THE INFLUENCE OF VARIATIONS IN THE THYROID STATE ON REPRODUCTION AND LACTATION 1N FEMALi ALBINO MiCE Thai: for flu Dam of M S. MICHiGAN STATE 'COtLEGE Diorofhy Hupp Ward 1950 This is to certify that the thesis entitled The Influence of Variations in the Thyroid State on Reproduction and Lactation in Female Albino Mice presented by Dorothy Hupp Ward has been accepted towards fulfillment of the requirements for 11.3. degree in PhXSiOlOSy Major professor Date December 4, 1950 0-169 . . 0 .. ... «u... sewn} . n y :0. l. - ‘. , c‘ I ’ u ‘ I .; 3.. .v \. m ”‘1. .J n . v.1 . . ,4... Jim/y, . u .-thi. . J‘lot ..‘A. V}. ..Fmasswu . ,. .1, \ ...‘\. r I. A’.u 40“} a. .91 . u. . \..1\J.n . . . t n .\.L‘ k: Pvt‘v V5 ”Mariam . .... 4 .. 6.. . mauveflwvfiu 894%.; {5&qu .. t , t - . . .. _ . . Us .. Tn: . u. .t.« V at.” .. t . . ._ . ,l..ti......a... t: t .4;- AL . “A. At... ....l...4..... .. t .7 .. ..., ,hmfinu? .... 3%. ..w . wartime.“ . ..I..:..tn.\t. .H, . . t. .1»: u. t . .. THE INFIUENCE OF VARIATIONS IN THE THXROID STATE ON REPRODUCTION AND IAC’IIAT ION IN FEMALE ALBINO MICE By Dorothy Huppkrd A TfiIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE ' Department of Physiology and Pharmacology 1950 THES!S DEDI CAT IONS This thesis is affectionately dedicated to my husband Whose constant understanding and helpful encouragement have made cmpletion of this work possible and to my parents, Mr. and Mrs. Charles D. Hupp, for their understanding guidance and inspiration. Dorothy Bupp Ward 244598 -11... ACKNOWLEDGHEmTS The writer wishes to express her sincere appreciation to Doctor B. P. Heineke, Professor of Physiology and Pharmacology, for his timely advice and guidance throughout this study and for the critical reading of this manuscript. Gratitude also is expressed to Mr. B. B. Rosebocm, former Head of the Department of Physiology and Pharmacology, and to Doctor B. v.111fredson, Head of the Department of Physiology and Pharmacology, for the provision of the materials, equipment, and facilities necessary for the completion of the study described herein; and to Mr. John Monroe and Mr.‘lilliam.Gunn for their valuable advice and assistance in handling and caring for the animals. - iii - TABLE OF CONTENTS INTRODUCTION KEVIN OF LIT ERATURE The Influence of Variations in the Thyroid State on Growth and on Food and Water Intake The Influence of Variations in the Thyroid State on the Estrous Cycle Smear Technigues and Normal Cycle Lengths Hypothyroidism Hyperthyroidism Mechanical Stimulation The Influence of Variations in the Thyroid State on Fertility Hypothyroidism Hyperthyroidism The Influence of Variations in the Thyroid State on Gestation Typcthyroidism Hyperthyroidism The Influence of Variations in the Thyroid State on Parturition and Litter Performance Normal Littering Responses Hypothyroidism Hyperthyroidism The Influence of Variations in the Thyroid State on Mammary Gland Development and on Lactation Transplacental and Transmammary Transmission of Thiouracil and of Thyroactive Substances Evaluation of Normal Lactation Hypothyroidism 11 ll 11 15 18 18 19 19 20 20 22 23 TABLE OF comsms (Cont.) Hyperthrcidism The Use of Thiouracil as a Goitrogen History Mode of Lot ion Its Use in a Method for Assay of Thyroid Secretion Rate advantages and Disadvantages PROCEIIIE RESULTS General Appearance Food and Water Intake Body Weight Estrous Cycles Gestation Litter Height and Size Nesting and Care of Litters Hair Coat and Opening of Eyes Sex of Litters lactation DISCUSSION sums! AND CONCLUSIONS LITERATURE CITED APPENDIX 26 28 28 29 30 31 34 39 39 41 43 47 47 49 53 54 54 56 58 68 70 79 INDEX OF TABLES AND FIGURES TABLE I Thyroxine Doses Used in Experiment I TABLE II Thyroxine Doses Used in Experiment II TABLE III Daily Food and Water Intake Calculated on Body Weight Basis TABLE IV Estrcus Cycle Length TABLE V Summary of Litter Data--Experiment I TABLE VI Summary of Litter Data--Experiment II TABLE VII Sex Ratio at Weaning TABLE VIII Lactation FIGURE I Percentage Weight Comparisons, Experiment I FIGUE II Percentage Weight Comparisons, Experiment II APPENDIX TABLE I Food and Water Intake--Experiment I APPENDIX TABLE II Food and Water Intake--Experiment II APPENDIX TABLE III Weight and Littering Data-«Group A, Control APPENDIX TABLE IV Weight and Littering Data-«Group B, Thiouracil APPENDIX TABLE V Weight and Littering Data--Group C, Thyroxine, 5 APPENDIX TABLE VI Weight and Littering Data--Group D, Thyroxine, 25 APPENDIX TABLE VII Weight and Littering Data-~Group E, throxine, 45 APPENDIX TABLE VIII Weight and Littering Data-«Group F, Thyroxine, 85 APPENDIX TABLE IX Ieight and Littering Data--Group G, Control APPENDIX TABLE 1 Weight and Littering Data-"Group H, Thiouracil APPENDIX TABLE XI Weight and Littering Data--Grcup I, Thyroxine, £1 APPENDIX TABLE XII Weight and Littering Data-~Group J, Thyroxine, _x_ 36 38 42 48 5O 51 55 57 45 46 80 81 82 83 84 85 86 87 88 89 90 91 INDEX or TABLES AND FIGURES (Cont.) APPENDIX TABLE XIII Weight and Littering Data-"Group K, Thyroxine, 255 APPENDIX TABLE XIV Weight and Littering Data--Group L, Thyroxine, 45 APPENDIX TABLE XV Estrous Cycle Length as Measured Between Peaks of Est rus--Experiment I APPENDIX TABLE XVI Estrcus Cycle length as Measured Between Peaks of Est rus--Experiment II APPENDIX TABLE XVII Estimate of Length of Gestation-- Experiment I APPENDIX TABLE XVIII Estimate of Length of Gestation-- Experiment II APPENDIX TABLE XIX Individual Litter Weight Data-~Experiment I APPENDIX TABLE XX Individual Litter Weight Data--Experiment II APPENDIX TABLE XXI Opening of Eyes and Appearance of Complete Eair Coat APPENDIX TABLE XXII Sex of Litters APPENDIX TABLE XXIII lactation APPENDIX TABLE XXIV Index of Lactation Performance 92 93' 94 95 97 98 100 101 10 3 104 105 107 INTROHJCTION Although considerable research has been reported in this field there is still much disagreement regarding the role of the thyroid secre- tion in reproduction in the female. Clinically it has been shown that many women‘with basal metabolic rates in the normal range, and with no outward signs of myxedema, have reproductional disturbances. These dis- turbances include menstrual irregularities, low fertility approaching sterility, and termination of pregnancy with abortion, miscarriage, or stillbirth. Correction or prevention of these disturbances is often ac- complished through constant medication with prophylactic or therapeu- _ tic doses of thyroxine or desiccated thyroid. It has been reported by various workers that induced mild hyperu thyroidism increases both milk and fat yield of lactating cows and goats. Many studies have been carried out on rate, rabbits, and guinea pigs re- garding the influence of’daily thyroxine treatment and the influence of hypothyroidism resulting from surgical ablation or pharmacological sup- pression of the thyroid gland on estrous cycle, length of gestation, resorption of fetuses, abortion, litter size, and lactation. The results reported, especially concerning the influence of vari- ations in the thyroid state on gestation, littering, and lactation often have been conflicting. Little experimentation on this subject has been reported in'which mioe'were the experimental subjects. Results of the existing work indicate that a definite interrelationship exists between the thyroid state and these processes. This study was initiated in an attempt to determine the influence hypothyroidism, produced by thiouracil administration, and varying de- grees of hyperthyroidism, resulting from.the administration of varied doses of d,l~thyroxine, exert on the physiological processes of repro- duction and lactation in female albino mice. REVIEH OF LITERATURE The Influence 2f Variations 3:2. the Thyroid State 22 Growth and en Food and Water Intake An excellent review on the effect of experimental changes of thyroid secretion status on various aspects of animal production has been made by Blaxter 3:11.. (1949). These aspects of production include growth and fattening, fertility, reproduction, mammary growth, and lactation. This report discusses the use of various thyroactive iodinated proteins, such as 'Protamone' and iodinated casein, and of various synthetic goitrcgens, such as thiourea and thiouracil, to change the thyroid se- cretion status experimentally in ruminants, swine, and poultry. Barker (1949) presented a good review of the influence of thiouracil on repro- duction and growth in the rat. The influence of variation in environmental temperature and thyroid status on growth, feed consumption, and sexual development in the growing male mouse has been studied by Magsocd and Reineke (1950a, 1950b). They reported that thiouracil feeding depressed both feed and water consumption in mice. Mice receiving graded amounts of thyroprctein in their feed showed an increase in food and water intake proportionate to the thyro- prctein dosage. Thyroprotein given as both 0.025 and 0.05 per cent of the ration caused highly significant weight gains. They found also that thiouracil administration caused a decrease in the weights of the testes and of the seminal vesicles; there were some atrophic and degenerative changes, with limited spermatogenesis. Mild hyperthyroidism stimulated spermatogenic activity in the testes and stimulated sexual development. Soliman (1950) found that mild doses of thyroprctein given to grating female mice produces in them a rate of growth exceeding that of the nondtreated controls. This indicates that the rate of thyroid hormone secretion in mice apparently is less than the optimal. The rate of growth and maturation are further decreased by thiouracil. Thiouracil decreased food and water consumption; thyroprctein increased the food and water consumption. The Influence 2.1: Variations in the Thyroid State 23}. the Est rous Eye 16 Smear Techniques and Normal Cycle lengths As early as 1917 Stockard and Papanicolaou made use of a small nasal speculum for vaginal examination of guinea pigs. The speculum permitted an examination of the entire surface of the vaginal canal and smears 'were made daily from the substances present in the lumen. These investi- gators studied the smears and observed the existence of a typical estrous cycle. Allen (1921, 1922) used the same technigue, slightly modified for use on.the mouse. He examined some animals three times daily to get the exact time relations of the various phases of cell changes occurring in their vaginal linings. One daily examination was found to be adequate for accumulation of data as to cycle length. He designated the various stages in.the cycle as diestrus, a period of relative Quiescence averaging from.cne to three days; proestrus, a period of augmented growth and con- gestion often lasting less than a day; estrus, the period of sexual ex- citement or heat usually lasting one or two days; and metestrus, divided into two stages each usually lasting a day and consisting of a return to the diestrus stage. The smears associated with the various stages of this cycle were described. He found the average deviation of the entire cycle to be from four to six days. Observations were made that ovulation in some mice is spontaneous at every estrus, and the ovaries are chiefly masses of corpora lutea. He found, however, that not all mice ovulate spontaneously during estrus. Some ovulate only sporadically and corpora lutea may be entirely absent. Normal estrous cycles were experienced in both types of mice. Long and Evans (1922) made a very complete study of sexual maturity in the female rat. They concluded that first estrus occurred at an average age of 92.7 days and that vaginal introitus occurred at an average age of 76.5 days with first ovulation in the majority of the females de- layed approximately five days after the occurrence of vaginal introitus. The first estrus cycle was found to be significantly longer than any suc- ceeding one. Blandau 33.213 (1939) studied the normal reproductive cycles of female rats by use of the copulatory response and found the average duration of heat periods to be 13.7 hours with the onset of three-Quarters of the heat periods studied occurring between 5 P. M. and 11 P. M. They found four-day cycles in 58 per cent of the cases studied and five-day cycles in 24 per cent of the cases. Blandau and Money (1943) found that the average age for onset of heat in 200 albino female rats was 49.4 days. The length of the first heat averaged 9.07 hours. They also observed that the first heat period was significantly shorter than succeeding ones and the first and second reproductive cycles were significantly longer than the remaining ones. The majority of the first heat periods began between 7 P. M. and 5.A. M. Blandau 32.333 (1941) reported that tests of estrous condition of the female rat can be made at freQuent intervals over a considerable period of time without disturbing the normality of the cycle. This was accomplished by ascertaining the degree of copulatory response to manual manipulation in the vulvar region. Indivudual animals tended to show a consistency in character of the cyclic behavior as measured by the, length of heat, the length of the cycle, and the time of day at which heat began. Lee (1927, 1928) reported an increased basal heat produc- tion in female rats during the last 10 hours of diestrus and in the first six hours of proestrus. Boughton and Stoland (1943) found that all the mature albino rate they used as experimental subjects exhibited estrous cycles at less than 25 weeks of age. Of these, 84 per cent were four-day cycles, and the seQuence of four-day cycles was Quite regular up to about 50 weeks of age, becoming increasingly irregular beyond this time. .An increasing proportion of the animals over 25 weeks of age failed to show evidences of heat on vaginal smears, and of those that did an ever larger propor- tion deviated from the four-day cycle. gypothyroidism Using thyroidectomized female rats Lee (1925) found that the first cycle following the operation and sometimes the second one was often much lengthened, however this also occurred after a sham operation only, apparently due to trauma or slight infection. The succeeding cycles showed an average lengthening of about one day above those of normal controls due to a lengthened diestrus. Freedman 23.213 (1935) confirmed this prolongation of the cycle in diestrus, with the lengthening of diestrus ranging from one to five days. Ross (1938) also found that the duration of the estrous cycles increased from the normal four or five days to seven or eight days in thyroideotomized female rats due to an extension of the diestrual phase. THE! Nelson and Tobin (1937) followed the vaginal smears of non-pregnant thyroidectomized rats and noted that the estrous cycles definitely were disturbed With a resulting tendency toward extended diestrus. Evans and Long (1921b) did not find permanent alteration in the length of the estrous cycles following thyroidectomy. They reported that the opera- tion usually was followed by a pause in the estrous cycles of six to 27 days; but this, in turn, was succeeded by normal estrous cycles. Dragstedt 33_al: (1934) reported that complete absence of the thyroid and the parathyroid gland in dogs did not prevent regular appearance of estrusin.the bitches. Krohn and White (1950) studied the effects of hypothyroidism on the reproductive cycle of female albino rats, using both surgical abla- tion and pharmacological suppression with thiouracil. They found that estrous cycles tended to be longer, especially in.the thyroidectomized animals, and more variable in length. Richter (1933) observed marked disturbances in the formerly very regular four to five-day reproductive activity cycles after thyroidectomy.’ The intervals between peaks of activity were found to vary from three to 25 days. During the period be- tween activity peaks the smears showed a consistently diestrous picture. 