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A statistical study of chick bioassays with vitamin D O'Neil, James Burton 1938

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A STATISTICAL STUDY OF CHICK BIOASSAYS WITH VITAMIN D. by James Burton O'Neil Thesis Submitted i n P a r t i a l F u l f i l l m e n t the Requirements f o r the Degree of MASTER OF SCIENCE IN AGRICULTURE I n the Department of P o u l t r y Husbandry The U n i v e r s i t y of B r i t i s h Columbia A p r i l 1938. ACKNOWLEDGEMENT The writer is indebted to Professor E.A. Lloyd and Mr. J, Biely of the Department of Poultry Husbandry for their helpful criticism in the preparation of this report. Thanks, are also due to Dr. A.E. Brandt, Senior Mathematical Statistical Analyst in the Department of Research of the Soil Conservation Service of the United States Department of Agriculture, for his keen interest and help in the stat-i s t i c a l interpretation of the data. The author wishes to express his appreciation to Mr. P.A. Boving of the Depart-ment of Agronomy for his aid in the revision of this report. • -. A STATISTICAL STUDY OF CHICK BIOASSAYS WITH VITAMIN D. TABLE OF CONTENTS I. Introduction - Vitamin D in Poultry Nutrition II. Discovery of Vitamin D 1 III. Vitamin D Content of Fish Oils . . . . . . . . . 4 IV. The Antirachitic Value of Cod Liver O i l . . . . 13 V. The Relation of Cholesterol and Ergosterol VI. Vitamin D Requirements- of Chicks . . . . . . . . . 34 VII. The Calcium-Phosphorous Ratio and i t s Relation L>O Rx c k G *t>s » © » » 9 • • • • .« • » © » • • • • 39 VIII. Etiology of Rickets . . . . . . . . . . . . . . 51 IX. Determination of Vitamin D 58 X. Experimental 1. Material and Methods . . . . . . . . . 64 2. St a t i s t i c a l Material and Methods . . . . 6 8 3. Explanation in Connection with FdC "tiiiQ-1 lDct"t&. « • • • • « • « • • + • » « 82* 4. Discussion of Results . . . . . . . . . 123 •A.-L • ou.ninictx,y • • » • • • « « • • • « > « • « • « » • 12 9 XII. List of References . . . . . . . . . . . . . . . 131 XIII. Appendix A. Weekly Weights of Chicks . . . . . . . . 138 B. Weights of Chicks at Four Weeks v and Their Average Tibiae , Weight Before and After Ashing . . . . . . . . 154 A STATISTICAL STUDY OF CHICK BIOASSAYS  WITH VITAMIN D. Introduction. It has been demonstrated that normal growth and egg production may be obtained with birds under confinement when they are fed a balanced ration. By such is meant one that contains the necessary amounts of carbohydrates, fats, proteins, minerals and vitamins. Among the latter, Vitamin D or the antirachitic vitamin, is the most important one for poultry. This vitamin assists in the assimilation of calcium and phosphorus, as e.g., the development of bone in growing chicks and formation of egg shell in laying birds. Partic-ular attention needs to be given to Vitamin D because i t is completely lacking in ordinary food stuffs. Under natural conditions poultry obtain their Vitamin D when exposed to sunshine while birds kept in confinement can only secure i t through the addition of special supplements to feeds such as fish oils or certain synthetic products. An animal receiving a. diet adequate in other res-pects but deficient In Vitamin D w i l l , upon the depletion of the reserve of this vitamin in the body, not only show infer-ior calcification of bone but w i l l also cease to show any growth whatever. ' In poultry, a lack of Vitamin D in the diet causes rickets or leg weakness in young birds and deformities in the skeletal structure in adult birds. The mineral content (ash) I I . of the ;leg bones of chicks of three to four weeks of age which are fed a diet completely deficient in Vitamin D is about 30-33 percent, while that of chicks which receive op-timum amounts of this vitamin is over 45 percent. In adult birds, a deficiency of-Vitamin D in the ration usually re-sults in faulty egg shells as well as lower egg production and hatchability. Since Vitamin D is absent in natural poultry feeds, symptoms of Vitamin D deficiency are much more prevalent than those of other vitamin deficiencies under conditions of confinement. Experimental work has definitely established the fact that the addition of Vitamin D to such natural poultry feeds as may be otherwise complete w i l l prevent rickets. During recent years attention has been drawn to the possibility of a wider distribution of the Vitamin-D factor in the liver and body fat of fish other than cod. It is now known that many fish o i l s , such as pilchard, men-haden and salmon are a rich source of Vitamin D. Referred to as the fat-soluble vitamin, D is found in greatest conc-entration in the livers and body oils of animals. The purpose of this experiment is to make a stat-i s t i c a l study of the data obtained from-' hi ©assays of Pilchard O i l conducted at the University of British Columbia.*-3— e DISCOVERY OF VITAMIN D The discovery of Vitamin D has been of the greatest importance to the p o u l t r y i n d u s t r y as w e l l as to mankind, be-cause i t has brought about the s o l u t i o n of the century-old pro-blem of the cause and prevention of r i c k e t s * To the E n g l i s h p h y s i o l o g i s t Mellanby belongs the c r e d i t of the discovery of the s p e c i f i c a n t i r a c h i t i c f a c t o r or v i t a m i n . His r e p o r t s , pub-l i s h e d i n 1918 and 1919, contain the f i r s t accounts of the undoubted production of true r i c k e t s i n an experimental animal-i n t h i s case the dog-and of i t s cure by d i e t a r y means. On d i e t s c o n s i s t i n g c h i e f l y of c e r e a l and s m a l l quan-t i t i e s of whole or skim m i l k , d i e t s which are now recognized as d e f i c i e n t i n Vitamin D and a l s o i n calcium, there developed i n Mellanby's puppies s o f t bones, bowed legs and other t y p i c a l deformities seen i n r a c h i t i c c h i l d r e n . More d e f i n i t e proof of the presence of true r i c k e t s was obtained from roentgenograms and from chemical a n a l y s i s of the dogs' bones, which were found to have a decidedly low calcium content. When a few cubic centimeters of cod l i v e r o i l was added to the d i e t , r i c k e t s f a i l e d to appear. Comparatively large amounts of b u t t e r f a t and suet prevented i t to some degree, but l a r d , cottonseed, o l i v e and l i n s e e d o i l s proved e n t i r e l y i n e f f e c t i v e . Here was proof p o s i t i v e that the cause of r i c k e t s was to be found i n a d e f i n i t e d e f i c i e n c y i n the d i e t and t h a t the cure l a y I n the a d d i t i o n of c e r t a i n s p e c i f i c foods to the r a t i o n . Since the f a t s that were most potent i n the preven-t i o n of r i c k e t s , cod l i v e r o i l and butter f a t , were a l s o r i c h 2. sources of the already known Vitamin A, Mellanby drew the con-clusion that the antirachitic factor was probably identical with fat-soiubie A. But American investigators were not w i l l -ing to accept this interpretation, and in 1922 McCollum and his co-workers at Johns Hopkins University published definite proof of the existence of two separate and distinct fat-soluble v i t -amins. Taking advantage of the fact, already established by Hopkins in England, that Vitamin A is readily oxidized, McGollum and his associates passed a stream of oxygen through cod liver o i l which was held at the temperature of boiling water for from twelve to fourteen hours. At the end of this time they found that the o i l s t i l l retained i t s antirachitic ' potency when fed to rats but had lost i t s power to cure xerophthalmia5 in other words, i t s t i l l contained the anti-rachitic factor but had lost i t s Vitamin A. This antirachitic factor McCollum named Vitamin D. For a number of years there had existed a more or less vague opinion that the-incidence of rickets was in some way connected with sunshine, fresh a i r and other hygenic factors in child's environment. That ultraviolet radiation consituted a specific cure for rickets, however, was f i r s t de-fi n i t e l y established by Huldschinsky, a German physician work-ing in Berlin during and directly after the World War. For some time scientists were puzzled to reconcile these two discoveries, that of a specific antirachitic factor in the food and that of the antirachitic action of ultraviolet rays. But the riddle was solved independently and almost 3 simultaneously by Hess of New York and by Steenbock of Wiscon-sin, who announced in 1924 that antirachitic potency could be developed In a number of different biologic materials by ex-posure to the rays of a mercury vapour quartz or a carbon arc lamp. Coincident with the discovery by Hess and Steenbock, i t was found that cod liver o i l contained Vitamin D. How two unrelated factors, such as cod liver o i l and ultraviolet light could each prevent or cure rickets remained a mystery for some time. However, i t was eventually found that ultraviolet light acts upon certain substances in the body which become convert-ed into Vitamin D. Extremely potent preparations of Vitamin D have been made in the laboratory by irradiating substances known as cholesterol and ergosterol, with ultraviolet light. Cholesterol and ergosterol are normal constituents of animal and plant cells, and are regarded as percursors of Vitamin D. However, irradiated ergosterol is a very poor source of Vita-min D for poultry. For this reason, fish oils are relied upon in poultry, when sufficient sunlight is not available, as a source of Vitamin D. Man has found that since he has no control over the hours of sunshine, the Vitamin D i n f i s h oils is to his advan-tage - he has free control over enormous quantities of liver i and body oils rich in Vitamin D, 4 VITAMIN D CONTENT OF FISH OILS. B i l l s et a l (16) conducted a taxonomic study of the distribution of vitamins A and D in one hundred species of fish. They found that for a given species, no relationship could be established between the vitamin A and D potencies of the liver or body o i l s . In general, but with many exceptions, liver oils rich in the one vitamin tended to be rich in the other, and the potency tended to vary inversely with the o i l content of the li v e r . The concentration of both vitamins was highest in the Percornorphi (mackerel, tuna, sea bass, sword-fish) and less high in the Cataphractl- (rockfish, sculpin). In the Iieterosomata (halibut and other f l a t fish) vitamin A predominated, and in the Holconoti (viviparous perches), vitamin D. Three quarters of the liver oils tested were more potent than cod liv e r o i l in vitamin D, and nearly a l l were more potent in vitamin A. The liver oils of the gray sole and sturgeon contained no vitamin D, and that of the basking shark no vitamin A and very l i t t l e vitamin Ds The prophylatic method of biological assay for vitamin D on some New Zealand fish oils was reported by Cunningham (33). The vitamin D content of the liver oils of three common New Zealand fish, ling (Genypterus blacodes), groper (Polyprion oxygeneios) and skate (Raiia nasuta), was estimated prophytactically on young rats by measurement of the bone ash. The oils were extracted.with petroleum ether from 5 the fresh-, minced livers which had been allowed to stand, except the ling liver o i l which separated on standing alone. The ether \fas removed by d i s t i l l a t i o n under reduced pressure. One sample of eel o i l and two of whale o i l were also tested. The potency in international units of vitamin D per gram of o i l was: ling liver o i l , 500, groper liver o i l (2 samples) 2,250 and 2,300$ skate liver o i l , 15$ whole fish eel oil,. 47; whale blubber o i l (2 samples), n i l and less than 1. Methods of promoting normal bone development were reported by Gutteridge (.48). Pilchard o i l , cod liver o i l , the radiation from a mercury vapour lamp, and sunlight, eiLther direct or after passage through certain glass substitutes, were found to be efficient in preventing rickets and producing normal bone "development in growing chicks, A special type of socket sunlight lamp was found to be useless for this purpose. Pilchard o i l , which is produced in large quantities on the Pacific coast of America, unlike cod liver o i l , was found to be a poor source of vitamin A. The.comparative antirachitic activity of several fish liver oils as compared to cod liver o i l was shown by Black and bassaman C17) • The vitamin D of blue f i n , yellow f i n , and striped, tuna liver oils was about half as active for chickens as that of cod liver o i l . The vitamin D'.'.of sword fish, halibut, mackerel, and different cod liver oils and of irradiated cholesterol a l l had about the ;-same activity for the chick. The vitamin B of irradiated phytosterol was of similar potency to irradiated ergosterol for chicks. 6 . •, Brocklesby and Large (24) reported preliminary laboratory and clinical, tests on blended fish, oils for medicinal purposes. A low priced, blended, medicinal o i l , of which the vitamin D and A potencies could be controlled, i s described and compared with & medicinal Norwegian cod liver o i l . The blended o i l consisted of.4.parts by volume of pilchard o i l with 1 part of .grey-fish liver o i l . Tables were given showing the chemical, analysis and Lovibond blue value of the three oils and the blended o i l . Tested c l i n i c a l l y , as a prophylactic for school children, the blended o i l and the cod liver o i l gave similar results» Stored in ordinary household conditions, the blended o i l and the cod liver o i l showed the same decrease in blue value after 7 weeks. Comparing the vitamin content :of certain eartilagin-our fishes with a commercial cod liver, o i l , Andre and Lecoq (1) interpreted their results on a percentage basis. The vitamin content of the, liver o i l of 3 cartilaginous fishes was. estimated by the methods of Simonnet for vitamin A and of Randoin and Lecoq for vitamin D. Compared with the value of a. commercial cod liver o i l , vitamin A contents of 90 per cent, 40 per cent, and 30: per cent, and,vitamin D contents of 40 per cent, 35 per cent, and 25 per cent, were found for the l i v e r oils of Centrina vulpecula (Rondelet), Scymnus Lichia (Muller and Eenle), and Raja bat is (Linn.) respectively, Dalmer, Werder and Moll (3.4) compared, biologically, the vitamin D i n f i s h liver oils and irradiated ergosterol. They found that the vitamin D obtained from fis h liver oils 7 C'oodj tunny and h a l i b u t ) by t r e a t i n g the non-saponifiable portions w i t h maleic anhydride had the same b i o l o g i c a l a c t i o n as the v i t a m i n D. i n i r r a d i a t e d e r g o s t e r o l . The a n t i r a c h i t i c potency was measured as the smallest dose r e q u i r e d to prevent r i c k e t s i n rats,•and. the t o x i c i t y as the minimal l e t h a l dose f o r mice. Observations were made by Eussehl and Ackersoh (80) on the e f f i c i e n c y of sardine o i l i n preventing r i c k e t s i n growing c h i c k s , both by observation, of growth curves, and by bone ash determinations. The b a s a l r a t i o n was c. omplete i n a l l e s s e n t i a l s except v i t a m i n D, which was s u p p l i e d by cod l i v e r o i l , by i r r a d i a t i o n with u l t r a v i o l e t l i g h t or d i r e c t sunlight,, or by sardine o i l . I t was found that 0.5 per cent of sardine o i l i n the d i e t provided adequate p r o t e c t i o n against r i c k e t s . I n close agreement with Muss.ehl and Ackerson were B i e l y and Palmer (12). They demonstrated t h a t the a n t i r a c h i t i c value of commercial p i l c h a r d o i l was equal t o , i f not b e t t e r than, that, of good medicinal cod l i v e r 611. The o i l was given at l e v e l s of 0.5 and 1.0 per cent of the d i e t and ash'analyses were c a r r i e d out on the t i b i a and f i b u l a of the c h i c k s . No b i o l o g i c a l t e s t s of the vitamin A value of the p i l c h a r d oil.were c a r r i e d out, but c o l o r i m e t r i c examinations showed 0.6, 2.1 and 2.4 blue units, per cent i n 10 per cent s o l u t i o n s of three samples of the: o i l , i . e . values consider-ably lower than that of cod l i v e r used. Conducting an experiment on f i s h o i l s as a source of v i t a m i n D f o r growing chicks under c o n t r o l l e d l a b o r a t o r y . 8 conditions. Carver et a l (26) summarized their work in the following points: 1. Of the several samples of sardine o i l and pilchard o i l tested on the W.S.C. all-mash biological ration,-0.25 and 0.5 per cent levels did not afford the chicks protec-tion from rickets in Assays 1 and 2. 2. In Assay 1, 1 per cent of pilchard o i l and sardine o i l added to the W.S..C. all-mash biological ration supplied complete protection from rickets. 3. One-eighth of one per cent of concentrated cod liver o i l added to the W.S.C. all-mash biological ration in Assay 1 and 2 provided the chicks with protection from rickets. 4. The W.S.C. commercial chick ration with i t s level of 0.8 to 1 per cent, phosphorus does not require the addition of any supplementary mineral containing phosphorus when the meat scrap used in the ration contains from 25 to 30 per cent,total ash. 5. The W.S.C. commercial chick ration required the addition of 1 per cent oyster shell flour in Experiments 1 and: 2, when the antirachitic factor was provided, In order to promote maximum growth and calcification. 6. Excellent results in growth and calcification were secured with a calcium level from 1 to 1.8 per cent and a level of phosphorus between 0.8, and.1 per cent. 7. V As a result of Experiments 1 and 2 i t appears that the. addition of 0.5 per cent of this sample of biologic-ally, tested sardine o i l to the total ration prevented the 9 occurence of rickets, when the ration used in : these experi-ments contained the necessary amounts of calcium and phosphorus.* • In his work on the comparison of sardine o i l and cod liver o i l , Tepper (.102) found that the New England College Conference poultry ration plus 0.5 per cent sardine o i l proved equally as efficacious as 1 per cent cod liver o i l . There was a slight growth retardation when only 0.25 per cent of the sardine o i l was used in the chick ration. The vitamin content of the body o i l of the herrings was noted by Schmidt-Nielsen (87). Values found previously for the vitamin A content of the oils from herring andtinned sprats were too high, owing to the absence of vitamin D from the experimental diet used. In fresh experiments to test the value of these materials for vitamins A and D, the following results were obtained. In tests for vitamin A the minimum daily dose for a rat of body o i l from fresh herring was 20 milligrams. In tests for vitamin D, cure of rickets was secured in rats with a daily dose of 5 or 6 milligrams of body o i l from some samples of kippered herring, with 10-12 milligrams from others. Body o i l from the large herring, fresh, cured rickets in a minimum daily dose of 4-10 milligrams. The o i l from the tin, in the case of tinned sprat ("bristling in olive oil") cured rickets i n a daily dose of 20 milligrams and the body fat i t s e l f i n a dose of 10 milligrams. The authors conclude that the vitamin D value of these products is good enough to make them suitable substitutes for cod liver o i l . 10 i The value of salmon o i l in. the treatment of infantile rickets was suggested by Eliot et a l (4.0) . They found that salmon o i l Jki daily doses of 10-20 cubic centimeters brought about healing in 13 children with active rickets. X-ray Examination and blood analysis showed that the response to treatment was very prompt,, and advanced healing took place in from 3 to 8 weeks." The oils used had been prepared from the waste products of three species of salmon, the Pink or Hump-back, the Chinook or Fling, and the Sockeye or Red;. Biological tests-on the three oils showed that the Sockeye and Chinook oils were nearly equal in.vitamin. A potency to.a composite sample made from nineteen medicinal cod liver o i l s , and that Pink o i l was distinctly inferior in vitamin A potency. The Sockeye and Pink oils contained twice as much vitamin D as the. cod liver o i l composite, and the Chinook o i l about 50 per .cent more than the same sample, of cod liver o i l . The authors point out that there is a potential supply of a million gallons of salmon o i l available through the salmon canning industries of the Pacific states and Alaska. They suggest that the o i l might be widely used as a rich and cheap source of vitamins A and D. The seasonal variation i n the o i l content of halibut liver was studied by B i l l s , Imboden and Wallenmeyer (15). Weekly records were taken of the fat, vitamin A, and vitamin D contents of the livers of halibuts caught near Seattle. The o i l content increased slowly ;from January (12 per cent) to June, there was then a sudden rise to a maximum value . (25 per cent) in August, followed by a slow decline during the 11 rest of the year. The vitamin A content ( Jan. 240,000, Aug. 35,000 international units per gram) and vitamin D content (Jan. 1,400.? Aug. 900 international units per gram) moved inversely with the fat content, although i n the case of vitamin .b. the fluctuation was wider than in the case of Vitamin D. In a tuna fis h liver containing only 4 per cent of o i l the very high concentration of 7,500 units of vitamin D per gram of o i l , was observed. The isolation of the antirachitic vitamin from tunny fish o i l was carried out by Brockmann (25). A vitamin concentrate from tunny liver o i l was partitioned between 90 per cent methanol and light petroleum, and the latter fraction was then partitioned between 95 per cent methanol and light petroleum.