t y per l b . of dry matter equivalent to 12 mcgm. of D.E.S. B. Methods; 1. Drying of Plant Material. Immediately after c u t t i n g , the fresh material was weighed, - 31 -spread one inch t h i c k on wooden drying trays and dried at 150°F. for approximately 20 hours. After t h i s period the dry weight and the per cent dry matter of the material were recorded. 2. Grinding of Dried Plant M a t e r i a l . Immediately after drying the plant material was ground i n a Wiley m i l l through a inch screen. 3. Storage of Ground Samples. A l l red clover samples and the f i r s t cuttings of second year growth of a l f a l f a , birdsfoot t r e f o i l , orchard grass, a l s i k e and white clover were stored i n one gallon glass jars with metal l i d s . The rest of the samples were stored i n one half gallon and i n one pint "seal t i g h t " cardboard containers. A l l samples were stored at room temperature and those i n the one gallon glass jars were exposed to daylight. 4. Extraction Procedure. One hundred and f i f t y grams of each dried sample were placed with 800 ml. of 95 per cent ethanol i n a 3000 ml. f l a s k and refluxed far one hour. After cooling, the ethanol was removed by suction f i l -t r a t i o n using No.-l Whatman f i l t e r paper. The plant material was returned to the f l a s k and refluxed for an additional hour with 700 ml. of ethanol. Again a f t e r cooling,the alcohol was removed by suction f i l t r a t i o n and the two alcoholic extracts were combined. These combined extracts were concentrated i n a flash-evaporator at 49°C. to approxi-mately 150 ml. This concentrate was mixed with approximately 140 gm. of the - 32 -control mouse diet and the remaining ethanol was evaporated by drying the feed-extract mixture at 85°c. f o r 15 hours. After drying,the mixture was ground, using a mortar and pestle and the weight adjusted with control d i e t , so that one gram of experimental diet corresponded to one gram of dried plant material. The feed-extract mixtures were stored i n one pint " s e a l t i g h t " cardboard containers and t h e i r estrogen-like a c t i v i t y determined as soon as possible. The diet remaining after the bio-assay was l e f t i n the containers and kept at room temperature for subsequent use. 5. Proximate Analysis of Samples. A complete proximate analysis was carried out on a l l red clover samples used i n the p e r i o d i c i t y experiment. From a l l other samples the moisture and protein content were determined. The analyses were done according t o the A.O.A.C. methods (83), and the re s u l t s recorded on a dry weight basis. 6. Housing and Care of Bio-assay Animals. In a l l cases the animals were housed i n 4" x 6" x 12" enamel lined crisper dishes. In a l l cases wood shavings were used f o r bedding. Each dish housed a group of 10-12 immature female mice for an 80 hour period. A l l experimental diets for bio-assay purposes were fed i n f i n e l y ground form. Each animal received f i v e grams of feed per 72 hours, divided into three equal portions over the three day period. The diets were offered i n four ounce glass jars placed within the crisper dishes. After having been on the diet f o r 72 hours, the animals were - 33 -fasted f o r eight hours. Water was supplied fresh d a i l y ad l i b i t u m i n one h a l f pint jars f i t t e d with rubber stoppers and glass delivery tubes. 7. S a c r i f i c e and Dissection of Bio-assay Mice. After having been on the experiment for SO hours the animals were s a c r i f i c e d with ether anaesthesia and weighed i n d i v i d u a l l y . The u t e r i were then removed (dissected at the uterine side of the c e r v i x ) , trimmed of extraneous t i s s u e , pressed between f i l t e r paper to remove free moisture and immediately weighed on a Ro l l e r - Smith balance. The weight of the uterus was expressed as a per cent of body weight. 8. Housing and Feeding of the Mature Mice on production Experiments Nos. I and I I . The mice used i n t h i s experiment were approximately 60 days old at the beginning of the experiment. The males weighed 24-27 gm. and the females 22-30 gm. These animals were also housed i n crisper dishes. During the pre-mating period f i v e females were housed i n one cage, t o which was added one male during the mating period. After termination of the mating period the females were separated and housed i n d i v i d u a l l y . Throughout the pre-mating and mating period animals received t h e i r experimental diets i n f i n e l y ground form. The diets were offered ad  l i b i t u m i n open s i x ounce glass jars placed within the cages, and the feed consumption recorded. Fresh water was supplied d a i l y ad l i b i t u m i n one half pint bottles as described previously. After the breeding period a l l animals received the mouse control r a t i o n (TABLE I I I ) i n the form of 3/16 inch p e l l e t s . - 3 4 -9 . Housing and Feeding of Guinea Pigs. During the 48 day pre-mating period the guinea pigs were housed i n d i v i d u a l l y i n wooden 18" x 9 " x 6 . 5 " boxes. At the start of the pr,e-mating period the animals received the control guinea pig r a t i o n (TABLE V). This period was designed to get the animals adjusted to t h e i r new surroundings and r a t i o n . The feed was supplied i n the form of 3/16 inch p e l l e t s during t h i s preliminary period. The remaining 3 3 days of the pre-mating time was used to study the influence of the estrogen-like substances of red clover on the growth of the guinea pi g . During t h i s period and during the mating period the animals received the feed - red clover extract mixture i n f i n e l y ground form. During the post mating period a l l guinea pigs received the control d i e t (TABLE V) i n the form of 3/16 inch p e l l e t s . Throughout the experiment ascorbic acid was added to the drinking water, at a l e v e l of 1.8-2 mgm. per 100 gm. of body weight. This l e v e l i s approximately three times greater than the amount required by the animal (29A). The reason f o r t h i s high l e v e l was that ascorbic acid i s broken down i n the water. During the entire experiment,feed consumption was recorded. The water -ascorbic acid mixture was made fresh d a i l y and supplied i n the pre-viously described one half pint b o t t l e s . 10. Mating Plan of Mice of Reproduction Experiment No. I. Twelve days p r i o r to mating, the male and the female mice were fed the p a r t i c u l a r experimental d i e t s . On the 12th day mating was i n i t i a t e d and the females were continued on the same diets during the 15 day mating period. The males which received the same r a t i o n as the females stayed with the females during the whole breeding period while males receiving a di f f e r e n t r a t i o n than the females were rotated. In the l a s t case two males were used f o r each group of females. After - 35 -having been with the females f o r two days the male was removed and put back on i t s p a r t i c u l a r ration f o r two days. The second male which had been on i t s p a r t i c u l a r r a t i o n f o r two days was then put with the same group of females'. TABLE VII Design of mating plan Group Mating 1 5 control females x 1 control male. 2 5 control females x 2 red clover males. 3 5 control females x 2 birdsfoot t r e f o i l males. 4 5 control females x 2 D.E.S. males. 5 5 red clover females x 1 red clover male. 6 5 red clover females x 2 control males. 7 5 birdsfoot t r e f o i l females x 1 birdsfoot t r e f o i l male. 8 5 birdsfoot t r e f o i l females x 2 control males. 9 5 D.E.S. females x 1 D.E.S. male. 10 5 D.E.S. females x 2 control males. After mating was completed, a l l animals, male and female, received the control r a t i o n i n pelleted form. Those females which did not have a l i t t e r three weeks after termination of the breeding period, were mated again to t h e i r o r i g i n a l males, i n order to check i f males and females had recovered from t h e i r p a r t i c u l a r treatments. This second mating period was also 15 days. 11. Mating Plan of Mice of Reproduction Experiment No.II. These mice received the guinea pig control diet plus red clover extract during the eight day pre-mating and the 13 day mating - 36 -period. The mating plan d i f f e r e d s l i g h t l y from that of Experiment I . In Experiment I I the males were not rotated every two days but the f i r s t .male stayed with the females f o r the f i r s t s i x days and the second f o r the remaining seven. TABLE VII I Design of the mating plan Group Mating 1 5 control females x 2 red clover males. 2 5 red clover females x 1 red clover male. After the mating period a l l animals received the mouse control r a t i o n i n pelleted form. Those females which did not l i t t e r within three weeks af t e r mating were bred again to the same males. This second mating period lasted f o r 14 days and was designed to see i f both sexes had recovered from the previous treatment. 12* Mating Plan of Guinea Pigs. Ten females and two males were used i n t h i s experiment. The females were divided into two groups of f i v e , one receiving control ration and the other control ration plus red clover extract (see Mater i a l s ) . One male received control diet and the other control plus red clover extract r a t i o n . After having been on t h e i r p a r t i c u l a r r a t i o n f o r 48 days the control females were mated to the control male and the "red clover" females to the "red clover" male. The mating period was terminated a f t e r thirty-one days. - 37 -VI. RESULTS AND DISCUSSION A. Development of the Bio-assay Procedure; Since the Permanent Commission on B i o l o g i c a l Standardization has not yet established an international standard f o r plant estrogens the author has expressed the potency of these substances i n terms of once r e c r y s t a l l i z e d D.E.S. This compound has been used widely i n the animal industry and i s r e l a t i v e l y stable. In developing a new bio-assay procedure i t i s int e r e s t i n g t o note Hartley's (76) comments when he reviewed the work of the Permanent Commission on B i o l o g i c a l Standardization. "Improvement i n ex i s t i n g methods of assay and the devising of new ones, and the progress of research, are more l i k e l y to be advanced by leaving to i n d i v i d u a l workers freedom of choice as to the method by which assays are carried out, rather than by insistence upon the d e t a i l s of a p a r t i c u l a r method which, on the one hand, may be d i f f i c u l t to describe adequately, and, on the other, may appear to give an a i r of f i n a l i t y i n a f i e l d of b i o l o g i c a l standardization i n which every encour-agement should be given for improvement and advance." In an a n a l y t i c a l d i l u t i o n assay the b i o l o g i c a l system plays an analogous r o l e to that of a balance i n weighing an object. The bio-l o g i c a l system i s an instrument and not a factor influencing the r e s u l t . Wood (114) gives the following description of t h i s kind of a bio-assay. "(a) that the response supposed to be produced by the known amounts of 'factor X' ( i . e . the effe c t i v e constituent of the standard preparation) i s ac t u a l l y due to the f a c t o r i t s e l f and not t o some other substance associated with i t , e.g., an impurity; and (b) that the res-ponse produced by the material to be analyzed i s also due s o l e l y to the presence i n i t of 'factor X', without augmentation, diminution, or modification by any other substance also present. In other words, i f we use the terms 'Standard Preparation' and 'Test Preparation' to denote respectively the solution of allegedly pure 'factor X» and .the solution prepared from the material t o be analyzed, we assume that the Standard Preparation contains no substance, other than factor X i t s e l f , c o n t r i -buting to the response we measure, and that the Test Preparation behaves for the purpose of the analysis so s i m i l a r l y to the Standard Preparation that i t may be regarded simply as a d i l u t i o n of the Standard Preparation i n a completely inert diluent." - 38 -In establishing a suitable bio-assay procedure f o r the quantitative determination of estrogen-like substances i n plant materials a number of preliminary experiments were carried out. 1. The Determination of Feed Intake of Immature Female Mice over a Three Day Period. TABLE IX shows that 21 day old female mice weighing 9-11 gm. consumed 7.9-9.2 gm. of control r a t i o n over a three day period. TABLE IX Feed intake of 21 day old Swiss albino mice receiving  control r a t i o n and water ad l i b i t u m . Group Number of Average Average Feed Intake Animals Body Weight Over 3 Day Period (gm.) 1 8 9.54 7.9 gm. 2 7 10.63 9.2 gm. 3 7 9.28 7.9 gm. Control rations containing plant extracts having high estro-genic a c t i v i t y tend to lower p a l a t a b i l i t y . This was also observed by Alexander et a l . (2) when dried "estrogenic" subterranean clover was fed t o guinea pigs. From these observations i t was decided not to feed at maximum intake l e v e l , but f i v e grams of diet per animal f o r the three day assay period. In doing so, a l l bio-assay animals consumed approximately the same amount of d i e t . 