THE THYHOID SECRETION M T S QF SHEEP AS AFFECTED BY: SEASON, AGE, BBEED, PBEGNANCY ADD XACTATION by Harold A* Henneman Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Animal Husbandry 1953 ProQuest Number: 10008329 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008329 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 THE THYROID SECRETION RATE OP SHEEP AS AFFECTED BY: SEASON, AGE, BREED, PREGNANCY AND IACTATION By Harold A. Henneman AN ABSTRACT 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 DOCTOR OF PHILOSOPHY Department of Animal Husbandry Year Approved 1953 Henneman This investigation was undertaken for two reasons. First, to develop a practical technique for determining thyroid secretion rates in intact, individual sheep. Second, to determine the effect of season, age, breed, pregnancy and lactation on the thyroid secretion rate of sheep. With the background of knowledge reported by Perry (l95l)» Wolff (l95l)» and Terry (1951)» preliminary investigations were conducted to develop a technique for measuring thyroid secretion in the live sheep. External thyroid counts were taken on two control ewes to determine the time after injection of 1*31 that a definite trend of output was es­ tablished. Efforts were made to maintain external thyroid counts at one hundred per cent of previous count by the subcutaneous injection of 1-thyroxin This procedure did not prove practical. An attempt was made to correlate percentage of previous count to milligrams of 1-thyroxine injected. However, there was an indication that when more thyroxine was injected than what is normally secreted by the thyroid, there was a great outpouring of l!31 from the gland. Two ewes were given overdoses of 1-thyroxine and this phenomenon was confirmed. A practical method was then devised to determine the daily thyroid secretion rate. Statistical methods were employed to extrapolate the daily secretion rate from the linear repression of 1-thyroxine injections on per cent of previous external count with individual sheep* One microcurie of I131 per pound of body weight was injected subcutaneously. At seven and ten days post injection, base counts were taken over the thyroid gland with the Gieger counter. taken on the outside of the foreleg. at three-day intervals. A background count was Four additional counts were taken For the first three-day period following the determination of the base counts, the sheep were handled the same as during Henneman the base period. For the second three-day period, a small quantity of 1-thyroxine was injected daily; during each successive three-day period the injections of 1-thyroxine were increased. It was important that the maximum amount of 1-thyroxine injected daily remain below the actual secretion of the sheep. The counts were then corrected for background and physical decay and the percentage of previous corrected count was computed. As the quantity of 1-thyroxine injected daily increased the percentage of pre­ vious count increased. The predicted daily secretion rate was then extrapolated by linear regression to the point where the percentage of previous count was one hundred per cent. This level of 1-thyroxine was then assumed to be the daily secretion rate. This procedure was used to determine the secretion rate of four groups of six sheep each and the differences were analyzed by the analysis of variance. The secretion rate in July (0.078 £ 0.020 mg.) was significantly different at the one per cent point from determinations made in January, March, May, September and December. Pregnancy in sheep does not cause a significant difference in thyroid secretion. Lactation creates increased demands upon the thyroid gland. The secretion rates in lactating, pregnant and non-pregnant ewes during March were; 0.5^1 - 0.096; 0.390 - O.O^; and 0.3^6 - 0.036 mg., respect­ ively. During the months of January, March, May, September and December the two-year old ewes secreted more thyroxine than four-year old ewes. Only during the month of September did there appear to be a difference between breeds; Hampshire, 0.350 * 0.083 mg. and Shropshire, 0.25h t O.O36 mg. REFERENCES Perry, W. , A Method for Measuring Thyroid Hormone Secretion in the rat with Its Application to the Bioassay of Thyroid Extracts. Endocrin., h8 t 64-3, 1951* Terry, W. ♦ The Effect of Constant Light or Darkness on the Thyroid Gland of the Sheep and on the Estrous Cycle. Thesis for M,S. degree. Michigan State College, 1951* 51 numb, leaves. Wolff, J* Some Factors that Influence the Release of Iodine from the Thyroid Gland. Endocrin., 28A, 1951* Harold A. Henneman candidate for the degree of Doctor of Philosophy Final examination, March 9* 1953* 1*30 P.M., 201 Agricultural Hall Dissertation: The Thyroid Secretion Bate of Sheep as Affected by: Season, Age, Breed, Pregnancy and lactation Outline of Studies Major subject: Animal Husbandly Minor subjects: Physiology, Statistics Biographical Items Bora, November 20, 1918* Belmont, Wisconsin Undergraduate Studies, University of Wisconsin, 1936-40 Graduate Studies, Michigan State College, 1940-42, cont. 1950—53 Experience: Graduate Assistant, Michigan State College, 1940-42, Member United States Army Air Forces, 1942-44, Member Agricultural Relations Depart­ ment, Manager Six-Horse Hitch, Wilson & Co., Inc., 1945-50, Research Graduate Assistant, Michigan State College, 1950-51* Assistant Pro­ fessor Animal Husbandry, Michigan State College, 1951-53 Member of American Society of Animal Production, Society of the Sigma Xi ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. R. H. Nel­ son, Head of the Animal Husbandry Department, for his continuous guid­ ance, unfailing Interest and encouragement during this course of study. His inspiration and help will always be sincerely appreciated* The writer is greatly indebted to Dr. B. P. Reineke for his con* stant help, guidance, supervision, criticism and