- . TRACER 5100mm BGNE MINERALEZATIQN m THE DEVELQPING .cwcxm EMB’Rvo Thais far thy Dear” “of Ph. D. MlCHlGAN STATE UNIVERSITY David Aévin Libby 1955 This is to certify that the thesis entitled Tracer Studies on Bone Mineralization in the Developing Chicken Embryo. presented by David Alvin Libby has been accepted towards fulfillment of the requirements for Ph D degree inwsbandry and Institute of Nutrition. @e/KM fir professor Date AUEIISt 18. 1955 0-169 TRACER STUDIES ON BONE MINERALIZATION IN THE EVELOPING CHICKEN EMBRYO By David Alvin Libby AN ABSTRACT Submitted to the School of Graduate Studies of Kichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Husbandry and Institute of Nutrition Year 1955 Date $7 /5’ Approved ’%~_ ' %ZM§ David A. Libby 1 The embryonate egg and the young chick were used to detenmine the effects of various compounds on the uptake and distribution of radio. active calcium. In an attempt to interfere with calcium supply, the complexing agents. boric acid. citric acid and ethylenediamine tetra acetic acid were used. Anti-vitamins for pantothenic acid, pyridoxine and biotin were utilized to impair the metabolism of ground substances. and the hormones glucagon and insulin were employed to alter the ener- gy supply to the developing bone. The Cans complexed with boric acid. sodium citrate or EDTA.has an increased mobility. This was indicated by more rapid uptake. trans- port and turnover of the tracer. In general. all tissues with.the ex. ception of the yolk sac ccntained.greater quantities of Cau5; the femur content was consistently doubled by these treatments. Yolk deposition of the isotope was prevented to a large extent by inJections of these compounds, thereby making more of the tracer available to the skeletal structures. In addition. these treatments caused greater quantities of Ga“5 to‘be returned to the gut for possible excretion. The Cau5 of embryonic excreta is recirculated. to a greater extent if ccmplexed, making the isotope repeatedly available to the embryo. Inhibitory vitamin analogues had little or no effect on embryonic bone uptake of Cans. Pantoyl taurine produced results similar to that of the complexing.agents. but to a much less marked degree. Statis- tically significant differences from control values were never obtained with any of the analogues. The results indicate that the use of the specific vitamin inhibitory analogues did not produce deficiency David A. Libby 2 conditions in the embryonate egg which are observedly similar to the deficiency conditions produced by maternal dietary deficiencies of pantothenic acid, pyridoxine or biotin. Glucagon was consistently effective in reducing the Cau5 content of embryonic and young chick bones. This probably was not the result of a reduction of glycogen.at the site of active calcium deposition. is in the case of the complexing agents, lesser quantities of the isotope were deposited in the yolk sac. Excretion of Ca95 may have been enhanced by glucagon injections. Insulin exerted no apparent effects on the Cans content of bone. Since calcium deposition was normal, additional glchgenesis at the site of mineralization will not increase deposition of calcium. TRACER STUDIES ON BONE MINERALIZATION IN'THE DEVELOPING CHICKEN EMBRYO 3! David Alvin Libby A THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pbultry Husbandry and Institute of Nutrition 1955 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. P. J. Schaible of the Department of Poultry Husbandry and to Dr. L. F. Folterink of the Department of Physiology and Pharmacology for their guidance, assistance and contributions which made this work possible The author is also indebted to Dr. H. C. Zindel, Head, Department of Poultry Husbandry and to Dr. D. V. Alfredson, Head, Department of Physiology and Pharmacology who graciously made available the laborb atory facilities where this work was conducted. The glucagon used in this study was supplied by Eli Lilly and Company, Indianapolis, Indiana. The author wishes to express his appreciation for being able to use this scarce hormone. Grateful acknowledgment is made to the Atomic Energy Commission, Division of Biology and Medicine for supplying the isotopes and for other assistance essential to this study. David Alvin Libby Candidate for the degree of Doctor of Philosophy Final examination, August 18, 1955, 9:00 A. M., Conference Room, Poultry Building. Dissertation: Tracer Studies on Bone Mineralization in the Develop- ing Chicken Embryo. Outline of Studies Major subject: Animal Nutrition Minor subjects: Physiology, Biological Chemistry Bi cgraphi cal Items Born, Movember 1, 1923, Marquette County, Michigan Undergraduate Studies, Michigan State College, l9nb-l950 Graduate Studies, Michigan State University, 1950-1955 Experience: U. S. naval Seabees, 19N3-l9u6, Graduate Teaching Assistant, Michigan State University, 1950-1951, Technician 2 A, Michigan State University, 1951-1953, Graduate Teaching Assistant, Michigan State University, 1953-195“, Graduate Research Assistant, Michigan State University, 1954-1955. Member of Poultry Science, American Association for the Advancement of Science, Society of Sigma Xi. TABLE OF CONT EN‘J‘ S A CKNOWLED MIIT S O O O O O O O O O O O C O O VITA LIST LIST I. II. III. Iv. VI. OF TABLES . . . . . . . . OF FIGURES . . . . . O O O C O . O O O 0 GENERAL INTRODUCTION AND OBJECTIVES . . DELINEATION OF THE PROBLEM . . . . . . . GENERAL PROCEDURE . . . . EVALUATION OF SPECIFIC PROCEDURES . . . . A. B. C. D. E. GENERAL EFFECTS OF THE EXPERIMENTAL TREATMENTS A. B. THE Site of Injection . . . . . . . . . . Age at Injection and Age at Harvest Egg-811611 31':de e e e e e e e e Whole Ash-ground.Aeh Correlation Oven Dry.ihole Ash Correlation . . Embryonic Mortality . . . . . . . . Femur Weights . . . . . . . . . l. Complexing Agents . . . . . . . 2. Vitamin Antagonists . . . . . 3. Hermones . . . . 4. Discussion . . . . . . . . . . EFFECTS OF COMPLEXING AND CHELATING AGENTS Survey of the Literature . . . . l. Boric Acid . . . . . . . . . ii iii viii 16 20 20 21 23 25 26 28 as 32 32 35 35 35 5 VII. VIII. Ix. X. B. C. D. THE A. B. C. D. 2. Citric Acid and Sodium Citrate . ....... 3. Ethylene diamine tetra acetic acid Procedure-socmplexing Agents . . . . . Results . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . EFFECT OF VITAMIN ANALOGUES . . . . . . Survey of the Literature . . . . . . . l. Desoxypyridoxine and Pyridoxine . . . 2. Pantothenic acid and Pantoyl taurine 3. Biotin and Desthiobiotin . . . . Procedurem-Vitamin Analogues . . . . Results . . . Discussion . . . . ...... . . . EFFECT OF HORMONES . . . . . . Survey of the Literature . . . . l. Glucagcn . . . . . . . . . . . . . 2. Insulin . . . . . . . . . . . . . Procedure--Hormones . . . . . . . . . Results . . . . . . . . Discussion . GENERAL DISCUSSION AND SUMMARY . SELECTED BIBLIOGRAPHY . . . . . O O O Page “3 1+7 50 51 59 a. an 65 71 7h 77 79 79 79 80 82 33 8b 90 98 10. 11. 12. 13. 1h. 15. 16. 17. 18. LIST OF TABLES Composition of all-mash layer-breeder ration . . . . . . Tissue uptake of Ca“5 in day old chicks in relation to site of injection . . . . . . . . . . . . . . Effect of age at injection on Cans uptake by 21-day bone. Effect of age at harvest on Cau5 uptake by bone . . . . . Cans recovery from shell of a developing egg . . . . Embryonic mortality due to experimental treatments . . . Embryonic mortality due to isotope solution . . . . . . . Effect of complexing agents on wet femur weight, mmisture and ash content . . . . . . . . . . . . . . . . . . . . Effect of vitamin inhibitory analogues on wet felmr weight, mmisture and ash content . . . . . . . . . . . Effect of glucagon and insulin on set femur weight, moist- ure and ash content . . . . . . . . . . . . . . . . . Summary of effects of treatments on embryonic femurs . . Effects of complexing agents on body weight, total bone size and relative bone size ... . . . . . . . . . . . . Control femur weights at different ages . . . . . . . . . Effect of boric acid on the as“5 distribution in the embryonate egg . . . . . . . . . . . . . . . . . . Effect of sodium citrate on Cau5 distribution in the embryonate egg . . . . . . . . . . . . . . . . . . . . Effect of sodium citrate on Ca)"5 distribution in the embryonate egg . . . . . . . . . . . . . . . . . . . . Effect of EDTA on Can5 distribution in the embryonate egg Effect of sodium citrate and/or EDTA on C‘“5 distribution in the young chick . . . . . . . . . . . . . . . . . . 17 20 22 33 33 3M 3M 36 37 38 51 52 53 5h 55 AM“ 7.. W. ..‘a 5... 19. 20. 21. 22. 23. 2h. 25. 26. 27. Effect of dietary sodium citrate, boric acid.and EDTA on Cau5 distribution in h seek chicks . . . . . . . . . Effect of pantoyl taurine on Ca1+5 distribution in the embryonate egg . . . . . . . . . . . . . . . . . . . . Effect of pantoyl taurine on Cau5 distribution in the embryonate egg . . . . . . . . . . . . . . . . . . . . Effect of desoxypyridoxine on Can5 distribution in the embryonate egg . . . . . . . . . . . . . . . . . Effect of desoxypyridoxine and desthiobiotin on Ca“5 distribution in the embryonate egg . . . . . . . . . . Effect of glucagon on Ca”5 distribution in the embryonate a“ O O O O O ..... O O O O O O 0 e o e O O O O 0 Effect of glucagon on Cau5 distribution in the day-old and 3'day (311161! e e e e e e e e e e e o e o e e s e e Effect of insulin on «“5 . . .. ..... .. . . . . Efffigt of insulin and glucagon on the distribution of in the embryonate egg . . . . . . . . . . . . . . 75 75 76 76 83 8M 8M 35 u- .‘ “5.1.11“. II. III. IV. V. VII. LIST OF FIGURES Yolk weight changes during incubation and after hatching . . . . . . . . . . . . . . . . . Effect of feed consumption on body and yolk.weight . Effect of feed consumption on percent yolk weight . . Correlation between counts on whole ashed femurs and on ground femur ash . . . . . . . . . . . . . . . Correlation between counts on whole ovenpdried femurs and on whole ashed femurs . . . . . . Cau5 content of various tissues in normal and in glucagon treated chicks . . . . . . . . . . . . Time curve of Cau5 deposition into control bone as related to several influencing factors . . . . . Page 10 12 12 26 88 91 I. GENERAL INTRODUCTION AND OBJECTIVES The embryonate egg is commonly used as a tool of research in virology, bacteriology, medicine, physiology and, of course, poultry husbandry. However, the use of radioisotope techniques in the devel- oping chicken embryo is a relatively more recent practice. The em- bryonate egg presents many advantages in tracer studies. lith proper care, the total quantity of an isotope administered to the embryonate egg will be retained. Thus radioactive contamination of the surround. ings is minimized and total recovery can be expected. Further, the density of the eggshell absorbs all alpha and many of the beta parti- cles. Only gamma radiations pass through readily. The chick embryo is relatively resistant to radiation damage and is not adversely affected by relatively large tracer doses. This is in spite of the fact that chick embryonic tissues have a high mitotic rate which is inhibited by radiation. Several factors contribute to the resistance exhibited by the chicken embryo (Dixon, 1952). The sterile environment of the embryo reduces subsequent infections which are a usual complication of radiation damage. The ready supply of fluid and food in the egg may well minimize dehydration and malnutri- tion mhich commonly follows post-radiation, gastro-intestinal damage in most animals. Dixon found that when radiation was given at a slow rate the embryo was able to tolerate very large doses. With an intra-yolk injection of 120 uc Phosphorus—32 at N days incubation age, a 15 per- cent hatch was obtained; these chicks were estimated to have received Hee' 2 “000 roentgen equivalents during the incubation period. A 30 percent hatch resulted from developing eggs receiving 2U00 roentgen equiva— lents and with 900 roentgen equivalents, a 50 percent hatch resulted. No deformities or malformations were produced which were attributable to the radiation. Karnofsky and coworkers (1950) reported that young embryos are very resistant to the acute lethal effects of thadiation. The LDSO of 2-day embryos at the rate of M3 roentgens per minute was greater than 2000 roentgens; for 5 day embryos the LD50 was 1500 roentgens. In the 8-18 day period, the LDBO varied between 750 and 900 roentgens. The 8-10 day period is the most sensitive. It is of interest to compare these figures with the roentgen equivalents in the experiments to be reported.here, and with the energies of the beta particles from,Calcium-u5. Phosphorus-32 emits a single beta particle with an effective energy of 0.68 Mev (Hand- book 52), whereas Calcium-#5 emits a single beta particle with.an effective energy of 0.085 Mev, only one-eighth.as great. The quan- tity of radioactive calcium used per egg in these trials was 8.0 uc. injected at either M or 1“ days incubation. Therefore, the eggs in- jected in this series of experiments were exposed to an average total dose somewhere between 13 and 3M roentgen equivalents ignoring the concentration of the internal emitter in the skeleton (Phosphorus—52 also localizes in the skeleton, however). In general, then, the total radiation dose given-in