2. The Time of Feed Passage i n 21 Day Old Swiss Albino Mice. The time of feed passage was determined f o r two groups of three mice. One group received the control r a t i o n with f e r r i c oxide added as an indicator at the 0.1 per cent l e v e l , and the other group received - 3 9 -the control r a t i o n with chromic oxide added at the 0.1 per cent > l e v e l . The results are shown i n TABLE X. TABLE X The time of feed passage i n 21 day old mice fed control r a t i o n and receiving water ad l i b i t u m Animal Indicator Time of Feed Passage F e r r i c oxide 1 8.30 hrs. 2 7.55 hrs. 3 Chromic oxide 8.10 hrs. 4 8.45 hrs. 5 8.15 hrs. 6 8.20 hrs. The average time of passage for the f e r r i c oxide group 7.98 hours and for the chromic oxide group 8.27 hours when control r a t i o n and water were offered ad l i b i t u m . However, t h i s difference i s not s i g n i f i c a n t . 3. The Influence of Time of Fasting on Uterine Weight of Intact Immature Female Mice. To obtain maximum values f o r uterine weight, when plant extracts were fed, i t was f e l t necessary to study the effect of time of fasting on the ultimate uterine weight of the assay females. From the results shown i n TABLE XI i t seemed reasonable to fast a l l b i o -assay animals for eight hours. This was approximately the same number of hours as necessary for the control r a t i o n to pass through the TABLE XI The influence of "time, of fasting" on uterine weight (Animals were fed 5 gm, of feed per 3 days and water ad libitum) Ration Group Number of Animals Hours. . of Fasting Av. Body Wt. at Zero hrs. Fasting (gm.) Av. Body Wt. at end of Fasting (gm.) Average Uterine Wt. (mgm.) Per cent Body Wt. Control 1 12 0. 9.99 9.99 19.90 0.199-0.008* 2 12 8 10.24 9.51 16.93 0.178±0.010 3 12 24 10.44 8.66 17.93 0.206+0.007 Control Ration containing 0.05 mcgm. D.E.S. per gm. of feed. 4 12 0 10.80 10.80 40.09 0.368+0.027 5 12 8 10.77 9.62 36.94 0.385*0.027 6 12 24 10.63 8.58 28.64 0.333-0.014 & Standard error - 41 -digestive t r a c t of the mouse. 4. The Length of the Bio-assay Period. An experiment was undertaken to study the number of hours required for the uterus to reach i t s maximum size when the mice were fed a r a t i o n containing estrogenic compounds. One l e v e l of D.E.S. and two red clover extract mixtures having di f f e r e n t estrogenic a c t i v i t y were fed to three groups of immature female mice. TABLE XII shows that i n a l l three cases the u t e r i reached t h e i r maximum weight at 72 hours. Stob et a l . (103) showed that a three day assay period was s u f f i c i e n t i n ovariectomized mice i n order to measure maximum a c t i v i t y of an estrogen-like substance. Alexander et a l . ( l ) observed that i n spayed guinea pigs much i f not a l l of the increase i n uterine weight occurred over the f i r s t two days when they were fed "estrogenic" sub-terranean clover. A short bio-assay period, using intact 21 day old female mice, also has the advantage that normal estrus does not e a s i l y mask the experimental r e s u l t s . From the res u l t s of TABLE XII i t was concluded that a 72 hours assay period would be used for a l l subsequent bio-assays. 5. The Eff e c t of Dehydration of the U t e r i on the Accuracy of the Bio-assay. A comparison of wet uterine weight, dry uterine weight and alcohol dehydrated u t e r i was studied. TABLE XII I shows that there i s an increase i n both t o t a l water and t o t a l solids of the uterus when estrogenic substances are fed to mice. These res u l t s confirm the work of Astwood (7) and C a r r o l l (33) who observed the same phenomenum. The influence of "assay length" on the uterine weight (Animals were fed 1.67 gm. of feed per day and water ad libitum) Ration Group Number Hours Hours Av. Body Wt. Average Per cent Body Wt. of on of at end of Uterine Animals Ration Fasting Fasting (gm.) Wt. (mgm.) Control 1 12 24 2 • 10 48 3 12 72 Control Ration 4 12 12 containing 0.05 (mcgm.) 5 12 24 D.E.S. per gm. of feed 6 12 43 7 12 72 i I 1 8 12 96 Control Ration 9 12 12 containing red clover 10 12 24 extract (1st cutting; 11 12 48 May 3, 1957) 12 12 72 13 12 96 Control Ration 14 8 48 Containing red clover 15 8 72 extract (2nd cutting; 16 8 96 July 18, 1956) 8 10.35 17.65 0.171*0.007 8 10.45 20.50 0.195*0.009 8 9.51 16.93 0.178±o.0l0 8 10.45 26.37 0.252±o.008 8 10.35 27.65 0.268±o.006 8 10.43 37.13 0.354*0.016 8 10.02 40.28 0.400±0.017 8 10.40 36.18 0.349*0.014 8 11.90 40.30 0.340*0.016 8 10.00 30.97 0.310*0.011 8 9.69 44.54 0.464*0.023 8 10.34 51.58 0.501*0.021 8 10.27 50.76 0.494*0.010 8 9.88 44.00 0.452*0.031 8 9.23 46.00 0.492±o.034 8 9.53 43.60 0.460±0.033 I - 43 -TABLE X I I I A comparison of wet uterine weights, dry  uterine weights and uterine weights of alcohol dehydrated  u t e r i (2 hrs. i n 95$ ethanol) Control Ration Control Ration 0.02 mcgm. D.E.S. per gm. of Feed 0.07 mcgm. D.E.S. per gm. of Feed ft Number of Animals 12 12 10 12 Mean Body Weight i n Grams 10.34 9.21 9.57 10.35 Mean Wet Uterine Wt. Mgm. 18.70 14.90 26.90 51.60 Mean Uterine Weight after ethanol Dehydration Mgm. - 5.70 - -Mean Dry Uterine Wt. i n Mgm. 4.70 " 3.00 6.20 9.00 Wet Uterine Wt. per Body Wt. x 100 0.180 0.164 0.281 0.A98 Et. OH Dehydrated Uterine Wt. per Body Weight x 100 - 0.062 - -Dry Uterine Wt. per Body Wt. x 100 0.045 0.033 0.064 0.086 ft A l l animals consumed 5 grams of feed per three days and received water ad libitum. For the control r a t i o n the standard errors of wet uterine weight, dry uterine weight and alcohol dehydrated uterine weight as a per cent of body weight were 5 . 8 , 5.2 and 5.2 per cent respectively, •"or the ra t i o n containing 0 .02 mcgm. of D.E.S. per gram of feed the per cent standard error for wet uterine weight as a per cent of body weight was 4 .3 ..and for dry uterine weight 5 . 9 . Corresponding figures for the rati o n containing 0 .07 mcgm. of D.E.S. per gram of feed were 3 .0 and 3 .0 per cent. These res u l t s indicate that neither drying nor alcohol dehydration of the u t e r i improve the accuracy of the procedure. Since both are time consuming, i t was decided t° use wet uterine weight expressed as a percentage of body weight as a measurement of - 44 -estrogenic a c t i v i t y . From the res u l t s obtained i n the preliminary experiments i t was decided to feed f i v e grams of die t per three days, to have an assay period of 72 hours, to fa s t the animals f o r eight hours, and to use wet uterine weight expressed as a per cent of body weight as the measurement of estrogenic a c t i v i t y of the experimental d i e t s . In a l l cases water was supplied ad libitum . B. Construction of the Dose Response Curve; The potency of the estrogen-like substances i n the plant material studied was determined r e l a t i v e to the a c t i v i t y of once r e c r y s t a l l i z e d D.E.S. Known quantities of t h i s compound, dissolved i n ethanol, were added to the standard mouse diet and fed to groups of randomly selected immature female mice. Groups of 10 mice were used for the f i r s t dose response curve and groups of 12 for the second. Two dose response curves were constructed since a s i g n i f i c a n t difference was established between the three l o t s of control r a t i o n . TABLE XIV Bio-assay results of the three l o t s of mouse control ra t i o n . Lot Date of No. of Mean Body Mean Per cent Feed Con-Assaying Mice Wt. (gm.) Uterine Body Wt. sumption/ Wt. (mgm.) 3 Days (gm.) 1 22-4-57 10 9.72 22.0 0.222+0.016^ 5 2 22-7-57 12 9.51 16.93 0.178±0.010 5 3 9-12-57 12 10.34 18.70 0.180±0.007 5 A P<0.05 - 45 -The f i r s t l o t of mouse control d i e t was only used to construct dose response curve 1 (FIGURE I ) , and to assay the second year growth of red clover harvested i n 1956. The data obtained f o r the construction of t h i s dose response curve are given i n TABLE XV. From these data a regression l i n e was calculated by the "method of least squares" (43)• Y = 0.206 + 6.139x Sy s = 0.035 x = mcgm. of D.E.S./gm. of feed y s uterine weight/body weight x 100 The second dose response curve and a l l other bio-assays and experiments were based on the second and t h i r d l o t of ra t i o n . Bio-assays of these two l o t s gave s i m i l a r results (TABLE XIV). The data f o r dose response curve I I (FIGURE II) are shown i n TABLE XVI. The equation of the regression l i n e again was calculated by the "method of least squares" and was Y =0.172 + : - 4 . 8 4 3 x sys = 0.017 This second dose response curve was used t o estimate the estrogenic a c t i v i t y of a l l samples with the exception of the previously mentioned red clover. C. Plant Extract Bio-assayst After developing a suitable bio-assay technique and construc-t i n g a dose response curve f o r the determination of estrogen-like sub-stances i n forage samples an experiment was designed to study the effect of length of time from cutting the forage i n the f i e l d to drying on these estrogenic substances. The results of t h i s study are shown i n - 46 -TABLE XV Tabulated re s u l t s f o r the construction of dose response curve I Group Mcgm. of D.E.S.