interest throughout the course of these investigations* The opportunity to have worked with him as teacher, scientist and friend, will always be remembered* Sincere thanks and acknowledgment are due to S. A. Griffin, who, working on the correlation of thyroid secretion rate and semen quality of rams, shared with the author the development of the technique* To the shepherd, R. L. Bell, and his assistant, L. W. Gearhart, the writer wishes to express sincere appreciation for their cooperation and help in working with the animals* The author deeply appreciated the financial support of Michigan State College through an assistantship and as a member of the staff* The writer is indebted to the Agricultural Experiment Station for the grant in aid which greatly facilitated this investigation, and, also to the departments of Animal Husbandry and Physiology and Pharmacology for the use of materials and equipment. TABLE OF COHTENTS Page I II III IV HJTRODDCTION 1 OBJECTIVES 3 REVIEW OF LITERATURE 4 EXPERIMENTAL PROCEDURE A« Development of a Technique for Measuring Thyroid Secretion Bate in the Live Sheep 1, Trial I — An Attempt to Maintain Thyroid Counts at One Hundred Per Cent of Previous Count 13 Trial II - Predicting Secretion Rate hy Linear Regression Using Three Groups of Eves 17 3* Trial III - The Effect of Large Daily Injections of 1-Thyroxine 18 2» 4* B* V VI VII 9 Trial IV - Extrapolating the Thyroid Secretion Rate from Varying Levels of Thyroxine Injection and a Series of External Counts on Individual Lambs Application of the Extrapolation Technique to the Study of Differences in Thyroid Secretion Rate 23 28 RESULTS ADD IHTERPEETAT ION A* Seasonal Difference 30 B. Effect of Pregnancy and Lactation 33 C. Effect of Age 35 D. Effect of Breed 38 SUMMARY AND CONCLUSIONS 43 BIBLIOGRAPHY 46 INTRODUCTION Research conducted along several different lines indicates that the thyroid secretion plays an important role in the physiology of many of the life processes important to animal production* It has been established that the thyroid hormone regulates the general metabolism of the body* Because of its action upon metabolic processes, it might be anticipated that the thyroid hormone would exert an important influence upon growth and differentiation* By de­ priving a young animal of thyroxine* growth is retarded and the sex­ ual organs remain infantile* Various experiments have been run in order to study the effect on growth and fertility of added thyroactive substances in the rations of animals* The data are very conflicting due in part* at least* to the varying levels of hyperthyroidism induced* This study has been designed to gain more basic information on normal thyroid secretion rates in sheep and the effect of various environ­ mental factors on the secretion rate* If thyroid secretion rates could be determined, a valuable landmark could be established for the physiolog­ ical dosage range in future studies of the effects of thyroidal stimula­ tion* Such data might also be useful in developing more efficient pro­ duction methods* At the present time the feeding of iodinated protein to lactating dairy cows has been shown to increase milk and fat produc­ tion* Indirectly* previous research has indicated that the thyroid gland is Involved in the problem of summer sterility in rams and may be a factor contributing to the seasonal breeding habit of the ewe* If suf­ ficient variation in thyroid secretion rates exist between individuals within breeds, it m y be possible to select on the basis of thyroid activity in an effort to increase growth rate and milk production and to overcome summer sterility in rams* All of these factors would be of importance in increasing the pro­ ductiveness and economic importance of sheep in the nation* s economy* It is also believed that the methods developed in this research will be adaptable for studies with other classes of livestock* OBJECTIVES To develop a technique for determining thyroid secretion rates in intact, individual sheep which would he practi­ cal for large animal research* To determine the effect of the following factors on the thyroid secretion rate of sheep* a* Season of the year h* Age of the sheep c* Breed d* Pregnancy e* lactation - 4 - HEYIEW OF UTEHATOSE Magnus-levy (1895) first observed, that the feeding of thyroidal materials caused an increase in the oxygen-carbon dioxide exchange* This observation has provided the basis for modem clinical metabolism determinations as one measure of thyroid status* MfSrch (1929) determined that the open-circuit type of respirometer was most suitable for the measurement of carbon-dioxide production* Teitelbaum and Home (194!) observed that the closed-circuit type of respirometer permitted direct measurement of the volume of oxygen con­ sumed* Many investigators have made use of these measurements of metabo­ lism as an indirect measure of thyroid status in laboratory animals or particularly for the assay of thyroidal materials* With the discovery of the goitrogenic compounds, a method of determining thyroid secretion rate was developed* Qoitrogens inhibit the formation of thyroxine, which permits the increased secretion of thyrotrophic hormone by the pituitary; this in turn stimulates the thyroid and causes the gland to increase in size* The enlargement of the thyroid can be prevented by the administration of thyroxine in sufficient amounts to bring the pituitary and thyroid back into normal balance* Dempsey and Astwood (1943) observed that the decrease in thyroid weight in thiouracil-treated rats bore a quantitative relation to - 5 - thyroxine dosage* The relationship between thyroid weight in milli­ grams per one hundred grams body weight and thyroxine dosage is a straight line. The point where the response curve intercepts the normal thyroid weight represents the amount of thyroxine required to maintain the normal thyroid-pituitary