METABOLIC EFFECTS OF DIETHYLSTILBESTROL GN GROWING SHEEP by JAMES ROBERT THOMPSON B.S.A., University of British Columbia, 1964 A THESIS SUBMITTED UJ PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in the Division of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1966 In presenting t h i s thesis 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 University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y aval]able for reference and study. I further agree that permission-for extensive copying of t h i s thesis f o r scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The feeding of 2.5 mgm. of diethylstilbestrol (DES) per head per day to growing wethers fed a dehydrated grass meal ration increased dry matter intake by 6 per cent and decreased dry matter di g e s t i b i l i t y by 2.4 per cent. The DES-treated lambs digested 5.4 cent (P<.05) more crude protein, 9.2 per cent (P<.20) more ether extract, 15 per cent (P< .01) less crude fibre, and 3.5 per cent (P<.01) less nitrogen-free extract. The use of a two stage in vitro digestion technique did not reveal any influence of a wide range of DES levels upon dry matter d i g e s t i b i l i t y . The implantation of 12 mgm. DES in growing ewes and wethers increased pituitary and adrenal gland weights by 46 per cent (P<.005) and by 23 per cent respectively. The thyroid gland weights were decreased 21 per cent (P<.005). The gross histology of the pituitary gland was not found to be changed by DES treatment The use of 1^1 did not reveal changes in the activity of the thyroid gland following the feeding of 1.0, 2.0, and 2.5 mgm. DES per day to growing wethers. Lambs fed an all-concentrate ration and implanted with 12 mgm. DES gained more rapidly and ef f i c i e n t l y than their controls The DES-treated lambs had a lower dressing percentage, and required less time to reach market weight. These results indicate that the action of DES in enhancing growth rate and efficiency of gain in the ruminaat appears to be mediated by one or more mechanisms. First, DES may alter the digestive activities of the rumen microflora by selectively promoting their proteolytic activity and inhibiting their c e l l u l o l y t i c activity. It may, nevertheless, allow for an increased absorption of the degrad-ation products of protein metabolism. These products may be present in a more readily digested form or may be more readily passed through the intestinal tract wall. Secondly, DES may stimulate growth of the anterior pituitary, resulting in an altered production of a number of the tropic hormones arising from this gland. With am increased growth of the anterior pituitary, an increased level of STH could account for a more efficient feed u t i l i z a t i o n . Control and DES-treated lambs u t i l i z e approximately the same amount of apparent digestible energy, however DES-treatment resulted in an increased growth rate. This must be due to an increased u t i l i z a t i o n of net energy, possibly associated with an alteration in tropic hormone production. TABLE OF CONTENTS Page I. Introduction 1 II. Literature Survey 3 1. Definition of an Estrogen 3 2. Compounds Possessing Estrogenic Activity 4 (a) Endogenous to the animal kingdom 4 (b) Obtained from synthetic sources 5 (c) Endogenous to the plant kingdom 5 3. Relative Activities of Estrogenic Compounds 8 (a) Relative potencies in vaginal smear tests 8 (b) Relative potencies in vaginal mitosis and epithelial thickness 10 (c) Relative potencies in stimulating . . vaginal metabolism 10 (d) Relative potencies in uterine weight tests 11 (e) Relative potencies according to a number of miscellaneous parameters 12 (f) Relative anabolic potencies in the ruminant 12 4. Structural Requirements for Estrogenic Activity 13 (a) In the steroid nucleus 13 (b) In the isoflavone nucleus 13 (c) In the sti l b e s t r o l nucleus 14 5. Action of Endogenous Mammalian Estrogens 15 (a) Metabolism in the animal body 15 (b) The action of steroidal estrogens in the animal body 17 (c) Natural animal estrogens in animal production 21 6. Action of the Synthetic Estrogen Diethylstilbestrol 23 (a) Metabolism in the animal body 23 (b) Action of the synthetic estrogens in the animal body. 24 1) F i r s t reported activity 24 2) I n i t i a l use in animal production 25 3) Use of diethylstilbestrol in swine production 26 4) Action of diethylstilbestrol in the ruminant 26 (a) I n i t i a l use 26 (b) Effect of diethylstilbestrol on rate of gain 26 (c) Effect on feed efficiency 29 (d) Effect on marketing 30 (e) Effect on carcass grade 31 (f) Effect on anatomy and physiology 32 (1) Blood composition 32 (2) Anterior pituitary and i t s secretions 33 (3) Thyroid and i t s secretions 34 (4) Adrenal cortex and i t s secretions 34 (5) Urogenital system 35 (6) Protein and nitrogen metabolism 37 (7) Calcium and phosphorus metabolism 38 (8) Digestibility of feed nutrients 39 (9) Rumen metabolism and microbial population 39 (c) The other synthetic estrogens 40 7. Action of Phytoestrogens 40 (a) Metabolism in the animal body 40 (b) Influence of phytoestrogens in livestock production 41 III. Study I.- Effect of Diethylstilbestrol on the Digestibility of Various Feed Components 44 1. Introduct ion 44 2. Materials and Methods 44 (a) Part A 44 (1) Experimental animals 44 (2) Housing 44 (3) Basal ration 45 (4) Experimental ration (preparation and feeding) 45 (5) Chemicals 46 (6) Experimental design 46 (7) Experimental methods 46 (b) Part B 47 (1) Incubation apparatus 47 (2) Source of rumen microflora 47 (3) Microbial substrate 48 (4) Chemicals 48 (5) Experimental methods 48 3. Results and Discussion 48 IV. Study II.- Effect of Diethylstilbestrol on Endocrine Function in the Fattening Market Lamb 60 1. Introduction 60 2. Materials and Methods 60 (a) Part A 60 (1) Experimental animals 60 (2) Counting apparatus 60 (3) Plateau considerations 61 (4) Radioactive sodium iodide 62 C ) Dilution of radioactive sodium iodide 62 (6) Method of injection into wethers 62 (7) Preparation of standard 62 (8) Method of measuring thyroid gland activity 63 (9) Expression of thyroid gland activity 64 (b) Part B 64 (1) Experimental animals 64 (2) Housing and feeding of experimental animals 64 (3) Experimental methods 65 3. Results and Discussion 66 V. Conclusions 75 VI. Bibliography 77 VII. Appendices 88 LIST OF TABLES TABLE I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. Median effective doses with 95 percent fiducial limits of a number of estrogens tested in the ewe Relative oral and subcutaneous activities of six estrogens given to spayed rats. The relative activity of various estrogens using estrone as a standard, when given intravaginally In one injection on the mitotic count and epithelial thickness tests. Optimal levels of oral and perenteral administration of estrogens to stimulate growth in cattle and sheep. Proximate analysis of the basal diet, dehydrated grass meal. Experimental design Average daily dry matter consumption of dried grass meal. Dry matter d i g e s t i b i l i t y , in vivo. Component digestion for wethers fed control ration Component digestion for wethers fed 2.5 mgm. diethylstilbestrol per day Summary of in vivo d i g e s t i b i l i t y study results. Dry matter d i g e s t i b i l i t y , in v i t r o . Average daily component intake of wethers fed control ration Average daily component excreta of wethers fed control ration Average daily component intake of wethers fed 2.5 mgm. diethylstilbestrol per day Average daily component excreta of wethers fed 2.5 mgm. diethylstilbestrol per day Composition of U.B.C. lamb ration (L-65) Composition of Bouin-Duboscq fixative Thyroid gland activity of wethers fed control and experimental rations. (Thyroid counts per minute as a percentage of standard counts per minute) Page 9 10 13 45 46 50 51 52 52 53 54 56 56 57 57 65 66 67 XX. Endocrine gland weights of lambs fed control ration XXI. Endocrine gland weights of lambs implanted with 12 mgm. of diethylstilbestrol XXII. Summary of lamb endocrine gland weight means XXIII. Summary of lamb feeding t r i a l data XXIV. Individual body weight records of wethers XXV. Percent absorbancy of dehydrated grass meal extracts at various wavelengths, (millimicrons) XXVI. Percent absorbancy of sheep feces extracts at various wavelengths (millimicrons) XXVII. Preparation of basal medium: Composition of in vitro fermentation medium XXVIII. Proximate analysis of feces from wethers fed control ration (26/9/65) XXIX. Proximate analysis of feces from wethers fed control ration (10/10/65) XXX. Proximate analysis of feces from wethers fed Ration No. 2 (2.5 mgm. diethylstilbestrol/ head/day) (11/11/65) XXXI. Proximate analysis of feces from wethers fed Ration No. 2 (2.5 mgm. diethylstilbestrol/ head/day) (13/11/65) XXXII. Summary of proximate analyses data for feces of individual wethers. XXXIII. Detection of plateau curve (counts per minute at various voltages obtained from a standard volume of I1"** solution) XXXIV. Individual market data for lambs fed control ration XXXV. Individual market data for lambs implanted with 12 mgm. diethylstilbestrol XXXVI. Diethylstilbestrol implant weights (mgm) XXXVII. Diethylstilbestrol implant residue weights 69 69 70 73 89 91 92 99 104 105 105 106 106 107 109 109 110 110 LIST OF APPENDICES Page Appendix I. 89 Appendix II. 90 Appendix III. 98 Appendix IV. 102 Appendix V. 104 Appendix VI. 107 Appendix VII. 109 Appendix VIII. 110 LIST OF FIGURES Page Fig. I. Absorption curves for acetone extracts of grass meal and sheep feces 93 Fig. II. Apparatus for the extraction of chromogens from sheep feces and grass meal 95 Fig.ICI. Counts per minute at various voltages 108 ACKNOWLEDGEMENTS The writer wishes to express h i s thanks to Dr. B. A. Eagles, Dean of the Facu l t y of Ag r i c u l t u r e and Chairman of the D i v i s i o n of Animal Science for allotting the use of the departmental f a c i l i t i e s . To Dr. W. D. K i t t s , Professor of Animal Science, the writer g r a t e f u l l y expresses h i s thanks f o r the encouragement, consultation, and guidance received throughout the course of t h i s study. The assistance of Miss Ann Warner, graduate student i n the D i v i s i o n of Animal Science i s g r a t e f u l l y acknowledged. Thanks are also extended to Mr. L e s l i e Holmes and Mr. C l i f f o r d Harvey of the D i v i s i o n of Animal Science for t h e i r assistance during the experimental work. 1. I. INTRODUCTION The synthetic estrogenic compound, diethylstilbestrol (DES) has found widespread use in the livestock industry. DES has been shown to improve rates of gain and feed efficiency, as well as to decrease production costs, in both cattle and sheep raised for meat. A number of detrimental effects are associated with the administration of DES to livestock. These effects include: urinary blockage, rectal and vaginal prolapse, urogenital hypertrophy, as well as general nervousness and "estrus-like"behaviour. Much work at present is concerned with the development of compounds that w i l l enhance growth in market animals without causing harmful secondary effects. Combinations of progesterone and estradiol benzoate have been shown to be most effective in meeting these requisites. Understanding the physiological mechanisms by which the known growth stimulants act is necessary before new and improved compounds can be produced. Since the growth stimulant DES is known to possess considerable estrogenic activity, i t has been suggested that i t may affect the growth and activity of the various endocrine glands. DES may also have a broad spectrum of activity on the rumen microflora. This study was designed to explore two aspects of the physiological activity of DES in the growing lamb. F i r s t , the effect of DES on the microbial activity of the rumen was studied in terms of its effect on the d i g e s t i b i l i t y of various feed components. Secondly, 2. the e f f e c t of the s y n t h e t i c hormone on the a c t i v i t y of the t h y r o i d 131 gland was s t u d i e d i n terms of I uptake. Endocrine gland weights, r a t e of g a i n and e f f i c i e n c y of g a i n i n both c o n t r o l and DES-implanted animals were compared. 