u—v- — *fi- w I '—_o a v... v_‘: we THE EFFECT 0F ccsmsmm mm cm DNiKNESE‘S ON THE": Tm‘xmaa GLAND OF “fa-4E $25229 may cm THE mmus. mm 'i'imm 1":er this Ehrgnm c9? ”71.. 5.. MSG-5.53533 EWAE’E USLLEGE ". ”Jinn. m m h o “ b. it! s- ‘u’VH difrfi‘f'fi'; :8. Wiztififi'ififl E '12?“ ‘f yam Jul "- ‘F—‘fi‘ I This is to certify that the thesis entitled presented by William Terry has been accepted towards fulfillment of the requirements for Date “11.13.4351— .JLdegree mmsbandry Major professor I'. 3. 3 1293 01001.." 7458 u I ‘flf'ffi (“fin—I'm 1': .11.. s....__' 1}}: *t-l! ‘_.4'..‘l..l_r_ ' 51 _' ‘52-} 0 ' The Effect of Constant Light or Darkness on the Thyroid Gland of Sheep and. on the Estroue Cycle . L ; 4,, J ' " ' I . . I l; 1 1 . o I I \ :- 'J E. » . I l ‘ l . l f} t 3 , 4 i ‘ i f I h 3. .‘ ,‘l r. . 7 E . i- .c ’ f ‘ . ’ ' b L ‘ '4 l‘ . n ‘I i ‘ i I , i 1 ;‘ ~—-r /' ‘ . n 8‘... '(f.—- .- l.' . rqj 3""- . u“ .C -I. Pl: ‘1' - ‘I ll. .duIIt.JIlII1 II. 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I .. t s i o | i ' O I o I C b | a‘ .l I I s, \I I ' r ‘3 if: L33 EFFECT OF CONSTANT LIGHT OR DARKNESS ON THE THYROID GLAND OF THE SHEEP AND ON THE ESTROVS CYCLE U1 William Anderson Terry M .ATHKHS 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 TUlS'I‘T-SR OF SCIEVCE Department of Animal Husbandry 1951 THESIS ACKNOWLEDGVENTS The work described in this paper was carried out with the cooperation of the Demlrtment of Physiology and Pharmacology. The author wishes to exnress his gratitude to Dr. B. V. Alfredson, head of the depart- ment, for the facilities and materials furnished. Thanks are most especially due to Dr. Joseph ”eites, Associate Professor of Physiology, for the invaluable advice and assistance he gave throughout the course of the work. Thanks are also tendered to Dr. R. H. Nelson, Head of the Department of Animal Husbandry, for providing the facilities of his department, and to Dr. Leonard Blakeslee, Associate Professor of Animal husbandry, for the cheerful assistance he gave in setting up the experiment. ********** ******** ****** **s* ** * TABLE OF CONTENTS PAGE IIJTRODITCTlchr‘IOOOOOOOO.0...0.0.0.009.........OOOOOOO......OOOOOOOO 1 8517.19]?! OER leE LITEEMTTTREOO00............OOOOOOOOOOOOOCOOO0...... 3 Effects Of Light On Thyroid Function....................... 3 Effects Of Light on GOMd mnctionOOOOQCOOOOOO0.00.00.00.00 8 Thyroid, Pituitary, And Gonad Interrelations............... l3 PROCEDTTREOOOOOOOOOOOO......OOOOOOOCOOOOOOOO......OOOOOOOOOCOOOO. 17 ‘H'ethOd Of Handling The SheePOOOOOOOOOCOOOOOOO......OOOOCCO. 17 Quarters Used For The Sheep................................ 20 Equipment And FeedingOOOOOOO..........OOOOCOOOOOOOOCOOOO... 23 Checking The Breeding Activity............................. 24 ChOCklnfl; Therid ACtiVityOOOIOOOO00000....0.000000000000000 26 Statistical Analysis Of Data............................... 27 REESTYLTSOOCOOOOOOOOOOOO0..........OOOOOOOOOOOOOOOOOCCOOOOOOOO.... 29 Effects Of Light On Thyroid Function....................... 29 Effects Of Light On Breeding Activity...................... 33 DISCTTSSICHRIO O O I O O O O O O O O O O O O O 0 O O O O O O O O O C 0 C 0 O O O O O O C O O O O O O O O O C O O O O O O 36 STI,.":‘:ARYO O O O O O O 0 O O O O I O O O O O O O O O O O O O O O I O O O O O O O 0 O O O O O O O 0 O O O O I O O O O O O 0 4O BIBLIOGMPHY. O O O O O O O . O O O O O O O O O 0 O O O O O O O O O O O O O 0 O C O O O O O O O O O O O O O O O O O 42 APPE‘IDIXOOOOOOO................OOOOOOOOOOOOOOOOO00.00.000.000... 45 IVTRCDVCTION Certain areas of the science of animal breeding present an increasing number of practical problems in physiology. This is especially true when the breeder desires to change the natural matinm season of a species. He finds that phases of endocrine function, which as yet are little understood, may play an important part in the seasonal rhythm of sexual activity in some species. The function of the various endocrine glands have been de- scribed in considerable detail by many workers. However, some of the environmental factors which may enhance or inhibit the functions of the endocrines have not been nearly so well investigated. Although considerable work has been done in the areas of temperature and nutrition, the light factor has not been held constant because of failure to recognize the profound influence it might have on the animal's endocrine system. As a result, some of the evidence ob- tained might be questioned in view of information now available. To illustrate this point, breeders and research workers have long theorized that summer heat is the limiting factor in sperm production in the ram. Rerliner and Warbritton (1937) were among those who advanced this hypothesis. They noted the low fertility of rams in the summer and suggested a relation to low thyroid activity because of the well established effects of heat in depress- in: thyroid function. It now seems highly possible, in view of \7 recent experimental evidence (Yeats 1949), that the increased length of daylight during the summer may also play a role in reducing thyroid activity, and hence partially account for the summer steril- ity in sheep. Photogenic influences on reproductive activity have been demonstrated by several workers (Rowan 1931, Brissonnette 1952, and others) but until recently little or no effort was made to determine the effects of light on the thyroid. Breeders have attempted to produce two lamb crops a year by various means, chiefly through the use of gonadotropic hormones. However, only limited success has been obtained even in the most re- cent trials. Since this method has not proven very practical, it seemed worthwhile to investigate further the effects of light on thyroid and reproductive function in the ewe. The primary purpose of the present work, therefore, was to determine whether light or dark- ness influences the thyroid activity and breeding