balance* These workers deter­ mined the thyroid secretion rates of rats at several different envi­ ronmental temperatures* They found that the thyroid secretion rate is increased markedly at low environmental temperatures and depressed at high temperatures* Mixaer et. al. (1944) observed that the thyroid of the chick re­ sponded in a similar manner and determined that female chicks had a higher thyroid secretion rate than male chicks* Monroe and Turner (1944) reported the secretion rate per unit of body weight is nearly constant in male rats* whereas in females the rate declines with increasing size* No significant differences from normal were found in pregnant or lactating rats* Reineke and Turner (1945a) observed a seasonal trend in the thy­ roid secretion rate of young chicks* The secretion rate in the spring and summer was only about one-half the winter level* A pronounced decline in thyroid secretion rates of mice were reported by Hurst and Turner (1945)* when the temperature was increased from 80 o o to 87 31# Differences were observed, also* in the secretion rates of three strains of mice* Schnltze and Turner (1945) determined that growing female goats averaging 20*4 kilograms secreted 0*64 mg* of D*I— thyroxine daily - 6 - while at a weight of 34*5 kilograms the secretion was 0*93 ®S« of D,b-thyroxine daily* Turner (1948) observed a pronounced decline in the thyroid se­ cretion rate of older hens as determined by the goiter-prevention method* ffor accuracy it has been suggested that five groups of animals with eight to ten animals per group be used in order to get a reli­ able figure for normal thyroid weight and also a good curve relating thyroid weight to thyroxine dosage* While this method is satisfactory for laboratory animals, it has definite limitations for use with large animals due to the expense of having to slaughter the animals in order to secure the thyroid weight* It is also impossible to determine in­ dividual secretion rates by this method* Since the discovery of iodine in the thyroid gland by Baumann (1896) and the fact that this iodine in the thyroid is protein-bound, (Baumann and Roos, 1896) a great deal of work has been carried out in an attempt to improve the chemical assay of thyroxine in the thyroid gland, (Hunter, 1910; Kendall, 1914; Earington, 1933» belaud and Ros­ ter, 1932; Blau, 1935* Beineke et* al., 1945b; Doery, 1945, Taurog and Chaikoff, 1946)* These methods as well as those of Harvey (1935)» McClendon (1928); Chaney (1940); Salter and Johnston (1948) and others have been used to determine the amount of protein-bound iodine in blood or blood serum as a measure of thyroid activity* This determination of proteiiv*boumd iodine in blood serum as an index of thyroid activity has been used by bong, et* al* (1951) for a - 7 - study In which a significant difference was found between breeds of dairy and beef cattle and also a difference due to age* Lewis and Ralston (1952) determined the plasma proteiBfbound iodine concentra­ tions of cows, before and after calving; calves at birth and again at twelve hours and three days; and colostrum as compared to milk* The above method shows promise as a practical way to measure thyroid activity* However* the question has been raised as to whether or not protein-bound iodine in the blood is actually a meas­ ure of thyroxine secretion* At the present time investigators who have used this method of protein-bound iodine determination would like a reliable method to measure thyroid secretion rate which could be used to check the results of the EBI method* 131 With the availability of the radioactive isotope I new meth­ ods have been suggested for determining thyroid activity* Scott et* al* (1951) have suggested a method of determination based on the Bed Blood Cell I131/Plasma I131 ratio. Perry (1951) measured the rate of loss of radioactive iodine from the thyroid gland of the rat with an external counter and de­ termined an index of the secretion of thyroid hormone* He checked the validity of this index by the effect of thyrotrophic hormone, goi- 131. H e was able to es­ trogens and thyroxine on the rate of loss of I tablish a relationship between dose of thyroxine and degree of inhibi­ tion of thyroid hormone' secretion* However, his thyroid secretion value shows poor agreement with results obtained by the goitrogen method* Wolff (1951) used a technique similar to Perry's in studying some factors that influence the release of iodine from the thyroid gland* Winchester and Davis (1952) destroyed the thyroids of chicks 'with radio iodine and then injected varying levels of D, L-thyroxine daily to different groups* They observed that birds receiving two and four micrograms most nearly equaled the performance of comparable controls* In this study it was hoped that sufficient support could be ob­ tained so that at least two methods of measuring thyroid activity could be run simultaneously, one as a check against the other* How- evert this objective was not achieved and the writer had to settle on one technique* The technique which Perry (1951) describes was se­ lected as a pattern from which our own technique was established by preliminary experiments* EXPMIMEHTA1 PROCEDURE A* Development of a Technique for Measuring Thyroid Secretion Sate in the Live Sheep Terry (1951) injected subcutaneous ly one microcurie of radioactive iodine per pound of hody weight of sheep and obtained external thyroid counts with the Geiger counter* v This level of was selected for injection subcutaneously in these experiments and was found to give reliable counts* All external thyroid counts were taken using a Bi*- clear Instrument and Chemical Corporation laboratory monitor with an •• end-window Geiger*-Muller tube* The counts were taken in the area of the neck where the highest count could be obtained* The same amount of pressure was used in putting the tube against the neck, insofar