-. The vitamin D remained in the last methanol fraction, freed from vitamin A and other contaminants. Further concentration was accomplished by chromatographic absorption on aluminium hydroxide. The concentrate, containing 25 to 30 per cent vitamin D, was then esterified with 3:5—dinitrobenzoyl chloride and the ester separated by fractional absorption. The 3:5-dinitrobenz.oate of vitamin D (vitamin D3) thus separated had a melting point of 128^-129 degrees Centigrade, ( ) n 100 degrees in chloroform, and proved to be identical with the ester of the product from the irradiation of 7-dehydrocholesterol. The free vitamin was an o i l with an absorption in the ultraviolet identical with that of the product from 7-dehydrocholesterol or from ergosterol, and a 1 2 Biological;activity for rats of 2 5 , 0 0 0 international units per milligram. 13 THE ANTIRACHITIC VALUE OF COD-LIVER OIL. Experiments were conducted by Holmes and Pigott (58) to determine the influence of cod-liver o i l on calcium metabolism i n growing chicks when supplementary calcium was obtained from different sources, fed at different levels, and was available in different ratios to phosphorus. Best growth was obtained when cod-liver o i l was added to a ration c ontain-ing from 1 to 4 per cent.Calcium and phosphorus having a maximum calcium and phosphorus ratio of 1.5-1.0. The most efficient utilization of calcium occurred when cod-liver o i l supplemented a ration in which the ratio of calcium to phosphorus was relatively low. Using cod liver o i l for brooder chicks, Kitto (66) reported that an experiment lasting 12 weeks, carried out on two groups of 81 chicks each, reared in an electric brooder, showed that no beneficial effect had resulted from the addition of cod liver o i l to the ration. Further work by the same author (67) substantiated his earlier findings. He stated that two groups of a hundred chicks each were kept under the same conditions, except that only one received cod li v e r o i l . Neither from the weekly weights nor from the general appearance of the birds could i t be said that the cod liver o i l fed group was superior to the control. Cxriem (45) reported on biological methods for determination of cod liver o i l in feed mixtures. Details were .14 given'of: a method of estimating vitamin D in eod liver o i l and poultry foods. "Dried cod liver o i l " was useless as an anti-rachitic agent. "A cod liver o i l f o r t i f i e d in vitamin D potency" was no more potent than a regular cod liver o i l distributed by the same dealer. Lachat and Halvorson (71) assaying reference cod liver o i l , found that the chick was useful for the estimation of vitamin D, on account of its great sensitivity to rickets. By using the U.S.P. reference cod liver o i l , containing 95 international units of vitamin D per gram, i t was found that 80-135 international units must be given to chicks during the f i r s t 4 weeks of l i f e to produce normal growth higher than 45 per cent. Graybill (44) in conducting experiments on rickets in poultry noted that there is insufficient sunshine in California to supply adequate vitamin D for poultry. The use of 1 per cent by.weight of cod liver o i l in the mash is recommended. The best phosphorus content of the ratio is about 0,8 per cent, and the most suitable calcium-phosphorus ratio is 2:1. Van Esveld (104) tested sixteen samples of cod liver o i l for the vitamin D content. Rats were used as the experi-mental animals and the content estimated from the results. The values ranged between 50 and 200 international units per gram. Cod liver o i l which was not sold in bottles was inferior in quality. It is considered desirable that medicinal cod liver o i l should be o f f i c i a l l y required to contain at least 100 international units of vitamin D per gram. ' 15 Roberts (85) extracted stearin from cod liver o i l and summarized Ms findings : 1. * Cod liver o i l stearin as was used in these experi-ments, when fed at levels of 1% or more of the ration, prevented visible external signs of rickets in chicks up to 8 weeks of age. 2. 2% of stearin in the ration was not sufficient to prevent rickets. 3. 1 or 2 % of the stearin did not retard the rate of growth or affect the mortality of growing chicks, but 4% apparently retarded growth to some extent. 4. Stearin extracted from cod liver o i l potent in vitamin D appears to be a practical source of the antirachitic factor for chicks- that must be grown away from direct sunlight. Stoeltzner (100) stated that propaganda originating from Norway has lately been appearing in German medical papers and in circulars sent round to the medical profession, suggesting that cod liver o i l is more effective and less dangerous as an antirachitic remedy than irradiated ergosterol. The author holds strongly the view that modern standardized preparations of irradiated ergosterol are much more effective and, i f intelligently used, no more dangerous, than cod liver o i l . Using comparative observations of viosterol and cod liver o i l , Prather, Nelson and Bliss (81) found that viosterol (an irradiated ergosterol preparation) contained vitamin D but not vitamin A. Its administration promoted calcification of 16 of the bones, but played no part in increasing the resistance of the patient to respiratory and other infections. Viosterol cannot, therefore, be regarded as a concentrated substitute for cod liver o i l , which contains both vitamins. The comparative effectiveness of viosterol and cod liver o i l in protecting the body against calcium deficiency in the diet was studied by Shelling and Asher (92). The authors stated that rats fed on a diet with very low calcium and optimal phosphorus often survived many"months with f a i r gain in weight, but growth and reproduction were improved by increasing the calcium and vitamin D of the diet.. Young rats were found more susceptible to a calcium deficiency than older rats ? but no difference was observed in the sparing capacity on body calcium of cod liver o i l and viosterol. The serum calcium remained normal on diets very low in calcium over long periods; i t was slightly reduced in rats receiving the diet from a very early age and was a l i t t l e higher in those receiving viosterol. A comparison of the antirachitic potency of cod liver o i l and irradiated ergosterol on a curative and preventive basis was made by Russell, Taylor and Wilcox (86). They stated that amounts of irradiated ergosterol and cod liver o i l which were antirachitically equivalent in curative tests on rats were also antirachitically equivalent in preventive tests on these animals, although i t is known that they are not equally effective in preventive tests on chickens. 17 The minimum curative dose for rats as determined by the line test, was insufficient for complete protection, as determined by estimation of the bone ash, when fed to rats on a preventive basis. 18 ••*•'= THE RELATION QF CHOLESTEROL AND ERGOSTEROL TO VITAMIN D, Angus et a l (2) in their paper on a crystalline antirachitic substance described the chemical and physical properties of ca l c i f e r o l . They reported i t as a .crystalline .substance of high antirachitic activity, which had been previously prepared in small quantities by the vacuum d i s t i l l -ation of the products of the irradiation of ergosterol, was now obtained in sufficient quantity to make a fuller study of i t s properties possible. Tested biologically, i t had twice the antirachitic potency of the standard preparation of irradiated ergosterol, issued by the Medical Research Council. . The melting- point was 123-125 degrees Centigrade. The sub-stance remained stable in dry ai r , at room temperature for six weeks. It is suggested that the substance is. an isomeride of ergosterol. •. It was toxic for rats in large doses, and caused calcium deposits in, the organs. The effect of ultra-violet Irradiation on the absorption spectrum was examined.. The substance differed i n certain particular^ from "vitamin. D"? i f that is taken to mean the substance which shows antirachitic activity in the normal products of irradiation, and i t is concluded that there are probably not less than'two substances, each possessing intense antirachitic activity. Windaus (108) described the chemistry of Irradiated ergosterol. The preparation i s described as a crystalline product, by interaction with maleic anhydride, in ethereal solution at room temperature. After the" portion which reacted 19 readily with this reagent had been removed with dilute potash, the solvent was evaporated from the original solution and crystalline material separated put. The crystals were long needles of melting point 122-123 degrees Centigrade; the main absorption was at 265-270 m^ . and, tested biologically, the antirachitic activity was about twice that of the standard solution of irradiated ergosterol, issued by the Medical Research Council. The material agreed in these points with the crystalline substance described by Bourdillon, et a l , but the two differed in their rotation. The author believes that in the photochemical transformation of ergosterol.into vitamin D, the molecular formula,, the alcohol group and the' three double bonds remain unchanged, and that a structural rearrangement takes place which leads to an increase in the spatial size of the molecule. Certain evidence is adduced in support of this view. A later paper by Windaus, Luttringhaus and Busse (109) summarized and discussed new observations on products of irradiation of ergosterol. The changes in absorption and optical rotation during irradiation of ergosterol are qualitatively in accordance with the following genetic relationship of the products of Irradiation: ergosterol -> lumisterol -> calciferol (vitamin B) -f tachysterol; calciferol->-suprasterols 1. and II. +• a substance with absorption maximum at 248 m^ . Taehysterol, not yet isolated, has a high negative optical rotation and intense absorption at 2 8 0 0 1 4 . It combines 20 rapidly with maleic anhydride, is oxidized readily, and so induces oxidative destruction of the vitamin. The changes observed on heating mixed products of irradiation can be interpreted in terms of the formation of pyrocalciferol and iso-pyrocalciferol from c a l c i f e r o l , and of pyrotachysterol (absorption maximum 290 m^ ) from taehysterol. The reactivation of heated products by irradiation is due to conversion of lumisterol into calciferol. The physiological properties of these products are not yet ful l y investigated, Acrude irradiation-product probably owes i t s toxicity to calciferol alone, but when over irradiated, toxicity due to "substance 248" and possibly to taehysterol is developed. The toxicity of crude irradiation products after reduction by sodium and alcohol is due to substances of obscure origin. The toxicity of products heated at 200 degrees i s principally due to products of decomposition of taehysterol. The sensitivity of mixed irradiation-products to oxidation and their possible content of toxic substances are strong arguments for the use of crystalline vitamin D for therapeutic purposes. The constitution of calciferol was described by Askew et a l (3). According to these investigators, the crystalline substance originally prepared, named calciferol, and considered probably not to be pure vitamin D, has been found to be a solid solution containing two biologically inactive impurities, named pyrocalciferol and "substance X" respectively, 21 which-together constitute about half of the original substance. The name calciferol is retained for the pure: vitamin D , which ..was isolated from the inactive constituents of the original preparation by preparing the 3:5-dinitro-benzoates. The pure c a l c i f e r o l is probably identical with the vitamin Dvp of Windaus and Linsert, and"substance X" with lumisterol, a constituent of Windaus' vitamin D-^ described by Luttringhaus. Details of the preparation and properties (including colour reactions) of calciferol, pyrocalciferol and "sterol X" are given. Experiments show that the toxicity of vitamin. D is an inherent property of the pure vitamin and is not due to a contaminant. . Some of the properties of ergosterol and, cal c i f e r o l were reported by Bacharach, Smith and Stevenson (7)* When care was taken to prevent decomposition, the melting-point of ergosterol was 162-164 degrees Centigrade, and that of calciferol, 115-117 degrees Centigrade. The degree of rotation varied considerably with the solvent, ergosterol giving the highest rotation when dissolved in chloroform and calciferol In absolute alcohol! the former was strongly laevo- and the latter strongly dextrorotatory. The ultra violet absorption at 281 m^ for ergosterol and^at 265 m^for calciferol was measured. The biological activity of calciferol was about 40x10 units vitamin D per gram. Chuang (28) reported-his work on the constitution of ergosterol. Oxidation of ergostane, C^g Hg^, by chromic acid yields norallocholanic acid, C^HggOg, identical with the product obtained from allocholanic acid by a Grignard reaction and oxidation of the resultant carbinol. It is concluded that ergosterol has a structure similar to cholesterol, but with a methyl group in position 24, and double linkings probably at 22:23, 9:11, and either 7:8 or 8:14. In their paper, Boer et a l (18) reported on a naturally occurring provitamin D. From a specimen of cholester ,01 containing as much as.4.5 per cent, measured spectroscopic-ally, of provitamin D, the provitamin was concentrated to 96 per cent as the acetate by repeated absorption on A I 2 O 3 A N ( 3 -elution with benz.ene and petroleum ether; i t was further repurified by crystallisation, and the free sterol was recovered by. saponificatioru It had a melting point in vacuo 150 degrees Centrigrade. Other physical properties, compos-ition of the dinitrobenzoate, and the- results of hydrogenation oz.onisation and irradiation with long wave ultraviolet light are described. Tested on rats, the antirachitic value of the isolated provitamin after ultraviolet international units of vitamin .D per gram. Tested on chicks, with X-ray of the knee joints and bone ash as c r i t e r i a , i t was as potent as cod liver o i l in rat dosage. . , From the observed properties a formula is pro-posed for the provitamin; while synthesis of such a compound was being attempted, the synthesis of a sterol with this formula, 7-dehydrocholesterol, was announced by• Windaus et a l . (Abst. 2561, Vol.5). It is concluded that the provitamin, 23 present as.an impurity in cholesterol, is identical with 7-dehydrocholesterol. Quoting B i l l s (13) on the biological effects of radiation, he says: "There is no good evidence that any vitamin except ° certain forms of vitamin D. are directly a photochemical product, and.it is by no means certain that a l l forms of vitamin D require radiant energy for their formation. It seems that the effect.of radiant energy on the vitamins is mainly a destructive one. It i s , however, a point of practical significance that in the momentary exposure to ultraviolet rays which is required to endow foodstuffs such as milk with vitamin D, no appreciable destruction of the other vitamins is evident. Research of the past decade have shown that ultra-violet rays, acting on the sterol-bearing fats of the skin produce a form of vitamin D which is similar in antirachitic action to the vitamin D contained in fis h o i l s . Work by Heilbron, Kami, and Morton and Fohl showed that ergosterol is the provitamin In cholesterol. However Waddell published experiments which indicate this is not the case. He found that irradiated cholesterol was many times more effective on chickens, rat unit for rat unit, than • ergosterol which had been irradiated either by i t s e l f or in the presence of cholesterol. The individual products of irradiation: 1... Ergosterol - parent substance. 24 i2. Lumisterol - appears to be i n i t i a l product of the irradiation of ergosterol. Is probably devoid of antirachitic action, but i t is converted by irradiation into vitamin D. 3. Taehysterol - probably devoid of antirachitic action, but i t may have a slight toxic effect. Great affinity for oxygen, f a i l s to crystallize. 4. Vitamin D C c a l e i f e r o l ) - the antirachitic potency of pure calciferol was found to be 40,000 international units per milligram. Higher animals obtain vitamin D in at least three ways, the relative importance of each must vary with habits, requirements, and opportunities: 1. Eating such foods as eggs,, fish, whole furred or feathered animals, and isolated dead vegetable tissues. 2. Ingesting Isolated sebaceous matter. 3. Directly absorbing the products of isolation formed on or in the skin. It is to be noted that in a l l of these sources, except possibly fish, the ultimate origin of the vitamin is traceable to sterols activated by light." Massengale and Hussmeier (76)' summarized their find-ings of the effect of activated ergosterol in-.'the chicken. Briefly they are: 1. In the serum of birds receiving a diet lacking in vitamin D and having a ratio of calcium to phosphorus of 8:1, the calcium increased during the f i r s t week and remained above normal for eight weeks; the inorganic phosphorus decreased 25 rapidly during the f i r s t 3 weeks and then remained approx-imately stationary. The addition of activated ergosterol (=4000 perce*nt cod liver oil) caused a marked increase of the phosphorus. When the ratio of calcium to phosphorus was 5:1, the calcium remained above normal for 5 weeks but there was a steady decrease of the phosphorus, though not as much as where the ratio was 8:1. The addition.of the activated ergosterol caused both calcium and phosphorus to become approximately normal,, 2. When activated ergosterol (= 4000 per cent cod liver oil) was fed to birds on high calcium ricket-producing diets, there were marked changes i n the calcium of the blood serum within 48 hours. 3. The serum calcium and phosphorus of birds of the age used in this experiment (18 weeks) remained approximately normal when they received a rickets-producing diet in which the ratio, of calcium and phosphorus was 1.6:1. The addition of activated ergosterol had no marked effect on either the calcium or phosphate of the serum u n t i l after 15 to 35 days, when an increase in serum calcium was noted. 4.. When the birds received a diet low in both calcium, and phosphorus, both the serum calcium and inorganic phosphorus decreased. On the addition of activated ergosterol there was a marked increase ins erum calcium but only a slight increase in phosphate. After 35 days the calcium of the serum was-approximately normal, yet the inorganic phosphorus remained subnormal. 26 ; The ergosterol requirement of growing chicks was reported by King, Eull and Hall (65). They stated that growing•chicks consuming a diet with a calcium;phosphorus ratio of 1.91:1, needed ten times as many rat units of vitamin D in the form of irradiated ergosterol as in the form of cod liver o i l to produce leg bones with approximately the same ash content. Both the sources of vitamin D were mixed with the diet. In a previous experiment the same amount of irradiated ergosterol given separately by mouth produced a higher ash content. Sjollema and Barbas (93) studied the antirachitic action of irradiated cholesterol in chicks. They found that cholesterol irradiated by ultraviolet light brought about heal-ing of rachitic lesions within 10 days in chicks on a diet of calcium content 0,74 per cent and calcium:phosphorus ratio 1:1, t,ut not in those on a diet of calcium content 0.19 per cent and calcium:phosphorus ratio 1:.5. When healing was complete, as estimated by radiography of the epiphyses, the ash content of the bones was s t i l l subnormal. The general condition and growth rate were more satisfactory in groups of chicks receiving 18.75 milligrams of irradiated cholesterol per day than in those receiving 41.75 milligrams, and better in a l l groups receiving the cholesterol than in one receiving cod liver o i l (approximately 200 International units per 100 grams of diet). In birds k i l l e d after one month's therapy with the cholesterol or cod liver o i l the blood inorganic phcs phorus was abnormally high. 27 .' iThe summary of Hathaway and Lobb's (51) paper oh provitamin D of heat-treated cholesterol is as follows: "1% Wadell's observation that irradiated crude cholesterol is more effective in preventing rickets in chicks than equivalent number of units of irradiated ergosterol has been confirmed* 2. This provitamin of crude cholesterol is destroyed by, purification through the dibromidej i.e., 20 rat units or 200 milligrams of purified cholesterol per 100 grams of ration showed only slight improvement over the basal diet alone. . 3. Heat treatment of the purified cholesterol increases its pro-antirachitic potency so that 2.5-5 milligrams per 100 grams of ration completely protect the chick. 4. Evidence i s given that irradiation of heat-treated cholesterol has formed a new form of vitamin D which has properties resembling those of the natural vitamin D of cod liver o i l more closely than those of the vitamin D of irradiated ergosterol, as shown by chick assay." Steenbockj.Kletzien and Halpin .