'fihen the animals were active, the estrous cornified cell stage had a tendency to be prolonged beyond the normal interval. Several workers observed the effect of administration of thiouracil on estrous cycles in rats. Nelson (1948) found that after either thyroidectomy or administration of thiouracil female rats show very ir- regular periods of estrus and they freguently may be absent for several weeks. Hana (1945) found disrupted estrous cycles in all rats given thiouracil. There were greatly lengthened periods between estrous stages, exemplified by 14-day periods between periods of cornification in some cases. He stated that the inhibitory action of thiouracil apparently is very rapid since some animals had only one estrous cycle after the addition of thiouracil to the diet. Hyperthyroidism Evans and Long (1921a) observed that estrous cycles usually were not greatly disturbed when rats normally showing four-day cycles were fed 0.25 to 1.50 gm. of fresh beef thyroid daily. However, when they were fed one-fourth to one-half of a gland daily the cycle was greatly lengthened or totally inhibited. Pronounced loss in body weight occurred and some animals died showing a degree of toxicity with this large dosage. Drill 32.213 (1943) found that female rats maintained continuous diestrus after 10 to 17 days of dosage with 100 mg. of thyroid gland in the daily diet. The administration of a yeast supplement during 79 days of thyroid feeding prevented this abnormality. Reiss and Pereny (1928) found that thyroid hormone inhibited heat in normal female rats and also suppressed the action of ovarian hormone applied to castrate animals. This result was confirmed by Van Horn (1933) ‘who found that approximately three times as much theelin was reguired to produce an estrous condition in a castrate rat made hyperthyroid by oral feeding of desiccated thyroid as was reQuired in an ordinary castrate animal. He believed that the increased metabolism induced by the hyper- thyroidism was instrumental in the elimination of theelin, thus keeping the amount of theelin in the body below that required for the estrous threshold. He also observed a prolonged diestrus in hyperthyroid rats and an irregularity of estrous cycles when sexually mature female rats with normal cycles were thyroideotomized. Hill (1948) studied the effect of both age and thyroid state on the response of ovariectomized rats to estrone. It was shown that young rats given tolerable doses of thyroxine, 7 ug. per 100 gm. body weight, for a 10-day period, show a decreased response to estrone. In old rats, 13 to 14 months of age, this same treatment increases the estrus response. Excessive doses of . thyroxine, 70 ug. per 100 gm. body weight, administered daily for 10 days to old rats decrease both estrus response and body weight. She stated that the sensitivity to estrogen decreased and the sensitivity to thyroxine increased in rats sometime between 11% and 14 months of age. Helpern and Hendryson (1935) raised the metabolic rate of female rats with dinitrophenol and found that heightened metabolism.22£;£2_did not lead to an increased rate of estrin elimination and had scarcely any effect on the estrous cycles, ovaries, and pituitaries. 0n the other hand there were marked changes in the estrous cycles, ovaries, and pitui- taries of thyroid-fed rats indicating that the thyroid has a specific action on them. 'Heichert and Boyd (1933) did considerable work on induced hyper- thyroidism in female rats and the ensuing diestrus. They showed that animals fed doses of 0.25 or 0.5 gm. of desiccated thyroid daily had none, one, two, or three regular estrous cycles after the beginning of thyroid feeding'followed by a prolonged diestrous period of 10 to 22 days in dura- tion. As long as thyroid feeding was continued, a series of such lengthened diestrous intervals occurred separated from each other by one or two normal estrous periods. Normal and regular estrous cycles followed again if thyroid feeding was discontinued during a prolonged diestrous period. These unusually long diestrous periods after thyroid treatment proved to ‘be pseudopregnancy since placentomata could be produced experimentally THE! during these periods. They also stated that it was possible for pseudo- pregnanoy to occur after mechanical stimulation of the vagina, even from taking smears . Mechanical St imulat ion Several groups of Workers found that mechanical stimulation of the vagina, as in swabbing or taking smears, often results in epithelial proliferation and cornification. Wade and Doisy (1935) used a small amount of cotton wrapped about the end of a toothpick and moistened with water to insert into the vaginal canal of spayed rats and rotate gently a few times to obtain smears. They found that the spayed animals which were neither smeared nor injected presented a thin, smooth vaginal epi- thelium, usually two cell layers thick When the vaginae were examined after the. animals were killed. A typical section from the vagina showed a progressive thickening of the epithelium up to twelve or more layers with the usual desquamation of the surface cells when the animals had been smeared once, twice, or three times daily. Likewise the vaginal smear underwent a progressive change. About one quarter of the spayed animals showed fully positive estrus smears on the third or fourth day of treatment when the smears were made three times daily. Emery and Schwabe (1936) agreed that frequent examination, four times a day, by this cotton swab method brought about positive estrus smears in the castrated rat instead of the usual negative diestrus smear. lactating mothers also were brought into an estrous-like condition by frequent examinations with the cotton swab. In normal virgin rats the estrous cycles became irregular and estrus occurred more frequently than normal when examinations were made four times a day With the cotton swab. They found, on the other hand, that the pipette and lavage method of TH E5 taking vaginal smears only rarely changed the diestrous smear of the castrated rat to a positive estrous-like smear. The cycles of normal virgin rats also were little affected by the lavage method. They con- sidered it necessary that extreme care be observed when taking daily smears with the pipette to insert only the tip into the vagina and not to move it back and forth while taking the smear. They warned that otherwise there may be sufficient pressure and irritation to give a positive smear. Rogers and Allen (1937) observed this same stimulation of the vaginal epithelium.by rotation of a cotton swab rotated several times in the vagina. They used a moist toothpick for taking smears and failed to observe these changes. The Influence g£_Variations inLthe Thyroid State 22 Fertility Hypothyroidism Hypothyroidism decreases fertility. Nelson and Tobin (1937) ob- served that non-fertile copulations were more frequent in thyroidec- tomized rats than in control rats. Litzenberg (1926) reported that myxedema is certainly a cause of sterility. He stated that lesser de- grees of hypothyroidism also cause or are an index of a cause of steril- ity. He found clinically that 39 per cent of 68 married women with low basal metabolic rates but without signs of‘myxedema were sterile. There 'were functional disturbances of menstruation in 40 per cent of 114 women, both married and unmarried, with low basal metabolic rates. 0f 44 sterile Women Who came for treatment half had basal metabolic rates of -10 or below. Thyroid medication was given to 18 of those who did have lower rates. Within two months six of them became pregnant. THEE ‘ r "...“...- ldfixa ‘ Litzenberg and Carey (1929) observed that in a large percentage of cases even a moderate degree of lowered metabolism.interferes with the reproductive function of women. Abnormal menstruation was found in one-third of all the women with low metabolic rates who were studied and in nearly two-thirds of the sterile wanen with low rates. Nearly half of all the women With decreased metabolism whom.they observed were sterile and more than half of the sterile women had below normal basal metabolic rates. These workers found that in many cases restoring the basal metabolic rate to normal by thyroid medication improved menstrua- tion and permitted conception. Foster and Thornton (1939) reported on the results they observed after treatment with desiccated thyroid on healthy women patients with normal pelvic findings who were seen because of menstrual irregularities and who had basal metabolic rates ranging from -1 to -33 with an average of -l5. Of 25 cases who complained of dysmenorrhea or difficult and painful menstruation 17 had complete relief and 5 had partial relief. Of 17 women with a scanty or infrequent menstruation or oligomenorrhea all experienced complete relief. Among 13 patients with metrorrhagia' or hemorrhage from the uterus 12 experienced complete relief. Six out of seven with excessively profuse menstruation or menorrhagia had com- plete relief. They believed that a therapeutic trial of thyroid is in- dicated if the basal metabolic rate is zero or below in those individuals complaining of menstrual irregularities who have no demonstrable patho- logical symptoms. Similarly, Winkelstein (1940) studied a group of sterile patients Who had been married at least one year with normal congress and Who had used no contraceptives. He found that a few of these women had slight - 11 - clinical hypothyroidism.and some had subclinical hypothyroidism. Some were overweight and were made to lose. All of them'were free of patho- logical symptoms. The normal range for basal metabolic rate was con- sidered by him to be -10 per cent to 010 per cent. He gave doses of desiccated thyroid starting with 1 grain per day and increasing the dose 0.5 to 1 grain per day at semiweekly intervals until a maintenance tolerance was reached and then continued indefinitely at this optimal dosage. Pregnancy occurred in most of these patients. Hyperthyroidism Gudernatsch (1915a, 1915b) found that female rats made hyperthy- roid by the feeding of fresh thyroid gland would not breed and pregnancy did not occur until a period of time had elapsed after the thyroid feeding was discontinued. Pregnancy did not occur in normal female rats when they were mated to hyperthyroid males. Clute and Daniels (1930) re- ported that a diminution of menstrual function and a lack of fertility often accompanies severe hyperthyroidism; in fact, the more severe the hyperthyroidism the less active is the ovarian function. The Influence of Variations in the Thyroid §tate 22 Gestation Hypothyroidism Long and Evans (1922) found that 90 per cent of the gestation periods in normal rats fall between 21.5 and 22 days in length. Ukita (1920) reported that rabbits thyroidectcmized from the seventh to the tenth day of pregnancy had their gestation period increased to 60 to 70 days in length. Krichesky (1935) disagreed with these results. He performed thyroidectomies on rabbit does from two hours to 12 days after their mating and found no apparent differences between the length of the gestation period in operated and unoperated does. Normal rabbits have an average 31-day gestation period ranging from 29 to 32 days; the thyroidectomized does ranged from 50 to 33 days in length of gestation period. Krichesky (1939) again reported that thyroid removal during the first 12 days of gestation in rabbits did not alter gestation length. Furthermore, these animals successfully terminated a second pregnancy within the limits of a normal gestation period. Litzenberg (1926) found that a normal basal metabolic rate apparently is necessary to a normal continuance of pregnancy. Clinically, he gave thyroid medication to 18 of 22 sterile women with basal metabolic rates of -10 per cent or beloW'and six or onedthird of them became pregnant and had normal full term pregnancies. Two of these women had had one or more previous pregnancies terminating in abortion, miscarriage, or stillbirth. One of these six women also had two normal pregnancies later while under thyroid medication. He suggested that women who habitually abort should have their basal metabolic rate measured. Litzenberg and Carey (1929) reported that 52 sterile women with a metabolic rate below normal were treated With desiccated thyroid to restore the rate to normal. Of these women 50 per cent became pregnant following treatment and had nor- mal full term pregnancies except two Who discontinued treatment after conception and aborted. Fifteen of these women who had normal full term pregnancies during treatment had aborted one or more times before treat- ment. Thus this work indicates that a normal basal metabolic rate should be maintained during pregnancy. These findings are not difficult to understand if it is true that reproductive tissue cells are more subject to deleterious influences than any other cells in the body. - 13 - The Japanese worker, Nojima (1933), thyroidectomized female rats and observed the effect on reproduction. He found that the mating rate decreased to 22.7 per cent of normal on the hundredth to the one hundred and fiftieth day after thyroidectomy, and the conception rate decreased to 33.3 per cent of normal on the eighty-eighth to the hundredth day after operation. Abortion occurred in a few of these thyroidectomized rats when pregnancy occurred and in those that did not abort there was a very slight tendency toward prolongation of the gestation period. If thyroidectomy were performed after the beginning of pregnancy it caused an increase in intra-uterine death rate, a decrease in fetal weight and size, and a hypertrophy of the fetal thyroid. Dragstedt 33.21: (1934) reported that complete absence of the thyroid and parathyroid glands in dogs did not prevent pregnancy. Nelson and Tobin (1937) found that thyroidectomy apparently has no harm- ful effects on pregnancy and parturition in rats and guinea pigs. Fifteen rats thyroidectomized during the last half of their gestation continued to term and delivered normal litters. Ten rats thyroidectomized prior to the incidence of pregnancy delivered one or more litters suc- cessfully, 17 in all. Parturition occurred in a normal fashion in six guinea pigs thyroidectomized during the last 10 days of pregnancy. Ross (1938) found that pregnancy with normal numbers of embryos occurred in three-quarters of the thyroidectomized adult female rats studied. There was, however, a decided increase in number of resorptions with only one third of the animals producing normal litters. King and Herring (1939) found that a low basal metabolic rate of -10 to -30 per cent in women often was followed by abortion. They even encountered abortion in cases where the husband had rather marked O s ... D a A n e e hypothyroidism. It was found advisable to give prophylactic doses of desiccated thyroid to pregnant women with a basal metabolic rate of -6 to -9 per cent, especially if'there'were a record of previous abortions. Sachs (1939) found that the basal metabolism of thyroidectomized rats falls severely within one month after the operation and, although fertilization occurred when the animals were mated, the advanced embryos died. Two parenteral injections of’l mg. of pure thyroxine before mating raised the basal metabolic rate to normal, and another such injection after mating led to normal pregnancy and normal birth, They found that additional injections of thyroxine on the thirteenth to the eighteenth day of pregnancy had no effect, indicating that thyroxine was no longer necessary after the thyroid gland of the embryo began to function. Folley gt a}: (1942) reported that pregnancy often was prolonged when thyroidectomized rats were mated. They delivered their young but seldom began to rear their litters. Jones 23.313 (1946) studied the effect of thiouracil-induced hypo- thyroidism.upon the reproductive system of the adult rat. Males did not lose their ability to sire litters. In female rats prolonged thiouracil administration interfered with continuation of gestation by causing resorption of embryos in all of the cases. If, however, the drug was given over a period of less than 100 days, some rats did do- liver litters which were normal in growth and development and reproduced normally. They judged the occurrence of pregnancy and resorption by finding sperm in the smear, followed by diestrus smears and finally bleeding. To be positive that this sequence of events was not given by pseudopregnancy they examined the animals by palpation for fetuses at the sixteenth day of gestation and performed laparotomies on seven animals. They found normally palpable fetuses in only one rat; the other rats had pregnancies which resorbed before the palpable stage. Of the laparo- tomized animals four had recognizable resorbing implantation sites, and the others had metrial glands (inflammatory areas). They concluded that treatment with.thiouracil for at least 100 days may be necessary to pro- duce severe hypothyroidism in rats. Krohn and White (1950) reported that hypothyroid rats conceived but due to resorption of fetuses at varying stages of gestation, they brought fewer young to term.than they had in a previous normal pregnancy, Whether they were made hypothyroid by surgical ablation of the thyroid gland or by pharmacological suppression with thiouracil. They also ob- served a slight prolongation of gestation. To be sure rats were hypo- thyroid at the time of mating, these workers waited at least 54 days after starting thiouracil injections and at least 28 days after thyroidec- tomy before mating. Preheim (1940) observed that gestation periods of thyroidectomized rats were prolonged approximately 24 hours. Hyperthyroidism Gudernatsch (1915a, 1915b) found that feeding fresh thyroid tissue delayed pregnancy. If