these experiments does not exceed five percent of the LD50 for single dose irradiation given at the most sensitive stage of embryonic development. The skeleton deserves further mention since it represents a rather special case, both from the standpoint of radiation sensitiv. ity and since it is the system with which the experimental work of this thesis is primarily concerned. In general, differentiated skeletal tissue is relatively resist- ant to radiation since considerably greater than whole lethal doses are required to produce demonstrable pathology (Hicks, 1953). It is primarily before the 8th day of incubation that radiation induces skeletal abnormalities. The important ossification centers at that time are those of the axial skeleton, with the long bones of the limbs starting to calcify at about the 8th day. After this time, although the bulk of the embryonic bone remains to be deposited, skeletal ab- normalities become increasingly rare. It is evident that the mmst radio-sensitive function during skeletal growth is the initial induc- tion of the skeletal anlage and that the calcification process itself, the laydown and turnover of matrix and the proliferation of epiphyseal plates with its accompanying mitotic activity are not particularly radio-sensitive despite the active metabolism associated with these processes. The initiation of the cartilagenous anlage in which ossification centers will subsequently develop is essentially completed by the 10th day of incubation. After that time growth occurs and differentiation precedes in an orderly fashion, first the cephalic end of the axial skeleton, then the vertebrae and finally the appendicular skeleton. This sequence has been described in detail by Lillie, (1952). The u femur, with which we shall be chiefly concerned, begins to ossify at about the 8th day of incubation, between the 10th to the 15th day heavy ossification begins, and thereafter, ossification continues until relatively late in life. Thus treatments previous to 7 days incubation will assure an effect on initial calcium deposition as oo- sification begins at about the 8th day of incubation. In view of this well worked out developmental pattern of bone formation, it can be seen that a great variety of experimental treat- ments are possible and that studies of bone metabolism in the chick embryo, can be of fundamental importance. Specific metabolites or antimetabolites can be utilized to alter or enhance the preformed nu. tritive supply of the embryo. The specific analogue of those vitamins which have been reported in the literature to be involved in bone de- velopment, can be administered to produce deficiency conditions (Cravens, 1952) and thereby directly or indirectly influence the nor- mal course of calcium metabolism. In addition, the use of antimetab- elites may enable one to ascertain the function of certain nutritive factors in embryonic development which would be difficult to determine by manipulation of the maternal diet. In the case of many B vitamins, exposing the dam to deficient rations will reduce egg production, as well as give considerable difficulty in designing adequate test rations. It is much more desirable to study develOpmental difficulties in the embryo directly, rather than to work indirectly through its parent. The transfer of calcium from the egg-shell to the skeleton of the develOping embryo can be either interfered with or increased by ’As’th _ 5 the use of appropriate chemical compounds. Certain complexing agents with an affinity for calcium will markedly alter bone developnent and calcium laydown (Landauer, 1952). Since many of these chelating agents are of current interest in the attempted removal of radioele— ments from the body (Foreman, 1953; Cohn _e_t_ 514 1953). these compounds can also be studied to advantage in the chick embryo. At various stages of development, hormones are elaborated which profoundly influence the pattern of bone mineralization (Landauer, 19147). Hormones may affect bone formation by several routes. In- directly, hormonal control of bone development is presumably mediated through control of vitamins, minerals, energy supplies and their necessary enzyme systems. In view of the very obvious points touched on above, the experi- mental work presented in this thesis was planned as a preliminary ex.- ploration to find those combinations of isotope techniques and experi. mental organs which might most productively be followed up to gain an understanding of those abnormalities in development which are the cause of reproductive wastage in the chicken. Depending on strain and con- ditions of management, from 10 to 1+0 percent of eggs set fail to re. salt in viable day-old. chicks. In cases where the primary cause is genetic, it is still of importance to try to understand how the genes do their damage. Probably of equal importance is the fact that after hatching a considerable amount of growth potential is lost to the poultry industry due to the incidence of leg disorders in poultry. Among these are "leg weakness" and swollen hock conditions in turkeys, and certain hock disorders such as perosis and a "crooked toe” condition in chickens. These leg disorders have been investigated by many workers and have been related to vitamin deficiencies, mineral inhalances and several other nutritional and physiologic disturbances, in addition to genetic influences and management practices. If these conditions can be re- produced in part in the developing embryo, there is no reason why the basic abnormal physiology of growing bones cannot be investigated as well before hatching as after hatching. This study, therefore, was proposed to investigate several of these factors, and supply a logical step towards solving problems of such vital concern to the poultry industry. II. DELINEATION OF THE PROBLEM In the laydown of bone, three components are indispensable. These are: first, an adequate supply of calcium and phosphorus which are the chief constituents of the mineral phase; second, an adequate organic ground substance or matrix containing collagen and chondroitin sulfate and possibly also citrate; and third, an energy supplyh-usually gly- cogen-—which can be utilized by the osteoblasts in the rapid metabolic turnover of the ground substances. Somehow, the general processes in skeletal development concerned with these biochemical systems are us- ually synchronized in time with the growth of the embryo as a whole in such a way that breakdown of a given phase of skeletal development is symptomatic of a more general breakdown in the over-all economy of the embryo. Thus, skeletal development is one indication of "growth”. In fact "skeletal age" is recognized as one of the more exact ways of designating develOpmental age--considerably better than over-all weight or length of the embryo. Since there are three processes in skeletal development, this thesis was planned to study each one in the same specific way and with the same common end point. Interference with the supply of calcium was attempted by the introduction of the complexing agents boric acid, sodium citrate and ethylene diamine tetra acetic acid. Interference with the metabolism of ground substances was attempted by introduction of the anti-vitamins pantoyl taurine, desoxypyridoxine and desthiobiotin which are known to affect the metabolism of cartilage and other connective tissures. Interference with the energy supply 8 was attempted by introducing the hormones glucagon and insulin known to be essential in the metabolism of carbohydrate and fat. In every case the effect of the treatment on radioactive calcium retention by the skeleton and certain selected tissues of 19 to 2k day surviviors was the end point observed. In brief, the procedure was to inject GauS and one of the chemical agents into a fertilized egg, wait until hatching and observe the effect of the agent on the tissue distribution of the isot0pe, paying particular attention to the femur. Although skeletal tissue is obviously of major importance in the calcium.metabolism.of the embryo, it soon became evident that the digestive tract, and particularly its primitive but persistent pre- cursor, the yolk sac were also significant. It must be borne in mind that the embryonate eggis a closed sys- tem, and once introduced, no Gau5 leaves. lxcreta.from the develop- ing embryo is deposited in the allantoic fluid, from which certain constituents can then be reabsorbed. This is probably true for most minerals but for calcium it is particularly important. Thus calcium is repeatedly available, either for incorporation into the skeletal system or for storage in the yolk sac. However, exchange of tracer calcimm with bone is rapid and equilibrium is probably established very soon after injection. The initial storage of Ca1+5 in the yolk sac and its long retention therein is of considerable importance. Apparently the yolk sac has ‘multiple functions. Early in incubation, the yolk is.a source of 9 critical nutrients to the embryo. Amino acids, vitamins and certain minerals are given up and water is taken in, thus conserving water which.might otherwise be lost (Romanoff, l9U3). Its content of potas- sium, sodium.and.chlorine increases early in incubation and decreases in the latter stages. It will be shown later in this thesis that the major elements calcium, phosphorus and iodine are stored in the yolk in the later stages of incubation to be utilised soon after hatching. The yolk contains 99.5 percent of the lipids of the egg, only one-third of which is converted into energy before hatching, chiefly in the latter stages of incubation. According to Romanoff (1932), the remaining two-thirds are utilised after hatching as a readily available source of energy for the chick while adjusting to its new environment. The very high metabolic rate of the chick requires a great deal of available energy during this critical period, which it cannot yet obtain from.the feed. It is obvious that calcium binding by the yolk may be associated with its lipid or phospholipid content and that calcium release may be a secondary Consequence of lipid ca— tabolism. According to Romanoff (19“3). the yolk begins to lose weight after the eighth day of incubation, and by the 21st day approximately 50 percent of the yolk is utilized. However, the most rapid absorption takes place in the early stages after hatching (Figure 1). Approximately 70 percent of the yolk material remaining at hatch- ing is absorbed by the third day, but the yolk alone can maintain the chick for up to six days. Although the chick loses 25 percent of its weight in this period, it will be shown that bone weight significantly 10 H oonlnom ”ho a.Um¢.F(Z( .MVO _ nquZ<0 Z. w0< ZO_Fmm _ ._ .2662, E; III udoz10 duozSzom 9:925 52, .OZ_IU._.> «30> .H m:. SWVH‘D NI .LHQIBM M‘IOA 11 increases during this period. Thus yolk utilization is associated with continuing skeletal growth even in the face of a loss in body weight. In an experiment on these points chicks were held without food or water to the third day after hatching and to the sixth day with only water, and the effect on yolk utilization investigated. It is clearly shown in Figures II and III that food consumption stimulates the absorption of the yolk sac, or conversely that starvation depresses it. Although the physiologic mechanism is obscure, this finding emph- asizes the practical importance of starting baby chicks on feed as soon as possible. The yolk may be absorbed by the chick through two pathways. First, and probably the most important, is the vascular route, and second is the direct connection by my of the yolk stalk into the lower digestive tract. Food passage through the digestive tract ap- parently stimulates the passage of yolk material through the duct into the lower intestine. At least this would account for the differences shown. Grossly, no marked differences in the vascular supply of the yolk sac could be found and it is assumed that the vascular uptake of yolk material remains constant, regardless of the fact that the chick is under considerable stress due to deprivation of food. In the absence of direct evidence however, this explanation must remain purely speculative. The percent of dry matter in the total yolk remains fairly con- stant at 50 percent throughout the entire incubation period and at least to the third day after hatching. mta were not collected on r... agenda-....“ . _\ YOLK PERCENT OF BODY WEIGHT Fig.3: EFFECT OF FEED CONSUMPTION ON BODY AND YOLK WEIGHT. 60 40 soov WEIGHT ‘ - ~-~ FULL FEED AND WATER \ DEPRIVED or FEED AND WATER—...... '0 _ YOLKK e WEIGHT\ \ I \ I 20 IN GRAMS WEIGHT k N PERCE Io ._ YOLK ...g..§ WEIGHT\\\ \ ‘b——-- —A# —-——————--4 -— —— — 7—— __._______.+____,ii.____c-- \ IO 20 I 2 3 4 5 AGIE. IN DAYS FigJII EFFECT OF FEED CONSUMPTION ON PERCENT YOLK WEIGHT. 