"I“: 2;?ECTW "3‘ i“3$3£ '3' '59 "FHZfL‘Sm 25L "‘” 222 E22323? “NM"? [55223: 2-24-3222? $5521.22? 2222: C': 2:22. Ema-REC? '?ha:§s £3: {rhea “€152:ng if? a. :‘AECH's-Gm‘i 3m 2:22;:‘3222322‘2’ Madmghwar 921222.? Sim-2:; 22:»; THES!S LIBRARY Michigan State University . THE EFFECT OF INJECTED THIOURACIL ON BODY WEIGHT AND HATCHABILITY OF THE CHICK EMBRYO BY MADHESHWAR DHARI SINHA AN ABSTRACT Submitted to the College of Agriculture Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SC IENCE Department of Poultry Science 1959 Approved: MADHESHWAR DHARI SINHA 1 This study was done to determine the effect of thioura- cil on early chick embryos. Most of the data published were mainly after thiourea or thiouracil were injected. First, different dosages of thiouracil were tried to determine the toxic level of this drug. Five milligrams of thiouracil were found to be the toxic level for the chick embryos and 2.0 mg. was found to be the maximum level to be used in any physiological work. One-tenth, 0.2, 0.5 and 2.0 mg. of thiouracil was injected into the small end of fertile eggs that had been incubated for h8 hours. Two groups of controls were kept. One group was injected with Ringer's solution and one group received no injection. The eggs were then incubated in forced draft incubator at 37.50 C and a relative humidity of 66%. The eggs were turned every two hours and were candled at LB hour intervals after the treatment. Between the 7th and the let day of incubation, twelve eggs from each of the treatments were randomly selected and sacrificed so embryos' body weights could be determined. The average body weights of all thiouracil injected embryos autOpsied on the 7th through the let day of incuba- tion were lower than those of controls. Significant weight differences between individuals receiving different levels of thiouracil were noted after the 11th day of incubation. MADHESHWAR DHARI SINHA 2 In each group, 12 fertile eggs were left in the incubator after the let day of incubation until they hatched or died. The experiment lasted for 6 more days and on the 27th day all the remaining eggs were sacrificed. Embryos receiving 0.1, 0.2, 0.5 and 1.0 mg. hatched by the 25th day of incu- bation. 0n the 27th day, some live embryos were found in the eggs which had received an injection of 2.0 mg. of thiouracil. Both groups of controls hatched on the 20th and let day of incubation. All of the control embryos showed re- traction of the yolk sac at hatching time; whereas in many of the treated embryos, yolk sacs were still outside the body cavity at hatching time. THE EFFECT OF INJECTED THIOURACIL ON BODY WEIGHT AND HATCHABILITY OF THE CHICK EMBRYO BY MADHESHWAR DHARI SINHA A THESIS Submitted to the College of Agriculture Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Poultry Science 1959 ACKNOWLEDGEMENT The author wishes to express his sincere appre- ciation and thanks to Dr. Robert K. Ringer for his invaluable assistance and guidance which enabled the completion of this study. He is indebted to Dr. Theo H. Coleman for his help and discussion on the thesis. The writer wishes to acknowledge his indebtedness to Mr. Kenneth Rood and Mr. E. W. Speakmann for their assistance in compiling and evaluation of the data. The author also wishes to thank Dr. H. C. Zindel for his c00peration in making facilities available for this study. 11 TABLE OF CONTENTS CHAPTER I. II. III. VI. VII. INTRODUCTION AND REVIEW OF LITERATURE History . . . . . . . . . . . . . . Mode of Action . . . . . . . . . . OBJECTIVE O O O O O O O O O O O O O 0 To determine the Dosage Level of Thiouracil . To determine the effect on the .aely Life the ChiCk Enbryo O C O O O O O C To determine the Effect on Hatchability . METHODS AND PROCEDURE . . . . . . . . EXperiment No. 1 . . . . . . . . . EXperiment No. 2 . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . The Influence of Different Dosages of Thiouracil . . . . . . . . . . . Effect of Thiouracil on Body Weight Effect of Thiouracil on Harching and Yolk Re traCtion e e o o o o o o o o o SUWARY o o o o o o o o o o o o o o BIBLIOGRAPHY . . . . O . O . . O O . APPENDIX . .2.. . . . . . . . . . . iii PAGE O‘O‘UIP \ONNOO 11 11 ll 19 22 38 TABLE I. II. III. .v. VI. VII. VIII. IX. LIST OF TABLES Effects of different dosages of thiouracil on chick embryos injected at us hours of incubation. O O O O 0 O O O O O O O O O O 0 Average body weight in gms. of chick embryos injected with thiouracil at 2h hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Effect of Thiouracil on length of incubation periOd O O O O O O O O O O O O O O O O O O 0 Body weight in gms. of 7-day old chick embryos injected with thiouracil at #8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 8-day old chick embryos injected with thiouracil at h8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 9-day