3. II. LITERATURE SURVEY 1. Definition of an Estrogen In 1923 Allen and Doisy used the term "estrogen" as applied to any pure chemical substance which has the a b i l i t y , when injected into the adult ovariectomized mouse or rat, to produce cornification of the vagina similar to that occurring during estrus in the normal animal (2, 3). This definition of an estrogen developed into the Allen-Doisy test and was based upon the observations of Stockard and Papinicolaou (197), who f i r s t reported the cyclic vaginal corn-i f ication of guinea pigs. The definition has since been extended to include estrogenic substances of unspecified chemical identity. The estrogenic activity of a pure chemical compound or crude material extract may be expressed as the increase of the total uterine weight in the intact immature mouse or ovariectomized immature rat. Approximately 300 compounds are known to satisfy these general definitions and, as far as is known, they a l l give a similar, characteristic response. Different estrogens do, however, vary greatly in potency. The estrogens may be classified into three groups: a) The true estrogens - those compounds capable of inducing the characteristic response as determined by the Allen-Doisy test (e.g., estradiol 17-^ Q). b) The impeded estrogens - which, though estrogenic, tend to inhibit the activity of a second estrogenic compound (e.g., e s t r i o l ) . 4. c) The pro-estrogens - those compounds which, when applied to the vagina, do not give the characteristic response un t i l they have been absorbed, metabolized in the li v e r , and returned to the vagina (e.g., androst-5 ene-3^, 17yJ d i o l ) . 2. Compounds Possessing Estrogenic Activity (a) Endogenous to the animal kingdom A l l the natural estrogens of animal origin isolated to date possess the cyclopentanoperhydrophenanthrene nucleus. In contrast to the androgens and progesterones, estrogenic steroids have a phenolic A-ring and a carbon atom in position 18, but not in position 19. The five major steroidal estrogens of animal origin are estradiol 17-j2, estrone, e s t r o i l , equilin, and equilenin. These compounds are a l l true estrogens, but in some instances act as impeded estrogens. Estradiol 17-ft, f i r s t isolated from sow ovaries by McCorquodale et a l . (135), is the most potent according to nearly a l l methods of testing. It is l i k e l y the only estrogen produced by the ovary; i t is also produced by the adrenals, placenta, and in the stallion, by the testes. Estradiol 17-e0.05) faster and more efficient rate of gain than control boars. The implantation of lighter animals has resulted in excessive fat deposition. (4) Action of diethylstilbestrol in the ruminant (a) I n i t i a l use DES was f i r s t used in ruminant nutrition in 1949 by Andrews et a l . at Purdue (6, 5). These workers found that the. implantation of 12 or 24 mgm. of DES subcutaneously in 70 pound feeder lambs resulted in faster gains and more efficient conversion of feed to body tissue. In 1950 i t was shown that implanting beef heifers with 42 mgm. of DES increased rate of gain, feed consumption and feed efficiency (63). The implantation of 60 and 120 mgm. of DES in yearling steers on a fattening ration, increased the rate of gain as well as the feed efficiency (7). (b) Effect of diethylstilbestrol on rate of gain It has been shown and accepted by most laboratories that either the feeding or Implantation of DES w i l l increase the rate of gain in ruminants. As a feed additive in fattening lambs, the recommended 27. l e v e l i s 2 mgm. per head per day. The implant should contain 12 mgm. of DES. The optimal l e v e l of DES to be fed to fattening beef c a t t l e i s 10 mgm. per head per day. The implant should contain 24 mgm. DES. The early workers at Purdue (6, 5) found that the implantation of either 12 or 24 mgm. DES i n lambs was equally e f f e c t i v e i n increasing rates of gain by as much as 30 per cent. These results have subsequently been confirmed (113, 52, 124, 205). A number of workers explored the use of 3 and 6 mgm. DES implants (115, 21, 70, 165). I t was found that a response i s often obtained but i s too irregular for commercial pro-duction. The response to implanted DES i n suckling lambs has not been found to be as great as the response i n weaned lambs (112, 167, 114, 70). The administration of one 12 mgm. implant, without l a t e r implantation, appears adequate for the market lambs (137, 20). L i t t l e sex i n t e r -action has been reported (50). The implantation of DES with progesterone has been shown to give results superior to low levels (6 mgm.) of DES only (82, 61, 21, 142). Testosterone propionate has a s i m i l a r effect (12). I n i t i a l l y 60-120 mgm. DES implants were used for stimulating the rate of gain i n beef c a t t l e (63, 7, 50). O'Mary et aJU (161) found that 24 mgm. implants were equally as ef f e c t i v e . Exhaustive studies by Fontenot i n 1959 (80) and Roubicek i n 1960 (186) showed that one 24 mgm. DES implant w i l l give optimal results i n fattening c a t t l e . Further increases i n rate of gain have not been obtained by giving a second implant 6 or more weeks following the f i r s t . Deleterious side effects have often been noted i n animals given a second implant (160, 162, 144, 186, 145). The implantation of suckling steer calves with 12 or 24~mgm. DES has resulted i n increased rates of gain, without effects on future performance (208, 143). 28. From a genetic point of view, i t has been noted that inherently fast-growing animals show a greater growth response to the implantation of DES than do inherently slow-growing animals (163, 71). Hale and co-workers (88, 89) f i r s t explored the po s s i b i l i t i e s of feeding DES to fattening lambs at the levels of 0.5 mcgm. to 1200 mcgm. per pound of ration. Increases in rate of gain were found at levels of 600 or more mcgm. DES per pound of ration. Later studies (90) showed the feeding of 2 mgm* (500 mcgm.) DES per head per day to increase gains by 22 per cent without any apparent reduction of carcass grades or harm to animal health. The feeding of 3.6 mgm. per head per day failed to promote as satisfactory increases in rate of gain, and tended to enhance a number of detrimental effects. DES has been shown to give best results when fed with a ration designed to meet the optimal requirements of the fattening animal (209, 172, 110). The actual source of ration ingredients, such as protein, does not appear to be influential (126). However, ration ingredients high in phytoestrogens (e.g., a l f a l f a meal) may tend to n u l l i f y the response to DES (45). Hartman et a l . have shown that a greater response to DES may be obtained with lamb rations containing higher roughage levels (92). The feeding of 1.0 mgm. testosterone with 2 mgm. DES has been shown to increase the rate of gain 14 to 20 per cent above that obtained with DES alone. Lambs fed DES during the hot summer months have not responded as well as those treated in the winter months (88, 1). A breed response to the feeding of DES has often been noted. Black-face lambs tend to respond more favourably than white-face lambs (39). Both ewes and wethers give a similar response to the feeding of DES. 29. Burroughs and co-workers, in 1954 (38) were the f i r s t to show the favourable effects of feeding DES to fattening steers and heifers. Both steers and heifers weighing 600 to 700 pounds or more, gained 20 per cent faster when fed 10 mgm. DES per head per day (38, 37). This treatment is effective throughout the fattening period and is particularly beneficial in animals fed high concentrate rations (38). Suckling steer calves have shown increases in rate of weight gain due to DES feeding. These results have been confirmed by a number of workers (138, 36). Feeding 10 mgm. DES per head per day has been shown to be superior to the implantation of 36 mgm. DES. As shown with lambs, ration components possessing estrogenic activity tend to n u l l i f y the response to DES (17, 134, 219). Feeding optimal protein levels to maintain rapid growth was " required to obtain best results from feeding DES (122, 183, 165). Previous DES treatment (e.g., with implants), reduced the expected response to DES feeding (219). The feeding of testosterone with DES tended to enhance the growth response (16). (c) Effect on feed efficiency Experiments considering feed efficiency have only compared the total amount of nutrients required per unit of gain between treated animals and control animals. The composition of these two animals differs at market weight with the treated animals containing less fat per unit body weight. Since these differences have not been considered, only a comparison of the gross feeding efficiency is possible. In feeding lambs, the gross feed efficiency ranges between 15 and 30 per cent greater than controls when these animals are fed or implanted with DES. In fattening steers, this value is lower, ranging from 9 to 12 per cent. Values in almost a l l experiments recorded have fallen within these ranges. Variations within a given range depend 30. upon the nutritional program. Ad libitum feeding gives results superior to restricted feeding (158, 138). High energy rations have been shown to give results superior to low energy rations for cattle (200). In attempts to analyze the apparent increase in feed efficiency more thoroughly, Clegg and Cole (52) found that 122 pounds of TDN (total digestible nutrients) are saved for each 100 pounds of gain with beef steers. Only 36 pounds of TDN were saved with beef heifers. Whiting et a l . (212) stated that the implantation of 12 mgm. DES in market lambs increased feed efficiency only because of an increase in o f f a l and tissue water content. Garret, in 1964 (83), found that improved feed efficiencies in DES implanted lambs were not real when placed on a caloric basis. (d) Effect on marketing In lambs, reduced carcass grades (measured by U.S.D.A. grades) following the implanting or feeding of DES are prevalent throughout the literature (104). This effect is most significant in implanted animals. Reduced carcass grades are not usually recorded in lambs fed up to 2 mgm. DES per day (90, 126). DES fed at higher levels tends to reduce carcass grades enough to be of economic importance (90, 60). Under a l l regimes of DES administration, percentage carcass yield is reduced 0.5 to 2.0 per cent (114, 61, 92, 68). Both live and carcass shrinkage in DES treated lambs is greater than in control lambs (114, 194, 172, 92). The incorporation of progesterone with DES implants has improved carcass yields and grades above those of lambs receiving control DES implants (82, 116). No effect on carcass shrinkage has been shown in DES implanted steers (160, 161, 145) or in DES fed beef steers (62) or heifers (32). 