performance of sheep. It was hoped that the knowledge thus obtained might lead to more practical methods for inducing breeding activity in sheep during the spring and summer months. -2- REVIEW OF THE LITERATVRE Effects Of light Cn Thyroid Function While the literature abounds with references concerning the effects of light on gonadal function of birds and mammals, very little work seems to have been done on the relation of the thyroid to photogenic influences. Host of the material available on the subject has been published in recent years, for it has long been assumed that heat and cold are the major external factors in thyroid regulation. “ills (191?) showed that high external temperature diminished the activity of the thyroid glands in rabbits as judged by both morphological and growth rate changes. Conversely, he found that a low external temperature increased the rate of growth in rabbits. The numerous experiments of other investigators of both earlier and later dates have quite firmly established the fact that the thyroid responds to thermal in- fluences. The following studies, made of light effects on the thyroid, have been far less uniform. They seem to indicate that in different species opposite thyroid responses may result at times when the animals are subjected to the same conditions. Dempsey (1943) studied the histological changes of the thyroids of twelve female rats, six of whom had severed pituitary stalks. They were kept under continuous light for one month. He measured -3- the change in cell height of the thyroids to determine the secretion of thyrotropic hormone by the anterior pituitary. He reported that the rate under continuous light showed a re- duction in thyroid cell height. He found the same histological changes in normal rats subjected to heat for an equal period. Simul- taneous exposure to both light and cold resulted in constant estrus in the female rat. He assumed that these results were due to altered pituitary function caused in part by the exposure to constant light. Dempsey (1943) further noted that all the results obtained, except the effect of cold, could be produced in the rats with severed pituitary stalks. This, of course lead to the assumption that the external stimulus of light reached the anterior lobe of the pituitary by a route other than the neural pathways through the stalk. Puntriano and Neites (1950) investigated the effects of con- tinuous artificial light or darkness for a twenty—eight day period on thyroid activity in 242 Rockland mice of both sexes. Four experi- ments were conducted with females and three with males. Thyroid activity was measured by determining (a) the weight increase of the thyroids following injections for ten days of a constant dose of thirouacil, (b) the normal weight changes of the thyroids of the mice kept under light or darkness, and (c) the uptake by the thyroid of a singly-injected tracer dose of radioactive iodine administered sixteen hours prior to sacrifice. Continuous light induced the following changes in the thyroids of male and female mice, respectively, as compared to controls maintained under the light conditions prevailing in the animal room. There was a decrease in thyroid weight of 11.30 and 12.37 percent; a reduction in thyroid response to thiouracil of 32.99 and 31.04 per- cent, and a reduction of 44.00 and 44.00 percent in thyroid uptake of radioactive iodine. The above are average changes based on a 100 gram body-weight basis. Continuous darkness induced the following average changes, which are also on a 100 gram bodydweight basis. As compared to the controls there was an increase in thyroid weight of male and female mice, re- spectively, of 17.24 and 18.71 percent; increases in thyroid weight of 22.70 and 30.43 percent in response to thiouracil, and increases of 41.40 and 57.43 percent in thyroid uptake of radioactive iodine. In a second group of experiments the effects of continuous light or darkness for twenty-eight days were determined on the gonadal function of 186 young female rats. Three experiments were carried out on Rockland and Carworth rats. During the last four days of the experiment each rat was injected with a constant dose of pregnant mares' serum. The results were as follows: under continuous light the Rockland rats showed average weight increases of ovaries and uterus in response to injections of pregnant mare's serum of 23.72 and 39.37 percent above the control. Continuous darkness produced no significant change in gonadal response to pregnant mares' serum. Neither continuous light nor continuous darkness appeared to in- fluence the response of the ovaries and uterus to pregnant mares‘ -5- serum.in the Carworth rats. From this they concluded that light did not induce changes in this strain of rats. They offered the hy- pothesis that continuous light increases the production of FSH by the pituitary and decreases the production of thyrotropic hormone. Stein and Carpenter (1943) reported on the behavior of the green salamander when exposed to normal daylight in the fall for forty days, and again to artificial light for 150 days. Under normal fall light the salamander showed an increase in thyroid activity as compared to the control which was kept in darkness. ‘When exposed to artificial light the thyroids showed a greater degree of stimulation. The controls kept in darkness had much less active thyroids. They found, however, that low temperature had a greater stimulating power than light. It was concluded from.the result that light and tempera- ture both effect the thyroid of the green salamander. It will be noted that the reaction of the thyroid of the salamander was just the opposite to mice (Puntriano and Tv'eites, 1950) when exposed to light. Kleinpeter and Nixner (1937) determined the effects of wave length (quality and quantity) of light on the thyroids of baby chicks. The chicks were kept under