old chick embryos injected with thiouracil at h8 hours of incubation compared with.controls injected with Ringer's solution or untreated . . . . . Body Weight in gms. of 10-day old chick embryos injected with thiouracil at #8 hours of incubation compared with controls injected with.Ringer's solution or untreated . . . . . Body weight in gms. of ll-day old chick embryos injected with thiouracil at h8 hours of incubation compared with controls injected with.Ringer's solution or untreated . . . . . Body weight in gms. of 12-day old chick embryos injected with thiouracil at h8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 13-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with.Ringer's solution or untreated . . . . . iv PAGE 21 #5 he in ta #9 50 TABLE XI. XII. XIII. IXV. XVI. XVII. XVIII. LIST OF TABLES Body weight in gms. of lR-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 15-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 'Body weight in gms. of l6-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 17-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with.Ringer's solution or untreated . . . . . Body weight in gms. of 18-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 19-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 20-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . Body weight in gms. of 31-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . PAGE 51 52 53 51: SS 56 57 58 LIST OF FIGURES FIGURE PAGE 1. Average body weight in gms. of 7-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated. . . . . . 23 2. Average body weight in gms. of 8-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 2R 3. Average body weight in gms. of 9-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 25 R. Average body weight of gms. of 10-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 26 5. Average body weight in gms. of ll-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with.Ringer's solution or untreated . . . . . 27 6. Average body weight in gms. of 12-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 28 7. Average body weight in gms. of 13-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 29 8. Average body weight in gms. of lR-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 30 9. Average body weight in gms. of 15-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected twith Ringer's solution or untreated . . . . . 31 10. Average body weight in gms. of 16-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . . 32 vi FIGURE 11. 12. 13. 15. LIST OF FIGURES Average body weight in gms. of 17—day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . Average body weight in gms. of 18-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . Average body weight in gms. of 19-day old chick embryos injected with.thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . Average body weight in gms. of 20-day old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with.Ringer's solution or untreated . . . . Average body weight in gms. of 21rday old chick embryos injected with thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated . . . . vii PAGE 31: 35 36 37 CHAPTER I INTRODUCTION AND REVIEW OF LITERATURE History. Certain drugs such as sulfocyanide, thiourea and thiouracil, inhibit the formation and secretion of thyroid hormone and thus are a powerful goitrogenic sub- stance in many different animals and man (MacKenzie, MacKenzie and McCollum, 19R1; Ritcher and Clisby, 19R1; Kennedy, 19R2; Astwood, Sullivan, Bissell, and Tyslowitz, 19R3; Mackenzie and Mackenzie, 19R3). These workers further reported that the characteristic morphological changes produced in the thyroid gland by these drugs are a loss of colloid, hyperemia, and enlargement (hypertrophy) and multiplication of cells (hyperplasia) leading to an in- creased size and weight of the thyroid. Adams and Bull (19R9) injected thiourea and thiouracil (Deracil) into yolk sac of developing chick embryos of White Plymouth Rocks. Dosages used were 0.5 m1. of a solution of 0.R% thiourea and 0.1% to 0.2% thiouracil on the 8th, 1Rth, and 18th day of incubation. They reported that the main effects were retardation of hatching in those eggs incubated to the 21st through.2Rth day and lack of retraction of the yolk, decreased body and limb growth, enlargement of the thyroid gland with typical hyperemia, hypertrophy and hyperplasia, a marked increase in height of follicular epithelium and colloid depletion. Grossowitez (19R6) found that chick embryos whose yolk sacs were injected with thiourea between the 7th and 17th days of incubation were delayed in hatching and their yolk sacs were not retracted. McCreight (1950) reported similar reaction in another strain of chicks. Adams and Buss (1952) working with White P1ymouth.Rock chick embryos which were given a single