31. The carcass grades of bulls implanted with DES have been shown to approach those of non-treated steers (121, 125, 42). Heifers, on the other hand, whether implanted (42, 144, 123) or fed (144) DES have shown slight reductions in carcass quality. The carcass grades of beef steers are not affected unless implants contain more than 24 mgm. DES (209, 218, 104). Cattle treated with DES and graded "on the hoof", often tend to grade lower because of the appearance of a typical "nymphomaniac stance" (104). Even though grades have been reduced by DES administration, the organoleptic quality of the meat judged by tenderness, juiciness and flavour, does not appear to be affected (42, 62, 191, 68). Some reports of decreased tenderness have been recorded (8), and are likely due to the increase of connective tissue. (e) Effect on carcass composition Thorough studies of the changes in lamb carcass composition following DES administration have been carried out by O'Mary ejt a l . (158, 159) and Wilkinson et a l . (214). These workers found a reduced thickness and weight of external fat, an increased weight of bone and connective tissue, an increased moisture content of external fat, an increased pelt weight, an increased carcass length and an increased weight of the viscera. Treated wethers tended to have less external fat containing more moisture than treated ewes (158). Limited feeding of wethers intensified the effect of DES in retarding the development of the late maturing tissues, particularly external fat (159). Although Wilkinson et a l . (214) did not demonstrate differences in the size of the longissimus dorsi muscle, Ellington (68) found a significant increase 32. in its size due to DES treatment. Other workers (114, 34) have shown a decrease in internal fat in treated animals. The iodine number of this fat has not been shown to change after DES administration. The response of cattle carcass composition to estrogen treat-ment is similar to, but not as apparent as, the response of the lamb carcass (104). Frequent reports have been made indicating a definite increase in the size of the longissimus dorsi muscle (217, 218). Most parameters indicate only slight deviations in carcass composition from control animals when either steers or heifers are administered DES at the recommended levels. In bulls, the administration of DES causes an increase in fat and a reduction of the edible portions of the carcass (42, 123). (f) Effect on anatomy and physiology 1. Blood composition: The i n i t i a l work of Dinusson et a l . (63) revealed that the implantation of DES in beef heifers reduced the erythrocyte count from 8.53 + 0.19 to 7.86 + 0.16 million per cc. Fletcher et aU_ (77) could find no consistency in hemoglobin or hematocrit values in beef calves implanted with 24 mgm. DES. Clegg and Cole (52) indicated that the implantation of DES caused a drop in the percentage of eosinophiles in both ewes and wethers. In 1954, Wilkinson et a l . (213) carried out an extensive study of the effect of a single 15 mgm. DES implant on finishing wethers. The blood was found to contain: a lower hematocrit, higher levels of plasma-free-cholesterol and fibrinogen, higher levels of globulin, higher total protein and lower NPN (non-protein-nitrogen) and phospho-l i p i d levels. No effect upon blood glucose, albumin, ester cholesterol, 33. total cholesterol or neutral fat was found. Work in 1965 by Johnston et a l . (108) showed that DES tends to depress serum alkaline phos-phatase activity. Work with beef cattle has shown very few changes in blood composition after DES administration. No influence has been shown on levels of glucose, NPN, serum protein-bound iodine, and sodium or potassium levels in Holstein heifers or beef steers (55, 77). The implantation of 36 mgm. DES tends to decrease blood urea levels (174), but has no effect on the circulating ©C- amino acid levels in steers (155). Clegg (51) found no changes in blood l i p i d s , indicating that no changes in fat metabolism are associated with DES administration in beef steers. Fomichou et_ a l . (79) have indicated that the blood level of unsaturated fatty acids in finishing steers implanted with 24 mgm. DES may drop as much as 21 per cent. It is evident that more work needs to be done in the f i e l d of blood chemistry. Work with blood lipids and phospholipids would be of particular importance. 2. Anterior pituitary and i t s secretions: Early studies (52, 41, 42) revealed an increased total pituitary weight in steers, bulls and heifers following treatment with DES. Clegg eJL a l . (54) demonstrated that the.dried weight of the anterior pituitary was increased 20 per cent in DES implanted lambs. Shroder in 1958 (189) suggested that the increase in pituitary gland weight was due to either hypertrophy or hyperplasia. The work of Hinds et a l . (100) clearly demonstrated that the increase in size of the pituitary from treated cattle was due to hyperplasia (increased DNA per whole pituitary) rather than hypertrophy (increased RNA per unit weight). 34. The potential for protein synthesis per unit weight (increased RNA per unit weight) was not influenced, neither was the amount of nitrogen per unit weight. The increase in total weight of the gland in DES treated animals is more extensive when the animal is on a high energy ration (172). Associated with the increase in anterior pituitary size is an increase in total STH (somatotropic hormone) production in cattle (52, 201, 99) and in sheep (190). Both size and total activity of the gland are increased with increasing levels of DES. The intramuscular injection of STH in fattening lambs with or without 2 mgm. DES fed per head per day increased nitrogen retention to the same extent as feeding the DES alone (200). This and other work on STH administration (201) suggests that DES activity i s mediated by causing the anterior pituitary to continuously increase i t s rate of STH production. Studies on bone growth (189) also indicated support for this thesis. Schroder et. a l . (190) found gonadotropin production was significantly (P> 0.01) increased in wethers. Burgers et a l . (36) found no difference in the gonadotropin content of the gland. No increase in production or storage of ACTH has been noted in cattle (52) or wethers (190). Studies have not revealed an increase in the thyrotropin content of the anterior pituitary of treated wethers (190). Burgess et a l . (36) found this gland to contain less thyrotropin when steers were implanted with 60 mgm. DES. 3. Thyroid and i t s secretions: The implantation of DES has been shown to reduce the weight of the thyroid gland in both bulls (42) and heifers (52). In steers, 35. no changes in thyroid weights have been recorded (52, 42, 36) after DES treatment. Burgess et a l . (36) demonstrated a mild state of hyper-thyroidism in beef steers treated with DES. Serum protein-bound-iodine levels have not been shown to be influenced by DES implantation in beef steers (55). 4. Adrenal cortex and i t s secretions: DES administration tends to increase the total adrenal weights in steers (52, 41), heifers (52), and lambs (41, 54, 172). Contrary to these findings, Schroder et a l . (190) did not find an increase in total adrenal weight, but did find an increase in adrenal cholesterol. This indication of a decrease in adrenal activity is inconsistent with the effect of DES on nitrogen metabolism. Ruliffson et a l . (187) found the level of adrenal cortical neutral 17-ketosteroids to increase in the urine of treated wethers. It is suggested that these male-like hormones are responsible for the stimulation of growth of the male accessory sex glands. 5. Urogenital system: The feeding or implantation of DES in both fattening steers and wethers produces widespread swelling of the various organs of the urogenital system. The seminal vesicles may increase in weight by as much as 300 per cent (160, 53, 162, 80). Hypertrophy of the kidney, bladder, ampullae, urethra, testes, prostate gland and bulbourethral gland also occurs (20, 194, 62, 117). The Cowpers gland is particularly sensitive in the wether and i t may form a "cul-de-sac" which often f i l l s with urine (20). Preputial and perineal swellings often occur in the 36. Implanted wether and may cause d i f f i c u l t y in urination (20). The enlarged prepuce may also become ulcerated and secondary infection may set in (90). The seminal vesicles of treated animals contain columnar epithelium, giving the appearance of actively secreting tissue, as compared with the low cuboidal type of epithelium found in control animals (53). An increased development of fibrous tissue has also been noted in this gland. Histological changes of the accessory sex organs have been shown to be the same regardless of the mode of administration (56). As a result of estrogen treatment, hypertrophy and inflam-mation of the urogenital system as well as urethral blockage have been noted (1). These problems, along with an increased incidence of urinary calculi, have been most commonly noted in wethers even when the recommended levels of DES are given (21, 117, 107, 39). Both heifers and ewes tend to display vaginal prolapse with the higher levels of DES (52, 144, 11). The incorporation of pro-gesterone in DES implants reduces the occurrence of prolapse although vulval swelling persists (142, 107). In beef heifers, ovarian weight has not been influenced by DES administration, although corpora lutea formation appears to be inhibited (52). Fuller mammary glands (114, 168, 96), longer teats (114, 168, 17, 80) and the presence of milk (114) are a l l common in both male and female market animals treated with DES. Edema of the anal area often occurs in both sexes as well as the occasional case of rectal prolapse (90). The incorporation of progesterone in DES implants reduces the occurrence of rectal prolapse. 