13 yolk dry matter after the third day. The physiology of absorption of the yolk is important in a study of calcium distribution as a consequence of two observations. First, a very large fraction of radioactive calcium injected either into the extra-embryonic body cavity or into the allantois finds its way to the yolk sac where much of it remains until very late in development, or indeed until after hatching. Second, after hatching, a marked shift in the Cans content of many tissues occurs. This is presumably related both to incorporation of yolk sac calcium.into the chick skeleton and to the now possible loss of calcium by defecation and urinary excretion. Excretion by the embryo and by the baby chick may play an impor— tant role in the calcium.economy. Recirculation of the isotope from the embryonic excreta has been mentioned. Before hatching, excreta calcium must be considered as highly available, as only slight amounts are ultimately lost in the feces or urine. The excreta in the period just prior to hatching contains very high tracer activity, and this activity decreases exponentially after hatching as the chick defecates and voids urine to the outside. After hatching, recirculation of calciumtbetween the bowel and body fluids contines to take place. This occurs in part through biliary excretion into the intestine. Although this section has presented several original data and Inns referred to findings chich will be documented later in.this thesis, it is essential to present them at this time to give the breadth of ‘perspective essential to a proper understanding of the over-all 1h biological situation. It is evident that in addition to the calcium pools ordinarily found in growing or adult birds, the avian embryo has at least one more in the yolk sac, another in the allantois and possibly others not yet defined in the extra embryonic body cavity or even, in the young embryo, in the albumin of the egg white. The egg ehell, as will be shown subsequently, does not accept Gau5 tracer, although non-radioactive calcium is absorbed from it during develop- ment. The immediate objective of this thesis, then is to determine whether treatment of the chick embryo with certain agents suspected to be of importance in skeletal growth will alter the distribution of c‘“5 between the various pools into which it would normally pass. Although we are interested primarily in the skeletal pools, it is well to remember that sufficient alterations in the physiology of other pools may well change the availability to the skeleton of the finite amount of calcium available. In.general. it may be remembered that if all the calcium in the egg were available to the embryo, no skeleton.abnormalities should exist. The question is, what limits calcium.availability to the bone? Is it a matter of competing metabolic systems? of calcium ”binding”? And what are the natures of the various defects in the metabolism of the developing bone itself which permit its successful maturation? It is in the light of these questions that the following experiments were begun. As mentioned, three classes of compounds were injected into de- Veloping eggs. Ghelating and complexing agents, vitamin inhibitory 15 analogues and hormones. Since the physiology of each is different, the thesis will be divided into three corresponding sections. Each will be preceded by a survey of the literature, general procedures and followed by a brief discussion, sumary and conclusions. 111. GENERAL PROCEDURE The eggs used for this study were from Single Comb White Leghorn pallets fed a complete all-mash ration, the composition of which is shown in Table I. They were collected daily, identified with the dam.number and the date, and incubated for various intervals. At 'FH suitable periods, the eggs were candled.and the most vigorous embryos selected. These were distributed as uniformly as possible by dam.and date into the experimental groups. Injections into eggs incubated less than one week were made into the extra.embryonic-body-cavity (EEBG). After one week of incubation, ‘ injections were made into the allantoic sac (AS). The eggs were first swabbed on the large end with alcohol, and marked for the injection site. Holes (1/32 inch) were then drilled through the egg shell, care being taken not to break.either the inner or outer shell membranes. The area around the drilled holes was then swabbed with mataphin. The air—cell membrane was punctured with a sterilized teasing needle to accomodate pressure changes due to injected volumes. Radioactive decay of the stock solution of Cans was calculated before each injection. An appropriate dilution was then made with distilled water so that 0.1 cc. solution contained 8.0 no high specific activity Clan5 (20+uc/mg Ga). Usual isotope handling tech- niques and safety precautions for soft beta radiations were observed at all times. In general, dosages of compounds selected for experimental work were calculated, from the literature, to be toxic to approximately 17 Table 1 Composition of all-mash layer-breeder ration. Ingredient Ihunds .__,a ,. ...v. a. rm. Ground yellow corn 363 Yellow hominy feed 50 Ground wheat 150 Pulverized oats 125 Wheat standard middlings 25 1 Wheat bran 25 ‘ Soybean oil meal 100 Alfalfa meal, 20% protein 25 Fish meal, 60 1 protein 25 Meat scrap, 50% protein 25 Dried skimmilk 25 Dried whey 25 Dicalcium phosphate 12.5 Ground limestone 12.5 Salt (iodissd) 5 Manganese sulfate, feeding grads 0.25 Antibiotic, 312 supplement 0-5 Vitamin mixturez .o.5 Vitamin 0.25 Vitamin D 0.6 1000.10 1Merck Propen ”2:3” 2Lederls 2—h90 3N'opcay "10', 10,000 A/gm. t‘Nopdex ”1500” 1500 D3/gm. 18 50 percent of the embryos, assuring that any effect of the compound on bone formation would probably be maximal. The compounds were weigh. ed on a quantitative balance, and dissolved in distilled water so that each treatment was delivered in 0.05 cc volumes. In the case of concurrent treatments, both substances were delivered in the same volume, but at no time was the isotope solution and the specific treatment combined in one injection. Two injections at the same thus were made by reinserting the needle into the same hole. If the pro- cedure required injections at several intervals during incubation, a new hole was drilled for each injection. The same air-cell hole was used on all occasions. The holes were then sealed with paraffin, and the eggs replaced in the incubator. Just prior to hatching, all eggs were placed in pedigreed hatching baskets lined with cheese cloth. This prevented any possible contamination of the incubator with radioactive excreta, down, shells etc. Aseptic conditions were maintained as nearly as possible. lhen tissues were taken from three- day chicks, the chicks remained in the lined.hatching baskets and re- ceived no feed or water. All tissues were harvested into tared crucibles. Each sample was weighed wet, dried for a minimum of 2M hours at 100°C and ashed at 600°C in a muffle furnace. Oven dried and ashed weights were re- corded from which water and ash percentages were calculated. After aching, each sample was pressed, ground.and spread evenly in the bot- tom of the cgflnible to insure a uniform counting geometry for all samples. Each sample was then counted on a standard sealer and all 19 calculations were made on an ash basis. The counting time was deter— mined by the activity of the sample, but was always sufficiently long to result in less than 5 percent error in the final activity. Abnormalities or teratological development, which might well be expected from toxic dose levels, were not looked for. Due to heavy mortality (see section V), the numbers of treated embryos harvested was always small. In general, the treated survivors did not exhibit significant differences from the controls in such characters as bone or body weight. The treated embryos which died.however, may have been considerably abnormal. The lhite Leghorns used were of the Creighton Strain of Leghorns and comprised birds of one pen on an all—mash, high-energy experiment being conducted at the Michigan State University Phultry Plant. Eggs were incubated in the Pbultry Building in an isolated Model 252 single stage Jamesway incubator with a 2520 egg capacity. The incubation temperature was 99.50! and remained the same for the hatch- ing period. Humidity was maintained at 86° and increased to 88° to 90° three days prior to hatching. Only experimental eggs were incubated and hatched in the same incubator, and a.maximum of 200 eggs in an ex- periment would be used. IV. EVALUATION OF SPECIFIC PROCEDURES A. Site g£_Injection Recovery of activity varies with the site of injection. Cal"5 injected into the extra-embryonic body-cavity is about twice as likely to end up in the femur as Can5 injected into the yolk sac. This is shown in Table 2 in which a comparison of injection sites was made. i Table 2 L Tissue uptake of semi in day-old chicks in relation to site of injection. 8.0 no Cau5 per egg at H days incubation. - '~ Site of injection Tissue Yelk sac EEBC cps/10 mg ash Femur 13.6 25.} Blood 1.“ 2.“ Muscle 8.5 1.u Yolk sac 551.9 216.8 Intestine 1.1 2.3 Cal"5 injected into the EEBC must pass through the various fluid compartments of the embryo before being deposited in the yolk sac. As previously mentioned, the yolk sac possesses the ability to im» mobilize and store large amounts of calcium as well as other minerals. Therefore, any Can5 injected into the yolk sac will be less likely to reach skeletal tissue than if injected into the EEBC. 21 B. Age at Injection_and Age at Harvest Recovery of Calcium-#5 quite naturally might be expected to differ if injected at different incubation ages or if the tissues are har. vested at different ages. The incubation age at the time of injection could be controlled to within a few hours. On several occasions eggs were injected after different incubation periods to determine the effect on recovery of tracer from tissues harvested at the same age. Table 3 Effect of age at injection on Cau5 uptake by 21 day bone. 8.0 uc/egg. Incubation age cps/10 mg. injected Treatment ash h 2.5 mg. Na citrate 33.6 15 2.5 mg. Na citrate 32.9 15 5.0 mg. Na citrate 33.0 N None 26.8 11 None 26.3 n 0.5 mg. desoxypyridoxine 2M.8 11 0.5 mg. desoxypyridoxine 28.1 Table 3 shows that between R and 15 days, the age at which the eggs were injected had relatively little effect on the final re- covery from the femur. This is undoubtedly the result of recircula- tion of the isotope in the closed system of the embryonate egg. The physiologic age of the embryos or chicks at the time of harvest was more difficult to control There are many factors which 22 speed up or delay hatching age in the chicks-individual (gentic) varia- tion, temperature, humidity and season in addition to the specific effects of the experimental treatments. Each might be expected to affect the specific tissue distribution of the total available calcium. It was therefore desirable that eggs injected at the same age be har- vested at different periods, particularly just before and after hatch- ing. Table h Effect of age at harvest on Cans uptake by bone. 8.0 uc/egg at M days. Age at harvest Treatment cps/10 mg. ash 21 day embryo (pip) None 16. 22 day embryo (day—old chick) None 21. 3 day chick None 2%.? 22 day embryo (day-old chick) None 23.9 3 day chick, None 51.3 day chick None 38. week chick None 0.2 It is evident from Table u that the age of the embryo or chick at the time tissues are taken will have a profoundly greater influence on the final results than will the age of the embryo at the time of injection, provided sufficient time has been allowed for the various calcium pools to come into equilibrium with the tracer. Several fac- tors govern the activity recovered from tissues close to or after hatching. Among them might be: (1) specific effects of the compound used, (2) actual size of the yolk sac reservoir, (3) concentration of __ _ _ . 23 isotope activity remaining in the yolk sac, (n) time period after hatching during which the chick was free to defecate, (5) quantity of feces and urine voided and (6) the retention of the isotope by the increasing soft tissue mass after hatching. It will be noted that recoveries are expressed per 10 mg femur ash. Since the femur calcium concentration does not vary appreciably in the times and under the control conditions tabulated, the figures are directly proportional to specific activities and.in most instances can be used as such. Thus the actual increment of calcium in the chick femur between 5 days and h weeks can be directly calculated, since the reduction in specific activity is due only to dilution. C. Eggrshel;_8tugy It is essential to determine whether or not calcium is exchanged between the egg shell and the developing embryo. If there is a con- siderable turnover between the embryo and the egg shell, an additional calcium pool would have to be considered. In addition, the possibility of contamination of the incubator during the hatching period would arise. Therefore, sections of egg-shell (0.5 cm?) were removed from the same developing egg which had received 8 no Can5 at u days incuba- tion at various intervals and these shell sections were counted both from the inside and the outside. Table 5 gives the results of this trial. These sections were counted at a geometry which was nine times as efficient as that for all other counts in this series of experiments. Since these counts are much lower than backgrounds it can be seen that Table 5 “5 Ca recovery from shells of a developing egg. 8.0 uc/egg. cps/0.5 cm? egg shell. 