injection of thiourea and :methyl thiouracil found that cell proliferation occurred in the thyroid‘gland. Vidal (1952) reported that thiourea injected at the 7th day and 15th day of incubation caused generalized retardation of growth with no specific abnormality but high.mortality. Romanoff and Lauffer (1956) reported the effect of injected thiourea on the develOpment of some organs of the chick embryi They confirmed some of the above findings and showed that the administration of thiourea resulted in prolongation of embryonic development and high.mortality. In all cases the thyroid was markedly hypertrophied, and increased in weight. ‘They also reported an increase in the weight of the adrenal and testes and a considerable decrease in liver and ovarian weights. Many investigators have reported hypertrophy and hyperplasia of the thyroid gland in fish, amphibians, reptiles, birds and mammals from.treatment with anti-thyroid drugs; with emphasis on the increased size of the follicular 3 cells and weight of the thyroid (Charipper and Gordan, 19R7; Adams and Bull, 19R9; Adams and Craig, 19R9, 1950, 1951; D'Angelo, Gordon and Charipper, 19R7; Domm and Beivaiss, 19R8; Goldsmith, 19R9; Lynn and Wachowski, 19Rl; McCreight, 1950). Mitotic activity shortly after injection of anti- thyroid drugs or goitrogenic compounds have been reported (Paschkis, Cantarow, Rakoff, and Rothenberg, 19R5; Adams and Bull, 19R9). Paschkis et a1. 19R5, reported that thioura- cil given daily to rats in drinking water produced a hyper- plasia as soon as 2R hours after treatment. Adams and Buss, 1952, determined the mitotic activity in chick embryos given one injection of an anti-thyroid drug on the 1Rth day of incubation and killed after 2R additional hours of incubation. The 1Rth day was chosen for the single injection because on the 13th day of incubation the follicles of the thyroid have become distinct and contain colloid (Bradway, 1929). The glandis completely developed by the 15th.day of incuba- tion (Sem, 1932; Martindale, 19R1; Bull, 19R8). The results from Adams and Buss (1952) using White P1ymouth.Rock chick embryos showed that counts of mitosis in comparable areas with cell proliferation began as early as the cellular hypertroPhy and colloid loss in the thyroids of the treated embryos. Twenty-four and forty-eight hours after injection mitosis in the injected embryos exceeded that in the controls. Thus they confirmed that the weight of the thyroid of chick embryos treated with anti-thyroid drugs was not only due to hypertrOphy of follicular cell but also due to a marked increase in the number of cells by mitosis. Working with day-old chicks, Astwood, Bissell and Hughes (19RR) found that when 0.1% thiouracil was given in the ration for 10 days the thyroid was enlarged 5 - 7 times more than that in the controls. Turner and Schultze (19R5) reported that the action of thiouracil on body weight is relatively greater in young, growing birds. Glazener and Jull (19R6) reported that thiouracil decreased the growth and feed consumption of broilers. Moreng and Shaffner (19R9); Shaffner (1951); indicated that thiouracil depressed weight gains in chickens except when fed at low levels (0.012 to 0.05 per cent). Thiouracil fed to hens at the level of 0.1 per cent for six months does not affect egg production or fertility and hatchability in chickens but the higher level (0.3 per cent) reduced these traits (McCartney and Shaffner, 191:9). Chicks hatched from thiouracil treated hens had enlarged thyroids and hypoactive thyroids (Wheeler and Hoffman, 19R8, 19h9, 1950; McCartney and Shaffer, 19R9; Booker and Sturkie, 19R9). These latter workers injected .thyroxine into these chicks hatched from the hypothyroid hens and reported a reduction in the size of the thyroid. Mellen (1957) working with metabolic rate (M.R.) of chicks found that thiouracil depressed the M. R. as long as the drug was given in feed, but lasted for only 18 hours when the drug was withheld after which the M.R. averaged the same as in the controls. ‘gggg 2: Action. The exact mode of action of anti- thyroid compounds is not known, but evidence supports the theory that the thiocarbamides among them prevent the iodination of tyrosine. Pitt-Rivers (1950); Larson, Keating, Peacock, and Rawson (19R5) found that thiouracil doesnot decrease the uptake of inorganic iodine, but prevents the gland from binding to protein or decreases the organic bond. Dempsy (19RR) by histological technique, found that the enzyme peroxidase is present in thyroid cells and disappears after thiouracil administration. The hypertrophy and hyper- plasia of the thyroid gland following treatment with anti- thyroid drugs evidently results from excessive secretion of thyrotrOphic hormone (T.S.H.) by the anterior pituitary gland. Stimulation causing this excessive T.S.H. results