* Number Mean Body Mean Pe r c e n t per gm. of Feed of Wt. (gm.) Uterine Body Wt. Animals Wt. (mgm.) 1 0.000 10 9.72 22.0 0.222 2 0.0025 10 8.94 19.6 0.218 3 0.005 10 9.45 23.3 0.246 4 0.010 10 9.68 26.5 0.274 5 0.015 10 9.72 30.9 0.31B 6 0.020 10 9.70 33.6 0.344 7 0.025 10 9.75 30.9 0.317 8 0*030 10 9.74 37.0 0.379 9 0.035 10 9.47 38.6 0.41O 10 0.040 9 9.48 39.6 0.416 11 0.045 9 9.31 45.9 0.495 12 0.050 9 9.91 46.6 0.468 13 0.055 10 10.16 58.5 0.578 14 0.060 10 9.74 55.4 0.576 15 O.O65 10 10.33 69.6 0.673 16 0.070 10 9.86 65.2 0.670 17 0.075 10 10.36 67.1 0.649 IS 0.090 9 9.89 72.1 0.726 ± A l l animals consumed 5 gm. of feed per three days and received water ad li b i t u m . o 3 5T •H u 0) M •H IV .CP 0.70 h 0.6o [-0.50 h 0.40 0.30 0.20 0.10 h 0.00 0.010 FIGURE I 0.020 0.030 0.040 0.050 0.060 0.070 Mcgm. of D.E.S./gm. of Control Diet DOSE RESPONSE CURVE I 0.080 0.090 0.100 - 48 -TABLE XVI Tabulated re s u l t s for the construction of dose response curve I I Group Mcgm. of D.E.S. Number Mean Body Mean Per cent per gm. of Feed of Wt. (gm.) Uterine Body Wt. Animals Wt. (mgm.) 1 0.000 12 9.51 16.93 0.178 2 0.0025 12 9.61 18.82 0.194 3 0.005 12 9.09 18.03 0.198 4 0.010 12 9.13 19.66 0.214 5 0.015 12 9.57 22.49 0.236 6 0.020 12 9.60 24.68 O.256 7 0.025 12 9.60 27.82 0.288 8 0.030 12 9.39 30.92 0.328 9 0.035 12 9.95 32.87 0.330 10 0.040 12 9.83 39.08 0.399 11 0.045 12 10.00 39.70 0.397 12 0.050 12 10.02 40.28 0.400 13 0.060 12 9.70 43.87 0.451 14 0.070 12 9.83 51.42 0.520 15 0.080 12 9.58 52.05 0.557 16 0.090 12 9.91 56.75 0.571 17 0.100 12 9.95 56.95 0.581 £ A l l animals consumed 5 gm. of feed per three days and received water ad lib i t u m . o •H 0) 13 4 3 x: •H Q) •P ZD "8 0.60 0.50 0.40 h 0.30 h 0.20 h 0.10 0.00 -p-vO _i 1 0.010 FIGURE I I 0.020 0.030 0.040 0.050 0.060 0.070 Mcgm. of D.E.S./gm. of Control Diet DOSE RESPONSE CURVE I I 0.080 0.090 0.100 - 50 -TABLE XVII. An analysis of variance revealed that the estrogenic a c t i v i t y of red clover was not s i g n i f i c a n t l y influenced (P^>0.05) by leaving the plant material on the f i e l d for 48 hours. Alexander et a l . (2) made a sim i l a r observation f o r the estrogenic a c t i v i t y of subterranean clover when the plant material was stored f o r 48 hours at 16 to 21°C. Next the effects of drying time and drying temperature on the estrogenic substances i n the feed-extract mixtures were studied. The results obtained by varying drying time between 7 and 24 hours and keeping the drying temperature constant at 65°C. are shown i n TABLE XVIII, while those obtained by varying drying temperature between 65 and 102°C. (drying time constant at 19 hours) are reported i n TABLE XIX. An analysis of variance revealed that the estrogenic a c t i v i t y of red clover was not s i g n i f i c a n t l y influenced by either of the two treatments. After these experiments were completed an extensive study of the effects of stage of maturity and frequency of cutting on the estrogenic substances of f i r s t and second year growth of certain forages was Undertaken. The resu l t s of t h i s study are given i n TABLES XX to LI. 1. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of A l f a l f a (TABLES XX TO XXVI And FIGURE I I I ) . Walker et §1. (109) postulated that the highest concentrations of estrogen-like substances i n forage plants occurred during the phase of rapid growth i n the spring, while other investigators (82) correlated the a c t i v i t y with the reproductive growth of the plant. The results i n TABLE XX show that second year growth of a l f a l f a i s not only high TABLE XVII The influence of time of curing on estrogen-like a c t i v i t y  of red clover ( f i r s t cutting of 1957 seeding, cut Sept. 24, 1957) Group Time between cutting and the beginning of drying at 65°C. Number of Animals Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Consumption per animal per 3 days (gm.) Estimated Potency per l b . of Dry Matter (mcgm.) 1 12 minutes 12 9.14 33.48 0.365*0.019 5.0 18 2 1.30 hrs. 11 9.46 34.11 0.36lio.017 5.0 18 3 26.00 hrs. 10 8.85 33.67 0.382io.025 4.4 20 4 48.00 hrs. 12 9.38 28.63 0.306±o.023 5.0 13 \J1 H TABLE XVIII The effect of drying time on the l e v e l of estrogen-like substances i n "control ration. - red clover extract" mixtures  (Red clover - f i r s t cutting May 3,-1957) Sample Treatment Mouse Bio-assay Sample Drying No. of Mean Body Mean Per cent Feed Con- Estimated Potency Time Temperature Animals Wt. (gm.) Uterine Body Wt. sumption per l b . of Dry Wt. per 3 days Matter (mcgm.) (mgm.) (gm.) IA 7 hrs. 65° C 11 9.84 40.62 0.413*0.007 4.3 26 ro IB 19 hrs. 65° C. 11 9.99 42.51 0.425*0.015 4.3 27 1 1C 24 hrs. 65° C. 10 10.54 45.05 0.426+0.017 4.8 25 TABLE XIX The effect of drying temperature on the l e v e l of estrogen-like substances i n "control r a t i o n - red clover extract" mixtures (red clover = f i r s t cutting May 3. 1957) Sample Treatment Mouse Bio-assay Sample Drying Time Temper-ature Number of ; Animals Mean Body Weight (gm.) Mean Uterine Weight (mgm.) Per cent Body Weight Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . of Dry Matter (mcgm.) 1 B 19 hrs. 65°C. 11 9.99 42.51 0.42510.015 4.3 27 1 D 19 hrs. 82°C. 12 9.94 39.62 0.400*0.014 4.7 23 1 E 19 hrs. 102°C. 12 9.38 35.30 0.378+0.026 43 22 TABLE XX The estrogen-like a c t i v i t y of f i r s t cuttings of a l f a l f a (Rhizoma) at different-stages of maturity (second year growth, 1957) Sample Description Mouse Bio-assay Plots Date of Cutting Stage of Maturity Per cent Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Feed Con-Body Wt. sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) A May 3 Vegetative 84.5 22.15 12 10.18 74.62 0.733*0.016* c 4.7 55 B June 3 Pre-bloom 74.8 18.71 12 10.08 30.17 0.298*0.011 5.0 12 C July 2 F u l l Bloom 66.0 13.72 12 9.96 • 21.85 0.218*0.011 5.0 4 D Aug. 1 Late Bloom 70.1 • 16.43 12 9.90 37.35 0.388to.028 4.8 21. . E Sept. 3 Past Bloom 63.6 7.93 12 9.81 34.27 0.350^0.020 5.0 17 ic Standard error TABLE XXI The estrogen-like a c t i v i t y of 1st. 2nd, 3rd and 4th cuttings of  A l f a l f a (Rhizoma) of plots A (second year growth, 1957) Sample Description Mouse Bio-assay Cuttings Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/. Potency Wt. 3 days per l b . Dry (mgm.) (gm.) Matter (mcgm.) 1 May 3 Vegetative 84.5 22.15 12 10.18 74.62 0.733*0.016 4.7 55 i 2 Jan. 2 Vegetative 81.2 22.34 12 10.46 33.54 0.322+0.021 5.0 Ik 1 3 July 2 Vegetative 76.2 22.75 11 9.71 21.95 0.226*0.007, 4.5 6 4 Aug. 15 Vegetative 79.5 18.53 12 9.92 59.34 0.598*0.024 4.6 43 TABLE XXII The estrogen-like a c t i v i t y of 1s t , 2nd and 3rd cuttings of  a l f a l f a (Rhizoma) of plots..B (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice' Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . (mgm.) (gm.) Dry Matter (mcgm.) 1 June 3 Pre-bloom 74.8 18.71 12 10.08 30.17 0.298+0.011 5.0 12 i 2 J u l y 2 Vegetative 80.0 22.12 12 9.74 19.51 0.169±0.008 4.3 0 ON 1 3 Aug. 15 Vegetative 78.2 25.40 12 8.79 29.14 0.332*0.032 4.3 18 The estrogen-like, a c t i v i t y of 1st, 2nd and 3rd cuttings of  a l f a l f a (Rhizoma) of plots C (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt, (gm.) Uterine Body Wt. sumption/ Potency Wt. (mgm.) 3 days per l b . (gm.) Dry Matter (mcgm.) 1 July 2 F u l l Bloom 66.0 13.72 12 9.-96 21.85 0.218*0.011 5.0 4 i 2 Aug. 1 Vegetative SI. 8 26.31 12 9.06 17.04 0.187*0.010 4.3 2 1 3 Sept. 3 Vegetative 76.6 22.75 11 9.17 17.64 0.194*0.012 4.6 2 TABLE XXIV The estrogen-like a c t i v i t y of 1st and 2nd cuttings of  a l f a l f a (Rhizoma) of plots D (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. .Wt. (mgm.) Feed Con- Estimated sumption/ Potency 3 days per l b . (gm.) Dry Matter (mcgm.) 1 Aug. 1 Late Bloom 70.1 16.43 12 9.90 2 Sept. 3 Vegetative 76.9 19.60 12 9.28 37.35 0.388+0,028 4.8 18.54 0.199*0.010 4.8 2 3 t» May 3 FIGURE I I I June 3 J u l y 2 Date of Cutting Aug. 1 Aug. 1$ Sept. 3 The Estrogenic A c t i v i t y i n Terms of D.E.S. of Second Year Growth of A l f a l f a at Various Stages of Maturity, and at Successive Cutting. TABLE XX? The estrogen-like a c t i v i t y of f i r s t , cuttings of a l f a l f a (Rhizoma) at different, stages of maturity ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Plots Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . (mgm.) (gm.) Dry Matter (megm.) A Aug. 15 Vegetative 84.1 24.28 12 9.40 15.75 0.168±0.008 5.0 0 B Sept, 25 Vegetative 64.1 6.58 12 9.12 15.90 0.171*0.009 5.0 0 (not many leaves pre-TABLE XXVI The estrogen-like a c t i v i t y of 1st and 2nd cuttings of a l f a l f a (Rhizoma) of plots A ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . (mgm.) (gm.) Dry Matter (mcgm.) 1 Aug. 15 Vegetative 84.1 24.28 12 9.40 15.75 G.16810.008 5.0 0 2 Sept. 25 Vegetative 75.1 22.85 12 9.09 19.99 • 0.220±0.015 5.0 5 - 62 -i n estrogenic substances i n the spring but that i t also contains sub-s t a n t i a l amounts a f t e r August 1 when the plants are past the period of rapid growth. The t h i r d cutting of plots B and the fourth cutting of plots A, both cut on August 15 also showed considerable a c t i v i t y . The l a s t two samples were i n the vegetative and not i n the reproduc-t i v e state. The results also contradict a t h i r d hypothesis stating that seasonal-variation i s due to a change of lamina to stem r a t i o (2). Furthermore, a l l samples of second year growth of a l f a l f a cut i n J u l y showed l i t t l e or no estrogenic a c t i v i t y . The plots of f i r s t year growth of a l f a l f a were heavily infected with leaf spots, and, as a r e s u l t of t h i s , most plants l o s t t h e i r leaves. F i r s t cuttings of these affected plots did not show any es-trogenic a c t i v i t y , but a second cutting of plot A showed some a c t i v i t y . This second cutting was affected to a much lesser degree by the above mentioned disease and hence the plants l o s t few leaves. 2. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of White Clover (TABLES XXVII t o XXXII and FIGURE IV). The growth pattern of white clover d i f f e r s considerable from that of a l f a l f a . This clover produces flowers throughout the summer and f a l l , i . e . , i t i s continuously i n i t s reproductive stage. However, the estrogenic pattern of white clover i s very s i m i l a r to that of a l f a l f a . The estrogen-like a c t i v i t y was again highest i n the f i r s t cutting of second year growth, cut on May'3. The potency was lowest i n the samples cut i n June and July, but not as low as the estrogenic a c t i v i t y of a l f a l f a samples cut on the same date. An exception to t h i s r e s u l t TABLE XXVII The estrogen-like a c t i v i t y of f i r s t cuttings of white clover at d i f f e r e n t stages of maturity (second year growth, 1957) Sample Description Mouse Bio-assay Plots Date of Cutting Stage of Per cent Maturity Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean . Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) A May 3 Vegetative 87.7 26.69 12 9.65 70.00 0.720*0.023* 5.0 57 B June 3 % F u l l Bloom. 84.0 23.43 12 9.53 25.94 0.27LT.0.012 5.0 9 C July 2 F u l l to Late 75.4 Bloom 20.65 12 9.77 21.53 0.221*0.010 5.0 5 D Aug. 1 Late Bloom 73.4 20.93 12 9.77 38.69 0.392*0.025 5.0 20 E Sept. 3 Past Bloom 59.1 (seed) • 18.63 12 9.46 36.25 0.381*0.031 5.0 20 & Standard error TABLE XXVIII The estrogen-like a c t i v i t y of 1st, 2nd, 3rd and 4th cuttings of white clover of plots. A (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . (mgm.) (gm.) Dry Matter (mcgm.) 1 May 3 Vegetative 87.7 26.69 12 9.65 70.00 0.720*0.023 5.0 57 i o> 2 June 3 \ Bloom 84.2 26.47 12 9.81 26.90 0.273±0.017 5.0 10 3 J u l y 2 5/6 Bloom 79.7 22.47 11 9.88 33.51 0.339±0.020 5.0 15 4 Aug. 1 Past F u l l 73.4 25.44 12 10.21 52.92 0.518*0.025 5.0 30 Bloom TABLE XXIX The estrogen-like a c t i v i t y of 1st, 2nd and 3rd cutting of  white clover, of plots. B (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No, of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt, (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . (mgm.) (gm.) Dry Matter (mcgm.) 1 June 3 % F u l l Bloom 84.0 23.43 12 9.53 25.94 0.271*0.012 5.0 9 O N 2 J u l y 2 g F u l l Bloom 78.3 23.34 12 9.80 41.82 0.426±o.029 5.0 24 3 Aug. 1 1/5 Bloom 81.2 25.50 12 10.67 65.73 0.61630.021 5.0 42 TABLE XXX The estrogen-like a c t i v i t y of 1st and. 2nd cutting of white clover of plots C (second year growth. 