2“ Incubation date Outside Inside 8 days 0.00 0.11 13 days 0.10 0.10 18 days 0.09 0.16 19 days 0.00 0.03 negligible amounts of Ca)"5 were deposited in the egg shell and this minimal activity could have no influence whatsoever on the final results. Egg shell calcium therefore, is involved in a one-way transfer from the egg shell to the embryonic system. Transfer in the reverse direction apparently does not occur. 35 D. Whole gag-ground 43.}; Correlation To assure uniform geometry in counting, samples were pressed, ground and distributed evenly in the bottom of the crucibles. It was found that after grinding bone samples. a much higher count was ob- tained. However, a precise correlation was found to exist between counts on whole (unground) ashed femurs and on ground femur ash (Fig. IV). fine increased activity is due to a greater surface area, 20 rxy= .sss 25 y= 355+.st y (cps PER l0 ".9 WHOLE AsHED FEMUR) IO 20 3O 4O 50 60 70 l l I l l l l , ab (cps PER lOm GROUND FEMUR AsH) F191! CORRELATION BETWEE COUNTS 0N WHOLE ASHED FEMURS AND ON GROUND FEMUR ASH. e.o_uc C145 PER EGG AT 4 one INCUBATION. reduced particle size and decreased sel f—absorpt ion. 26 E. Oven-dry Whole Ash Correlation~ On several occasions the same femur samples were counted before and after ashing. A very close correlation was found to exist between these counts as shown in Figure V. Variations are undoubtedly due to differences in self—absorption in the tissures, and the expected 5.0 percent counting errer resulting from the counting technique. go . ' Pa}: .906 Q = -o.I2+I.Is «- D s 35 Ill 0 IL 0 '1‘. a: 0 3° I O 2 Is. > 0 e a, :5 £- " ' O " ee I: , ' ‘i’ .'.° . . (I) ll. 0. 3 '- >315 5 I0 lb 20 as so as I I I I I I I ¢(CPS PER IO ms WHOLE AeHEo FEMUR) Fig.1 CORRELATION BETWEEN COUNTS ON WHOLE OVEN- DRIED FEMURS AND ON WHOLE ASHED FEMURS. e.o_uc ca45 PER ecc AT 4 one INCUBATION. 27 In view of the excellent correlations demonstrated in this and in the previous section, the time required to obtain femur data can be greatly reduced by counting the oven dry femurs directly without “5 ashing or grinding. Injection of 8.0 uc Ca per egg normally gives sufficient activity in a single embryonic femur such that long count- ing times are avoided. Considering normal variation between individual embryos, the slightly greater accuracy given by ashed.and ground samples is rarely required. Appropriate linear correction factors are still required for absolute comparisons, however. V. GENERAL EFFECTS OF THE EXPERIMENTAL TREATMENTS A. Embryonic Mortality The embryonic mortality which occurred in all experiments is shown in Tables 6 and 7. The data presented in Table 6 are from those ex- periments reported in this thesis. The data presented in Table 7 are from trials not otherwise reported for reasons to be discussed later. As can be seen in Table 6. eight microcuries of Calcium-i6 did not produce a marked mortality among the control groups of embryos as 71 percent of all the inJected controls survived. A considerable number of the embryos which did die, died within #8 hours of the in- Jection, indicating that the immediate effects of injection, and not radiation was the major cause of embryonic mortality in the control groups. In comparing this data with that of Dixon, (1952) mentioned earl- ier, the embryos in these experiments were calculated to have received not more than 9.8 roentgen equivalents if injected at 16 days incuba— tion, and 33.3 roentgen equivalents if injected at 4 days incubation. The observed mortality agrees fairly well with.a semi-logarithemic ex- trapolation of Dixons' data if allowance is made for the absence of plain water injected controls in the present series. It is clear for the dosage calculation and from.the early mortality, when such occur- red, that skeletal development could not have been affected by radia- tion in these experiments. “(AIL-1. a 29 .suasusu u emu .somsodam u osao .saaowpoanpeoe “upon .osauocanhmhuomoe u eon .oqaasea Amoumsm u an .uwoe owpeos saves eawasuu omoammao u «BAH .eaoe cannao u.asm vouoodw~ hawdeaaoa nuobwbndm dopoowwm veneerudn cocoons“ Inseam acoonom aceonem owd owe Hensoawemmmw Hoaasoo souosnsosu IIIIIIIIIMIIIII.” I a... «escapees» aeaaoaaneaxe ea use headaches oasohu93fi a sense 3o oceanom masoao n ma .saaaema H «mm .uowsosam "Honda .mauompoflnpeou a non .oqwuouwnhAhKomou u some mu cod 0 mm cod 0 on II :a mu euH case mm m mm om H on I- an saga mm ~ mm om H OH II :H omma 00H 0 mm 00H 0 ca II ma w m mom son OCH 0 mm 00H 0 Mg II AH w : season headaches encambnam eoaoonma muwaeouoa euobarnam deacon!" .cooeeraem doaoofisu Inseam «soouom ameoaom ew4 ems .H! mewpmnsbmn Heumoanuemwfi Acheson ii I! III wwe mom mzeo on w .mofiamaoe oaopoeu on one auuueonoa cwsohnpafl N canes 31 Table 7 shows the results of several trials which produced exces- sively high mortality. These trials all received Ca1+5 from the remain. der of a stock solution which had decayed to the extent that little or no dilution was required.to deliver eight microcuries per egg. Injec- tion of this solution definitely increased mortality in both the con- trol and experimental groups, possibly because of the larger amounts of nonpactive calcium chloride in the undiluted stock solution, or because of its low pH. It is also possible that after this time, bac- terial contamination of the stock solution may have occurred.a1though the pH was in the neighborhood of 2.5. It must be kept in mind that the mortality in these control and experimental groups is not representative of normal mortality in an average setting of eggs. An excess of eggs was always incubated, and on the day of injection all eggs set were candied. All infertile eggs and dead or weak embryos were removed and.only the most vigorous embryos were selected for the experiments. Very likely such selected embryos would all have resulted in.healthy chicks after the usual in- cubation period. Therefore, the observed mortality should be attrib— uted entirely to the injections. Consequently death must have resulted either from the injection procedure (though no bacterial contamination was ever observed) or from.the materials injected. Radiation effects having been reasonably eliminated, there remains the small amount of calcium chloride in very weak acid solution (except in the set of dis- carded data), the distilled water diluent and the experimental mater- ials. Obviously, the experimental materials were injected in toxic 32 amounts. No controls were run to check the calcium chloride, the acidp ity or the distilled water. B. Femur Heights The femur weights, moisture and ash contents presented in Tables 8, 9 and 10 include the bone data from all the experiments presented with one exception. In this case, the first glucngon trial, the bones were inadvertently allowed to dry out somewhat before the first weighing, resulting in inaccurate data for wet weights, moisture and ash content. The dissection procedure was as follows. As soon as possible after drawing the blood sample, one leg was removed from the embryo, the flesh stripped from the femur as cleanly as possible, the tendons cut and the bone gently wiped with cheese cloth. The femur was then weighed, dried and ashed according to the described procedure, 1. Complexing agents In general, the wet weight of the femurs is reduced by about 10 percent following treatment with boric acid or EDTA but is not affected by sodium citrate. The water and ash content of the femurs is not af- fected by the complexing agents (Table 8). Thus in the survivors of a rather toxic dose of these agents, femur development proceeds in.a surprisingly normal manner insofar as total weight, water and ash content are concerned. aw. -... . i" “a... 33 , -l'u” ' {IE :t'i' "'t". ' ‘t "E'- '1'..- 'En’.’ ' 's .I' ..l.’ 'b‘.’ ’ .7 ".’:':-' o.aa o.~m we H mood as pose an «ens ma.o m.~a m.~m ma H :mm me pose an «o ma.o a.aa ~.Ha 0.0m o.~m me H ones a» n coma me pose an 4m an.o e m.m m.o~ m m aha mm seam ms mm.a ones»? oz ms mm; 9w 9w QB 93 a H :3 m H n: mm auntie ez ms mam m me To T2 TE m n m? .1." men am «So me mum : Tm To w.$ 92 a. H Ra m n 2 cm 33:0 oz ms m.m m 92 13 TR mm: o a ”ma m a ma ones»? we as m.m m 92 3: TS 1.3 D H m? m a mom am So» 3.8: we mm; a .aaaxm aouasoo .Hpnxm Honasoo amusoaaaoanm Honpnoo chagao mwe sea ucoapocua undue linen agonMI. Ilvooos umeonom IgImdmuflowIMflJ-Wg mo 03. amonsoo nus one 0.33.38 3:32. gm not so Snows M55338 mo poomum w e anus 3% m ens m oases» as some» usage». I!’ (I: ”| (I?) I” II (III ’I' )r t I!" 'iIIII'Y'Eé..- m.oa e.aa ~.n~ m.m~ :a a one u n one am sausage page m.o IQOMGOHHHM M5. m M o.aa m.a~ e H sow am assumes "page m m.o~ a.m~ Hm.“ om am genomes moans m m.oa :.m m.o~ ~.ma w e am ma m new Hm .eaaemae sees a m m.m o.m o.me m.mo V on n awn ma n ems em «cocoons me oh a.m e.m :.m~ m.:~ e H mma m H one am someones me an a IiwflnwmllflIApQHfi HOHvQOO Illddiflinwflflflwtonmkfi Hahunoo ohhpEWIIIIIIIWIMO HOMIvdoEOGNMIB Adakvar- Mmmmmymmmwm "uweammzwnmmwom “mm“ aAMwos mason no: no 0M4 if, 3", ' ' " 'I” .0; ..- ’ "r IIUWIIIIINHI; III IE»... I. nooosoo men one ousamwoa .eanwaos none“ as: so unwound use sowsosaw no mooowmm on canes ~.N m.m~ m n_ema ma espoapoaanmoe we oo.m m; e; NA; 0.3 s n o? m n e2 2 oqnsoeetaewaoe ea 8.0 a m.~ o.w m.o~ m.o~ m H man me u we” Hm oaauaeasaaauomoe ma cm.o m s.» o.m m.aa m.o~ m H man an H and aw oneness ascends ma mm.o m w.oa m.oa e.a~ m.a~ an a mma o u mma am ceases» fineness ma oo.a H H,.auaxm «anymoo .auanm Hmmmwoo «mucosaaoaxfi aospnoo oxygen mmo sea unoaeeosa Henna nus ucooaom noawaymmwonom “um“ omummw uses“ pom 'II " l ’r' I? "' I’IV'I' E 'D , ’ ' I, 'P )l'IP"' "'Il’ P"':'-§-:"-:E:'Ei assumes men one easpedoa .onwaos meson new no eosMerse anoaapamsw magnum» mo «vacuum m oases 35 2. Vitamin antagonists The data presented in Table 9 show that the vitamin deficient conditions, as produced by the specific inhibitory analogues for that vitamin have no effect on either the total size of the embryonic femur, or its water or ash content. Again, if the embryo survives at all, the weight and general composition of its femur is surprisingly normal. 3. Hormones In all cases, the glucagon and insulin were injected into the de- veloping egg at 1“ days incubation. Although the hormones were admin— istered after bone devlopment was well under way, sizable differences resulted from insulin treatment as shown in Table 10. Bone weight and water content was reduced. Ash content was increased. Glucagon was variable in effect on these end points. h, Discussion The general effects just described are summarized in Table 11. Boric acid and insulin, which have been studied in considerable detail by Landauer (1952, 1952a), produced their anticipated effects. In addition, 2.5 mg EDTA significantly reduced femur weight although the amount of chelating agent is able to bind only about 0.5 mg of calcium (Merck Index, 1952). A single femur at 21 days incubation contains about 5 mg calcium. It is tempting to calculate that a 10 percent reduction in femur size implies about 0.5 mg less calcium in ”I A T? “‘i‘ 36 that single bone. It is absurd to assume, however, that the entire effect of the EDTA injection is exerted on only one of the bones of the entire skeleton. What this finding really implies is that the supply of calcium which is actually available for skeleton development at any one time is limited indeed. Despite the large total calcium supply in the egg, it is apparently doled out to the embryo by a very slow process. The type of metabolism associated with calcium release and transport to the embryo apparently conserves calcium for the im- mediate post-hatching period when the total demand, before the feed- ing mechanism is adequate to take over, is considerably greater. This again suggests a possible role for the unabsorbed yolk. A more com» plete picture will emerge when the data for the radioactive tracer calcium.are presented. Table 11 Summary of effects of treatments on embryonic femurs remur Percent Percent Treatment wet weight water ash Beric acid reduced no effect no effect no citrate variable no effect no effect EDTA. reduced no effect no effect Pantoyl taurine no effect no effect no effect Desoxypyridoxine no effect no effect no effect Desthiobiotin no effect no effect no effect Glucagon variable no effect no effect Insulin reduced reduced increased —_ i ‘ - — v ‘ j——‘ The values tabulated above are absolute values not adjusted for differences in size of the entire embryo. Whole embryo weights were not obtained in this study. Consequently, it is impossible to say 37 whether or not the embryonic skeleton was specifically affected by the treatments or whether the results were simply associated with altered whole embryo weights. In the case of trial 6, Table 8, complexing agents were added to the ration after hatching. Table 12 illustrates that between hatching and 4 weeks of age such dietary additions reduced femur weights more than they reduced body weights. Consequently, it may be assumed that the complexing agents, at least, have specific effects on skeletal 1 growth over and above their effects on general body growth. Table 12 Effects of complexing agents on body weight, total bone size and relative bene size. v——-“vw‘— "fl wr '— - "W-”W- “fl—“w ' w w w—w—w ' -‘u— Bedy Femur mg femur per Group Addition to diet weight weight 100 gm body wt. gm m8 1 None 178 1660 2 0.1% Na citrate 165 1030 623 3 0.1% Boric