from.the decreased amount of thyroid hormone in the chulat- ing blood. CHAPTER II OBJECTIVE Thyroid inhibiting drugs have been used in an attempt to measure the relative requirement for the thyroid hormone in varying physiological conditions. The objective of these experiments were: 1. To determine the dosage level of thiouracil for further physiological and embryological exPeriments and to determine the level of toxicity of this drug. 2. To determine the effect on subsequent liviability and growth after injections of thiouracil into R8 hour embryos. In.most reports in the literature, injections have been made after the 7th day of incubation; therefore after the thyroid has started forming. No previous workers have determined the effects of injections of thiouracil into R8 hour embryos. 3. To determine the effect on hatchability, after injections of thiouracil into R8 hour chick embryos. CHAPTER III METHODS AND PROCEDURE Experiment No. 1. — To Determine the Toxicity Level of —ThIouracilon the Chick Embryo. Twelve dozen fertib eggs of White Leghorn hens (Gallus gallus d.) were obtained from the Michigan State University Poultry Research Farm. The eggs were selected for good shell texture and uniformity of size and weight. The eggs were incubated in a force-draft incubator at 37.5°C and a relative humidity of 60%. The maintenance of temperature and humidity at this level is recommended by the manufacturer of the incubator and also by Romanoff (1956) who states that at this level the maximum hetchability is obtained. The eggs were turned every two hours and were candled at R8 hour intervals after treatment started. After R8 hours of incubation the eggs were divided into 7 groups, each group containing 12 eggs. The groups were injected as follows: 0.1 mg. of thiouracil, 0.5 mg. of thiouracil, 1.0 mg. of thiouracil, 2.0 mg. of thiouracil and 5.0 mg. of thiouracil; and two controls--one with the injection of Ringer's solution and one without any injection at all. ' The mode of injection was via the Yushok methods (1950). This constitutes an injection being made into 8 the albumin at the small end of the egg so as not to strike the major allantoic blood vessel area. The thiouracil was difuted in Ringer's solution (sodium chloride, 9 gm., potassium chloride, 0.R2 gm., calcium chloride, 0.29 gm. and distilled water, 1000 m1.). The solutions were sterilized by autoclaving for 30 minutes. The small ends of the eggs were washed with 70% ethyl alcohol and dried. The Operator's hands were also scrubbed and washed with ethyl alcohol. The egg shell was pierced by a needle which was flamed and inserted at the small end. Then 0.5 ml. of sterile thiouracil was injected for each of the different treatments and 0.5 ml. of Ringer's solution was injected to one of the controls. Then the hole made by the needle was sealed with liquid paraffin. All eggs were then incubated. As a spot check to test for con- tamination and whether or not the treatments had killed the started embryos, eggs were candled 2R hours after injections were made. The eggs were left in the incubator up to the 21st day and then opened. Results of the above experiment at let day of incubation are shown in Table I. The embryos in Group 5 which contained 5 mg. thiouracil, started to die R8 hours after injection and all the embryos were dead on the 9th day of incubation. TABLE I Effects of different dosages of thiouracil on chick embryos injected at R8 hours of incubation. No. of No. of No. of Dead Eggs LIve 95222 Dosages Embryos .Sgt Embryos 1 0.1 mg. of thiouracil ‘ 3 12 9 2 0.5 mg. of thiouracil 2 12 10 3 1.0 mg. of thiouracil R 12 R 2.0 mg. of thiouracil 7 12 5 5 5.0 mg. of thiouracil 12 12 0 6 Control with 0.5 ml. of Ringer's solution» 1 12 ll 7 Control without injection 2 12 10 This demonstrated that 5.0 mg. of thiouracil was toxic thus inhibited embryonic development and completely suppressed body metabolism and the death of the embryos resulted. Experiment N9. g. - Effect of Thiouracil on Body Weight and Hatchability. As the 5.0 mg. was toxic to the embryos the dosage level used in this experiment was: 0.1 mg. of thiouracil, 0.2 mg. of thiouracil, 0.5 mg. of thiouracil, 1.0 mg. of thiouracil and 2.0 mg. thiouracil. Controls consisted of uninjected eggs and eggs injected with Ringer's solution. 