1957) Sample Description Mouse Bio-assay Cutting Date of ' Cutting Stage of Maturity Per cent Moistur e Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean' Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 Jul y 2 Full-Late 95.4 20.65 12 9.77 21.53 0.221*0.010 5.0 5 Bloom • 2 Aug. 1 1/8 Bloom 80.2 23.00 12 9.44 26.52 0.286+0.017 5.0 11 May 3 June 3 J u l y 2 Aug. 1 Sept. 3 Date of Cutting FIGURE IV The Estrogenic A c t i v i t y i n Terms of D.E.S. of Second Year Growth of White Clover at Various Stages of Maturity and at Successive Cuttings. TABLE XXXI The estrogen-like a c t i v i t y of f i r s t cuttings of white clover at d i fferent stages of. maturity ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Plots Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . (mgm.) (gm.) Dry Matter (mcgm.) A Aug. 15 Vegetative 90.4 26.50 12 9.60 16.74 0.174*0.003 5.0 0 B Sept. 25 Vegetative 88.1 22.68 11 8.93 10.69 0.1201:0.005 5.0 0 / / / / / TABLE X X X I I The estrogen-like a c t i v i t y , of 1st and 2nd cuttings of white  clover of, .plots.. A ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Cutting Stage of Per cent Maturity Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 Aug. 15 Vegetative 90.4 26.50 12 9.60 16.74 0.174*0.003 5.0 0 2 Sept. 25 Vegetative 87.1 25.10 12 9.51 15.01 0.158IQ.003 5.0 0 - 70 -was the second cutting of plots B, cut on J u l y 2. After August 1 a l l samples showed an increase i n estrogenic a c t i v i t y . This increase was i n most cases of a greater magnitude than the increase i n estrogen-l i k e substances i n corresponding a l f a l f a samples. The samples from f i r s t year growth which were i n the vegetative state and cut between August 15 and September 25 did not show any estrogenic a c t i v i t y . 3. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of Red Clover (TABLES XXXIII to XXXVII and FIGURE V). Red clover produces flowers throughout the summer and f a l l s i m i l a r to white clover. However, i t s estrogenic pattern d i f f e r s considerably from the l a t t e r . A l l samples of f i r s t and second year growth showed considerable estrogenic a c t i v i t y with highest potency i n the spring and decreasing towards the f a l l . Samples of f i r s t year growth were r e l a t i v e l y higher i n a c t i v i t y , ; than the samples from second year growth cut at the same time. However, the former samples were i n the reproductive state, while the l a t t e r were past bloom. Legg et a l . (85) made si m i l a r observations on red clover i n Great B r i t a i n . These workers postulated that the presence of estrogenic substances was associated with reproductive growth. This hypothesis may hold true f o r red clover but c e r t a i n l y not f o r the other forages studied. 4. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of Birdsfoot T r e f o i l (TABLES XXXVIII to XLIV). The only sample of birdsfoot t r e f o i l that showed considerable TABLE XXXIII The estrogen-like, a c t i v i t y of f i r s t cuttings of red clover at different.stages of maturity (second year growth, 1 9 5 6 ) Sample Description Mouse Bio-assay -Plot Date of Cutting Per cent Moisture Per cent Protein No. of Mice.V Mean Body Weight (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption per 3 days (gm.) Estimated Potency per l b . of Dry Matter (mcgm.) A May 2 8 2 . 6 2 2 . 0 0 8 8 . 4 6 4 7 . 0 0 . 5 6 + 0 . 0 2 * 3 . 8 3 4 B May 2 5 7 3 . 8 1 9 . 5 2 8 1 0 . 6 2 62.6 0 . 5 9 + 0 . 0 1 3 . 9 3 6 l t 1 C June IS 8 4 . 2 1 6 . 0 3 8 8 . 7 2 4 1 . 7 0 . 4 8 + 0 . 0 1 3 3 3 1 1 D July 2 7 3 . 2 1 5 . 8 7 8 9 . 0 9 4 4 . 1 0 . 4 9 + 0 . 0 2 3 . 5 3 0 E July IB 6 9 . 5 1 4 . 6 2 8 9.63 3 7 . 6 0 . 3 9 * 0 . 0 2 5 1 4 F Aug. 1 8 5 8 . 8 13.26 8 - - 0 . 2 8 * 5 6 G Sept, 2 56.9 1 4 . 6 5 8 9.36 23 .0 0 . 2 5 + 0 . 0 1 5 < 5 ± Standard error TABLE XXXrV The estrogen-like a c t i v i t y of f i r s t , second and t h i r d cuttings of red clover of plots B (second year growth. 1956) Sample Description Mouse Bio-assay Cutting Date of Cutting Per cent Moisture Per cent Protein No. of Mice Mean Body Weight (gm.). Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption per 3 days (gm.) Estimated Potency per l b . of Dry Matter (mcgm.) 1 May 25 73.8 19.52 8 10.62 62.6 0.59*0.01 3.8 36 2 J u l y 18 75.9 17.71 8 11.11 61.9 0.57*0.04 3.9 34 3 Sept. 2 57.9 14.87 8 10.36 43.8 . 0.42+0.02 5.4 15 TABLE XXXV The estrogen-like a c t i v i t y of f i r s t , second and t h i r d cuttings of red clover of plots (second year growth, 1956) Sample Description Mouse Bio-assay Cutting Date of Cutting Per cent Moisture Per cent Protein No. of Mice Mean Body Weight (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption per 3 days (gm.) Estimated Potency per l b . of Dry Matter (mcgm.) 1 June IS 84.2 16.03 8 8.72 41.7 0.48*0.01 3 33 2 J u l y IS 75.6 17.90 8 9.53 43.6 0.49*0.03 5 21 3 Aug. 18 70.2 15.76 8 9.60 31.1 0.33*0.02 5 9 o 0 A A P l o t s A B C D E F JL -L .ay 2 FIGURE V May 25 June 18 J u l y 2 J u l y 18 Date of Cutting Aug. 18 Sept. 2 The Estrogenic A c t i v i t y i n Terms of D.E.S. of Second Year Growth of Red Clover at Various stages of Maturity, and at Successive Cuttings. TABLE XXXVI The estrogen-like a c t i v i t y of f i r s t cuttings of red clover at dif f e r e n t stages, of maturity ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Plots Date of Cutting Stage of Maturity Per cent Moisture Per cent Protein No, of Mean Body Mice Wt. (gm.) Mean Per cent Feed Con- Estimated Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . Dry (mgm.) (gm.) Matter (mcgm.) A Aug. 15 Early Buds 86.7 B Sept. 25 F u l l Bloom 68.1 21.75 12 15.74 11 10.18 36.87 0.362*0.019 4.8 9.46 34.11 0.361^0.017 5.0 18 18 VJ1 TABLE XXXVTI. The estrogen-like a c t i v i t y of f i r s t and second cuttings of  red clover of plots. A ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice "Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . of (mgm.) (gm.) Dry Matter (mcgm.) 1 Aug. 15 Early Buds 86.7 21.75 12 10.18 36.87 0.362+.Q.019 4.8 18 2 Sept. 25 Few Flowers 76.4 21.45 11 8.79 28.23 0.321±o.Ol6 4.7 15 TABLE XXXVIII The estrogen-like a c t i v i t y of f i r s t cuttings of birdsfoot t r e f o i l at d i f f e r e n t stages of maturity (second year growth, 1957) Sample Description Mouse Bio-assay Plots Date of Stage of Per cent Per cent No of Mean Body Mean Per cent Feed Con- Estimated Potency Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body VJt. sumption/ per l b . Dry VJt. (mgm.) 3 days Matter (mcgm.) (gm.) A May 3 Vegetative 87.7 24.81 11 9.26 32.71 0.364*0.026 4.1 22 B June 3 1/3 Bloom 77.5 17.62 10 10.10 16.44 0.163*0.006 4.8 0 C July 2 Late Bloom 73.6 IS. 45 11 9.59 17.85 0.188±0.006 4.8 1 D Aug. 1 Late Bloom 67.6 16.34 11 8.83 14.38 0.163^0.006 3.6 0 (second wave of flowering) E Sept. 3 Past Bloom 61.4 11.31 9 8.73 17.53 0.201±0.011 3.9 4 TABLE XXXIX The estrogen-like a c t i v i t y of 1st, 2nd, 3rd, 4th and 5th cuttings of  birdsfoot t r e f o i l of plots A (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Cutting Stage of Maturity Per cent Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Per cent Uterine Body Wt. Wt. (mgm.) Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 May 3 Vegetative 87.7 24.81 11 9.26 32.71 0.364*0.026 4.1 22 2 June 3 Early Bud-ding 84.1 27.37 10 10.08 18.15 0.179±0.009 4.8 1 3 July 2 Vegetative 82.3 20.25 11 8.58 13 .'78 O.l6l±0.008 4.5 0 4 Aug. 1 Early Bud 83.9 26.25 11 8.75 12.26 0.154*0.006 4.6 0 5 Sept. 3 Vegetative 67.6 17.63 9 9.20 12.30 0.133*0.007** 4.7 0 * S i g n i f i c a n t l y decreased P<0.05 ** " " P< 0.01 TABLE XL The estrogen-like a c t i v i t y of 1st, 2nd, 3rd and 4th cuttings of birdsfoot t r e f o i l of plots B (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Cutting Stage of Maturity Per cent Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 June 3 1/3 Bloom 77.5 17.62 10 10.10 16.44 0.163*0.006 4.8 0 2 July 2 Very Early Bud 81 .9 21.19 11 9.37 14.39 . 0.154*0.003* 4.9 0 3 Aug. 1 Late Budding Early Flower-ing 82.2 26.25 11 9.03 13.35 O.L46±0.006* 4.3 0 4 Sept. Vegetative 69.4 17.22 10 9.71 13.21 0.137*0.004** 5.0 0 * S i g n i f i c a n t l y decreased P<'0.05 ** « " P<0.01 TABLE XLI The estrogen-like a c t i v i t y of 1st, 2nd and 3rd cuttings of birdsfoot t r e f o i l of plots C (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Cutting Stage of Maturity Per cent Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 July 2 Late Bloom 73.6 18.45 11 9.59 17.85 0.188*0.006 4.8 1 2 Aug, 1 Budding 83.4 27.62 10 8.56 14.80 0.171^0.010 4.1 0 3 Sept. 3 Vegetative 78.6 20.22 12 8.80 14.18 0.l6l±0.008 4.2 0 03 O TABLE XLII The estrogen-like a c t i v i t y of 1st and 2nd cutting of birdsfoot t r e f o i l of plots D (second year growth. 1957) • Sample Description Mouse Bio-assay -Cutting Date of Cutting Stage of Per cent Maturity Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Feed Con-Body Wt. sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 Aug. 1 Late Bloom 67.6 (second wave of flowering) 16.34 11 8.83 14.38 O.I63+O.OO6 3.6 0 1 o> H 1 2 Sept. 3 Vegetative 77.7 17.71 11 9.17 13.95 0.151+0.007* 4.2 0 it S i g n i f i c a n t l y decreased P < 0.05 ±k " " P<0.01 TABLE XLIII The estrogen-like a c t i v i t y of f i r s t cuttings of birdsfoot t r e f o i l at di f f e r e n t stages of maturity (1st year growth. 1957) Sample Description Mouse Bio-assay Plots Date of Cutting Stage of Maturity Per cent Per cent No.of Mean Body Mean Per cent Moisture Protein Mice Wt. (gm.) Uterine Body Wt. Wt. (mgm.) Feed Con- Estimated sumption/ Potency 3 days Per l b . (gm.) Dry Matter (mcgm.) A Aug. 15 Vegetative (few buds) B Sept. 25 Vegetative (few pods) 86.6 16.32 12 9.44 78.0 10.82 9 10.20 14.97 0.157±0.007 4.8 15.77 0.154*0.007 5.0 0 0 00 ro TABLE XLIV The estrogen-like a c t i v i t y of 1st and 2nd cuttings of birdsfoot t r e f o i l of plots A (1st year growth, 1957) Sample Description Mouse Bib-assay Cutting Date of Cutting Stage of Maturity Per cent Moisture Per cent Protein No. of Mean Body Mice Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Feed Con-Body Wt. sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 Aug. 15 Vegetative 86.6 16.32 12 9.44 14.97 0.157*0.007 4.8 0 (few buds) 2 Sept. 25 Vegetative 87.1 . 18.18 12 9.84 14.44 0.148*0.008* 4.5 0 4 S i g n i f i c a n t l y decreased P<0.05 " " P<0.01 - 84 -estrogenic a c t i v i t y was the f i r s t cutting of second year growth, cut May 3. None of the other samples analyzed showed s i g n i f i c a n t amounts of estrogen-like substances as measured by the increase i n uterine weight of immature female mice. However, i t i s interesting t o note that i n many bio-assays the average uterine weight and the average feed intake of the mice were s i g n i f i c a n t l y reduced. This decrease i n uterine weight may be explained by assuming that birdsfoot t r e f o i l contains a com-pound or series of compounds which lower d i r e c t l y or i n d i r e c t l y the weight of the u t e r i of immature mice, since starvation i t s e l f does not lead to a decrease i n uterine weight as great as resulted from the feeding of birdsfoot t r e f o i l extract. 5. The Effect of Stage of Maturity and Frequency of Cutting on the Estrogenic A c t i v i t y of Orchard Grass (TABLES XLV to L ) . The f i r s t cutting of orchard grass harvested on May 3 was the only sample of orchard grass analyzed that showed s i g n i f i c a n t estro-genic a c t i v i t y (P-<0.01). This confirms the results given i n the l i t e r a t u r e . Estrogenic a c t i v i t y of orchard grass has been reported i n very few cases (82)j most investigators (82, 48) did not detect any estrogen-like compounds i n t h i s plant species. . 6. The Effect of Stage of Maturity on the Estrogenic A c t i v i t y of the Second Year Growth of Alsi k e Clover. From May 13 to August 1 different plots of the second year growth of a l s i k e clover were cut at two week i n t e r v a l s . The estrogen-like a c t i v i t y of the samples i s recorded i n TABLE LI . As compared to white and red clover samples, the a l s i k e samples showed very l i t t l e or no estrogenic a c t i v i t y . However, i t i s TABLE XLV The estrogen-like a c t i v i t y of f i r s t cuttings, of Orchard Grass  at di f f e r e n t stages of maturity (second year growth, 1957) Sample Description - Mouse Bio-assay Plots Date of Cutting Stage of Per cent Maturity Moisture Per cent Protein No. of. Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) A May 3 Vegetative 81.6 11.75 12 10.35 23.15 0.224*0.008 5.0 5 B June 3 \ Bloom 73.6 11.47 12 9.84 16.95 0.171*0.010 5.0 0 C July 2 F u l l Bloom 63.9 11.14 12 8.93 U.05 0.158*0.010 5.0 0 D Aug. 1 Late Seed (much under-grass) 59.8 6.22 11 9.17 15.17 0.l64±o.008 5.0 0 E Sept. 3 Seed (much undergrass) 45.9 5.81 10 8.80 17.56 0.201*0.007 5.0 3 A Standard error TABLE XLVI The estrogen-like a c t i v i t y of 1s t , 2nd and 3rd cuttings of Orchard Grass of plot A (second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Cutting Stage of Maturity Per cent Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm. 1 May 3 Vegetative 81.6 11.75 12 10.35 23.15 0.224*0.008 5.0 5 2 June 3 Vegetative 80.4 13.90 11 9.82 18.27 0.18610.008 5.0 1 3 July 22 Vegetative 71.7 8.43 12 9.34 16.19 0.173*0.010 5.0 0 TABLE XLVII The estrogen-like a c t i v i t y of 1st and 2nd cuttings of Orchard Grass  of plots B (second year growth. 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/ Potency per Wt. (mgm.) 3 days l b . Dry (gm.) Matter (mcgm. 1 June 3 g Bloom 73.6 11.47 12 9.84 16.95 0.171*0.010 5.0 0 2 J u l y 22 Vegetative 71.9 7.62 12 9.19 15.64 0.169*0.006 5.0 0 TABLE XLVIII The estrogen-like a c t i v i t y of. 1st and 2nd cuttings of Orchard Grass of plots C.(second year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Cutting Stage of Per cent Maturity Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) 1 Ju l y 2 F u l l Bloom 63.9 11.14 12 8.93 14.05 0.158±0.010 5.0 0 2 Aug. 1 Vegetative 76.5 1 S .25 12 8.68 15.93 0.183*0.005 5.0 1 TABLE XLIX The estrogen-like a c t i v i t y of f i r s t cuttings of Orchard Grass  at d ifferent stages of maturity ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Plots Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . Dry (mgm.) (gm.) Matter (mcgm.) A Aug, 15 Vegetative - 13.25 12 9.94 17.13 0.172*0.007 5.0 0 B Sept. 25 Vegetative 66.9 9.50 10 10.32 19.35 0.189*0.009 5.0 2 TABLE L The estrogen-like, a c t i v i t y of 1st and 2nd cuttings of Orchard Grass of plots A ( f i r s t year growth, 1957) Sample Description Mouse Bio-assay Cutting Date of Stage of Per cent Per cent No. of Mean Body Mean Per cent Feed Con- Estimated Cutting Maturity Moisture Protein Mice Wt. (gm.) Uterine Body Wt. sumption/ Potency Wt. 3 days per l b . Dry (mgm.) (gm.) Matter (mcgm.) 1 Aug, 15 Vegetative - 13.25 12 9.94 17.13 0.17210.007 5.0 0 2 Sept. 25 Vegetative 68.1 10.30 12 9.62 15.21 0.157*0.008 5.0 0 TABLE LI The estrogen-like a c t i v i t y of f i r s t , cuttings of a l s i k e clover  at d i f f e r e n t stages of maturity. (second year growth. 1957) Sample Description Mouse Bio-assay Plots Date of Cutting Stage of Per cent Maturity Moisture Per cent Protein No. of Mice Mean Body Wt. . (gm. ) Mean Uterine Wt. (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . of Dry Matter (mcgm.) A May 13 Vegetative 84.9 19.97 12 9.86 25.23 0.257+0.'010* 5 8 B June 4 F u l l Bloom 82.5 18.06 12 10.43 20.20 0.194+0.006 5 2 C June 18 L i t t l e Past 80.4 F u l l Bloom 16.00 10 9.38 14.74 0.158+0.010 5 0 D July 2 Late Bloom 6 9 . 1 15.91 10 9.25 13.43 0.145+0.006 5 0 E Jul y 22 Past Bloom 48.9 (moldy) 14.00 10 10.32 21.11 0.205+0.010 5 3 F Aug. 1 Past Bloom 28.7 (moldy) 16.75 10 9.99 25.38 0.256+0.018 5 8 4 Standard error - 92 -interesting to note that the l a s t sample cut on August 1- showed s i g n i f i -cant amounts of these compounds to be present. The plant material i n t h i s sample was i n the drought stage, had a dark brown color and was moldy. 7. The Influence of Length of Daylight on the Estrogenic A c t i v i t y of Red Clover. An experiment was designed to study the effect of exposure t o d i f f e r e n t lengths of daylight (Photoperiod) on the l e v e l of estrogen-l i k e compounds i n second year growth of red clover. The r e s u l t s ob-tained i n t h i s investigation are given i n TABLES LII to LLX. On May 3 a l l plots were cut and samples for analysis were obtained by pooling the plant material of the four ultimate A^ p l o t s , A 2 p l o t s , p l o t s , etc. (See "Materials. u.) Proximate analysis of these samples were carried out (TABLE L I I I ) . The data reveal no s i g n i f i c a n t difference i n composition and estrogenic potency between the samples. From May 3 to June 4 a l l B and C plots were covered with wooden boxes (See "Materials."') from 4:30 P.M. to 8:30 A.M.; i n other words, these plants received only eight hours of daylight per day. On June 4 the ultimate plots designated with odd numbers were harvested. Duplicate samples were obtained by pooling plant material of two corresponding odd numbered ultimate p l o t s . S i x samples were obtained i n t o t a l , two of which had received f u l l daylight and four of which had been r e s t r i c t e d to eight hours of daylight per day. The proximate analyses of these samples are given i n TABLE LIV and the bio-assay r e s u l t s i n TABLE LV. The plant material from the control plots A-^  was. phy s i o l o g i c a l l y older than that of the plots (Bj and Cj) which had been under reduced TABLE L I I The estrogen-like a c t i v i t y of f i r s t cuttings of red clover  (second year growth, 1957) Sample Description Mouse Bio-assay Plots Gutting* Length of Stage of Per cent Per cent No. Mean Mean Per cent Feed Con- Estimated Daylight Maturity Moisture Protein of Body Uterine Body Wt. sumption/ Potency Mice Wt. Wt. 3 days per l b . (gm.) (mgm.) (gm.) Dry Matter (mcgm.) A1*A2 1 F u l l Day Vegetative 86.8 23.47 10 10.08 49.82 0.495*0.026 5.0 3 6 Aj|.*A2 1 F u l l Day Vegetative 86.8 23.56 10 9.97 50.82 0.512*0.025 4.3 37 B-L + B 2 1 F u l l Day Vegetative 86.7 23.25 10 10.27 54.60 0.532*0.019 4 .4 39 \+%2 1 F u l l Day Vegetative 87.3 23.28 12 10.34 51.58 0.501-0.021 4.3 36 ~ C . ^ 1 F u l l Day Vegetative 86.7 23.31 11 10.21 50.94 0.500+0.021 4.2 37 1 F u l l Day Vegetative 87.2 23.53 H 9.72 46.51 0.480*0.025 4.3 3 4 ft Cut on May 3, 1957. TABLE L I I I Proximate analyses of f i r s t cuttings of red clover (second year growth, 1957) Sample Description Proximate Analyses Plots Cutting * Length of Daylight Stage of Maturity Per cent Dry Matter Per cent Protein Per cent Fat Per cent Crude Fiber Per cent Ash Per cent N-free Extract Estimated Potency per l b . of Dry Matter (mcgm.) V A 2 i J L F u l l Day Vegetative 13.2 23.5 6.0 12.7 8.5 49.3 36 A l + A 2 1 F u l l Day Vegetative 13.2 23.6 6.0 13.8 8.2 48.4 37 V B 2 1 F u l l Day Vegetative 13.3 23.3 5.3 13.5 9.1 48.8 39 V B 2 1 F u l l Day Vegetative 12.7 23.3 5.9 12.6 9.0 49.2 36 1 F u l l Day Vegetative 13.3 23.3 5.3 12.5 8.5 50.4 37 C l + C 2 1 F u l l Day Vegetative 12.8 23.5 5.3 13.4 8.7 49.1 34 ft Cut on May 3, 1957. TABLE LIV" Proximate analyses of second cuttings of plots of red clover which were exposed to different periods of daylight (second year growth,  growing period - May 2 to June 4 , 1957) Sample Description Proximate Analyses Plots Cutting Length of Stage of Daylight Maturity Per cent Dry Matter Per cent Protein Per cent Fat Per cent Crude Fiber Per cent Ash Per cent N-free Extract Estimated Potency per l b . of Dry Matter (mcgm.) A l 2 F u l l Day Very Early Bud 15.9 21.4 5.1 14.0 8.3 51.2 38 A l 2 F u l l Day Very Early Bud 14.2 22.2 5.4 14.9 8.5 49.0 36 B l 2 ft 8hr. Day Vegetative L4.7 29.3 5.1 13.3 9.4 42.9 34 B l 2 8 hr. Day Vegetative 15.3 28.3 5.1 14.2 9.8 42.6 29 c l 2 8 hr. Day Vegetative - 27.2 5.5 13.2 9.1 45.0 25 D l 2 8 hr. Day Vegetative 15.5 28.9 5.7 13.2 9.9 42.3 35 ft Daylight from 8:30 A.M. to 4:30 P.M. TABLE LV The estrogen-like a c t i v i t y of second cuttings of plots of red clover which were exposed to different periods of daylight  (second year growth, growing period - May 2 to June 4. 1957) Sample Description Mouse Bio-assay Plots Cutting Length of Stage of Per cent Per cent No. of Mean Mean Per cent Feed Con- Estimated Daylight Maturity Moisture Protein Mice Body Uterine Body Wt. sumption/ Potency Wt. Wt. 3 days per l b . (gm.) (mgm.) (gm.) Dry Matter (mcgm.) A l 2 F u l l Day Very Early 84.1. 21.37 12 9.39 51.04 0.545*0.014 4.6 38 Bud A l 2 F u l l Day Very Early 85.8 22.22 12 10.09 53.14 0.526±o.Ol6 4.6 36 Bud B l 2 8 hr. Day* Vegetative 85.3 29.25 12 10.68 56.98 0.535*0.023 5.0 34 B l 2 8 hr. Day Vegetative 84.7 28.31 11 10.04 48.47 0.483*0.025 5.0 29 G l 2 8 hr. Day Vegetative 81.0 27.16 12 10.53 45.74 0.397*0.024 5.0 25 C l 2 8 hr. Day Vegetative 84.5 28.90 11 10.30 54.28 0.530*0.026 4.8 35 * Daylight from 8:30 A.M. to 4:30 P.M. - 97 -daylight. The f i b e r content of the samples of the former p l o t s was higher and the protein content lower. The average potency of the con-t r o l samples corresponded to 37 mcgm. of D.E.S., and that of the samples taken from plots which had been r e s t r i c t e d to eight hours of daylight to 31 mcgm. Since the difference between 37 mcgm. and 31 mcgm. can be accounted for by chance, no d e f i n i t e conclusion can be reached as to the effect of length of daylight on the subsequent estrogenic a c t i v i t y . From June 4 to June 20 only the C plots were r e s t r i c t e d to eight hours of daylight. On the l a s t mentioned date the plant material from the ultimate plots designated with even numbers was harvested. By that time the plants on plots B 2 had reached approximately the same physiological age as those of plots A]_ on June 4. Again, duplicate samples were obtained by pooling plant material of two corresponding ultimate p l o t s . Proximate analyses of these samples are given i n TABLE LVII. The estrogenic potency of the control plots had dropped from 37 to 29.5 mcgm. of D.E.S., while that of the C 2 p l o t s , which had been r e s t r i c t e d i n daylight over the whole growing period, had dropped from 31 to 28.5 mcgm. of D.E.S. Restoring plots B 2 to f u l l daylight for the l a s t 16 days of the growing period resulted i n an increase from 31 to 33 mcgm. of D.E.S. per l b . of dry matter. However, t h i s decrease i s not s t a t i s t i c a l l y s i g n i f i c a n t . The physiological age of the plant decreased from plots A 2 to C 2, while the protein content increased i n the same order. The t h i r d part of t h i s experiment was designed to study the effect of r e s t r i c t i n g the daylight received by the plants over the f i r s t part of the growing period as compared to r e s t r i c t i n g them over the TABLE LVI Proximate analyses of second cuttings of plots of  red clover which were exposed to different periods of daylight (second year growth, growing period - May 2 to June 20. 