artificial illumination for fourteen-day periods. Controls were maintained under normal light conditions. While they found that increased light increased the thyroid activity slightly, they could find no relationship between the wave length and thyroid function. -6- Reineke and Turner (1947) studied the seasonal variation in the thyroid hormone secretion of the chick. They found that in both sexes thyroid secretion reached a maximum level in Cctober and November. It declined during February and early ”arch. In the latter part of March it declined further and remained at a low level until the follow- ing August. During the fall thyroid secretion rose again to the levels observed during the previous year, indicating a definite rhythm of activity throughout the season. It seems that the increasing light in the spring may have had some influence on thyroid activity. Turner (1948) found that old White Leghorn hens were stimulated to increased egg production in the summer months if thyroprotein was fed. He could find no beneficial effects from feeding thyroid hormone in the winter months. From.this he concluded that thyroxine secretion in the White Leghorn was already at maximum levels during the winter months. He also noted that an increase in gonadotropic hormone was shown by the enlarged combs of the birds fed thyroprotein. This de- cline in thyroid secretion rate in the summer may indicate the pos- sibility that light as well as increased temperatures influence the thyroid of the fowl. Berliner and warbritton (1937) commented on the low fertility of Inns during the summer months and suggested that it might be due to low thyroid activity resulting from the high summer temperatures. Their hypothesis was at least partially confirmed by Bogart and “ayer (1946). These investigators noted that high summer temperatures -7- caused a decline in spermatogenesis in the ram. They found that when thyroprotein or thyroxine was given to the rams during the summer months, breeding capacity was restored to near normal levels. All these workers attributed the hypothyrodism observed to the high summer temperature. However, it has been demonstrated several times (Cole and Miller, 1935, and Yeats, 1949) that ewes can be induced to breed in the spring and summer if the amount of light received each day is gradually reduced by artificial means. It seems logical to assume that the ranzwould be influenced by the same conditions. Therefore, light may be an important factor in the summer decline in ram fertility. Effects Of Light On Gonad Function A great deal of evidence has been.accunmlated showing that light exerts an effect on the reproductive cycle, especially in avian species. No extensive review of this tremendous amount of literature is contemplated here since such reports are already available in other papers. Yeats (1949) gives a very complete summary of the progress made in determining light influenced, sexual changes in birds and mammals. However, a brief survey is indicated by the nature of the experiment being reported. Rowan (1929) observed that in the junco changes took place in the testes just prior to its northward migration in the spring. The interstitial tissue and germinal cells of the testis and ovaries reached maximum development during migration, and reproductive ability -9- was thus assured by the time the northern breeding grounds were reached. He found a second period of gonad interstitial activity in the fall. This hyperfunction of the gonads seemed to occur just before and during migration. Later Rowan (1931) artificially altered the daylight period of several migratory species. He found that by doing this he could cause a bird to come into full song and breeding condition during the winter months. From the observations he had made Rowan (1931) postulated that the migration of birds is stimulated by the hormone elaborated in the interstitial tissue of the testis and ovary, its elaboration being caused by the increase or decrease in normal day- light. Van Oordt and Danste (1939) observed similar results when they studied the reaction of the greenfinch to light and darkness. Birds in full song (”ay) were placed in darkness and killed at varying intervals. While in the dark both the testes and ovaries decreased in size and spermatogenesis ceased. The birds also began to molt, a process which usually takes place in August. The greenfinches were then exposed to increased light in August, after being in the darkness. Their gonads initiated spermatogenesis and increased in size. Song was restored. Benoit (1936, 1957) observed that ducks ceased to breed when the eyes were removed. However, when artificial light was directed into the eye sockets by quartz rods, breeding capacity was restored. The reports on avian response to light led to work designed to show whether mammals were influenced in the same manner or degree. Brissonette (1932) first showed a similar increase in gonad response to light in the ferret. Marshall (1940) also subjected female ferrets to different degrees of light intensity by placing them at various distances from a 1000 watt light bulb. He found the response (accel- eration of the estrus cycle) was correlated somewhat with the light intensity. T’Iorgan (1949) observed the same results when he studied the female opossum's reaction to light in its non-breeding season. He found that the increase in the size of the reproductive tract was directly proportional to the amount of light received from an arti- ficial source. Sykes and Cole (1944) attempted to alter the breeding season of eight ewes, five of which were of Rambouillet breeding. The other two ewes were of the English breeds, one being a Southdown, the other a Hampshire. The sheep were placed under artificial light in March and the lighting was increased gradually until three hours had been added to the normal day. Light was then decreased in one