acid 158 951+ 599 u 0.1% EDTA 157 1005 5‘” From the foregoing data, a summary of control femur weights at the ages studied may be constructed. This appears in Table 13. In this comparison, it is especially evident that groups of normal con- trol embryos of given chronolegic age vary considerably in femur characteristics just as they do in other characters. Highly signifi- cant differences between control groups was frequently found. Table 13 Control femur weights at different ages *— w “V V w“ w w v“ "ifi Wm Compl exi ng Vi tamin Age agents analogues Hormones an an an 19 day 105 i 5 136 I 5 20 day 131+ .t 3 21 day 152 i 3 15M 2 11 151 t 5 15331; 158:: b 2m115 176 I 15 280 i 8 23 day 173 t 3 2 day 18b it 12 49 day 1160 1 7 --mmw — w v ‘ “m It will be recalled that eggs were selected for these experiments so as to obtain groups of equivalent composition, at least to the de- gree of securing groupings from a single or from a restricted number of dams. Since these dams did not always produce sufficient eggs at the right time, it was necessary then to include more than one group. Apparently the genetic differences between these groups was considerable. Actually it is amazing that the femur weights of 21 day embryos, for example, were not statistically different in 5 or 8 groups tabu- lated. This degree of homogeniety could only be achieved by control of the parent stock. In two trials in which the embryos were treated with hormones, tibiae rather than femurs were taken, which accounts for the large al-day bones. Althoughfemur size differed, as indicated, the percent water and Percent ash at different ages was much more uniform, as might be expect ed. 59 Returning to the summary of Table ll, perhaps the most significant finding of this preliminary compilation is not that a certain few dif- ferences due to treatment were observed. Rather, it is remarkable that more frequent and larger differences were not induced. Generally speaking, the treatments did not greatly influence the femur of the chick embryo in the ways studied. Consequently, with the exceptions noted, it will be possible to describe the radioactive tracer data as though the over-all femur characteristics were the same in both control and experimental groups. VI. COMPLEXING AND CHELATING AGENTS A. Survey g_f_ the Literature l. Boric acid Boron is essential for normal growth in higher plants and occurs extensively in animal tissues, but a requirement for boron in animals has not been established. Theresi (191W) prepared a ration low in boron and although the ration was not adequately consumed, the results suggest that 0.6 gamma of boron daily would probably satisfy the normal growth requirement of the rat. Boric acid has been used therapeutically in a variety of manners. If used in minor concentrations borates are considered as beneficial, but continuous usage, or following boric acid intoxication, an accumu- lation of boron occurs in the central nervous system and in the liver (Pheiffer 235.2}:- l9li5). Mulinos (19511) reports that there has been ample clinical proof that boric acid poisoning is not rare. It is more than coincidental that boron accumulation is at the site of Principal pathological changes described in riboflavin deficiency by 33881 91 a_l_., (1910). Since the discovery by Beeseken (19149) that boric acid forms complexes with various polyhydoxy compounds, Frost (1942) reported that such complex formation occurs in the presence of fiborlavin, the reaction involving the hydroxyl groups of the ribityl “€19 Chain of riboflavin. However, the boron-riboflavin complex was shown to have full biological activity in microbiological assays and in growth studies in rats and dogs. 0n the other hand, Roush and 1 a A 1 ‘ t o .a jle-. 1+1 Norris (1950) found that borates inhibit the riboflavin containing xanthine oxidase, presumably by interaction with the ribityl group of riboflavin. Kuhn (19N3) has reported the inactivation of other bio- lOgically important reactions. Very few reports could be found in the literature connecting boric acid to bone formation. The fact that it is a potent teratogenic GONb pound, has been investigated extensively by Landauer (1952) and is the chief reason for using it here. It was found.that the injection of 2.5 milligrams of boric acid into the yolk sac of each embryonate egg at 8M and 96 hours of incubation led.to malformations of the face and extremities. The most common facial abnormality was a combination of shortened lower beak, cleft palate and coloboma of the face. The skeleton of the wings was generally normal. In the legs, the femur and tibia were generally normal except that the tibia was occasionally bent. The tarsometatarsus was frequently very short and bent at a right angle, resulting in a club footed condition. The feet were often poorly'developed. Most comonly, the fourth toe was shortened or en- tirely lacking; the first too was next frequently affected; in extreme cases, all toes were missing. Shortening of the toes was often asso- ciated with syndactylism. Average embryonic body weight was below normal, the more serious the malformations generally being associated with greater reductions in weight. Curled toe paralysis was found in many of those chicks which hatched subsequent to boric acid treatment at any stage during the first four days of incubation, but in no case was clubbed down observed. The livers of boric acid treated and he morphologically abnormal embryos were deficient in riboflavin. (Engel gt_§l,,(l9NO) also reported that in riboflavin deficient embryos, the livers were deficient in riboflavin). Supplementation of boric acid treatment at 2“ hours with sodium pyruvate resulted in significant lowering of certain abnormalities. Supplementary nicotinamide at 96 hours significantly decreased the incidence of facial defects, lowered to a doubtful extent the appendicular malformations, but raised the incidence of curled toe paralysis. Riboflavin dissolved in boric acid greatly reduced the teratogenic properties of boric acid, pre- sumably due to a sparing action. Continuing this work, Landauer (1952a) found that water soluble boron compounds, other than borates share the teratogenic qualities of boric acid, and concluded that this effect is general of boron and not specific for boric acid. It was found that complex formation with polyhydroxy compounds (D.ribose, pyridoxine hydrochloride, D-sorbitol hydrate) reduces or abolishes the teratogenic qualities of boric acid. Landauer theorized that boric acid interferes with normal development by complex formation in ovo with polyhydroxy compounds, thereby pro- ducing symptoms resembling riboflavin deficiency. Additional evidence suggesting that boric acid acted on coenzymes rather than enzymes was presented. Riboflavin is not the only vitamin which is complexed by boric acid. In his review on boric acid, Zittle (1951) revealed that vitap Min 312, pantothenic acid, pyridoxine and possibly inositol will react With boric acid, although the vitamin-boric acid complex may not alter 143 the biological value of the vitamin. The interference of boric acid with so many biologically important (polyhydroxy) compounds may account for the fact that riboflavin would not completely correct the appen- dicular deformities produced by Landauer (log. git.). Further, that the boron-vitamin complex does not always totally inactivate the vita- min may account for the failure of boric acid to produce the clubbed down which is typical of a natural riboflavin deficiency. Boric acid ”complexes” with calcium by forming calcium borate salts, and it would be logical to assume, that the formation of calcium borates do not decrease the biolOgical activity or availability of such calcium. 2. Citric acid and sodium citrate The involvement of citric acid in bone metabolism was first shown by Dickens (l9ul), who reported that over 90 percent of the citric acid in the body was contained in the skeletal tissue. Approximately one percent of the bone was accounted for by citrates. Under suitable lg_zi§gg.conditions, a precipitate similar to that found in bone was formed by combinations of calcium, phosphate and citric acid (Kuyper, 1938). While it has been established that citric acid participates actively in bone formation, Gomori and Gulyas (lguh) produced evidence of decalcification of bone in dogs, and increased urinary calcium fol- lowing injections of citrate. Considering the above reported functions of citric acid, it naturally follows that this compound occupies some vital role in the bk pathology of rickets. Lecoq (1949), observed a distinct recalcifica- tion in young rachitic rats after treatment with citric acid. The im- provements in bone lesions took place in alkalotic but not acidotic rickets. Citric acid was shown to improve rickets under certain con- ditions (Shohl, 1937; Day, l9h0) which conditions apparently were de- termined by the calcium: phosphorus ratio. It has been shown that the serum citrate of rachitic infants is reduced, and when large doses of vitamin D are administered the serum citrate increases markedly (Nutrition Reviews, 1952). Greenwald (192b) found that diffusible calcium in the fluids of the body is partly ionized, and partly bound to some citrate-like com- pound. In l93h, Hastings gt_al., investigating the state of calcium in the body fluids, described the nature of the calcium-citrate com» plex. The probable formula is Ca3Cit2, with a primary and secondary dissociation. However, not more than five percent of sermm calcium is normally associated with citrate. During an investigation concerning the role of thiamin in citric acid metabolism, the excretion of citric acid in the urine was shown to decrease during a thiamin deficiency (Sober g£_§l,, 19h0) but this has been shown to be due to a reduced food intake. Kuyper and Mattill (1933) found that urinary excretion of citric acid invariably rose after a meal regardless of the dietary, however high levels of carbo- hydrates favored citric acid excretion, presumably as a result of stepped up carbohydrate metabolism. High levels of starch promoted the highest rate of excretion of citric acid, sucrose was intermediate 145 and the glucose effect was practically negligible. Unfortunately, (malcium excretion was not investigated. The high rate of absorption of citric acid was demonstrated by Sherman at E. (1936). Less than two percent of orally administered citric acid was recovered in the urine. The majority of the citric acid.was probably converted into glycogen by the liver or rapidly oxidized. Being an integral functioning component of intermediary carbo~ hydrate metabolism, citric acid is universally distributed throughout the body (Thunberg, 1953). It is logical to assume that the production and.distribution of citric acid in the body is controlled by certain enzyme systems. Principally this is regulated by the pantothenic acid containing Coenzyme A in a condensation reaction involving activated acetate and oxalacetate (Williams, 1950). In an attempt to evaluate the roles of certain enzymes in the citric acid cycle on the availa- bility of citrate in bone and mechanisms of calcification, Dixon and Perkins (1952) found that the addition of 0.0005 molar sodium citrate markedly inhibited calcification of rachitic rat bone slices ig_zltgg, This excess of citrate limited the availability of calcium for bone formation by binding a portion of the ionized calcium. Relative ac. tivities of specific enzymes involved in the citric acid cycle found in bone favor the synthesis of citric acid, as compared to degradation and removal (Nutrition Reviews, 1953). The greatest quantities of the enzymes responsible for formation of citric acid, citrogenase and M6 aconitase, were found in.areas where calcification was preceeding ac. tively. These conditions would seem to be those which would permit the accumulation of citrate in local areas of new formation. It might be reasonably concluded that the increased concentrations of citrate made available at the site of new bone formation facilitates the depo- sition of calcium. 0n the other hand, citric acid may, by binding calcium, limit its availability for deposition in bone, or, if present in excess, may act to mobilize calcium, and by this means, cause dis- solution of bone. Perkins and Dixon (1953) reported that in parathyroidectomized rats, the bonecesses. 