as humanly possible* 131 The radioactive I serves only to tag or mark the thyroid gland and its secretion so that it can be measured with a G-eiger tube and monitor* 127 It was assumed that the large amounts of I present in the 131 system perform exactly as the small amount of I which was measured* 60 A standard source of Co was used to check the machine at the time counts were taken on the sheep* This served to eliminate any error due to variable efficiency of the Geiger tube and monitor, as each day* s readings were corrected to the standard* A shield was used on the Geiger tube when all standard, background and external thyroid counts were taken so only gamma rays were counted* - 10 The background counts were taken on the outside of the foreleg of the sheep and these were subtracted from the external thyroid counts* This procedure corrected approximately for any gamma emis- 131 sions from I in the blood stream* lastly* the external thyroid counts were corrected to zero time for physical decay of the isotope* All of the sheep were fed iodized salt* free-choice, in an at­ tempt to insure uniform iodine intake* The corrected external thyroid counts per minute are recorded in Table I, and are shown graphically in Figure 1* for two control ewes* The curves in Figure 1 are typical of the normal uptake and out131 put curves of I in sheep* By computation the Hampshire ewe was secreting daily* 12*27 per cent of the iodine in the thyroid gland, while the Shropshire ewe was secreting daily, 7*80 per cent* In this study we were concerned with the thyroid output and, from Figure 1, seven days after injection of 1* ^ was selected as the earliest 131 reliable time that sheep reach a rather steady output of I , - 11 - TABLE I EXTERNAL THYROID COUNTS PER MINUTE OF TWO CONTROL EWES Time after Injection Hampshire Ewe Shropshire Ewe 6 hours 797 409 20 hours 1636 1102 hours 2314 1488 2 days 2770 2167 3 days 5332- 3538 4 days 3587 3697 5 days 2966 3418 6 days 3365 3498 7 days 2639 296? 8 days 2565 3128 10 days 2013 2368 days 1416 1533 16 days 892 1400 days 841 1186 22 days 565 925 25 13 17 - 12 - 5200 1*000 u 2000 X 1000 U1 800 600 400 300 200 100 0 5 10 15 20 DAYS Fig. 1. External thyroid counts on two control ewes - 13 - Trial I - An attempt to maintain external thyroid counts at one hundred -per cent of previous count. Perry (1951) maintained external counts at one hundred per cent in rats "by a daily injection of 15 mi­ crograms of 1-thyroxine• In this trial with sheep a "base reading was taken on the third 131 day after injection of I • This was considered zero time and all thyroid counts were corrected for physical decay hack to this time* On the seventh day after injection of 1*31 a q^a^ity 0f l-thyroxine was injected subcutaneously. The l-thyroxine injected was expected to exert an effect on the level of the next day* s count* At this time the l-thyroxine was used in the form supplied by Glaxo labora­ tories, Greenford, Middlesex, England* The dry thyroxine was weighed on an analytical balance and dis­ solved in distilled water which was made slightly alkaline with NiaOH, Then enough acid (HCL) was added to make the solution slightly cloudy* This solution was then stored in the refrigerator and used within two weeks* Four ewes were used in this trial and the data are presented in Tables II and III; also in graphic form in Figures 2 and 3* It was assumed in this trial that when the count remained at ap­ proximately one hundred per cent of previous count, the injected l-thyroxine was sufficient to replace the normal thyroid secretion* In other words, the animal1s thyroid gland was not secreting any thy­ roxine* - 14 - TABLE II EFFECT OF 1-TEYRCKINE INJECTIONS ON EXTERNAL THYROID COUNTS OF SHROPSHIRE AND OXFORD EWES f --- Days Shropshire Oxford Following Mg. of $ of Mg. of Thyroid Thyroid $ of Injecyjn 1-Thyroxine Counts Previous 1-Thyroxine Counts Previous In.iected Per Minute Count Injected Per Minute Count 3 (Zero time) 4 5^10 4580 4548 4185 5 5893 4363 6 5369 4705 7 0.8 5241 8 0.8 5458 9 0.8 10 0.8 11 0.9 12 0.9 13 0.9 14 1.0 0.7 4369 104.1 0.7 4013 97.1 0.7 5148 97.1 0.7 3810 93.5 4665 90.6 0.8 3285 91.9 88.7 0.8 3671 88.7 0.8 3276 91.8 4026 109.6 0.9 3244 107.8 15 3242 80.5 3069 87.3 16 2798 86.3 2195 83.4 17 2630 93.9 1940 86.8 21 1664 9 0 .2 93.5 91.8 1566 - 15 - TABLE III EFFECT OF 1-THYRQXINE INJECTIONS ON EXTERNAL THYROID COUNTS OF DORSET AND RAMBOJILEET EWES Rambouillet Dorset Days $ of Mg. of Thyroid Following Thyroid $ of Mg* of Previous IhjecJ^n 1-Thyroxine Counts Previous 1-Thyroxine Counts In.iected Per Minute Count In.iected Per Minute Count 3 (zero time) A 4410 7280 3974 6292 5 4363 7156 6 4945 6683 0.8 6960 126.4 0.9 6283 95.4 0.9 4515 95.4 0.9 5497 93.5 4492 99.4 1.0 5055 91.9 93.0 1.0 3884 93.0 1.0 4263 9 1 .8 4298 110.6 1.1 4599 1 07.8 15 3258 75.8 4016 87.3 16 2680 82.2 3350 83.4 17 2408 89.8 2911 86.8 21 1744 7 0.6 3923 8 0.5 4959 9 0.5 10 0.5 11 0.6 12 0.6 13 0.6 14 0.7 90.2 93.5 91.8 1780 M I NUTE — 16 " 6000 5000 0 ]»jeste< PE R 3000 COUNTS 2000 OXF )RD 1000 DAYS Fig. 2. Effect of l-thyroxine injections on external thyroid counts. 