hour steps during late “arch, April, and early May. Services by the rams turned with the ewes were observed in the case of five of the ewes, and five ewes gave birth to normal healthy lambs. The Hampshire ewe did not breed. One of the two Rambouillet ewes who failed to lamb was suspected of having aborted. These lamb- ings occurred about five months previous to the usual time. However, -10.. none of the ewes had lambed the previous year so direct comparison could not be made. Yeats (1949) demonstrated that the sexual season of the sheep, which normally breeds in the fall, could be successfully reversed by altering the light conditions. Using two groups he placed one on increasing light in the spring while the other was subjected to de- creasing increments of light each day. He found that if the decreas- ing light treatment was continued about two months the ewes showed estrus and accepted the rams. Normal lambs were born in the fall during the usual season of mating. He noted that while temperatures ranged in the high summer levels the sheep on decreasing light showed no depression of sexual activity. Conversely, when the group on de- creasing light was changed to increasing light in the fall (normal breeding season) no estrus cycle was shown and the sheep refused to breed, although a group on the normal decreasing level of light bred as expected. He therefore concluded that decreasing light in the fall of the year, rather than the onset of cooler weather (as had been widely susposed), acted as the stimulus for breeding. Once the fact had been established that light influenced the breeding season of birds and mammals, investigators attempted to determine the manner in which that influence was transmitted to the gonads. All of the evidence gathered to date points to a primary effect on the anterior pituitary. Whitaker (1940) demonstrated that the white-footed mouse can show no cyclic sexual activity after it has been blinded by removal -11... of the eyes. ”ice kept in constant darkness also showed a reduced and non-cyclic sexual activity. If light of low intensity (one foot- candle) was used, breeding took place throughout the year and the mice did not go into anestrum. Low temperature did not effect this constant estrus. Fiske (1959) observed that female rats kept in constant light showed long periods of estrus and diestrus. Other groups of rats kept in constant darkness were found to display a long period of metaestrus. When the pituitaries of rats under constant light were assayed they were found to contain large amounts of FSH hormone. Those of the rats kept in constant darkness showed high concentrations of luteinizing hormone. Adult males kept in constant darkness had larger pituitaries and gonads as compared to males under constant light. Truscott (1944) showed that sexual maturity in rats was delayed after the optic nerve was severed, even.though constant lighting was used. In normal rats constant lighting was found by both Truscott (1944) and Fiske (1959) to hasten the advent of sexual maturity. Pomerat (1942) stated that the pituitaries of rats kept in con- stant darkness for one and one-half months resembled those of young castrated females. This condition persisted for as long as a year if the animals were kept in darkness, but the changes became less pronounced. There are many mammals and fowl whose sexual cycle is not influ- enced by light. These are primarily of tropical origin. The guinea -12.. pig was reported by Dempsey (1954) to be uneffected by light changes. Since it normally lives where the days are twelve hours long through- out the year, this is not surprising. In summary, it can be seen that many animals and birds of the temperate zone exhibit cyclic sexual activity. This activity of the gonads has been shown repeatedly by many workers to be influenced by seasonal changes in the daily level of light. The light is conducted to the pituitary through the eyes causing that gland to increase its secretion of gonadotropic hormones. Thyroid, Pituitary, And Gonad Interrelations It has been well established that there is an interrelationship between the endocrine glands of the body. The relation of the thyroid to the gonads has been studied by many workers, and the results furnish ample proof that altered thyroid condition causes a change in gonadal function. In 1925 Jaap reported on the effects of feeding dessicated thyroid tissue to ”allard drakes. This species normally shows an increase in testis size in the late winter and spring. He noted that an increase of as much as ten times over the controls could be induced with the thyroid treatment at this season. He attributed this increase to the higher metabolism of the birds which resulted in greater loss of testis hormone from the body. This loss, he felt, would allow greater elaboration of gonadotropic principle by the anterior pituitary lobe, causing gonad stimulation and increased spermatogenesis. -13- Cole (1925), another early investigator, stated that a group of five to eight year old fowls underwent rejuvenation in feathering and increased in egg production during a period of thyroid hormone feeding. Van Horn (1955) investigated the effect of excessive amounts of thyroid substance on the physiological activity of estrogen. He re- ported that approximately three times as much estrogen was necessary to induce artificial estrus in castrated white female rats after they had been placed in a hyperthyroid condition. He further stated that when hypophyseal implants from hyperthyroid female adult rats were implanted in immature females, they caused an increase in gonad stimulating power of the hypophyses ranging from 15 to 65 percent. ”eites and Chandrashaker (1949) studied the effects of induced hyper- and hypothyroidism on the response of the gonads to a constant dose of pregnant mares' serum in immature male rats and mice. They produced