pregnancy did occur, it resulted in abortion or the young died soon after birth. In rabbits cretinized by thyroidectomy at three weeks of age, and then after several months made severely hyperthyroid by feeding desiccated thyroid, Kunde gt Elf (1929) found, in most instances, that although fertilization and implantation occurred the young were never born. Instead, resorption of all or many of the fetuses occurred during the latter two-thirds of pregnancy. Finding of sperm on the vaginal smears of isolated does placed in mating cages no longer than one hour and palpation of fetuses about eight days later was taken as proof of'pregnancy. In some of the rabbits laparotomy incisions were made and fetuses were counted on the seventh to fifteenth day of gestation. It was reported by Rose (1947) that muscular maturation of the fetal rat may be delayed if the mother is made hyper- thyroid by feeding desiccated thyroid during the early portion of the pregnancy. Reichert (1930) noticed a prolonged gestation period in pregnant rats fed 0.25 gm. desiccated thyroid daily. He stated that in normal rats 90 per cent of parturition occurs at 21.5 and 22 days but one of these hyperthyroid animals delivered on the twenty-third day, three on the twenty-fourth, three on the twenty-fifth, and only one on the twenty- first day of gestation. He believed this prolongation to be due to inability of the rats to deliver rather than to their failure to come to full term.at the normal time, possibly because of inability of the estrus-producing hormone to act on uterine muscles to produce the nor- mal contractions of parturition. Possibly the increased metabolism is instrumental in eliminating this hormone. Three guinea pigs given 300 mg. thyroid daily from the tenth, the twentieth, and the thirtieth days of pregnancy were found by Carloni (1930) to have gestation periods of 61, 60, and 57 days, respectively. Young were born alive, and the litters of the first two animals showed no effects of the thyroid administration. The guinea pigs in the third litter were hyperactive. The length of - pregnancy.was 55 days, 51 days, and 53 days when 400, 500, and 600 mg., respectively, of thyroid were administered daily from the thirtieth day of gestation. The guinea pigs from mothers fed thyroid were longer and thinner than normal, and litters were all small. - 17 - Danforth and Loumos (1936) found that pregnant rats are more tolerant to desiccated thyroid than are non-pregnant rats. Danforth.gt_alf (1937) administered large doses of estrous, estriol, emmenin, progesterone, and the anterior pituitary-like factor of pregnancy urine to female albino rats for a week and then fed 100 mg. of desiccated thyroid to each rat daily for two weeks. There was significantly less increase in the oxygen consumption rate in all these doubly-treated groups than in control rats receiving only thyroid. This inhibition of the metabolic effect of orally administered thyroid indicates the reason for the ability of pregnant rats to tolerate doses of desiccated thyroid which are toxic when fed to non-pregnant animals. Bodansky and Duff (1936) reported the remarkable tolerance to thyroxine of pregnant rats. The number of young per litter was approximately the same in treated and untreated animals. They found, however, that a greater incidence of still-births occurred in the thyroxinedtreated rats than in the normal controls. They observed a normal gestation period in a considerable proportion of those rats treated with thyroxine. Normally 21.5 to 22 days is the gestation period in rats. They found, however, that two of their six untreated controls delivered on the twenty-third day and the rest by the twenty-fourth day. Of the thyroxine-treated rats, three delivered on the twenty-third day, two on the twenty-fourth day, and one on the twenty- sixth day. Since they had made no attempt to fix the actual insemination time, these results are questionable. ‘Weichert and Boyd (1934) began giving a daily dose of 0.5 gm. desiccated thyroid mixed to a thin paste with water and fed directly to the rat by a medicine dropper as soon as sperm were found in the vaginal smears indicating fertile copulation. 0f 21 thyroid-fed animals used 10 -18- had to be discarded due to resorption of embryos during the first half of pregnancy. They did not observe any prolonging of gestation, pro- bably due to their general practice of discontinuing thyroid feeding some time before parturition. The Influence 9.5. Variations 2 the Thyroid State .93}. Parturition and Litter Performance Normal Littering Responses Eat litters normally are born in the late afternoon of the twentieth day of gestation or, more often, on the morning of the twenty-first day according to Hall and Kaan (1942). Blandau and Money (1943) reported that the average litter size for first litters in normal female rats was six with a range of three to 10. Blandau and Soderwall (1941) made direct observations on parturition in normal rats. They found that the interval between delivery of the first and last members of a litter may vary considerably, even in animals bearing the same number of young. The largest number of pregnant mothers littered between 12 noon and 6 P. M. and delivery was complete in less than 70 minutes in the majority of cases. Sturman-Hulbe and Stone (1929) reported that the parturient rat normally manifests a strong nest building tendency closely associated with the birth and care of her young. This maternal nesting response lasts until the young begin to leave the nest at 17 to 20 days after parturition. Crozier and Enzmann (1935) reported that mice giving birth to more young than they could adeQuately suckle killed a proportion of the litter. During pregnancy there was a permanent increase of the weight of the mother depending upon the size of the litter carried. ‘After birth - 19 - of the litter, the mother killed off part of her litter on reaching the limits of her milk-producing capacity. Falconer (1947) also ob- served that the practice of infanticide seems to be rather widespread among mice. He found that individual weights in litters of normal mice at 12 days of age were greatest in litters of two, three, or perhaps four, gradually decreasing with increasing litter size, the reduction being approximately 0.075 gm. for each additional mouse in the litter. Hypothyroidism Nelson and Tobin (1937) found that 15 rats thyroidectomized during the last half of pregnancy delivered normal litters. This was also true of_10 rats thyroidectomized prior to the onset of pregnancy. Nojima (1933) reported an unusual result of thyroidectomy. He observed in rats that conception on the twenty-fourth to the thirty-fifth day after thyroidectomy produced 80 per cent of females in the litter, instead of the normal 48.5 per cent. He did not attempt an explanation. Folley gt_al, (1942) found that although thyroidectomized rats mated and de- livered their young, they seldom began to rear their litters. Hyperthyroidism ‘Weichert and Boyd (1934) observed that feeding of 0.5 gm. of desic- cated thyroid daily to pregnant rats for as long as 18 days after fer- tilization resulted in failure of the animals to care for their young. 'fleichert (1930) also observed that hyperthyroid rats not only did not care for their young but also ate many of them. Kraatz (1939) reported a larger litter size in rats made slightly hyperthyroid for a short time. ‘An average litter size of 13 resulted when adult female rats were treated with.dai1y administration of 0.25 to 0.3 gm. of thyroid substance for three to five days and then mated within four days to normal males as compared to an average litter size of nine in untreated littermates. ‘An increase in the amount of thyroid given proved deleterious to repro- duction. The Influence 23 Variations in the Thyroid State gnluammary Gland Development and _o_r_1_ lactation Transplacental and Transmammary Transmission EffThiouracil and of Thyroactive Substances Several workers reported transplacental transmission of thiouracil. 'Williams (1944) found significant Quantities of thiouracil in the fetuses of rats given 0.1 per cent thiouracil in their drinking water during gestation. Kauffman gt a}: (1948) presented evidence indicating that thyroid follicle and colloid formation began in the mouse on the sixteenth day of fetal life. They observed depressed colloid formation and in- hibited follicle formation in the fetal thyroids when the mother had re- ceived feed containing 0.2 per cent thiouracil. These workers believed this effect to be due to placental transfer of thiouracil. Goldsmith 22.21? (1945) reported an enlargement of thyroids in fetuses from rats fed thiouracil attributed either to the result of passing of thiouracil, of the thyrotropic hormone, or of a combination of both across the placenta» Tobin (1941) concluded that thyrotropin» did not pass across the placenta since it is a very large protein molecule. He determined the number of implantation sites in pregnant rats by laparotomies on the tenth day of pregnancy and intraperitoneal injections of thyrotropic hor- mone were begun the next day. On the sixteenth day the animals were sacri- ficed and the number of living and dead embryos were counted. He found -21.. that the percentage of living embryos was inversely proportional to the duration of the thyrotropin administration rather than to the dosage. Although the maternal thyroids showed histological signs of stimulation when the animals had been treated for two days or more, the thyroids of the embryos failed to show such signs. He attributed the embryonic deaths to secretion from the stimulated maternal thyroid. Hughes (1944) observed both transplacental and transmammary effects of thiouracil. Thiouracil was administered in the drinking water of pregnant female rats beginning four to eight days before parturition and continued after the birth of the young. The young of thiouracil- treated mothers appeared to be normal but showed thyroid hyperplasia as early as one day of age and definitely retarded development at 10 days. Freiesleben and Kjerulf-Jensen (1946, 1947) also observed both transplacental and transmammary transmission of thiouracil derivatives. Fetuses of pregnant rats which were given a diet containing 4-n-propyl- 2-thiouracil were mixed in the food of normal rats. This food-fetus mixture brought about thyroid hyperplasia in rats eating it. ‘When 6-methy1-2-thiouracil was administered to suckling rats immediately after parturition, thyroid hyperplasia was found in their young within one week and a maximal hyperplasia at 12 to 14 days. Goldsmith 3331;. (1944) found that young rats, suckled by mothers maintained on a labora- tory stock diet containing 0.5 per cent thiourea, had hyperemic and en- larged thyroids. 'Williams'gtnal. (1944b) reported that infant rats, whose only pos— sible source of thiouracil was the mother's milk, showed a thiouracil concentration of 5.6 mg. per cent in the blood While that of the mother's blood was 12 mg. per cent. williams gt Elf (1944a) reported that human patients receiving from 0.2 to 1.2 gm. of thiouracil daily showed a thiouracil concentration in the blood varying from 0.8 to 6.4 mg. per cent and about three times that concentration in their milk. Reineke and Turner (1944) conducted experiments to determine whether cows fed 'Protamone' for stimulating increased lactation would transmit any thyroidal substance in their milk. They fed guinea pigs milk from.'Protamone-fed' cows and found no changes in basal metabolic rates. In a similar experiment on goats thyroidectomized when young there was no improvement of the cretinism and no metabolic stimulation. However these animals responded markedly to 0.25 gm. 'Protamone' fed directly. Similarly, two human beings showed no elevation of basal metabolic rate as the result of daily consumption of one Quart of milk from 'Protamone-fed' cows but did have significant elevation after receiving 0.5 to 1.0 grain of 'Protamone‘ per day orally. They con- cluded that the mammary gland does not permit passage of biologically detectable amounts of thyroidal hormone when 'Protamone' is fed to lac- tating cows. Lukacs (1930) found that six litters (51 rats) suckled by mothers made hyperthyroid by thyroid feeding gained only 46 to 69 per cent as much as did the young in four control litters (31 rats) suckled by normal mothers. Lukacs regarded this as evidence that the thyroid hormone produced in the maternal organism.was secreted in the milk. This seems to be fallacious reasoning. The cause of the smaller weight gain observed in the young rats suckled by the hyperthyroid mothers probably was decreased lactation. Evaluation of Normal lactation Falconer (1947) found that milk production of mice could be evaluated satisfactorily from measurements of litter growth. He used the 12-day _~V7 total weight of the litter suckled as a convenient measure of milk production. The weight was taken at this age since young mice begin to take solid food when their eyes open at about 13 to 15 days of age and thus 12 days after birth was considered to be the latest time at which a measurement Would represent growth due only to milk consumed. To allow for difference of litter size he used an index of performance independent of the size of the litter suckled to express the milk production of a mouse. This index of performance was obtained by expressing the litter weight produced by a mouse during a particular lactation as a percentage of the average weight of litters of that number. Of course, the index measures only the response of a mouse to a standard stimulus and gives no indication of the maximum capacity of the mouse. Even with the in- dexes of performance it is uncertain whether the comparisons of the lactation of mice having different numbers of'young are completely valid and Whether real differences of performance will be detected. Hypothyroidism Several studies have been made concerning lactation in thyroidec- tomized animals. Grimmer (1918) reported a temporarily diminished milk secretion believed due to operative trauma following thyroidectomy in the goat. Graham (1934b) found that thyroidectomy of cows caused a marked lowering of milk fat secretion as well as a diminution in milk secretion but a similar decrease accompanied control operations. Dragstedt 33.323 (1934) reported that complete thyro-parathyroidectomy in dogs did not prevent the hypertrophy of the mammary glands during pregnancy or the secretion of’milk after delivery provided tetany tas controlled. Nelson and Tobin (1937) found that rats thyroidectomized during the last half of pregnancy suckled one or more litters successfully. After thyroid removal during the last 10 days of pregnancy, guinea pigs lactated in a normal fashion. Preheim (1940) found that thyroidectomy in rats reduced the growth of their offspring 12 per cent, indicating that lactation was affected. Davenport and Swingle (1927) found that thyrocauterized mothers lactated poorly. Karnofsky (1942) observed that the thyroid gland was not essen- tial for lactation in the rat. However, thyroidectomy before conception, during gestation, or immediately after delivery was found to diminish lactation. Folley (1938) likeWise found that removal of all the thyro- parathyroid tissue from lactating rats caused an immediate marked decline in lactation as judged by growth rates of sucklings. Then these operated rats were subseguently remated, parturition was normal but lactation was definitely subnormal. Folley'gt_alf (1942) confirmed this work. They were, however, able to maintain lactation partially in thyroidectomized rats by injecting them daily with parathyroid extract. They concluded that the observed failure of lactation after thyroidectomy is due, at least in part, to parathyroid deficiency. Smithcors and Leonard (1942) thyroidectomized normal immature male rats and observed an inhibition of.mammary duct growth and a stimulation of alveolar development as compared with their normal controls. In young female rats Leonard and Reece (1941) observed a thickening of the mammary gland ducts and an increase of the number of lateral and buds after thyroidectomy. Nelson and Hickman (1937) also found that mammary deve- lopment may occur in the absence of the thyroid. Mammary glands of thyroid- ectomized rats receiving estrone showed marked development as did normal controls receiving estrone. Oddly enough Smithcors (1945) reported that thiouracil administered to normal or castrated male and female rats failed to produce the lobule- alveolar growth in the mammary glands similar to that obtained following thyroidectomy. Since the period of thiouracil treatment was for only 35 days he believed that possibly the period of treatment was not suf- ficiently long for the changes to occur. Meites and Turner (1947) ad- ministered O.1 per cent thiouracil in the feed of young female rats for 24 days. They observed that this treatment reduced the lactogenio hor- mone content of the pituitary below that in the case of normal rats.