7000 <000 3 5000 1 ng. Inje 4000 3000 2000 1000 DAYS Fig. 3. Effect of l-thyroxine injections on external thyroid counts. - 1? - However, as the trial progressed it appeared less and less likely that a very good estimate of daily thyroid secretion rate could he made* It was more or less a trial and error method and it was obvious that a great many readings and injections would be necessary before a reasonable estimate of secretion rate could be made* At the start of the trial it was believed that if more than the daily output of thyroxine was injected the counts in the thyroid would remain at one hundred per cent* However, with the Bambouillet and Shropshire ewes especially, the per cent of previous count was decreas­ ing as the thyroxine injections were increased* Also, when all four ewes received their largest injections of thyroxine the greatest drop in counts was observed* Probably, in the case of the Oxford ewe, the daily secretion rate was approached when 0*8 mg* of 1-thyroxine was injected daily* How­ ever, the method did not appear to be very practical and therefore a second trial was started to test a different hypothesis* Trial II - Predicting secretion rate by linear regression using three groups of ewes* In the second trial twelve ewes were injected 131 with one microcurie of I per pound of body weight and seven days later a base count was taken on the thyroid gland* The four ewes in Group A were then injected daily for three days with 0*A mg* of 1-thy­ roxine* The four ewes in Group B received a similar schedule of 1-thy­ roxine injections of 0*8 mg* daily and Group C received daily 1-thy­ roxine injections of 1*2 mg* daily* Injections of 1— thyroxine were made for three days in order that the thyroid gland might adjust its secre- 18 tion to the daily injections* taken of 1^31 ^ Three days after the "base counts were the thyroid, a second external count was taken and corrected for physical decay* The percentage of previous count was then determined for each ewe and the predicting equation was deter­ mined* In this trial the predicting equation was Y « -*504 + 1*264 x* When x is equal to 1 (or 100$), Y * 0*76 mg* with a standard error of the predicting line of 0*28 mg* (Figure 4)* This value of 0*?6 mg* was then assumed to he the average daily thyroid secretion rate of these twelve ewes* However, from the data, which appear in Table IV and Figure 4, it appeared that, at the highest level of 1-thyroxine injection (1*2 mg* daily), the animals' thyroid glands were actually secreting as much or more 1 ^ ^ than when only 0*8 mg* of 1-thyroxine was injected* From the plotted data in Figure 4 it appears that when the injections of exogenous thyroxine surpass the normal daily secretion the data no longer fit the straight line* This same observation was made in Trial I and therefore a third trial was started to check this assumption* Trial III - The effect of large daily injections of 1-thyroxine. Continued external thyroid counts were taken on two sheep that were in­ cluded in the previous trial* These data along with the 1—thyroxine in­ jections are included in Table 7 and Figure 5* Shropshire 215 received 1- thyroxine injections of 0*8 mg* daily for three days and the thyroid counts remained at approximately one hundred per cent of previous count* Then the 1-thyroxine injections were increased to 1*5 mg* daily and the external counts fell rapidly, even more rapidly than when no thyroxine was injected* - 19 TABLE IV MILLIGRAMS OF 1-THYROXIHE INJECTED M I IT AND PERCENTAGE OF PREVIOUS COUNTS FOR THE TWELVE EWES Group A Mg. of 1—Thyroxine Injected Daily Per Cent of Previous Count Shropshire 209 0.4 90.7 Hampshire 0.4 91.9 Shropshire 9^9 0.4 102.2 Shropshire 916 0.4 86.6 Average______________ 92*0 Group B Dorset 0.8 115.7 Shropshire 215 0.8 121.5 Shropshire 911 0.8 96.7 Shropshire 923 0.8 97.3 Average 112.3 • Group C Rambouillet 1.2 119.2 Oxford 1.2 87.1 Shropshire 940 1.2 106.8 Shropshire 952 1.2 122.8 Average__________ 106.4 - 20 - Y * - 0.504 + 1.264 X Standard error - 0.28 mg* If X * 100$; Y * 0.76 mg. Q 1.2 ui i“ o U) z 1.0 Ui 2, x o 0.8 Q£ >X Ii 0 .6 u. o < K -i s 0.0 6o 0 PER CENT 80 OF 120 P R E V I O U S C O O N T Fig. 4. Predicting equation using data obtained on twelve ewes - 21 ~ TABLE V EXTERNAL THYROID COOTS OF TWO EWES RECEIVING LARGE DOSES OF INJECTED 1-THYROXINE Date Shropshire 215 Mg. of Thyroid 1-Thyroxine Counts Injected Per Minute Shropsh Lre 209 Mg. of Thyroid 1-Thyroxine Counts Per Minute Injected Nov. 3 1340 2798 Nov. 4 1140 2630 Nov. 8 1664 0.4 1400 Nov. 9 0.8 Nov. 10 0.8 0.4 Nov. 11 0.8 0.4 Nov. 12 1.5 Nov. 13 1.5 Nov. 14 1.5 759 1.5 827 Nov. 15 1.5 577 1.5 722 903 918 1.5 1057 1.5 Nov. 16 428 542 Nov. 17 375 469 - 22 - sooo S H fO P S IIR E 3000 ui l- 2(9 2000 A ig* Injcctet 2 M X of ui a. 1000 v> 6oo I- 500 2 900 800 700 S H iO P S IIR E 215 O O 300 200 100 0 1 2 3 4 5 6 7 8.9 10 11 D A Y S Pig* 5 * S m c t of large dally inject lone of 1- thyroxine on external thyroid counts* 23 - Shropshire 209 received only 0*h mg* of 1-thyroxine daily by in­ jection and as expected the counts were not held at one hundred per cent* However* when this ewe> received 1*5 of thyroxine daily the percentage decline in counts was even greater* These results confirmed earlier suspicions that when more thyrox­ ine is injected than what is normally secreted by the thyroid gland* 131 from the thyroid. Cornwall (1950) there is a great outpouring of I observed that the secretion rate of chicks receiving one microgram of exogenous thyroxine was inhibited more than in groups of chicks in­ jected with two and three microgram doses* Therefore* it was concluded that in order to predict the daily thyroid secretion rate by correlat­ ing daily injections of 1-thyroxi ne to percentages of previous count* it would be necessary to inject thyroxine in quantities which were be­ low the daily thyroid secretion rate* Thyroxine secretion could then be estimated by extrapolating the prediction line so obtained to the 131 point where no further discharge of I was indicated* Trial IV - Extrapolating the thyroid secretion rate from varying levels of thyroxine in.lection and a series of external counts on indi­ vidual lambs. Previous to the start of this trial the stock supply of 1-thyroxine was farther purified by Dr. S. P. Reineke* The activity of the 1-thyroxine increased, necessitating a lower level of thyroxine injections if reliable estimates of secretion rate were to be obtained* Pour lambs were used in this trial. 