hypothyroidism by feeding 0.1 percent thiouracil in the ration for four to twenty day periods, and hyperthyroidism by feeding thyroprotein in various concentrations for ten day periods. They found that thiouracil or thyroprotein, when fed alone, had no effect on the weight of the seminal vesicles and coagulating glands. When pregnant mares' serum was injected, the response of the seminal vesicles and coagulating glands was inhibited by all except the lowest levels of thyroprotein in the rat, while in the mouse gonadotropic response was increased by all except the highest concentrations of thyroprotein. Thiouracil increased the gonadotropic response in rats -14- by as much as 500 percent, while the response in mice was reduced by as much as 75 percent. This work was repeated with female immature rats and mice by Johnson and Ueites (1950) and the same results were obtained. They concluded that young rats secreted more than an optimal amount of thyroid hormone whereas young mice secrete less than an optimal amount of thyroid hormone. Reineke, Bergman, and Turner (1941) studied eight thyroidectom- ized male kids for lactogelic, thyrotropic, and gonadotropic hormones. They found that both lactogenic and thyrotropic hormones were present in normal amounts in the cretinous pituitaries. The gonadotropic hormones were low and the testes were lighter than those of the con- trols. P'an (1940) reported that the gonadotropic content of the pitui- tary of normal and castrate rats and rabbits was decreased after thy- roidectomy. Evans and Simpson (1929) reported that the gonad-stimu- lating ability of the anterior pituitary was increased in hyperthyroid rats, while the glanis from hypothyroid rats were less effective than the controls. Chu (1944) found that in thyroidectomized rabbits the ovaries contained many more large follicles than normal controls. He stated that the hypophysises of the thyroidectomizei rabbits were free of ovulating hormone, whereas the follicle stimulating hormone was con- siderably increasei. -15.. While there are apparent contradictions in some of the works cited, the fact stands out that the thyroid does influence the gonads directly. It also causes the gonadotropic secretion of the pituitary to change markedl‘. The pituitary, thyroid, and gonads are thus seen to be so interrelated that any alteration in one causes a secondary effect in the others. PROCEDURE Wethod 0f Handling The Sheep The sheep used were from the Michigan State College Experimental Flock. This group is composed of purebred ewes of several breeds, with the Rambouillet predominating in numbers. The flock has been used in part for lamb and wool production but its main function is to serve for experimental research of various types. Its size is main- tained by shifting into it the poorer types from all the college flocks. The sheep in the experimental group are not culls, however, in the usual sense of the word, but rather the poorer individuals from an excellent college flock. A group of about fifty ewes was available from which to select the animals needed. However, those of Dorset breeding were eliminated since they were considered undesirable in an experiment where out- season breeding was one of the factors to be considered. Other sheep, some of which were bred, and others of excessive age, were also eliminated. When all the undesirables had been culled out there re- mained only thirty—two ewes from which to select. From.these, twenty- four were picked on the basis of breed, age, weight, and previous lambing history. The ewes finally selected for the work were of Rambouillet and Shropshire breeding. It was realized that the Rambouillet ewe some- times breeds in the summer months and might show less reaction to light and darkness than the English breeds. Their inclusion was neces- sary since they were the only breed available in sufficient numbers. -17— TABLE I SHEEP REC-0 R113 011 THE E‘Ml USED, SHO'NING THEIR LA‘flII‘IG HISTORY FOR THE TWO YEARS PRECEDING THE EXPERIMENT Control Ewe Lambed in Lambed in No. Breed ‘Weight Age 1950 1949 215 Shropshire --- 5 ------- March 17 27 Shropshire 118 5 Apr. 1 march 8 541 Shropshire 119 2 ------- Yearling 555 Shropshire 119 2 Apr. - Yearling 589 Rambouillet 159 - Jan. 19 Yearling 691 Rambouillet 99 Yr. 591 Rambouillet 150 2 Nov. 28 Yearling 574 Rambouillet 115 2 Jan. 25 Yearling 277 Rambouillet 124 5 Jan. 22 -------- Constant Darkness 502 Shropshire 120 2 mar. 14 Yearling 526 Shropshire 156 2 mar. 10 Yearling 517 Shropshire 126 2 Apr. 5 Yearling 590 Rambouillet 129 2 Nov. 24 Yearling 584 Rambouillet --- 2 Jan. 19 Yearling 162 Rambouillet --- - --------------- 684 Rambouillet 107 Yr. 145 Rambouillet 128 9 Jan. 7 Jan. 26 Constant Light 558 Shropshire 115 2 Jan. 8 Yearling 546 Shropshire 115 2 Mar. 8 Yearling 252 Shropshire 125 5 Mar. 12 Apr. 7 92 Rambouillet 117 - Apr. 16 Feb. 26 176 Rambouillet 114 6 Jan. 22 Jan. 27 679 Rambouillet 114 Yr. 585 Rambouillet 127 2 mar. 15 Yearling 575 Rambouillet 128 2 Jan. 24 Yearling The age and weight were considered very important. No valid comparison could be made of thyroid activity if the experimental animals were of several extremes of age. The weight was held as nearly equal for the groups as possible in order to simplify the calculations of the results. The ewes were divided into three groups with five Rambouillets and three Shropshires in each group. They were then designated as a control group, a constant light group, and a constant darkness group. A Shropshire ewe (No. 215) entered the control group pen from the pasture through an unlatched gate and was allowed to remain for the rest of the experiment. Her results are averaged in with the rest of the control sheep. The selection of rams was very limited. Only a small number were available for use and most of these were found to be in a low state of fertility when their semen.was examined. Two Hampshires