- When estrogen and thiouracil together were administered to rats for 10 days subseQuent to a two-week preliminary treatment with thiouracil a- lone, the estrogen and thiouracil failed to maintain even the normal level of lactogenio hormone. Trentin.gt_alt (1948) reviewed the effects of thiouracil on repro- duction and growth in mice and rats with special attention to mammary growth. They concluded that experimentally induced mild hyperthyroidism is conducive to enhanced mammary responsiveness in the mouse, Whereas the opposite is true of hypothyroidism. In the rat, on the contrary, ex- perimentally induced hypothyroidism is conducive to mammary gland growth and responsiveness. work which these workers did confirmed these facts. They administered diethylstilbestrol to one group of castrate male rats and thiouracil and diethylstilbestrol to another group of male castrates and then compared these groups with a group of control castrate male rats. These receiving both thiouracil and stilbestrol showed extensive alveolar development with good duct extension and a striking advancement of mammary development as compared to the controls and a less marked im- provement over those receiving only stilbestrol. They repeated the experiment on intact male albino mice and detected no difference in the response to estrogen in the stilbestrol and thiouracil treated mice from those treated only with estrogen. Hyperthyroidism The influence of the thyroid gland on lactation has been clearly established by administration of thyroid hormone to pregnant and lac- tating animals. 'Weichert and Boyd (1934) observed that experimental hyperthyroidism in pregnant rats resulting from the feeding of 0.5 gm. desiccated thyroid daily starting on the first day of pregnancy caused earlier mammary development than occurred in controls. They also re- ported that the thyroid-fed rats had an earlier appearance of secretion in the alveoli than did the controls. While working with the lactating cow in declining lactation, Graham (1934a, 1934b) found that thyroxine injections or thyroid feeding Quickly caused a marked temporary increase in milk fat production and a lesser increase in milk secretion to a higher level than previously after which the gradual fall, which is normal during this portion in the lactation curve, continued. During the early stages of lactation following parturition such treatment had no apparent effect on the amount of milk secreted. He also observed that feeding excessive Quantities of thyroid to a thyroidectomized animal caused a diminution in milk secretion and an even greater diminution in fat secretion. In the goat de Fremery (1956) found that low thyroxine dosage pro- duced no demonstrable change in the milk output. On the other hand, high dosage of thyroxine, 15 mg. daily, caused severe hyperthyroidism with reduced milk output. Contrary results were reported by Reineke and Turner (1942). They fed thyrolactin, an artificially formed thyroprctein, to goats in the declining stages of lactation at the rate of 5 to 10 gm. daily. There was an increase in milk production ranging from 0.8 to 40.8 per cent and averaging 10.51 per cent. An average in- crease in milk yield of 8.59 per cent occurred in cows fed 50 to 100 gm. of thyrolactin daily. There also was an average increase of 6.77 per cent in milk fat percentage and an increase of 13.9 per cent in fat yield. Gardner (1942) fed small amounts of desiccated thyroid to intact male mice and reported that their mammary glands showed duct proliferation and hyperplastic end buds; no mammary growth showed in castrate male mice similarly treated. Mixner and Turner (1942) reported that an optimal dose of thyroxine increased by about 25 per cent the efficiency of mini- mal doses of progesterone and estrone in stimulating mammary lobule- alveolar growth in castrate female mice. On the other hand, thyroid- ectomy inhibited the ability of these mice to respond to progesterone and estrone. 'Weichert gt_al. (1934) reported the initiation of mammary secretion in a normal virgin rat fed desiccated thyroid and occurring only during the pseudopregnant period induced by the experimental hyperthyroidism. Gudernatsch (1915a, 1915b) reported a decrease in milk output from feeding thyroid to rats. ‘When rats were extremely hyperthyroid the young in their litters showed a diminished ability to groW'and stayed behind the normally-fed rats of litters of corresponding age in relative size. The subject of lactation was adequately reviewed by Folley (1940). He summarized the influence of the thyroid gland on lactation by stating that the thyroid gland is essential for normal lactation and that thyroxine stimulates milk secretion in the cow. In many species lactation can pro- ceed in the absence of the thyroid, but only to a slight degree. —‘UU- The Use 33 Thiouracil 33 g Goitrogen Histogy Until recently it was necessary to perform thyroidectomy to pro- duce experimental hypothyroidism. Kennedy (1942) reported that an at- tempt to isolate the goitrogenic substance in rape seed suggested that it might be a derivative of thiourea. ConseQuently he gave daily doses of 20 mg. allyl thiourea to rats for eight weeks. Upon examination ‘ these rats had thyroids three to four times the normal weight with the glands showing extreme hypertrophy and hyperplasia and almost complete lack of colloid. His experiments with thiourea produced similar results. Several different groups of‘workers reported experiments testing the effect of thiourea on the thyroid gland during the next year. The Mackenzies (1943) observed the production of a hyperemia and an enlarge- ment of the thyroid with a reduction in colloid and an increase in the height of the thyroid epithelium accompanied by severely lowered basal metabolic rates in rats, mice, and dogs administered sulfonamides and thiourea. The reaction did not occur in chicks or guinea pigs. The effect of the drugs was not prevented by a number of dietary factors including liver, p-aminobenzoic acid, vitamin C, and iodine. However, thyroxine at proper dosage level prevented the effect entirely. Astwood gt :1. (1943) confirmed these results and reported that the goitrogenic activity of thiourea was higher than that of any of the sulfonamides tested. Williams and Bissell (1943) used thiouracil, a thiourea deriva- tive, to treat thyrctcxicosis effectively. £033 2!. Action Thiouracil apparently lowers the basal metabolic rate by a de- crease in the production of thyroid hormone in the same manner as do thiourea and sulfonamides. Glock (1949) fed 0.1 per cent thiouracil in \the diet to a litter of puppies and to adult dogs. Although there were no outward manifestations of hypothyroidism in any of the dogs or puppies, their thyroids were considerably enlarged with marked hyperplasia and the loss of colloid generally was relatively small. Many investigators have attempted to determine the mechanism of thiouracil action. Malkiel (1946) found that thiouracil does not in- activate preformed and circulating thyroxine and is not antagonistic to it. Bawson'gt_gl3 (1944) found that thiouracil acts to block the usual easy and ready uptakeof iodine by the thyroid, that is, thiouracil acts to interfere with the iodination of the thyroglobulin molecule preventing the elaboration of any physiologically active thyroid hormone. Astwood and Bissell (1944) found that the administration of thiouracil to young rats was followed by a nearly complete disappearance of iodine from the thyroid gland as well as by a threefold increase in the size of the gland. Franklin.gt_3£: (1944) showed that thiouracil fed to rats inter- fered with.the incorporation of injected radioactive iodine into diiodotyd rosine and thyroxine by their thyroid glands. Jandorf and Williams (1944) found that subcutaneous injection of thyrctropic hormone into rats caused an increase in the rate of oxygen consumption of all organs studied and that especially liver and muscle showed increased metabolism, This effect was abolished by simultaneous treatment with thiouracil. The hyperplasia of the thyroid was greater when the two were given simultaneously than when either was given alone. They concluded that this effect was due to the inhibition of thyroid hormone formation by the thiouracil. Himsworth (1943) also concluded that thiourea acts by interfering with the synthesis of the thyroid hormone. Keston.gt a}: (1944) reported that analyses showed no appreciable amounts of radioactive iodine in any form in the thyroid glands of thiourea- treated rats. Its Use in a Method for Assay_2£ Thyroid Secretion Rate ’Several workers have reported the use of thiouracil together with thyroxine as a method for assaying thyroid hormone secretion rate. Dempsey and,Lstwocd (1945) reported using the method as a basis for an assay procedure for thyroid hormone. They found that maintenance or restoration of normal thyroid weight by simultaneous administration of 0.1 per cent thiouracil in the drinking water and of graded doses of thyroxine by injections to rats gave a measure of the amount of thyroid hormone secreted by the thyroid gland. At room temperatures averaging 25° C. a Quantity of thyroid hormone eQuivalent to 5.2 ug. of thyroxine was required daily to maintain a thyroid of normal weight in young male rats. Reineke 32.233 (1945) used this method and found that, when young male albino rats were given 0.1 per cent thiouracil in their drinking water for two weeks, daily injections of 4.75 ug. of d,l~thyroxine re- turned their metabolic rate to normal and daily injections of approximately 4.8 ug. were reQuired to return the thyroid weight to normal. Hurst and Turner (1947) determined the thyroid secretion rate in mice using the simultaneous thiouracil-thyroxine administration method of assay. They found that mature female mice kept at an.average temperature of 80° F. produced the eQuivalent of 5.5 ug. of d,l-thyroxine per 100 gm. body ‘weight daily. a...— ...-m;» If . Reineke and Turner (1945) determined the comparative thyroidal activity of l- and of d,1-thyroxine by their relative ability to reduce the thyroid weight of thiouracil-treated chicks and rats to normal. They found that l-thyroxine produced twice the effect of the racemic mixture in every species tested. Advantages and Disadvantages The effects of thiouracil and thyroidectomy were compared by Gordon 22.2}! (1946). Both methods reduced food and water consumption and in- hibited body grthh of rats. In the thyroidectomized group these effects occurred more rapidly and were more pronounced. Oxygen consumpticn'was depressed to approximately similar levels by both methods of thyroid ablation. [Lstwood (1943) tested the relative effectiveness of 106 chemical compounds in inhibiting the function of the thyroid. He found Z-thiouracil the most effective compound tested and stated that 3 to 4 mg. daily of thiouracil induced maximal responses in young rats. There are several advantages in the use of thiouracil over thyroidec- tomy for producing hypothyroidism experimentally. Meyer EEHElF (1944) reported that thiouracil is relatively non~toxic. Krohn and “hits (1950) found that pharmacological suppression of the thyroid was more effective in causing a consistent depression of metabolic rate than was surgical ablation. Meyer and Ransom (1945) reported that thiouracil is more re- liable than thyroidectomy in producing reduction in metabolic rate. Thiouracil acts on any thyroid tissue in the organism, irrespective of location, and thus is effective in animals which show no reduced basal metabolic rate after thyroidectomy due to the presence of accessory 'thyroid tissue. Chapman (1941) found that small amounts of thyroxine -O‘- apparently can be formed even in the complete absence of the thyroid gland. From his experimental data on thyroidectomized animals re- ceiving an iodine supplement, he concluded that iodine may play a role in body metabolism in the absence of thyroid tissue, possibly by pro- ducing a thyroxine-like substance in the tissues. Ely 32 a}: (1948) studied the absorption and elimination of thiouracil in calves and goats receiving oral and subcutaneous doses of the drug. + Maximum blood plasma thiouracil concentrations were reached four to eight hours after oral administration. 'Thiouracil was practically elimi- L nated from the blood stream within 24 hours after cessation of adminis- tration. These results give both an advantage and a disadvantage of thiouracil suppression of the thyroid. It is advantageous that the blood plasma so Quickly after oral administration has a maximum concen- tration of thiouracil acting to suppress further formation of thyroxine by the thyroid and it often is an advantage to be able to restore the experimental animals to normal. This is impossible with thyroidectomized animals except by constant thyroxine administration. One disadvantage of thiouracil is that it must be continuously administered to be ef- fective. Barker (1949) observed in rats that even after 16 months of depressed thyroid function, withdrawal of thiouracil resulted in tem- porary renewal of growth, and reproductive activity was restored. Hughes (1944) listed disadvantages of thiouracil administration by daily subcutaneous injection as the freQuent handling necessary, the trauma incident to the daily injection, and the necessity for constant and continuous treatment. He noted that if growing animals stunted by prolonged treatment with the drug were accidentally without it for a day they would show a subseQuent gain. Jones 32.333 (1946) concluded - 33 - as a result of their experimental observations that 100 days of treat- ment with thiouracil may be necessary to produce severe hypothyroidism in rats and that there may be only a borderline hypothyroidism if treat- ment with thiouracil has been for fewer than 100 days. 1"! - 34 - PROCEDURE Female albino mice were used in this series of experiments. The 30 animals in the first experiment were from a group of mature mice averaging 33 gm. body weight and all of which previously had borne one or two litters. These mice originally were obtained from Rockland Farms, New City, N. Y., and had been maintained in this laboratory since reaching sexual maturity. The 60 animals used in the second experiment were obtained especially for these experiments from Rockland Farms and averaged 20 gm. body weight at the time of their arrival. Since female mice are considered to ap- proach sexual maturity at around 25 gmt body weight, this was the aver- age body weight of the animals at the time the experiment was started. The mice were fed a balanced stock diet"I ad libitum, and drinking 'water was available at all times. They were kept in wire cages measuring 16;}- in. by 92- in. by 7 in. high with five mice per cage. The laboratory 'was maintained at a constant temperature of 75 degrees Fahrenheit through- out all of the experiments. In the first experiment 30 mice were divided into six groups of five animals each and labeled as Groups A through F. In the second ex- periment 60 mice were divided into six groups of 10 animals each labeled as Groups G through L; each group of 10 animals was subdivided into two cages of five mice each. Each individual mouse in a group was identified by means of a system of marking with picramic acid. In both experiments each mouse was weighed to the nearest tenth gm. once a week. A record of the food and water consumption for each * - The stock diet consisted of ground or whole Purina Laboratory Chow. - 55 - group was maintained to the nearest gm. during the period previous to breeding. During the time records of intake were being made, the food was ground chow and was placed in special food-saving dishes to prevent wastage. These dishes were of metal with a sliding inverted-V-shape top in which there were eight small holes, four on each side, of a size such that the mice could get their heads in to get food but could not get their front paws in to scratch out the food. It was found that the mice wasted some food even from these dishes if they were filled com- pletely. Consequently, at no time were they kept more than half full. Experiment I was started on April 16, 1948, at which time the re- cords of the 30 mice and of their food and water consumption were started. Beginning June 20, 1948, vaginal smears were taken on all 30 mice twice daily commencing at 8:30 14M. and at 4:30 P.M. each day through July 22, 1948, 29 days comprising two weeks both before and after injections were started. ‘A glass medicine dropper pipette with its tip pulled out fine and fire-polished was used for taking the smears. A.small amount of dis- tilled water was drawn into the medicine dropper, washed in and out of the vagina, and transferred to a slide to dry. The slides were flamed slightly to fix the smear. Staining was accomplished by means of imp mersion in 0.1 per cent methylene blue solution for two minutes, after “which the slides were washed with distilled water, dried, and examined at a magnification of 100 times. Subcutaneous injections were started for each of the 30 mice on July 8, 1948, and were administered once daily throughout the remainder of the experiment. Each mouse received 0.1 m1. of solution in each in- jection. Group A was the control group; Group B was the thiouracil group deprived of thyroid activity by virtue of a 0.1 per cent thiouracil solution received in place of their drinking water starting simultaneously with the injections and continuing for the remainder of the experiment. These two groups received daily injections of physiological saline (0.85 per cent). The other four groups received varying daily doses of d,l-thyroxine a solution as shown in Table I. A concentrated stock solution was made up using 70 mg. of d,l-thyroxine powder per 100 m1. of solution. The average body weight of the mice considered in making up each of the thyroxine solutions was 33 gm. ' } TABLE I Thyroxine Doses Used in Experiment I ’dgl-thyroxine used'in—the injections Group Rate Per 100 gm. bodygweight Per each mouse (in ug.) 4—11“ ug.) 0 g_ 5.5 1.8 ‘ D %E 11.0 3.6 E #5 22.0 7.2 F 85 44.0 14.4 Under rate in.Table I let 5 stand for the rate of thyroxine secretion found by Hurst and Turner (1947) to be normal for albino mice; that is, 5.5 ug. of d,l~thyroxine per 100 gm. body weight. After 14 days of injection males were placed with each of these six groups. One week later these males were replaced with different males. The second male was removed after a second seven-day period*. In both experiments each male placed with a thiouracil group was given a daily injection of thyroxine, x dose, to prevent his becoming hypothyroid from the thiouracil drinking water. Records of food and water consumption * ~4L11 the males used in_both experiments were proved fertile. - 57 - were discontinued upon the placing of males with the groups. The special food-saving dishes were removed, and the mice received whole chow cubes. 