(.98) compared the reaction of the chicken to Irradiated ergosterl and irradiated yeast as contracted with,the natural vitamin D in fish liver oils.. Samples of irradiated ergosterol and irradiated,.yeast were, .less effective in promoting calcification in chickens than in rats, while cod l i v e r o i l and burbot-liver o i l were equally effective for the two species. It was therefore, concluded that either vitamin D produced by irradiation is different from 28 that contained in cod liver o i l , or that cod;liver o i l con-tains something which has an effect upon the efficiency with which vitamin D functions. As, however, irradiated ergosterol i s equally effective (both in calcifying properties and in toxic properties) whether given to the birds in solution in corn o i l or in cod liver o i l , i t is concluded that cod liver o i l does not contain a substance which makes the vitamin D in i t more effective, and the only conclusion to be drawn from the observation is that the vitamin D of irradiated ergosterol i s a different substance f rom the vitamin D occurring naturally in liver o i l s . The action of irradiated ergosterol on rats and chickens was demonstrated by Hess and Supplee ,(55) . The following is their summary: "Recently Chickens have been used by many in the study of rickets. For the past three years we have been carrying out experiments to ascertain t he a.ction of ultraviolet light, or irradiated ergosterol, and of cod liver o i l on these animals. Without going into detail in regard to these experi-ments, we wish to point out in this connection two significant differences between chickens and rats in their reaction to specific antirachitic-substances. In the f i r s t place i t was found that whereas chickens: are,regularly protected against leg weakness by an addition of 1 per cent of cod liver o i l to their ration, a supplement of irradiated ergosterol equivalent to many times this amount failed to afford protection. It may be added that the ash of the bones was found to be comparative-29 / ly low-'in,, the animals to which irradiated ergosterol had been given. This lack of response was a i r the more surprising, as chickens werje found to respond, readily to mild intensities of ultraviolet radiations and showed a high percentage of bone ash after an experimental period of 10 weeks." Seal and Biely (9) reported that a dose, of 1/40 cubic centimeters of viosterol, having a potency of 250 D, is as effective as 1 cubic centimeter of cod liver o i l (112 units per gram) in prevention of rickets. The effect of large doses of irradiated ergosterol upon the ash content of the femora of young and adult rats was shown by Jones and Robson (62). In their findings they : reported that adult rats, receiving toxic :doses of irradiated ergosterol for seven to twenty days, showed calcium, deposits in the soft tissues but no significant difference i n bone calcification when compared with l i t t e r mate controls. The absolute amount of ash was not decreased though there was sometimes a decrease in the relative amount. When non-toxic doses- were given there was an increase in calcification. The diet contained 5 per cent whole milk powder and o,5 per cent calcium lactate. No support was given to the theory that there is withdrawal of^minerals from the skeleton in hypervitaminosis D. .The-'calcification, of .the bones of. rats on a diet low in ergosterol was described by Hume and Smith; (61). The rats were.fed on a diet made.as low as possible in ergosterol. Half received added ergosterol. It was sought, by irradiating • 3 0 the animals with ultraviolet light, to ascertain whether cal-cification of the bones (estimated by ashing) was limited by restriction* of the intake of ergosterol and therefore, indirectly, whether synthesis of ergosterol can take place in the animal body. No evidence of such limitation was obtained but i t i s pointed out that the existence of capacity for synthesis is only one of several possible explanations. In the course of the experiment the rats developed a scaly con-dition of the t a i l . The toxity of irradiated ergosterol was reported by Duguid, Duggan and Gough (39). They showed that irradiated ergosterol was more toxic to rats fed on a vitamin-free synthetic diet, of which high calcium content, than to rats fed on a normal stock diet of bread and potatoes. When the calcium content of the stock diet was raised to that of the synthetic diet there was l i t t l e difference in the toxic effect. The high calcium content v>ras the most potent factor in determining toxicity, and on such a diet the presence or or absence of vitamins, other than vitamin D, made no differ-ence. When the calcium and phosphorus content of the syn-thetic diet was much reduced the addition of such vitamins appeared to diminish the toxicity, but the evidence is not accepted as convincing. Arterial lesions are considered to be a reliable criterion of irradiated ergosterol poisoning in the rat. Renal lesions occurred in two forms, as calcif-ication of the renal arterioles and as calcareous cast formations in-the tubules. The former were part of the 31 general arterial involvement , the latter were f ound to be of common occurrence in the r at, independently of irradiated ergosterol administration, and could not be admitted as reliable evidence of toxicity. Loureiro (73) studied the vitamin D of cod liver o i l and irradiated ergosterol* He found that when acetic acid extract of cod liver o i l was treated with petroleum ether, vitamin D remained in the acid, while vitamin A passed into the petroleum ether. On- evaporation a semi-solid brownish material with a strong characteristic odour was obtained. This contained much ozone, was soluble in fat solvents, and partly insoluble in saturated hydrocarbons. 40 per cent of this fraction was insoluble in petroleum heptane, and i t was the insoluble portion which showed the antirachitic power. It is pointed out that the acetic acid fraction had physical properties very different from those of irradiated ergosterol, and i t i s suggested that the vitamin D of cod liver o i l and irradiated ergosterol are separate entities, Dols (37) conducted a comparative investigation on rats and chickens of the identity of the a r t i f i c i a l anti-rachitic vitamin (irradiated ergosterol) and the natural vitamin U of cod liver o i l . He found that young albino rats and day old leghorn chickens were mainta.ined on the same rachitogenic diet. The c r i t e r i a of rickets were X-ray exam-inations of the hock, and post mortem examination of the skeleton with some ash determinations. Chickens were pro-tected from rickets by 2 per cent of cod liver o i l in-the 32 ration but-.not by irradiated ergosterol equivalent to 20 per cent of cod liver o i l , as determined on rats. The unsaponi-fiable fraction of cod liver o i l was as effective for chicks as the corresponding amount of whole o i l . The value of irradiated ergosterol for chicks was not enhanced by dissolving i t in the saponifiable fraction of cod liver o i l . A tunny liver o i l concentrate was nearly as effective as crystalline calciferol for rats, and as effective, in rat dosage, as cod liver o i l for chicks, whence i t i s argued that the material having antirachitic potency in the tunny concentrate like that in cod liver o i l could not be the same as crystalline c a l c i f -erol. Irradiated cholesterol, on the other hand, in rat dosage was as effective, for chicks as cod liver o i l , whence i t is argued that the material activated was not ergosterol. In terms of international units, 250 per 100 grams of ration,• in the form of cod liver o i l , tunny liver o i l concentrate or irradiated cholesterol, sufficied f o r chicks, while 2500 as calciferol was insufficient. Schonheimer, Behring and Gottberg (88) conducted a series of experiments with ergosterol on various animals. Mixtures of ergosterol and Cholesterol dissolved in fat were fed to mice, rats, rabbits and dogs. The sterols were extracted from the whole bodies of the mice and the rats after removal of the alimentary canal, from the brains of the rabbits, and from the blood and thoracic duct lymph of the dogs, four hours after feeding the sterols. The ergosterol content of each sterol preparation was determined 33 spectrographically. In every case i t was extremely low (about 0,05 per cent), and did not differ significantly from that found in control animals. The results with mice, rats and rabbits thus showed that feeding ergosterol did not increase the amount stored, while the results with dogs showed that ergosterol was absorbed, i f at a l l , a.t less than one thousandth the rate of cholesterol. 34 • ' ' VITAMIN D 'REQUIRBMEMTS OF CHICKS , The v i t a m i n D requirements of growing chicks were estimated by Couch (29) on two very d i f f e r e n t b a s a l d i e t s . : I t was found" t h a t f o r normal growth, f e a t h e r i n g , prevention of r i c k e t s and crooked breast bones, and complete c a l c i f i -c a t i o n , of bones as estimated by the ash method, 41.0. Steen-bock u n i t s per 100 grams of d i e t were r e q u i r e d on one r a t i o n , and 55,8 u n i t s on the other. The lower v i t a m i n D requirement on the f i r s t r a t i o n was probably due to a higher percentage of lime. I n a l a t e r paper Couch, Fraps and Sherwood (30) studie d the v i t a m i n D requirements of chickens grown i n the absence of s u n l i g h t . Their experiments i n d i c a t e d t h a t chick-ens f e d a r a t i o n with the proper amounts of calcium and phos-phorous need no a d d i t i o n a l , v i t a m i n D to make good growth f o r the f i r s t 6 weeks even i n the absence of s u n l i g h t . The v i t -amin requirement of the coc k e r e l appears to be higher than th a t of the p u l l e t . The vi t a m i n D requirements of chickens vary with the p a r t i c u l a r c r i t e r i o n used i n determining the e f f e c t of the v i t a m i n B supplied. Maximum g a i n i n weight re-q u i r e d more v i t a m i n D than any one of the other f a c t o r s stud-i e d . The number of u n i t s of vi t a m i n D re q u i r e d by growing chickens appears to depend upon the nature of the r a t i o n , e s p e c i a l l y the percentages of calcium and phosphorous con-t a i n e d i n i t . Chickens f e d a r a t i o n containing 1.48 percent 35 calcium and 0.65 percent phosphorous required 12.3 Inter-national Units of vitamin D from cod liver o i l per 100 grams of feed for*maximum growth and best u t i l i z a t i o n of the feed, and 3.1 International Units of vitamin D from cod liver o i l per 100 grams of feed for the prevention of rickets and crook-ed breast bones and calcification:of the bones at 12 weeks. Chickens fed a ration containing 0.96 percent calcium and 0.66 percent phosphorous required up to 12 weeks 50.2 Inter-national Units of vitamin D from cod liv e r o i l per 100 grams of feed for maximum growth, best u t i l i z a t i o n of the feed, and calcification of the bones, and 12.3 International Units of vitamin D from cod liver o i l per 100 grams of feed, for the prevention of rickets and crooked breast bones. This high vitamin D requirement was probably due to the.low calcium content of this :ration.; Chickens fed a ration containing 1.26 percent calcium and 0.77 percent phosphorous required,up to 12 weeks 6.7 International Units of vitamin,D from cod liver o i l per 100 grams of feed for maximum.growth, prevention of crook-ed breast bones, best u t i l i z a t i o n of the feed, and calcifiea-, tion of the bones, and 3,4 International Units of vitamin D per 100 grams of ration from cod liver o i l for the prevention of rickets. The maximum level of vitamin D may not have been fed with this ration. Chickens fed a ration containing. 1.36 percent calcium and 0.78 percent phosphorous required up to 12 weeks 6.7 International Units of vitamin D from cod liver o i l per 100 grams of feed for maximum growth and-calcification 36 of the hones5 and 3.4 International Units of vitamin D from cod liver o i l per 100 grams of feed for the prevention of rickets and*crooked breast bones and for best uti l i z a t i o n of the feed. The maximum level of Vitamin D may not have been fed with this ration. Carver et a l (27) reported the vitamin D require-ments of chickens. According to the authors, chicks hatched and kept in standard conditions, received a ration of which the Ca.:P ratio was 2.7:1. As judged by c r i t e r i a of growth, ash analysis of bone and line test at 8 weeks of age, such chicks, confined without sunshine, required 17 International Units of vitamin D, as cod liver o i l , per 100 grams of ration from 1 to 16 weeks of age, and 8 units from 16 to 24 weeks. Exposure to sunshine promoted adequate calcification between May and September but growth was enhanced by addition of cod liver o i l . The chicks stored vitamin D and the reserves Were not exhausted u n t i l after 3 months' egg laying. Pullets, receiving an all-mash laying.ration, with Ca:P ratio 2.3:1, required 67 International Units of vitamin D per 100 grams of ration for maximum egg production, as judg-ed by number and size of eggs and stoutness of shell, when confined, and 17 Units when allowed access to sunlight. Eggs from confined hens failed completely to hatch i f less than 17 Units were given, and 135 Units were needed to obtain maximum hatchability. When the hens ha.d access to sunshine a l l the year round, 34 Units were needed. 37 " Murphy5 Hunter and Knondel (77), i n their experi-ment with White Leghorn chicks deprived of sunshine found that the minimum protective level of f o r t i f i e d cod liver o i l (270 International Units of vitamin D per gram) was one-six-teenth percent* Up to one-quarter percent? on the other hand, was necessary to maintain normal body weight, egg pro-duction, egg size and quality and hatchability of egg. It Is calculated that in order to secure satisfactory performance a yearly Intake of approximately 25,000 International Units of vitamin D i s necessary. There was no difference in the performance of two groups of birds on free range, one of which received one-eighth percent of f o r t i f i e d cod l i v e r o i l . The comparative vitamin D requirements.of growing chicks, turkeys and pheasants were noted by Baird and Greene-(8). They state that the antirachitic requirements of turk-eys and pheasants can be met adequately by the feeding of fo r t i f i e d cod liver o i l . Chickens require a minimum of ap-proximately 18, Units (U. S.P.). vitamin D per 100 grams of feed to 12 weeks of age when provided with a ration complete in other essentials, and which results in a weight average of 1,000 grams or better at 12 weeks. Turkeys require a minimum of approximately 60-70 Units (U.S.P.) vitamin D per 100 grams of feed when given a ration complete for a l l other essentials and which results in a weight average of 1,900 grams or better at 12 weeks. Pheasants require a minimum of.approximately 50-60 Unit's (U.S.P.) vitamin D per 100 grams of feed when given a ration complete in a l l other essentials and which re-suits in a weight average of 600 grams or better at 12 weeks. 39 THE' CALCIUM-PHOSPHORUS RATIO AMD ITS -RELATION TO RICKETS. ' H©lmes and Tripp (59) reported on the f a c t o r s which i n f l u e n c e the e f f e c t i v e n e s s of a rachi t o g e n i c r a t i o n * They concluded t h a t there appeared to he considerable v a r i a t i o n i n the ash content and Ca:P r a t i o of d i f f e r e n t batches of Steenbock's r a t i o n 2965 ? prepared under ordinary l a b o r a t o r y conditions» I n v e s t i g a t i o n s were made on various samples of yell o w corn which i s the main c o n s t i t u e n t of the d i e t * I t was found t h a t the ash, calcium and phosphorous content of corn from d i f f e r e n t areas v a r i e d m a t e r i a l l y * Other i n v e s t -i g a t o r s had shown tha t the p r o t e i n content and also the v i t -amin A content were extremely v a r i a b l e * I t was p o s s i b l e also t h a t the v i t a m i n D content was not constant* Thus the e f f -ectiveness of the r a c h i t o g e n i c r a t i o n was dependent on the v a r i a b i l i t y of the m a t e r i a l s used i n making the d i e t * These f a c t o r s would have to be c a r e f u l l y c o n t r o l l e d , when two or more l a b o r a t o r i e s wished to conduct p a r a l l e l experiments on the v i t a m i n D content of a given product* Watkins and M i t c h e l l (106) studied the value of c o n t r o l l e d experimental feeding and concluded t h a t the great-er the consumption of a rac h i t o g e n i c d i e t the slower the r a t e of c a l c i f i c a t i o n of the bones* I n general i t appears that the greater the consumption of a d i e t unbalanced i n some p a r t i c u l a r , the poorer nourished w i l l be 'the animal with r e f -erence to the f u n c t i o n with respect to which the r a t i o n i s unbalanced. 40 Cruickshank (32) found t h a t the calcium and phos-phorous requirements of the growing chick have y i e l d e d some-what c o n t r a d i c t o r y r e s u l t s , which i s not s u r p r i s i n g seeing t h a t at the present time many of the f a c t o r s a f f e c t i n g u t i l -i s a t i o n of these elements are unknown. The data a v a i l a b l e i n d i c a t e t h a t the d i e t a r y f a c t o r s concerned i n producing sat-i s f a c t o r y growth and c a l c i f i c a t i o n of bone are the Ca:P r a t i o , the absolute amounts of., calcium and phosphorous and the l e v e l of v i t a m i n D, . The a c i d base balance also appears to be of importance although growing c h i c k s are able to adapt them-selves to wide v a r i a t i o n s i n d i e t a r y conditions without ap-p r e c i a b l e m o d i f i c a t i o n of the a c i d i t y of the m a t e r i a l i n the d i g e s t i v e t r a c t . The optimum Ca:P r a t i o i s v a r i a b l e , since i t i s i n -fluenced,by the supply of the a n t i r a c h i t i c f a c t o r and the l e v e l of calcium and phosphorous i n the ration® I t i s impro-bable, t h e r e f o r e , t h a t there i s a Ca.:P r a t i o which i s optimum f o r a l l d i e t s . I n t o t a l or p a r t i a l absence of v i t a m i n D, no Ca:P r a t i o w i l l produce both good growth and normal bone, but with c e r t a i n r a t i o s the e f f e c t of the v i t a m i n d e f i c i e n c y i s l e s s marked. Conversely an adequate supply of v i t a m i n D l e s -sens the e f f e c t of v a r i a t i o n i n the CasP r a t i o . : With an optimum amount of v i t a m i n D i n the d i e t , the calcium requirement of growing chicks has been estimated a t approximately 0.65 percent of the r a t i o n , and the phosphorous requirement at 0,4 to 0.5 percent. These are minimal r e q u i r e -41 . ments, however, and some workers advocate l a r g e r amounts, es-p e c i a l l y of calcium. - M i t c h e l l , Card and Hamilton, working with White Leghorn c o c k e r e l s , c a l c u l a t e t h a t , at 0.5 l b . body weight,'-a d a i l y intake of 0.11 grams of calcium i s r e q u i r e d f o r growth and maintenance, while at 2-3 l b . , 0.24-0.25 grams i s necessary. The r a t e of. growth i s doubtless of primary im-portance i n determining the amounts of calcium and phosphor-ous required* The r a p i d c a l c i f i c a t i o n , which takes place during the f i r s t 6 weeks under optimum growth c o n d i t i o n s , n e c e s s i t a t e s a high d a i l y i n t a k e , w h i l e , with l e s s ' r a p i d growth, good c a l c i f i c a t i o n w i l l be secured with smaller am-ounts of these elements. Deobald et a l (35) summarized t h e i r work on the a v a i l a b i l i t y of calcium s a l t s f o r bone formation and r i c k e t prevention i n chicks as f o l l o w s : • 1. Chicks fed a b a s a l r a c h i t o g e n i c r a t i o n supplem-ented 'With a v a r i e t y of common calcium s a l t s and varying l e v e l s of cod l i v e r o i l showed th a t the,ash content of the f a t - f r e e bone was higher when.the supplement contained phos-phorous i n conjunction with calcium. The calcium i n these s a l t s appeared to be equal l y a v a i l a b l e f o r bone formation i n s p i t e of the d i f f e r e n c e s i n s o l u b i l i t y of the supplements. 