and one Shropshire were finally chosen. They all showed good libido and the semen check indicated that they were capable of breeding any ewes which might come in heat. A fourth ram was introduced into the experiment on August 10th. This is the last Hampshire listed in Table II. A routine semen check on this date showed the Shropshire used originally to be completely sterile and he was therefore replaced. Semen checks were made on all the rams several times during the course of the summer to determine the level of their fertility. The TABLE II BREED, AGE, AND SEVEN QUALITY OF THE RAMS USED Ram Age in Semen Quality No. Breed Years Scale from I to V 754 Hampshire Yearling V (June 29) 77 Hampshire 5 IV (June 29) Chatman Shropshire 2 V (June 29) --- Hampshire - IV (Aug. 10) semen was collected by the artificial vagina method, which was found to be very satisfactory. It was judged on motility, volume, concen- tration, and amount of abnormal spermatozoa present. The semen was then rated on an arbitrary scale from one to five; one being consider- ed extremely poor and five excellent. Quarters Used For The Sheep A basement type barn was selected which had three doors opening on the north side of the structure. A floorplan of this basement is given in Figure 1. It can be readily seen from.this plan that plenty of air and light were available in all parts of the structure. Three pens were partitioned off in the basement. Each was acces- sible from the outside by one of the doors, and each was equipped with a door in the ceiling through which hay could be dropped from the mow overhead. The first pen was used for the control group. The two large doors at opposite ends of the pen were left open at all times. The sheep were restrained by low gates across these exits. Plenty of normal daylight and good ventilation were thus assured. Except for the fact that they were confined to dry feed, these ewes were under almost exactly the same conditions as the sheep on pasture. 'The center pen was lightproofed by covering the walls with tar paper. However, due to the rough nature of the structure absolute exclusion of light was not obtained. It is possible that the stray rays of light may have influenced the activity of the thyroids of this group. A small lightproof ventilator was installed in the darkened pen after the experiment had been in progress for approximately forty-five days. Before this ventilator was installed a strong ammonia smell was noticeable and the humidity was somewhat high. This condition markedly decreased once the fan was installed. No respiratory trouble was noticed among the sheep, however. The third pen was maintained under a condition of constant light. This was effected by using two ordinary household lightbulbs of 150 watts each. These lightbulbs were located on the ceiling about one- third the distance in from either end of the room. They were allowed to burn for the entire time the experiment was in operation, and thus insured that the sheep were never in darkness at any time. No re- flectors were used on the lights and in some corners the intensity was rather low. Hurdles were used to force the sheep into the better lighted areas of the pen so that they were constantly subjected to the -21... 6A 7’ E / wnvoan ( I I! ——-.-’-‘————I1 I I I I I ,J T" K \ \ \ \ \ I l y wwnowa' pt X _\ 30’ <>1 FA cgmfitrA/vr ucHr H I ,I’ (Kg-“Hr P RAM PEN ear}: 70 CONSTANT DARK/VI“ RAM PEN WA TER—‘H 1 CONTROL GROUP RAH PIN 6A TE Etna RACK Pi ure 1 Fleet-pun of the sheep quarters. Note that. 111 LEE pens except the one in constant darkness were well supplied with light and air. Forced ventilation was pro- Vided in the dark pen. The ewes Were housed on one side of the feed racksend the rattan the other. Feed was scored in the loft overhead. The dotted lines in the lighted pen Indicate hurdles placed to force the sheep into the better lighted are: of the room. light. The windows in this pen were open at all times to give venti- lation for the quarters and to take advantage of the normal light during the day. Except for the light the ewes were kept exactly as were those in.the control group. The three groups were placed in their respective pens on the 22nd day of June. They were released on August 29th when the experi- ment was terminated. Temperature measurements were made in the three pens daily. The temperature was never found to vary over three de- grees from one pen to another. The pen in constant darkness averaged about 10 C. higher than the control pen. Equipment And Feeding A large feed rack, accessible from both sides, was placed in each of the pens. This rack was of sufficient size to hold a day's supply of feed and long enough to allow the entire group to feed from one side without crowding. Fresh hay was placed in this rack each morning after the unpalatable residue of the previous day was removed and spread for bedding. Water was piped to each pen from a nearby well. The pens were cleaned each day as much as seemed necessary, and 'were completely bedded once each week. A phenothiazine and salt mix- ture was kept in each pen at all times to control internal parasites. The entire barn was sprayed once during the summer with a D.D.T. water base solution to reduce the fly population. -23- Since the conditions, except for lighting, were held constant for all the groups the sheep were not turned out on pasture at any time during the experiment. They depended entirely on new hay for their ration. This hay was, for the most part, a bromegrass, clover, and timothy mixture. The sheep were always supplied with all of it they could clean up. All of the sheep were weighed at the start of the experiment and again about forty-five days later to determine any change in condition. Table III shows the results of these weight checks. It will be noted that there were no significant weight changes in any of the groups. TABLE III THE