'When the breeding perioi was over and the males were removed, each mouse was placed in an individual littering cage. These wire cages measured 4%-in. by 7 in. by 5 in. high and were fitted with metal trays in the bottom to facilitate the preparation of nests. Shavings were filmished e As soon as each litter was observed, each member of the litter was 'weighed to the nearest mg. The young were weighed again in this same manner at one week of age. At two weeks of age the young were weighed to the nearest tenth gm. The dates on which the young mice had complete hair coats and on which they had their eyes open were recorded. ‘The litters were sexed as soon as a partial hair coat was present so that the teat! of the females were perceptible and the females were marked 'with picramic acid. AAt three weeks of age the litters were weaned, and the mothers were sacrificed. Females that did not litter and that obviously were not pregnant were sacrificed with ether and autopsied three weeks and one day after the second male had been removed from the groups. Practically the same procedure was followed in Experiment II with the 60 younger mice. Vaginal smears were taken once daily during the fourdweek period from July 13, 1948, through August 11, 1948, commencing at 4:15 P.M. each day and comprising 30 days with two weeks before and two during injections. Food and water consumption were recorded during this time. The first males were With these groups for six days starting .August 12, 1948. Males were rotated after six and 12 days, respectively. 'The last males were removed August 26, 1948, and the 60 mice were placed in individual cages for littering. ....n IA\!. - 38 - Daily subcutaneous injectionS'were started July 29, 1948, two weeks after the experiment started, and were continued throughout the remainder of the experiment. The groups and the injections were as follows. As in Experiment I daily injections were 0.1 ml. in size. Group G was the control group and received exactly the same treatment as that of Group Am Group H was the hypothyroid thiouracil group and was treated in the same manner as Group B. The remaining four groups were treated with d,l-thyroxine as shown in‘Table II. A.concentrated stock solution was prepared in the same manner as in the first experi- ment. The average weight of these mice used in preparing the thyroxine .solutions for each of the groups was 255m. TABLE II 'Thyroxine Doses Used in Experiment II d,1-thyroxine used in the injections Group Rate For 100 gm. body weight Per each mouse (in ug.)‘ (in ug.) I 55 2.75 0.7 J .1- 505 104 L if 22.0 5.6 As before, let fibe the normal thyroid secretion rate as found by Hurst and Turner (1947). Statistical methods used in all calculations are from Mode (1946) and Snedeoor (1946). The values of t were compared for significance with the values in‘Table 3.8 (Snedeoor, 1946). £ESULTS General Appearance The 30 mice in Experiment I Showed more definite changes in physi- cal appearance due to the experimental variations in the thyroid state than did the 60 younger mice in Experiment II. .About 10 days after in- jections were started the animals in Group B, the thiouracil group, began to appear and feel greasy. Their hair coats became matted and dirty. The entire cage surface was greasy and dirty. Within five days these symptoms disappeared and the mice were clean and appeared no dif- ferent from the controls. The animals in Group C,‘§ thyroxine mice, had the general appearance of good health. Their hair coat was very sleek, glossy, and clean. They were alert and active. Their general appearance seemed even better than that of the controls. As the dose of thyroxine increased, its effects became more ap- ‘ parent. The animals receiving 21 thyroxine, Group D, became more ex- citable than usual. They were somewhat nervous and scratched in the litter more often than did the controls. They were more busy exploring their cage as well as more curious than usual and they often started immediately to crawl out of the cage when it was opened. The Group E mice, 4§ythyroxine, showed definite symptoms of hyper- thyroidism. They breathed rapidly, almost panting, and were nervous and easily excited. Their eyes seemed Quite bright, and their move- ments were very jerky. They were irritable and very apt to bite when being injected or when vaginal smears were being taken. They did not bite when being handled before injections were started. The 55 thyroxine animals, Group F, evidently were suffering from thyrotoxicosis. Most of the time they made little excited chattering squeaks. Their movements were very nervous and jerky and they always were in motion while they were awake. They scratched nervously and often stuck their noses through the holes of the wire sides of their cages. Their eyes were bright and glassy. Their breathing was extremely ...-M3333": _ rapid and shallow even while they were sleeping or resting. Their at- titude while resting or sleeping was not that normally'taken by mice. They stretched out completely With their paws extended instead of being hunched up with their paws under them as is usually the case. “1" These thyrotoxic symptoms increased throughout the period of thyroxine treatment. The thyrotoxicosis was fatal to all except one of the mice. One mouse in the group died nine days after treatment started. .Lnother died after 15 days of thyroxine treatment. After 20 and 29 days of in- jections, respectively, two others died. The one remaining animal was sacrificed after 51 days of injections, this being 21 days after the breeding period ended. Before this mouse was sacrificed she had a very shabby, scraggly hair coat, especially around the face. She had pro- truding exophthalmic eyes. She lay resting in the cage looking very dejected with her ears laid back, her back hunched up a little, and her eyes open, panting and making no unnecessary movements. She was gradually losing weight.» An autopsy showed that the animal had not been pregnant. The thiouracil mice of Experiment II, Group H, exhibited a greasy appearance for several days approximately two weeks after the treatment was started.” The thyroxinedtreated animals in this second experiment did not show any definite changes in appearance. - 41 - Food and Water Intake Records of food and water intake before and during injections are presented in detail for Experiments I and II in Appendix Tables I and II, respectively. .Analysis of the records of food and water intake before the beginning of the injections shows acme puzzling results. In both experiments there were initial differences between each of the r: groups and their control group. Many of these differences in Experi- ment I were significant at the one or five per cent levels of proba- bility. The differenceefbund in this regard in Experiment II were not I significant. A clue to these puzzling differences is found in the group mean weights at the beginning of the experiments. Although the mice in each experiment were chosen from animals of the same age and approximately the same weight, they were not grouped to give exactly the same initial group mean'weight. These mean weight differences were taken into ac- count in the food and water intake data by determining the ratios be- tween the mean food weights and mean water weights for each group and the group mean body weights of all animals in the group for the period under consideration. These ratios and the percentages of change in mean food and water intakes during injection from these intakes previous to injection are presented in Table III. The Group 0 mice, 5 thyroxine, showed little change in food and water intake as a result of the injections. The animals in the thyro- toxic Group F, 65 thyroxine, showed both a decreased food and water in- take. The two intermediate thyroxine doses, 25 and fig, given to Groups D and B, respectively, increased both food and water intake, with the -42.- .... HYufiIi‘! unit; r .«. «.3 «.8 2. ....2 0...: Me .oqsnprga a ... 15 0.8 .3. ”.3 «.2 .m« 85x82“. a a- a.o« «.«.« T 92 E: m 638.29 H. a. «.3 «.3 e. «.3 «.3 Mm 335...»: H 2.. «.3 m.«« e- at: 9.3 Seasofla m ««. 0.3. o....« «a. «.3 92 33:8 a E 0.83.3 ...- 0...”... «.3 3.. «.2 0.2 mm 633.29 a 2. ~33 . a.«« 2. «.3 92 m. 855.39 m m. o.»« «.3 m. as: «.«H m« .ofineafi a n. . «.o« «.3 «u 0.2 ”.2 m. 655.2“. o S- 2: 0.3 S. ..«H «.3 dengue“. m 3.. 12 n.«« 2.. 92 9: Hogans 4 H sausages La es and? Ge commas 8 Es 8- as Es 02 :3 09:26 unoavoonnu escapee 3" 09330 odcflpoo sH escapee an Damages macho 9.3% on? . mama when“ .smm em. 3.3 £3... .33 no e8.§3«8 3.35 .3»... en. econ 5:8 H: mam: larger dose causing the greater increase. The animals in Experiment II receiving these same thyroxine doses, Groups K and L, showed corresponding results. The smaller thyroxine doses in Experiment II, %E_and‘xg injected daily into Groups I and J, respectively, did not produce much change in food and water intake. The daily food and water intakes were slightly increased for Group I and slightly decreased for Group J. Both control groups, A and G, showed increased food intake. Their water intakes during treatment differed. The Experiment I animals showed decreased water intake, While the mice in the second experiment had in- creased water intake. The Experiment II thiouracil group, H, showed both decreased food and water intakes. Group B in Experiment I had decreased water intake but a very slightly increased food intake. ggdypweight Only the weights of mice that littered were considered in deter- mining the group mean weights for each.Week of both experiments, except that in Group F‘which had no litters, the weights of all animals in the group were averaged each week. Appendix'Tables III, IV, V, VI, VII,and VIII, part A, summarize the mean weights of the animals in Experiment I and Appendix Tables IX,.X, XI, XII, XIII, and XIV, part.A, give the cor- responding results for the Experiment II animals. Littering dates and the dates of last weighing are presented in part B of the same tables. Record of infanticide, death, or sacrifice is also found in these tables. All other mothers raised some or all of their young to two weeks of age and at that time their injections and weekly weighing were discontinued. ~. ‘ ‘- n. I - _..I‘ ii- - _--_.:- - 44 - The relative weight changes of each of the groups in Experiments I and II are shown graphically in Figures I and II, respectively. The means for Groups F and L represent lessening numbers of animals as is indicated on the figures. Infanticide was the cause for this decrease in number of animals considered in the Group L means and death from thyrotoxicosis was the cause for it in Group F. Figure I clearly shows the effect of the thyrotoxicosis of the Qi'thyroxine dosage on the weight of the animals in Group F. .After only two days of thyroxine treatment the mean weight of the group had started to drop and at no time afterward did the weight reach that of the beginning. The control group, A, showed an initial decrease in weight, possibly due to handling. As the animals grew accustomed to daily handling their weight began to rise. They showed a general in- crease in weight parallel with their gestation periods as did all the other groups which had litters, and a gradual slight decrease in weight following birth of their young, to a weight about five per cent above their starting weight. Groups B, C, D, and E, showed a permanent in- crease in weight after pregnancy which was greater than that of the control group. The 5 thyroxine group, C, gained weight even after parturi- tion to levels above those reached during gestation as compared to de- creases in weight during lactation evidenced by Groups A, B, and D, and a slight increase at a lower level by Group 2. Examination of Figure II shows that all groups in the second experi- ment had terminal weights 40 to 55 per cent higher than their original weights. Groups H and K, thiouracil and Qi'thyroxine, respectively, showed slight increases in weight during lactation but their weights were below those recorded during gestation. All other groups showed slight decreases in weight during lactation. rh—a -45- l40r GROUP A r GROUP 8 /\ , < —_—G'ROLP C . ’ l"\ I30» ~ camp 0 I \,’ ‘ GROLPE Ix. . ..me f: . ‘ . :3 ° I L/ S STARTED '~i 60* m INJECTION o i é 5 2: 3 6 ‘1 a. weexsouexpr. FIGUIE _I_ Percentage Weight Comparisons, Experiment I -45- I60 f v - - T . , . . I6)l\ I? . , II GROUP 5 , I70’ -—-—GROUP I GROUP J GROUP K GROUP L 7 I60 3 I40’ - + ‘~ ~ ........ “‘ "\.\ .0 Do .0 L WEIGHT - 9b 0F ORBINAI. E3 0 ‘-.\\ \ II 0 * g? I A. . I '00 ‘ \/ WITH 1 MALES ‘ maecnous ’ STARTED 9°o—r-7-T-i—s—r-v—r-c— WEEKS ON EXPT. FIGURE _l_Z_I_ Percentage Weight Comparisons, Experiment II -47.. Estrous chles The vaginal smears were rated carerIly as to the stage in the estrous cycle evident in each. The number of days between the peaks of the estrous stages both before and during injections and the mean cycle length for each mouse are presented in.Appendix Tables XV and XVI for Experiments I and II, respectively. The average lengths of estrous cycles for all groups before and during injection are summarized in Table IV. The analyses of estrous cycle lengths for Experiment I suggest that hypothyroidism in mice may lengthen estrous cycle and hyperthyroid- ‘F-anrA-nm ism may decrease the length of estrous cycles. The control group and the §_thyroxine group showed no change in length of cycle. The GE, 45? and G: thyroxine groups had a progressive decrease in cycle length. The thiouracil group exhibited a greatly lengthened estrous cycle. Gestation It is almost impossible from these experiments to state whether or not the length of gestation in albino mice is affected by changes in the thyroid state. Since no definite copulation plugs were observed and vaginal smears were discontinued as soon as males were with the females for mating, it is impossible to state With any certainty when fertilization occurred. Appendix Tables XVII and XVIII summarize de- tailed data concerning birth of litters for Experiments I and II, res- pectively. To attempt to estimate the length of gestation in each mouse, the average estrous cycle length for mice in her group, or a multiple of this length, was subtracted from the number of days elapsing between the last recorded estrus and the date of littering. These estimated -43- TABLE IV Estrcus Cycle Length Mean Length of Est rous Cycles Group Treatment Before Injections During Injections Chs. e “‘33) (daysT 1 Experiment I p ‘ Control 507 508 .2 B Thioun°11 500 808 .76 C Thyroxine, 5 6.3 6.5 03 D Thyroxine, 2g 6.0 5.5 -8 E TWI‘OXi-no, 2 707 600 -22 F Thyroxine, 8_x_ 7.0 6.0 -14 Experiment II G Control 6.2 6.8 910 H Thiouracil 6.2 6.0 -8 I Thyroxine, $3 6.6 5.8 -12 J Thyroxine, 5 6.1 6.2 e2 K Thyroxine, 2_x_ 5.4 6.1 e18 L TWroxino, 4-:- 606 503 -5 ‘lfio ..