131 One microcurie of I per pound of body weight was injected subcutaneously* At seven (zero time) 131 ariid ten days, post injection of I , base counts were taken over the 24 - thyroid gland with the Geiger counter* on the outside of the foreleg* day intervals* A "background count was taken Tour more counts were taken at three** For the first three-day period following the determina— tion of the "base cotints the sheep were handled the same as during the "base period; that is* no 1-thyroxine was injected* For the second three-day period 0*1 mg* of 1-thyroxine was injected daily and a read­ ing of external counts was made* During the next three-day period 0*25 mg* of thyroxine was injected daily into each lamb and then for the last three-day period* 0*5 mg* of thyroxine was injected* These counts were then corrected for background and physical de­ cay* to zero time* and the percentage of previous count was computed* As the quantity of 1-thyroxine injected daily was increased, the per­ centage of the previous count increased, which indicated that the thy­ roid itself was secreting less thyroxine* The data are presented in Table VI* TAB IE VI MILLIGBAMS OF 1-THYRQXINE INJECTED M I IT AND PERCENTAGES OF PREVIOUS COUNT OF FOUR IAMBS .... r . ... i-----i $.... Iamb Previous Count No Thyroxine Injection Previous Count 0*1 mg* Thyroxine Previous Count 0*25 mg* Thyroxine Previous Count 0*5 mg* Thyroxine Shropshire 72*2 90*9 93.8 81.2 Cross Bred 52 71.2 84.5 66*3 93.8 Cross Bred 69 80*6 66.3 80.6 92.6 Hampshire 59.<* 63*7 80.7 82.1 — 25 •• By combining the data of all four lambs there were sixteen ob­ servations which could be analyzed statistically and an average se­ cretion rate for the entire group determined* This procedure was fol­ lowed and is expressed graphically in Figure 6* From the predicting equation* T * -*585 + 1*03 x, the average daily secretion rate for the four lambs was found to be 0*445 per cent* when x was equal to one hundred The standard error of the predicting line was 0*15 mg* The + standard deviation of the Mbw value was - 0*37 mg* and BbM was signifi­ cantly different from zero at the two per cent level* The correlation coefficient between milligrams of 1-thyroxine injected and per cent of previous count was 0*552, which was significant at the five per cent level* Without question, this method of analysis would be the most ac­ curate for predicting an average daily secretion rate for a group of sheep, as there are more observations to support the predicted value* However, in the study to be presented it was mandatory to obtain pre­ dicted values for individual sheep so that an estimate of variation within groups could be obtained* This could be used as a basis for testing significance between group means by the method of analysis of variance* It was fully realized that the predicted values for individ­ ual sheep, which were based on only four observations, were not very reliable* By computing the predicted values for each of the four lambs the secretion rates were as follows: 0*428 mg*; Crossbred 69: Shropshire: 0*263 mg*» Crossbred 52: 0*503 ®g*» and Hampshire: 0*695 Hone of - 2 6 - *y x * b = ,60 0.552* 1.03* ± 0.37 m g. Y * - o^a* + l- n t x / Standard error * 0.15 m g. If X * / / 100$; Y = 0.445 m g. / >50 7n / / / - , / / / .4 0 / t / / / i / > >30 / z i re / . / / Z0 / / / / r '1y / / / / / / / / 10 / • / / > / / / / / / / / - / 00 — ( 20 P E R 6 L / 40 CENT 60 OF P R E Y J O U S 80 loo C O U N T . Predicting daily thyroid secretion rate from per cent of previous count on a group of four lambs. - 27 the Bbn values were significantly different from zero at the five per cent level, nor were the correlations, between injected thyroxine and per cent of previous count, significant at the five per cent level* However, the average daily secretion rate for the group as determined from the individual predicted values, was 0*472 mg* as compared to 0*445 mg* which was obtained by the previously mentioned statistical method* This method of procedure for obtaining and analyzing the data was accepted as the one to use in studying the differences in thyroid secretion rate in sheep* From the data to be presented in the next sec­ tion it will be apparent that this extrapolation technique gave good repeatable results* The results obtained are in line with variations in secretion rate which have been reported in other species where other techniques for estimating secretion rate have been used* The extrapolation technique is well suited to the study of thy­ roidal secretion in large animals as the productive capacity of the animal is not impaired* 28 B» Application of the Extrapolation Technique to the Study of Differences in Thyroid Secretion Bate Twenty-four sheep were selected at the beginning of the year to he used in studying the differences in thyroid secretion rate due to season, age* breed* pregnancy and lactation* They were divided into four groups of ewes with six animals in each group as follows: 6 Open Shropshire ewes dropped in 1950* average weight 114 pounds* 6 Bred Shropshire ewesdropped in 1950* average weight 117 pounds* 6 Bred Shropshire ewesdropped in 1948, average weight 139 pounds* 6 Bred Hampshire ewes dropped in 1950* average weight 141 pounds* All of the Shropshire ewes dropped in 1950 were sired by the same ram* All of the Hampshire ewes were sired by the same ram* It was im­ possible to select six 1948 ewes from the seme sire* Dae to deaths of lambs or ewes during the year the number of sheep in each group was not maintained* Half of the open ewes were acciden­ tally bred and, therefore, only three ewes remained in the open group for the determinations in January and March* When these ewes lambed, the lambs were immediately removed and the ewes were considered as dry while the other ewes were milking* The entire group of ewes were handled in one flock under similar feeding conditions and iodized salt was fed free-rihoice* When the external thyroid counts were made the sheep were selected in the same order each time and the readings were taken at the same hours