1957) Sample Description Proximate Analyses Plots Cutting Length of Daylight Stage of Maturity Per cent Dry Matter Per cent Protein Per cent Fat Per cent Crude Fiber Per cent Ash Per cent N-free Extract Estimated Potency per l b . of Dry Matter (mcgm. A2 2 May 4-June 20 F u l l Day 1/4 Bloom 18.5 17.9 4.7 20.0 7.3 50.1 28 A2 2 May 4-June 20 F u l l Day 1/5 Bloom 18.1 20.3 4.1 19.0 7.1 49.5 31 B2 2 May 4-June 4 8 hr. Day* Very Few Buds 14.7 22.3 5.2 14.4 9.5 48.6 30 B2 2 May 4-June 4 8 hr. Day Vegetative 14.2 23.3 5.2 13.9 7.9 49.7 36 G2 2 May 4-June 20 8 hr. Day Vegetative 15.5 24.7 5.2 14.5 9.4 46.2 27 C2 2 May 4-June 20 8 hr. Day Vegetative 15.2 27.8 6.1 14.3 8.8 43.0 30 A- Daylight from 8:30 A.M. to 4:30 P.M. TABLE LVTI The estrogen-like a c t i v i t y of second cuttings of plots of  red clover which were exposed to different.periods of daylight  (second year growth, growing period - May 2 to June 20, 1957) Sample Description Mouse Bio-assay Plots Cutting Length of Stage of Per cent Per cent No. Mean Mean Per cent Feed Con- Estimated Daylight Maturity Moisture Protein of Body Uterine Body Wt. sumption/ Potency Mice Wt. Wt. 3 days per l b . (gm.) (mgm.) (gm.) Dry Matter (mcgm.) A 2 2 May 4 - £ Bloom 81.5 17.99 12 9.80 42.90 0.444*0.033 4.5 28 June 20 F u l l Day A 2 2 May 4 - 1/5 Bloom 81.9 20.28 12 9.06 45.82 0.508±o.031 4.5 31 June 20 F u l l Day B 2 2 May 4 - Very few 85.3 22.29 11 9.41 46.57 0.492*0.027 5.0 30 June 4 £ buds 8 hr. Day B 2 2 May 4 - Vegetative 85.8 , 23.13 12 9.03 46.30 0.516*0.025 4.5 36 June 4 8 hr. Day C 2 2 May 4 - Vegetative 84.5 24.74 11 9.93 45.16 0.460*0.030 5.0 27 June 20 8 hr. Day C 2 2 May 4 - Vegetative 84.8 27.75 12 10.26 50.68 0.494*0.022 5.0 30 June 20 8 hr. Day & Daylight from 8:30 A.M. to 4:30 P.M. TABLE LVIII Proximate analyses of t h i r d cuttings of plots of red clover which were exposed to d i f f e r e n t periods of daylight. (second year growth, growing period - June 4 to Ju l y 3. 1957) Sample Description Proximate Analysis Plots Cutting Length of Stage of Daylight Maturity Per cent Dry Matter Per cent Protein Per cent Fat Per cent Crude Fiber Per cent Ash Per cent N-free Extract Estimated Potency per l b . of Dry Matter (mcgm.) A l 3 June 4-July 3 Budding F u l l Day 15.1 22.3 6.1 13.7 8.5 49.4 29 A l 3 June 4-July 3 Budding F u l l Day 15.9 21.1 5.4 13.9 8.0 51.6 25 B l 3 June 4-June 20 Budding F u l l Day June 20-July 3 8 hr. Day* 13.1 26.8 5.9 12.3 8.4 46.6 28 B l 3 June 4-June 20 Budding F u l l Day June 20-July 3 8 hr. Day 12.5 26.7 5.1 15.7 9.3 43.2 23 c l 3 June 4-June 20 Vegetative 8 hr. Day June 20-July 3 F u l l Day 15.2 26.0 5.9 12.3 8.4 47.4 32 c l 3 June 4-June 20 Vegetative 8 hr. Day 13.8 26.6 5.6 12.5 9.0 46.3 22 June 20-July 3 F u l l Day A Daylight from 8:30 A.M. to 4:30 P.M. TABLE LLX The estrogen-like a c t i v i t y of t h i r d cuttings of red clover which were exposed to different periods of daylight  (second year growth, growing period - June 4 to July 3, 1957) Sample Description Mouse Bio-assay Plots Cutting Length of Daylight Stage of Maturity Per cent Moisture Per cent Protein No. of Mice Mean Body Wt. (gm.) Mean Uterine Wt. (mgm.) Per cent Feed Con-Body Wt. sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) A l 3 June 4 -J u l y 3 F u l l Day Budding 84.9 22.25 12 9.46 42.39 0.447*0.027 4.5 29 A l 3 June 4 -July 3 F u l l Day Budding 84.1 21.12 12 9.21 39.08 0.425*0.016 4.8 25 B l 3 June 4 -June 20 Budding 86.9 26.76 12 10.00 46.59 0.463*0.019 4.8 28 F u l l Day June 20 -J u l y 3 * 8 hr. Day 1 B 1 3 June 4 - Budding 87.5 26.72 12 9.51 39.48 0.418*0.019 5.0 23 June 20 F u l l Day June 20 -July 3 8 hr. Day C 3 June 4 - Vegetative 84.8 26.00 12 9.60 45.13 0.470*0.021 4.3 32 1 June 20 8 hr. Day June 20 -July 3 F u l l Day C 3 June 4 - Vegetative 86.2 26.56 12 10.26 41.40 0.406*0.025 5.0 22 1 June 20 8 hr. Day June 20 -July 3 F u l l Day A Daylight from 8:30 A.M. to 4:30 P.M. - 102 -l a s t part of the growing period. Plots were r e s t r i c t e d i n daylight from June 4 t o June 20, while the plots had eight hours of daylight from June 20 to J u l y 3. On J u l y 3 the plots were harvested ( t h i r d cutting) and the samples analyzed (TABLES LVIII AND LIX). Since there i s so much difference between duplicates as far as the estrogenic a c t i v i t y i s concerned, no r e l i a b l e conclusions can be reached on t h i s part of the experiment. The proximate analyses show, as expected, that the greater the physiological age of the plant, the higher the f i b e r content and the lower the protein content. TABLES LTV, LVI and LVIII show that there i s no correlation between estrogenic a c t i v i t y on the one hand and on the other hand protein, f a t , f i b e r , ash or nitrogen free extract content of the samples. D. The S t a b i l i t y of the Estrogen-like Substances of Plant Origin: L i t t l e l i t e r a t u r e i s available on the s t a b i l i t y of the estro-gen-like substances i n plant materials. Alexander et a l . (2) observed that storage of subterranean clover over a period of 48 hours at 16-21°C. did not affect the estrogenic a c t i v i t y . Prolonged storage at room temperature i n a i r , and i n the dark also did not affect the potency of dehydrated clover. However, the dehydration process i t s e l f lowered estrogenic a c t i v i t y of the clover sample. The s t a b i l i t y of estrogenic-like substances i n dried ground a l f a l f a , red clover and white clover, as w e l l as the s t a b i l i t y of these compounds i n feed extract mixtures was studied. Samples of plant material taken at d i f f e r e n t stages of maturity and samples of sequential cuttings were studied. The results of t h i s investigation are recorded i n TABLES LX to LXV. - 103 -TABLE LX shows the s t a b i l i t y of the estrogen-like sugstances i n f i r s t cuttings of ground dry a l f a l f a and i n dry feed extract mixtures of the same samples. The sample of plot A was cut on May 3* extracted on May 20 and the feed-extract mixture assayed on May 22 and November 14. On May 22 the sample had an estrogenic a c t i v i t y corres-ponding to 55 mcgm. of D.E.S. and on November 14 a potency of 42 mcgm. of D.E.S. This shows that the estrogenic a c t i v i t y of the feed-extract mixture decreased when i t was stored f o r s i x months i n the dark and at room temperature. The dry ground sample of the plant material was stored i n glass bottles with metal l i d s , at room temperature and i n daylight during t h i s s i x month period. On November 7 part of t h i s sample was extracted and assayed for estrogenic a c t i v i t y on November 10. The bio-assay revealed that no estrogenic substances were present at that time. Similar results were obtained when sequential cutting of a l f a l f a were assayed i n the above manner (TABLE LXI). A study on the s t a b i l i t y of the estrogen-like compounds i n white clover gave s i m i l a r results to those from the a l f a l f a samples, the longer the samples were stored the lower the hormone-like a c t i v i t y . However, here again the breakdown of the active compound i s much more rapid i n the dried ground plant material than i n the dried feed ex-tra c t mixtures (TABLES LXII and LXIII). The s t a b i l i t y of the active compound i n red clover d i f f e r s considerably from that of a l f a l f a and white clover. The estrogen-l i k e substances i n red clover are much more stable, l i t t l e breakdown occurs when the ground dry material i s stored for a f i v e months.', period at room temperature and subjected to daylight. Here the break-down of the estrogenic compounds i n the feed-extract mixture was of a TABLE LX The s t a b i l i t y of the estrogen-like, compounds, i n 1st cuttings of ground dry a l f a l f a (RHIZOMA) and i n the dry free-alcohol extract mixtures of the same samples (second year growth, 1957) Sample Description Time of Storage Mouse Bio-assay Plots Date of Stage of Per cent Per cent Date of Date of No. of Mean Mean Per cent Feed con- Estimated Gutting Maturity Moisture Protein Extra- Assaying Mice Body Uterine Body Wt. sumption Potency ct i o n Wt. Wt. 13 days per l b . (gms) (mgms) (i pi.) Dry Matter (mcgms.) A May 3 Vegetative 84.5 22.15 May 20 May 22 12 10.18 74.62 0.733+0.016 4.7 55 Nov. 14 10 10.49 61.98 0.591+0.018 5.0 42 Nov. 7 Nov. 10 12 9.92 16.93 0.170+0.011 5.0 0 B June 3 Pre-Bloom 74.8 18.71 June 14 June 25 12 10.08 30.17 0.298+0.011 5.0 12 Dec. 4 10 9.72 31.22 0.318+0.029 4.7 15 Oct. 7 Oct. 15 11 9.64 22.96 0.237+0.015 5.0 6 Nov. 7 Nov. 11 12 9.20 18.08 0.196+0.008 4.9 2 C Ju l y 2 F u l l Bloom 66.0 13.72 J u l y 9 July 20 12 9.96 21.85 0.218+0.011 5.0 4 Dec. 5 10 9.30 22.48 0.230+0.010 5.0 6 Nov. 11 Nov. 13 12 8.35 13.39 0.161+0.009 5.0 0 D Aug. 1 Late Bloom 70.1 16.43 Aug. 27 Aug. 28 12 9.90 37.35 • 0.388+0.028 4.8 21 Dec. 3 11 8.81 27.40 0.307+0.022 3.9 16 Nov. 7 Nov. 17 12 10.29 17.08 0.168+0.007 5.0 0 E Sept. 3 Past Bloom 63.6 7.93 Sept. 5 Sept. 9 12 9.81 34.27 0.350+0.020 5.0 17 Dec. 4 9 10.81 37.27 0.345+0.027 4.8 17 Nov. 10 Nov. 17 12 10.39 22.07 0.210+0.023 5.0 2 O TABLE 1X1 The s t a b i l i t y of the estrogen-like compounds i n sequential cuttings of dry ground alfalfa.(RHIZOMA), and, i n the dry feed-alcohol extract  mixtures of the same samples, (plots A. second year growth, 1957) Sample Description Time of Storage Mouse Bio-assay Cutting Date of Stage of Per cent Per cent Date of Date of No. of Mean Mean Per cent Feed Estimated Cutting Maturity Moisture Protein Extrac- Assaying Mice Body Uterine Body Wt. Con- Potency t i o n Wt. Wt. sumption per l b . (gms.) (mgms.) 3 days Dry Matter (gms.) (mcgms.) 1 May 3 Vegetative 84.5 22.15 May 20 May 22 12 10.18 74.62 0.733+0.016 4.7 55 Nov. 14 10 10.49 61.98 0.591*0.018 5.0 42 Nov. 7 Nov. 10 12 9.92 16.93 0.170+0.011 5.0 0 2 June 3 Vegetative 81.2 22.34 June 14 Ju l y 2 12 . 10.46 33.54 0.322+0.021 5.0 14 Dec. 26 12 9.40 19.30 0.207+0.010 5.0 3 Dec. 27 Jan. 2 11 8.98 18.49 0.209+0.015 4.7 4 3 July 2 Vegetative 76.2 22.75 Ju l y 20 Aug. 2 12 9.71 21.95 0.226+0.007 4.5 6 Dec. 26 12 9.61 19.91 0.209+0.010 5.0 3 Dec. 27 Jan. 2 9 9.20 18.32 0.201+0.015 4.1 3 4 Aug. 15 Vegetative 79.5 18.53 Aug. 28 Sept. 2 12 9.92 59.34 0.598+0.024 4.6 43 (border Dec. 26 12 9.39 4 .20 0.469+0.018 5.0 28 effect) Dec. 27 Jan. 2 12 9.50 34.96 0.371+0.019 4.3 22 TABLE LXII The s t a b i l i t y of the estrogen-like compounds i n f i r s t cuttings  of dry ground white clover, and i n the dry feed-alcohol extract mixtures  of the same samples (second year growth. 1957) Sample Description Time of Storage Mouse Bio-assay Plots Date of Stage of Per cent Per cent Date of Date of No. Mean Mean Per cent Feed Estimated Cutting Maturity Moisture Protein Extrac- Assaying of Body Uterine Body Wt. Con- Potency t i o n Mice Wt. Wt. . sumption/ per l b . (gm.) (mgm.) 3 days Dry Matte: (gm.) (mcgm.) A May 3 Vegetative 87.7 26.69 May 16 May 22 12 9.65 70.00 0.720+0.023* 5.0 57 Dec. 5 12 10.42 55.03 0.527*0.018 5.0 33 Nov. 11 Dec. 2 12 10.57 18.65 0.176*0.011 5.0 0 B June 3 Nearly 84.0 23.43 J u l y 19 July 30 12 9.53 25.94 0.271*0.012 5.0 9 F u l l Bloom Dec. 5 10 10.29 19.10 0.185*0.011 5.0 1 Oct. 7 Oct. 15 10 9.10 12.80 0.151*0.008 4.6 0 Nov. 11 Nov. 18 12 9.84 15.76 0.161*0.006 5.0 0 C July 2 Full-Late 75.4 20.65 July 29 Aug. 12 12 9.77 21.53 0.221*0.010 5.0 5 Bloom Dec. 6 11 10.17 22.37 0.220*0.009 5.0 4 Nov. 12 Dec. 2 10 9.76 19.94 0.204*0.013 5.0 3 D Aug. 1 Late Bloom 73.4 20.93 Sept. 6 Sept. 17 12 9.77 38.69 0.392*0.025 5.0 20 Dec. 6 11 10.33 30.10 0.291*0.013 5.0 11 Nov. 12 Nov. 30 11 10.01 25.30 0.253*0.011 5.0 8 E Sept. 3 Past Bloom 59.4 18.63 Sept. 7 Sept. 17 12 9.46 36.25 0.381*0.031 5.0 20 (seed) Dec. 9 12 10.22 21.96 0.215*0.010 5.0 4 Nov. 13 Dec. 2 12 9.83 25.00 0.252*0.015 5.0 8 ft Standard error TABLE LXIII The s t a b i l i t y of the estrogen-like compounds i n sequential cuttings of dry ground white clover, and i n the dry feed-alcohol extract mixtures of  the same samples (plots A. second year growth, 1957) Sample Description Time of Storage Mouse Bio-assay Gutting Date of Stage of Per cent Per cent Date of Date of No. Mean Mean Per cent Feed E s t i -Cutting Maturity Moisture Protein Extrac- Assaying, of Body Uterine Body Wt. Con- mated t i o n Mice Wt. Wt. sumption/Potency (gm.) (mgm.) 3 days per l b . (gm.) Dry Matter (mcgm.) 1 May 3 Vegetative 87.7 26.69 May 16 May 22 12 9.65 70.00 0.720*0.023 5.0 57 Dec. 5 12 10.42 55.03 0.527*0.018 5.0 33 Nov. 11 Dec 2 12 10.57 18.65 0.17610.011 5.0 0 2 June 3 % Bloom 84*2 26.47 J u l y 8 July 10 12 9.81 26.90 0.27310.017 5.0 10 Dec. 26 12 9.42 20.18 0.218+0.012 5.0 4 -Dec. 27 Jan. 2 10 9.93 18.33 0.185*0.009 5.0 0 3 July 2 5/6 Bloom 79.7 22.47 July 30 Aug. 27 11 9.88 33.51 0.339*0.020 5.0 15 Dec. 26 12 9.96 20.50 0.229*0.010 5.0 5 Dec. 27 Jan. 2 10 10.12 15.14 0.149*0.006 5.0 0 4 Aug. 1 1/5 Bloom 73.4 25.44 Aug. 29 Sept. 2 12 10.21 52.92 0.518*0.025 5.0 30 Dec. 27 12 10.78 49.79 0.460*0.022 5.0 27 TABLE LXIV The s t a b i l i t y of the estrogen-like compounds i n ground dry red  clover and i n the feed-extract mixture of the same sample  ( f i r s t cutting of 1956 seeding, cut May 3. 