37. Shortly a f t e r treatment, a r e l a x a t i o n of the lumbar ligaments occurs, causing the angle formed by the lumbar and s a c r a l vertebrae to become more acute. The manifestation of the e f f e c t i n the l i v e animal is an elevation of the tail-head and a depression of the l o i n , g i v ing the appearance of a t y p i c a l "nymphomaniac stance". This condition occurs in steers, h e i f e r s , and b u l l s (200,117, 80). A genetic p r e d i s p o s i t i o n as well as the age at the time of treatment, both influence the extent of t h i s response. A l l of the above p h y s i o l o g i c a l and anatomical responses normally occur s u b c l i n i c a l l y i n DES treated animals. Only when high l e v e l s are administered or other estrogens (such as the phytoestrogens) are present, do these responses become noticeably harmful to the animal. The genetic p r e d i s p o s i t i o n of the animal i s also c r i t i c a l . Sheep, in general, appear very s e n s i t i v e to estrogen administration. Great care must be taken i n using DES or other estrogens to stimulate the growth rate of sheep. Three days a f t e r estrogen administration, animals tend to become r e s t l e s s and " r i d e " (1). Since t h i s occurs l a r g e l y with lambs, i t does not present much of a problem. These animals also tend to be more aggressive feeders than c o n t r o l animals (126). 6. Protein and nitrogen metabolism: The protein requirements of animals treated with DES have not been shown to be increased to maintain the increased growth rate (124). Nitrogen retention, however, i s increased by as much as 200 per cent i n treated beef steers (159) and lambs (211, 175, 210). Nitrogen retention i s increased by increasing the l e v e l of DES fed (137). 38. Once DES treatment has been discontinued, there i s no carry-over e f f e c t on nitrogen metabolism (18, 43, 19). Nitrogen r e t e n t i o n may be decreased by feeding low l e v e l s of pr o t e i n (e.g., 6 per cent) (199, 204). McLaren et. a l . (97) studied nitrogen metabolism i n the DES-fed wether, and found that metabolic f e c a l nitrogen, endogenous urinary nitrogen, and c r e a t i n and a l l a n t o i n excretion were not ef f e c t e d . No r e l a t i o n s h i p was found between nitrogen retention and PBI (protein-bound iodine) l e v e l s , i n d i c a t i n g that the thyroid is l i k e l y not instrumental i n th i s response. STH and DES have been shown to be equally e f f e c t i v e i n enhancing nitrogen r e t e n t i o n , i n d i c a t i n g a possible r o l e for the anterior p i t u i t a r y (200). 7. Calcium and phosphorus metabolism: The administration of DES to the ruminant has been shown to increase the retention of both phosphorus and calcium (211, 175). Treated animals consuming low lev e l s of protein (6 per cent) tend to r e t a i n less calcium and phosphorus than c o n t r o l s . With an adequate l e v e l of protein (10 per cent) DES tends to increase calcium and phosphorus retention (204). Shroder et a l . (189) made an extensive sgudy of calcium and phosphorus metabolism i n the wether fed 2 mgm. DES per day. DES was shown to have no influence upon blood l e v e l or upon rate of disappearance of C a ^ of P^. The absorption of calcium and phosphorus from the gut was shown to be increased as urinary excretion of the two elements was decreased. Increased reabsorption and de-creased re-excretion increased the true d i g e s t i b i l i t y of calcium by 32.6 per cent and of phosphorus by 34.1 per cent. The retained elements are l i k e l y incorporated into the skeleton as t h i s organ i s found to be e a r l y maturing i n DES treated animals. 39. The retention of sodium i s also increased by DES administration (175). 8. D i g e s t i b i l i t y of feed n u t r i e n t s : In almost every laboratory, the feeding or implantation of DES i n c a t t l e or sheep has shown no influence upon the d i g e s t i b i l i t y of dry matter, crude f i b r e , ether extract or even crude protein (73, 19, 200, 210). Story et a l . (198) found a small increase i n dry matter d i g e s t i b i l i t y i n lambs and att r i b u t e d i t to increased crude pr o t e i n d i g e s t i o n . V i r t u a l l y no work has been done with DES treated ruminants, to study t h e i r water metabolism. M i t c h e l l et a l . (138) noted that steers fed 10 mgm. DES per head per day consumed 5.33 gallons of water per day whereas t h e i r controls consumed 4.93 gallons per day. 9. Rumen metabolism and microbial population: Brooks et a l • (34, 35) reported that the addition of 10, 15 and 20 mcgm. of DES i n v i t r o to a r t i f i c i a l rumens s i g n i f i c a n t l y (P>0.05) increased c e l l u l o s e d i g e s t i o n by the microorganisms. No work has been produced to confirm these f i n d i n g s . Recent work by Christiansen et a l . i n 1964 (48), demonstrated that the feeding of DES prevents the disappearance of rumen protozoa i n lambs changed from a high roughage d i e t to a high concentrate d i e t . DES was also found to increase protozoal counts i n the rumens of lambs consuming p e l l e t e d and non-pelleted r a t i o n s with and without concentrate. Rumen protozoal counts tended to p a r a l l e l the rate of gain and gross feed e f f i c i e n c y of these lambs. 40. (c) The other synthetic estrogens A number of other synthetic compounds possessing estrogenic a c t i v i t y have been used to promote growth i n the ruminant. Included in t h i s group are: hexestrol, d i a l l y l h e x e s t r o l , d i e n e s t r o l , t r a n s - s t i l b e n e , dihydroxystilbene and d i a l l y l s t i l b e s t r o l . Their r e l a t i v e potencies have been discussed. Of these compounds, hexestrol i s the only one that has been used to any commercial extent. I t i s s l i g h t l y less potent than DES, but i s favoured because i t tends to have fewer c l i n i c a l e f f e c t s than DES at i t s optimal l e v e l f o r use i n l i v e s t o c k production (168, 10, 11). Hexestrol i s used to a large extent i n Great B r i t a i n while DES i s l a r g e l y used i n North America. Dienestrol was used to some extent u n t i l about 1954 (104). This compound di d not always give the expected anabolic response and was required at r e l a t i v e l y high l e v e l s . 7. Action of Phytoestrogens (a) Metabolism i n the animal body Very l i t t l e work has been done on the metabolism of the phyto-estrogens i n the ruminant. Ni l s s o n (147, 150) has demonstrated that biochanin A and formononetin are demethylated by the rumen mi c r o f l o r a to form g e n i s t e i n and daidzein, r e s p e c t i v e l y . Red clover ( T r i f o l i u m pratense), containing r e l a t i v e l y large amounts of the very weak estrogen formononetin, would increase i n estrogenic a c t i v i t y to a considerable extent once con-sumed by the ruminant. The demethylation of r e l a t i v e l y i n a c t i v e forage phytoestrogens may be of considerable p h y s i o l o g i c a l importance. Nilsson (148, 150) has also studied the metabolism of t r i t i u m l a b e l l e d biochanin A and formononetin in r a t s , and found that demethylation 41. occurs in the liver by means of enzyme systems located in the micro-somes . (b) Influence of phytoestrogens in livestock production Although the phytoestrogens are 10"^ as active as DES in stim-ulating increases in uterine weight, they are of considerable importance in livestock production because of the large amounts consumed by grazing animals. The f i r s t observations of the effect of the phytoestrogens on livestock were made in Western Australia during the period 1940-1951. A large percentage of sheep grazing subterranian clover (Trifolium subterraneum - var. Dwalganup) suffered reproductive disorders such as failure to conceive, s t i l l - b i r t h , early death of lambs, uterine inertia, and vaginal prolapse (22). Lambing percentages dropped at times to a low of 8 per cent (23). The problem was controlled to a considerable extent by increasing the intake of grasses which are known to be low in estrogens. Cattle and horses maintained on the same pasture showed no c l i n i c a l symptoms (24). Curnow et a l . (58) clearly demonstrated that the causitive agent was a phytoestrogen. Bradbury and White, in 1951, (28) found this phytoestrogen to be genistein. Since this early work with subterranean clover, a number of workers have reported that phytoestrogens influence cattle and sheep physiology. Engle et a l . (72) reported that ewes on ladino clover con-ceived three weeks later, on the average,than did comparable animals grazing a l l season on bluegrass. The average f e r t i l i t y of these animals was also found to be less. Fox et a l . (81) demonstrated that ewes grazing red clover pasture 17 days before and 51 days after breeding had a considerably longer lambing period than did ewes fed either a l f a l f a or clover hays. Their total lamb crop was also lower. Bartlett et a l . (15A) j 42. suggested that the non-fatty solids content of cows milk may be increased by phytoestrogens. A large number of other reports on the action of phytoestrogens have appeared in the literature and have been reviewed by Pope (170, 171). Once the feeding of the synthetic estrogen DES was shown to enhance the growth rate of market animals (104), i t was suggested that the consumption of phytoestrogens may have a similar action. As early as 1952, Cheng et a l . (45) demonstrated that the implantation of lambs with DES, when fed estrogenically-active a l f a l f a and clover hays, did not increase rate of gain above that of their controls. A number of other workers since this time have shown that the response to fed or im-planted DES is reduced or n u l l i f i e d when these animals are consuming spring pastures high in legume content. In 1966, Oldfield et a l . (157) reported an extensive study of coumestrol in a l f a l f a as a factor in growth and carcass quality in lambs. Coumestrol levels ranging from 18.5 ppm to 152 ppm were used. A general trend toward a positive growth response to elevated coumestrol levels in wether lambs, but not in ewes, was recorded. Marked increases in teat length and in seminal vesicle and pituitary weights were obtained in animals fed higher levels of coumestrol. Organoleptic tests consist-ently demonstrated improved tenderness and juiciness scores for lamb roasts from animals fed high-coumestrol diets. These results confirm those of Oldf ield et aJL_ (156) and Johnston et al_^ (108) with lambs, and those of Matsushima and Clanton (134) with steers. An anabolic response has not always been obtained after feeding forages with high estrogenic activity (205). This variation is possibly due to phytoestrogen inhibitors produced during certain growth phases 43. of plants. Evidence for anti-estrogenic activity in a number of forages ha8 been recorded by Biely and Kitts (26). Noteboom and Gorski (153) have reported that injected coumestrol diacetate stimulated, similarly to estradiol 17-/3 , the incorporation of labelled precursors into protein, l i p i d and RNA in the cells of the rat uterus. Oldfield e_t a l . (157) interpret these results plus the observation that there was no additive effect to estradiol 17-^ and coumestrol acetate, to suggest that coumestrol performs similarly to estradiol 17-/Jin triggering the anabolic response. 