GAIN OR LOSS OF THE GROUP IN WEIGHT OVER HOST OF THE COURSE OF THE EXPERIMENT. AS WILL BE NOTED THERE WAS LITTLE CHANGE 'Group Group Pounds Weight 0n Weight 0n Gain June 22 August 10 or Loss Control 961 lbs. 961 lbs. 0 Constant Darkness 4 982 lbs. 1010 lbs. +28 Constant Light 955 lbs. 949 lbs. -4 Checking The Breeding Activity It was originally planned to check the estrus dates occurring in each ewe while the experiment was in progress. The brisket of each ram was kept well marked with grease and ochre, and any activity by -24- the rams was observed when they were turned with the ewes in the even ning. (The rams were kept penned separate from the ewes during the day.) Any service marks appearing on the ewes' rumps in the morning were recorded when the ram were removed. This plan was finally abandoned after a trial period because the results were so unreliable. Several workers have used this method of checking oestrus and have re- ported excellent results. In this experiment, however, the results were highly unreliable at best. It was found that when the ewes were closely penned with an active ram too many false markings were ob- served to allow any reasonable emphasis to be placed on those ewes who were marked during the night. In view of the lack of reliability which could be placed in the service marks, it was decided to determine breeding activity by re- cording all lambs dropped within 147 days after the close of the ex- periment. Ewes who lamhed within this period were presumed to have been bred while under the experimental conditions. Since the fertility of the rams declined as the experiment pro- gressed into the summer they were rotated from pen to pen during the last month of the trial in an effort to keep a fertile ram with each ewe most of the time. This rotation.was made once every two days and as a result no data was gathered on the effect of light on spermatcgenesis or thyroid activity in the ram. Checking Thyroid Activity The ewes were checked for thyroid activity by injecting a tracer dose of 1131 on the morning of August let, sixty-two days after the experiment was started. The exact amount of radio-activity in the iodine used was computed by the formula - -Rt N - No e where NO 3 origional activity 6 = base of natural logarithm a. = decay constant of the iodine t = time A dosage of one microcurie of iodine per pound of body weight was injected subcutaneously in the rear flank. The iodine collected by the thyroid was measured by counting the radioactivity over the gland. A Geiger—Muller type ionization tube attached to a portable amplifier and recording circuit was used for counting. The tube was used with an aluminum shield in place so that only gamma photons were measured. A11 counting was done while holding the tube directly against the skin over the thyroid isthmus. The wool had been clipped from the necks of all the animals before the 1131 was administered so that the tube could be placed as close to the thyroid gland as possible. The radiation was counted over the gland for a four minute period. This length of time seemed to give a reasonably accurate count. Sev- eral of the ewes in each group were recounted after the group had -25- been checked and the results were always within one or two percent of the originnal count. The background count was determined for each ewe at the time the thyroid count was made and this figure was subtracted from the thyroid count. Six counts were made on each of the groups at intervals of twenty-four hours to determine the level of iodine collection in the gland. The counts were made at four, twenty-four, forty-eight, seventy-two, ninety-six, and one-hundred-ninety-two hours. Statistical Analysis Of Data All of the data obtained on thyroid function was treated statis- tically. The standard error of the group means was computed by the formula 3.3. = d2 . n(n - 17 The significant differences between the means were determined by the formula :3 . E13 Hoar-J +. £13 Hm The regression lines for the plotted curves of thyroid activity were also computed using the formula y = a + b log x, where -27- I 2y 10:2; x - (By) (210;: X) b = n 2 2 2(log x) - (2109; x) n in which y counts per minute log x 3 the log of the time n = number of cases. a is found by the formula a = y - b'Togjf . 66, the standard error of estimate for the lines, was computed by the following fo rmula: Zyz - (2302- b [2(y 109; x) - {22) (210g xl] 6" = n < > n n - 2 . The significant dif ferences between the regression lines was computed by using the formula where 6b is obtained by the formula 1 6b - 6. V2(10g :02 -_jzlog4()2 n -28- RESULTS Effects Of Light On Thyroid Function It can be seen in Figure 2 that a consistent difference exists between the thyroid function of the three groups. The group kept in constant darkness averaged a higher count than the controls, while in the group kept in constant light less iodine was collected by the thy- roids as compared to the controls. The averages given in Table IV show, however, that when the standard error is taken into consideration the apparent difference tends to disappear at some of the time inter- vals measured. TABLE IV THE AVERAGE COUNT FOR EACH GROUP COWPWTED ON THE BASIS OF AVERAGE COUNTS PER HVNDRED POWNDS OF BODY WEIGiT. THE STANDARD ERROR OF ESTIMATE IS ALSO GIVEN Time in Hours Average Counts Per Minute After Injection 2 the Standard Error Control Dark Light 4 234.9 2 34.1 283.1 I 30.6 176.1 t 42.5 24 685.1 3 60.2 762.3 1 72.8 578.0 1 106.8 48 762.3 3 69.6 1013.3 1 83.7 604.2 1 149.7 72 871.2 2 101.6 964.5 t 104.6 610.1 1 169.1 96 976.5 t 96.5 990.9 t 86.4 743.0 1 34.9 192 591.7 3 93.4 647.9 1 49.4 491.1 2 144.3 ....a tats 434.33.): Car‘s 232: «.2 5.... L235 Sarita 3.323 19.6 \o :53; g kid Lt‘k ”IOU doth IOU WHHIK‘ ‘Q L z‘k HKOU 3 MN $~ Iianw 811/ 3'1 N775) 86x . a ago we can zuyiaen hoocoaouuwv acuo«u«¢mmu 05% undo» . Avm>uood vaoahzo no no>sao 0:» o» wocvuu umcmn oAmIQLuu .zom on on vgaou ad: nocmw ... ... 