1957) Sample Description Time of Storage Mouse Bio-assay Stage of Per cent Maturity Moisture Per cent Protein Date of Extraction Date of Assaying Number of Animals Mean Body Weight (gm.) Mean Uterine Weight (mgm.) Per cent Body Wt. Feed Con-sumption/ 3 days (gm.) Estimated Potency per l b . Dry Matter (mcgm.) Vegetative 86,8 23.44 May 15 May 15 10 10.08 49.82 ' 0.495*0.026 5.0 36 Dec. 2 9 9.75 39.52 0.406+0.024 4.2 26 Oct. 21 Oct. 24 12 9.69 48.50 0.50310.018 5.0 31 -109 -1 greater magnitude than that i n the dry ground plant material, (TABLE LXIV). In other feed - red clover extract mixtures stored over longer periods of time , the breakdown was less than that which occurred i n the above sample. Cheng et a l . (38) demonstrated the presence of estrogenic substances i n a l s i k e , red and white clover and a l f a l f a hay. Pieterse et a l . (92) also found estrogen-like substances i n certain samples of a l f a l f a hay. However, no information was given i n regard to time of cutting, stage of maturity and time of storage of these samples. Pieterse et a l . (91) further showed that the estrogenic a c t i v i t y of a l f a l f a silage was s i g n i f i c a n t l y greater than that of fresh a l f a l f a . This type of storage seems to increase the potency as a res u l t of fermentation. E. The Effect of Plant Estrogens on Certain Laboratory Animals: Apart from the publications on sheep, grazing on subterranean clover i n Western A u s t r a l i a , l i t t l e l i t e r a t u r e i s available on the effect of plant estrogens on reproduction and growth. Engle (61) studied the conception rate i n three groups of Columbia ewes grazing on ladino clover, birdsfoot t r e f o i l and blue grass. The ewes grazing on blue grass conceived on the average three weeks e a r l i e r than those grazing on the other two pastures. S i x t y - s i x per cent of ewes grazed on blue grass lambed to f i r s t service, while on ladino clover the figure was 41 per cent and on lotus 31 per cent. The majority of the ewes on the legume pasture conceived after i t had been subjected to a k i l l i n g f r o s t . Mouse bio-assays revealed the presence of estrogenic compounds i n ladino clover and birdsfoot t r e f o i l but not i n blue grass. In humans, Coussens and Sierens (41) reported that a war-time - 110 -diet of t u l i p bulbs caused aberrations i n the reproductive cycle of Dutch women. Estrogenic substances were shown to be present with the aid of a mouse bio-assay technique. The influence of estrogenic sub-stances on milk production i n the cow has been postulated (10, 98) but no direct proof has been obtained. Mouse reproduction experiments Nos. I and I I and the guinea pig experiment were designed to obtain more detailed information on the effects of the plant estrogens on the reproduction and growth of animals. 1. Mouse Reproduction Experiment No. I. The plant material used i n t h i s experiment i s described under "Material" while the feeding and mating procedures are f u l l y discussed under "Methods". The mean i n i t i a l body weight, the mean body weight at mating and the average food consumption per day are shown i n TABLE LXV. The feed consumption of the mice receiving control ra t i o n with red clover extract was s i g n i f i c a n t l y decreased. The same was the case with mice receiving control r a t i o n plus birdsfoot t r e f o i l extract. As a result of t h i s reduced feed intake most mice on these rations decreased i n body weight. The effects of the d i f f e r e n t rations on the reproduction i n the mouse are given i n TABLE LXVI. In the discussion of the results the different groups of animals w i l l be i d e n t i f i e d by the rations they were receiving, e.g., "red clover females" are a group of female mice receiving the control diet containing red clover extract. "Control females" bred to "control males" showed 100 per cent f e r t i l i t y , i n d i -cating that the control diet did not i n t e r f e r e with reproduction i n the TABLE LXV Mean i n i t i a l weight, mean body weight at mating  and mean d a i l y feed consumption of mice on reproductive experiment 1 Females Males Mean I n i t i a l Body Weight (gm.) Mean Mating Body Weight -(gm.) Mean Feed Consumption per day (gm.) I n i t i a l Body Weight (gm.) Mating Body Weight (gm.) Mean Feed Consumption per day (gm.) Control 26.0 25.2 7.55 25.3 26.4 7.12 Control + Red Clover Extract 27.1 23.8 4.02** 25.3 21.0 5.61* Control + Birdsfoot T r e f o i l Extract 26.2 22.3 3.45^ 26.3 22.7 4.88 M Control + D.E.S. 26.1 25.9 7.41 26.7 26.7 6.97 ± P < 0.05 A* P < 0.01 TABLE LXVI Experimental data of female mice on reproductive experiment 1 H , u _ , _ . F i r s t Mating Second Mating Group Mating No. of Fe-males Bred No. of Fe-males L i t t e r i n g No. of Young Born Ali v e • Mean No. per L i t t e r Per cent Fer-t i l i t y No. of Fe-males Bred •No., of Females L i t -t e r i n g No. of Young Born A l i v e Mean No. per L i t t e r Per cent Fer-t i l i t y 1 Control Females x Control Male 5 5 48 9.6 100 0 0 0 0 0 2 Control Females x Red Clover Male 5 - 3 28 9.3 60 0 0 0 0 3 Control Females x Birdsfoot T r e f o i l Male 5 3 27 9.0 60 2 1 10 10 50 4 Control Females x D.E.S. Male 5 5 47 9.4 100 0 0 0 0 0 5 Red Clover Females x Control Male 5 0 0 0 0 5 4 41 10.3 80 6 Birdsfoot T r e f o i l Females x Control Male 4* . 1 9 9.0 25 3 3 25 8.3 100 7 D.E.S. Females x Control Male 5 2 17 8.5 40 3 3 32 10.7 100 8 Red Clover Females x Red Clover Male 5 0 0 0 0 5 5 35 7.0 100 9 Birdsfoot T r e f o i l Females x Birdsfoot T r e f o i l Male 3 M 0 0 0 0 3 3 34 11.3 100 10. D.E.S. Female x D.E.S. Male 5 3 17 5.7 60 2 .. 2 21 10.5 100 ft ftft One animal died during the pre-mating period as a result of the rati o n . Two animals died during the pre-mating period as a resul t of the ration. Remaining two animals of Group I I had a skin i n f e c t i o n and were removed from the experiment. - 113.-mou.se. Three groups of f i v e "control females" bred to "red clover males," "birdsfoot t r e f o i l males" and "D.E.S. males" showed 60, 60 and 100 per cent f e r t i l i t y , respectively. This shows that the males receiving the "D.E.S, ra t i o n " were l e s s affected by t h e i r diet than males on "red clover" or "birdsfoot t r e f o i l rations." Mating "control males" to groups of "red clover females," "birdsfoot t r e f o i l females" and "D.E.S. females" resulted i n 0, 0 and 40 per cent f e r t i l i t y . This indicates that the reproduction i n the females was affected to a much greater extent than that of the males. By mating "red clover females" to "red clover males," " b i r d s -foot t r e f o i l females to birdsfoot t r e f o i l males" and "D.E.S. females" to D.E.S. males, the per cent f e r t i l i t y of the groups was 0, 0 and 60, respectively. Bio-^assays of the feed - red clover extract mixture and the rati o n containing D,E,S. resulted i n the same increase i n uterine weight. However, the red clover extract mixture interfered with the reproduction i n the mouse to a much greater extent than did the r a t i o n containing 0.068 mcgm. of D.E.S. per gram of d i e t . This result i s doubly i n t e r e s t i n g i n that the estrogenic a c t i v i t y of the red clover extract expressed i n terms of D.E.S. was 0.068 mcgm, per gram dry matter. Those females which did not have a l i t t e r three weeks af t e r the mating period was terminated were bred again to the same males i n order to determine i f both males and females had recovered from the previous treatment. With the exception of one "control female" of group 3 and one "red clover female" of group 6, a l l females and males recovered t h e i r reproductive a b i l i t y . - 114 -Similar results were obtained by Fox et a l . (71) who incor-porated clover hay and fresh red clover i n a mouse diet at the 40 per cent dry matter l e v e l , and found that females and males f a i l e d to re-produce on these d i e t s . East (54) showed that when genistein, the estro-genic compound of red clover, was added to a mouse die t at the 0.2 per cent l e v e l i t lowered the number of l i t t e r s born, but not the number of young per l i t t e r . The present re s u l t s i n connection with red clover extract confirm these observations. However, East (54) also stated that genistein affected f e r t i l i t y i n the males to a greater degree than i n the females. This contradicts the results obtained i n t h i s experiment. Emmens (59) injected e s t r a d i o l benzoate into male, and female mice and observed that females were much more susceptible than males to t h i s synthetic estrogenic hormone. These observations and those of Bennetts (18) i n connection with rams grazing on "estrogenic" subter-ranean clover are more i n l i n e with the author's r e s u l t s . The results of t h i s study also confirm observations made by Emmens (59) and Fox et a l . (71) that male and female mice recover normal reproductive power after the hormone administration stops. 2. Mouse Reproduction Experiment No. II. The clover used i n t h i s experiment had a potency corres-ponding t o 12 mcgm. of D.E.S. This was approximately 60 per cent lower than that of the red clover used i n mouse reproductive experiment No.I. The control ration and the control r a t i o n containing red clover extract were the same as those used i n the guinea pig experiment. This was done to determine i f di f f e r e n t species of animals reacted d i f f e r e n t l y to the same l e v e l of estrogen-like substances. Housing, feeding and the mating plan of the animals are f u l l y discussed under "Methods." - 115 -The mean i n i t i a l body weight, the mean body weight at mating and the mean d a i l y feed consumption over t h i s period are given i n TABLE LXVII while the results on the reproductive performance of these animals are given i n TABLE LXVIII. Mating "control females" to "red clover males" resulted i n 60 per cent f e r t i l i t y , while "red clover females" mated to "red clover males did not reproduce at a l l . These results are i d e n t i c a l to those obtained i n mouse reproduction experiment No. I. I t should be noted that even though the results are the same, the r e l a t i v e potency of the red clover was 60 per cent lower than i n the f i r s t experiment. Again the males were affected to a lesser extent than the females. The mean d a i l y feed consumption of the animals receiving red clover extract was s i g n i f i c a n t l y lowered, as was the case i n mouse reproduction experiment No. 1. After the animals were returned to control diet they recovered quickly as i s shown by the number of l i t t e r s produced from the second mating (TABLE LXVIII). 3. The Guinea Pig Experiment. L i t t l e l i t e r a t u r e i s available on the influence of the plant estrogens on the growth of animals. Certain effects of estrogens i n pasture plants on fattening lambs have been postulated (38) but no direct proof has been given. Carter et a l . (34) showed that the growth of mice from three to seven weeks was not affected by either soybean o i l meal or genistein. This l a s t compound i s the main source of estro-genic a c t i v i t y i n red clover. A guinea pig experiment was designed to acquire more information TABLE LXVH Mean i n i t i a l weight, mean body weight at mating and. mean d a i l y feed consumption of mice on reproductive experiment I I Females Males Ration Mean I n i t i a l Body Wt. Mean Mating . Body Wt. Mean Feed Consumption per day (gm.) I n i t i a l Body Wt. (gm.) Mating Body Wt. (gm.) Mean Feed Consumption per day (gm.) Control 23.9 25.2 7.60 25.2 26.4 7;. 