44. III. STUDY I. THE EFFECT OF DIETHYLSTILBESTROL ON THE DIGESTIBILITY OF VARIOUS FEED COMPONENTS. 1. Introduction Study I was designed to explore the effects of diethylstilbestrol (DES) on the activity of the rumen microflora. It was divided into two parts. Part A involved an in vivo study of the effect of DES on the di g e s t i b i l i t y of various feed components in the growing lamb. Part B involved a study of the effect of a wide range of levels of DES on dry matter d i g e s t i b i l i t y in an in vitro system. 2. Materials and Methods (a) Part A (1) Experimental animals: Four growing wethers, numbers 172, 174, 159 and 162 were used throughout this study. The average i n i t i a l weight of these animals was 92 pounds. A complete record of their body weight gains is given in Appendix I. (2) Housing: The animals were kept in individual 44" x 30" x 18%" metal cages throughout the bimonthly experimental periods. The cages were kept in bright, well-ventilated rooms. The animals were removed from these cages only for a weekly weighing and again, each day during the last week of each experimental period, for isotope determinations (Study II). 45. (3) Basal ration: The wethers were kept on a diet of free choice pelleted, dehydrated, grass meal, iodized cobalt salt, commercial mineral mix and water throughout the study. The dehydrated grass meal consisted almost entirely of orchard grass, Dactylis glomerate, cut in early June, 1965 when approximately eight inches in height. Proximate analysis indicated the following composition of the grass meal: TABLE V. Proximate analysis of the basal diet, dehydrated grass meal. Dry Matter % 92.6 100 Ash % 8.9 9.6 Crude fibre % 21.8 23.5 Ether extract % 7.0 7.6 Protein (N x 6.25) % 18.8 20.3 (4) Experimental rations (Preparation and feeding): Three experimental rations, numbers 1, 2, and 3, were made up containing 1, 2.5, and 2 mgm. DES per pound grass meal respectively. These rations were made up as follows: Sufficient DES for a total of 500 pounds was accurately weighed out and dissolved in 100 ml. acetone. Three to four pounds of dehydrated grass meal, ground in a ba l l - m i l l , were placed in a 5 5/8" x 13%" x 1 1/8" pyrex tray and soaked with acetone so that a layer of acetone always covered the ration. The DES solution was then added, thoroughly mixed, and allowed to dry under a 46. heat lamp with occasional sti r r i n g . The dry grass meal was then accumu-latively mixed with more ground grass meal unt i l a total weight of 50 pounds was obtained. This premix was taken to a commercial feed manu-facturer, where an additional 450 pounds of ground grass meal was added to make a total of 500 pounds of ration, which was then pelleted. (5) Chemicals: A l l chemicals used met the American Chemical Society specif icat ions. (6) Experimental design: The four wethers were treated identically during treatment periods of approximately one month in length. The treatments were alternated on a monthly basis (Table VI). After 10 days on each treatment, fecal samples were collected twice during the subsequent 20 day period for both proximate analysis and dry matter d i g e s t i b i l i t y determinations. TABLE VI. Experimental design Treatment Month Control 1 Ration No. 1 2 Control 3 Ration No. 2 4 Control 5 Ration No. 3 6 (7) Experimental methods: The determination of dry matter d i g e s t i b i l i t y was performed by the chromogen method f i r s t proposed by Reid et a l . in 1950 (181). The procedure followed was that outlined by Brisson and Hatina (33) 47. with some modifications; for details see Appendix II. The proximate analysis of both grass meal and sheep feces was performed as outlined in the AOAC Methods of Analysis, 1960 (154). (b) Part B (1) Incubation apparatus: The incubation apparatus consisted of a 26" x 12" x 7" deep, Labline, water bath set at 39.5 + 0.25° C. A wooden test tube rack with a maximum capacity of 72, 90 ml. centrifuge tubes was submerged in the water. The rack was adequately perforated to permit sufficient movement of the water to maintain a uniform temperature throughout. The complete digestion process was carried out in 90 ml. Pyrex centri-fuge tubes. During the i n i t i a l 48 hour incubation period, anaerobic conditions were maintained by continuously bubbling carbon dioxide through the fermentation medium. Carbon dioxide entered the centrifuge tubes by means of a glass capillary tube (i.d. 1 mm.; o.d. 6.5 mm.) which passed through a number 6 stopper to within 2 mm. of the bottom of the tube. Excess gas escaped through the l i p of the centrifuge tube. The "bubbling" action of the gas prevented the precipitation of substrate particles. For each experiment, a new 50 pound carbon dioxide cylinder was required. A flowmeter regulated gas flow from the cylinder to a manifold lining the top of the water bath. Individual rubber tubing connections led to each of the capillary glass bubblers. (2) Source of rumen microflora: Three sheep, one Rambouillet-Romney Marsh cross-bred range wether, and two grade Shropshire rams were fistulated to provide 48. direct access to the rumen. The fistulation technique is outlined in Appendix IV. The animals were fed a diet of dehydrated grass meal, water and iodized cobalt salt, similar to the wethers in Part A of this study. In Part B, however, grass meal intake was limited so that the animals would not become too fat. (3) Microbial substrate: The microbial substrate for the in vitro studies was ground grass meal with DES added as indicated in Table XII. (4) Chemicals: A l l chemicals met the American Chemical Society specifications. DES was supplied by Sigma Chemical Company, St. Louis 18, Missouri, U.S.A. The pepsin (1:10,000) was supplied by Nutritional Biochemicals Corporation, Cleveland, Ohio, U.S.A. (5) Experimental methods: The in vitro d i g e s t i b i l i t y studies were performed as outlined by T i l l e y and Terry (203) with two major alterations. F i r s t , the fermentation medium used was that of Quicke et a l . (178), as modified by Donefer et a l . (67), rather than that of McDougall et a l . (136). Secondly, continuous bubbling with carbon dioxide was maintained rather than i n i t i a l l y gasing and then allowing the microorganisms to provide their own anaerobic conditions. The method used in this study is out-lined in detail in Appendix III. 3. Results and Discussion The wethers were continuously growing throughout the study period. The gain in body weight during each bimonthly experimental 49. did not exceed 10 pounds, consequently increases in body weight were not considered in measuring the various parameters. The feeding of 2.0 and 2.5 mgm. DES per head per day resulted in an increased dry matter consumption of 0.22 and 0.24 pounds per head per day, respectively (Table VII). This represents a six per cent increase in consumption. A significant (P<.01) overall decrease in dry matter d i g e s t i b i l i t y resulted from the feeding of DES. A significant (P<.05) decrease in dry matter d i g e s t i b i l i t y resulted from the feeding of either 2.0 or 2.5 mgm. DES (Table VIII). Proximate analysis values of the feces of wethers fed control ration and control ration plus 2.5 mgm. DES per head per day are outlined in Appendix V. Wethers fed 2.5 mgm. DES displayed a 15 per cent (P<.01) reduction in crude fibre d i g e s t i b i l i t y and a 3.5 per cent (P<.01) reduction in nitrogen free extract (N.F.E.) d i g e s t i b i l i t y . Crude protein (nitrogen x 6.25) and ether extract d i g e s t i b i l i t i e s were in-creased 5.4 per cent (P<.05) and 9.2 per cent (P<.20), respectively. The low level of significance for the increase in ether extract digest-i b i l i t y is due to the inherent v a r i a b i l i t y in the method and the small number of animals used (Tables IX, X, XI). The in vitro dry matter d i g e s t i b i l i t y studies did not reveal any influence of DES upon the activity of the rumen microflora (Table XII). Remarkably consistent results were obtained with the in vitro technique for a period ranging over 11 months. The average percentage dry matter digested was 71.9 + 1.0 per cent, which was 6.4 per cent higher than the average in vivo value. A plausible explanation for the higher percentage digestion with the in v i t r o system is that the dehydrated grass meal was subjected to digestive activity for a longer period of time (96 hours) than in the in vivo system. TABLE VII. Average daily dry matter consumption of dried grass meal. Treatment Average Daily Dry Matter Consumption (Pounds) Sheep Sheep Sheep Sheep No. 159 No. 171 No. 174 No. 162 Mean Ration No. 1 (1 mgm. DES*/head/day) Control Ration No. 2 (2.5 mgm. DES/head/day) Control Ration No. 3 (2 mgm. DES/head/day) 4.00 3.85 4.11 3.26 3.72 4.03 4.05 4.36 3.94 3.87 3.19 3.32 3.72 3.54 3.70 3.74 3.67~ 3.76 3.39 3.70 3.74 3.72 3.96 3.53 3.75 * DES = diethylstilbestrol TABLE VIII. Dry matter dig e s t i b i l i t y , In vivo. Percent Dry Matter Digested Date of Experiment Treatment Sheep No. 159 Sheep No. 171 Sheep No. 174 Sheep No. 162 Mean Treatment Mean 4/ 8/65 Control 66.5 67.1 67.3 66.6 66.9 66.9 25/ 8/65 Ration No. 1 (1 mgm. DES*/head/day) 63.1 64.6 62.5 64.2 63.6 12/ 9/65 Ration No. 1 (1 mgm. DES/head/day) 66.1 66.2 65.0 66.3 65.9 64.83 26/ 9/65 10/10/65 Control Control 66.8 69.0 65.6 68.3 65.0 68.0 63.4 68.3 65.2 68.4 66.8 11/11/65 Ration No. 2 (2.5 mgm. DES/head/day) 63.1 63.7 64.0 66.7 64.4 13/11/65 Ration No. 2 (2.5 mgm. DES/head/day) 59.71 63.2 64.4 65.4 64.3 64.42 16/ 2/66 Control 71.7 69.4 67.7 67.7 69.1 69.1 19/ 3/66 Ration No. 3 (2 mgm. DES/head/day 64.4 66.0 63.9 65.6 65.0 3/ 4/66 Ration No. 3 (2 mgm. DES/head/day 64.7 65.8 64.8 67.2 65.6 65.32 Average 66.2 66.0 65.3 66.0 65.8 1 Value not included, animal scouring. 2 Means are significantly (P<.05) lower than the mean for animals consuming control ration. 