- - 49 - gestation periods also are shown in Appendix Tables XVII and XVIII. Mean lengths by groups of the estimated gestation periods are in- cluded. All of these estimated periods range from 17 to 22 days in length with the majority 18 to 20 days in length. Group mean lengths of estimated gestation range from 18.5 to 20.4 days. .Lnimals which did not litter were autopsied in an attempt to de- termine whether fertilization and implantation had occurred with re- sorption of fetuses interrupting gestation. None of the autopsied ani- mals in Experiment I showed any evidences that fertilization had oc- curred. The one 8§_thyroxine animal, Group F, which survived apparently had not mated. Since the mice in Experiment II never had been mated previous to their use in this experiment, uteri normally were small if there had been no gestation. If the uterus of any autopsied animal of this ex- periment was larger, it was considered evidence of possible interrupted gestation. .Lnimals J3 and J4, mice that received §_thyroxine doses, K'2, a mouse that received gi'thyroxine, and two animals that received e§_thyroxine, L1 and L'l, had large uteri and may have had interrupted pregnancies. A.control animal, G1, and a thiouracil animal, H'l, also had rather large uteri. No uterus Which was examined was inflamed. It is impossible to state With any certainty that pregnancy had occurred in any of these animals. Litter'fieight and Size Data concerning litter weight and size are presented in Appendix Tables XIX and.XX for Experiments I and II, respectively. These data are summarized in Tables V and VI. In Experiment I litters from the con- ‘trol group averaged seven young at birth; the thiouracil group, eight UK: -.' - -50- L eonwaos.»oc gonna” one a .. apmnapmpOtm ea H.».H,Mm on» as peaoamaemam - * mNm.o a.» a.Ne «Hm.o mH.o Na.o «ea.o m.H m.o N mw...c«uosana u oeN.o o.a e.«n .«om.N eN.o Ne.» NN¢.o e.N n.e » mm..oaaueaaga a neo.a e.« N.Nm .mNN.N mH.o Na.m ooo.N N.” N.N e n .onauouana o we».o N.N ”.mn mam.a on.o an.» ”mm.o o.o o.» n HaoaasoNaa m H.nH N.o» NN.o m«.e a.” a.¢ N dungeon 4 .3 a. £8 3 p4 noN.H sow.» wao.on .Nm¢.¢ 000.0 Ndo.¢ eea.o o.H m.m N we ..cauouaaa u NeN.o anm.ea awa.NN NNN.H NNo.o com.» mwm.o o.¢ a.» ..n mm...=aaosana a new.” New.» NNo.Nn eeH.N mad.o hoe.» NNo.N N.H «.N m a ..euuouana o mN¢.o nmN.e oNN.NN eon.a NeN.o NNN.» Nam.o m.o m.o ..n Haoaeeoaaa m maa.a mNN.aH ma~.o oNN.» N.” a.« a “chance 4 owd Mo when 5 ad III 0..- ...... III II... II... 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Ho has—ism mo .0: HP and“. -52.. and a half; E_thyroxine, 10.4; 85 thyroxine, 11; and if. thyroxine, six and a half. In no case was the number significantly different from that of the control group. ‘When comparisons were made between groups of the number of survivors at seven and at 14 days of age, there likewise were found to be no significant differences. The mean litter size for the various groups in Experiment II at birth were eight for controls, 7.8 for thiouracil, 7.4 for-fix thyroxine, 6.7 for 3‘. thyroxine, 6.6 for 25thyroxine, and 9.8 for 45 thyroxine. There was no significant difference in litter size at birth between the control and any other group, but the dx_thyroxine group showed a highly significantly greater number of survivors at seven and at 14 days of age. In both experiments the decreases in litter size at one and two weeks of age were due to infanticide. In many of the litters some of the young which were killed were partially or completely eaten by the mothers. There were no significant differences in individual birth.weignts in Experiment I. At seven days of age the mean individual weight of young in Group E, 4§_thyroxine, was significantly greater than that of the control group, A. At 14 days of age the mean individual weights of young in the _x_ and 235 thyroxine groups, C and D, were significantly less than that of Group A. In experiment II the mean individual birth weight of the %§_thyroxine group, I, was significantly greater and of the xpthyroxine group, J, was highly significantly greater than that of the control group, G. At seven days of age the mean individual weight of young in the %x thyroxine group, I, was highly significantly greater ' (7| CR I than that of the control group. .At 14 days of age the mean individual weight of the thiouracil group litters was highly significantly lower and that of the 4x thyroxine group litters was significantly lower than that of the control group. Since total litter weight reflects litter size and mean individual weight, variations are easily of large magnitude. In Experiment I the ‘5 thyroxine litters led the list in this measurement throughout the lac- tation period. The 4x'tnyroxine group also had large total litter 'weight values at seven and at 14 days of age. In no case were the dif- ferences significant. In the second experiment at seven days the-fix thyroxine group had significantly greater and the 4x thyroxine group had highly significantly greater total litter weights than those of the control group. At 14 days the thiouracil group had significantly smaller ‘total litter weights and the fix and QE-thyroxine groups had highly sig- nificantly greater total litter weights than those of the controls. Nesting and Care of Litters In Experiment I the mice in the x and 8x thyroxine groups were ex- cellent mothers and the 45 thyroxine mice were good mothers. The mothers in these three groups manifested very strong nesting tendencies and .formed large deep nests. The x thyroxine animals kept their nests Quite clean by pushing feces and waste food out of the cage with a rooting procedure. The thiouracil-treated animals piled up fairly good ‘nests. The control mice did not exhibit very strong nesting tendencies. Axflmmls CS, C4, and ES carried their young back to the nests in their xnouths if any were out. Group C,‘§ thyroxine, seemed to enjoy nursing sand seemed Quite relaxed. ‘The animals in other groups did not stretch out completely relaxed while nursing. . Tar-a1 if“! In Experiment II very little difference in nesting tendency was noted. All groups seemed to contain egually good mothers. G2, a con- trol animal, was nervous and restless and was a very poor mother. She ate six of her ten young. G'5 was a good' mother. One of the 5 thyroxine animals, 1'2 was an excellent mother. She kept her nest very clean and carried her young back if they were out of the nest. She also attempted to bite when her young were removed from.the cage for weighing. Animal L3, a 4§_thyroxine mouse, killed all of her young and "ate some of them. One thiouracil mouse, H'4 had a very well-made neat 116813 e Hair Coat and Opening 23 fiyes In Experiment I the numbers of animals in each group having litters that were raised to weaning age were only two or three. Therefore, hair coat and eye opening were not recorded. Appendix Table XXI shows a record of the number of days of age at which hair coat became complete and at Which all young in the litter had their eyes open for Experiment II. There were no significant differences apparent between each of the treated groups and the control group. The average age at which hair coat was complete was nine and a half days with a range of nine to 9.8 days. The average age for opening of eyes was 14.5 days with a range of 14.2 to 14.6 days. §_e_x_ _o_i_'_ Litters The litters were sexed as soon as the teats of the females were perceptible. At three weeks of age the litters were separated from their mothers. The sexes of the young of each litter at weaning are given in.tppendix Table XXII. The sexes of the young at weaning are presented by groups in Table VII. The mean sex ratio of all groups in -55... TABLE VII Sex Ratio at Weaning Sex Distribution afieaning . Sex Ratio Group Treatment Males Females Melee Females (77 (75) A Control 9 5 64 36 B Thiouracil 8 10 44 56 c Thyroxine, 5 13 24 35 65 D Thyroxine, 25 10 9 53 47 s Thyroxine, 45 7 6 54 46 Totals Experiment I 47 54 46.5 53.5 . G Control 6 19 24 76 I H Thiouracil 19 18 51 49 I Thyroxine, g3 29 22 57 46 J “thyroxine, i 18 20 47 53 K Thyroxine, 2_x_ 15 15 5O 50 L Thyroxine, 4_x_ 19 20 49 51 Totals Experiment II 106 114 48.2 51.8 - 55 - Experiment I was 46.5 males to 53.5 females and of all groups in Ex- periment II was 48.2 males to 51.8 females weaned. The control group in the first experiment weaned 64.0 per cent males. In Experiment II the controls weaned 24.0 per cent males. This was the lowest ratio of males in both experiments. The fig thyroxine group had a higher ratio of males, 57.0 per cent, than any other group in the second experiment. The sex ratios indicate no definite trend or relationship between thyroid state and sex of litters. Lactation The data on lactation are given by individuals in Appendix Tables It“: no. .mbl’ 3 .1. I XXIII and XXIV. These data for Experiment II are summarized by groups in Table VIII. The total weight of mice in the litter at 14 days of age was used to measure the growth due to milk consumed. The total litter weight at 14 days of age was expressed as a percentage of the average total weight of all litters of that number at 14 days. This ratio is the index of performance. Indexes of performance were not considered valid for litters in which the number of young at 14 days of age differed from the number at birth since some of the milk produced went to young which died or were killed and were not considered in the total litter weight at 14 days. The indexes of performance of the Experiment I animals are not summarized by groups because there were only seven animals which had the same numbers in their litters both at birth and at 14 days of age. In the second experiment the index of performance for the thiouracil group was less than that of the controls. All four groups which re- ceived thyroxine had higher indexes of performance than did the controls. In no case was this difference significant. ‘2” All“? IA” -57.. 000.0 0.0H 0.N0H 0 n. 65.80000 0 000.H 0.0 0.00H 0 MN 65.3.35 0 00N.H 0.0 0.02 0 M assuage 0 0N0.N H.0 0.00H a M 600.5000. H 000.H 0.0 0.00 0 26.3.30 0 m.» >.ma o flamenco a Lab lob » E! use: 080305” Mo 000.52 9:06:39 @595 eosgonmaom Mo No.95“ meanness HHH> Eqmdy DISCUSSION The more clear-cut changes in physical appearance due to experi- mental variations in the thyroid state exhibited by the 30 older mice used in the first experiment suggest declining thyroid secretion in mice With increasing age. Hill (1948) found that the response to thyroid in rats normally increases with increasing age. The excellent general appearance of the Group C, 35 thyroxine, mice indicate that this dosage is EMT optimal one for older mice. These mice showed better performances by most measures used than did the con- trol animals. Larger doses of thyroxine gave increasing symptoms of :- hyperthyroidism, including thyrotoxicosis at the 83‘. dose of thyroxine. The sizes of the thyroxine doses used in the second experiment conse- Quently were halved for the four thyroxine groups. The results on food and water intake during injection shown in Table III in general parallel those er Soliman (1950) and those of Magsood and Reineke (1950a). The x thyroxine group in Experiment I and the if and 5. groups in Experiment II showed little change in food and water intake as a result of the injections. These facts indicate that the estimated thyroid secretion rate of 5.5 ug. per 100 gm. of body weight per day for mature female albino mice found by Hurst and Turner (1947) and used as the _x_ thyroxine dose is near the normal thyroid secretion rate of the mice used in both experiments. The increased food intakes exhibited by both control groups pos- sibly may be explained by the increased handling after injections were begun. The apparent partial loss of appetite of the Group F, 835 thyroxine, mice shown by the reduced food and water intake is an - 59 - indication of the severity of the thyrotoxicosis. Thompson (1942) lists loss of appetite and loss of weight among the symptoms of crisis in toxic goiter. I qusood and Reineke (1950a) reported depression of both food and water consumption by thiouracil feeding and reported that food and water intake increased proportionately with graded increase in thyroprctein dosage. Gordon 22 El! (1946) reported reduced food and water consump- tion after thiouracil feeding. Soliman (1950) also found that food and water consumption were increased by thyroprctein administration while both were decreased by thiouracil administration. These results are confirmed by these experiments. The rise in mean weight of all groups after gestation to a weight above their original weight agrees with the work of Crozier and Enzmann (1935) shOWing that pregnancy in mice results in a permanent increase in the weight of the mother. The very decided increases in weight of the Experiment II groups after gestation reflect the fact that these were the first pregnancies and the first litters for these mice. The increase in weight during lactation exhibited by Group 0,.5 thyroxine, Group E, §x_thyroxine, and Group K, §x_thyroxine, may be a characteristic associated with the increase in milk secretion resulting from.the thyroxine injections although this is a quite variable response. This increase in milk secretion has been reported by Graham (1934a) to occur in cattle when the thyroxine is injected during the period of de- clining lactation but not if the treatment was given during the early stages of lactation following parturition. The variations of mean estrous cycle length before injections, from 5.0 to 7.7 in the first experiment and from 5.4 to 6.6 in Experiment II possibly may be explained by the mechanical stimulation of the vagina occurring when the smears were taken. Emery and Schwabe (1956) found irregular estrous cycles occurring in normal virgin rats as a result of freQuent vaginal examination by the cotton swab method. They considered extreme care necessary in taking daily smears by the pipette and lavage method to avoid false smears from mechanical stimulation. The smaller ”my I vaginae of the mice made it extremely difficult to avoid some mechani- cal stimulation. 'Wade and Doisy (1935) found a progressive change in the vaginal smears of spayed rats smeared omce,twice, or three times ' ’2‘3‘.