2. When the l e v e l s of c o d , l i v e r o i l added to the r a t i o n were c r i t i c a l the s o l u b l e s a l t , calcium l a c t a t e , pro-duced b e t t e r growth and bone formation than the i n s o l u b l e salt", p r e c i p i t a t e d calcium s i l i c a t e . The d i f f e r e n c e was not 42 apparent, however,.when cod l i v e r o i l l e v e l s were higher, • 3. When extremely i n s o l u b l e calcium s i l i c a t e s were fed t h e i r a v a i l a b i l i t y was p r o p o r t i o n a l to t h e i r s o l u b i l i t y i n d i l u t e a c i d * Increasing the cod l i v e r o i l had no e f f e c t on the a v a i l a b i l i t y of the calcium i n such compounds. Wilgus (107) i n two experiments comprising 880 day-old White Leghorn ch i c k s d i v i d e d i n t o twenty experimental l o t s and c a r r i e d out over a p e r i o d of s i x t e e n weeks, con-cluded t h a t the c h i c k ' s requirement f o r calcium and phos-phorous ,is s a t i s f i e d i f the r a t i o n , s u i t a b l e i n a l l other respects? contains: at l e a s t 0„67 percent. calcium and 0.50 : percent phosphorous? with a Ca:P r a t i o varying from 1 to 2 92:1. A r a t i o " o f 2«5:1 appears b o r d e r - l i n e , judged by the widening of the Ca:P r a t i o t h a t occurs i n the blood serum and the cheek i n growth, and on s i m i l a r evidence a Ca:P ra.tio of 3,3:1 i s d i s a s t r o u s . This l a t t e r f i n d i n g c o n f l i c t s with the conclusions of Bethke and h i s a s s o c i a t e s , who found the optimum r a t i o to l i e between 3:1 and 4:1, and those of Hart and h i s co-workers, who found the optimum r a t i o to l i e be-tween 2:1 and 4:1. The i n f l u e n c e of e r g o s t e r o l on b i r d s f e d a rach-i t i c d i e t was demonstrated by King and H a l l (64). They showed tha t normal b i r d s could be produced by the a d d i t i o n of 1 drop d a i l y of 250 D e r g o s t e r o l per b i r d to an otherwise r a c h i t i c d i e t . The a d d i t i o n of oatmeal to the d i e t induced a more severe r a c h i t i c c o n d i t i o n of the b i r d s . Vitamin D 43 (in the f6rm of 1 drop of ergosterol---250 D daily) tended to overcome the ra.chitogenic Influence of the oatmeal. The ad-dition of vitamin A did not appear to improve the condition of the birds. Prolonged boiling of the oats with hydrochlor-ic acid (withoutsubsequent dialysis) appeared to render the anti-calcifying substance in the oats more available. Hall and King (49) conducted experiments with chi-ckens to demonstrate the effect of irradia.ted ergosterol on the calcium-phosphorous metabolism* Three groups of 16 week old chickens on a rickets-producing ration received varying daily doses of irradiated ergosterol (Group 1, normal, 0.008-0*032 milligrams; Group 2, hypervitaminosis, 0.60-3*60 mil l -igrams j Group 3, rachitic, basal ration only). • Group 3 developed 100 percent rickets, but was con-sistently heavier than Group 1 Which showed normal growth rate. Group 2 grew Well to the 9th.week, then rapidly de-clined with onset of cachexia and anorexia® The values for serum calcium and phosphorous of Groups 1 and 2 remained at a steady level after a preliminary decline, Group 2 showing a slight tendency to hyperCalcaemia in the later stagesj Group 3 rapidly declined from 15.5; mill-igrams calcium per 100 cubic centimeters serum to a f i n a l valir, ue of 7975 milligrams at 12 weeks. The inorganic phosphorous of the three groups remained steady for 9 weeks and then di-verged, Group 1 remaining steady at 6.5 milligrams, Group 3 rising to 8 milligrams and then becoming sub-normal, and • 44 Group 2 f a i l i n g to the low value of 3,75'milligrams at 15 weeks. The phosphatase content of the hone tissue by Kay's method was: Group 1, 1.97 units; Group 2, 0,33 Units; Group 3, 4,30 units. I t i s suggested that "leg weakness" i n conditions of hypervitaminosis D i s due to depletion of this enzyme in the bone tissue accompanied by hypercalcaemia, whereas in con-ditions of hypovitaminosis D the exact opposite is the case. No evidence of calcification of tissues of Group 2 was obtained except in the case of one bird. Hyperplasia of the testes of birds in Group 3 was noted. The changes in,bone tissue, :characteristic of conditions of hypo- and hyper-vitame inosis D, are carefully described. • • • Experimenting with modifications of the calcium-phosphorous of a specific ration for gro?;ing chicks, Mussehl and Ackerson.(79) concluded that a ration having a Ga:P ratio of 1:0.9 was not improved for growth to 8 weeks of age by ad-ditions of calcium carbonate or phosphate salt's, making ratios of 2:1 and 1:2 respectively. With heavy breeds the addition of dibasic sodium ortho phosphate gave the poorest growth. With single comb White Leghorns ammonium ortho phosphate was slightly detrimental, and sodium ortho phosphate more so. There were no marked differences in the bone analj^ses, but dibasic sodium orth© phosphate in the case of the heavy breeds, and sodium ortho phosphate in the Leghorns,, caused a; rise, in plasma inorganic phosphorous up to 7.7 and. 9,6 m i l l -45 igrams per 100 cubic centimeters r e s p e c t i v e l y . Griem et a l (47) studied the e f f e c t of calcium and phosphorous v a r i a t i o n s and sources of experimental chicks on the assay of v i t a m i n D carriers.. The e f f e c t s of. v a r y i n g .the calcium and phosphorous content of Hart, K l i n e , and Keenan's f a c h i t o g e n i c d i e t was studied by estimation of the ash con-te n t of the; bones of chicks^ R a i s i n g or, lowering the l e v e l s beyond those eommonly present : i n the commercial i n g r e d i e n t s c o n s t i t u t i n g the r a t i o n , had no appreciable e f f e c t on the development of r i c k e t s or the response to cod l i v e r o i l . Low phosphorous content (0.48 percent) and a Ca:P, r a t i o of .1,988 prevented optimum c a l c i f i c a t i o n at the higher l e v e l s of cod l i v e r o i l feeding. .•: Chicks from f i v e d i f f e r e n t commercial hatch e r i e s showed very good agreement i n t h e i r responses to the b a s a l r a t i o n alone or to three l e v e l s of cod l i v e r o i l supplements According to Querioda (.83) r a t s showed normal growth without the a d d i t i o n of v i t a m i n JD , .on a d i e t of low m i n e r a l content, c o n s i s t i n g of sodium chloride. 1, y e a s t 3, egg albumin 10, maizena 100, and containing 0.0 6 percent calcium and 0*12 percent phosphorous. V a r i a t i o n of the amount of calcium by • adding calcium carbonate, and of phosphorous by adding d i b a s i c sodium ortho phosphate gave r e s u l t s confirming those of Shohl et a l * With a d i e t containing 0.12 percent phosphorous and a Ca:P r a t i o of 1, r i e k e t s could h a r d l y be prevented w i t h a l -most t o x i c amounts of v i t a m i n D, up to 5 V of c a l c i f e r o l d a i l y ; 46 w i t h a r a t i o of Ca:P of 4 the s e v e r i t y of the r i c k e t s i n -creased hut could be prevented by r e l a t i v e l y small amounts • < of v i t a m i n D (0.06^ of c a l c i f e r o l ) . With a d i e t containing o,-35 percent of phosphorous and,a Ca:P. r a t i o of 1, no v i t - . . amin D was re q u i r e d ; with a Ca:P r a t i o of 4'. and the same am-ount of phosphorous, r i c k e t s occurred but could be prevented by 0.016 to 0,033^ of c a l c i f e r o l d a i l y . No r e l a t i o n was found between the s e v e r i t y of r i c k e t s and the ash content, of the bones. I f , before the p e r i o d on ra c h i t o g e n i c d i e t with 0,12 percent of phosphorous -and a Ca:P r a t i o of 1,. the r a t s were given d i e t s r i c h i n calcium (0,7 to 0.3 percent) and phos-phorous (0,6 percent) the otherwise almost unpreventible r i -ckets coulc be prevented by the l a r g e dose of 1^ d a i l y of c a l c i f e r o l . • -• I t i s suggested i n the d i s c u s s i o n , t h a t r i c k e t s i n r a t s produced on a d i e t containing 0,2 percent phosphorous , and a Ca:P r a t i o of 1, more, c l o s e l y resembles the human d i s o r -der, .than r i c k e t s produced with a Ca:P r a t i o of 4, The e f f e c t of v i t a m i n D intake of.the hen on the. bone c a l c i f i c a t i o n of the chick was shown by Murphy, Hunter and Knandel (78). From the bone photographs and bone ash de-terminations of a t o t a l of 346 sets of bones evidence was se-cured to i n d i c a t e t h a t v i t a m i n D i s transmitted i n a quanti-t a t i v e way from the hen to the chick. Evidence to show the vit a m i n D reserve of the chick i s depended upon the vi t a m i n D intake of the parent stock can be secured when the chicks are 47 from one to f o u r weeks o l d . The r e s u l t s of minor v a r i a t i o n s i n the calcium-* phosphorous-vitamin D complex were reported by Dovms (38).. Groups of, young r a t s were fe d f o r 28 days on a s e r i e s of 33 d i e t s i n which the main v a r i a b l e s were the Ga:P r a t i o and the presence or absence of v i t a m i n D. At the end of t h i s p e r i o d there were no.gross d i f f e r e n c e s ' i n the appearances of the jaws or t e e t h i n any of the groups 5 and i n no animals was d e n t a l c a r i e s found. S l i g h t h i s t o l o g i c a l changes i n the d i r -e c t i o n of d e f e c t i v e c a l c i f i c a t i o n were found i n animals whose d i e t s had had a Ca:P r a t i o greater than 2.0 or l e s s than 0.5, The a d d i t i o n of v i t a m i n D to d i e t s low i n phosphorous intens-i f i e d the changesj whereas i t s a d d i t i o n to d i e t s low i n c a l -cium prevented the changes. - - The s i g n i f i c a n c e of the calcium and phosphorous con-t e n t , of food was shown by Waltner (105). He found t h a t r a t s weighing 36 to 100 grams were fe d f o r a l t e r n a t i n g periods of 1 to 14 days on a high Ca: low P d i e t and on a low Ca: high P d i e t over a t o t a l p e r i o d of 40 weeks. When no v i t a m i n D was fed, the animals gained weight on the f i r s t d i e t s but l o s t on the second.• This e f f e c t was apparent even when the d i e t s were changed d a i l y , though i n t h i s case the r e s u l t i n g g a i n i n wei-ght a f t e r 16 weeks d i d not f a l l much below that of c o n t r o l r a t s r e c e i v i n g optimum amounts o f calcium and phosphorous each day. When the d i e t s w.er'e changed every three days, every week or every f o r t n i g h t , the weight f l u c t u a t i o n s were more marked, 48 and In the last two groups the total increase after 16 weeks was insignificant. When vitamin D in addition was fed, flue-tuations in weight were no longer observed, though the rats receiving the low Ca: high P diet for a fortnight at a time, did not increa.se in weight as much as the controls. It is therefore concluded that when the vitamin D intake is defic-ient, periods: of deficient calcium Intake may have a deleter-ious effect on the organism, even though the average calcium intake i s the optimum, Querido (82) working on the influence of calcium and phosphorous of the diet on the vitamin D requirements, formulated several opinions. He suggests that the develop-ment of rickets in rats, on a diet with a Ca:P ratio of 1 and a phosphorous content,of 0.12 percent, could not be com-pletely; prevented by administration of as much as .5. of cal-c i f e r o l daily. On a diet with a Ca:P ratio of.4 and.the same phosphorous content, 0.06^ of calciferol was an adequate pro-phylactic dose. The rats did not develop•;• rickets when the CatP ratio was 1 and the phosphorous content 0.35 percent. Mild rickets, developed with this amount of phosphorous and a Ga:P ratio of 4, but vvas readily preventible by minute doses of vitamin D, Heubner (56) worked with various types of expert-menta.l animals to show their reaction to treatments of irrad-iated ergosterol. Preparations of known antirachitic potency were administered and determinations, made of the Inorganic . 49 phosphate 'in the blood (modified Bell-Doisy-Briggs method) and the serum calcium (method of de Waard or after ashing). Gats receiving doses of 50,000 "rat units" showed a rise in their blood phosphorous in proportion to the dose, and a max-imum was reached in about 2 days. A similar rise was obser-ved with rabbits, and in spite of considerable'.individual var-iation was generally;proportional to the dose. With weaker antirachitic preparations, the effect on the blood phosphate Was not apparently parallel with the antirachitic potency. No characteristic changes in the ordinary calcium, balance were caused in dogs or rabbits by the administration of i r -radiated ergosterol. There was, however, an increased excre-tion of calcium in the urine with the appearance of a milky precipitation of Cadcium salts. No change in the anatomy or in the calcium content of the organs 'was observed in rabbits treated with irradiated ergosterol. Similar' work with toxic and sub-toxic doses was car-ried out by Freeman and Farmer (43). Sub-toxic doses of i r -radiated ergosterol, fed to dogs on a diet of commercial dog food and bone meal (diet A), had l i t t l e effect on serum phos-phatase, Hypercalcaemia and/or hyperphosphataemia, following administration of toxic doses, was accompanied by a decrea.se in serum phosphatase activity. Substitution of a bread and corn meal diet (high carbohydrate) for diet A in cases of hy-percalcaemia caused a drop in serum calcium co-incident with elevation of serum phosphatase. In dogs not receiving irrad-50 iated ergosterol a beef heart (high protein) diet induced a low? and a bread and corn meal diet a high? phosphatase act--a i v i t y . The highest acid soluble organic phosphorous values for whole blood were associated with the lowest serum phos-phatase activity and vice versa. In their studies on hypervitaminosis D? Harris? Harris and Maitland (52) noted the"influence of variations in the calcium and phosphorous Intake upon the effects produced by overdose of irradiated ergosterol on rats. The i l l effects with deposition of calcium in the organs were enhanced by an increase In the calcium or i n the Ca:P ratio of the diet. On diets almost devoid, of calcium or phosphorous? symptoms also developed i f sufficient irradiated ergosterol was given but cal-careous deposits in the soft tissues did not then occur; there was? however, great rarefaction of the bone. Excess of vitamin D was Interpreted as promoting hypercalcaemia through Increased absorption of calcium from, the gut? or i f the cal-cium in the diet were low, by resorption of calcium from the bones. With increasing doses urinary excretion overtook gut absorption and with very high doses? when the general cond-ition of the animals became bad and the food intake began to f a i l , gut absorption also was diminished. 51 ETIOLOGY OF RICKETS. Ia their work with nutritional conditions in chick-ens? Hughus and Titus (60) asked the question "should leg weakness in growing chicks be called rickets?" They summar-ize their findings into the following points: 1. The preponderance of evidence indicates that the etiology of leg weakness in chicks and rickets in mammals is the same. 2. The chemical analysis of the blood shows the two conditions to be alike so far as the calcium and phosphorous content is concerned. 3. The general pathology of the two conditions is alike in that i t is characterized in both by a faulty metab-olism of calcium and phosphorous resulting in an improper cal-cification of the bones. 4. It is suggested that the difference in the hist-ological bone lesion in leg weakness in growing chicks and rickets in rats described by Pappenheimer and Dunn may be due to the difference in the two species of animals. 5S If the term rickets can properly be used to des-ignate any disease in.a growing organism characterized by a faulty metabolism of calcium and phosphorous resulting from a lack of vitamin D and ultra-violet light, then' the experiment-a l data justify diagnosing leg weakness as rickets. Smellie (94) briefly reviewed the pathology of di f f -erent types of rickets. In his interpretation, he said that 52 simple rickets i s attributed to failure of phosphorous absor-ption and renal rickets to failure of phosphorous excretion,, The bone changes in renal rickets are of the experimental low-calcium rickets type, and i t is possible that calcium is mob-il i z e d from the bones to maintain the normal serum calcium level as a, defensive mechanism against tetany. The acidosis associated with chronic nephritis may be another factor tend-ing to raise the^ serum calcium in renal rickets. Renal rick-ets and experimental " strontium rickets" resemble each other in the histological bone changes, and in the fact that neither respond to the administration of vitamin D. Administration of vitamin D is contra-indicated In renal rickets, since, by in-creasing phosphorous absorption, i t w i l l aggravate the bone lesions, • B i l l s et al (14) made an attempt to produce a curve from which.the potency of an antirachitic substance, compared with a standard cod liver o i l can be assessed from the results of line tests made with a single dose. The probable error of assay with the "line test" was estimated as 31 percent v/hen 4 rats are used, 10 percent with 20 rats, and 4 percent with 100. I t appeared that neither sex, colour, weight, nor the total amount of food Ingested during the test were of impor-tance. The amount of food taken on the f i r s t day of the test, however, was of significance. Five days was the optimum dur-ation of the test. The alternative methods of either giving the curative substance as a proportion of the basal 53 d i e t or -of' administering i t as a separate dose produced e r r -ors of equal magnitude but of opposite e f f e c t . The; e f f e c t of an i n s u f f i c i e n t supply of-vitamin D on the growth of the s k e l e t o n and i n t e r n a l organs of chickens was demonstrated by Shelby and S h e l l (91). They concluded t h a t a d e f i c i e n c y of the a n t i r a c h i t i c f a c t o r produces i n fowls a c o n d i t i o n , 'known'-as l e g weakness, s i m i l a r to r i c k e t s i n mam-• mais. I n the present i n v e s t i g a t i o n the e f f e c t s were studied of l i m i t i n g the v i t a m i n D supply of young chickens to a quan-> t i t y s u f f i c i e n t to prevent obvious r i c k e t s but i n s u f f i c i e n t f o r a l l b o d i l y requirements. Under such conditions the chick-ens developed crooked breasts, reduced s i z e of skeleton and i n t e r n a l organs, and softening, of the bones. The chest c a v i t y was, d i s t o r t e d and reduced i n s i z e and the lungs under-develop-"- ed. A subsequent p l e n t i f u l supply of v i t a m i n D f a i l e d to i n -duce normal development, although the p r e v i o u s l y formed s o f t bone hardened to normal consistency. I t i s concluded t h a t chickens, which s u f f e r at an e a r l y age from ;:even a s l i g h t degree of v i t a m i n B d e f i c i e n c y , develop- body organs sub-normal both i n s i z e and i n e f f i c i e n -cy, and do not subsequently reach the l i m i t of t h e i r i n h e r i t -ed c a p a c i t y , e i t h e r as egg producers or as carcases of meat. Dhar (36) studied the occurrence and e t i o l o g y of r i c k e t s . H i s conclusions were t h a t r i c k e t s i s due to a de-crease i n the pH of the blood, t h i s l e a d i n g to a decrease i n the degree of supersaturation of calcium phosphate and a dim-54 i n i s h e d tendency to bone formation* He suggests t h a t the an-t i r a c h i t i c a c t i o n of v i t a m i n D i s due to i t s a s s i s t i n g the complete o x i d a t i o n of f a t s and preventing the accumulation of thp acids which.are formed by incomplete o x i d a t i o n * Photographic recording of l i n e t e s t s were o u t l i n e d by Stevens and Nelson (99) and Bacharach et a l (6), The l a t -t e r authors give a d e t a i l e d account of the technique of the photographic recording of r e s u l t s i n l i n e t e s t s f o r vi t a m i n D. The bones were prepared by the ordinary method, except that a f t e r washing they were•"fixed" f o r a few minutes i n 25 per-cent sodium thio s u l p h a t e s o l u t i o n . F i l m packs were used i n preference to p l a t e s , and an I l f o r d micro 5 Orange f i l t e r was found to give good c o n t r a s t without s a c r i f i c e of d e t a i l . The method seemed1'to permit of diagnoses, at l e a s t as accurate as those made by d i r e c t observations on the bone sec t i o n s . S i m i l a r work was conducted by McGowan and Emslie (74). They were p a r t i c u l a r l y i n t e r e s t e d i n the nature and pathogenesis of r i c k e t s . I n t h e i r summary, they stated t h a t i t i s very u n l i k e l y , owing to the type of food u s u a l l y given, t h a t chickens w i l l s u f f e r from a d e f i c i e n c y of phosphate but almost c e r t a i n t h a t calcium w i l l be l a c k i n g . This l a t t e r de-f i c i e n c y i s exaggerated and aggravated by the osteoporotic c o n d i t i o n of the body of the chicken on hatching, a circum-stance which n e c e s s i t a t e s a very a c t i v e calcium metabolism to meet i t . Vitamin D has only a very s l i g h t i n f l u e n c e i n com-pensating f o r t h i s l a c k of calcium. -In the u s u a l amended 5 5 diet of chickens, large quantities of calcium, generally as calcium carbonate, are incorporated but, owing to the alkal-osis induced by such foods, the calcium is not absorbed and metabolised, and the condition of osteoporosis, already pres-ent, persists and. i s • exaggerated. It is doubtful whether1,' ev-en' i f regard be had to the exact chemical combination of cal-cium given and the amount of i t supplied, calcium in i t s e l f as such and,without vitamin D could be administered so as to avoid this complication. Administered in amounts larger than those associated,with the appearance of osteoporosis, calcium by interfering with the- absorption and metabolism:of phosphor ous, produces r i c k e t s " Vitamin D by i t s fundamental action prevents, on the one hand, the occurrence of osteoporosis, due to deficiency of available calcium, and on the other, rickets,-due to phosphorous deficiency, J,An important pre*-cursor of the condition, perosis, is the feeding of large: quantities of, calcium oxide and phosphate in chemical union with one another,, Thoenes (103) studied the question of hypervitamih-osis due to: vitamin D. In his work he found that rats fed on a diet containing only a small amount of.