'hether or not the metabolic failure in the embryo is due only to the blocking action of the inhibitor is not known. Umbreit and Waddell (1919) suggest simultaneous phosphorylation of desoxy- JPJVIfiidoxine and pyridoxal, both of which compete for combination with 301118 newly formed apoenzyme with which this coenzyme functions. If the apoenzyme becomes saturated with pyridoxal phosphate, the desoxy- Pyridoxine phosphate, even if formed could not then. exert its inhibi- tory action. It may also be that the quantity of the apoenzyme has greatl.y increased and more of the inhibitor would be required for com- ‘birultion.with it to block the reaction which the enzyme catalyzes. In a recent review on vitamins in metabolic functions, Snell (1955) lemmas.» b8 emphasized the importance of pyridoxine in protein metabolism. In this respect, pyridoxine participates in several major enzyme systems. Among these are the amino acid decarboxylases, transaminases, dehydra- ses and desulfhydrases in addition to several enzymes specific for individual amino acids. It is understandable therefore that protein in excess will aggravate a pyridoxine deficiency. Gheldelin and Williams (191+2) reported that the pyridoxine con. tent of the egg is approximately 0.22 micrograms per gram of egg. 2. Pantothenic Acid and Pantoyl taurine In the first study relating pantothenic acid to hatchability of 0838. Lepkovsky gt a}: (1938) was unable to demonstrate that this '1 tanin was essential for the normal hatchability of fertile eggs, al- though a relationship was established between the level of pantothenic aCid in the breeder ration and in the egg. However, in 1939, Bauern. feind and Norris did show that pantothenic acid was necessary for hatchability using a heat-treated diet. This treatment, however, may have produced a multiple vitamin deficiency through destruction °f riboflavin and biotin in addition to pantothenic acid. Gillie 9t '11- (19143) found that the quantity of pantothenic acid in the chick is dependent on the amount in the egg, and the chicks which did hatch frOm eggs laid by hens deficient in pantothenic acid were inferior in quality, exhibiting general debility, muscular incoordination and 3'0 11 en ho cks. Approximately 800 micrograms of pantothenic acid per 100 grams 69 of diet was required for good hatchability of eggs and the survival and rapid growth of the chicks (Gillie gt‘ a_l_., 19147). Embryonic mor- tality due to pantothenic acid deficiency was confined almost entirely to the last two or three days of incubation, and consequently did not result in characteristic embryonic deformities. The production of Perceis in growing chicks on pantothenic acid low and deficient diets 'as described by Libby (1952). The incidence of perosis was shown to be determined by the level of pantothenic acid in the diet. Panto- thenic acid was further shown to be influential in growing bone by Patrick and Schweitzer (1952). The addition of 10 ppm pantothenic acid increased the bone mineralization of young growing chicks on a Practical type diet. The method of measurement has been discussed (Pyridoxine section). The combination of pantothenic acid with other B vitamins also increased the mineralization of bone when fed a sim- Plified semipurified type diet. There is no difference in the pantothenic acid content of eggs and the pantothenic acid content of newly hatched chicks according to Pmarten _e_t_ 31.. (19%). It is apparent therefore, that there is no Significant change in the pantothenic acid content of eggs during the incIllnetion period. The necessity of pantothenic acid for embryonic deVGIOPment, and the fact that it does not decrease during incubation Sugs’a‘ésts that pantothenic acid enters into an enzyme system essential for embryonic development. The occurrence of 'bound' pantothenic acid in coenzyme A has been shown by Lipman gt 3;]... (1945. 1945. 1950)- 70 An increase in embryo survival from eggs with an increased panto- thenic acid level was demonstrated by Taylor gt 31, (1941). Increased Pantothenic acid content of eggs, either by injection or through a supplemented maternal diet was associated with an increased hemoglobin concentration of the blood in the twelve day chick embryo. From this work it appears that during early embryological development, the chick embryo is highly responsive to vitamin imbalance induced by moderate increases of pantothenic acid. Riggs (1948) reported that rats on a pantothenic acid free diet Show a decreased ability to acetylate para-amino benzoic acid as deter- mined by the levels of acetyl compound in the urine after a test dose 0f the free acid. Snell and Quarles (191a) reported that the average freshly laid egg contains 11 to 11+ micrograms of pantothenic acid per gram of egg. The first growth inhibition specifically and competitively re- Versed by pantothenic acid was reported by Snell (1941) who prepared and tested pantoyl taurine as an inhibitory analogue of pantothenic acid for lactic acid bacteria. Brackett (l9’+'o) produced death in chicks when a pantoyl tauramid was included in the diet at 0.5 percent, but at 0.1 percent only slightly reduced weight gains resulted, and 1“ 0.025 percent level did not effect the chicks. The toxicity of even 0.5 percent of the analogue in the diet was prevented by supple— r"“5-’311‘l‘da.i:.ion with 0.025 percent of calcium D—pantothenate. Snell e_t_ a}; (1943) reported that a pantothenic acid deficiency could be produced in mi (:9 by long-continued feeding of pantoyl taurine. but this could 71 not be substantiated. No reference could be found in which typical pantothenic acid deficiency symptoms were reported in the chick by the use of pantoyl taurine. From his experiments, Mcllwain (1916) concluded that pantoyl taurine acts as a bacteriostatic agent by preventing the conversion of Putothenic acid to a functional derivative in susceptible bacteria. 3. Biotin and Desthiobiotin The importance of biotin for the hatchability of fertile chicken eggs was first demonstrated by Cravens 23:. 11: (1942), who reported that a maternal deficiency of biotin in the chicken results in a rapid decline in hatchability. The effect of the biotin deficiency on em- bryonic development was further reported by Cravens gt a_1_. (191th). The production of biotin deficient eggs and subsequent deficient em... bryos resulted in a marked increase in embryonic mortality on about the third day of development. Another mortality peak occurred during the last three days of incubation, and the greater the degree of bio— tin deficiency, the greater will be the early mortality peak. The “1081: critical need for biotin is during early development, and adequate in; actions of biotin after 120 hours of incubation are of little bone. fit to the deficient embryo. Injections prior to incubation will cor- rect the deficiency however. In this work, skeletal abnormalities were described due to the biotin deficiency. Many embryos exhibited a Parrot-beak condition associated with severely crooked tibiae and much shortened and twisted tarsometatarsi. syndactyly or webbing 72 between the third and fourth toes was also observed. Couch and Cravens (19“?) observed perosis in the embryo and in the day old chick due to maternal biotin deficiency. The legs were extended to the rear, the hooks were stiff and the chicks were unable to draw the legs up under the body to assume a standing position. These symptoms were not cor- rected by oral or injected biotin. A very severe perosis was also observed by Couch 22.2l: (19u9) in chicks from eggs which had been injected with ten gamma of biotin at 96 hours of incubation, It was suggested that this vitamin there- fore, in required for some reaction which becomes critical with regard to gastrocnemius metabolism at about 96 hours of incubation. Biotin is generally accepted as an integral member of the anti- perosis complex. Jukes and Bird (1942) revealed that biotin would prevent perosis under certain conditions, and.McElroy and Jukes (19N0) reported perosis to accompany the egg white injury syndrome in chicks, which suggests that this symptom is the result of induced biotin de— ficiency. In the work of Patrick g£_§l, (1943), biotin was found to be an anti-perosis factor and was highly successful in preventing perosis when in combinations with choline and brewers' yeast. The production and subsequent protection against perosis was shown to be determined by the level of biotin in the diet (Libby and Groschke, 1951) The injection of 25 or 100 micrograms of biotin per egg at the start of the incubation period did not offset the skeletal abnormal— ities produced by five units of insulin injected into the 120 hour embryo (Couch gt El: 19u9, see hormone section). 73 The yolk of the freshly laid chicken egg is reported to contain 0.3? micrograms of biotin per gram (Snell gt.alfi,1940) and the albumin contains 0.05+ micrograms per gram (Gyorgy and Rose. 19M2). ‘An early observation by Sumnerson _e_t__ a}, (19414) that biotin is concerned in.the oxidation of pyruvate to lactate has been further localized by Lardy gt 5;, (19M?) to a function in carbon dioxide fix» ation by which pyruvate is converted to oxalacetate. Lichstein and Umbreit (19“?) have demonstrated the need for biotin in the decarboxyl- ation of oxalacetate, and Ochoa gt_a;, (19h?) have found that the liver enzyme causing oxalacetate decarhoxylation is decreased in biotin deficient turkeys. Lichstein.and Umbreit (l9u7a) have suggested a second role for biotin involving the enzyme system concerned with de- amination of certain amino acids, including aspartic acid. Desthiobiotin was found to possess growth promoting activities comparable to biotin for Saccharomyces cerevisiae by Melville gt_al, (19u3), but this same chemical was found to competitively prevent the utilization of biotin by Lactobacillus casei by Dittmer 22.2l: (l9uh). and for several other bacteria by as many authors. Desthiobiotin is also reported to have an inhibitory effect on the growth of certain types of tumors which normally contain very high amounts of biotin (Keresztesy gt_al,, lghb). The many interesting papers which deal with the anti-biotin ac- tivity of avidin, and its role in the egg white injury syndrome which eventually was instrumental in the identification of biotin will not be reviewed. B. Procedure--Vitamin Analogues Four experiments were conducted with the analogues of the three vitamins, pantothenic acid, pyridoxine and biotin. In all experiments, each egg received 8.0 uc CauS. In the first two experiments, pantoyl taurine was injected after 4 days incubation and the tissues harvested after 22'days incubation (day-old chicks). In the first experiment, 1.0 mg pantoyl taurine was injected into each egg and in the second experiment, 0.25 mg pantoyl taurine per egg was used. In the third experiment two settings of eggs were incubated, and an injection of 0.5 mg desoxypyridoxine per egg was administered to one setting at h days incubation, and the same quantity administered to the other setting at 11 days incubation. Tissues were harvested from dayaold chicks. In the fourth experiment, 0.5 mg desoxypyridoxine was injected into one experimental group, and H.O ugm desthiobiotin were injected into the other group, both injections given at 1b days incubation. Tissues were harvested from 19-day embryos. C. liesults Results obtained from specific vitamin antagonists were not large and generally lack statistical significance. Experiment 1. The injection of 0.25 mg of pantoyl taurine at h days incubation produced no statistically significant effects (Table 200, although the mean recovery from treated bone was unusually high. 75 Table 20 Effect of pantoyl taurine on Cau5 distribution in the embryonate egg 0.25 mg per egg1 Tissue Control fantoyl taurine Bone “9.82: 7.0 58.1%1 3.03 Blood 2.5 t 0.5 2.7 t 0.1 Flesh 0.7 t 0.1 0.7 t 0.01 Yolk uh.0 i 7.0 31.7 t in.a Excreta° 13.2 3h.8 l. Injected at M days, harvested at 22 days incubation. Experiment 2. Four times the injected quantity of pantoyl taurine used in experiment 1 did not appreciably alter the earlier results. Again, the bone (“Jan5 was unusually high in those embryos treated with pantoyl taurine. Table 21 shows that the injection of 1.0 mg pantoyl taurine at M days increased the Cal“5 content of femur by 10 percent (compared to a 14 percent increase in experiment 1) and blood values were correspondingly high. Table 21 Effect of pantoyl taurine on Cau5 distribution in the embryonate egg 1.0 mg per eggl Tissue control Pantoyl taurine Bone uh.921 15.03 53.531 19.63 Blood 1.1 i 0.03 1.2 i 0.02 Flesh 0.3 r 0.01 0.“ t 0.01 Yolk 7.u.t 2.9 7.3 t 1.5 l. Injected at U days, harvested at 22 days incubation. 76 Experiment 3. It can be seen in Table 22 that no differences were produced by treatment of u day embryos with 0.5 mg desoxypyridoxine. Table 22 Effect of desoxypyridoxine on Cau5 distribution in the embryonate egg 0-5 me per essl Tissue Control Desoxypyridoxine Bone 26.523: 1.53 26.13: 2.83 Blood 1.} i 0.1 1.2 i 0.1 Flesh 1.0 i 0.2 1.0 .t 0.2 Yolk 9.9 t 2.8 9.2 :t 3.6 Excretac #57 115 1. Injected at h days, harvested at 22 days incubation. Experiment N. The eggs of this experiment were injected at 16 days of incubation and tissues harvested from 19 day embryos (Table 23). The difficulties in obtaining blood samples from embryos of this age accounts for the absence of blood samples in this experiment. Table 23 Effect of desoxypyridoxine and desthiobiotin on Cau5 distribution in the embryonate egg 0-5 me per 0531 5 uem per eesl Tissue Control Desoxypyridoxine Desthiobiotin Bone 26.12: 7.23 27.12: 3.53 22.l+a.t 3.03 Flesh 2.1 :t 0.3 1.6 i 0.14 2.2 1 0A Yolk 7.1 i 2.3 10.8 it 14.0 6.7 It 1.3 l. Injected at 16 days, harvested at 19 days incubation. No significant differences due to desoxypyridoxine or desthio- biotin were observed. 