3 Means are significantly (P^.01) lower than the mean for animals consuming control ration. * DES = diethylstilbestrol 52. TABLE IX. Component digestion for wethers fed control ration. Percent Digested Animal No. Dry Matter Crude Protein Ether Extract Crude Fibre Nitrogen Free Extract Percent Ash Retained 159 67.9 67.9 62.1 64.4 73.5 56.8 171 67.0 67.1 61.3 64.2 71.5 59.0 174 66.5 67.2 56.0 61.5 72.3 56.3 162 65.9 66.7 60.7 62.8 71.3 54.3 Average 66.8 67.2 60.0 63.2 72.2 56.6 TABLE X. Component digestion for wethers fed 2.5 mgm. diethylstilbestrol/day. Percent Digested Animal No. Dry Matter Crude Protein Ether Extract Crude Fibre Nitrogen Free Extract Percent Ash Retained 159 63.1 68.7 61.3 54.6 68.9 48.7 171 63.5 68.5 63.6 52.9 70.0 52.4 174 64.2 72.4 67.9 52.9 69.0 52.8 162 66.1 73.7 69.0 54.5 70.7 58.3 Average 64.4 70.8 65.5 53.7 69.7 53.1 53. TABLE XI. Summary of in vivo d i g e s t i b i l i t y study results. Treatment Percent Digested Dry Matter Nitrogen Percent Intake Dry Crude Ether Crude Free Ash (Pounds) Matter Protein Extract Fibre Extract Retained Control 3.72 66.8 67.2 60.0 63.2 72.2 56.6 Ration No. 2 1 3.96 64.4 70.8 65.5 53.7 69.7 53.1 Difference +0.242 -2.42 +3.62 +5.5 -9.53 -2.53 -3.5 1 2 3 Dried grass meal plus 2.5 mgm. diethylstilbestrol/head/day. Difference between control and Ration No. 2 is significant (P< .05). Difference between control and Ration No. 2 is significant (P< .01). 54. TABLE XII. Dry matter d i g e s t i b i l i t y , in vitr o . Percent Dry Matter Digested Date Treatment Replicate No. 1 Replicate No. 2 Replicate No. 3 Mean 27/ 7/65 Control 71.2 70.5 70.7 70.8 0.5 mcgm. DES* 70.3 72.3 72.9 71.8 1.0 mcgm. DES 68.4 71.7 71.7 70.7 2.0 mcgm. DES 69.4 70.6 70.9 70.3 4.0 mcgm. DES 71.1 71.6 69.9 70.9 6.0 mcgm. DES 70.7 69.8 71.2 70.6 15/ 6/66 Control 71.9 71.0 72.9 71.9 6.0 mcgm. DES 73.3 73.1 71.8 72.7 10.0 mcgm. DES 72.9 72.4 71.1 72.1 16.0 mcgm. DES 73.1 72.1 72.7 72.6 20.0 mcgm. DES 72.3 72.8 72.0 72.4 30.0 mcgm. DES 72.3 72.3 72.0 72.2 40.0 mcgm. DES 72.8 73.4 73.7 73.3 24/ 6/66 Control 72.4 71.7 - 72.1 5.0 mcgm. DES 72.5 74.3 72.6 73.1 15.0 mcgm. DES 73.9 73.6 71.5 73.0 * DES = diethylstilbestrol 55. Assuming the gross energy of the dehydrated grass meal to be 2000 kcal* per pound of dry matter, wethers fed control ration and wethers fed 2.5 mgm. DES per day retained an average of 4840 and 5027 kcal.. digestible energy per day, respectively. This amounts to a 2.6 per cent increase in energy intake, which does not account for the increase in body weight gains expected with DES feeding - i f this increase was of similar composition to the increase in control lambs. Analysis of lamb carcasses has indicated that DES-fed animals have a lower percentage fat and a higher percentage protein than control animals (104). A unit of weight gain in DES-fed lambs, requires less energy than a corresponding unit weight gain in their controls. These findings substantiate the work of Garrett (83) who found that improved feed efficiencies in DES-implanted lambs were not real when placed on a caloric basis. The increase in crude protein digestion is real. A number of workers (104) have shown that DES administration increases nitrogen retention in lambs. McLaren et a l . (136A) showed that the amount of metabolic fecal nitrogen i s not altered by feeding DES to wethers. The increase in percentage crude protein d i g e s t i b i l i t y can be accounted for in a number of ways. When the ruminant consumes pelleted, finely ground rations the protozoal population tends to extinction. Christiansen et a l . (48) showed that the feeding of DES w i l l prevent this extinction and enhance the maintenance of near normal rumen protozoal populations. The importance of a well faunated rumen i s emphasized as protozoal protein is more readily digested in the lower gut than i s bacterial protein. The decrease in crude fibre d i g e s t i b i l i t y is hard to rationalize in these terms as Yoder et a l . (220) have recently shown that a substantial rumen protozoal population i s required to maintain the eellulolytic activity of the rumen microflora. From Tables XIII to XVI, the relative proportions of ether extract, crude fibre, and N.F.E. retained, to protein retained 56. TABLE XIII. Average daily component intake of wethers fed control ration Total Average Dally Intake in Pounds Nitrogen-Dry Crude Ether Crude Free Animal No. Matter Protein Extract Fibre Extract Ash 159 3.85 0.78 0.29 0.90 1.51 0.37 171 4.05 0.82 0.31 0.95 1.58 0.39 174 3.32 0.67 0.25 0.78 1.30 0.32 162 3.67 0.75 0.28 0.86 1.43 0.35 TABLE XIV. Average daily component excreta of wethers fed control ration Total Average Daily Excreta.in Pounds Nitrogen^ Animal No. Dry Matter Crude Protein Ether Extract Crude Fibre Free Extract Ash 159 1.24 0.25 0.11 0.32 0.40 0.16 171 1.34 0.27 0.12 0.34 0.45 0.16 174 1.11 0.22 0.09 0.30 0.36 0.14 162 1.25 0.25 0.11 0.32 0.41 0.16 57. TABLE XV. Average daily component intake of wethers fed 2.5 mgm. diethylstilbestrol per day. Total Average Daily Intake in Pounds Animal No. Dry Matter Crude Protein Ether Crude Extract Fibre Nitrogen-Free Extract Ash 159 4.11 0.83 0.31 0.97 1.61 0.39 171 4.36 0.89 0.33 1.02 1.70 0.42 174 3.72 0.76 0.28 0.87 1.45 0.36 162 3.76 0.76 0.29 0.88 1.47 0.36 TABLE XVI. Average daily component excreta of wethers fed 2.5 mgm. diethylstilbestrol per day. Total Average Daily Excreta in Pounds Animal No. Dry Matter Crude Protein Ether Crude Extract Fibre Nitrogen-Free Extract Ash 159 1.52 0.26 0.12 0.44 0.50 0.20 171 1.71 0.28 0.12 0.48 0.51 0.20 174 1.74 0.21 0.09 0.41 0.45 0.17 162 1.62 0.20 0.09 0.40 0.43 0.15 58. can be calculated. Allowing the value for crude protein to equal 1.0, the values for these components for control animals and DES treated animals are: 0.35, 1.09, and 2.07; and 0.35, 0.88 and 1.98 respectively. These values indicate possible changes in the microbial activity subsequent to DES administration. Although protein digestion has significantly (P^.05) increased the digestion and retention of the more readily avail-able energy sources, ether extract and, to a lesser extent, N.F.E., have paralleled this change. The retention of the less readily available energy source, crude fibre, was dramatically reduced. A possible explanation for this observation is that DES present in the rumen selectively stimulates the growth of proteolytic organisms, possibly protozoa, which may use N.F.E. and ether extractable nutrients as their major energy source. Organisms u t i l i z i n g relatively more crude fibre for their energy requirements may be selectively inhibited. The f u l l significance of protozoa to the nutrition of the sheep is not clear, though increased growth of lambs with protozoa, as compared with lambs without c i l i a t e protozoa has been noted (49). Greater concentrations of protozoa resulting from feeding antibiotic to lambs (120, 177), suggest that protozoa may be involved in the favourable response to antibiotic exhibited by growing animals. DES may be involved in a similar mechanism. Increased nitrogen retention i s also demonstrated in DES-implanted wethers. Even though DES has been shown to be recirculated through the saliva, the amount involved would be too small to give the response found. Possibly associated with i t s endocrinological response, DES may control the movement of amino acids and other nitrogenous sub-stances across the gastro-intestinal (G.I.) wall. Amino acids have been shown to be actively transported across this barrier. DES may act by stimulating the enzymatic mechanism involved. Most amino acids and 59. smaller nitrogenous digestion products are water soluble and pass through the G.I. tract wall mainly by passive transport. Once through, these compounds are transported by the circulatory system to the liver where they are metabolized. DES may enhance the retention of the nitro-genous compounds by enhancing their fixation in the l i v e r . The liver has been shown to increase in size after DES treatment. Neither of these mechanisms can explain the influence on the di g e s t i b i l i t y of the other feed components. Ether extract, crude fibre, and N.F.E. d i g e s t i b i l i t i e s have not been shown to be influenced in the implanted animal. It appears that in the DES fed animal part, and possibly - though not li k e l y - a l l , of the DES response is mediated through rumen microbial activity. 60. IV. STUDY II. THE EFFECT OF DIETHYLSTILBESTROL ON ENDOCRINE FUNCTION IN THE FATTENING MARKET LAMB. 1. Introduction The effects of DES on the endocrine system of the fattening lamb were investigated. This study was divided into two parts. The 1 investigation of thyroid I uptake by the wethers in Study I, Part A, is reported in Part A. Part B involved a study of the influence of DES on the total weights of the anterior pituitary, thyroid and adrenal glands in 42 fattening lambs fed an a l l concentrate diet. A report and discussion of the overall gross effects of DES implantation in these animals is included in Part B. 2. Materials and Methods (a) Part A (1) Experimental animals: The experimental animals used in this study were the same four wethers that were employed in Part A of Study I. Their housing and feeding has been outlined in the previous study. Parts A of both Study I and II were carried out simultaneously. (2) Counting apparatus: A Nuclear-Chicago Corporation model DS-5-1 versatile s c i n t i l l a t i o n detector was used, with a model 151 decade scaler and T^ timer. Pre-set time counting was followed at a l l times. A s c i n t i l l a t i o n counter was employed, since these instruments are 30 to 50 times more efficient than the G.M. tube. A 2" x 2" thallium activated sodium iodide crystal was used with a G5S focusing collimator. 61. The Intensity of radiation i s inversely proportional to the square of the distance between the source of radiation and the point 131 measurement. Precision measurements of thyroidal z should be made at as great a distance as the sensitivity of the detector w i l l permit. Perlnutter et a l . (166) have shown that at a distance of 30 cm. between the radioactive source and the receptor, a variation in location of one cm. produces a mean of three per cent difference, compared with a mean 42 per cent difference at a distance of one cm. The s c i n t i l l a t i o n probe was fixed to a flexible arm allowing i t s movement to a horizontal position. In order to maintain accurately a standard distance between the radioactive source and the receptor, a metal spacer was attached to the G5S focusing collimator. Three steel rods, 5 mm. in diameter, and 30 cm. in length, were fixed at each end to steel rings 9 cm. in diameter to form the spacer. (3) Plateau considerations: Sc i n t i l l a t i o n counters develop pulses of varying heights. These heights are determined by: (a) The multiplication factor of the phototube, which depends upon . - the construction of the tube and the voltage applied to the tube. (b) The amplification of the preamplifier. (c) The energy of gamma rays and beta particles. In order to establish reliable and reproduceable operating con-ditions, counting rate was regressed on applied voltage when a standard radiation source was placed at a fixed distance from the crystal (Appendix VI). A l l countings in this study were carried out at 1350 volts. 