8.6.1.3643 ... «will. k tflx fl k‘vfi 9‘69 uCtECSOU 99u3¢§thek§4 TABLE I or EACH ENE THROUGH THE FIRST THE GROT'P AVrJRAGES, AND THE AMOUNT BY WHICH EACH EWE'S COUNT DIFFEHED FEOM THE TJEAN OF THE GROUP ARE ALSO GIVEN control (Four Hour Check) Ewe Total Counts Difference Group No. Counts/Vin. Per 100 Lbs. From Ween Ween 27 200 183.5 51.4 234.9 541 520 412.7 177.8 215 255 212.5 22.4 555 335 270.2 35.3 691 420 385.3 150.4 589 215 149.3 85.6 574 255 214.3 20.6 277 170 136.0 98.9 591 200 148.2 86.7 Constant Darkness (Four Hour CheCET 502 360 318.6 35.4 283.2 517 325 266.4 16.8 526 290 204.2 79.0 145 430 286.7 3.5 162 120 96.8 186.4 584 300 245.9 37.3 590 440 341.1 57.9 684 385 356.4 73.2 -46.. TADLE I - Continued INDIVIDUAL DATA ON THEiHADIOACTIVE COVNT OF EACH EWE THROUGH THE FIRST FIVE OBSERVATIONS MADE OF THYROID ACTIVITY. THE GROUP AVERAGES, AND THE.A”OUNT BY WHICH EACH ENE'S COUNT DIFFERED FROW'THE VEAN OF THE GROUP ARE.ALSO GIVEN Group III (Four Hour Check) Ewe Total Counts/@fina Difference No. Counts/Vin. Per 100 Lbs. From T‘ean Count of Group 558 325 295.5 119.4 585 150 114.5 61.6 176 460 403.5 227.4 575 90 69.8 106.3 Group Mean 679 70 58.3 117.8 762.4 92 180 156.5 19.6 232 285 254.5 78.4 546 215 182.2 6.1 Group I (24 Hour Check) 27 850 779.8 94.7 541 1440 1142.9 457.8 215 710 591.7 93.4 555 850 685.5 0.4 Group ”ean 691 810 743.1 58.0 685.1 589 795 552.1 133.0 574 490 411.8 273.3 277 640 512.0 173.1 591 1000 740.7 55.6 Group II (24 Hour Check) 502 920 814.2 59.1 517 1070 877.0 166.6 526 925 651.4 110.9 145 745 496.7 265.6 Group Mean 162 465 375.0 387.3 762.3 584 975 799.2 36.9 590 1215 941.9 179.6 -47- TABLE I - Continued INDIVIDUAL DATA ON THE RADIOACTIVE COUNT OF EACH EWE THROUGH THE FIRST FIVE OBSERVATIONS MADE OF THYROID ACTIVITY. THE GROUP AVERAGES, AND THE AMOUNT BY WHICH EACH EWE'S COUNT DIFF iED FROM THE DEAN OF THEiGROUP ARE ALSO GIVEN Group III (24 Hour Check Ewe Total Counts Win. Difference No. Counts/Min. Per 100 Lbs. ‘ From ”ean Count of Group 585 425 324.4 253.6 176 1335 1171.1 593.1 575 285 220.9 357.1 679 625 520.8 57.2 Group Mean 92 700 608.7 30.7 578.0 232 800 714.3 136.3 546 1000 847.5 269.5 558 660 600.0 22.0 Group I (48 Hour Check) 27 1070 981.7 219.3 541 1100 873.0 110.7 215 740 616.7 145.7 555 1330 1072.6 310.2 Group Ween 691 825 756.9 5.5 1013.3 589 1260 875.0 112.6 574 605 508.4 253.9 277 575 460.0 302.4 591 935 692.6 69.8 Group II (48 Hour Check) 502 1300 1150.4 137.1 517 1205 987.7 25.6 526 1330 936.3 76.7 145 950 633.3 380.0 Group Kean 162 730 588.7 424.6 1013.3 584 1365 1118.9 105.5 590 1320 1023.3 9.9 684 1285 1189.8 176.5 h TABLE I - Continued INDIVIDUAL DATA ON THE RADIOACTIVE COUNT OF EACH ENE THROUGH THE FIRST FIVE OBSERVATIONS WADE OF THYROID ACTIVITY. THE GROUP AVERAGES, AND THE AMOUNT BY WHICH EACH NE'S COUN'I‘ DIFFERED FRO“! THE MEAN OF THE GROUP ARE ALSO GIVEN Group III (48 Hour Check) Ewe Total Counts/kin. Difference No. Counts/Nin. Per 100 Lbs. From Mean Count of Group 585 480 366.4 237.8 176 1835 1609.7 1005.4 575 285 220.9 383.3 679' 655 545.8 58.4 Group Nean 92 690 600.0 4.2 604.2 232 555 495.5 108.7 546 905 766.0 162.7 558 685 622.7 18.5 Group I (72 Hour Check) 27 895 -821.1 50.1 541 1870 1484.1 612.9 215 770 641.7 229.5 555 1175 947.6 76.4 Group Mean 691 1100 1009.2 137.9 871.2 589 990 687.5 183.1 574 1075 903.4 32.2 277 570 456.0 415.2 591 1300 963.0 91.8 Group II (72 Hour Check) 502 1075 951.3 13.2 517 1335 1100.7 136.1 526 1065 750.0 214.5 145 1168 778.7 185.9 Group Nean 162 610 491.9 472.6 964.2 584 1090 893.4 71.1 590 1325 1027.1 62.6 684 1360 1259.3 294.7 TABLE I - CONTINUED INDIVIDUAL DATA ON THE RADIOACTIVE COUNT OF EACH ENE THROUGH THE FIRST FIVE OBSERVATIONS FADE OF THYROID ACTIVITY. THE GROUP AVERAGES, AND THE AT‘IOUNT BY ‘v‘vHICH EACH “HE'S COUNT DIFFERED FROM THE VEAN OF THE GROUP ARE ALSO GIVEN GrQUDIII (72 Hour Check) Ewe Total Counts/Ein. Difference No. Counts/Fin. Per 100 Lbs. From Vean Count of Group 585 640 488.6 121.6 176 1400 1228.1 617.0 575 480 372.1 238.1 679 415 345.8 264.3 Group Kean 92 990 860.9 200.7 610.2 232 435 388.4 221.8 546 855 724.6 114.4 558 935 850.0 239.8 Group I (96 Hour Check) 27 680 623.9 32.1 541 1580 1253.9 662.3 215 490 408.3 183.4 555 790 637.1 45.4 Group Nean 691 345 316.5 275.2 591.7 589 590 409.7 181.9 574 635 533.6 58.1 277 390 312.0 279.7 591 1050 777.7 186.1 Group II (96 Hour Check) 502 885 769.6 121.6 517 550 450.8 197.1 526 635 447.2 200.8 145 1085 723.3 75.4 Group Wean 162 635 504.1 143.9 647.9 584 695 569.7 78.3 590 1045 810.1 162.1 684 535 495.4 152.6 -50.. TABLE I - Continued INDIVIDUAL DATA ON TH“ RADIOACTIVE COUNT OF EACH ENE THROVGH THE FIRST FIVE OBSERVATIONS MADE OF THYROID ACTIVITY. THE GROUP.AVERAGES, AND THE mom? BY amen EACH arm's comn‘ DIFFERE‘) FRO?! THE JEAN OF THE GROUP ARE ALSO GIVEN Group III (96 Hour Check) Ewe Total Counts Min. Difference No. Counts ”in. Per 100 Lbs. From Mean Count of Group 585 285 217.6 273.5 176 1680 1473.7 982.6 575 335 259.7 231.4 679 350 291.7 - 199.4 Group Vean 92 630 547.8 56.7 491.1 232 445 397.3 93.8 546 740 627.1 136.0 558 485 440.9 50.2 -9"- ~’ n ‘ "" " ”WY-an; mtflfiiqug 34“ (I3 5794 (IL/«[20 L78 ""8““ “’78: X may 1.3544484 L c . ‘ I 7 _ ‘ I \l . 1 fiTx' o H " _— a k . 4 I . M 1 ‘_ ‘ l ,. _. I . . ’ I v . I . I I . . I x $0 ‘ .1. J: ‘ . t “ . '\ I . I - .‘ ,' l I‘ I | ‘ ‘ . A f ‘ . . 1 r ' ‘ . 1- A. n ’ ., l . . | . ‘ u ., . ' ' ‘ 1 ‘ ‘ A. l \ u . r .‘ ‘ ‘- “‘ Y ‘ I I ' r ' "‘ r -' ‘4 A 1 "k i I . ‘ D (V I \. y 1 1' .‘ . 1' A . 2‘. ,; l .. .24» , . .1 I y V , ‘ fl . l. v ' V (I L r. - ‘ . Q" ‘ ‘> 2 ¢‘. 'A . . l ' 1‘ I. ’ I. 1 I I V I. ‘ -‘. \ _. ~ ' ‘ r 7’ \- . . ,- i . it I , ' , . .. "V‘ ‘ a . _ I. l I ' _ ' 5 , ’ v, I I l 1 . | . ‘.‘ f' l I . V r _ _ I - ' 0“ I ’ 4‘ ' ‘ ~v, ‘ v'. w . m, r .7 7. r ' I I , I" . ‘r X f. *fl WM I . _ .. ’ I I . 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