77 0. Discussion Patrick and Schweitzer (1952) cite data (without standard errors) which indicate that in chicks on a multiple deficiency, calcium panto- thenate, and pyridoxine return tibial Phosphorus-32 retention from subnormal to normal values. Under the conditions of this present study, it is impossible to confirm the general conclusions that "Calcium pantothenate, niacin, para-amino benzoic acid, pyridoxine, vitamin 312 and folic acid play a role in bone mineralization”, as far as pantothenic acid and pyridoxine are concerned. Since the experiments here were designed to test the rather broad generalization of Patrick and Schweitzer in a different situation and with different methods, it is important to determine whether such differences contribute to the divergent results. First, with respect to the data of Patrick and Schweitzer, it may be noted that (a) no statistical evaluation of the results is possible and (b) their data are expressed simply as percent of dose recovered from both tibiae. No data on either body weight, tibia weight or tibia ash is presented, although they must undoubtedly have gathered this data. Consequently, the precise effect of their treatment can neither be determined nor statistically evaluated and one is left with a claim based on interesting but isolated data. Their claim, however, is that they observed an effect which one might very well expect simply from what is known about the growth promoting effects of these vitamins. If the chicks grOW, they should accumulate more phosphorus in their bones and less should be found in their excreta. 78 Second, with respect to the chick embryo data presented herein, it may be noted that (a) the small numbers employed increased standard errors, possibly to the point of obscuring real differences, (b) ex— tensive loss via total excreta was impossible since recoveries were measured in day-old chicks, (c) Cans contributed by the yolk sac toward the end of the incubation period may obscure the picture, and (d) the vitamin analogues may not be acting only through mechanisms involving production of a true vitamin deficiency. It is obvious then, that although the bone data presented, as far as they go, do not qualitatively confirm the report of the Tennes- see investigators, it is impossible to draw rigorous conclusions from them. It is fruitless to indulge in speculation when what is required is more extensive data. The problems raised by data of both types are of such major gen— eral importance that it is to be regretted that simple solutions are apparently impossible. A rigorous study on these points alone should be undertaken both in the embryo and in the growing chick. VIII. THE EFFECT OF HORMONES A. Survey o_f_ the I.iter_a;t51_r_e l. Glucagon Although a hyperglycemic factor was postulated not much later than the discovery of insulin, the hyperglycemic factor of the pancreas 'has until recently received but scant attention. As the name indicates, it is a principle which raises the blood sugar levels. It is appar- ently produced in the alpha cells of the islets of Langerhans, al- though the possibility that glucagon or materials with similar prop- erties may also be produced in other cells resembling alpha cells (most of which occur in the gastric and duodenal mucosa) cannot be omitted.(Pincus and Rutman, 1953). Kimball and Murlin (1923) reported fihat acetone precipitation of their pancreatic extract yielded a sub- stance that caused a marked hyperglycemia in dogs and rabbits. This substance was called glucagon, but it is also called by several other names (H. G. factor, H. G. F., hyperglycemic-glycogenolytic factor). Cori (1952) and Sutherland (1952) using the liver slice technique found that glucagon stimulates the phosphoylase system in liver slices by converting phosphorylase to the active form of the enzyme. Under certain conditions glucagon may prevent the fall in blood sugar which normally follows administration of a small amount of insulin (Pincus and Rutman, 1953). In many of their actions on carbohydrate metabolism, glucagon and 80 epinephrine are similar. Both cause hyperglycemia by activating liver phosphorylase. Kirtley g£.al, (1953) interpreted the degree of rise they observed in blood glucose after glucagon administration as being related to the amount of liver glycogen immediately available. Gluca— gon is antagonistic to the hypoglycemic action of insulin, but this is not a true antagonsim as the two substances have different sites and modes of action (DeDuve, 1953). The effect of glucagon on growth has not been reported. However, Elrick (1953) has shown that administration of glucagon will increase the width of the epiphyseal cartilage of the hypophysectomized rat. It would appear therefore, that glucagon plays at least a secondary role in osteogenisis. Its action also appears to be involved in some manner with the somatotrophic (growth) hormone of the anterior lobe of the pituitary. Since its chief known effects are on carbohydrate metabolism (breakdown of glycogen through increased phosphorylase activity), Elricks observations emphasize again the role of carbohydrate metabolism in bone growth. 2. Insulin Insulin has been shown to have a profound influence on bone de. velopment. While insulin in some manner must play a role in normal calcification of bone, the literature reports only adverse effects re- sulting from the administration of insulin. Abnormalities of the beak, eyes and extremities occur during the embryonic development of the chick if five units of insulin is injected into the incubating egg at 81 120 hours of incubation (Landauer, 19M7). In an attempt to relate this phenomenon to the congenital abnormalities produced by a maternal bio- tin deficiency (see vitamin section) Couch et_al, (l9h9) produced skeletal deformities, syndactyly, buphthalmia, notched eyelids, perosis and abnormalities of the beak in the developing embryo by the injec— tion of five units of insulin at 120 hours of incubation. The skeletal r“‘ deformities produced are as follows: (1) The femur is shortened, - thickened and bent near the proximal end. (2) The tibiotarsus is shortened and thickened and the distal end is bent posteriorly. (3) L The tarsometatarsus is shortened. (h) The humerus, radius and ulna are decreased in length. (5) The distal end of the humerus is bent posteriorly. (b) The radius and ulna are bent and curved outward. However, the blood glucose values of chicks with insulin-induced anomalies did not differ from those of normal day old chicks. By regulating the proportion of carbohydrate oxidized, insulin affects the oxygen consumption and of course the respiratory quotient. Hall (195h) demonstrated that the oxygen consumption of diabetic rab— bit muscle slices was increased by 63 percent by insulin, whereas no significant differences were shown on the oxygen consumption of normal muscle slices treated with insulin. A combination of citrate and in- sulin,however, increased the oxygen consumption of diabetic muscle by 127 percent, indicating a synergistic relationship between these two compounds. It appears then that the carbohydrate metabolism of the developing skeleton needs investigation and that the effects of glucagon and insulin on Ca)"5 uptake might be informative. 82 B - Per 0.....e dszs- fiasasngg Four experiments were conducted using glucagon and insulin. All eggs received 8.0 no Ca“5 A8 at 1% days incubation. In the first experiment.group 1 served as the controls, group 2 received 10 ugm glucagon A5 at 14 days, groups 3 received 20 ugm glu— cagon in 2 injections at 1h and 16 days incubation and group n received 30 ugm glucagon in 3 injections at 1h, lb and 18 days incubation. All tissue samples were harvested at the pipping stage (approximately 21 days incubation). In experiment 2, all eggs were injected at 1“ days with 10 ugm glucagon and the calcium tracer or with.the tracer alone. The chicks were allowed to hatch, and one-half were sacrificed for tissue analy- sis soon after emergence. The remaining half were sacrificed at 3 days of age. Experiment 3 was carried out in the same manner as experiment 1, except that l, 2 and 3 units of insulin replaced the glucagon at the same times and all chicks were allowed to hatch before sampling (22 days incubation). Experiment M involved the concurrent use of glucagon and insulin. At 1“ days incubation, in addition to 8.0 uc Cans, one experimental group received 5 ugm glucagon plus one—half unit insulin. All eggs were allowed to hatch. Tissues were removed and Ca)45 activity after 22 days incubation (day—old chicks) was determined. :m.m-m.u . r - I 53 C. Eesults Experiment 1. As can be seen in Table 2“, 10, 20 or 30 micrograms h of glucagon decreased the uptake of Ca 5 in all tissues but not in the excreta, the smallest differences being usually on the border line of Table an F #5 *- Effect of glucagon on Ca distribution i in the embryonate egg t 1 10 ug/«ags1 20 ug/eggl 3o tug/eggl . Tissue Control glucagon glucagon ‘ glucagon f Bone 160722.: 2003 8 821‘ 101‘} 9052: 0033 11.“?! 2003 L Blood 2.0 t 0.1» 0.3 t 0.2 1.2 “t 0.3 0.7 t 0.1 Flesh 1.1. i 0.3 0.3 i 0.2 0.6 f 0.1 0.3 t 0.1 Yolk 6.1+ :t Lu the i 1.9 3.6 i 1.0 3.9 :r 0.8 Excreta° 272 285 322 275 1. Injected at 1” days, harvested at 21 days incubation. statistical significance. This decrease amounted to 30-47 percent in bone, 36-- 66 percent in blood, H3 - 79 percent in flesh and 25 - N5 percent in the yolk. The differences in excreta values are not con- sidered to be significant. Also, the differences between 10, 20 and 30 units of glucagon are not significant. Experiment 2. The results shown in Table 25 show that in general, it was not possible to prove that the differences produced by glucagon prior to hatching are maintained for three days after hatching. Hows ever, in the bone, the Can5 uptake was apparently decreased by 22 per- cent at the time of hatch, and after three days, the difference due 8% Table 25 Effect of glucagon on Can5 distribution in the embryonate egg and young chick fi V v ‘— —_ 'm" h— ‘— 22 day embryos1 3-day_old chicksl Tissue 10 ugfegg— 10 ug/Fsg Control glucagon Control glucagon Bone 21.53: 6.23 1o.72+ 3. s3 2h.731 5.23 19.62+ 7.13 Blood 1.3 I 0.1 0. 9+ - 0.1 1.3 i 0.3 1.2 3 0.1 p**““ Liver 0.7 I 0.10.6 i 0.1 0.7 I 0.1 0.1; '3: 0.1 I Flesh 2.0 i 0.3 1. 5 i 0.7 i 0.1 0.5 i 0.1 i mm 1auiab u8.uiL3 1&u1a2 mbioe ' Excretac N9 M3 11 3 hi - -_ a“ 1. Injected at 1“ days incubation, harvested as shown. 10:-“‘sio'1ffii'1"" ‘ to glucagon was still 21 percent. Statistically these differences are not significant, but the general trend of results found in experiment 1 still persists. Experiment 3. In this experiment, eggs were treated with l, 2 and 3 units of insulin, However, mortality was excessive in the groups Table 26 Effect of insulin on ea“5 distribution in the embryonate egg ~ 7. “v"_‘ V ' Tissue Control Insulin1 Bone 21.723 1.73 20.5%: 1.53 Blood 0.8 i 0.1 0.9 i 0-1 Liver 0.3 i 0.1 0.2 i 0.03 Flesh 1.0 i 0.2 1.1 i 0.2 Yolk 5.5 t 1.8 4-3 i 0.9 Excretac 17 12 k 1- Injected at 1” days, harvested at 21 days incubation. w.“ .. -..-H's..- --m'- --“ -m—O —. .5".' ~0- recoiving 2 and 3 units of insulin, so the remaining eggs of these two ”f- ‘1 “h,- _ 85 groups were grouped and averaged with the group of eggs which received only one unit of insulin. It was felt justified to do this, as the values were Very similar. The data, presented in Table 2b, show that the injection of insup lin into 1“ day embryonate eggs does not significantly affect the Ca1+5 content of any of the tissues. re Experiment h. The combination of one-half unit of insulin and 5 micrograms of glucagon gave results which were typical of 10 micro- grams of glucagon alone. It can be seen in Table 27 that a reduced g'mn.‘— .- ...“ . Cah5 content was produced in every tissue. The femur content was significantly reduced by “3 percent. The blood, yolk, liver and ex- creta counts were reduced by 37, 27, 5N and M5 percents respectively. Flesh samples also showed reduced activity, although this difference is not significant. Table 27 Effect a; glucagon and insulin concurrently on the Ca distribution in the embryonate egg .W" “-‘------.’---- m--“--«w “— “-— ‘C.5 units insulin- Tissue Control 5 ug glucagon per e Bone 20.02: 2.53 11.13: 1.93 Blood 2.3 i 0.3 1.5 i 0.1 Flesh 1.2 i 0.2 1.1 '1: 0.5 Yolk 6.5 i 1.5 11.7 t 1.0 Excretac 2h 11 Liverc 0.3 002 —— —"'—-‘-.'-‘-‘ -~--~ mmm-m -- -mm--~-- 1. Injected at 1“ days, harvested at 21 days incubation. 