10 One hundred and twelve dozen fertile eggs were obtained from Michigan State University Poultry Research Farm. All the eggs were from White Leghorn hens (Gallus gallus d.). They were selected for uniformity of size and shape. Random selection during the setting of the eggs in the incubator trays was used. Each group was set with 16 dozen eggs in the forced draft incubator under the previously stated condition of temperature and humidity. Eggs were again turned every 2 hours. Following the technique of Yushok (1950) as previously described, injections were made after 2R hours of incubation. After 2R hours of incubation the eggs were candled by an ordinary egg candler to check fertility. On each day from the 7th through the 21st day of incubation, twelve eggs from each treatment were randomly selected and sacrificed at which time the embryo body weights were taken. In addition to body weight, the number of live embryos was recorded so as to determine fertility. After the let day one dozen of the eggs in each group was allowed to remain in the incubator until they hatched or died. This procedure was used to determine the effect of an anti» thyroid drug (thiouracil) on the delay of hatching as reported for thiourea (Grossowicz, 19R6; Adams, 19R9; Adams and Buss, 1952; Vidal, 1952). 11 CHAPTER IV RESULTS AND DISCUSSION A. The Influence of Different Dosages of Thiouracil:- Before starting the final experiment, the toxic level of thiouracil was determined as discussed in Chapter III. . The experiment showed that 5.0 mg. of the thiouracil when injected into chick embryos resulted in 100% mortality after 9the day of incubation. 2.0 mg. was less toxic to the embryos, thus it was included in the experiments. (Table I) *' Romanoff and Lauffer (1956) injected 2, 3, 5 and 10 mg. of thiourea and obtained 100% mortality in the 5 and 10 mg. of treated embryos. Similar results were obtained by Vidal (1952) with 5.0 mg. of thiourea. There are no data available regarding the toxic level of thiouracil. B. Effect of Thiouracil on Body Weight:- From an examination of Table II it is evident that in general the average body weight of chicks injected with , thiouracil was less than that of the controls injected with.Ringer's solution or left untreated. The embryo weights of the eggs treated with thiouracil were less than 12 the controls, until the 11th day of incubation at which time the thyroid had'become functional (Hansbrough and Khan, 1951). Before the 11th day there is no significant differ- ence between the thiouracil treatments (except the 0.1 mg. 1evel--the low level) but all the treated embryos with thiour- acil are significantly different than the controls. Thus it indicates that the earlier injection, i.e., injection of different doses of thiouracil, does inhibit the body weight before the thyroid is completely formed. This suggests that thiouracil inhibits body development even though the thyroid per Se is not functional and indicates that colloid, which is present at the seventh day, may be involved in develoPment. There are no published data to show the effect on embryos of injections of.thiouracil before the deve10pment of the thyroid except Vidal (1952) who injected high levels of thiourea to determine any abnormality in development and mortality. Vidal (1952) mentioned that injecting thiourea at O or R8 hours of incubation caused no abnormality in develOpment although it did cause high mortality: however, he did not study the effect on body weight and hatchability. His primary object was to determine the effect of the injections on subsequent livibility of embryos. With the exception of the controls, most of the embryos died before the 11th day of incubation. 13 As shown in Table II there is a change in signifi- cant differences with individual treatments after the 11th day of incubation, the period when the thyroid is completely formed (Khan, 1951). Reduction of body weight caused by 2.0 mg. of thiouracil was significantly greater than the reduction caused by 1.0 mg. of thiouracil. But there is no significant difference between treatments of 0.1, 0.2 and 0.5 mg. of thiouracil when injected into chick embryos. The greatest difference is between the controls and treated embryos. The average body weights of all embryos aut0psied on the 7th through the let days of incubation were lower than those of controls. From the 17th day of incubation through the 2lst, those of the embryos injected with thiouracil were consistently lower than those of the con- trols. The difference between the higher concentration injected (2.0 mg. of thiouracil) and controls are approxi- mately 13 gms. on the let day, 10 gms. on the 20th, 8 gms. on 19th, 6 gms. on the 18th and 5 gms. on the 17th day of incubation. There was a pronounced difference in weight between individuals receiving different treatments of thiouracil. There is an approximate difference of 6 to 7 gms. between 2.0 mg. and 0.1 mg. of thiouracil injected chick embryos on the 2lst day of incubation. There is a difference in body weight of approximately 3 -'5gns. between the 2.0 mg. and 0.1 mg. treatments from the 17th to 2lst day. omen awe: oesuapaoo Hm.mm as.sm od.am He.om :o.ma mo.ma ::.ma eHo been me ad.om mm.oa No.sa oo.ea om.ea J~.ma oa.ma eao name we wd.mfl ma.ma mm.bH mm.:a \-.ma me.ma om.ma eao ease ea ~m.ma em.ea ~m.:a me~ww o~.ma no.HH Ho.HH eao