‘ '. J . -.K .51.” !LA. daily. The larger variation in mean cycle lengths in Experiment I may be explained by the fact that smears were taken twice daily in that experiment and only once daily in the second experiment. Allen (1921, 1922) observed that one daily examination was adequate for accumulation of data as to cycle length. He found the average deviation of the en- tire cycle in mice to be from four to six days. The groups in Experi- ment I averaged about six and a half days for cycle length before treatment started and the average for Experiment II was six days. Another possible explanation of the larger variation in cycle lengths for the older mice used in the first experiment is their ad- vanced age. Boughton and Stoland (1943) found that an increasing pro- portion of mature albino rats over 25 weeks of age failed to show evi- dences of heat on vaginal smears, and an ever larger proportion of those Which did deviated from the four-day cycle observed in the majority of the rats beloW'25 weeks of age. Beyond 50'Weeks of age the seguence of four-day cycles became increasingly irregular. Since smears were taken for only two weeks before and two weeks after the beginning of experimental treatment the results on the estrous - 61 - cycle length are not conclusive. Lee (1925), Freedman.gt_al: (1935), and Ross (1938) found an average lengthening of estrous cycles in thyroidectomized female rats due to a lengthened diestrus with the pro- longation ranging from one to five days or with the duration increased from the normal four or five days to seven or eight days. Nelson (1948) and Mann (1945) both observed very irregular or greatly lengthened . vi! periods of estrus in rats given thiouracil. Krohn (1947) found that daily subcutaneous injections of propylthiouracil disturbed the normal 1'?! Li ti _ estrous rhythm of mature albino mice, causing lengthening, irregularity,. or complete disappearance of the cycles. The lengthening of mean cycles VIE-IL. - in the Group B thiouracil animals during treatment confirm these results. ‘The progressive decrease in estrous cycle length with increasing doses of thyroxine observed in Experiment I is contrary to the results reported by most investigators. Evans and Long (1921a) observed little disturbance of the normal four-lay cycles of rats fed small amounts of fresh beef thyroid daily. With the administration of large amounts of thyroid they found the cycle was greatly lengthened or totally inhibited. Continuous diestrus in rats was maintained with 100 mg. of thyroid gland daily (Drill 33 El}, 1943). Reiss and Pereny (1928) and Van Horn (1933) observed that the thyroid hormone inhibited heat and was antagonistic to the action of ovarian hormone. In analyzing the effects of the varying doses of thyroxine used in these experiments on gestation it must be remembered that the thyroxine doses used throughout the two experiments were based on average weights of 33 gm. and 25 gm., respectively, and weights in both experiments in- creased permanently during gestation. The duration of gestation in mice normally is 19 days, varying from 18 to 20 days (Parkes, 1926-1927, and Asdell, 1946). MacDowell 22.3lf (1927) stated that most litters are born before the end of the nineteenth day and Enzmann.gt_glf (1932) reported that the period of gestation in non-lactating mice is 20 days. In these experiments the exact time of fertilization was not ob- served, but the estimated gestation periods ranged from 17 to 22 days in length with the majority between 18 to 20 days suggesting that there were no effects of variation in the thyroid state on gestation length in these experiments. There are many conflicting results in previous work. Ukita (1920) reported greatly lengthened gestation periods in thyroidectomized rabbits, but Krichesky (1935, 1939) found no alter- ation of gestation length in rabbits after thyroid removal. Nelson and Tobin (1937) found no harmful effects on pregnancy and parturition in rats and guinea pigs from thyroidectomy and Nojima (1933) found a slight tendency toward prolongation of the gestation period in thyroidec- tomized female rats which did not abort when pregnancy occurred. Wbichert (1930) observed a prolonged gestation period in pregnant rats fed desiccated thyroid daily. On the other hand, weichert and Boyd (1934) observed no prolongation of gestation, possibly because they discontinued thyroid feeding some time before parturition. The normal duration of gestation in guinea pigs is about 67 to 68 days (Asdell, 1946) but Carloni (1930) observed gestation periods in guinea pigs of 51, 53, 55, 57, 60, and 61 days under different conditions of thyroid adminis- tration during pregnancy. Many workers have reported resorption of embryos in rats or rabbits both from.thiouracil feeding or thyroidectomy and from thyroxine administration. Jones E: El. (1946) reported resorption in rats after prolonged thiouracil administration of at least 100 days. If treat- ment were for less than 100 days previous to mating some rats did de- liver normally. Krohn and thite (1950) also found resorption of fetuses when rats were mated at least 54 days after starting thiouracil in- jections or at least 28 days after thyroidectomy. Chu (1945) found that thyroidectomy in the rabbit at an early stage of pregnancy caused resorption and abortion of the embryos, while still-born young were de- livered if the operation was performed at a late stage of pregnancy. Possibly the fact that the Group B and Group H mice had received thiouracil in their drinking water for only two weeks previous to mating explains the fact that there were no definite evidences of resorption for any of these mice. Kunde gt_513 (1929) found resorption of fetuses in severely hyper- thyroid rabbits and Bodansky and Duff (1936) found a greater incidence of still-births in thyroxine-treated rats than in normal controls. The average number of young for first litters in normal rats is six with a range of three to 10 (Blandau and Money, 1943). Kraatz (1939) found an average litter size of 13 in rats made slightly hyper- thyroid for a short time before mating as compared with an average size of nine in untreated littermates. In mice Asdell (1946) reported that mean litter sizes vary from four and a half to seven and a half with the second litter the largest and a steady decrease afterwards. For both experiments the mean litter sizes at birth for each group showed no significant differences from the control groups. These ex- periments do not confirm the result of Kraatz (1939) that hyperthyroidism in rats increased average litter size at birth, possibly because thyroxine dosage was continued throughout gestation in these experiments While it was discontinued after mating in his experiments. The fact that the fig and the §_thyroxine dosages in the second ex- periment resulted in increased individual weights of young at birth shows that these administrations were beneficial to gestation. This effect did not carry through in the 14-day lactation. It is interesting to note that thiouracil and if thyroxine administration exerted parallel effects on lactation as observed from the lower mean individual weights '1‘! ' .1 Va.“ A- of young at 14 days of age in both these groups. Parkes (1926) found that the rate of growth of young albino mice during the suckling period was inversely proportional to the size of the litter. Falconer (1947) also observed that individual mouse weights at 12 days of age were greatest in small litters falling off gradually with increasing litter size with the reduction being about 0.075 grams for each additional mouse in the litter. The average age for opening of the eyes of the young was 14.5 days and no differences appeared from the experimental variations in the thyroid state of the mothers. This age was within the normal age range for mice. Falconer (1947) stated that the eyes of young mice open from 13 to 15 days of age. The average age at which hair coat was ob- served to be complete was nine and a half days with no appreciable dif- ferences between groups. Many workers have reported transplacental and transmammary transmission of thiouracil. Transplacental transmission was observed in rats by Williams (1944) and Kauffman.gt Elf (1948). Williams 22.31, (l944a, 1944b) found transmammary transmission of thiouracil in human patients and in rats, resPectively. Hughes (1944) - 55 - and Freiesleben and Kjerulf-Jensen (1946, 1947) observed both trans- placental and transmammary transmission of thiouracil. Under the con- ditions of these experiments transplacental and transmammary transmission of thiouracil are not demonstrated by these measures of physical develop- ment. The varying doses of thyroxine given the mothers are not reflected in the rate of hair coat growth or the age of opening of the eyes of ‘l—‘q. ‘ their young. This is in agreement with the observations of Reineke and Turner (1944) that the mammary gland does not permit passage of biologi- cally detectable amounts of thyroid hormone when thyroprctein is fed to lactating cows. 1? In rats conceiving 24 to 55 days after thyroidectomy Nojima (1953) observed 80 per cent of females in the litters instead of 48.5 per cent females as in litters of normal rats. Parkes (1926-1927) agrees with this ratio as normal for mice. He reported about 52 per cent males normally in mice. It must be remembered that the sex ratios given in these experi- ments are for the young weaned. No records were made of the sex ratio at the time of sexing, and some of the young were killed by their mothers between this tits and the weaning time. Some young likewise were killed at birth or before sexing was possible. The fact that the controls in the first experiment were 36 per cent females at weaning and the con- trols in the second experiment were 76 per cent females indicates that any conclusions drawn from these data as to the effect of variations in the thyroid state on the sex of mice litters would be invalid. The practice of infanticide in mice at birth of the litter or during the suckling period was observed by both Crozier and Enzmann (1935) and Falconer (1947). This practice occurred whenever the mother - 55 - reached the limits of her lactating capacity. No attempts were made in these experiments to record infanticide at birth. In Experiment I both of the Group E, 45 thyroxine, animals which littered had the same number of young in their litter at 14 days as at birth. In each of the other groups some animals had the same number of young at 14 days as at birth and some did not. In the second experiment each group con- tained both animals without infanticide during the suckling period and animals with infanticide during suckling. These results do not indicate that infanticide among mice is made any more or any less prevalent as a result of changes in the thyroid state. The numbers of young in the Ir . ‘v '. two Group E litters were five and eight. Neither of these litters was large in comparison with the mean litter sizes in mice of four and a half to seven and a half reported by.Asdell (1946), so the fact that there was no infanticide during suckling in these two litters does not necessarily signify that these mothers had a larger capacity for milk production. There are many conflicting reports concerning the effects of hypo— and of hyperthyroidism on milk secretion. Nelson and Tobin (1937) found that thyroidectomized rats and guinea pigs lactated in a normal fashion. Contrary to this was the work of Preheim (1940), Davenport and Swingle (1927), and Folley (1938) who found poor lactation in thyroidectomized rats. .Graham.(1934a, 1934b) found temporary increases of milk fat production and milk secretion in lactating cows injected with thyroxine or fed thyroid during declining lactation but no such effect during the early stages of lactation following parturition. 3x- cessive quantities of thyroid caused a diminution in both milk and fat secretion. Reineke and Turner (1942) observed the same general results -67- with goats as the experimental animals. 0n goats de Fremery (1956) observed contrary results. Low thyroxine dosage produced no change and high dosage reduced milk output. The validity of the indexes of performance for the evaluation of lactation in these experiments is Questionable since an insufficient number of animals were used. Means of 14-day litter weights were cal- culated from litters in all groups, regardless of treatment, having the same number of young. ConseQuently the group indexes of performance actually are compared to the mean for all groups rather than to the mean of the control group alone. These indexes of performance do, however, indicate possible trends of decreased lactation due to thiouracil hypothyroidism and of increased lactation due to thyroxine administration provided the dosage is not sufficiently large to be thyrotoxic to the animal. The group mean litter weights also represent a measure of lactation performance. These values for Experiment II show trends similar to those indicated by the indexes of performance. -68- SUMMAAY.LNU CONCLUSIONS ‘The effects of thiouracil-induced hyp0thyroidism and of varying degrees of hyperthyroidism produced by graded doses of thyroxine on the food and water consumption, body weight, estrous cycles, gestation, littering, and lactation in female albino mice were observed. 1. Food and water consumption were depressed by thiouracil feeding and increased proportionately with graded increase in thyroxine dosage, With the exception of dosage at the estimated level of eight times the normal thyroid secretion. 2. Dosage at the estimated level of eight times the normal thyroid secretion was thyrotoxic. 3. All mean group body weights were permanently increased after gestation. 4. Estrous cycles were not significantly altered by thiouracil or by the thyroxine dosages used in this experiment. 5. Dosage at the estimated level of the normal thyroid secretion and at half that level was beneficial to gestation in young mice having their first litter. 6. Thiouracil and thyroxine dose levels used in this experiment did not significantly alter the number of young in litters at birth. 7. Mice receiving thyroxine at the estimated level of four times the normal thyroid secretion showed a highly significantly greater number of survivors in their litters at seven and 14 days of age than did the controls. 8. The thiouracil administration used in this experiment did not significantly alter mean individual weights of the young at birth. 9. 'Bhyroxine administration at the estimated level of the normal thyroid secretion and at half that level, respectively, produced highly significantly greater and significantly greater mean individual weights of the young at birth in young mice having their first litter. 10. 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Obst. and Gynec. 12: 706-709. Litzenberg, J. C., and Carey, J. B. 1929. The relation of basal metabolism to gestation. Am. J. Obst. and Gynec. 17: 550-552. Long, J. L., and Evans, H. M. 1922. The oestrous cycle in the rat and its associated phenomena. Experimental studies in the physio- logical anatomy of reproduction. Memoirs Univ. Calif. 6: 1-148. Lukacs, J. 1930. Geht das Schilddrflsenhormon in die Milch fiber? Arch. f. Kinderh. 91: 9-13. (Cited from Biol. Abs. 6: 816, 1932.) MacDowell, E. C., Allen, B., and MacDowell, C. G. 1927. The prenatal gromh Of the mouse. J. Gen. PhySiOle 11: 57-700 Mackenzie, C. G., and Mackenzie, J. B. 1943. Effect of sulfonamides and thioureas on the thyroid gland and basal metabolism. Endocrinology 32: 185-209. Malkiel, S. 1946. .A note on the mode of thiouracil action. Endocrin- ology 38: 58-59. Mann, C.‘W. 1945. The effect of thiouracil upon the heart rate, estrous cycle, and spontaneous activity of the white rat. J. Psychol. 20: 91-99. Maqsood, M., and Beineke, E. P. 1950a. Influence of variation in en- vironmental temperature and thyroid status on growth and feed consumption of the male mouse. Am. J. Physiol. 160: 253-258. Maqsood, M., and Reineke, E. P. 1950b. Influence of environmental tem- peratures and thyroid status on sexual development in male mouse. Am. J. Physiol. 162: 24-30. Meites, J., and Turner, C. h. 1947. Effect of thiouracil and estrogen on lactogenio hormone and weight of pituitaries of rats. Proc. Soc. Exper. Biol. and Med. 64: 488-492. Meyer, A. 3., Collins, M. B., and Marine, D. 1944. Toxicity of thiouracil in normal and thyroidectomized rats. Proc. Soc. Exper. Biol. and Med. 55: 221-222. Meyer, A. B., and Ransom, G. V. 1945. The metabolism of rats after thyroidectomy or during thiouracil treatment, and the effect of thyroid feeding. Endocrinology 36: 259-265. - 75 - Mixner, J. P., and Turner, C. W. 1942. Influence of thyroxine on mammary lobule-alveolar growth. Endocrinology 31: 345-348. lode, E. B. 1946. The elements of statistics. Prentice-Hall, Inc., New York, N. Y. Nelson, W. 0., and Hickman, J. 1937. Effect of oestrone on hypophyses and reproductive organs of thyroidectomized rats. Proc. Soc. Exper. Biol. and Med. 36: 828-830. Nelson, 3. 0., and Tobin, C. E. 1937. Lactation in thyroidectomized rats and guinea pigs. Endocrinology 21: 670-676. Nelson, W. 0., and Wheeler, H. E. 1948. Effect of iodine in hypothyroid mtSO Fade Proc. 7: 85-860 Nojima, K. 1933. Experimental study on the effects of thyroidectomy to pregnancy. Jap. J. Obst. and Gynec. 16: 483-501. (Cited from Biol. Abs. 10: 3471, 1936.) Parkes, A. S. 1926. The growth of young mice according to the size of litter. .Ann. Appl. Biol. 13: 374-394. (Cited from Biol. Abs. 1: 2512, 1927.) Parkes, L. S. 1926-1927. Brit. J. Exper. Zool. 4: 93-104. (Cited by ASdell’ Se A0, 1946.) Preheim, D. V. 1940. Studies on thyroidectomized rats with special reference to lactation and growth. 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APPENDIX - 30 - .r.-- apaaanenoam mo Hopoa ed p. peaoaaaewam . .. apaammepocm to H.>.H.eo a. sneoamflewwm.. * aa.e - ...Ho.¢ ow.» om.He o” ..nma.» N¢.H oo.e¢ ad m ..canoaasa m m¢.mfl. ..eme.m mm.H mm.~e «a .omm.~ mm.a «m.an ma mw ..aaxouaaa a mm.m . ..eme.m ow.” ao.oe «a mae.a ma.a ne.en me me ..eauosaga a an.” . ..oee.a mo.~ n¢.m¢ ea ..Hma.m mm.o mm.ee ea .m...eaxoaana, o wfl.en- mmo.o mm.H ae.e~ a ..enm.e mm.a aa.ae ma flaouaeoaaa m mm.wa- .. 