1‘5 ......AurlfluHs ... .. J 86 D. Discussion Treatment of in day embryonate eggs with glucagon significantly reduces Cans uptake by the embryonic femurs. It is obvious, therefore, that incorporation of tracer into bone is greatly reduced following a general but temporary mobilization of tissue glycogen. On the other hand, uptake of non-radioactive calcium appears to be unaffected. This result is essentially opposite of that obtained following the ad— ministration of complexing agents. With insulin, no effect is observed, either on Cau5 or on the non—radioactive Cane. When insulin and gluca- gon are given concurrently, only the glucagon effect appears. From.the tracer results, it mightbe inferred that increased glycogenolysis (induced by glucagon) depresses calcification but that increased glycogenesis (induced by insulin) has no effect on the skeleton. However, Gutman and Yu, (1950) have summarized a number of types of evidence which indicates that bone salt cannot be laid down unless glycogenolysis is at least adequate. It would then have to be postulated that glucagon so completely removes glycogen from.the pro-osseous cartilage that it cannot calcify to as great a degree (this concept can also be expressed in more adequate kinetic terms). This is not likely since Caho appears to go in normally and only Ca]+5 is ”excluded" by glucagon. There must be enough glycogen in the pro. PBSeous cartilage to get non-radioactive calcium in. Why then doesn't the tracer calcium enter? The only logical answer is that they must (nuns from two different sources, most of the tracer calcium coming IHTJbably from the yolk with the bulk of the femur calcium coming either 87 fromithe shell or from the albumen, in any case from.a more labile sup- ply. In view of the glycogenolysis induced by glucagon, fat and phos- phoprotein metabolism in the yolk.would probably be spared. Less Ca1+5 should then pass from yolk to embryo, which was the observed finding (note particularly the yolk data 3 days after hatching, Table 25). The principle hypothesis which appears, from the data, to be re— quired then is that glucagon has a different effect on calcium in» mobilized in the yolk than it has on calcium in other pools available to the embryo and that insulin does not have this differential effect. This viewpoint can be very easily tested in a straight forward bio- chemical manner and should lead to a considerably clearer understand- ing of the relative roles of each hormone in normal metabolism. Excretion of radioactive calcium due to glucagon was increased to a slight extent prior to hatching. At this time, the large bowel is ;practically gorged with excreta with a very high radioactivity. After the chick has been allowed to defecate, total excretion of can5 is de- creased in the glucagon treated embryos. This is logical, as less isotope was present in the yolk initially, and therefore less is avail- able to the chick;after hatching. In experiment 3 in which the em- bryos were treated with insulin, less Cah5 was excreted into the gut Prior to hatching. The effect of glucagon on the 08“5 content of ex- creta, yolk and bone is shown in Figure VI. The tracer calcium fixed in bone is increased daily through addi- tdxan of high specific activity calcium from the mineral stores in the 88 400 NORMAL GLUCAGON TREATED __ _ ... E XCRETA coum's PER SECOND PER IO m9 ASH O ' 1| 12 31 AGE IN DAvs E39321 Ca 4” CONTENT or VARIOUS TISSUES IN NORMAL AND IN GLUCAGON TREATED CHICKS. acyc Ca45 AT :4 DAYS. 89 yolk. The bone increases in size by 20 percent or more in the 3 day period despite the fact that the chicks received no feed or water in this period (see Table 11). Thus the total calcium stored in the yolk must have been considerable. The three-day increase in bone size does not represent all the yolk calcium since only approximately 75 percent of the yolk remaining at hatching is absorbed by the third day. Since in experiment 1 all tissues except excreta showed a de- creased Ca“5 uptake due to glucagon, there must have been some un- sampled pool or pools with enough activity to add up to the 100 per- cent introduced. Consequently it was decided to sample the livers in experiments 2 and 3 due to their responsiveness, both in fat and carbohydrate metabolism, to hormone treatments. Since calcium appears in the bile, it is possible that the liver plays an important role in calcium distribution. This supposition was not borne out, however, as recovery of the isotope from liver, muscle and brain was very sim- ilar, and did not appear to be importantly involved in calcium distri- bution. This point is by no means closed, however, since Cans was recovered from bile but the effect of glucagon, if any, was not con- stant. In one case, a small amount of abdominal fat from a glucagon 1”5 treated embryo had a very high Ca activity. Data on brain, bile and fat are not included in the tables. I.(_‘A_-—a_-‘—4_-_. .a- i ...—uh Lu-gt '\ IX. GENERAL DISCUSSION AND SUMMARY In this thesis research, a series of experiments were conducted to determine the effects of several classes of compounds on embryonic and.young chick bone development and mineralization using radioactive calcium as a tracer. The compounds used were selected on the basis of reports in the literature which showed them either to be involved in normal bone development or to be capable of altering skeletal de- velopment. From the data secured (and as much from the data from control groups and from supplemental experiments as from data from !—Y_"'—‘_.W-Trn_L—ZTLTI‘ experimental groups), a new insight into the biology of the chick em. bryo has resulted. This is summarised in Figure VII where the incu- bation period is divided into a number of successive stages as described below. In the construction of Figure VII, twentyhone day embryonic bone activity is taken.as 100 percent, and bone activity at other stages as a percent of the 21 day bone. lhenever possible, data from bone harvested at different intervals fromTthe same experiment were used. In all cases data were taken from eggs injected with 8.0 no Cau5 at u days incubation age. From the resultant plot the following stages in calcification of the femur may be noted. Period A.- Embryonic bone - the femur begins to calcify at about the eighth day from very high specific activity calcium, probably do- rived from the yolk. Days 8-1%. .1. OJ .m>(o. V L.( 860 01.0.0 .mmOFUUW O._. m ....ZOU OFZ. ZOELwOQUO CU ...—O u>m3U NET... a. gn— ombadx m< mmazummmfimz. mo< xoio . . 2... 8:5 2. uo< 05.92”. Lt>_._.o< as so cazua coo IZ 'JNEDUBd OOI=OA88W3 AVG OAHGWB AVG I? :10 lVHJ. JO .LNBOUBd 9V OBSSBHdXB AllMLDV 92 Period B - Developing bone - dilution of the highly labeled calcium takes place in the growing femur. There must then be a persistent pool of calcium which was not labeled by the injection of Gan5 at four days and which is drawn on by the femur during this stage. The egg shell is the obvious guess but the other possibility, the calcium pool in the egg white, has not been carefully studied. Days lu.lS. Period 0 - Beginning of active absorption of calcium (well labeled with Gaufi) from the yolk. It can be calculated.that both the tem- porary slow down in femur growth at this time and.the influx of additional Cans from the yolk:are required to halt the drop in femur specific activity. Days 18-20. Period D - Hatching - possibility of calcium.removal from either shell or albumen to embryo is lost. Chick becomes an open system with excreta no longer available for recirculation unless droppings are eaten. my 21. Period E - Neo-natal - establishment of feeding and beginning of actual use of the bowel as a significant medhanism for absorption. Skele- tal calcium and energy supplied simultaneously by absorption of yolk sac. Bone specific activity rises. Days 1-3. Period F - Normal growth - Dilution of labeled Ca in the skeleton by food calcium and perhaps loss of Cau5 from the bones as they re— . model. The latter would give rise to labeled endogenous fecal cal- cium. m"..— "l' 93 Several things should be said with respect to the absolute values for the Cans activity of various samples removed from embryos in these studies. From the tabulations of Ewing (19W), the total ash (exclud— ing shell) in an egg is about 550 mg of which about 3’40 mg is calcium. Approximately 0.1+ mg of calcium was injected with the “’45. The total activity injected was slightly less than 15,000 cps. Consequent- ly, if the activity were uniformly distributed in the ash, all ash i‘ should count about 270 cps per 10 mg of ash. Only occasionally were ; such high activities recovered (and then only in yolk or excreta sam- 1. ples). This indicates that (a) injected ““5 is not uniformly distri- buted in the embryo and (b) most of the activity recovered must have ;-____ been greatly diluted by non-labeled calcium. The white contains only about 100 mg of ash (approximately 6 mg calcium) which is entirely in- adequate to give a dilution sufficient to account for the low activi. ties per unit ash found in this study. It is necessary then to con- sider that, in all probability, the shell was the major source of the unlabeled diluting calcium. Ewing reports more than 6000 mg of ash in the whole egg, which by difference gives about 5500 mg of ash for the shell alone, of which in turn more than 2000 mg is calcium. Although such arithmetic is inexact, to say the least, the prob- able picture is quite clear and is stated above. Complete analytical data which would allow exact description of the true situation were not obtained. Their importance in the next stage of investigation is obvious, however. In a very recent study, the Oak Ridge group (Johnston, Com, e_t_ El.» in press) have studied normal chicks (no treatments; 0.16 only) 9h injected into the white, not into the E.E.B.C. Their data are not in- consistent with those reported here although differences in mobility due to a different site of injection are striking. Since only parts of their data have been carefully compared with the data summarized above, it is sufficient to state that (a) apparently'no serious die- crepancies exist and (b) there will soon be in the literature a beau. tiful summary of normal chick Ce.“5 recoveries at each day of incubation. f This thesis, however, is devoted primarily to the effects of certain : agents and not to the simple description of the normal day-to-day picture. The two studies are complementary. 2 lith respect to the agents employed in this series of experi- j ments, certain generalizations may be drawn: The Cah5 complexed with boric acid, sodium citrate or EDTA had an increased mobility. This was indicated by more rapid uptake, trans- port and turnover of the tracer. In general all tissues, with the exp ception of the yolk sac contained greater quantities of Cans; the fe- mur content was consistently doubled by these treatments. ‘Yolk de- position of the isotope was prevented to a large extent by injections of these compounds, thereby making more of the tracer available to the skeletal structures. In addition, these treatments caused greater quantities of Guns to be returned to the gut for possible excretion. The Cau5 of embryonic excreta is recirculated, to a greater extent if complexed, making the isotope repeatedly available to the embryo. Inhibitory vitamdn.analogues had little or no effect on embryonic bone uptake of Cans. Pantoyl taurine produced results similar to that 95 of the complexing agents, but to a much less marked degree. Statisti- cally significant differences from control values were never obtained with any of the analogues. The results indicate that the use of the specific vitamin inhibitory analogues probably did not produce defic- iency conditions in the embryonate egg which are observably similar to the deficiency conditions produced by maternal dietary deficiencies of pantothenic acid, pyridoxine or biotin. Glucagon was consistently effective in reducing the Ca.“5 con- tent of embryonic and young chick bones. This probably was not the result of a reduction of glycogen at the site of active calcium de- position. As in the case of the complexing agents, lesser quantities of the isotope were deposited in the yolk sac. Excretion of Can5 may have been enhanced by glucagon injections. Insulin exerted no apparent effects on the Cau5 content of bone. Since ealeimm deposi- tion was normal, additional glycogenesis at the site of mineralisation will not increase deposition of calcium. These findings offer, in addition to their technical value to the Physiologist and embryologist, certain encouragement to the practical Poultry husbandryman. In many ways they confirm the very eommon obser- vation, that if a chick can hatch, it is probably all right, or will be all right as soon as it eats. Although this rough generalization is certainly too broad, the fact remains that in these experiments, many chicks survived very serious challenges to their existence with no apparent change in either their skeletal physiology, or in the mineral metabolism of a number of their tissues. Although the injection 96 of antivitamins killed most of the treated embryos, those that were able to survive were essentially normal, insofar as Can5 uptake was concerned. Perhaps Cau5 uptake is a weak.point upon which to base such conclusions. ane-the-less, looking at the thesis data as a whole, one is impressed by how much truth is contained in the common observation. Ehysiologically, the effect of such findings is to emphasize the importance of compensatory mechanisms in development. There must exist a great reserve capacity which is able to counteract or temporarily nullify serious stress situations. Until the reserve is gone, the whole living system.continuee to operate in fairly good shape, although the compensatory mechanisms can be easily demonstrated, but if the re- serve is depleted, everything else rapidly fails and death ensues. Mortality is then such an early consequence that data on the exact mechanisms of death are difficult to obtain. Quite naturally, the embryonate egg of the chicken has been a- volved with very excellent homeostatic mechanisms, much.as the heart and circulatory system of adult animals has been evolved with a built in ”cardiac reserve“ which is able to compensate for long periods of time without immediate malfunction. 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