ease ma smdma mw.ma em.m mo.» owes 1:~.e eo.e mac ease ea m~.oa em.oa .me.b 1wo.e om.m two.m em.: eHo ease ma 1mmm.s macaw ema.: www.mi cem.w wso.m om~.m eHo ease ma moc.m mom.m‘ www.mv oao.n sem.m ms».m mem.m eHo ease HH mmmed wmmqmu. mom.m Hmm.m omm.m Ham.a omm.H. see same OH mam.m maa.w, mme.H Hme.a NJ:.H wom.a emm.a eao ease a sem.ai ~:0.m wmm.a meH.H wfla.a mda.a ‘wmo.a oao name a mmm.o pem.o mme.o Hoe.o mme.o mem.o om:.o eHo name A .Ghuaoo Hohpzoo . assessoasa no meson eseaeneao seat moasnsm scene no names; .cepeoapss ho Goausaon n.nownfim spas mopoonna maonpsoo spat veneqaoo soapenwoaa ho manon w: as HHOeHBOHmp_£pHB beacons“ nomanae Means Mo .eaw a“ pnmfieh huon owwaopm HH mgmda 15 Ho>oa mm as ugeoamadwfimuwoq memos mafia eaaom n soapsHOm m.nowsam go .HE mo. oepoonnH u N Hohpnoo cepsompnb n H donunoo «Newmi ee.owq om.mm ee.mm eH.Hm mm.ma merma mm.mm me.mm mm.:m mmeww, w ms.am md.om . se.ma mm.smV . me.~m Ho.:m em.mm m:.am mm.om inasmw m. m. .ma.a.o .ms m.o .mm w.o .ms o.H .ms o.m Hoaunoo Hoapaoo assessoasa to demon cassettes seat moaaosm scene so named; Apessatsoov HH memes eHo ease Hm eHo mace om eao mace ma owaasm.mm ems 16 The difference is approximately the same in all the treatments from 13th to the let day of incubation. The retardation of growth with exposure of chick embryos to anti—thyroid compound, was first studied by Andrews and Schnetzler (19R5) who raised chicks from eggs laid by hens given thiouracil. They demonstrated the presence of the drug in the yolk of such eggs, but they did not describe any modification in development of the embryos. The second paper, that of Grossowicz (19R6) already men- tioned, emphasized retardation of hatching and lack of retraction of yolk sac. Adams and Bull (19R9) injected 0.R% of thiourea on the 1Rth day of incubation and embryos killed daily thereafter showed a statistically significant differ- ence. They also found that when a series of chick embryos were injected with thiourea on the 8th, or 8th and 1Rth, or 8th, 1Rth and 18th days of incubation, the body weights at autopsy on the let day were consistently less than those of the controls. Decreased body weight in New Hempshire chicks after doses of thiourea and thiouracil have been reported (Macright, 1950). These chicks were injected on the 6th, 12th and 18th days of incubation and killed at daily intervals thereafter. Some papers, however, include no data on body weights or growth of chicks injected with thiourea or hatched from eggs laid by hens fed thiouracil. Romanoff and Lauffer (1956) injected a single dose of 5.0 mg. of thiourea into different groups on successive 17 days from 0 to 18 days of incubation. All the embryos died except for eight, which hatched on the twenty-first day of incubation from eggs injected on the 18th day. The body weights of dead embryos were less than controls. This in- dicated that 5.0 mg. of thiourea was toxic to chick embryos. The above results are in agreement with data presented in Table II. There are several reports on the administration of goitrogenic drugs to very young chicks from one day to a week old, that have a bearing on the possible influence of such compounds on early growth. In these experiments, the anti-thyroid drug, thiourea, or thiouracil was included in the food or drinking water for varying periods of time and in varying quantities (Astwood, Bissell and Hughes, 19RR; Mixner, Reineke and Thrner, 19RR; Schultze and Turner, 19R9; Briggs and Lillie, 19R6; D'Angelo, Gordon and Charipper, 19R7). One day old chicks of several breeds usually responded to two weeks of age on 0.1% thiouracil in their food by slight losses in body weights (Mixner, et. al., 19RR). Thiouracil (0.1% to 0.8%) included in food or White Leghorn chicks for two weeks from the day of hatching lowered the body weights approximately 10 gms. compared with the con- trols (Sultze and Turner, 19R5). Weak solutions of thiourea (0.05% or 0.07%) caused only negligible reduction in the weight of White Rock chicks that were fed the drug for 3 18 weeks from hatching. However, thiourea of 0.1% strength reduced the weight of female chicks R8.8 gms. (37.2%) and of males 3R.8 gms. (28.2%) below those of their controls (Schultze and Turner, (19R5). There was a reduction of 27.2% in chicks of a New Hampshire cross given 0.5% thiouracil in their food for 5 weeks (Briggs and Lillie, 19R6). _Body weights lower than those of their controls were also re- ported in chicks of a Barred Rock X New Hampshire cross fed 0.1% thiourea in the ration for 20 weeks (D'Angelo, Gordon and Charipper, 19R7). ' In these experiments, the general effects upon the body weight of newly hatched chicks of treatment with moderately strong solution of anti-thyroid drugs were: (1) long treatment of 3 to 20 weeks usually reduced it. (2) short ones sometimes reduced it and sometimes did