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Animals Preg. Littered lean Std. Error Change Inc-7 (no-T Trio.) (gm-T (gm? (3%) June 26 O 4 31.42 1.59 July 3 l 4 29.60 1.42 ~5.79 July 10 2 4 28.78 1.10 -8.40 5“ July 31 5 4 3 30.28 0.30 -3.63 tug. 7 6 4 4 32075 1062 .4023 Aug. 14 7 4 2 2 34.10 4.19 +8.53 . Aug. 21 8 4 l 3 33.80 5.20 +7.57 !. Aug. 28 9 4 4 33.15 4.38 05.51 i Sept. 4 10 l l 43.20 -- -- ; Sept. 11 11 1 1 41.40 -- -- ' B Mouse Date of Littering _ Last Date Weighed 11* Aug. 15 Aug. 28 A2“I Aug. 22 Sept. 11 AS" Aug. 14 Aug. 28 A41? Aug. 14 Aug. 28 (Ate her young) L5 -- July 17 (Died) * - Only those that littered were used in determining mean weights. - 83 - APPENDIX Wkight and Littering Data Group B, Thiouracil 1. looks on ‘Ani- TABLE IV Weight Date Expt. male Prgg. Littered Mean Std. Error 1: Change (n00 (noj (no-T “(gm-7 (gm-1 (35) June 26 0 4 33.60 1.83 1.187 July 10 2 4 31.30 0.73 1.909 -6.84 July 17 3 4 33.02 1.04 1.804 -1.73 July 24 4 4 33.50 1.06 1.455 -0.30 July 31 5 4 33.92 1.36 2.614* 40.95 Aug. 7 6 4 4 34.95 1.61 0.963 +4.02 Aug. 14 7 4 4 37.78 2.20 0.778 012.44 Aug. 21 8 4 3 1 45.22 3.94 1.750 934.58 Aug. 28 9 3 3 39.37 1.39 1.354 .1701? Sept. 4 10 3 3 37.47 0.29 -- ~- Sopt. 11 ll 2 2 37.05 3.95 -- -- * - Significant at the 5%ilevel of probability B 1 House Date of Littering Last Date'fibighed 81¢ Aug. 22 Sept. 11 B2* Aug. 24 Aug. 21 (Died) 83* Aug. 23 Sept. 11 B4 -- Aug. 21 (Sacrifioed) 35* Aug. 21 Sept. 4 * - Only those that littered were used in determining mean wéights. - a4 - APPENDIX TABLE V Weight and Littering Data Group C, Thyroxine, _x_ A. ieeks on .Lni- fibight Date Expt. male Preg. Littered Mean StJ. Error t Change (110-) (rm-T (Inc-T (gm-1 (gm-1 (75) June 26 0 5 35.26 1.06 2.010 July 3 1 5 35.20 1.17 3.044* -0.17 July 10 2 5 35.40 1.51 3.544** 00.40 July 17 3 5 34.40 1.31 2.384* ~2.44 July 24 4 5 35.38 0.94 2.890* 40.34 July 31 5 5 4 35.04 1.60 2.924* -0.62 Aug. 7 6 5 5 40.46 1.82 3.164* 914.75 Aug. 21 8 5 5 42.02 0.51 1.573 919.17 Aug. 28 9 5 5 44.44 1.47 2.444: 426.04 Sept. 4 10 1 1 52.40 ." -- -- * - Signi?ioant at’the‘5%fllevel of probability **- Significant at the 1% level of probability B Mouse Date of Littering Last Date Weighed G]. Aug. 21 Sept. 4 02 Aug. 18 Aug. 28 (Killed her young) 03 Aug. 14 Aug. 28 04 Aug. 12 Aug. 28 C5 .Lug. 14 Aug. 28 All animals were utea'in‘aetermining mean weigfite. -35.. APPENDIX TABLE 71 Weight and Littering Data Group D, Thyroxine, 235 A fieeke on An}.- figight‘_» . Date Expt. male Preg. Littered Mean Std. Error t Change ~ (no-7 (nm) (no-1 Tgmo) Tam-T (3’?) Jun. 26 0 3 35040 0.68 20302 ‘ July 3 1 3 35.70 1.25 3.224: 00.85 '- " n July 10 2 3 36.77 0.78 5.925** 03.87 July 17 3 3 34.40 0.76 2.956* -2.82 July 24 4 3 1 35077 0074 30485‘ .1004 July 31 5 3 2 36.13 0.70 7.681** 02.06 Aug. 7 6 3 3 40.03 3.15 2.055 013.08 Aug. 14 7 3 1 2 43.67 2.11 2.040 023.36 Aug. 21 8 3 3 . 40.77 2.64 1.195 015.17 Aug. 28 9 3 3 39.50 3.48 1.135 011.58 _ Sept. 4 10 1 1 52.40 -- -- -- ' t - Significant at the 575 level of probability u - Significant at the 1% level of probability B Mouse Date of Littering Last Date Weigheg D1 -- Aug. 28 (Saorifioed) D2"I Aug. 11 Aug. 28 D3. Aug. 21 Sept. 4' D4"I Aug. 14 Aug. 28 D5 - July 17 (Died) * - Only those that littered were used in determining mean weights. TABLE VII - 86 - APPENDIX Weight and Littering Data Group E, Thyroxine, 45 A. Weeks on Ani- lie—ight Date Expt. male Preg. Littered Mean Std. Error t Change Inool (rm-1 (no-1 ism-T Tam-T (5%) July 3 1 2 32.60 0.40 2.054 -2.83 July 24 4 2 l 34.45 0.45 2.758 02.68 Aug. 7 6 2 2 37.10 6.00 0.700 010.58 Aug. 14 7 2 1 1 46.05 6.05 1.624 037.26 Aug. 21 8 2 2 41.00 0.00 1.385 922.21 Aug. 23 9 2 2 42.00 0.18 2.016 025.19 It - Significant at the 5% level of probability B Mouse Date of Littering Last Date Weighed Bl -- Aug. 28 (Saorifioed; 32 -- Aug. 28 (Sacrificed E3 -- July 23 (Died) E4"I Aug. 15 Aug. 28 E5* Aug. 11 Aug. 28 * - Only those thatjitteref were used in determining mean weights APPENDIX TABLE VIII 'Ieight and littering Data Group F, Thyroxine, 85 A. Weeks on .Lni- ubight Date Expt. male Preg. Littered Mean Std. Error t Change ( no .) (n07) lno .) (€167 72am (3) June 26 0 5 36.26 2.10 1.838 July 3 1 5 36.98 1.33 3.793** 01.99 July 10 2 5 36.46 1.47 4.183** 00.55 July 17 3 5 33.06 2.29 1.137 -8.82 July 24 4 4 35.55 2.18 1.742 -1.96 July 31 5 2 34.40 2.60 1.574 -5.13 Aug. 7 6 1 29.30 -- 2.130 -19.19 Lug o 14 7 1 34 0 30 -" 0 0048 -5 e40 . Aug. 21 8 1 36.00 -- 0.423 -0.72 Aug. 28 9 1 33060 "" 00103 '7034 00 - Significanffat thefilfl level of PFOBEEZIIEY 3 Mouse Date of Littering Last Date'Weighed F1 -- July 31 (Died) 22 -- July 17 (010(1) F3 -- July 23 (Died) F4 -- Aug. 28 (Sacrificed) F5 -- July 23 (Died) All animals living were used in determining mean weight: - 33 - APPENDIX TABLE 11 Weight and Littering Data Group G, Control A. Week—s on Ani- flight Date Expt. male Preg. Littered Mean Std. Error Change (no-7 1110-) (Two) T811107 (Sm-7 l'xo July 15 0 5 22.52 1.42 July 20 1 5 22.44 1.94 -0.56 July 27 2 5 24.44 1.10 08.53 Aug. 5 5 5 24.46 1.10 08.61 ‘uge 10 4 5 24042 1029 .8044 .Aug. 17 5 5 5 24.64 1.10 .10.50 Aug. 24 6 5 5 27034 0092 .21040 Aug. 51 7 5 5 33.84 1.65 .50.27 Sept. 7 8 5 2 5 55.92 2.62 050.62~ Sept. 14 9 5 5 52.50 0.45 445.45 Sept. 21 10 1 1 54.50 -- -- B Mouse Date of Littering Last Date‘fieighed Gl -- Sept. 14 (Saorifioed) 62. Sept. 5 Sept. 14 (Died) G3 -- Sept. 14 (Saorifioed) G4 -- Sept. 14 (Sacrifioed) 05* Sept. 10 Sept. 21 0'1 -- Sept. 14 (Sacrificed) G'2* Sept. 3 Sept. 14 G'3* Sept. 9 Sept. 14 (killed her young and died) G'4 -- Sept. 14 (Sacrificed) G'50 Sept. 5 Sept. 14 * - Only those animals that littered were used in dEtermlng mean Weight - 39 - APPENDIX TABLE I Weight and Littering Data ...—...... Group H, Thiouracil A Weeks on Ani- J'lfei’gl'xt Date Expt. male Pre . Littered Mean Std. Error t Change Inca (no-Y lie-T (Sm-3 (Sm-l 1%) July 15 0 5 25.10 0.76 0.358 July 20 1 5 25.54 0.74 1.495 010.56 Aug. 3 3 5 26.24 0.89 1.258 013.59 Aug. 10 4 5 26.62 0.56 1.564 .15.24 Aug. 24 6 5 50.74 1.10 2.5710 .55.07 Aug. 31 7 5 5 39.46 2.33 1.889 970.82 Sept. 7 6 5 5 55.14 0.51 0.292 043.46 Sept. 1‘ 9 5 5 34096 Oe92 20619. .51034 1" - Significant at the 5% level of probability B louse Date of Littering Last Date Weighed H1 -- Sept. 14 (Sacrifioed) 32* Sept. 5 Sept. 14 B3 -- Sept. 14 (Saerifioed) H4 -- Sept. 7 (Died) H5“ Sept. 1 Sept. 14 H'l -- Sept. 14 (Saorificed) H'2t Sept. 4 Sept. 14 H'3* Sept. 3 Sept. 14 (Died) 3". Sept. 3 SOPte 14 3'5 -- Sept. 14 (Sacrifieed) * - Only those animals that littered were used in determining mean weights e u e 0 e I e . _ . _ _ . . e u 1 e o . . e . a a e e c e 0 O 0 o | 2 a e i I J a e 0 v 0 0 I e 0 a C O . 1 . I I 4 ‘ dew . .W 0‘1 n0... 01.: APPENDIX TABLE II Weight and Littering Data Group I, Thyroxine, 11535 A Weeks on AniT fei'ght Date Expt. male Preg. Littered Mean Std. Error t Change 17107 (no J (110-) (Smo) (Sm-7 We) July 13 0 7 25.31 0.74 1.742 July 20 l 7 26.29 0.77 1.845 03.87 July 27 2 7 27.17 0.71 2.085 07.35 ‘ug0 3 3 7 27077 0060 20642‘ .9072 Aug. 10 4 7 28.19 0.73 2.5441!I 011.38 Aug. 17 5 7 3 27.80 0.67 2.298!I 09.84 Aug. 24 6 7 7 51014 0074 50218“ ’23005 Aug. 31 7 7 6 l 35.84 1.58 0.822 041.60 Sept. 7 8 7 2 5 37036 1050 10139 .4706]. Sept. 14 9 7 7 37.16 0.93 4.743011- 046.82 Sept. 21 10 3 3 35.30 2.48 0.323 -- * - Significant atfithe 5,3. level of prb‘iability 00 - Significant at the 1% level of probability B House Date of Litteringg Last Date Weighed 11 -- Sept. 14 (Sacrifioed) 12* Sept. 9 Sept. 21 13 -- Sept. 14 (Sacrifioed) 14‘ Aug. 31 891750 14 15* Sept. 3 Sept. 14 I'l -- Sept. 14 (Saorificed) 102.: Sept. 6 Sept. 14 1'3* Sept. 1 Sept. 14 1'41' Sept. 10 Sept. 21 1'53 Sept. 7 Sept. 21 * - Only tboee animals that littered were used in determining mean weights - 91 - APPEN DIX TABLE III Weight and Littering Data Group J, Thyroxine, _x_ A i. eeks on Ani- Weight Date Expt. male Preg. Littered Mean ”Std. Error t Change (moo) (no.) (neo) (gmoT Tgmo) (7%) July 13 0 6 24.93 0.99 1.392 July 20 1 6 26.03 0.80 1.711 04.41 July 27 2 6 26.90 0.74 1.856 07.90 Aug. 3 3 6 27.55 0.58 2.48510I 010.51 Aug. 10 4 6 27.67 0.97 2.014 010.99 Aug. 17 5 6 3 27.80 0.60 2.36213 011.51 Aug. 24 6 6 6 31.28 0.71 3.390" 025.47 Aug. 31 7 6 6 36.48 1.36 1.150 046.33 Sept. 7 8 6 2 4 37.48 2.74 0.939 050.34 Sept. 14 9 6 6 36055 0094 40111‘* ’46061 Sept. 21 10 3 3 40057 0064 -- -- 0 - Significant at the 53§level of probabilfiy M: - Significant at the 1% level of probability B House Date of Littering Last Date Weighed J1 -- Sept. 7 (Died) J2. Sept. 8 SOPt0 21 J3 -- Sept. 14 (Sacrifioed) J4 -- Sept. 14 (Sacrifioed) JB‘ Sept. 11 Sept. 21 J'l. Sept. 7 Sept. 21 J'2t Sept. 3 Sept. 14 J13. Sept. 3 Sept. 14 J“‘ Sept. 4 Sept. 1‘ J'5 -- Sept. 14 (Sacrificed) I" - Only tEose animals thtjittered were used in Remining mean weights r ljnz‘.‘ I' TABLE XIII APPENDIX ‘Height and Littering Data Group K, Thyroxine, 25 A. Weeks on [Ani- Weight Date Expt. male Preg. littered Mean —Std. Error t Change (n6775(no-) (neo) ‘Tgmi) (sm4) 1%} July 13 0 5 23.52 0.43 0.741 . a July 20 1 5 24.32 0.51 0.937 «2.93 [ >€ ‘Lng. 3 3 5 24.04 0.50 0.348 01.78 Aug. 24 6 5 5 31.92 0.54 4.29344 435.14 Aug. 31 7 5 5 40.00 1.22 2.780* 069.35 Sept. 7 8 5 5 33.84 0.76 0.029 .43.27 Sept. 14 9 5 5 36032 1014 30299. ’5307? 3; 0 - Significant at the 5%'1evel'of probability 0* - Significant at the 1% level of probability B —_4 _ Mouse Date of Littering Last Date'fieighed Kl. Sept. 4 Sept. 14 K2* Sept. 1 Sept. 14 K3* Sept. 3 Sept. 14 K4* Sept. 3 Sept. 14 K5. Sept. 1 Sept. 14 K']. "' Sept. 7 (Died) K'Z -- Sept. 14 (Saorifioed) K'3 -- Sept. 14 (Sacrificed) K'4 -- Sept. 14 (Sacrifioed) K'5 -- Sept. 14 (Sacrificed) * - Gfily those animals thatfilitteredfiWere used Ihfdétermining mean ‘Ieights APPENDIX TABLE XIV 'Weight and Littering Data Group L, Thyroxine, 45 A. _fiieeks on .Ani- ‘Jigight ‘*' Date Expt. mals Preg. Littered Mean t . Error t Change THO-701m) inc-T (gm-T (Em-T 1%? W July 13 0 6 23.53 1.31 0.523 July 20 1 6 25.87 1.13 1.528 09.94 July 27 2 6 26.63 1.09 1.414 013.17 Aug. 3 3 6 27.15 1.15 1.690 015.38 ting. 10 4 6 28.63 1.43 2.186 021.67 Aug. 17 5 6 4 29.02 1.47 2.277* 023.33 Aug. 24 6 6 6 32.68 2.12 2.311* 038.89 Aug. 31 7 6 6 42.02 4.23 1.772 078.58 Sept. 7 8 5 1 4 37.26 2.47 0.928 058.35 Sept. 14 9 4 4 41.52 3.14 2.9090 076.46 * - Significant at§%1evel of probability B Mouse Date of Littering Last Date'fieighq1_ L1 -- Sept. 14 (Sacrificed) 102‘ Sept. 3 Sept. 14 L3"I ‘Lug. 31 Sept. 7 (Killed her young) L4 -- Sept. 14 (Sacrifioed) L5 -- Sept. 14 (Sacrificed) L'1* Sept. 12 Sept. 14 (Killed her young) L'2* Sept. 6 Sept. 14 113* Sept. 3 Sept. 14 L'4* Sept. 4 Sept. 14 L'5 -- Sept. 14 (Sacrifioed) * - Only those animals that littered were used in determining mean weight; - 94 - APPENDIX 4 -2 TABLE X7 Estrous Cycle Length as Measured 1 Between Peaks of Estrus---Experiment I a Treatment wTime between Peaks _~_Mean Cycle Length : and.Lni- Before During Before During ; mal No. Injections Injections Injections Injections (days) Maya) @578? (days) Control Al 3, 7 10, 4 5.0 7.0 12 5, 5 8, 5 5.0 6.5 A3 3, 5, 4 5, 4, 6 4.0 5.0 A4 4, 5 6, 4, 4 4.5 4.7 L51I 10 8, 4 10.0 6.0 Thiouracil Bl 2, 5, 5 7 4.0 7.0 B2 5, 5 6, 5, 5 5.0 5.3 B3 it 18 -- 18.0 B4. 5 10, 6, 5 5.0 7.0 B5 6, 6 7, 6 6.0 6.5 Thyroxine, x 01 " 6 11, 5 6.0 8.0 02 8 7, 6 8.0 6.5 03 7 5, 5, 6 7.0 5.3 04 8, 3 8, 6 5.5 7.0 05 4, 6 6, 5 5.0 5.5 Thyroxine, 2: 014 "' 6 6, 5, 5 6.0 5.3 D2 4, 9 4, 4, 5 6.5 4.3 D3 5 6, 5 5.0 5.5 D4 4, 9 7, 5 6.5 6.0 D5* 6 8, 5 6.0 6.5 Thyroxine, 4x 31* "' 7, 6 7, 5 6.5 6.0 32* 10 7, 5, 5 10.0 5.? E3. 9 7, 5 9.0 6.0 B4 6 6, 5, 5 6.0 5.3 B5 7 5, 6, 7 7.0 6.0 Thyroxine, 85 £10 0* 7, 7 -- 7.0 F2‘ 4, 6 5, 7 500 6.0 F3: 9 8, 3, 4, 9.0 4.2 F4# 8 10, 4, 6 8.0 6.7 F5* 6 6, 6, 7 6.0 6.3 0 canimal had no litter #0 - Only one estrus during period of examination - 95 - APPENDIX TABLE XVI Estrous Cycle Length as Measured Between Peaks of Estrus--Experiment II Treatment 41156 between Peaks Mean Cycle Length and Ani- Before During Before During mal No. Injections Injections Injections Injections. (dayS) (days) (days)_' (day677 ' Control 01* 6 8 6.0 8.0 02 5 11, 6 5.0 8.5 G3* 6, 5 5, 7, 4 5.5 5.3 04* 7 6, 5, 7 7.0 6.0 G5 7 7, 5 7.0 6.0 G‘1* 6, 5 4, 6 5.5 5.0 G'2 6 6, 6 6.0 6.0 0'3 7 9, 6 7.0 7.5 0'41 7 8, 5, 5 7.0 6.0 G'5 6 9 6.0 9.0 Thiouracil H1* 6 3, 9 6.0 6.0 H2 5, 5 5, 6, 5 5.0 5.3 H3* 8 6, 7, 4 8.0 5.7 34* 6 6, 6 6.0 6.0 BB 6 5, 7, 6 6.0 6.0 H'l* 5, 5 5, 5, 6 5.0 5.3 3'2 6, 5 5, 5, 6 5.5 5.3 H'3 4, 7 7, 6, 4 5.5 5.7 H'4 7 6, 10 7.0 8.0 H'5t 8 7, 7 8.0 l 7.0 Thyroxine, t 11* 5, 5 6, 5 5.0 5.5 12 10 10, 4 10.0 7.0 13* 7, 7 6, 5 7.0 5.5 14 7 5, 4, 5 7.0 4.7 15 10 5, 6 10.0 5.5 1110 5, 6, 4 6, 7 5.0 6.5 1'2 5, 8 7, 6 6.5 6.5 1'3 5 5, 6 5.0 5.5 1'4 5, 5 6, 5 5.0 5.5 1'5 5, 5 5, 6 5.0 5.5 -96- APPENDIX TABLE XVI (Continued) fitment Time between Peaks hfiean 01:016 Length and Lni- Before Daring Before During mal No. Injections Injections Injections Injections (days) (days) (days) (days) Thyroxine, 1 J1* "' 5, 5, 5 6, 7 5.0 6.5 J2 6, 7 6, 7 6.5 6.5 J31 6, 5 5, 5 5.5 5.0 J41!I 7 5, 5, 5‘ 7.0 5.0 J5 6 9, 7 6.0 8.0 J'l 6, 5 5, 5, 5 5.5 5.0 J'2 7 11, 6 7.0 8.5 J'3 6, 7 6, 5 6.5 5.5 J'4 7 4, 5, 6 7.0 5.0 J'5* 5, 5 8, 7 5.0 7.5 Thyroxine, 235 K1 5, 6 5, 5, 5 5.5 5.0 K2 3, 5, 5 5, 7 4.3 6.0 K3 5, 5 8, 6 5.0 7.0 K4 5, 5 9 5.0 9.0 K5 6 7, 5, 4 6.0 5.3 101* 5, 6 7, 5 5.5 6.0 K'2* 4, 4, 5 6, 5 4.3 5.5 K'3* 7 8, 8 7.0 8.0 K'4* 6, 5 5, 7 5.5 6.0 105* 6 6, 3, 3, 2, 3 6.0 3.4' Thyroxine, 4_x_ Lllt 5 6, 7 5.0 6.5 L2 6 5, 5, 4 6.0 4.7. L3 5, 6 8, 5 5.5 6.5 L4!I 4, 5 6, 6 4.5 6.0 1.50 4, 5 5, 4, 5 4.5 4.7 U1 5, 5 6, 6 5.0 6.0 DZ 5, 5 5, 6, 4 5.0 5.0 U3 7, 6 6, 5 6.5 5.5 U4 7 9, 6 7.0 7.5 L'51II 7, 7 6, 6 7.0 6.0 * - Animal haTno litter. u ...”P.§>..’Ir> Eu :3 .3 em. 0 2: we cook a no? 0 02.3 @0330 Hon.“ 9030.3 00333.8. .3 cache 25.5: no a 9w." ”mono“ on» 59060229990 hm upeanwwe I ...... 903.3 on .252» on» a." 90.3% ... ... 3.2 2 .2 on 2 .24 «a .22. m 2 2 2 3 2 .63 2 22. 0.5 m 3 av 3568.223 8 2 2 2 .mp4 2 22. a «a 0.2 2 2 a» 3. 33. on 22. a 8 2 .2 on 2 .43 2 22. 3.3 m. 8 . Na .0568??? mm mm 2 2 .28 2 22. m we om .8 2m 2 .22 .2 .22. . um 05 «.8 2 N... 2 2 .564 mm 22. ..m .3 .m no 2 2 8 2 .24 2 22. m «o . S 2 en 3 .33 2 .22. 3.0 ml 8 N .0303.“th mm .2 S S .23 S 22. x mm 22 ON .8 S 2 .23 2 .22. m 2 2 ...» 2 4m .53 2 .22. .m «m 8 on a... 2.. 33 2 22. m.» m 3 238525. 3 .2 am 2 .24 2 22. 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Sex of Litters fiatment irreat ment and Ani- Sex at Weaning 1nd Ani- Sex at Weaning mal No. Males Tie—males mal No. Males Females Experiment I Experiment II Cont r01 Cont r01 A1 4 3 G2 2 2 AZ 4 2 G5 0 6 A3 1 0 0'2 2 5 G'5 2 6 Thiouracil B). 4 3 Thiouracil B3 2 4 H2 4 4 85 2 3 H5 4 5 3'2 4 3 Thyroxine, x H'3 3 6 c1 "’ :5 9 H'4 4 0 C3 4 6 C4 2 4 Thyroxine, fix cs 4 5 12 " 5 4 I4 2 5 Thyroxine, 2x 15 6 2 D2 " 6 5 1'2 4 2 D3 3 3 P3 3 4 D4 1 1 I'4 5 1 1'5 4 4 Thyroxino, 41: E4 - 3 5 Thyroxine, 1: E5 4 1 J2 "’ 3 5 J5 4 4 J '1 4 4 J'Z 4 2 J '3 0 3 J '4 3 2 Thyroxine, 21: K1 " 2 5 K2 4 2 K3 1 4 K4 1 1 K5 7 3 Thyroxine, 4:: L2 " 6 4 L'2 4 5 L‘3 6 4 U4 3 7 - 105 - APPENDIX TABLE XXIII Lactation No. in Litter Litter‘Weight Index of Mouse Treatment Birth 14 Days Individ. Mean* Performance (ng (gm-7 (2’9 Experiment I A3 Control 4 1 (4.2) -- -- D4 Thyroxine, 35. 12 2 (12.8) -- -- B5 Thiouracil 10 5 (39.1) 105 E Thyroxine, 43_ 5 5 37.1 37.1 100 A2 Cont r01 6 6 4O .9 106 B3 Thiouracil 6 6 31.4 82 C4 Thyroxine, x 6 6 43.2 112 D3 Thyroxine, 2'35 8 6 (41.2) 38.5 107 Al Control 8 7 (45.6) 128 Bl Thiouracil 7 7 35.5 35.5 100 E4 Thyroxine, 45’ 8 8 50.2 50.2 100 C5 Thyroxine, 35 11 9 (54.7) -- -- C3 Thyroxine, £_ 14 10 (49.6) -- -- D2 Thyroxine, 25 13 11 (49.1) -- -- Cl Thyraxine,‘§_ 12 12 64.2 64.2 100 Experiment II K4 Thyroxine, '5' 2 2 15.0 15.0 100 J'3 Thyroxine, §_ 3 3 26.0 26.0 100 G2 Control 10 4 (26.3) 105 H5 Thiouracil lO 4 (18.2) 72 H'4 Thiouracil 4 4 25.1 100 J'4 Thyroxine,l§_ 5 4 (29.4) 25.1 117 K3 Thyroxine, %§ 8 5 (22.6) -- -- G5 Control 7 6 (41.6) 99 I'2 Thyroxine, ~13 6 6 40.3 96 1'4 Thyroxine,-§£ 6 6 48.4 116 J'2 Thyroxine, x 6 6 38.4 92 142 Thyroxine, 23$ 6 6 40.2 41.8 96 - 106 - APPENDIX TABLE XXIII (Continued) No. In Litter Litter j{flight Index of Mouse Treatment Birth 14 Days Iniivid. Mean"I Pe rformanoe (gm-1 (gm-T (313 0'2 Control 7 7 34.7 91 3'2 Thiouracil 7 7 40.9 107 I4 Thyroxine, ’- 7 7 32.4 85 1‘3 Thyroxine, g 7 7 45.5 119 K1 Thyroxine, 25 7 7 38.1 38.3 99 0'5 Control 8 8 39.8 90 HZ Thiouracil 8 8 34.4 78 I5 Thyroxine, _ 8 8 43.0 98 1'5 Thyroxine, _ 8 8 52.2 118 J2 Thyroxine, 35 10 8 (38.8) 88 J5 Thyroxine, _:_:_ 8 8 51.1 116 J'1 Thyroxine, _x_ 8 8 44.0 44.1 100 11'?!» Thiouracil 10 9 (27.0) 61 12 , Thyroxine, $35 10 9 (50.8) 114 L'2 Thyroxine, 45 9 8 44.6 44.6 100 K5 Thyroxine, 2: 10 10 47.7 115 L2 Thyroxine, 45 15 10 (50.0) 121 L'3 Thyroxine, 43; 10 10 35.0 41.4 85 L'4 Thyroxine, 4x 10 11 (64.9) -- -- * - Only those litters having same number of young at 14 days as at birth were used in this average. Litter weights in parentheses were not averaged. - 107 - APPENDIX TABLE XXIV Index of Lactation Performance No. in Litter Treatment Mouse ’Firth “14 Daxs Index of Performance (7%) Experiment II Control GZ 10 4 105 G5 7 6 99 0'2 7 7 91 G'5 8 8 90 Thiouracil H2 8 8 78 HS 10 4 72 H'2 7 7 107 H'3 10 9 61 H'4 4 4 100 Thyroxine, £05 12 10 9 114 I4 7 7 85 I5 8 8 98 1'2 6 6 96 1'3 7 7 119 1'4 6 6 116 1'5 8 8 118 Thyroxine, _1_:_ J2 10 8 88 J5 8 8 116 J'1 8 8 100 J'2 6 6 92 J‘4 5 4 117 Thyroxine, 25. K1 7 7 99 K2 6 6 96 K5 10 10 115 Thyroxine, 45 L2 15 10 121 L'2 9 9 100 L'3 10 10 85 ROOM USE ONLY O y \ \ ale-Ir...- . .ll .... . .4 a! ‘r. .\ 0 . ... . ,. (..- sacs -\ '.P ‘0 ‘ O 0 ‘lhb'. 0.- IIIllNlfllllulflfllfllllfllfljlllmujllll| munum: O