62. (4) Radioactive sodium iodide: 131 The I was obtained from Atomic Energy of Canada Limited, Commercial Products Division, Ottawa, Canada, in quantities not exceed-ing one millicurie per month. Its chemical form was N a l 1 3 1 in a sodium bisulphite carrier, with one millicurie in a total volume of 0.3 to 0.5 ml., stored in one-quarter ounce sealed glass bottles. The hour and date at which the material would represent one mill i c u r i e was stated. The sample was stored in a lead container. A l l work involving this material was carried out in an isolated laboratory. A l l necessary precautions were observed. (5) Dilution of radio-active sodium iodide: Approximately three ml. of d i s t i l l e d water were added to the sample bottle, which was quantitatively emptied into a 50 ml. volumetric flask. Additional small amounts of d i s t i l l e d water were added to the bottle, with swirling, u n t i l the f i n a l volume was obtained. The f i n a l concentration of I 1 3 1 was equivalent to 20 microcuries per ml. (6) Method of injection into wethers: The wethers were injected 30 days after i n i t i a t i o n of the feeding of the experimental rations. Feces samples had been collected by this time. A total of 50 microcuries was injected subcutaneously into each animal. This amount produced readings ranging from 10,000 to 50,000 counts per minute, the range which can be most accurately measured. (7) Preparation of standard: An amount identical to that given to the wethers was injected into a 25 ml. volumetric flask and made up to volume with 63. d i s t i l l e d water. The flask was fixed securely in a holder designed to f i t the end of the spacer. The holder was r i g i d l y secured on a tripod to provide a standard amount of reflected radiation. (8) Method of measuring thyroid gland activity: Prior to treatment, the neck region surrounding the cricoid cartilage was clipped clean of fleece. During the measurement of thyroidal activity, the wethers were held in an upright sitti n g position on the floor. A stool for the handler was pressed firmly against the animal's back. The handler was able to hold the sheep in place with his knees and was able to t i l t the head back with his hands. The s c i n t i l l a t i o n probe was turned to a horizontal position and moved forward u n t i l the outside ring of the spacer surrounded the cricoid cartilage. The anatomical guide used in a l l t r i a l s was the cricoid cartilage located in the center of the outside ring, with this ring pressed firmly against the neck. The highest counts were obtained at this position. Before injection of the isotope, the activity of the gland was measured to ensure that no residue activity from previous experiments remained. The f i r s t measurement was made eight hours post-injection. Subsequent measurements were made every 8 hours for a period of 40 hours, after which readings were made every 16 to 24 hours un t i l 128 hours post-injection. Standard and background counts were determined whenever the thyroidal activities were determined. A l l activities were expressed as counts per minute. Three one-minute counts were collected in series and averaged to give the individual values. Mean background counts were subtracted from both standard and treatment values. 64. (9) Expression of thyroid gland activity: The activity of the gland was expressed as: Thyroid counts per min. - background counts per min. x ^QQJ Standard counts per min. - background counts per min. This is the only means by which the results can be compared on a month to month basis. Many of the values obtained exceed 100 per cent because the radiation from the standard was reduced by a lead shield which acted as protection. (b) Part B (1) Experimental animals: A total of 42 cross-bred wether and ewe lambs from the U.B.C. flock were used in this part of the study. The i n i t i a l average weight was 56 pounds (range-36 to 76 pounds). On April 15, 1965, the lambs were divided into two groups, each possessing a similar weight range and average. The control group consisted of 8 wethers and 14 ewes. The treated group consisted of 11 wethers and 9 ewes. These animals were implanted with a single pellet containing 12 mgm. of active DES. Implant ation was made subcutaneously near the base of the non-tagged ear. At this time, a l l lambs weighing 40 pounds or more were separated from their dams. Those weighing less than 40 pounds remained with their dams an additional week, at which time they too were heavy enough to wean. (2) Housing and feeding of experimental animals: The lambs were kept in 12' x 24' pens containing not more than 12 animals. Treated animals were separated from control animals. The heavier animals in each group were separated from the younger, but sexes were not separated. Fresh, clean, bedding was supplied in the form of 65. Water and iodized cobalt salt were provided "free choice". For the f i r s t two days after weaning, approximately 3 pounds of a l f a l f a hay were fed with the experimental ration. The experimental ration (U.B.C. L-65) was fed throughout the experimental period to a l l animals (see Table XVII). Maximum ration consumption was insured by feeding no. more than would be consumed before the next feeding. TABLE XVII. Composition of U.B.C. lamb ration (L-65). Amount Present Component ; (Pounds) Rolled barley 1635 Dehydrated grass meal 100 Fishmeal 90 Molasses 150 Tricophos 15 Iodized salt 10 Vitamin A (dried) (10,000 IU/gm.) 0.5 Aureofac AO 0.625 Calcium pantothenate 0.25 Total Digestible; Protein (TOP) 12.8% Total Digestible Nutrients (TDN) 69.7% Total Digestible Dry Matter (TDDM) 87.9% Total Calcium 0.33 % Total Phosphorus 0.50 71 Digestible Energy 1394 kcal/lb. (3) Experimental methods: Daily feed composition records were kept throughout the study. A l l lambs were weighed weekly and were marketed when their weight reached 100 pounds. Seventy-two hours prior to shipping to a local abbatoir, the DES implant was removed,.dried and weighed on a torsion balance. At the time of slaughter whole heads, thyroid glandsj and adrenal glands from each animal were collected. Live weights and warm carcass weights were recorded to determine the individual dress-ing percentages. The heads were later opened to recover the pituitary gland. 66. The fresh pituitary, thyroid and adrenal glands were stripped of adhering adipose and connective tissue and weighed on a torsion balance. The glandular tissues from each lamb were then placed in individual 100 ml. beakers f i l l e d with freshly prepared Bouin-Duboscq fixative (Table XVIII.) for three days. The tissues were then washed with water and stored in 50 per cent alcohol u n t i l imbedded, sectioned, and stained by the periodic acid-Schiff reaction. TABLE XVIII. Composition of Bouin-Duboscq fixative. Component Amount 80% ethyl alcohol 150.0 ml. formalin 60.0 ml. glacial acetic acid 15.0 ml. picri c acid crystals 1.0 gm. 3. Results and Discussion Table XIX presents the results of Part A of this study. Expressing counts per minute from the thyroid, as a percentage of the counts per minute obtained from the standard, did not reveal any effect of DES on this gland. The thyroid gland i s known to vary in activity with length of day. These results tend to verify this well-known phenomenon. Burgess et a l . (36) reported a mild state of hyper-thyroidism in beef steers treated with DES. The literature contains l i t t l e evidence of DES affecting the activity of the ruminant thyroid gland. Ostrovsky and Kitts (164) have shown consistent increases in 131 thyroid uptake of I in female intact and ovariectomized rats receiving small doses of red clover extracts possessing estrogenic activity. Non-active timothy grass extracts failed to e l i c i t such TABLE XIX. Thyroid gland activity of wethers fed control and experimental rations. Thyroid counts per minute as a percentage of standard counts per minute. Injection Animal Date No. 8 16 24 32 40 48 56 64 72 80 88 .96 104 112 120 128 Ration 20/ 7/65 171 47 45 46 45 43 40 40 39 38 39 35 20/ 7/65 174 91 105 110 110 107 110 103 106 100 107 95 Control 20/ 7/65 159 53 56 57 62 65 59 57 54 54 50 44 20/ 7/65 164 49 42 46 43 45 43 41 39 40 38 - . . _ 36 X 60 62 65 65 65 63 60 60 60 59 53 30/ 8/65 171 69 67 65 72 69 69 66 64 59 Ration No. 1 30/ 8/65 174 106 128 135 145 139 128 124 126 104 (1 mgm. DES*/ 30/ 8/65 159 125 136 124 125 108 104 94 89 71 head/day) 30/ 8/65 164 117 131 123 126 126 121 116 113 99 X 104 116 112 117 111 106 100 98 83 30/ 9/65 171 65 73 80 85 82 84 82 74 80 78 30/ 9/65 174 43 47 52 58 58 57 61 57 60 58 Control 30/ 9/65 159 52 59 68 77 76 78 79 81 86 79 30/ 9/65 169 48 54 60 61 61 64 64 67 67 62 X 52 58 65 70 69 71 72 70 73 69 2/11/65 171 47 50 48 55 66 57 53 51 48 50 2/11/65 174 71 76 75 92 96 94 96 84 87 85 (2.5 mgm. DES 2/11/65 159 51 62 70 81 83 82 85 93 91 90 /head/dav) 2/11/65 164 40 41 44 48 50 46 47 44 47 46 X 52 57 59 69 74 70 70 68 68 68 16/ 2/66 171 102 121 139 136 141 145 135 135 126 Ration No. 2 16/ 2/65 174 97 127 142 144 140 144 141 135 128 Control 16/ 2/65 159 47 56 '67 75 76 79 75 82 80 16/ 2/65 164 139 166 204 210 196 187 183 200 172 X 96 118 138 141 138 139 134 138 127 23/ 3/66 171 127 146 163 192 172 188 170 174 164 Ration No. 3 23/ 3/66 174 68 88 95 106 108 111 110 108 98 (2 mgm. DES/ 23/ 3/66 159 49 64 80 88 85 94 88 98 89 head/day) 23/ 3/66 164 74 100 116 139 134 140 131 136 127 X 80 100 114 131 125 133 125 129 120 * DES = diethylstilbestrol 68. a response. These workers also found orally administered DES to stimulate a pronounced increase in thyroidal I 1 3 1 uptake in these animals. Noach (151, 152) showed that estradiol benzoate treated castrate male rats displayed an increased thyroid weight and an in-131 creased thyroid I uptake. From his own results and literature survey, Noach suggests that estrogens may decrease thyroid stim-ulating hormone (TSH) release from the anterior pituitary as well as intensifying the action of TSH on the thyroid gland. From these results and those discussed below, i t is f e l t that more work should be done in this f i e l d , to determine whether or not DES activity may be mediated to some degree through i t s activity on the thyroid gland in the ruminant. A large number of replicates are required because of the variation from animal to animal. A l l treatments must be carried out at the same time of the year. The feeder lambs in Part B of this study were found to grow at an extremely rapid rate when fed the all-concentrate ration. L i t t l e d i f f i c u l t y was experienced in keeping them on f u l l feed provided no more than one-quarter of a pound of feed was found remaining in the feed trough 24 hours after feeding. The implant-ation of 12 mgm. of active DES induced a number of secondary effects: nervousness, riding, d i f f i c u l t y in urination, rectal swelling, and one case of vaginal prolapse (ewe No. 502). Diffi c u l t y was experienced in obtaining the glands from the abbatoir as a large proportion of them were destroyed while the lambs passed along the r a i l . The fresh weights and the fresh weights in grams per kilogram metabolic size (body weight in kg?