-vitamin A sometimes developed keratomalacia when given large doses of irradiated ergosterol, although controls on the diet without this addit-ion showed no symptoms of vitamin A deficiency. Vitamin A de ficiency and vitamin D overdosage led to the same type of calcium deposition in the kidneys-. It i s therefore suggested 56 that there i s an intimate relation between the functions of vitamins A and D, The toxic effects of irradiated ergosterol in the rat 'could be largely prevented by including butter in the diet, but this protective effect was not due entirely to vitamin A, for margarine had a similar, though much less pro-nounced effect. Working -with hens Seifried and Heidegger (90) con-cluded that excessive feeding of vitamin D to hens results in •abnormal calcium storage and a condition corresponding to hyp ervitamino sis D in mammals. Hens are less sensitive. De-ficiency of D also leads to excessive calcium.storage. The work of Ham and Lewis (50) was very interesting. They showed, that young rats, receiving very large daily doses of vitamin D showed rachitic lesions in their long" bones af-ter three "weeks. As the matrix which formed in the bones during the experiment was very poorly calcified i t was con-cluded that the administration of large amounts of vitamin D inhibited the normal calcification process in bone. As os-teoclasts did not form a prominent part in the histological picture, the poor calcification of bone could not be attrib-uted to them. The phenomena observed in this experiment can best be explained, i t is thought, by the theory which considers vitamin D to act by increasing in some way the attraction of the blood for calcium. The results are compatible with, a l -though they do not directly support, the theory that vitamin 57 D acts through the intermediary of the parathyroid mechan to control a fraction of the serum calcium. DETERMINATION OF VITAMIN D. Investigations v/ere conducted by Schultzer (89) on the comparison between preventive and curative methods in the determination of vitamin D. Groups of about 10 rats, not s t r i c t l y comparable in l i t t e r composition, were kept on McCol-lum's rachitogenic (3143) diet and received graduated doses of the same cod-liver o i l throughout, both in preventive and in curative tests* In the case of the former tests, the same an-imals were examined by X-ray, microscopically at autopsy, and by.estimating the percentage ash in the. fresh bone. In the case of the curative tests, the same examinations were made but, in addition, an X-ray photograph was taken at the begin-ning, of the curative period 1as well as at the end of the ex-periment, and the line test was also -carried,•,©Uty.V,-;. ; It was concluded that the preventive method was su-perior to the curative, i t being claimed that the former gave less variation in the responses of individual rats to the, same dose of cod-liver o i l , and that bone analysis did not give more uniform results than X-ray examination. Key and Morgan (63) extended and confirmed the work of Dyer, viz. the comparison of the antirachitic potency of two substances by f i r s t determining the average amount of healing produced by a convenient dose of each when given to several rats (assessed by comparison with a, scale showing gra-ded degrees of healing) and then, determining the relative am-[ ounts of vitamin D which would bring about these average am-59 ounts of nealing (by reference to a curve showing the relat-ion between degree of healing and dose of vitamin D given) e The authors show how small an alteration in the Ca:P ratio of the diet may give a result which may be erroneously ascribed to the presence of vitamin D. The antirachitic factor balance in the growing chick was studied by Klein and Russell (68). Two groups of newly hatched White Leghorn chicks were kept for 4 weeks on the same basal ration,with supplements of 23 rat units (1 cubic centi-meter) cod- liver o i l , and 75 rat units (1cubic centimeter) corn o i l solution) of irradiated ergosterol respectively, per .100 grams food. The amounts of antirachitic vitamin present in the, bodies of the chicks, and that present, in the collected droppings.,, were then estimated by assay on rats. Ho trace of the antirachitic factor could, be .found in the bodies of either group of chicks, but 43.1 percent in rat Units of the vitamin D fed as cod-liver o i l , and 26.5 percent in rat units of that fed as, irradiated ergosterol, was recovered from the drop-pings*1 The experimental data do not account for the fate of the remainder of. the antirachitic vitamin fed to the chicks. Amounts of the antirachitic factor of less than 1 rat unit per chick were detected in the bodies of unfed newly hatched chicks similarly investigated. In their studies relative to the estimation of v i t -amin D, Lachat, Halvorson and Palmer (70.) found that day-old chicks were suitable animals for testing the,vitamin D content 60 of .food'"materials. T h i r t e e n r a c h i t o g e n i c d i e t s were tested. Those cont a i n i n g skim m i l k or "buttermilk powders allowed poor growth and appeared to r e t a r d the development of r i c k e t s . The most s a t i s f a c t o r y , which was used i n the subsequent exper-iments, c o n s i s t e d of yellow corn, 59; wheat f l o u r middlings, 25; crude c a s e i n , 12; calcium carbonate, 2; d r i e d baker's yeast, 1; sodium c h l o r i d e , 1, An experimental p e r i o d of 4 weeks was found to be as s a t i s f a c t o r y as one of 5 weeks, neg-a t i v e c o n t r o l s averaging 34 percent and p o s i t i v e c o n t r o l s , 48 percent bone ash. The b a s a l d i e t of the negative c o n t r o l s was supplemented with 1 per cent corn o i l (mazola), and the same o i l was added to the f i s h o i l r a t i o n s to make them up to 1 percent of the d i e t . By t h i s method 22 commercial products were t e s t e d ( l l cod l i v e r o i l s , 4 cod l i v e r o i l concentrates, 2 p r o p r i e t -ary feed supplements, 1 sardine o i l , 1 burbot l i v e r o i l , 1 cod l i v e r meal, 1 mineral feed concentrate, 1 cod l i v e r o i l stear-ine) and considerable v a r i a t i o n was found i n the vit a m i n D content, /' The q u a n t i t a t i v e determination of v i t a m i n D by means o f i t s growth-promoting property was conducted by Coward, Key and Morgan (31), A method has been worked out f o r the quanti-t a t i v e determination of v i t a m i n D by means of growth curves of young r a t s on a p l a n s i m i l a r 'to that used f o r t e s t i n g v i t a m i n A, The r e s u l t s of the growth t e s t v/ere found to be comparable with-those obtained by the l i n e t e s t but the l a t t e r i s more 61 suitable"for routine testing as the preparatory period is on-ly 3-3-2 weeks and the test period 10 days, whereas in the growth test, the preparatory period may he 1-3 months and the te,st period is 5 weeks. In the growth test bucks respond more readily to the dose of vitamin D. It "was calculated that the accuracy of the method for vitamin D was only about one third of that for Vitamin A with bucks, while the degree of accuracy was slightly greater in vitamin D tests than in vitamin A "when do-es were the test animals. A comparison was made by Griem (46) of the antirach-i t i c activity of two proprietary feeds, claiming to contain vitamin D, by feeding young chicks with the mash i t s e l f or with the o i l extracted from the mash. The ash content of the fat free tibiae of the chicks, after a 4 weeks' prophylactic test, was the criterion of vitamin D potency. An unexplained loss of activity was found for both samples in the extracted o i l , as compared with the mash i t s e l f , so that feeding of the mash' was recommended as the better method* Randoin (84) in his report states that the biolog-i c a l activity of the original international standard of v i t -amin D was found to be the same as that of the second one, i s -sued in 1932. The relationship between the international unit of vitamin D and the physiological unit of Randoin was determin-ed on young rats receiving the rachitogenic diet of Randoin 62 and Lecoq, to which the test material was added after a week. The degree of rickets was assessed by X-ray, By this method one Randoin'unit was equal to 5 International Units, According to Beccari (10) intensive growth and change in ash constituents are not considered to be exact c r i t e r i a for vitamin D. Lachat and Halvorson (72) reported the effect of seasonal variation upon vitamin D assay. They found that the ash content of the tibiae and the growth response produced by doses of the U.S.P, reference cod liver o i l showed that chicks required slightly less vitamin D during late summer than dur-ing the spring, The use of a standard preparation of vitamin D in a l l tests on poultry is recommended® 63 EXPERIMENTAL In papers and reports by Asmundson, Allardyce and Biely (5) 3 Asmundson and Allardyce (4) and Biely and Palmer (.12.) ? i t was shown that British Columbia Pilchard O i l is a valuable source of Vitamin D. These investigators found that chicks fed on a Vitamin D free ration containing one half of one percent Pilchard O i l showed normal calcification. The purpose of the present experiment was to find out whether or not a significant difference would result between the average weights and percentage of ash of different lots of chicks receiving graded amounts of fish o i l at closer levels than those used by earlier investigators. These have shown that significant differences could be recorded In the average weights and calcification of chicks receiving 1/8%-1/4%-and 1/2% of fish o i l . I t was thought that the points of differentiation might be even closer, and on this assumpt-ion, i t was decided to lengthen the scale of gradation by injecting quantities of 3/16% and 3/8% of fish o i l . The com-plete series therefore included tests with l/8%-3/16%-l/4%-3/8%-1/2% and 1% of both'Reference Cod Liver O i l and Pilchard Oi l . It was deemed advisable in the present experiment to ascertain, i f possible, a significant* difference among lots which received 1/8% and 3/16% and 3/16% and 1/4% fish o i l res-pectively. 64 The same method as employed by Biely and Palmer (12) in brooding and feeding the chicks was used i n this experiment and the data* consequently may be comparable. Experimental Material and Methods % Day-old single comb White Leghorn chicks were placed in battery brooders under s imilar conditions as described by Biely and Palmer (12). The basal rationrecommended by Hart, Kline and Keenan (54) was fed in this experiment. I t consisted of J Yellow corn Wheat.middlings Crude casein Calcium carbonate Calcium phosphate Salt Yeast In this ration the supply of calcium and phosphate is so regulated that in the absence of vitamin D, rickets may easily develop in three weeks, while, i f this vitamin is supplied i n satisfactory quantities, normal bone calcification takes place. 59 Parts 25 . 12 1 1 1 1 6 5 ; As indicated above, the basal ration was supple-mented as follows: Lot 1 1 / 8 % Reference Cod Liver Oil n 2 3 / 1 6 % tt n I I ti 3 1 / 4 % tt n it i i ti 4 3 / 8 % u tl ii it it 5 . 1 / 2 % it tt n it it 6 1 / 8 % Pilchard O i l ( 1 0 ) x t! 7 3 / 1 6 % u t.i tt tr 8 1 / 4 % tl: ti t* tr Q 3 / 8 % tt ti it ri 1 0 1 / 2 % tt t! u I! 1 1 1 % tt 11 ti tl 1 2 1 / 8 % Pilchard O i l ( 1 4 ) x It 1 3 1 / 4 % tt u. tt 1.1 1 4 1 / 2 % !t it it IT 1 5 1 % tt » ii tt. 1 6 Control The control chicks received the basal ration to which was added 1 / 2 % of Wesson O i l . The supplements of Lots 1 - 4 receiving 1 / 8 % , 3 / 1 6 % , 1 / 4 % and 3 / 8 % Reference Cod Liver Oil respectively were diluted with Wesson O i l to 1 / 2 % . Similarly the supplements of Lots 6 - 9 which received 1 - 8 % , 3 / 1 6 % , 1 / 4 % and 3 / 8 % Pilchard O i l ( 1 0 ) were diluted to the same level. Lots 1 2 and 1 3 which were supplemented with x The two f i s h oils used, and designated by numbers 1 0 and 1 4 respectively, were obtained from the Western Chemical Ind-ustries. 66 1/8% and .1/4% Pilchard O i l (14) were treated in a like manner. No Wesson O i l was added to Lots 5, 10, 11, 14 and 15 as they already contained 1/2% or 1% of o i l . As soon as the chicks had been placed in the brooders, they were supplied with feed and water. For the duration of the experiment they had free access to the feed and water at a l l times. To insure ample light during the experimental period, a r t i f i c i a l illumination was provided by means of electric lights. In this way the chicks had twelve hours of light and twelve hours of darkness. When a day old, the chicks were banded on the leg, weighed and placed in the battery brooders. Each succeeding week the birds were weighed and the weights recorded (Table 1 and Appendix). The method of assay was essentially the same as recommended by Lachat (69). The chicks were killed at the end of the experimental period and the right and l e f t tibiae of each bird removed and cleaned. The bones of each tibiae were then cut into three or four pieces and placed in individual extractionthimbles in a Soxhlet apparatus. The fat was extracted by the use of hot 95% ethyl alcohol for 48 hours with a change of alcohol after 24 hours, ' The fat-free bones, s t i l l i n the thimbles,were then dried in a moisture oven, transferred into crucibles, weighed and ignited in a muffle furnace at approximately 850 degrees Centigrade for one hour. The percentage of ash was determined after reweighing the 67 the moisture and fat-free bones. The average weight of the tibiae and the average percentage q»f ash was determined for each chick. 68 Statistical Material and Methods: The weights of the chicks and the average weight of their tibiae before and after ashing were analysed by coyariance. Fisher's (41) definition of variance as described by Snedecor (96) "is. a technique for segregating from compar-able groups of data the variation traceable to specified sources". This is a term applied to a st a t i s t i c a l method used to reduce the data from experiments involving one variate measured in several groups. Following from this, the reduc-tion of data from experiments involving two or more variates measured in several groups would be called "covarlance". The purpose, then, of the present work is to find the cause of the variation in the percentage of the ash of the chicks at four weeks of age. The subsequent calculations illustrate the method of procedure." From these are derived Tables 10-13. For convenience the following abbreviations are used: N = number of chicks in each lot. 3W = summation of the weight of each chick. • SB = summation of the average weight of the tibiae of each chick. SA - summation of the average weight of the ash of each chick, Si'f3 » summation of the weight of each chick squared. SB2 = summation of the average weight of the tibiae of each chick squared. SA2 r summation of the average weight of the ash of 69 . of each chick squared. SWB - summation of the weight of the chick times the average weight of its tibiae. S\VA = summation of the weight of each chick times the average weight of its ash. SBA = summation of the average weight of the tibiae of each chick times the average weight of its ash. (SW)2 = (SW)CSW) ; N N (SB) 2 = (SB) (SB) . F N (SA) 2 = (SA)(SA) •N N S(W) (B)- (SW)(SB) N N S(W) (A) = (SW)(SA) N. N S(B)(A)= (SB)(SA) N N df - degrees ; Using the above terms the follo\¥ing records were obtained: Lot; SW SB SA 1 14 2548 7.3670 : 3,0201 '2 12 . 3002 •9.0602 4.0918 3 12 2288 7.1012 3.0886 4.- 13 2998 9.6299 4.3951 5 13 3214 10.4969 4.8838 6. 16 2782 8.4172 2.8981 7 14 2258 6.6091 2.4187 8 18 4100 12.4836 5.0676. 9 15 3304 9.7150 4.2346 10 18 . 43.14 13.9745 6.4306 11 15 2996 9.4925 4.3321 12 ' " 13 . 1994 .": 5.4848 1.7919 13 12 2296 ; 6.6473 2.3202 14 13 2382 . 7.2299 . 3.0331 15 13 2594 .. 7.9563 3.6098 16 r ' ' 13 • 1906 5.5236 1.7939 224 44.976 -. 137.1890 57.4100 71 Lot S W 2 S B 2 S A 2 1 : '495088 4.26032308 0.72100327 2 .764372 7.01415556 1.44669358 3. 449472 4.41014192 0.83677936 4. 732308 . 7.73208445 ' .1.62103927 5 : 819788 8.75291019 1.89681890 6 507092 ' 4.82975194 0.57267717 7 375560 3.24100623 0.43437965 8 955664 8.90184004 1.48315152 9 754544 6.68836280 1.31218050 10 1074900 11.46097715 2.41861900 11 .. 663316 7.19167975. •1.50901199 12 322772 2 .49520320 0.26238975 13 448136 3.77268201 0.46073500 14 : 461780 4.27403067 0.75385645 15 534780 '5.09785377 1.05135798 16 285044 2.37598588 0.25062475 9644616 92.49898864 17.03131814 72 Lot SIB SWA SBA 1448o2666 . 457.4700 1.74542738 / 2 2311.7914 1045.3008 •. 3.17848443 3 1401.1730 607.5248 1,91721752 4 2370.4226 1053,8016 3.53786584 5 2667.5436 ' 1239.7348 4.06900934 6 1557.2120 536.6180 1.65983324 7 1099.6612 610,3910 1.18300145 8 2909.9286- 1195.3624 :3.62109560 9 .2238.6636 984.7952 2.95338065 10 .3500.7562 ' 1608,0416 5,26229030 11 2171.0504 953.3136 "3.29335456 , ' 12 891.9864 278.2842 0.80672487 13 1297.8308- 453.2754 1.31586280 .'• 14 1354.5456 569.9268 1.78749421 . 15 1648.7556 747,9102 2.31354517 16 816.2254 264.1372 0.76917636 29685.8130 -12605.8876 39,41376372 The correction terms obtained (for detail explanation see Page 85 ) were as follows: for W : (44976)2 ©.030,538.29 224 ' for B : (137.1890) 2 - 84.02152554 ' 224 for A : (57.4100)2 =. 14.71387544 .224 f ° r im.y : (44.976) (137.1890) B- 27,545.5913 224 . for WA : (44976)(57.4100) = 11,527.1078 224"' .. for B4 : (137.1890)(57.4100) = 35.16080575 224 ; •* . 74 Lot (SW) 2 (SB) 2 (SA)2 K N 1 463,736.00 3.87662064 0.65150028 2 751^000.33 6.84060200 1.20916708 3 436,245.33 4.20225341 > 0.79495415 4 691,384.92 7.13345953 1.48591569 5 . 794,599.69 8.47576230 1.83473092 6 .•• 483,720.50 4.42807849 0.52493647v 7 364,939.14 3.12001448 0.41786497 8 933,888.89 8.65779272 1.42669988 9, 727,761.07 6,29208166 1.19545580 10 1,033,922,00 10.84925834 2.29736755 11 598,401.67 6.00717041 1.25113933 12 305?848.92 2.31407931 0.24699273 . . f 13 439,301.33 3.68221644 0.44861066 14 436,455.69 4.02088107 . 0.70766889 15 517,602.77 4.86943920 1.00185838 16 279,448.92 2.34693515 0.24754440 9,258,257.17 87.11664515 15.74240718 75 Lot S(W)(B) N S(W)(A) K S(B)(A) N 1 1340.7940 549.6582 1.58921976 2 2266.5600 1023.6319 3.08937716 1353.9621 588.8931 1.82773052 4 2220.8030 1013.5776 3.25572103 5 ,2695.1566 1207.4256 3.94344309 6 .1463.5406 503.9071 • 1.52461795 7 1065.9534 597.9400 . 1,14181644 8 2843.4866 1154.2868 ,3.51454952 • 9 . 2139.8906: 932.7412 2.74260926 10 3349.2218 1541.2004 4.99246776 11 1895.9686 852.6477 2.74149728 12 841.2839 274.8499 0.75601639 t: 13 1271.8500 V 443.9316 1.28525545 14 1324.7401 555.7572 .1.68684689 15 1587.5878 720.2939 2.20928090 16 809.8447 263.0133 0.76221431 28370.6438 12153.0584 37.06266371 76 Sum of squares and products between lots gave the following results: For W. :•• 9.,258,257.17 -9,030,538.29 227,718.88 For B s 87.11664515 ^84.02152554. 3.09511961 For A : 1 5 • 7 4 2 4 0 7 1 8 • -14.71387544-1.02853174 For WB : 28,370.6438 -27,545.5913 825.0525 For WA : 12,153.0584 -11,527.1078 625,9506 For BA : 37.06266371 -35.16080575 1.90185796 7 7 Lot S W 2 - ( S 1 ) 2 S B 2 - ( ; S B ) 2 S A 2 - ( S A ) 2 U N N •1 3 1 . 3 5 2 , 0 0 0 . 3 8 3 7 0 2 4 4 0 . 0 6 9 5 0 2 9 9 2 ' 1 3 , 3 7 1 . 6 7 0 . 1 7 3 5 5 3 5 6 0 . 2 3 7 5 2 6 5 0 ' 3 1 3 , 2 2 6 . 6 7 0 . 2 0 7 8 8 8 5 1 0 . 0 4 1 8 2 5 2 1 4 4 0 , 9 2 3 . 0 8 0 . 5 9 8 6 2 4 9 2 0 . 1 3 5 1 2 3 5 8 5 2 5 ^ 1 8 8 e 3 1 0 . 2 7 7 1 4 7 8 9 0 . 0 6 2 0 8 7 9 8 6 2 t 3 5 3*71 © 5 0 0 . 4 0 1 6 7 3 4 5 0 . 0 4 7 7 4 0 7 0 •7 1 0 , 6 2 0 . 8 6 0 . 1 2 0 9 9 1 7 5 0 . 0 1 6 5 1 4 6 8 / 8 2 1 , 7 7 5 . 1 1 0 . 2 4 4 0 4 7 3 2 0 . 0 5 6 4 5 1 6 4 . 9 2 6 , 7 8 2 , 9 3 0 . 3 9 6 2 8 1 1 4 , 0 . 1 1 6 7 2 4 7 0 1 0 4 0 , 9 7 8 . 0 0 0 . 6 1 1 7 1 8 8 1 0 . 1 2 1 2 5 1 4 5 1 1 . * 6 4 , 9 1 4 . 3 3 , 1 . 1 8 4 5 0 9 3 4 0 . 2 5 7 8 7 2 6 6 1 2 1 6 , 9 2 3 . 0 8 0 . 1 8 1 1 2 3 8 9 6 . 0 1 5 3 9 7 0 2 . • 1 3 8 , 8 3 4 . 6 7 0 . 0 9 0 4 6 5 5 7 0 . 0 1 2 1 2 4 3 4 1 4 2 5 , 3 2 4 . 3 1 , 0 . 2 5 3 1 4 9 6 0 0 . 0 4 6 1 8 7 5 6 1 5 1 7 , 1 7 7 . 2 3 0 . 2 2 8 4 1 4 5 7 0.0494.9960 , 1 6 5 , 5 9 5 . 0 8 0 . 0 2 9 0 5 0 7 3 0 . 0 0 3 0 8 0 3 5 3 8 6 , 3 5 8 . 8 3 ^ 5 . 3 8 2 3 4 3 4 9 ' 1 . 2 8 8 9 1 0 9 6 78 Lot £WB~S('€) (B) H Sm-S(W)CA) N SBA-S(B) (A) 1 107.4726 -92.1882 0.15620762 2 • 45.2314. • 92.3660 0.08910727 3 47.2109 18.6317 0.08948700 4 149.6196 40.2240 0.28214481 5 72.3870 ' 32.3092 0.12556625 6 , 93.6714 32.7109 0.13521529 7 33.7078 12.4510 0.04118501 8 66.4420 41.0756 0.10654608 9 • 98.7730 52,0540 0.21077139 10 151.5344. 66.8412/ ' 0.26982254 11 275.0818 100.6659 . 0.55185728 12 50.7025 \ 3.-4343- 0.05070848 13 \ 25.9808 9.3438 • 0.03060735 14 . 29.8055 14.1696 ; . 0.10064732 15 61.1678 27.6163 0.10426427 16 .6.3807 1.1239 0.00696205 1315.1692 - 452.8292 2.35110001 79 Total sum of squares and products resulted in for W : 9,644,616.00 386,358.83 -9,030 ,,538^ 29 227,718.88 614,077.71 -614,077.71 for B 92.49898864 -84.08152554 8.47746310 5,38234349 3.09511961 8.47746310 for A 17.03131814 -14.71387544 2.31744270 1.28891096 1.02853174 2.31744270 for IB 29,685.8130 •27,545.5913 2,140.2217 1,315.1692 825.0525 2,140,2217 for WA 12,605.8876--11,527.1078 1,078.7798 625.9506 452.8292 1,078.7798 for BA 39.41376372 -35.16080575 4.25295797 2.35110001 1.90185796 4.25295797 BCL i-H m >! 