not; breeds differed in their responses; and thiourea was more effective than thiouracil. In mammals many instances of retarded growth after administration of anti-thyroid drugs have been reported (Astwood, et. al., 19R3; Christensen, 19R5; Dempsy and Astwood, 19R3; Fitzhugh and Nelson, 19R7; Goldsmith, Gordon and Charipper, 19R5; Higgins, 19R5; Hughes, 19RR; William Weinelass, Bissell and Peter, 19RR).' Only dogs and monkeys seem to be refractory to the treatment with these drugs (Aranow, Engle and Sperry, 19R6; Donowski, Man and Winkler, 19R6; Mayer, 19R7). These drugs inhibit metamorphosis in 19 anuran and urodele tadpole (Gordon, Goldsmith and Charipper, 19R3), but in some instances body growth in the anurans has continued although limb growth has not (Adams and Craig, 19R9a). C. Effect of Thiouracil on Hatching and Yolk Sac Retraction In each group, 12 fertile eggs were left in the incubator after the 21st day of incubation until they hatched or died. Chick embryos receiving the different dosages of thiouracil were delayed in hatching if the eggs containing them.were incubated to hatching dates or beyond. However, no chicks injected with thiouracil hatched on or before the 2lst day of incubation with the exception of 3 chicks out of 12 fertile eggs of Group 1 receiving 0.1 mg. of thiouracil and R chicks in Group 2 receiving 0.2 mg. of thiouracil and 2 chicks from Group '3 receiving .5 mg. of thiouracil. The results of this phase of the experiments are shown in Table III. The results show that chicks receiving 2.0 mg. of thiouracil had not hatched at the 27th day of incuba- tion. At this time they were sacrificed and embryos were removed. Four embryos were alive and eight were dead. In Groups I to IV all the chicks hatched by the 25th day of incubation. The controls receiving Ringer's solution and without ' any treatment hatched on the 20th and let days of incubation; 20 and the yolk.sacs were entirely within the body. This effect was similar to that reported by Grossowicz (19R6) but less striking since the eggs were only allowed to go six days beyond normal hatching, compared with ten days in his experiment. He reported a correlation between the time of injection (the 7th to the 17th day) and dosages administered (0.3 to 3.0 mg. of thiourea) and the degree of retardation. Adams and Bull (19R9) used 2.0, R.0 and 6.0 mg. of thiourea and found no differences between treatments. Beginning treat- ment on the 8th or 1Rth day made no apparent difference. In further studies by Adams and Buss (1952), they found no chicks injected with thiourea or propyl thiouracil hatched on or before the let day of incubation. Romanoff and Lauffer (1956) compared the effect of different doses when eggs were injected with 2, 3, 5 and 10 mg. of thiourea on the eleventh day of incubation (a state of incubation at which the thyroid is known to be functional). Observation showed that the administration at this time re- sulted in prolongation of embryonic development by 3 to 10 days beyond the normal period of 21 days and increased pre- natal mortality up to 100% in some cases, as compared to 25% mortality among the controls. ' Similar results were obtained in the present experiment when the chicks received thiouracil before the formation of thyroid. There is no information in the literature on the action of anti-thyroid drugs administered before thyroid 21 formation, i.e. before the 11th day of incubation. The present experiment shows that thiouracil, when injected into R8 hour embryos will prolong the incubation period and will delay yolk sac retraction. TABLE III Effect of Thiouracil on Length of Incubation Period Days 0.1 28° 0.2 gg. 0.5 gg. 1.0 mg. 2.0 gg. Control 21st 3 R 2 - - 10 22nd 1 O l - - 23rd R 6 5 6 .- 25th 3 l 2 R - 27th - - - - l2 (sacrificed) 22 CHAPTER V SUMMARY Thiouracil, in amounts varying from 0.1 to 2.0 mg., was injected into the albumen of chicken (gallus gallus d.) eggs after 2R hours of incubation. The embryos were sacri- ficed or were permitted to deve10p until they hatched or died. The controls received Ringer's solution or were not injected. ' Observation showed that administration of 5.0 mg. of thiouracil after R8 hours of incubation caused 100% mortality by the ninth day of incubation. ' There was a highly significant loss of body weight of all the treated chick embryos compared to controls. Signifi- cant differences between individual treatments were noted after the 11th day of incubation. Experiments showed that the administration of thiouracil resulted in prolongation of embryonic development beyond the normal period of 21 days and lack of retraction of the yolk sac. Body FIGURE 1 23 Weight _. .. 