of the day* Thus, all readings were 72 hours apart* Dae to the fact that no previous estimates were available regarding secretion rates* the problem of how much 1-thyroxine to inject, could only be estimated* However* the calculations were made immediately so that if it was obvious that too much thyroxine was being injected, ad­ ditional three-day trials were run with a lower level of injection* Daring September too small amounts of thyroxine were injected to give a good spread in percentages of previous count and, therefore, an addi­ tional three-day trial was run using a higher level of injection* Dur­ ing July it appeared that the thyroid counts remained on a plateau for a longer period and the steady output curve was delayed* Thus, another reading was required* These problems made it necessary to use good unbiased judgment in regard to reliable readings for use in the statistical calculations and analysis of variance* 30 RESULTS AND INTERPRETATION A. Seasonal Difference in Thyroid Secretion Bate The group of open Shropshire ewes were used to study the effect of season of the year upon daily thyroid secretion rate to alleviate any differences which may he due to pregnancy and lactation* To study the differences in thyroid secretion rate during differ­ ent months of the year the analysis of variance was used as illustrated in Tahle VII. TAB IE VII AKAUfS IS OF VARIANCE Source Degrees of Freedom Sum of Squares Total 22 0.2565 5 0.1611 0.0322 17 0.095A 0.0056 Months Error Mean Squares Experimental Error 0.075 The “F“ value ({j*83j§ = 5*7**) indicates a significant difference between at least two of the means at the one per cent level. The stand­ ard deviation of the means and standard deviation of the difference of the means was computed* The “Student t“ tahle was then used to test for the significance of differences between the means. There was a highly significant difference between the daily thyroid secretion rate in July as compared to the other months of the year in which readings were taken, (Table VIII), All of the college sheep were shorn in February which may account for the slight increase in secretion rate during March (0,3^6 mg,) as compared to January (0,287 mg,). Even though the secretion during May was not significantly dif­ ferent from the March reading there seems to be a trend downward in secretion rate. Also, in September, (0,286 mg,) when Shropshire ewes are generally entering their normal breeding season, their thyroid secretion rate was significantly greater than in July, (0,078 mg,) when very few ewes of the Shropshire breed have been observed to come into estrus. These results were in line with previously reported research, Reineke and Turner (1945&) observed a seasonal trend in thyroid se­ cretion rate of young chicks, the rate in spring and summer being only about one-half the winter level, Brody and Procter (1932) reported a seasonal energy metabolism rhythm in sheep which they believed to be associated with the sea­ sonal sex rhythm, Brody (1933) has also observed this seasonal meta^* bolic rhythm in young goats prior to the first lactation, Meites and Chandrashaker (19^9) and Johnson and Meites (1950) con­ cluded that thyroprotein treatment increased gonadotrophic response in the female mouse, Evans and Simpson (1930) observed a reduced secre­ tion of the pituitary gonadotropins from thyroidectomized rats, p n ri Heineke (1 9 5 0 ) Maqsood reported that modifications in the thyroid status and environmental temperature can have a pronounced effect on sexual function as well as body growth. December — 32 ■" d © a) M On PA • 4- O PA • .112 (H © rg a 4©3 va .285 £ NO va pH • O C CM M • -dNO CM • NO 00 CM • o NO O• £ © 03 PA 0PA 0 • J>» £ 0OC M • VA 0 C0 O • EH # 00 CM VA On ON CM On CO O. 03 * - 33 - Berliner and Warbritton (1937)* and Bogart and Mayer (1946) con­ cluded that high summer temperatures were a major factor in the pro­ duction of summer sterility in rams and, also, the thyroid was of major importance in the reproductive physiology of the ram* Apparently, high summer temperatures caused a reduced thyroidal secretion which was responsible for lowered sexual activity* While a great many experiments have been reported on the effect of added thyroxine on semen quality: Berliner and Warbritton (1937); Anderson (1945); Bogart and Mayer (1946); Warwick et* al* (1948); and Eaton et* al* (1948), the results are very conflicting* This may be due to the varying levels of thyroxine administration employed by the different investigators* The experiment reported herein should pro­ vide a valuable landmark in determination of the physiological dosage range for different seasons of the year in studying the effects of thyroidal stimulation* B* The Effect of Pregnancy and lactation on Thyroid Secretion Bate Comparisons between ewes that were not pregnant and ewes that were pregnant, during the months of January, March and December, in­ dicate that there was no significant difference in the daily thyroid secretion rate* This would indicate that pregnancy does not make great demands on the thyroid gland for increased secretion. The data for making these comparisons between open, pregnant and lactating ewes are given in Tables VIII and IX* Daring March, there was a significant difference at the five per cent level between non-pregnant ewes (0*346 mg*) and lactating ewes *d ® rg § d> O ® NO vo IV S* CSI 00 Cn» • • • • • CA ♦ On CM CM (*> O frl 9 fl 3 CM « & i