4 n o O: Si H 13 <; H I O PQ o CO 03 fa O M W CO fa g m o o CO H <ti! o PQ i • * M PQ H lO C5 H m co -P: o d 13 o k a 0: CO CD cd d ol CO o CO ita' PQ d o o •H o> -p o d o ctf ca > co H IN 0s o a> o o o 10 o LO 00 I—1 0> H H CO o LO LO 0s CO CXI * e H cxj CO CO 00 o 0s CXi IN ov 00 IN e • • LO •PS CO CXI LO !N CO O LO CO .Is-CO: 0) H LO CO • CO o H' CO » « • LO LO o CO H CO CO • H H CO IN H CO LQ CO CXj o d. CD CD IS •P CD PQ to 0s CO 00 CO •H •H O EN CO IN CO e H * H e CO H Ol o CO H 0s- CO ro H •sF CO H CO LO CO •IN 0s 00 IN O CO 0 « « CO LO CO 00 CO H oo 00 IN e e • CO 00 IN H LO IN IN CO o CO co 00 H CXJ CO CO LO CO ,co H o CO CO CO CO -p CO o -p H o H I—1 a -P o =i3 PQ < H d o o •H CD -p O cd U *H d h o aj CO > CO £N 00 CO LO CO CO o CD CO LO CO LO i—I CO -p o H d CD <D t> -P CD PQ (N 00 CO CD 0^  H CD O O CO 0s H IN 00 LO H 00 i—I co -P o H d •H 'Xi P •H CO Oi CO LO CD o o 0. o e H H H o LO CO CD H CD ^ t> CO CO 00 Q CD LO O CO LN CO o • 0 « o o IN CN O) CO Is-H ft CO :pq 81 Within W . Calculation of correlation coefficients. lots: A , B. A . Total: II. • •W. " B . 386,358.83 (WW) 1,315.1692 ( W B ) 452.8292 (WA) 621.5776 (al) 1,442.05244 (bl) 705.67835 (b2) 0.9120120 (cl) 0.6416934 (c2)| 5.38234349(BB) 2.35110001(BA^ 2.3199878. (a2) 2,63388702(b3)| 0.8926351(c3) 1.28891096(AA: 1.1353021 (a3) W. 614,077.71 783.63110 IB.. B , 2,140.2217 2,281.62681 0.9380244 .8.47746310 2.9116083 A. 1,078.7798 1,192.93329 0.9043086 4.25295797 4.43238469 0.9595191 2.31744270 1.5223149 8 2 Calculation of the standard partial reg ression coef f icients.v Within lots; • fim •¥ 0 . 9 1 2 0 1 2 / 4 B = 0 . 6 4 1 6 9 3 4 ( 1 ) 0 . 9 1 2 0 1 2 ^ 4 - /5AB = 0 . 8 9 2 6 3 5 1 ( 2 ) Divide (2)by 0 . 9 1 2 0 1 2 , subtract ( 1 ) from ( 3 ) '/3k% + 1 - 0 9 6 4 7 6 ^ 3 = 0 . 9 7 8 7 5 3 6 ( 3 ) A w + 0 - 9 1 2 0 1 2 / 4 B = 0 . 6 4 1 6 9 3 4 CD 0 . 1 8 4 4 6 4 ^ A B M 0,3370602 /3AB - 1 . 8 2 7 2 4 1 0 ' 0 . 9 1 2 0 1 2 / ^ W = - 1 . 8 2 7 4 1 0 -h 0 . 8 9 2 6 3 5 1 - - 0 . 9 3 4 7 7 4 9 . . A w - - 1 . 0 2 4 9 5 8 9 •Total: • • . /5AW •f 0.9380244/5AB - 0 . 9 0 4 3 0 8 6 ( 1 ) 0 . 9 3 8 0 2 4 4 ^ A ¥ '•+••' A B = 0 . 9 5 9 5 1 9 1 ( 2 ) •;' Divide ( 2 ) by 0.9380244,- subtract ( 1 ) from(3) /#AW 4- 1,.Q66070./3A-B- - 1 . 0 2 2 9 1 4 8 ( 3 ) . -r 0 . 9 3 8 0 2 4 / 3 A B = 0 . 9 0 4 3 0 8 6 'CD . 1 2 8 0 4 6 y^AB = 0 . 1 1 8 6 0 6 2 0 . 9 2 6 2 7 8 0 0 . 9 3 8 0 2 4 4 / | W = 0 . 9 5 9 5 1 9 1 - --0.9262780 = 0 . 0 3 3 2 4 1 1 /5AW = . 0 3 5 4 3 7 3 5 83 Calculation of multiple correlation coefficients. Within lots: , H 2 ,= (-1.0249589) (0.6416934) (.1.827241) (0.8926351) = 0.97335009 E = 0.9865 Total: R 2 r (0.03543735)(0.9043086) CO.9262780)(0.9595191) =0.92082773 R = /0.9595 . 3 4 a o-• H to CQ : O) CD JH CD H ft •ri -P H d a CD Xi -P ? H o CD O a o. •r-t <h •H C| bO • H ' CQ ' CD x l <P u o CQ CD + 3 CD Xi CD C0 SH CO cO CO l > H C0 D •sf1 CO CQ 05 H 05 o a o o o o CD © o o CQ ^ CD O cO S d d CT] ra CQ d o H i-P cO • H 54 cO >' FH o CD O f-f d o fco LQ H 00 o CO o CO LO CO CO co o © o Ii CO CO 05 !> co 00 H o o Ii >—X H 05 r> 05 CO <tf CO CO IN CO H CO 05 o [> • Q o o e o CO '05 IN q CO H 05 00» IN 00 rrl CO' CO H CO CQ P CQ O -P H lo cl CD d CD • H H Xi cO +3' P -P CD • H o P Q ( 3 * CO CO CO e '05. LO CO LO 00 H •sT 01 05 o: O J o H O I O lo o o II 85 Explanatory notes for terms and methods in the calculations: • • . page 70 SW* - the total weight of a l l the chicks in each lot is added. 2 Page 71 SW - the weight of each chick is squared and the cumulative total of each lot is obtained. Page 72 . SWB - the weight of each chick is multiplied by the average weight of its own tibiae, and the cum-ulative total of each lot is obtained. Page 73 The correction term for W is derived by squaring the sum of SW and dividing by N. Page 74 (SW)2- care must be taken to distinguish between N SW^  and (SW) . This figure is obtained by squar-ing S?/ in each lot, dividing by N in each lot '. and then adding the lots. Page 75 S(W)(B) - Multiply SW in each lot by SB in each lot, N divide by N in each lot and then add. Page 76 Sum of squares between lots for W: subtract the correction term for W from the sum of (SW)2 . Sum of products between lots for WB: subtract the correction term for WB from the sum of S(W)(B). Page 77-8 As a check,, i t should be noted that a l l the values under the sums of squares, ie, SW -(S¥) are N positive. If any negative values are obtained, check the work for an error. However, in the sum of the cross products, ie, SWA-S(W) (A) i t is possible to have either positive or negative values. 86 Page 79 ; To obtain the total sum of squares for W: subtract the correction term for W from the sum. of SW2, . Total, sum of products for WB: subtract the correction term for WB from the sum of SWB. On the right hand side of the page there appears another set of figures that, when added to-gether, yield the same answer as on the le f t hand side. They are merely a check. To obtain the total, sum of squares for ¥ by the check method, add SW2-(SW)2 aid CSV/)2 N N - correction term for W . Page 80 By turning back to page 76 , i t w i l l be seen that the figures obtained there are the'sum of squares and cross products for "Between lots". On page 79 are the figures for the sum of squares and cross products for "Total". The figures for "Within lots" are obtained by . r subtracting the "Between lots" from the "Total". As there are 16 lots, the degrees of freedom for the "Between lots" w i l l bel5. Similarily with 224 chicks the degrees of freedom for the "Total" w i l l be 223. The mean square values for the "Between lots" are obtained by dividing W, B and A by the degrees of freedom - 15. The same method is used for the "Within lots." 87 .The F value, is derived by dividing the "Between lots" by the "Within lots". By consulting Fisher's Tables for F values (41) i t is noted that these values are highly signif-icant. This is one method for finding the correlation between W and B , W and A, and B and A. The figures for the "Within lots" are from page 80 . They are arranged under their appropriate headings, e.g., W under W and opposite W . The figures in brackets have nothing to do with the calculations - they are merely a guide. a l , ,aS, a3 - the square roots of WW. BB and AA respectively b l - (al)(a2) b2 = (al) (a3) b3 = (a2)(a3) c l = WB_= correlation between W and B. b l c2 = WA = correlation between W and A. b2 c3 = BA = correlation between B and A . b3 The application of the principle of lease squares leads to a pair of simultaneous normal equations, / 5 A W + rwB/^AB - rAW R WB/4w •+- /^AB = rAB 88 ; in which/^. and^? B represent the standard partial regression coefficients. Page 83 The multiple correlation coefficient is arrived at by the formula R2 =/^AWrAW+AB^AB from whence R is calculated. It is always less than unity, but greater than either of the coefficients of rAw and r AB? a fact which i s sometimes valuable in detecting errors in calculation. Page 84 The part of the total sum of squares of A^ indepen-dent of regression i s found by the formula (1-R 2)A 2 The degrees of freedom, N-3, are less than N by the number of variates. The deducted degrees of freedom correspond to the partial regression coefficients used. 89 Explanations in connection with Factual Data*. Table 1 shows the number of chicks started and the number of chicks at the end of four weeks as well as their average weekly weights. Growth curves are shown in Figures 1-3. In each case the comparison is made with the control lot. On examination of the figures in Table 1, i t w i l l be seen that the growth was f a i r l y uniform for the f i r s t week. By the end of the second week, differences in the average weight of the lots became apparent. From this period and until the end of the experiment, these differences become more marked. Figures 1-3 illustrate this point in graphical form. During the last week of the experiment, the comparatively small gain made by the rachitic chicks is evidenced by a "flattening" of the growth curves. In addition these chicks showed the characteristic symptoms of avitaminosls D during this period. The s t a t i s t i c a l analyses of the weights of the chicks are given in Tables 2 and 3. In a l l cases but four.the differences of the average ?/eights are s t a t i s t i c a l l y s i g n i f i -cant. It may be noted that the difference in the average weight between 1/8% and 1/4% Reference Cod Liver O i l i s not significant. A similar situation exists between 1/8% and 3/16% Pilchard O i l (10) and between 1/8% and 1/2% Pilchard O i l (14). Singularly enough, the same lack of significance is found between the weights of chicks obtaining 1/8% and 1% Pilchard r-i 13 a PQ E-l cn o t l r—1« o o CO EH P £3 C3 CO CD CD pS CO 6 *C0 CQ SQ ,y CO CD CD d *S •H -P •d CO .b( •H CXI CD CD CD f5 ; •' CD tK 03 031 U M CD I—1 CD > CD 3 is-CO H & o CO O O •H x l u o d CO CD •• £ o -p izi co ^ co M o • H X l O -P o Ctj .«H rH _ 'd • d CD CO CO • H O -H CO a x co -cd d «H CO CD • o M -P . • O SH •' » -H Crf >• P Jteq O. 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CfJ. tf> O O O O O r-i r-i r-i r-i r-i O O Q-O-O-00 !>- CO O CO o CO rH CO r-i CO LO • • • CO CO CO CD LO H r-i Ol-H « • • CM CO CO H H H .+1 +1 +1 LO LQ LO 01 Ol r-i • • # t> -01 cD; co co ^ r1 ^ <sH ^ rirlrH o o o * 0 • P-* fa:fa: C&Sfife% H^tHWrH rH rH r—I O O O PH PM..P-!. 96 Oil ( 1 C J . ; Tables 4-6 contain the results of the individual ash analysis of -the chicks and Tables 7-8 the st a t i s t i c a l analyses of the average ash content. The weights of the chicks receiving graded amounts of fish o i l at close levels are analysed sta t i s t i c a l l y and the results are shown in Table 9. It is interesting to note that the average weight of the lot receiving 3/16% Reference Cod Liver O i l i s greater than the average weight of the lot receiving 1/4% and this difference is sta t i s t i c a l l y significant. By comparing Tables 8 and 9, i t may be noted that even though there is no significant difference between the weights of the lots receiving 1/8% and 3/16% Pilchard O i l (10), there is a significant difference, nevertheless, in the per-centage calcification. A similar situation is evidenced between 1/4% and" 3/8%, and 3/8% and 1/2% Pilchard Oil (10). Also i t is seen that the same condition exists between 1/4% and 1/2% and 1/2% and 1% Pilchard O i l (14). The difference between the weights of the lots receiving 3/16% and 1/4% Reference Cod Liver Oil differ significantly,but when the difference between the ash of these two lots Is compared, i t is seen that there is no sta t i s t i c a l significance. Similarily the difference between the weights of the lots supplemented with 1/8% and 1/4% Pilchard O i l (14) is significant while the difference between the ash of these two lots is not. 22. to o u • P a o o HCOCOLQ'HHCOCDCDO CO LQ 0} 05 (X) O O 05 CO 1 co co co LQ CO H COCOC^0)OCOCOCOCO^LQLQC)O CO C0C0C0COCOCQC0COC0COC0CO o LQ © CO CO p H <J H H > PH O P<4 H o CO \ H CO \ CO 05 tD LQ o~5 CD LQ o CO CO <sf CO « J O H t O W ^ O l C O C O O l C O C O C O O l H CO CO ^  ^<qHLOLOLQcDCDCDCO[> CO ^  t> t> H IN CO CO 05 05 CO -Nfi <£> [ > - ^ L ^ O O l L Q 0 i L Q C O O C O ^ L Q e © 6. • . » » « . e e e <» • * CO C0CO^^LQLQCOtO[>l>l>-LQ to St1 LQ c o O LQ ^1 PP <; EH o o o r—I CO CO LQ CO 05 H ^ CO CD 05 00 CO CO CO to t> IN O "st1 00 CO CO ^ CO 'rH CO CO CO <sjH^ CO ^ ^ <fl ^ -vf LQ LQ tO CO ^ ^ ^ Q to e CO <! 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'. «jM^Mc<l CO P . a CD « • « • H o o o o a » • * • a f  fa fa fa d « • c a ; O O O O fa" fa fa fa ' fcR 5R -P +3+3 +3 +3 p p p d d d d d d d d 05 o5 a j o5 a j a j a j o5 O O O O t! o o o • H > H - H - H - H • H - H - H Vl Vl Vl VI Vl Vl Vi Vi • H -ri -ri - H -ri • H - H - H d d d d d d d d bO bO Sb bO -bO bo bo bo • H - H - H - H - H • H - H > H CO CO CO CO CQ CQ CO CQ +3+3+3 +3 P o o o O O d d d d d O 00 CO CO o H 00 00 [N O - 01 C0 O L Q o \r CO t> D- O O L Q ^ r-i CO CO O CO CO Is- 01 r-t CO ' • r-i t—I 0101H O CO L Q ^ L Q ^ 01 • o • • * CO CO [> 01 01 H CO H CO O O O O O • • • • • fa fa fa fa fa ^ o o CO CO 00 CO O 01 r-i 01 L Q H O O O r H C O C O O H LO t^* o r-i 01 CO • • • +1 4-1+1 +1 +i -f-J+l +1 L Q O O 01 r-i CO 00 CO CO i—I o o o o b sf| ^  r-i r-i r-i H H rH H H O O O « • 0 fa fa fa; rH H | ^ M H O O O O O rH r-i r-i r-i H ^ <tf <tf r-i H H O O O O O o o o faP^fafafa fa fa fa 103 > The results of the assay of each lot show t hat the median in a l l the lots containing less than 1/2% of fish o i l is above the*mean. The lots containing 1/2% or 1% of fish o i l follow the normal curve more closely. Tables 10-13 are derived by using the average weight of the ash of the chicks as the independent variate and the weight of the chicks and their average tibiae weight as the dependent variates. The highly significant value of F in Table 13 indicates that the different average weights of the ash is not due to the chicks alone. Analysing this data in more detail, the chicks are grouped according to the o i l fed. In this case the controls are classed as a group. The results from this analysis are shown in Tables 14-16. Once again a highly significant value for F Is obtained. It i s evident, therefore, that the different average weights of the ash is not due entirely to the fish oils fed. * Tables 17-20 are obtained by analysing the relation-ship existing betv/een the independent and two dependent variates for each level of o i l fed. The results show that the different average weights of the a sh of the chicks receiving Pilchard O i l (10) i s due to the levels of o i l fed. Similar results are found in Tables 21-28. It becomes evident from the corresponding figures that the various levels of Pilchard O i l (14) and Reference Cod Liver Oil are responsible for. the different average weights of the ash. -104 TABLE 10 x DATA FOR 16 LOTS OF CHICKS. u . - Sums (In grams) enick Average Average numbers weight .tibiae weight ash weight 1 1- 14 2548 7.3670 3.0201 2 15- 26 3002 9.0602 4.0918 3. 27- 38 2288 7.1012 3.0886 4 39- 51 2998, . 9,6299 4.3951 5 52-.64 3214 10.4969 . 4.8836 6 65- 80 . 2782 8.4172 2.8981 7 ... 81- 94 2258 6.6091 2.4187 ; 8 95-112 4100 12.4836 5.0676 9 113-127 r 3304 . 9.7150 4.2346 10 128-145 4314 13.9745 6.4306 11 146-160 2996 9.4925 4.3321 12 161-173 1994 5.4848 1.7919 13 174-185 2296 6.6473 2.3202 14 •„ , 186-198 2382 7.2299 3.0331 15 199-211 2594 7.9563 3.6098 16 ,212-224 1906 5.5236 1.7939 x For data oh each lot refer to the appendix. 105 H H a pq E H -P KO > CQ CD Cti bO "i in o CD > +? •§)• •H CD CD 05 • H CD ,Q hO-H.pq 05 -P in > O B o •H -P. . c d •H !H oi > <+H o CD O U o co CO CO 00 CO CO [> CO 01 CO to 01 CO ^ CO o CM CM 01 co o O CO o H CO CO CO CO CO H L> 01 01 •HH rH CM -HH 01 « © © © e • • • CQ o 00 'CM O' 01 to ^ ^ O O OJ r- oi co co CO CO O H CM rH H rH rH H H H O 01 rH CO O CM 1> to to e • ' « © ,y to CO o- CO o H CO t> CO -P -H CO to O 1> P ,B •'• tr-. m bO 6 is •H ^ CO H CD p . • „ to o to pis O: 01 01 CO CO o -p CD |0J. rQ.. cn -p . ,, «-~ O *fC0 pq -H £5 -P! ;§> CQ O CQ CD ' *- U *E\3 C O M C M £ ^ o- £ CO OrCQ CO > £3 H CM CO ^  LQ O CO t> oo o 0. L Q ^  to CO rH H CO 00 00 H !> H rH 01 CO CO CO 01 CO t> H CO L Q CO to 01 ^ O •^ 01 « «r e> © © c « © © CO L Q O H O co CO CQ 00 ^ ^  00 01 CO tO L Q H CM, 05 D- CO CO CO CO to a u CD -P CQ . CO B o pq +3 cn o co CD C O S H C O , pq o ,_ co o -fi • I C O CO "ffl H CM CO'-^  tO pq CO 01| H CO! CO HI O £> © ©I ! > ' H H H H! <sF CO O CM cH w CO L Q © © C O H U CD HP B' o •rH -P o CD H MM O o5 C O CQ O CQ CQ H C O CO <cf 106 Ml •P • H CD &: CD CO bo cti • cti <; CD O > < • a o • H -P aS • H Jh 05 > .<+-) O CD O u d o to 0 0 CD 01 O IN".LO o oi: 0 0 L Q IN W O C O C O fN 01 00 M C O CN "NF oo co co « e e CO L Q O 12 ° •p •£ bo CD IN L Q •H CO H CO CD -H CO L Q CO O •H • e e CD Hp pQ O L Q fao St] CO 05 «H H 00 SH O ' CD CO > C O f^! O 01 C O C O C O L Q r—I rH O 01 • • « L Q CO O H <cfj CO *H H H C O IN. 1 H 00 CO CN to ' IN .00 00 L Q M e e o "IN 03 CO H +3 - H IN H L O CO P ,d • Q.IN. CO C D ho o. • H ^ IN C O -CD C H . H CO CO "3S o C O CO CO CO CD d •H rH • H H • CD H -p • s • o u C H H o5 co -P -P o o EH P co ! xll to p 0 > - £ 0 1 C O C O H L Q O C O 01 e • •sf H H 01 C D rH CO CO L Q CO 01 CO C O 00 • © • •CO CO o ^ H IN L Q IN 01 ^ O 00 C O C O 01 C O 01 C O rH C O C O L Q CO C O CD a • H O rH -P CD H * CO *^ rH -p .—» o CO CD H .rQ H to • d 05 • H -P X) -P £ . 6 • H to o u <++ CO -p oi H CO to 3 • H -pbo to o •Hi P. ICO to H 05 co •P -P o o . . EH P in pq ^ L Q IN 00 H CO CO O © © CO H oo S3 CO CO CO H e e H H CO CD d • H H rH H CD H 0 5 P> s o M <H rH o5 to +3 .p o o EH A < CO •p o d • H -P • H CO o p o oJ to 107 co ri fa I M <; o m E H Q IS H fa o 1. fal fa1 Ol fa o H fa l o * t CO CO P H O. fa E H ^ IS ri E H Ol fa fa •••cn ri II t o fa I fa O s IP CO 0| cO o fa 0 d SH d as CD 0 1 CQ CM CD C Q CO CD O CD CH rrj SH O CD hi) 0 0 f-i fa ".<n CO, fa CO '••erf Vl 0 PH o p X! IK •H 0 CO «H 0 0 O S-i b£ erf erf d d SH d a 0 ca ca > < CO "d 0 o 0 V Tj O 0 hO 0 0 J 4 fa <+-i d o 0 - H U P U V erf d o -H o FH 0 1 erf > cd ri erf P o H co ri o o o » o LO - co CO 00 CO ri o © » o o ri CP CO o CO CO rH O P4 o i 01 o o o ri 01 ri « o LQ ri CO LQ CO CO o CO CO t> 01 01 e « o o CO CO CO CO o CO o o e a o 01 LQ CO 00 CO CO co1 o • ri CO oo " LQ cd o ri CO CO CO p o fa 108 Table 14. DATA FOR 15. LOTS OF CHICKS IH 4 GROUPS Lot Chick, numbers Sums (in grams) Average Average Weight tibiae weight ash weight 1- 5 1- 64 6-11 65-160 12-15 161-211 16 212-224 14050 19754. 9266 1906 43,6552 60.6919 27.3183 5.5236 19,4794 25.3817 10.7550 1.7939 109 L Q H m o H r-M o CO 3 H co rH o fa H i fa ,EH H CO rH CQ rH fa H O H fa fa fa O o fa o fa J2J o H EH -f.-r fa ' E H O H fa O fa ei EH O fa fa o fa fa fa is ; ,3 !fa o o b fa o H :EH |fa CO oi •fa| o to fa fa Q isi H 001 -p •ri CD X ! •*3 CD tsOV ' Crf O •fc CD > <4 P •§» • H CD CD erf X 5 CD . H h O P fa •OJ U Vi CD O > -4,. • Vl O CQ . ^ P O hOxi ri O CD fed O • H P Crf • H JH erf .;> v o CD O SH d o 02 OO O 05 CO r - H r - H » e CO 05 C-00 O H CO H CD CO O CO Q 05 CD CQ CO © e © 05 05 o 00 G5 CO 05 t > o HcO 00 C Q co H o o <*o H CO e-* CO 1 H 05 C"-C0 e © !>C0 o-cd O C D ^ H Hco C O CD d ; •rH H OH CO 05 H H CO CO ,05 O CO « 0 C Q 05 H CO #\ CO CO L Q O P CD H H CD H fa erf P a'' o Vl CQ . PCO o HI p • H X5 00 d+-> 01 CQ M. :•* obo H erf co p p • O O E H fa U " > . U • H | XI CO 05 001 CO L Q L Q 05 CO CD e • "ST O H 05 L Q O 00 C D L Q H L Q L Q £ > 05 © • * CO CO o 00 \ r o 3 8 00 O - C O Cd ' o- q O H 05 <tf H co t> © •» e e « o 00 H !> CO CO CD d n H H CD H fa erf p a o f-i vt H erf cQ +3 P o o CH fa fa CO p o HPO erf t o d • H X i P •rH CO s CQ'V' fa, <* obo o > CO t> HI H CO e • CO o | CO t> CD 05 F- CD 00 CO © o H H CO CD d . 3 H H CD H fa erf P a o u v H erf CQ P P o o tH fa 4 cd P o H d • H X i P • H OO erf -5 -110 •pq EH to O] EH S rH! P i i < p ; O; P o •is; ! Pq O , to H P iS to EH O P H o H i CM OJ o p EH EH to PH pq o to P i o pq Pq O cu; 1$ |3 ICO IP, o| i to p o u \u m CD' cd .cd 0 to P O CQ SH 0 O 0 . T 3 P-l p <D hO o 0 CD U p P pq CO 0 cd p cd P cd d o 0 a CQ -p p tic •H 0 IS CO P 0 0 O ?H hi- cd ed B d u d C T X ! CO > CO El 0 ' O 0 P.rH, O 0 0 0 " S4 P • P 8) d o 0 -H O P P P cd d o -H O CH ai • cd > H O J CM H cd P o EH CO t> o o o 00! H| C M ! o U M CO o o o o 0 C Q H C O PH C O 00 C O cd 00 C O CM d H H o D © • © CO a o o co C O H 00 •IN o C O CM H 01 01 © c a ' o C O . H 00 IN o o H © © o O CM CM IN C O ' H H IN C O 00 e * « C M H O C O o ,; cp CM CM CM CM CO -p o p I l l -ft TABLE 37. DATA FOR 6 LOTS OF CHICKS -RECEIVING PILCHARD" OIL (10)'. Sums (in- grams) Chick Average Average Lot numbers Weight tibiae weight - ash weight .6. 65-80 2782 8.4172 2.89.81 7 81- 94; .'• .2258 6.6091 2.4187 8 95-112 4100 12.4836 5.0676 9 113-127 3304 9.7150 4.2346 10 128-145 4314 13.9745 6.4306 11 146-160 2996. 9.4925 4.3321 112 •123 O M EH 8 m E H o B S PH 02 fa o CQ xo o EH fa O o H E H fa 6 > H fa O fa fa CQ fa O H « o o fa H o 01 EH fa O H fa O <0 fa fa fa E H O fa £H H O fa EH -4 fa fa fa CO EH is; fa t [ 1 1 u H 1 fa fa ' J fa O u p •H CD CD CQ bi) cd cd • '« > -4 -p fa •• bO •H CD CD cd •S-ri X3 CD -H • hop fa ctJ CD O > CQ O -P -H Xi Xi bO o •H CD CH «: O a o •H P cd •H SH cd > <+H O CD O U o CQ ^ LQ 01 O- ^ 01 LQ 01 01 O C-CO .CO CD CO o co co CO -cf O o o 03 H co r> co Is- -tf OJ O CO CO H H 00 CO 01 01 01 o 01 O 01 © e © © © • e • % © • © H CO 00 o o O-cO H CO O 0- CD H CM 00 oo H rH £> CO <xf< LQ LQ LQ O £> C O C 0 | O- CO C O CD © © t> CO D- 00 *V IT CO CO H H CO 01 CO 01 CO "sf •xf1 CD CD CD O 01 © © D oo o 00 CO 01 o r-i. CO CO o o O CO o o o o o • CD r> 01 CD t> 01 r- H O !> CO LQ H V CD CO CD CD CO O CO o p CD XI CO ,Q £3 01 •U , •~/ < & CD; cd •~N )~- P pSM 00 01 fS Pi 01 o •«-—' • te; p' ^  jg: co o :o ft CD . «•> *-• u *po *• C O P H C O £ fa •m o & co 01 0< '.QS^ fn CD C0! CO 01 H CD CO CO © © CO 00 <xr CO CO CO CO LQ -H 00 01 01 © e CO H p t 01 co 6 U CD P a o cd oi fa 01 cd^ P X3 o o i . CD -~ Sn T O CO inCO " fa O X 3 02 O ' C Q ^ r-i CO CO •xti LQ H CO CO -cf LQ fa CO LQ 01 01 H Q O O CO < i CD P o •H P O CD JH fnCO O cdl cd 01 oo o co > r-i CO CO "vt* s i 10 -p xi ho •H CU £ Xj CO • cu cd <cj hO cd CH (4 o > -p • a - • ' •H CD CD CfJ tes-H • X5 CD -H • cd SH «H CD O > CO o •H x ! ho o g •H CD CH S - O El O •H -P cd •H PH cd > o CD O M o CQ 0) CO 01 H CO LN CO O « © OO CX! 00 CQ •xfi H: ^ 00 CD 00 CO ^ 01 •sf 00 CXI CQ 01-e © « c o H o c o ^ c o c o c o 01 01 CQ ^ CO CO \fi © • CO 01 CO CD 01 CO ^ ^ CO O H H H CQ CD CO CO 01 © © © 01 c o o H "sj* O CN 0 CO e « 01 o CN CO CO o CD IN CO IN CX) CO CD S3 •H H CD H XJ cd P> S o u <u H k cd to -P -P o o E-f |4 oo 01 CO CO •EN CD » © CO HJ. 01 CO H HJ 01 CO H o CD cd o CO H CQ a >_> •rH >^ >* X) -P. •H CQ E§ to M •* pq CQ S3 CO CO CO tN CO CO IN CD CO CD H CO O CO 01 co CD CO CO H rH CQ CN H o rH CO 01 CD CO CO 01 D CD LN « » « • © © • • © H O H H O H o o o CO CQi CO <^ l CO 01 01! » ©i CO o i CO CO 01 o LQ CO 01 LN © © CO H CO CD a •rH H CO rH CD H X) cd -P pi o SH CO o|co HI •Hi "H •H| Es to P ' o, •rl rl cd CQ CO CX) X3-CQ H" cd CO -P P 1 o o. EH J U " > U CO CD a •H H 00 H CD H X) cd -p B o £ H -cd co -p -p o o H J cO -p o H a •H x -p •H CQ P. o p o cd cQ 114 o CO P M P Q E H K t o PHI 01 E H _ CO E H Q CO l& IH CJJ HI P H :OI m\ t o 51 —t H P 4 P H o CO H •8! o H CO © P H O P H E - i < H E H CO fx] P H O CO K o SH P H O cu] i+pi . 