1,0 L 1 l 1 1 1 T 1 ' A 1 Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 7 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated. FIGURE 2 Body Weight db 2.5 \ A A I T l I I 1 l I I w Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg #1 # 2 Dosage Thiouracil injected Average body weight in gms. of 8 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with.Ringer's solution and untreated. FIGURE 3 Body Weight L3.5 L 1 1 J a L Central Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 9 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution and untreated. Body Weight 26 FIGURE L 21.5 1‘ 1 A A t C ntrol Control 0.1ng. 0.2ng. 0.5 $3. 1.0 mg. 2.0 mg. # 1 # 2 Dosage Thiouracil injected L Average body weight in gms. of 10 day old chick embryos in- jected with Thiouracil at RB hours of incubation compared with controls injected with Ringer‘s solution and untreated. 27 FIGURE 5 Body Weight it 6.0 .. 3.0 at 2.5. \ Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # 1 # 2 ' Dosage Thiouracil injected Average body weight in gms of 11 day old chick embryos in- jected with Thiouracil at i118 hours of incubation compared with controls injected with Ringer's solution and untreated. 28 FIGURE 6 Body Weight l l V I l P I Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 12 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution and untreated. 29 FIGURE 7 Body Weight "r 11.0 1- 9.0 it 7.0 J A L .1 I U ' j ' v w Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 13 day old chick embryos in- jected with thiouracil at RC hours of incubation compared with controls injected with Ringer's solution or untreated. 3O t 12.0 *- 10.0 A l l a l 1 CU w ' w t ' ‘— L U Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 1R day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated. 31 Body Weight FIGURE 9 - 17.0 1 C ntrol Control 0.1'nlg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Doses of Thiouracil injected Average body weight in gms. of 15 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated. 32 FIGURE 10 Body Weight «L 19.0 «1- 17.0 L " ISeo . 13.0 \ l l a l L J Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in pms. of 16 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated. 33 FIGURE 11 Body Weight .L21.0 'Control Control 0.1Img. 0.2 mg. 0.5 kg. 1.0 kg. 2.0 mg. # 1 # 2 Dosage Thiouracil injected Average body weight in gms. of 17 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated. _ 3h FIGURE 12 Body Weight 25.0 .. 211.0 m 23.0 t 22.0 4t- 21.0 ¢ 20.0 +.19.0 L L J A —v c ntrol Control 0.1Ing. 0.2r-g. 0.5 mg. 1.0'ng. 2.0 ;g. # 1 # 2 Dosage Thiouracil injected Average body weight in gms. of 18 day old chick embryos in- jected with Thiouracil at RR hours of incubation compared with controls injected with.Ringer's solution or untreated. 35 FIGURE 13 Body Weight it 27 l l #L_ s : i . . a Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 19 day old chick embryos in- jected with Thiouracil at;RB hours of incubation compared with controls injected with Ringer's solution or untreated. Body 36 w °13ht FIGURE 1R '1' 30e0 a a l A l 1 fl. Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0;g. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 20 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with.Ringer's solution or untreated. 37 FIGURE 15 Body ,Weight +- 33 w- 30 w 27 1 2R 1'. 21 i i 2 4 is % Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg. # l # 2 Dosage Thiouracil injected Average body weight in gms. of 21 day old chick embryos in- jected with Thiouracil at R8 hours of incubation compared with controls injected with Ringer's solution or untreated. we. was 38 BIBLIOGRAPHY Adams, A. E. "The effect of the administration of dicltryl shilbestrol on adult normal and castrated males." 'Q. Rev. Biol., 21:1. 19R6. Adams, A. E. and Alice L. Bull. "The Effects of Anti-thyroid Drugs on Chick Embryos." Anat. Record, 10R:R21. 19R9. Adams, A. E. and M. Craig. 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Poultry Science, 25:186-187. 19h6b. , VanderLaan, W. P. and A. Bissell. "The influence of selected goitrogenic compound on the th oid gland of chicks." Endocrinology, 38:308-3lh. 19 6a. . ’ ‘ffiififl' . "Effects of propyl thiouracil and of potassium .51 thiocynate on the uptake of iodine by thyroid gland Q ‘ of the rat." Endocrinology, 39:157-160. 19h6b. r ‘ Vidal, A. "Influence de la thiourie sur le developpement del embryon de poulet." Ann. Endocrinol. 13(6): {» 982-990. 1952. - g Willer, B. H. "The endocrine glands and development of the chick." Am.‘g. Anat., 33:67-103. 192h. Williams, R. H., A. R. Weinglass, G. W. Bissell and J. B. Peters. "Anatomical effects of thiouracil." Endocrin- ologl. 3u=317-328. lath. Woodside, G. L. "Experimentally induced h per-thyroidition in the chick embryo." Anat. Rec., 67: 3-h30. 1937. x Yushok, W. D. "Effect of anti-thyroid drugs on chick embryo." Thesis C. V., 1950. _ APPENDIX Hobea &H be unconnecmem ammmeao. 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