^) are recorded in Tables XX and XXI. Expressed by the latter method, the average weight of the control pituitary glands was 0.0309 + 0.0078 gm. 69. TABLE XX. Endocrine gland freights of lambs fed control r a t i o n . .PituJLfe«ry;:' Thyroid Fresh Wt. (g^jc) Fresh Wt. (g§ Animal Pitnitary per kg?n flhysroid Ho. Wt. (ma.) Body: Vtf.. . ' 3, to enhance precipitation, was not found to be required. The tubes were removed from the water bath, allowed to cool, and were centrifuged at 2400 r.p.m. for 20 min. The supernatant was poured off. The precipitate was resuspended in 50 ml. of pepsin solution and aerobically incubated for 48 hours. Occasional swirling was required to keep a l l substrate particles in solution. After 48 hours, the incubation tubes were centrifuged at 2400 r.p.m. for 20 min. The supernatant was discarded. The precipitate was quantitatively washed into a dry pre-weighed 100 ml. beaker, which was placed in a force-draft oven, set at 105° C, and dried to constant weight. Total dry matter residue was then calculated. Preparation of Pepsin Solution; The pepsin solution was made up by dissolving 2.0 gm. of 1:10,000 pepsin in 850 ml. demineralized water. One hundred ml. of N HG1 was added and the solution then made up to 1 l i t r e with d i s t i l l e d water. Preparation of Diethylstilbestrol Solutions: One hundred mgm. of DES was accurately weighed into a 10 ml. volumetric flask and made up to volume with ethanol. Two ml. of this .101. up to volume with demineralized water. A small amount of ethanol may be added at this step to obtain the fine emulsion desired. This is the standard solution (200 mcgm. DES per ml.) from which dilutions may be made for additions to the centrifuge tubes. Diethylstilbestrol was added to the incubation tubes as 2 ml. aqueous suspensions. The ethanol content of these suspensions never exceeded 0.1 per cent. 102. APPENDIX IV. FISTULATION TECHNIQUE AND CARE OF ANIMALS The cannula was essentially a s t i f f plastic washer (interior washer) 9 cm. in diameter. Into the centre and protruding out one side was fixed a.6.5 cm. long threaded plastic tube ("neck"), with an inside diameter of 2.5 cm. and a screw cap. The exterior (removable) washer was made of the same s t i f f plastic. The three sheep were fistulated by a qualified veterinarian. Rather than use a "single-incision" method, which was used on the wether, the rams were fistulated by a "double-incision" method. Briefly, after the animals were prepared, a 4-inch incision was made below the l e f t transverse process of the sixth lumbar vertebra, through the abdominal musculature to the rumen wall. A second 2-inch incision was made about 8 inches cranial from the f i r s t . In both cases the abdominal musculature was sutured to the rumen wall to prevent contamination of the peritoneal cavity. The rumen wall was now opened and the complete cannula (with-out exterior washer) was passed completely through the the f i r s t incision. The "neck" of the cannula, only, was pulled tight through the second incision. The f i r s t incision was completely sutured, while the second incision was sutured about the cannula neck. The external washer was now screwed snuggly in place, but not tightly. The wound was cleaned and dressed daily for the f i r s t 10 days. After this time, the exterior washer was tightened and with care, rumen contents could be removed. The fistulation operation did not appear to have any adverse effect upon the growth and performance of the animals. It was found that once every four weeks, the exterior washer 103. should be loosened, the area around the f i s t u l a washed and shaved. This procedure prevented leakage, infection, and the possi b i l i t y of the interior washer cutting into the rumen wall. 104. APPENDIX V. TABLE XXVIII. Proximate analysis of feces from wethers fed control ration (26/9/65). Percentage of Total Dry Hatter Nitrogen-Animal Replicate Crude Ether Crude Free No. No. Protein Extract Fibre Extract Ash 159 1 21.0 8.9 24.9 32.2 13.0 2 21.0 7.9 25.3 32.6 13.2 3 21.0 10.3 24.8 30.7 13.2 X 21.0 9.0 25.0 31.8 13.1 171 4 20.6 8.7 25.1 33.9 11.7 5 20.0 9.0 24.5 34.7 11.8 6 20.4 9.8 24.5 33.6 11.7 X 20.3 9.2 24.7 34.1 11.7 174 7 19.2 8.0 27.0 32.9 12.9 8 19.2 7.4 25.5 35.3 12.6 9 19.1 8.1 26.3 33.9 12.6 x" 19.2 7.8 26.3 34.0 12.7 162 10 20.4 8.1 24.6 34.0 12.9 11 20.1 8.2 25.2 33.3 13.2 12 20.0 7.6 25.2 34.1 13.1 x" 20.2 8.0 25.0 33.8 13.1 Average 20.2 8.5 25.2 33.4 12.7 105. TABLE XXIX. Proximate analysis of feces from wethers fed control ration (10/10/65). Percentage of Total Dry Matter Nitrogen-Animal Replicate Crude Ether Crude Free No. No. Protein Extract Fibre Extract Ash 159 13 19.7 8.3 26.9 32.1 13.0 14 19.6 7.9 26.7 32.5 13.3 15 19.8 8.1 26.3 32.8 13.0 X 19.7 8.1 26.6 32.5 13.1 171 16 19.7 9.5 25.5 33.4 11.9 17 19.7 8.8 25.8 33.8 11.9 18 19.7 7.9 25.4 35.1 11.9 x" 19.7 8.7 25.6 34.1 11.9 174 19 20.0 9.4 30.8 28.1 11.7 20 19.7 8.5 27.2 32.8 11.8 21 19.4 8.8 26.6 33.5 11.7 X* 19.7 8.9 28.2 31.5 11.7 162 22 19.7 9.6 26.2 32.1 12.4 23 19.5 9.3 26.2 32.5 12.5 24 19.6 8.1 26.6 33.2 12.5 X 19.6 9.0 26.3 32.6 12.5 Average 19.7 8.7 26.7 32.7 12.3 TABLE XXX. Proximate analysis of feces from wethers fed Ration No. 2 (2.5'mgm. diethylstilbestrol /head/day) (11/11/65), Percentage of Total Dry Matter Nitrogen-Animal Replicate Crude Ether Crude Free No. No. Protein Extract Fibre Extract Ash 159 1 16.6 7.5 29.1 34.0 12.8 2 16.6 7.6 29.5 33.6 12.7 3 16.5 6.7 29.2 34.7 12.8 X 16.6 7.3 29.3 34.1 12.8 171 4 16.0 6.6 30.9 33.2 13.3 5 16.0 6.4 31.0 33.8 12.8 6 15.7 6.3 31.3 34.1 12.6 X 15.9 6.4 31.1 33.7 12.9 174 7 15.8 9.0 30.6 32.6 12.0 8 15.7 6.2 31.4 34.7 12.0 9 15.6 7.6 32.3 32.6 11.9 X 15.7 7.6 31.4 33.3 12.0 162 10 18.1 7.1 29.1 33.1 12.6 11 18.1 7.5 29.2 32.7 12.5 12 17.8 7.5 29.4 32.5 12.8 X 18.0 7.4 29.2 32.8 12.6 Average 16.5 7.2 30.3 33.5, 12.6 106. TABLE XXXI. Proximate analysis of feces from wethers fed Ration No. 2 (2.5 mgm. diethylstilbestrol /head/day) (13/11/65) Percentage of Total Dry Matter Nitrogen-Animal Replicate Crude Ether Crude Free No. No. Protein Extract Fibre Extract Ash 159 1 18.2 8.2 27.5 32.2 13.9 2 18.2 8.6 28.1 31.3 13.8 3 18.1 7.9 28.3 32.0 13.7 X 18.1 8.2 28.0 31.8 13.8 162 1 17.2 8.0 30.0 32.5 12.3 2 17.2 8.0 32.1 30.3 12.4 3 17.1 7.3 33.4 29.9 12.3 x- 17.2 7.8 31.8 30.9 12.3 Average 17.7 8.0 29.9 31.4 13.1 TABLE XXXII. Summary of proximate analyses data for feces of individual wethers. Percentage of Total Dry Matter Nitrogen-Animal . Crude Ether Crude Free No. Treatment Protein Extract Fibre Extract Ash 159 ^•Control 20.4 8.6 25.8 32.2 13.1 2Ration No. 2 17.4 7.8 28.7 33.0 13.2 "x 18.9 8.2 27.3 32.6 13.2 171 Control 20.0 9.0 25.2 34.1 11.8 2Ration No. 2 15.9 6.4 31.1 33.7 12.9 18.0 7.7 28.2 33.9 12.4 174 ^Control 19.5 8.4 27.3 32.8 12.2 2Ration No. 2 15.7 7.6 31.4 33.3 12.0 X 17.6 8.0 29.4 33.1 12.1 162 1Control 19.9 8.5 25.7 33.2 12.8 2Ration No. 2 17.6 7.6 30.5 32.2 12.5 X 18.8 8.1 28.1 32.7 12.7 Dehydrated grass meal Dehydrated grass meal plus 2.5 mgm. diethylstilbestrol/head/day. APPENDIX VI. 107. TABLE XXXIII. Detection of plateau curve (counts per minute at various voltages obtained from a standard volume of I**! solution) Length of Voltage Count (min.) c .p.m. 500 10 25 600 10 40 700 10 63 800 10 155 850 10 618 900 10 6,473 950 10 9,364 1,000 10 12,200 1,050 10 13,988 1,100 10 15,177 1,150 10 16,298 1,200 10 16,624 1,250 10 16,912 1,300 10 17,245 1,350 10 17,279 1,400 10 17,297 1,450 10 17,413 1,500 10 18,699 1,550 10 21,721 108. FIG. III. Counts per minute at various voltages obtained from a standard volume of 1*31 solution. /SSI Volts 109. APPENDIX VII. TABLE XXXIV. Individual market data for lambs fed control ration. I n i t i a l Market Total Total No- Dressed Average Animal Weight Weight Gain Days On Weight Dressing Gain/Day No. Sex (lbs.) (lbs.) (lbs) Treatment (lbs.) % (lbs.) 278 F 62 108 46 52 55 51 0.89 505 F 70 114 44 52 59 52 0.85 509 F 72 108 36 52 56 52 0.69 504 M 72 102 30 38 51 50 0.79 279 F 70 105 35 52 53 51 0.67 272 M 76 100 28 38 49 49 0.74 510 F 52 98 46 52 49 50 0.88 260 M 56 99 43 66 50 51 0.65 512 F 61 99 38 66 53 54 0.58 506 F 60 100 40 73 56 56 0.55 508 F 52 100 48 66 49 49 0.73 345 M 69 106 37 38 53 50 0.97 524 F 51 98 47 66 49 50 0.71 503 F 54 97 43 73 51 53 0.59 259 M 52 102 50 66 51 50 0.76 507 M 56 103 47 59 53 51 0.80 516 F 54 100 46 80 50 50 0.58 526 F 36 100 64 80 49 49 0.80 531 F 38 99 61 88 45 45 0.69 519 M 40 99 59 88 50 51 0.67 518 F 44 93 49 88 41 44 0.56 515 M 40 97 57 73 47 48 0.78 X 56 101 45 64 51 50 0.72 TABLE XXXV. Individual market data for lambs implanted with 12 mgm. diethylstilbestrol Weight at Market Total Total No. Dressed Average Animal Implantation Weight Gain Days On Weight Dressing Gain/Day No. Sex (lbs.) (lbs.) (lbs.) Beatment (lbs.) 7. (lbs.) 263 M 66 106 40 52 53 50 0.77 262 M 64 102 38 38 51 50 1.00 264 F 67 104 37 52 49 47 0.71 267 F 62 103 41 52 52 51 0.79 266 M 76 105 29 38 51 49 0.76 577 M 62 105 43 52 50 48 0.83 517 M 60 103 43 52 48 47 0.83 520 F 62 101 39 66 50 50 0.59 258 F 58 104 46 52 47 45 0.88 276 F 62 105 43 52 49 47 0.83 523 F 50 101 51 66 49 49 0.77 522 M 54 102 48 52 50 49 0.92 501 M 50 99 49 59 49 50 0.83 521 F 54 100 46 52 50 50 0.88 511 M 60 97 37 59 48 50 0.63 502 F 37 76 40 59 36 47 0.68 530 M 43 102 59 80 49 48 0.74 529 M 37 98 61 88 44 45 0.69 514 F 36 96 60 88 48 50 0.68 525 M 38 101 63 73 47 47 0.86 X 55 101 46 59 48 48 0.78 110, APPENDIX VIII. TABLE XXXVI. Diethylstilbestrol implant weights.(mgm.). Individual Implant Weights (mgm.) 14.6 15.0 14.7 13.8 14.5 14.7 14.6 14.4 15.3 15.0 15.7 14.8 14.8 15.0 14.7 15.0 14.6 14.9 14.8 14.9 14.7 14.6 14.8 15.4 14.2 14.1 14.5 14.5 14.3 15.3 x - 14.7 + 0.4 TABLE XXXVII. Diethylstilbestrol implant residue weights. Implant Average Pituitary DES Weight Animal Res idue Days on Gain/Day 1 Weight Retained No. (mem.) Treatment (pounds) (mgm.) (mgm.) 521 4.4 48 0.88 0.858 10.3 276 4.3 48 0.83 0.765 10.4 517 6.2 48 0.83 1.047 8.5 264 7.0 48 0.71 0.693 7.7 263 6.9 48 0.77 0.569 7.8 258 4.1 48 0.88 0.814 10.6 522 5.8 48 0.92 0.960 8.9 502 5.0 55 0.68 0.799 9.7 523 7.5 62 0.77 1.810 7.2 520 5.6 62 0.59 0.790 9.1 525 4.0 69 0.86 0.819 10.7 530 3.9 76 0.74 0.620 10.8 529 4.0 84 0.69 - 10.7 X 9.4 + 1.3