1 I-PI I co I cul Leo PH o , 1 CU! PH d crj cd d a CO co <H CU O PH Cd H d 3 CTj co co CO cu o| cu TO PH P CD ho o cu CD . PH « <+H CXI •PH XI CO cd u o -p x ! cu CD cd PH CD > CD PH d cd cd CD CO <H CU O PH . cd s d d rjl co co co S cu o cu PH P CD hO O CD cp O <t-i d o CU -H O - P PH MH Cd d O -H O PH : Q cd > o> H H 05 O H O H o O 0 © o O CO H CO CO f> 05 t> H CQ o O o e © • o o o CQ 05 LQ 05 CO 00 O 05 C O H 01 CO CO CO CO r> 05 05 ' © e o o 05 CQ CO o o CO o o « « o o CO CO t> 05 CO co H H o o CO © 0 o H o o C O H PH crj O CO - P PH •p o PH o EH pq H" 115 -ft TABLE 21 DATA FOB 4 LOTS OF CHICKS •••••• RECEIVING PILCHARD OIL (14) Sums: (in grams) Lot Chick Average numbers. -Weight tibiae weight Average Ash weight . 12 161-173 1994. 5.4848 1.7919 13 174-185 2296 6.6473 2.3202 14 186-198 2382 7.2299 3.0331 15 199-211 2594 7.9563 3.6098 116 o r—i i 0 1 o H fa o CO fa o H O C O E - i o B o , fa fa fa o t o I fa o C O .g 'DO! I o | E H I O !C0 Q M fa r-H o : In ifa fa O fa C O E - i H fa H O •-•4 o fa H fa fa O H E H P . i H : f a . fa iO -p fa bO • r i 0) * a CD CQ &0 a j CD O > p • £ "• •too--•ri CD 0'ctS 'H « fa fa 0 -H UOP 05 FH < H 0 O , 5 . CQ o P -ri ti fa • 'ri , 0 V) S O o • r i •P . a j • r i in a j > o 0 o in d ! o col xf CO rH LO: L Q co: co o ; o c O "xf1 CO LQi O 01| CM H CO 01 H LO -HH O H CO CM co co r> • • • CO CD O O -cf rH H CO LO CO o <xr LQ -Cv CO CO 00 L> CO H H CM 01 CO 00 01 CM CO CO C O o p 0 (CO c o ti DO C O C O +->. p: or/) FH 0 - H * | C M CM SHCM " O " fa H CO CO xf L Q CO 01 t> C- CO CO CM CO o CO C- H CO CM CM CO CO O H CO t> CM O- LQ01 LO CO o « a « 0 CO LO o o o CO CO d H CO CO LO O CO LO » e o o xf H CO CO CO D- H CD CO o 00 «xf o 01 CO CD H CO LQ CO Oi CO CO O H CO [> r> r> CD CD O © © © ft © © • « CO CO 05 o o LQ <xfi H 01 CO CO 01 H H H 01 coco LQ <C~H CO CO O H 01 CD o o 0 1 O « • • © CQ E4 in 0 P p o aj c o C O a - ° , fa P vD ^ C O O C O CD ** •x U -ICO CO FHCVI fa O fa -a C O C O O C O O C O >> fn Ej F-t 0 P El O •rH P O 0 CO fnCM <cj o aj CO O CQ • 2 H CO CO "xf IQ fa H CM CO -cf' 117 co co P P EH -p P ho • H CD P ^ 03 Cij • CU <rf hop cd o P CD > CQ P -P O P - H .hop • H O ! tt) O • H - P cd • H U cd > p o 0 o d o C Q CD 00 00 CO CO I> LQ CO CO 00 O 05 CO CD rH 05 H CO H CO r> CO O-00 CO-'O" ^ H » « e e CO O LO H o O O O H^ CO 05 H •P P hO •H 0 0 cd CO IN CO. CO «? -H oo rH CD G> LQ O LQ CO 0 -H pq f> rH CO t> hO-P © © ft 0 cd 05 CO L> CD fri P CO CO CD CD 0 o CO H CO t> .; <! • 05 o>; 01 CD * 0 CO CO CO o 05 L> CO CQ 00 rH CO 0 d • H H 00 O CD CO CO ©' • C73 rH LQ CD CO CO CO CO CO CO 0 rH p cd -p o -p 0 CQ »•» •p —< o H p rH cd CQ EH p H d • H p -p • H p CQ % CQ CQ M -Ot\J o , C Q .. ffl CO O LQ CO H CO O H4 CO CO 05 o © • o o o CO LQ CO C0| O CQ O CO © © H Ol H 00 co r> LQ CD r > c o ©- « o o CO 0 d •H H CO CO 0 rH p cd -p O SH P to -p.. •H •5l cd cQ P CQ CQ P , * obo • H P P C Q rH Cd CO -P -P O O _ _ E i P O ^ ! 4 P H H+ CO CO CO CO, CO HI • ©I O O l o-.ro co o CO LQ H CO ' © « o o CO 0 d •H rH CO CO CO p 0 o P H P cd d P - H p a -p " O - H P £ P CQ rH P cd CO O -P P - H cd O O P C Q E-i p O >i • 118 •oo fa E-I o o •ol E-i fa1 00 I EH O fa U ' H o o fa" CJ> M f-3 CD < fa: O fa o H fa fa Oj OO H Si fa $ fa o fa H E H ltd jfa ifa | o CO Q fa fa o CD U d cvS CD -P CD D 1 S CQ • H P CQ CD CQ Vl ^ Vl O FH O cd B d CQ d a U 00 CQ o F-) JH fa CQ s CD o CD \ % S uo CD fa ? H V CO fa X! CQ Ctf Vl o -p fa s> CD CD cd in C D > CQ Vl CD CO CQ C Q S3 C D O C D C H r r j U O CD hO CD CD FH P •' Vl d o CD -H O P F-. V cd d O -H O U co cd > CO CO 05 o o o H o O © o o o CO 00 CO o CO H CO o o o » e « o o o CO o LO LO LQ 05 LQ CO o CO CO H 00 05 0 © o o CO CO o o • © o H o CO 00 LQ CO CO H 0 « o o Or "Cr CO LQ o » o CO « o CO rH U cd O C Q P U P o U o EH fa fa 119 TABLE 25 DATA FOR 5 LOTS OF CHICKS RECEIVING ~. ,RI-F-RlHCE COD LIVER OIL. ~ Suras(in grams) Chick Average Average Lots , numbers Weight tibiae weight ash weight 1-14 2548 7.3670 3.0201 15-26 3002 9.0602 4.0918 27-38 2288 7*1012 3.0886 39-51 2998 9.6299 4.3951 52-64 3214. 10.4969 4.8836' 120 S I Hi & P4 01 8 o P CO 'EH O D 8 PH P :< to to O: to o P H > H (Hi ! o PH PH1 CO P o <~r' t - M to PH O, tn EH O. p to P4 o p H O p pq > H P « O O IP O M l P • 3 P o p o tH EH P O P •6 to % P H lO IH P P P O o IP p PH -P P to • H CP je P CQ CD cd< cd P PH O CD > p p • bO CD • H Cd CD T\ P • ^ - H - p q CD - P bO cd P PH O CD > to H H o o O CQ CO o o O CO p e • * « o oo <F ^  o -P - H « 00 H H CO P P 53 O H H tO H H bO o • CD P rH >vft CO CD CO O " s o , CO O CO CO H a o • H P cd • H PH cd > P o CD o PH d o :o to o C Q r> rH COl 05 CO « » o to to t> C Q CO ^ SHH; CO CO CO CO CO t o L> CO o CQ r> OO » o o CO 0 0 o OO CO oo c o to •a PH CD -P O -P CD tn p j to tn <ci a o •H-QU +> £ > PH o cn CD •VSH HCO p CO PHCO v;: o si a . H CO 00 H^  L Q OH oo L>' H^  co: "sH co S 3 05 H^  CO COCO o H H H CO CO to 05 t> CO CO H H 05 L Q 05 « e o H H O toco co tO CO CO o CO CS 05 t> O CO O L> 05 O H 05 GO rH L Q H^  CO t> 05 H CO L Q 05 05 • H L> CO e « © « © • © © « 05 00 CO 05 O : CO 05 CO05 00 H ^  CO CO H L Q t o t o O 05 rH CQ s PH CD P | C 0 cd 0 0 to p CO d o • H cd^ --p p > o co CD « " p «C0 CO PHCO W o p C O C Q C O C O O 05 r> L Q r> © © o o p to CO s PH CD -P d o • H P o CD PH PHCO O CO tn Go o x>>. PH to o tn^L, H cn O tn > H CO CO "sf* LQ e m © « « e H CO CO 'H 121 o CO fa fa fa 3 H 01 H o fa 'fa o fa fa o . ' H fa o fa 01 rH O B o fa fa p «j 01 s Oil fai o 01 H o H . f a Hlfa fa 01 01 fa o H fa o fa o 01 rH o l fa! -P %> • r i CD £xi CQ CD Ctf -uo CO <+H M O CD > P Xi ho CD •ri Cd CD -ri •Sfa •ri 0 p fa Cd Vi FH O 0 > CQ P O Xi -ri CiOxl 'ri O «5 O d o •ri p cd • r i FH cd > Vi O CD O FH d o :o CD xfl CO oil D? xf' c6,co . 0 © CO CD, oi o; CO H CO 01 CO 01 H E> CO H H O- CO l>-• © © ^ ° o 00 CD rri CO O CH O CO « © ^ oo HI CO CD CO CD CO O.iO H CO 0 d •ri H CO CD CO CD 0 CO © © H CO 01 LO O CO -xT CO CD CO © H fa cd P S o FH Vi H cd 0 P P o o H fa O P 0 CQ , P O H d •ri fa p •ri 'CO fa Ol co Ol £5 fa O > ? H 01 CO O 00 CD H H HH « © H O L O L Q xf CO CD "Cr x-H "xf 00 O- 01 t>. e » © o o o CD H L Q <xH o 01 01 O 01 CD LO H !> L Q H O H O H 01 oo "eft co 01 L Q sj< CO LO L Q 01 CO 01 CO E> CO CO ©' © « © © © « © © L Q CO H H O CO o H H H 05 CO L Q CD H "xf< <tf CO 0 d •ri fa L Q xf CO LN Ol "Cf L Q O © © O o H 01 CD CO xf 01 L Q • © O O CO 0 d •ri H •x, CD CO 0 p 0 o H H cd d P • r i .Xi >—i P o •ri U E» V 0 H fa cd 0 opo p p o o cd co H fa O ^ 122 c o CM PQ CO <j Pq] EH H -fxl CJ3 m < CO IO M LQ H CO o H H ~ i I - H o St* CO Pq o pq PH O EH PH SI > Pq] OJ CO H ^, H EH to pq pq O CO O pq pq o CU a SH cd cd CD CQ cd a •ri -P CQ CQ <4H CU CD O in ^ cd «H £3 d O 3 D CO CQ CQ FH O FH CQ a FH •CD . O fxl CD < H FH O CD ao CD CD H PH < H CO CQ cd CD FH o a cd cd d -p CD CT Xi CQ h( • H CD & CQ CD <H CD •he O FH cd cd FH a d CD d g > to 4 co a CD O CD td FH P CD hO O CD CD : FH O <H d O CD -H O -P FH P cd d O -rH o FH CO cd > f > CO H co O O o o o e • P o CO CO CO 01 05 05 CO o CM o o o * © © o o o H CO o CO H CO 05 CO CO 05 LO CO CO *o St* CO » CO o o H o o 05 LO sf* CO 01 05 H o H o O © © o O St* D -st*. O © sF H FH cd O CQ -p FH •P o FH O EH x! 123 Discussion of Results: The average weights of the chicks obtained at the end of four weeks compare favorably with those of Halvorson and Lachat (71), Asmundson and Aliardyee (4), Gutteridge (48) and Biely and Palmer (12). The results of the ash analyses are very similar to "those obtained by Griem (45). According to Snedecor (96), the range of the ash analysis in each lot is not unexpected. With groups consisting of approximately sixteen experimental animals, the range i s three and one-half times the standard deviation. It has been worked out by Brandt, Irwin and Nelson (19). that with the differences recorded sixteen experi-mental, animals in a .group are sufficient. As the Reference Cod Liver O i l contained 95 Inter-national Units of vitamin D i t was possible, by comparing the average ash of the chicks fed Pilchard Oil (10) aid (14) with the average ash of the chicks receiving the Reference O i l , to obtain an approximation of the potency of these o i l s . From the results i t Is seen that the basal ration supplemented with 3/8% Reference Cod Liver O i l insured normal calcification. .As, however, 1/4%.of this o i l produced a slightly lower percentage of c alcification, i t would seem that 1/4 of 95 or 23.75 International Units §.re not quite sufficient to insure normal calcification, whereas 3/8 of 95 or 36 International Units are sufficient. According to Lachat (69) the minimum requirements for chicks are about 27 Inter-124 national;Units. Since 1/4% of the o i l was not sufficient to insure normal calcification, and whereas 3/8% was ample, i t might be concluded that a more closely graded dose, i.e., 5/16% would be nearer the minimum requirements. If there was no s t a t i s t i c a l significance between the average ash of 1/4% and 3/8% Reference Cod Liver O i l , a more closely graded dose would not be acceptable. However, the difference between the average ash of these two levels is significant (Table 8). Thus i t is evident that the feeding of 5/16% Reference Cod Liver O i l would be justified. It i s apparent that the samples of Pilchard O i l vary considerably in their vitamin D content. By comparing Pilchard O i l (10) with Reference Cod Liver Oil, one feels justified in assuming that 1/2% Pilchard O i l (10) was equal on a Unit basis to 3/8% Reference Cod Liver O i l . Thus the vitamin D potency of Pilchard O i l (10) would be at least 72 International Units. As 1/2% Pilchard O i l (10) gave a slightly higher and 3/8% a somewhat lower percentage of calci-fication than 3/8% Reference Cod Liver O i l , i t would seem that the use-of a more closely graded dose, le., 7/16% would give a closer approximation of the potency of Pilchard O i l (10). The highly significant difference between the average ash of 3/8% and 1/2% Pilchard O i l (10) as shown in Table 8 would warrant the feeding of 7/16%. Table 6 contains the average percentage calcific a -tion of the various levels of Pilchard Oil. (14) • It i s evident that more than 1/2% and less than 1% would be required 125 to insure normal calcification. Referring to Table 8, i t is -' noted that the difference in the average percentage ash between 1/2% and 1% Pilchard O i l (14) is highly significant. Thus to obtain the potency of this o i l - i t would be necessary to feed at several levels between 1/2% and 1%. Based on the results obtained. Pilchard O i l (14) contains at least 36 International Units of vitamin D. In evaluating the relationshipsamong the weight, of the chicks and the 'average weight of their tibiae before and after ashing, the table on page 81 i s the f i r s t point of interest. The calculations on pages 70-80 are merely preliminary. The correlation coefficients between the weight of the chicks and the average weight of their tibiae, the weight of 'the chicks and the average weight of their ash and the average w eight of the tibiae of the chicks and the average weight of their ash show the relationship existing between these variates. In solving the multiple regression equations, and/%3 represent the standard partial regression coefficients that are used to compute the multiple correlation coefficients. A convenient method for presenting and studying some of the statistics of multiple regression has been devised by Wright (110). In the following figure the straight lines representstandard regression coefficients, while the curved lines • indicate correlations;. 126. 127 i s called the path to Y from X]_, the direction being -"indicated by the arrow. There i s a second path from X-]_ to Y -by way of XgV Its value is defined as the product, rTO = (0.9380).(0.9263) = 0.8689 The relations among three variables are not superficial - the , real contribution of X x to Y is evaluated by the path. While the path coefficient is not a correlation coefficient, i t does evaluate the influence of X on Y independent of units of measurements. Multiple regression is used to evaluate individuals and to compare them oh the basis of their deviations from average performance?due account being taken of known inform-ation contained in the independent variates. This coefficient measures the success of estimating the averagew eight of the ash from the two preliminary items of information, There are two objectives in computing Table 12. The f i r s t is to calculate the,multiple regression of the entire set of data and the second is to computate the three correlation coefficients. The test of significance is made with the two sums of squares, total and error. The remainder, 0.14912784 with df =15,. i s appropriate for testing the differences among lot mean ash adjusted to the weight of the chicks and their average tibiae weight. -The differences among the adjusted lot means prove to be significant, P =0.0099/0.00017 & 59.6234 being well beyond.the 5% point.;(41). 128. ; ,The next procedure in the calculation of the data was to group the chicks according to the fish oils fed (Table 14) . * Group 4, the Controls, received the basal ration only but, nevertheless, they were considered a part of the data and consequently treated as such. The test of significance is made in the same manner as described above. The value, F =- 11.6389, proves that the differences among the adjusted lot means is highly significant (Table 16) . The data was then segregrated according to the levels of each fish o i l fed. Pilchard O i l (10) supplemented at six levels, was tested in the same manner as above F = 61.3771 is highly significant. Similarily Pilchard O i l (14) fed at four levels was tested. Once more a highly significant value, 46.1769, is obtained for F. Reference Cod Liver O i l was fed at five levels and the data analysed in the same manner. The value for F, 43.0588, being beyond the 5% point is highly significant. As the f i r s t part of the analysis of this data was described in detail, i t is not deemed necessary to describe the latter parts. The F values in a l l cases are obtained by the' same method of calculation and are interpreted in the same manner. 129 ' - SUMMARY 1. There were 224 day-old single comb White Leghorn chicks reared in battery brooders t i l l four weeks of age. They were divided into sixteen groups of sixteen to twenty-two chicks each. 2. The ration recommended by Hart, Kline and Kennan (54) was fed. I t was supplemented with 1/8%, 3/16%, 1/4%, 3/8%, 1/2% and 1% Pilchard O i l (10), 1/8%, 3/16%, 1/4%, 3/8% and 1/2% Reference Cod Liver O i l and 1/8%, 1/4%, 1/2% and 1% P i l -chard O i l (14).. One lot of chicks received only the basal ration. 3. A comparison was made of the rates of growth and the calcification of chicks fed rations to which various levels of Pilchard O i l (10), Pilchard O i l (.14) and Reference Cod Liver Oil were added. 4. The tibiae of a l l the chicks were ashed in duplicate. The method of assay was essentially the same as the A.O.A.C. method. The lots receiving the basal ration supplemented with 3/8%, 1/2% and 1% Pilchard O i l (10), 1/4%, 3/8% and 1/2% Reference Cod Liver O i l and 1% Pilchard O i l (14) showed nor-mal calcification. 5. Assuming that 27 International Units of Vitamin D are sufficient for normal calcification. Pilchard O i l (.10) con-tained at least 72 International Units of Vitamin D when com-pared with Reference Cod Liver O i l . 6. Using the same basis of comparison, Pilchard Oil (14) contained at least 36 International Units of Vitamin D. 1 3 0 7. ; The data show that tests with more closely graded doses of o i l would be justified i n order to obtain a more accurate estimate of the Vitamin D potency of an unknown o i l in comparison with Eeference Cod Liver O i l . 8. When Pilchard O i l (10) and Pilchard Oil (14) were fed at the same unitage as Reference Cod Liver O i l , they promoted the same degree of calcification. 131 LIST OF REFERENCES. 1. Andre, E. and Lecoq, R„, - C. R. Acad. Sci., 194: 912, 1932. 2. Angus, I. C. , Asken, F.A., Bourdillon, R.B., Bruce , II.M., Callow, R.K., FIschmann, C., Philpot, J. St. L. and T.A. Webster, Proc. Roy. Soc. (B),109: 488 - 506, 1931-32. 3. Askew, A. et a l , , - Proc. Roy. Soc. (B), 109: 488 - 506, 1931-32. 4. Asmundson, V .S. and V/. J . Ailardyce, - Sci. Agric., 13: 749, 1933. 5. Asmundson, V. 3 ., Ailardyce, W. J. and J.. Biely, - Sci. A g r i c , 9: 594, 1929. 6. Bacharach, A.L.., Allchorne, E., Hazley, V.and 3. C. Stevenson, - Indust. Eng. Chem. (Analytical Ed.) ,5:12-14,1933. 7. Bacharach, A. L., Smith, E. L. and S. 6. Stevenson, - Analyst, 58: 128 - 135, 1933. 8. Baird, F. D. and D. J. Greene, - Poultry Sci., 14: 70 - 82, 1935. 9. Beall, D. and J. Biely, Trans. Roy. Soc, Canada, 26: 57-60, 1932. 10. Beccari, £ ., - Giorn, Biol, Appl. Ind. Chem., 2.:' 174 -181, 1932. 11. Biely, J., Lloyd,. E. A, and W$ Chalmers, - Worlds Poultry Congress,'1936. 12. Biely, J.-, and V. E. Elvira Palmer, - Sci. Agric,,.14: 136, 1933. 13. B i l l s , C. E., - Research Lab., Mead Johnson and Co., Evansvilie, ind., U .3 .A. , DC, 323 - 340, 1936. 14. B i l l s , C E.,Honeywell, Edna M., Wirick, Alice M. and Mildred Nussmeier. - J. Biol. Chem., 90: 619 - 636, 1931. 132 15. B i l l s , C. E., Imboden, M. and J. C. Wallenmeyer, - J. Biol. Chem., 105: Proc. X., 1934. 16. B i l l s , 0. E., McDonald, F. G., Massengale, 0. N., Imboden, M., Hall, H., Hergert, W. D.~ and J. C. Wallenmeyer, - J. Biol. Chem., 109: Proc. VII., 1935. 17. Black, A. and H. L. Sassaman, - Amer. J. Pharm., 108: 237, 1936. 18. Boer, A. G., Beerink, E. H., Van Wijk, A. and J. Van Nierkerk, - Proc-. Kon. Akad. We tens ch. Amsterdam, 39: 622 - 632, 1936. 19. Brandt, A . E., Irwin, Margaret H. and P. Mabel Nelson, - J. Biol. Chem., 88: 461 - 470, 1930. 20. Brocklesby, H. N., - Can. Chem. and Met., March, 1929. 21. Brocklesby, II. N., - Can.. Chem. and Met., August, 1929. 22. Brocklesby, H.N,, - Can. Chem. and Met., January, 1930. 23. Brocklesby, H. N.,and O.F. Denstendt, - Bio. Board of Can., Bull. 37. 24. Brocklesby, II. N. and A. G . Large, - Canad. Med. Assoc. J., 32: 292, 1935. 25. Br o c kman, II., - Roppe-Seyler's Ztchr., 241: 104, 1936. 26. Carver, J. S., Brunstad, A», St. John, J. L., Frasier,.F. W. and W. Athow, - Wash. State Exper. S.ta., Tech. Bull. 284, 1933. 27. Carver, J. S., Robertson, E. I., Brazie, D., Johnson, R. II. and J-f L. St. John, ' . - Wash. Agric. Exper. Sta. Bull 299, (1934). 28. - Chuane-, C. K., - ' K I H I . Der Chem., 500: 270 - 280, 1933. 29. Couch, J . R., - Poultry Sci., 13: 306-307, 1934. 30. Couch, J. R., Fraps, G. S. and R. M. Sherwood, - Texas Agric. Exper. Sta. Bull. 521 (1935). 133 31. 'Coward, K. H. , Key, K. M. and B. G. E. Morgan, - Biochem. J., 26: 1585-1592, 1932. 32. Cruickshank, Ethel M., *- School of Agriculture, Cambridge. 33. Cunningham, M. M.., - H. Z. J. Sci. Techno1, 17: 563, 1935. 34. ' Dalmer, E. r V/erder, F. V. and T. Moll, - Ztschr. f a Physiol. Chem., 224: 86, 1934. 35. Deobold, Ii. J., Slvenjem, C. A., Hart, E. B. and J. G. Halpin, - Poultry Sci., 15: 42 - 47, 1936. 36. Dhar, 11. R., - J. Phys. Chem., 36: 1846 - 1850, 1932. 37 . Do Is , M. J. L., - Ztschr. Vitaminforsch., 5: 161 - 169, 1936. 38 . Downs , W. G. , - J . Dent. Res., 12: 363 - 373, 1932. 39. Duguid, J. B., Duffgan, 11. H. and J. Go ugh, - J. Pathol, and -Bacterid., 35: 209 - 218, 1932. 40. Eliot, M. M., Heison, E. M., Souther, S. P. and M. K. Cary, - J . . Amer. Med. Assoc., 99: 1075, 1932. 41. - Fisher, R. A., - Statistical Methods for Research Worders, Oliver and Boyd, London, 1936. 42. Fraser, E. B„, Stothard, J. G. and H. S. Gutteridge, - Dora. Dept. of A g r i c , Pamphlet 163, Hew Series. 43. Freeman, S and C. J. Farmer, - Amer. J. Physiol., 113: 209 - 220, 1935. 44. Graybill, H. W., - Cal. Dept. of A g r i c , Monthly Bull. 23 (1934). 45. Griem, W. B., - J. Assoc. Off. Agric. Chem., 16: 184-187, 1933. 46. Griem, v/. B., - J. Assoc. Off. Agric. Chern., 13: 341-346, 1935, 134 47. , Grtem, W. B., K i l l i a n , M. J., Clifcorn, L . £., Thompson, W. S. and E. Gundlach, - J. Assoc. Off. Agric. Chem., 18: 471 - 475, 1935 48. Gutteridge, H. S., " - Sci. A g r i c , 12: 327, 1931-32. ,49. Hall, G. E. and E. J„ King, - Poultry Sci., 10, 132 - 153, 1931. 50. Ham, A. W. and M. D, Lewis, - Reprint British Jour, of Exper. Path., 15: 228, 1934. 51. Hathaway, Millicent L., and Dorothy E. Lobb, - J.'Biol. Chem., 113: 105 - 110, 1936. 52. Harris, L. J. and I., J. R. Maitland, - Biochem. J., 25: 367 - 390, 1931. 53. Harshaw, H. M., Fritz, J. C. and H..W. Titus, - J. Agric. Res., 48: 997 - 1008, 1934. 54. Hart, E. B., Kline, 0. L. and J. A. Keenan, - Sci., 73: 710- 711, 1931. 