20 Control + Red Clover Extract 23.6 23.2 4.20 28.3 27.7 5.04* i H H O ± P 0.05 ±k P 0.01 TABLE LXVIII Experimental data of female Swiss albino .mice fed control ration containing red clover extract (mouse reproductive experiment II) F i r s t Mating Second Mating Group Mating No. of Females Bred No. of Females L i t -t e r i n g No. of Young Born Alive Mean No. per L i t t e r Per cent F e r t i l -i t y No. of Females Bred No. of Females L i t -t ering No. of Young Born Ali v e Mean No. per L i t t e r Per cent Fer-t i l i t y 1 Control Females x Control Male 5 5 48 9.6 100 0 0 0 0 0 2 Control Females x Red Clover Male 5 3 '34 11.3 60 2 2 15 7.5 100 3 Red Clover Females x Red Clover Male 5 0 0 0 0 5 5 58 11.6 100 - 118 -on the effects of plant estrogens on growth and reproduction of animals having a digestive t r a c t comparable to that of a ruminant. Growth curves obtained during the pre-mating period of the females receiving control r a t i o n are given i n FIGURE VII and those of the animals receiving control r a t i o n containing red clover extract i n FIGURE VII I . S t a t i s t i c a l analysis revealed that the growth of guinea pigs weighing 500 to 600 grans was not s i g n i f i c a n t l y influenced by adding red clover extract to the r a t i o n . Feed consumption was a l i t t l e increased as compared to the animals receiving control r a t i o n (TABLE LXIX). However, t h i s increase was not s i g n i f i c a n t . TABLE LXIX Mean i n i t i a l body weight, mean body weight at mating, and mean feed consumption per day of female  guinea pigs over pre-mating period (33 days). Group Ration Mean I n i t i a l Mean Body Mean Feed Con-Body Wt. (gm.) Weight at sumption per day Mating (gm.) (gm.) 1 Control 548 565 22.8 2 Control Con-tain i n g Red Clover Extract 553 575 24.7 Five"control females" were mated to a "control male", and f i v e "red clover females to a "red clover male." In the f i r s t group two out of f i v e females conceived and i n the second group three out of f i v e were pregnant, (TABLE LXX). 119 -Growth Curves of Female Guinea Pigs Receiving C o n t r o l D i e t Containing C o n t r o l D i e t . - 120 . QQ | 1 1 1 1 1 1 ^ W 5 10 15 20 25 30 Time i n Days FIGURE VIII , f Growth Curves of Female Guinea Pigs Receiving Control Diet Containing Red Clover Extract. - 121 -TABLE LXX Conception rate of guinea pigs I n i t i a l K u f f i b e r Q f F e m a l e s No. of Mean No. No. of — — —— ; Fetuses per Ration Females A l i v e A l i v e at at end Female Time of of Pregnant Mating Mating Period 1 Control 5 5 4 2 a i i 2 Control + 5 5 5 3 9 3 Red Clover Extract A One early abortion Since the f e r t i l i t y i n the control group was poor and that of the "red clover group" below average no d e f i n i t e conclusion can be reached i n regard to the effect of red clover extract on reproduction i n the guinea pig. East (53) showed that ingestion of subterranean clover caused i n f e r t i l i t y i n female guinea pigs, and that t h i s was due to f a i l u r e of conception rather than to f a i l u r e of implantation. However, the results of t h i s experiment show that the repro-duction i n the guinea pig i s affected t o a much lesser extent i f not at a l l , by red clover extract as compared to that of female mice receiving the same rati o n . - 122 -VII. SUMMARY AND CONCLUSION To determine the estrogenic a c t i v i t y i n certain forage samples a suitable mouse bio-assay procedure was developed. This pro-cedure consisted of having an assay period of 72 hours during which each animal received f i v e gm. of d i e t , of fasting the animals for eight hours, and of using uterine weight expressed as a per cent of body weight as the measurement of estrogenic a c t i v i t y of the experimen-t a l d i e t s . A dose response curve was then constructed using once r e c r y s t a l l i z e d d i e t h y l s t i l b e s t r o l (D.E.S.) as the reference compound. From t h i s curve the estrogenic potency of the samples of plant material was estimated, and expressed i n terms of D.E.S. Before studying the effect of stage of maturity, frequency of cutting and length of daylight (photoperiod) on the estrogen-like compounds of plant material, a number of preliminary experiments were carried out. The data of these experiments revealed that estrogenic a c t i v i t y of red clover was not s i g n i f i c a n t l y influenced by leaving the plant material on the f i e l d f o r 48 hours, before i t was dried at 150°F. for 18 hours. Furthermore, the estrogenic a c t i v i t y of feed -red clover extract mixtures was not s i g n i f i c a n t l y influenced by either varying the drying time between 7 and 24 hours (drying temperature constant at 65°C), or by varying drying temperature between 65 and 102°C. (drying time constant at 19 hours). The studies of the effects of stage of maturity and frequency of cutting of a l f a l f a , white clover, red clover, birdsfoot t r e f o i l and orchard grass on the estrogen-like compounds i n these species, revealed - 123 -that there was a great difference between samples taken from d i f f e r e n t plant species at the same time of the year (PLATE I ) . A seasonal v a r i a t i o n i n the estrogenic a c t i v i t y occurred i n the f i r s t cuttings of second year growth of the different plant species (FIGURE V I I I ) . A l f a l f a and white clover possessed high estro-genic a c t i v i t y i n the spring (May), showed a sharp decrease i n June and July, while after August 1st, the active substances were present once again i n considerable quantities. The only samples of birdsfoot t r e f o i l , orchard grass and al s i k e clover that showed s i g n i f i c a n t estrogenic a c t i v i t y were the f i r s t cuttings of second year growth harvested on May 3. An exception to t h i s was the sample of als i k e clover cut on August 1st. This plant material was i n the dough state and appeared moldy. However, i t i s possible that i n t h i s case the estrogen-like substances were produced by the molds, since certain micro-organisms are capable of producing these estrogenic compounds. Red clover d i f f e r e d greatly from the previously mentioned species. Again samples taken i n May were highest i n potency, but there was no sharp decrease during June and July as was the case with a l f a l f a and white clover. Nevertheless there was a gradual decrease towards the f a l l . Samples of f i r s t year growth were r e l a t i v e l y higher i n estrogenic a c t i v i t y than the samples from second year growth cut at the same time. Here again red clover d i f f e r e d considerably since none of the samples of f i r s t year growth of the other species studied showed sig n i f i c a n t amounts of estrogen-like substances. The hypothesis that estrogenic a c t i v i t y i s associated with reproductive growth (82) holds true f o r the samples of red clover studied. However, t h i s hypothesis i s not v a l i d for a l f a l f a and white clover, since these - 124 -THE INFLUENCE OF ESTROGENIC EXTRACT CF DIFFERENT SPECIES  OF FORAGE ON UTERINE WEIGHT A i \ CONTROL * Uterine weight 19.60 mgm. % Body weight .179 ORCHARD GRASS 1st cutting, 3 r d May, 1957 Vegetative Uterine weight: 21.00 mgm. % Body weight .195 LOTUS 1st cutting, 3rd May, 1957 Vegetative Uterine weight: 38.35 % Body weight .372 A RED. CLOVER 1st cutting, 3rd May, 1957 Vegetative Uterine weight: 54.05 mgm. % Body weight: .524 ALFALFA 1st cutting, 3rd May, 1957 Vegetative Uterine weight: 60.15 mgm. % Body weight: . 5 8 I A WHITE CLOVER 1st cutting, 3rd May, 1957 Vegetative Uterine weight£3.05 mgm. % Body weight: .612 * A l l animals received 5 gm. of feed mixture/3 dayi and were fasted f o r 8 hours. PLATE I • A l f a l f a o White clover © — • — Red clover o A l s i k e clover A Birdsfoot t r e f o i l A Orchard grass May 2 May 13 May 25 June 3 June 18 J u l y 2 J u l y 18 Aug. 1 Date of Cutting 3ept. 3 FIGURE VI The Estrogenic A c t i v i t y of Second Year Growth of D i f f e r e n t Species of Plants at Various Stages of Maturit y . - 126 -species were low i n estrogenic a c t i v i t y when they were i n the repro-ductive stage (June and July) and showed an increase i n the " l a t e bloom" and "past bloom" stages. The results also indicate that estrogenic a c t i v i t y i s not s o l e l y associated with the phase of rapid growth, since the samples of f i r s t cuttings of second year growth of a l f a l f a and white clover harvested i n August and September showed higher estrogenic a c t i v i t y than second, t h i r d and fourth cuttings taken at the same time. Furthermore, f i r s t cuttings of f i r s t year growth of a l f a l f a and white clover did not contain any estrogen-like substances. The r e s u l t s also disprove a t h i r d hypothesis stating that seasonal v a r i a t i o n i s due to a change i n lamina to stem r a t i o (2). The data obtained by varying the number of hours of daylight (photoperiod) received by the plant seem to indicate that the estro-genic a c t i v i t y i n red clover decreased when the photoperiod was shortened. Proximate analysis of the samples revealed further that estrogenic a c t i v i t y was not correlated with the nitrogen content of the sample. The study on the s t a b i l i t y of the estrogen-like compounds i n a l f a l f a , white clover and red clover suggested that the estrogenic a c t i v i t y of a l f a l f a and white clover was due to a d i f f e r e n t compound than that of red clover. This observation has been proven to be correct by Bickoff et a l . (24) who isolated coumestrol from ladino clover, a l f a l f a and strawberry clover. This compound seems to be e a s i l y destroyed during storage (TABLES LX to LIV) while genistein of red clover i s much more stable (TABLE LXIV). This may be due to the - 127 -fact that coumestrol contains i n i t s chemical configuration an oxygen bridge which could be e a s i l y destroyed. I t i s interesting to note that estrogen-like compounds of a l f a l f a and white clover are not as e a s i l y destroyed i n the feed - extract mixtures as i n the dried ground forage sample. The reason f o r t h i s may be due to the presence of a compound(s) which degrades the active substance. The animal reproduction experiments showed c l e a r l y that red clover and birdsfoot t r e f o i l extract interfered with the reproduction i n the mouse. In both cases the females were more severely affected than the males. Furthermore, i t was int e r e s t i n g to note that the reproduction i n the mouse was influenced to a greater extent when red clover extract was fed than when a corresponding quantity of D.E.S. as measured by the uterine weight technique, was added to the d i e t . In nearly a l l cases both males and females recovered t h e i r reproductive a b i l i t y quickly when the feeding of estrogen-like compounds ceased. In female guinea pigs weighing between 500 and 600 gm. the growth rate was not s i g n i f i c a n t l y influenced by the addition of red clover extract to the d i e t . - 128 -V I I I . BIBLIOGRAPHY 1. ALEXANDER, G., and WATSON, R. H., " 1 The assay of estrogenic a c t i v i t y of Trifolium. subterranean L. by increase i n uterine weight i n spayed guinea pigs.' I Characteristics of dose-response relationship." Australian Journal of A g r i c u l t u r a l Research. 1951, 2: 457-479. 2. ALEXANDER, G., and WATSON, R. H., !' 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