rv • CNJ C^ C'«V vo r-l rv & — O NO NO 8 A •d s « 41 si • • °Q &\ 5 r • • ON •d fc o h m a MILLIGMMS 00 f< m MI IT BT PHEGMUT AUD IACTATDJG 1950 SHROPSHIRE EWES - 3^ - o ON cn SM C rv • •as cm CO VA rv • VO CM CA • VA «H cn • s a g CO CM VA ON On CM 0v On r j ON ON CM O ON 3 ON va S ON O On ON £ o a iri o H *H a •H S'S s O o CA • Rr On II * M4i»fPj> - 35 - (0*541 mg*) of the same age and breed* In May* the difference be" tween open ewes and lactating ewes was not as great* but there was a difference at the ten per cent level of significance* Also* during March* and at the ten per cent level of significance there is a dif— ference between pregnant ewes (0*390 mg*) and lactating ewes (0*541 mg*) of the same breed and age* Very few data appear in the literature in regard to this prob­ lem* Monroe and Turner (1944) reported no significant differences from normal in pregnant and lactating rats* Hugh (1951) observed that the mouse thyroid was protected from radiation damage by the process of lactation indicating there was a greater output of radio­ active iodine from the thyroid gland* The literature regarding the response of lactation to the feed­ ing of thyroidal materials has been recently reviewed by Blaxter et* al. (1949)* It was shown by Graham (1934a, 1934b) and others that thyroid feeding or thyroxine injection increased both the milk and fat yield of dairy cows* The effects of iodinated casein feeding were first reported by Eeineke (1942)* The increase in milk yield ob­ tained by feeding thyroidal materials has amounted to as much as fifty per cent, (Heineke, 1943; Archibald, 1945). Where milk yield in­ creased five to twenty per cent, fat yield was increased twenty—five to fifty per cent* (Vaniandingham et* al** 1944)* C* The Effeet of Age on Thyroid Secretion Hate In studying differences in thyroid secretion rate due to the ef­ fect of age, the group of Shropshire ewes that was dropped in 1950 _ 36 — was compared to the group of Shropshire ewes that was dropped in 1948* All of these ewes were bred, lambed and nursed their lambs* By use of the analysis of variance* significant differences were obtained between these two different age groups except in the month of July when all of the sheep were secreting thyroxine at a very low level* In January, there was a difference between the means which was significant at the five per cent level between bred 1950 ewes (0*303 mg*) and bred 1948 ewes (0*167 mg*)* In March* the difference between the bred ewes was significant only at the ten per cent level; however, the difference in secretion rate for the two groups of lactat­ ing ewes was significant at the one per cent level* The data used for the age comparisons are presented in Tables IX and X* During the month of May, while the ewes were nursing lambs, the younger ewes, dropped in 1950, secreted more thyroxine (0*330 mg*) than the ewes dropped in 1948 (0*114 mg*). The difference was significant at the one per cent level* By considering all ten of the 1950 Shropshire ewes as one group (0*270 i 0*045 mg*) and comparing them to the mean secretion rate of the 1948 Shropshire ewes, there was a significant difference between the means at the five per cent level of significance during September* Also, in December after the ewes were bred, there was a significant difference due to age, at the five per cent level* Turner (1948) observed a pronounced decline in the thyroid secret tion rate of older hens* The medical profession has also observed a 37 - ® •d g pq 00 un H• -3F Q H• O N• xo 4* ON ® 00 • • • cm Jt on H• R »H • *O • U ® g 1 <§• I O H i H « |H PN ON O r—I 4 iH CO ! i 0 o •H 43 4 •d 2 m fl & «d ® 1 * CNJ © CM • 8 H On On O ON O • • • PN -4 IN CM © • • CM NO iH & ® U g s-s s ■P J* 03 5S(H UN CM UN UN 00 UN UN CM © UN UN 43 d © ► co R 43 - 38 - lowering of the metabolism in humans with advancing age# By use of the protein-bound iodine technique# long et* al* (1951) has observed differences due to age in cattle# Here again it would be very important to take into consideration the age of the animal if experiments are designed involving added thy­ roxine or the feeding of iodinated casein# D# The Effect of Breed on Thyroid Secretion Bate Two breeds of sheep were used in this study in an attempt to de­ termine whether or not there was any difference in daily thyroid se­ cretion rate due to breed# The Hampshire and Shropshire ewes were all dropped in 1950# and were bred# lambed and nursed their lambs# Bata on the Shropshire ewes are presented in Table IE# and data on the Hampshires in Table XI* While the thyroid secretion rate of the Hampshire ewes was slightly higher than that of the Shropshires at the time of every determination# a significant difference at the ten per cent point was reached only during the month of September, (Hampshires 0*350 mg#, Shropshires 0#25h mg#)# A higher level of significance was not obtained even when all ten Shropshires were considered as one group# Apparently, differences between these two breeds in thyroid se­ cretion rate are not as large as the differences due to season, age and lactation# In order to detect the smaller differences, either more sheep should be included in each group, or a more refined technique for estimating secretion rate will need to be designed# 39 - h 9 H© 8 O © P c*n O• 4r\ i-•t 0 o •H

PN • 00 CO n • CM CM 4 • CM CM Jfr © VN • • • N rN c*N PN Os vo o • TABIE A ON C*N a o •H 4> © O » VN » A •s u NO $ •d a u p •0 s p CM r-t Cw jJ" p• 3*• \0 o PN • Ci- CM «N r \ • H s NO On £> £1 cio *3 ON IV Jfr V? O © c\ •-2F • vn • -3F » s O *H VN P « on “ £ rt t £ ft oo ON © oo *0 CO «) was significantly greater at the ten per cent level than Shropshires* (0*254 £ 0*036 mg#)* 46 — BIBLIOGBAPHY Anderson, J* nation* The Semen of Animals and Its Use for Artificial Insemi­ Edinburgh, 1945* 141 pp* Archibald, J* G* Some Effects of Thyroprotein Milk. Jour* Dairy Sci*, 28: 941, 1945* ontheComposition of Baumann, E* Ueber das Hormale 7orkommen von Jod im Thierko’rper. Seyler1s Z* Physiol* Chemie, 21: 319, 1896* Hoppe- and E. loos* Ueber das Normale Vbrbommea des Jods im Thierkorper. Hoppe-Seyler1a Z* Physiol. Chemie, 21: 481, 1896* Berliner, 7* and 7* Warbritton* The Pituitary and Thyroid in Delation to Sperm Production in Bams* Amer* Soc* An* Prod., Proc., pp* 137-142, 1937. 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