55. Hess, A. F. and G. C. Supplee, - Proc. of the Soc. for Exper. Biol and Med., 27: 609 - 10, 1929 - 30. 56. Heubner, W., - Schweiz. med. Wochenschr., 62: 369, 1932. 57. Holmes, A. D., - Hew England Poultryrnan, June 15, 1929. 58. Holmes, A. D. and M. G . Piggott, - Indust. Eng. Chem., 23: 190 - 196, 1931. 59. Holmes, A. D. and F. Tripp, - J. Biol. Chem., 97: Proc. IX, 1932. 60. Hughus, J. S. and R. V/. Titus, - J. Biol. Chem., 69: 289 - 294, 1926. 61. Hume, Eleanor M. and Hannah II. Smith, - Biochem. J., 25: 292 - 299, 1931. 62. Jones, J. II. and G. M. Robson, - J. Biol. Chem., 91: 43 - 56, 1931. 63. Key, K. M. and B. G. E. Morgan, '- Biochem. J., 26: 196 - 207, 1932. ' 135 64. ; King, E. J. and 6. E. Hall, - Poultry Sci., 10.: 332 - 352, 1931. 65. King, E. J., Hull, H. and G. E. Hall, . *- Poultry Sci., 12: 129 - 132, 1933. 66. Kitto, H. A., - N. Z. J. A g r i c , 45: 42 - 43, 1932. 67. Kitto, H. A . , - N. Z. J. A g r i c , 46: 350 - 351, 1933 68. Klein, D. and W. C. R ussell, - J. Biol. Chem., 93: 693 - 704, 1931. 69. Lachat, L. L., - Poultry Sci., 13: 353 - 357, 1934. 70. Lachat, L. L., HaIvorson, H. A. and L. S. Palmer, - J. Assoc Off. A g r i c Chem., 15: 660-675, 1932. 71. Lachat, L. L. and H. A. Halvorson, - Sci. , 80: 342, 1934. 72. Lachat, L. L. and'H. A. Halvorson, - Poultry Sci., 15: 362 - 368, 1936. 73. Loureiro, J. A. de, - C. R. Soc Biol., 106: 555 - 557, 1931. 74. McGowan, J. P. and A. R. G. Emslie, - Biochem. J . , 2 9 : 1503 - 1512, 1934. 75. Manning, J. R., Nelson, E. K. and C. D. Tolle, - U. S. Dept. of Comm. Invest. Report No. 3. 76. Massengale, 0. N. and Mildred Nussmeier, - J . Biol. Chem., 87: 415 - 422, 1930. 77. Murphy, R. R., Hunter, J. E. and H. C,, Knandel, - Penn. St a. Bull. 303, 1934. 78. Murphy, R. R., Hunter, J. E. and H. C. Knandel, - Poultry Sci., 15: 284 - 289, 1936. 79. Mussehl, F. E. and C. W. Ackerson, - Poultry Sci., 11: 293 - 296, 1932, 80. Mussehl, F. E. and C. W. Ackerson, - Poultry Sci., 12: 31 - 33, 1933. 81. Prather, E. 0. Jr., Nelson,M. and A. R. Bliss, - Amer. J. Bis. Child., 42: 52 - 56, 1931. 136 82. ,Querido, A., - Acta brev. neerland. Physiol., 5: 9 - 11, 1935. 83. Querido, A „, *- Arch, neerland. Physiol., 20: 487 - 538, 1935. 84. Randoin, L., - Bull. Soc. Chim. biol., 16: 428 - 439, 1934. 85. Roberts, R. E., - Poultry Sci., 9: 102 - 106, 1929. 86. Russell, W. C , Taylor, M. W. and D. E 6 Wilcox, - J . Nut., 9: 567 - 574, 1935. 87. Schmidt-Nielsen, S. and S., - Kongelige Norske Videnskabers Selskab, Forh, 3: 74 - 77, 1930. 88. Schonheimer, R., B eh ring, PI. V. and K. V. Gottberg, - Nut. Abs and Reviews 2: 975, 1932. 89. Schultzer, P., - Biochem. J., 25: 1745 - 1754, 1931. 90. Seifried, B. and B. Heidegger, - Tierarztliche Rundschaw, 39: 172, 1933, 91. Sheehy, .E. J. and Shell, K. - Sci. Proc. Roy. Dublin Soc, 20: 173, 1932. 92. Shelling, D. H, and D. E, Asher, - Bull. Johns Hopkins IIosp., 50: 318 - 343, 1932. 93. bjollema, B. and ¥. G. Barbas, - Nut. Abs. and Reviews, 5: 617, 1932. 94. Smellie, J. M., - J. State Med., 39: 718-727, 1931. 95. Snedecor, G. W., - Calculation and Interpretation of Analysis of Variance and Covarlance, Collegiate Press, Ames, Iowa, 1934. 96. Snedecor, G. W., - Statistical Methods, Collegiate Press, Ames, Iowa, 1937. 97. snedecor, G. W. and H. A. Wallace, - Correlation and Machine,Calculation, Collegiate Press, Ames, Iowa, 1931. 137 98. * S.teenbock, H., Kletzien, S. W. F. and J. G. Halpin, - J. Biol. Chem., 97: 249 - 264, 1932. 99. Stevens, H. and E. M. Nelson, Indust. Eng. Chem., 4: 200 - 201, 1932. 100. Stoeltzner, V. L., - Munch, med. Wochenschr., 78: 1290, 1931. 101. Supplee, VI. C. and C.F. Lee, - U. of Maryland Agroc. Exper. Sta., Bull.389, 1935, 102. Tepper, A. E., - N. H. Agric. Exper. Sta. Bull. 284, 1935. 103. Thoenes, F., - Jahrb. f. Kinderheilk., 131: 1, 1931. 104. Van Esveld, L. W., - Nut. Abs. and Reviews, 5: 2581, 1936. 105. Waltner, K., - Nut. Abs. and Reviews, 5: 3805, 1936. 106. Watkins, W. E. and H. H. Mitchell, - Poultry Sci., 15: 32 - 41, 1936. 107. Wilgus, H. S. Jr., - Poultry Sci., 10: 107 - 117, 1931. 108. Windaus, A., - Nut. Abs and Reviews Is 233, 1932. 109. Windaus, A., Luttringhaus, A.and P. Busse, - Nut. Abs. and Reviews 2: 949, 1932. 110. Wright, Sewall, - Annals of Math. Stat., 5: 161, .1934. 138 TABLE 1' WEEKLY WEIGHTS OF LOT 1 - 1/8% COD LIVER OIL Day o l d 1 Week 2 Weeks 3 Weeks 4 Weeks 36 Gm * 72 Gm 106 Gm 148 Gm 214 Gm 36 64 102 154 210 34 68 116 168 162 36 80 120 172,: 236 34 72 118 162 240 36 72 114 172 258 34 52 76 102 134 40 56 64 88 120 40 62 9 4 120 162 36 62 88 114 148 40 68 90 128 150 36 56 84 110 164 38 64 96 152 238 34 34 58 88 112 40 72 102_ 140 x 38 60 96 136 X 40 74 106 158 X 36 68 94 132 X 38 74 104 146 x 36 58 96 148 x 34 66 92 138 X 34 60 102 152 X No. 22 22 22 22 14 Avr.36063 Gm 64*82 Gm 96.27 Gm 137.64 Gm 182.00 Gm x K i l l e d at 3 week s f o r ash a n a l y s i s . 139 TABLE 11. fEEKLY WEIGHTS .OP LOT 2: - $/l6% COD LI'rER OIL old 1 week 2 Weeks 3 Weeks 4 weeks 34 Gm * 62 Gm 84 Gm 118 Gm 180 Gm 34 70 112 164 2 50 38 74 112 166 260 34 76 122 172 282 36 68 90 120 184 40 7 2 . 106 158 240 36 82,- 120 184 276 40 70 122 178 278 3.6;- :' 36 130 162 252 34 34 94 94 270 38 38 114 164 248 36 36 112 176 282 38 60 94 152 X 34 66 94 142 x 36 56 108 158 x 36 72 96 138 X 34 58 92 136 X 34 78 114 156 X 34 60 88 132 X 36 •'. 74 114 132 X 38 ' 74 112 178 X X 34 X X 22 21 21 21 12 5 9 9 0 Gm 69,71 Gm I O 6 019 Gm 151„43 Gm 250.16 G x K i l l e d at 3 weeks for ash a n a l y s i s , xx Died 140 TABLE 111. JST-6. f WEEKLY- WEIGHTS 0E LOT 3 - i% COD LIVER OIL o l d 1 'Week; 2 Weeks 3 Weeks 4 Weeks 38 Gm * 70 Gm 94 Gm 128 Gm 178 Gm 36 72 88 142 194 3 4 60 90 126 178 36 66 108 168 236 34 56 70 98 144 36 64 108 166 230 40 60 94 146 216 34 56 70 94 124 36 58 76 112 182 40 70 112 • 162 222 40 60 94 144 214 38 60 82 118 ' 170 34 50 84 136 X 38 68 96 152 X 34 70 96 144 x 34 62 94 130 X 36 64 104 148 x 38 66 104 156 X 36 60 88 132 X 40 62 84 132 X 40 52 76 X X 38 X X 22 21 21 20 12 36.63 Gm 62.20 Gm 91.04 Gm 136.70 Gm 1 9 0 0 66 i l l e d at 3 weeks f o r ash a n a l y s i s . xx Died. 141 TABLE IV. • ; WEEKLY WEIGHTS OF LOT 4 - 3/8% COD 'LIVER OIL Day o l d 1 Week 2 Weeks 3 Weeks 4 Weeks 38 Gm* 72 Gm 118 Gm 182 Gm 278 36 76 1166 172 248 36 76 104 120 160 36 60 82 122 182 34 36 52 50 58 80 76 126 90 192 40 76 112 174 252 34 66 112 168 256 40 78 108 186 292 38 74 116 186 270 36 72 128 198 276 40 66 64 182 266 34 66 104 156 236 38 60 78 130 x 34 62 88 144 x 36 64 104 156 X 36 66 102 158 X 38 64 94 144 x 36 58 94 140 x 36 62 100 154 X 36 64 86 128 x 40 58 114 xx Ho. 22 22 22 21 13 Avr.36.72 Gm 6 ^ . ^ Gm 98,27 Gm 152.48 Gm x K i l l e d xx K i l l e d at 3 weeks for ash a n a l y s i s , s u f f e r i n g from a blood c l o t 9 142 TABES T i •> WEEKLY WEIGHTS OE LOT 5 - i% GOD LIVER OIL. Day o l d 1 Week 2 Weeks 3 Weeks 4 Weeks 34 Gnf 60 Gm 108 Gm 180 Gm 284 Gm 36 70 110 190 284 40 68 120" 194 292 40 70 104 164 248 36 60 90 114 133 38 66 104 168 244 36 60 72 118 188 36 72 114 188 280 34 56 92 158 226 36 70 110 172 228 40 74 110 158 232 40 76 122 196 280 40 70 120 194 290 36 60 90 130 x 36 60 98 134 X 40 62 86 130 X 36 58 92 150 X 36 72 106 156 X 38 56 90 142 x 34 68 92 144 x 36 62 96 150 X 36 66 78 X X No-. 22 22 22 21 13 Avr.37.OO Gm 65.27 Gm 100.18 Gm 158.57 Gm 247.23 Gr x K i l l e d at 3 weeks f o r ash a n a l y s i s . xx Died. 1 4 3 T A B L E VI* . WEEKLY WEIGHTS OF LOT 6 - l/Q% PILCHARD OIL (10) Day o l d 1 Week 2 Weeks 3 Weeks 4 Week 38 Gm* 62 Gm 96 Gm 122 Gm 142 Gm 40 68 96 124 160 36 48 68 92 126 36 58 86 118 160 40 74 122 190 256 40 64 102 146 184 38 56 76 102 128 34 54 82 120 140 38 62 104 148 228 36 , 72 122 168 190 34 54 68 108 158 36 72 108 146 200 40 56 72 96 142 38 68 114 154 208 38 52 74 96 140 34 62 110 162 220 38 64 100 142 x 38 70 100 136 X 40 56 102 138 X 34 58 90 136 X 38 X X 36 X X 22 20 20 20 16 37.27 Gm 6l,50 Gm 94, 60 Gm 131.90 Gm 173.87 Gm l i e d at 3 weeks f o r ash a n a l y s i s xx Died 144 TABLE V l l . WEEKLY WEIGHTS 03? LOT 7 - 3 / l 6 f PILCHARD OIL (10) Lay o l d 1 Week 2 Weeks 3 Weeks 4 Weeks 38 Gm. 60 Gm. 80 Gm 120 Gm 184 Gm 40 78 114 156 214 38 60 84 122 162 34 54 88 114 156 40 52 80 102 134 38 54 78 120 180 40 60 74 108 140 38 54 76 102 124 40 60 78 104 . 160 38 62 88 124 168 36 52 84 126 180 38 62 66 9 9 110 40 72 108 142 196 40 66 94 122 150 36 60 88 134 X 38 60 96 136 X 38 62 88 128 X 36 68 102 138 X 34 46 X X 34 46 X X 36 X X 34 X X 22 20 18 18 14 27 Gm 59.40 Gm 87.00 Gm 1 2 1 . 5 5 Gm 16 l.28 Gm x K i l l e d at 3 weeks for ash analysis, xx Died. 145 TABLE V l l l . •WEEKLY WEIGHTS OE LOT 8 - l / 4 # PILCHARD OIL ( 1 0 ) . Day o l d 1 Week 2 Weeks 3 Weeks 4 Weeks .* 36 Gm 58 Gm 88 Gm 2 1 4 Gm 312 Gm 34 60 82 120 168 34 64 1 0 2 154 208 38 76 1 2 0 1% 244 36 66 98 148 2 1 4 34 68 1 0 4 146 204 3 6 66 114 168 2 2 0 38 78 1 2 4 184 262-34 60 1 0 4 162. 2 4 0 36 76 128 194 288 34 66 9 4 •L £w ^ 5 194 36 70 3. «L 2 164 234 36 58 94 158 230 40 70 n o 1 2 2 196 38 80 144 166 238 38 78 136 182 238 38 64 88 144 180 36 62 102 154 230 36 56 100 134 X 36 62 96 136 X 34 68 102 142 X 36 58 92 I 3 8 X 22 22 22 22 18 36„ 09 Gm 66„ 55 106.09 Gm 156.00 Gm 227.77 Gm x K i l l e d at 3 weeks for ash a n a l y s i s . 146 TABLE IX. WEEKLY WEIGHTS OE LOT 9 - PILCHARD OIL (lO) Day old 1 Week 2 Weeks 3 Weeks 4 Weeks 34 Gm 62 Gm 108 Gm 168 Gm 242 *36 58 76 112 150 34 63 112 180 266 40 76 116 180 ' 2 52 33 64 114 158 212 36 58 78 128 180 36 70 118 184 284 36 56 98 153 238 34 66 96 126 170 38 62 90 128 130 36 46 74 106 150 34 6o 106 168 238 34 66 108 172 260 36 62 98 176 262 34 68 102 162 212 34 62 92 138 X 38 64 112 134 X 34 • 6o 94 134 X 34 54 90 142 x 36 48 74 108 X X 33 xx 40 xx 22 20 20 20 15 Avr .35.9O 61,50 Gm 97.80Gm ' 148,10 Gm 220 , 2 6 Gm x Killed at 3 weeks for ash analysis, xx Died. 147 TABLE X. k WEEKLY WE IGHT S 03T LOT 10 .-. PILCHARD OIL (10) . Day o l d 1 Week 2 Weeks 3 Weeks 4 Weeks 36 Gm 56 Gm 84 Gm 112 Gm 152 Gm 36 66 88 118 182 38 84 128 190 260 34 62 108 188 276 40 60 86 130 218 38 62 104 158 238 36 54 86 126 188 38 78 130 200 308 36 74 116 162 230 36 58 90 148 228 38 66 88 150 230 36 74 124 163 256 38 74 128 196 280 36 72 116 172 256 40 80 140 218 328 34 70 126 186 276 36 68 116 174 260 36 60 80 108 148 36 60 98 138 X 34 62 92 134 X 38 64 98 136 X 36 64 98 136 X 22 22 22 22 18 36,63 Gm 66.73 Gm 105.18 Gm 157*63 Gm 239.66 Gm K i l l e d at 3'weeks f o r ash a n a l y s i s . 148 TABLE X L . •WEEKLY WEIGHTS OE LOT 11 - 1% PILCHARD OIL (10). D a y 0 l & 1 Week 2 Weeks 3 Weeks 4 Weeks * 34 Gm 56 Gm 88 Gm 154 Gm 214 34 52 60 86 126 36 56 84 192 278 34 58 74 108 • 154 34 50 64 72 94 40 54 72 108 148 40 50 70 96 132 34 70 108 164 2 58 34 68 112 172 248 36 66 112 182 274 34 58 88 100 130 36 68 108 164 248 34 70 120 173 254 36 74 124 198 294 38 68 102 116 144 36 70 104, 144 x 38 62 98 148 x 40 70 92 130 X 34 68 124 136 X 36 46 62 X X 36 X X 34 X X 22 20 20 19 15 Avr. 35„8l Gm 61.70 Gm 93»30 Gm 139.37 Gm 199,73 Gm x Killed at 3 weeks f or ash analysis. xx Died. 149 TABLE i l l . WEEKLY IIGHTS OP LOT 12 - l/Q% PILCHARD OIL (14) Day o l d 1 Week 2 Weeks 3 Weeks 4 Weeks * 38 Gm 4 8 Gm 64 Gm 76 Gm 82 34 5 8 96 118 138 34 58 7 8 112 130 34 60 84 154 210 36 60 78 82 126 34 54 106 158 206 4 0 46 72 104 182 36 . 54 8 6 114 144 36 56 106 152 2 0 4 34 62 9 8 120 136 40 62 8 4 116 152 34 60 88;: 118 154 36 62 9 6 108 130 36 60 92 132 X 34 42 72 126 X 40 58 96 152 X 36 50 8 6 128 X 34 6o 1 0 8 94 X X 1 3 18 18 18 13 32»00 Gm 56011 Gm 88,33 Gm 120*25 Gm 153»38 x K i l l e d at 3 weeks f o r ash analysis,, xx Died* 1 5 0 TABLE X l ' l l . 'WEEKLY WEIGHTS OE LOT 13 - l/A-% PILCHARD OIL (14). Day o l d 1 Week 2 Weeks 3 Weeks 4 Weeks 34 Gm. . 50 Gm 82 Gm 116 Gm 162 Gm 34 68 114 158 206 36 62 102 148 214 34 56 96 144 202 36 88 138 196 250 32 60 90 142 180 32 56 88 128 172 32 68 112 154 196 32 56 92 124 162 32 58 100 152 212 32 56 98 140 192 32 62 82 114 148 38 56 92 134 X 40 52 86 136 X 32 58 98 132 X 32 60 96 134 X 16 16 16 16 12 32.00 Gm 60 .38 Gm 97.87 Gm 140»75 Gm 191.33 Gm x K i l l e d at 3 weeks f o r ash a n a l y s i s . 151 TABLE XIV. WEEKLY WEIGHTS OP LOT 14 - ±% PILCHARD OIL (14). Day ©Id 1 Week 2 Weeks 3 Weeks 4 Weeks 32 Gm 70 Gm 120 Gm 170 Gm 242 32 58 92 124 192 32 60 80 92 96 32 66 96 112 158 32 70 110 102 152 32 68 106 144 184 32 50 82 156 220 32 64 108 164 240 32 50 74 102 124 32 58 92 148 222 32 60 86 96 142 32 62 108 164 218 32 56 80 118 192 32 54 74 140 X 32 62 98 144 X 32 56 92 136 X 32 62 96 138 X 17 17 17 17 13 AYI -0 32.OO Gm 6o„35 Gm 93„76 Gm 126,47 Gm 18 3.-23 Gm x K i l l e d at 3 weeks f o r ash a n a l y s i s . 152 TABLE XV, WEEKLY WEIGHTS OE LOT 15 - 1% PILCHARD OIL (14) pay o l d I Week 2 Weeks 3 Weeks 4 Weeks 32 Gm 64 Gm 100 Gm 160 Gm 226 32 58 84 118 182 32 66 110 162 244 32 46 62 80 106 32 60 86 122 180 32 56 88 126 192 32 60 82 128 210 32 60 104 174 262 32 56 88 126 196 32 72 108 124 186 32 60 92 148 208 32 52 78 122 182 32 58 94 148 220 32 66 102 140 x 32 66 94 144 x 32 58 104 162 X 32 54 88 144 x 32 48 104 xx 18 18 18 17 13 00 Gm 58089 Gm 92e66 Gm 135.41 Gm 199«53 x Killed at 3 weeks for ash. analysis xx Died. 153 TABLE XVI, WEEKLY WEIGHTS OP LOT 16 « CONTROL. Day old. 1 Week 2. Weeks 3 Weeks 4 Weeks . 38 Gm 66 Gm 88 Gm 104 Gm 132 Gm 34 68 100 124 166 34 48 60 78 102 36 64 96 118 150 38 68 102 124 164 40 64 88 120 158 38 58 86 120 180 34 62 88 116 154 36 • 64 88 108 146 34 60 90 108 150 38 60 80 98 110 36 58 76 104 142 40 64 84 106 152 40 76 114 146 X 38 64 90 128 X 38 68 96 126 X 38 70 110 136 X 38 66 106 136 X 18 18 18 18 13 37.11 Gm 63.78 Gm 91*22 Gm 116.66 Gm 146. 6l Gm x K i l l e d at 3 weeks for ash a n a l y s i s . 154. TABLE XVII. •ft WEIGHT OF CHICK, AVERAGE WEIGHT OF TIBIAE AHDOF ASH IN GRAMS" LOT 1 - 1/8% REFERENCE COlTTlVER OIL n u . . Average Average Chick Tibiae Ash Number Weight Weight Weight 1 214. 0.6905 0.2758 2 210 0.6643 0,2272 3 162 0.4838 0.1880 4 236 0.6708 0.2598 5 240 0.7140 0.3086 6 258 0.7546 0.3139 7 134 0.3137 0 o 1382 8 120 0.3050 0.1221 9 162 0.4484 0.1991 10 148 0.3607 0.1501 11 150 0.4413 0.1489 12 164- 0.5033 0.2355 13 238 0.7223 0.3273 14 112 0.2943 0.1256 155 TABLE XVIII. 4. WEIGHT OF CHICK, AVERAGE WEIGHT OF TIBIAE. ANDuF ASH IN GRAMS" LOT 2 - 3/16% REFERENCE COD LIVER OIL Average Average C h l c k Tibiae Ash Number Weight Weight Weight 15 180 0.5282 0.2439 16 250 0.7689 0.3350 17 260 0.8379 0.3592 18 282 0.8482 0.3836 19 184 0.5602 0.2554 20 240 0.6589 0.2826 21 276 0.8210 0.3751 22 278 0.8512 0.3590 23 252 0.7589 0.3439 24. 270 0.8086 0.3771 25 248 .0.6734 0.2841 26 282 0.944.8 0.4929 156 TABLE XIX. WEIGHT OF CHICK. AVERAGE WEIGHT OF TIBIAE AND OF ASH IN GRAMS LOT 3. - 174% REFERENCE COD LIVER OIL Average Average Chick Tibiae Ash Number Weight Weight Weight 27 178 0.4836 0.2037 28 194 0*6184 0.2646 29 178 0.5818 0.2692 30 236 0.7857 0.3243 31 144 0.4139 0.1898 32 230 0.6908 0.3040 33 216 0.7007 0.3216 34. 124 0.3222 0.1433 35 182 0.6740 0.2880 36 222 0.5856 0.2126 37 214 0.7298 0.3388 38 170 0.5147 0.2287 157 TABLE XX, WEIGHT OF CHICK. AVERAGE WEIGHT  OF TIBIAE AND OF ASH. IN GRAMS LOT 4 - 3/8% REFERENCE COD LIVER OIL , . Average Average Chick - Tibiae Ash Number Weight Weight Weight 39 278 0,9282 0,4348 40 248 0.7859 0.3727 41 160 0.4348 0.1768 42 182 0.5141 0.2421 43 90 0.1869 0.0824 44 192 0.7515 0.3524 45 252 0,8595 0.3735 46 256 0.9065 0.4231 47 292 Q.8661 0.3893 48 270 0,8970 0.4281 49 276 0.8689 0.3959 50 • 266 0.8291 0.3560 51 236 0.8014 0 .3680 158 TABLE XXI, WEIGHT OF CHICK. AVERAGE WEIGHT  OF TIBIAE AMD OF ASH IN GRAMS LOT 5 - 1/2% REFERENCE COD LIVER OIL Average Average Chick Tibiae Ash Number Weight Weight Weight 52 284 0.9062 0.4095 53 284 0.9434 0^4449 54 292 1.0036 0.4492 55 248 0.8382 0.3820 56 138 0.4060 0.1788 57 244 0.8217 0.3718 58 188 0.7698 0.3537 59 280 0.7358 0.3286 60 226 0.7700 0.3442 61 228 0.7322 0.3909 62 232 0,7573 0.3901 63 280 0.8275 0.3761 64 290 0.9852 0.4640 159 TABLE XXII WEIGHT OF CHICK. AVERAGE WEIGHT QF TIBIAE AMD OF ASH IN GRAMS LOT-6 - 1/8% PILCHARD OIL (LOT. A v e r a g e Average Chick Tibiae Ash Number Weight Weight Weight 65 142 0.4474 0.1309 66 160 0.4889 0.1622 67 126 0.3294 0.1199 68 160 0.4925 0.1723 69 256 0.8976 0.3077 70 184 0.5473- 0.2010 71 128 0.3112 0.1159 72 140 0.4227. 0.1460 73 228 0.6895 0.2552 74 190 0.6118 0.2154 75 158 0.4225 0.1535 76 200 0.5635 0.1933 77 142 0.3297 0.1173 78 208 0.6690 0.2379 79 140 0.4493 0.1388 80 220 0.7449 0.2308 160 TABLE XXIII ?ffiIGHT OF CHICK. AVERAGE WEIGHT OF TIBIAE AND OF ASH IN G RAMS  LOT 7 - 3/16% PILCHARD OIL (lO) Average Average Chick Tibiae Ash Number Weight Weight Weight 81 184 0.5279 0.2089 82. 214 0.6488 0.2252 83 162 0.4896 0.1750 84 156 0.4287 0.1895 85 134 0.3882 0.1372 86 180 0.4721 0.1793 87 140 0.4440 0.1417 88 124 0.3712 0.1380 89 160 0.4080 0.1548 90 168 0.4922 0.1693 91 180 0.5501 0.2024 92 110 0.3095 0.1018 93 196 0.6403 0.2222 94 150 0.4385 0.1734 161 TABLE XXIV. WEIGHT OF CHICK. AVERAGE WEIGHT OF TIBIAE ANDOF ASH IN GRAMS" LOT 8 - l7i% PILCHARD OIL (10) Chick Number Weight average Tibiae Weight A v e r a g e Ash Weight . 95 312 0.9388 0.4149 96 168 0.5345 . 0.1797 97 208 0.6685 0.2458 98 244 0.7838 0.3062 99 214 0.6830 0.3096 100 204 0.58 63 0.2116 101 220 0.7100 0.2513 102 . 262 0.7666 0.3295 103 240 0.7322 0.2884 104 288 0.9010 0.3489 105 194 0.4925 0.1948 106 234 0.7104 0.3180 107 230 0.6787 0.2815 108 196 0.6142 0.2778 109 238" 0.6131 0.2725 110 238 0.8211 0.3121 111 180 0.5590 0.2284 112 230 0.6899 0.2966 1 6 2 T A B L E XX V . WEIGHT OF C H I C K . A V E R A G E WEIGHT OF T I B I A E A N D O F A S H I N GRAMS L O T 9 - 3 / 8 % P I L C H A R D O I L ( 1 0 ) Average Average Chick Tibiae A s h Number We ight Weight Weight 1 1 3 2 4 2 0 . 7 2 1 1 0 . 3 5 1 8 1 1 4 1 5 0 0 . 4 1 4 5 0 . 1 6 1 5 1 1 5 2 6 6 0 . 8 9 0 3 0 . 4 1 8 4 1 1 6 2 5 2 0 . 7 7 0 0 0 . 3 3 8 9 1 1 7 2 1 2 0 . 6 4 8 4 0 . 2 6 6 6 1 1 8 1 8 0 0 . 5 0 8 4 0 . 2 2 7 4 1 1 9 2 8 4 0 . 9 5 2 9 0 . 4 5 2 7 1 2 0 2 3 8 0.6902 0 . 3 1 8 7 1 2 1 1 7 0 0 . 4 5 6 8 0 . 2 0 2 9 1 2 2 1 8 8 0.4799 0 . 2 0 7 4 1 2 3 1 5 0 0 . 4 4 1 0 0.1460 1 2 4 2 3 8 0 . 7 2 2 7 0 . 3 0 8 9 1 2 5 2 6 0 0 . 7 3 0 9 0 . 3 0 2 2 1 2 6 2 6 2 0 . 7 6 3 2 0 . 3 3 5 6 1 2 7 2 1 2 0 . 5 2 4 7 0 . 1 9 5 6 163 TABLE XXVI. WEIGHT OF CHICK, AVERAGE WEIGHT OF TIBIAE AMD OF ASH IN GRAMS  LOT 10 - 1/2% PILCHARD OIL (107 Chick Number Weight Average Tibiae . Weight Average Ash Weight 128 152 0.4225 0.1985 129 182 0.5313 0.2494 130 260 0.7983 0.3673 131 276 0.8891 0.4101 132 218 0.8106 0.3845 133 238 0.7540 • 0.3575 134 188 0.5454 0.2521 135 308 " 1.1110 0.4944 136 230 0.7290 0.3069 137 228 0.7466 0.3695 138 230 0.7392 0.3435 139 256 0.7696 0 . 3551 140 280 .0.9169 0.4236 141 256 0.9147 0.4214 142 328 1.0068 0.4616 143 276 0.9278 0.4253 144 260 0.9138 0.4012 145 148 0.4479 0.2087 164 TABLE XXVII. WEIGHT OF CHICK, AVERAGE WEIGHT OF TIBIAE AMD OF ASH IN GRAMS  LOT 11 - 1% PILCHARD~5lL (10) Average Average Chick Tibiae Ash Number Weight Weight Weight 146 214 0.6583 0.2929 147 126 0.3372 •0.1641 148 278 0.9437 0.4385 149 154 ' 0.4365 0.1983 150 94 0.2095 0.0944 151 148 0.4393 0.2063 152 132 0.3304 ' 0.1439 153 258 0.7497 0.3448 154 248 0.8351 0,3841 155 274 1.0273 0.4819 156 130 0.3425 0.1521 157 248 0.8079 0.3578 158 254 0.8709 0.3997 159 294 1.1006 0.5044 •160 ' ' 144 0.4036 0,1689 165 TABLE XXVIII. -a WEIGHT OF CHICK. AVERAGE WETCHT OF TIBIAE AMD OF ASH IM GRAMS-LQT_lg__- .1/8% PILCHARD OIL (l 4) Chick Humber Weight Average Average Tibiae Ash Weight Weigh t 161 82 0.1915 0.0715 162 138 0.4481 0.1250 163 130 0.3497 0.1325 164 210 0.5611 0.1855 165 126 0.2675 0.0998 166 206 0.6262 0.2027 167 182 0.4060 0.1277 168 144. 0.3974 0.1273 169 204 0.5894 0.1859 170 136 0,4217 0.1378 171 152 0,4133 0.1228 172 154 0.4686 0.1446 173 130 0.3443 0.1288 166 TABLE XXIX. WEIGHT OF CHICK. AVERAGE WEIGHT OF TIBIAE AND OF ASH IN GRAMS  LOT 13 - 1/4% PILCHARD OIL (14) Average Average Chick Tibiae Ash Number :Ieight Weight Weight 174 162 0.4806 0.1627 175 206 0.5960 0.2073 176 214 0.5821 0.2065 177 202 0,5314 0.1992 178 250 - 0.7778 0.2577 179 180 0.5451 - 0.1881 180 172 '0.5061 0.1676 181 196 0.5135 0.1655 182 162 0.4987 0.1619 183 212 0.6040 0.2402 184 192 0.6002 0.2111 185 148 0.4118 Q.1524 167 TABLE X)CX. WEIGHT OF CHICK, AVERAGE WEIGHT OF TIBIAE AND OF ASH IN GRAMS  LOT 14 - 1/2% PILCHARD OIL (14) Average Average Chick Tibiae Ash Number IMght Weight Weight 186 242 0.7302 . 0 . 3 2 0 1 187 192 0.5489 0.2207 188 96 0.2789 Q.1283 189 158 0.5073 0.2006 190 152 Q.4519 0.2156 191 184 0.5243 0.2235 192 220 0.7095 0.2956 193 240 0.7950 0.2828 194 124 0.6658 0.2574 195 222 0.5182 0.2748 196 142 0.7218 0.2913 197 218 0.3377 0.1188 198 192 0.5404 0.2036 168 TABLE XXXI. -a WEIGHT OF CHICKv AVERAGE WEIGHT  OF TIBIAE AKDOF ASH IH~GMMS~ LOT 15 - 1% PILCHARD OIL (14) Average Average Chick Tibiae Ash liumber Weight Weight; Weight 199 226 0.6898 0.3210 200 182 0.5278 0.2426 201 244 0.7455 Q.3142 202 ' 106 0.3050 0.1405 203 180 0.5160 0.2411 204 192 0.5639 0.2456 205 210 0.6095 0.2783 206 262 0.8845 0.4197 207 196 0.6383 0.2851 208 186 0.5928 0.2760 209 208 0.6620 0.2872 210 . 182 0.5274 0.2402 211 220 0.6938 0.3183 169 TABLE XXXII, LOT 16 - CGlvTKnr~~ Chick average Average Number « o n-„u iibiae Ash J i e i ^ L _ _ _ i^ e i e-ht Weight 212 132 0.4192 0.1478 213 166. 0.4916 0.1619 214. • 102 0.3648 0.1295 215 150 0.4689 0.1546 216 164 0.4925 0.1385 217 158 0.4940 0.1658 218 180 0.4194 0.1415 219 154 0.4356 0.1242 220 146 0.4029 0.1226 221 150 0.4001 . 0.1261 222 110 0.4136 0.1327 223 142 0,3582 0.1368 224 152 0.3628 0.1119 

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