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A study of some chemical constituent levels in the blood of fowl Kare, Morley R. 1948

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^£3 S7 A STUDY OF SOME CHEMICAL CONSTITUENT LEVELS IN THE BLOOD OF FOWL by Morley R. Kare A Thesis Submitted i n P a r t i a l F u l f i l m e n t of The Requirements f o r the Degree of MASTER OF SCIENCE IN AGRICULTURE In the Department of POULTRY HUSBANDRY The U n i v e r s i t y of B r i t i s h Columbia June, 194-S. ABSTRACT As very l i t t l e information on the subject was available, a study was undertaken of some chemical constituent levels in the blood of fowl. The normal range of nonprotein nitrogen, urea nitrogen, creatinine, uric acid and total chlorides in the blood plasma of 137 14 month old White Leghorn pullets and cocke'rels was determined, and a study was made of the blood constituent levels in some poultry disease. Diets containing different mineral supplements at levels considered to constitute a stress were fed to eight experimental lots of four White Leghorn pullets over a period of six months. Some of the blood chemical constituent levels of the birds were determined at regular intervals and post mortem investigations on fa t a l i t i e s were carried out to determine i f the diets could e l i c i t a condition parallel to pullet disease. The mineral supplements included in the ration failed to produce pullet disease. Effects of these minerals on plasma protein, haemoglobin, glucose,' and on egg production were studied. ACKNOWLEDGMENT The author wishes to acknowledge h i s indebtedness to Pr o f e s s o r J . B i e l y , under whose d i r e c t i o n the experimental work was done, f o r h i s advice and encouragement. To the Head of the P o u l t r y Husbandry Department, P r o -f e s s o r E. A. L l o y d , whose hearty enthusiasm f o r p o u l t r y work has been a constant source of s t i m u l a t i o n , a p p r e c i a t i o n i s s i n -c e r e l y tendered f o r h i s reviewing of t h i s manuscript and the countless^ instances o f h i s valuable counsel. To the emotional feathered colleagues, the b i r d s , who co n t r i b u t e d again and again u n s t i n t i n g l y to t h i s p r o j e c t , the author's sympathy i s o f f e r e d , w i t h th© hope that they w i l l judge t h e i r r o l e i n the s p i r i t i n which the work was done. TABLE Of CONTENTS Part 1(A) - The normal blood levels of some chemical constituents 1 Introduction 1 Review of the literature 4 Materials and methods 7 Procedures 7 Results 12 Observations and discussion 17 Part 1(B) - Blood constituent levels in some poultry disease 21 Introduction 21 Materials and methods 25 Procedures 26 Results 27 Observations and discussion 42 Conclusion 47 Part II - A study of the effects of a mineral stress 50 Introduction 50 Materials and methods 55 Procedures 59 Results 60 Observations and discussion 71 Conclusion 73 References A STUDY OF SOME CHEMICAL CONSTITUENT LEVELS IN THE BLOOD OF FOWL PART 1(A) - The Normal Blood L e v e l s of Some Chemical Con s t i t u e n t s I n t r o d u c t i o n The c l i n i c a l d iagnosis of diseases i s r o u t i n e l y aided "by the a p p l i c a t i o n of chemistry t o examinations of blood and other body f l u i d s . The present i n t e n s i v e use made of c l i n i c a l chemistry has been made p o s s i b l e by the development of s a t i s -f a c t o r y methods f o r the analyses of small amounts of blood. In t h i s respect the micro methods developed by F o l i n , B e n e d i c t , Van Slyke and other outstanding biochemists are now i n d i s p e n -sable to modern c l i n i c a l s t u d i e s of b l o o d values i n p a t h o l o g i -c a l c o n d i t i o n s . The f a i l u r e t o make wide use of t h i s mode of ap-proach to p o u l t r y pathology work may be p a r t i a l l y a t t r i b u t e d to the l i m i t e d value of an i n d i v i d u a l s i c k fowl. At the same time, s t i l l a s s essing the problem on an economic b a s i s , c l i n i -c a l i n v e s t i g a t i o n s of fowl blood where f i n d i n g s would be ap-p l i c a b l e t o whole area populations of f o w l , c o u l d be f i n a n -1 d a l l y j u s t i f i e d . A l a r g e number of methods f o r human bl o o d chemistry i s a v a i l a b l e , some of which w i t h minor adaptation, are a p p l i -cable t o fowl blood analyses. Since the u r i n e and faeces of the b i r d are voided together and cannot be separated without employing elaborate techniques, methods already developed f o r the chemical study of these body products are o f l i m i t e d value i n p o u l t r y work. Blood chemistry i n v e s t i g a t i o n s can be c a r r i e d out w i t h a view to a i d i n g d i f f e r e n t i a l d iagnosis or as an adjunct to a determination of the cause of a p a t h o l o g i c a l e n t i t y . To date i t s use i n p o u l t r y pathology work has been extremely l i m i t e d . The normal chemical composition of the blood can be a l t e r e d by p h y s i c a l and metabolic f a c t o r s . The former i s ex-e m p l i f i e d i n those cases of r e t e n t i o n due to a l t e r a t i o n or d e s t r u c t i o n of permeable membranes i n the e x c r e t o r y organs, such as the lungs, kidney and l i v e r . The l a t t e r i s demonstra-t e d where there i s an increased or diminished formation or u t i l i z a t i o n of the v a r i o u s c o n s t i t u t e n t s of th© blood. An example i s th® accumulation of glucose i n diabetes m e l l i t u s . Some c l i n i c a l e n t i t i e s create an abnormal blood c o n d i t i o n which i s the r e s u l t a n t of both p h y s i c a l and metabolic disturbances. C l i n i c a l chemistry t e s t s are not an end i n them-s e l v e s . The d i a g n o s t i c value of b l o o d chemistry i s circum-s c r i b e d . Since a l l determinations o f d e v i a t i o n from normal must be compared to a range, a p a t h o l o g i c a l l e v e l of a b i r d 3 whose normal i s at one extreme of the aocepted a r b i t r a r y l i m i t s c o u l d be masked. A wide normal range, a c h a r a c t e r i s t i c of some p o u l t r y b l o o d values, would enhance t h i s p o s s i b i l i t y . Often abnormal f u n c t i o n i n g of one organ i n v o l v e s a body network, or may occur simultaneously w i t h the ma l f u n c t i o n i n g of an e n t i r e l y separate organ, f o r example, l i v e r and i s l e t s of langerhan. Such a combination of c o n t r i b u t i n g f a c t o r s would complicate the f i n a l e f f e c t upon bl o o d c o n s t i t u e n t l e v e l s . The compensatory nature of the body, i n some oases, allows remaining t i s s u e of a p a r t i a l l y destroyed organ to maintain the homeostasis of the body i n s p i t e of a p a t h o l o g i c a l disturbance., To i l l u s t r a t e , r e n a l impairment i s o f t e n accompanied by an accumulation of nitrogenous wastes i n the blood. S u c c e s s f u l nephrectomies are common and can be fo l l o w e d by normal nonprotein n i t r o g e n l e v e l s . S l i g h t impairment of one kidney, t h e r e f o r e , does not necessar-i l y r e s u l t i n a r e t e n t i o n of nonprotein n i t r o g e n i n the blood. On the other hand, abnormal l e v e l s of a c o n s t i t u e n t i n the b l o o d can be a m a n i f e s t a t i o n of a multitude of e n t i t i e s . For example, increased nonprotein n i t r o g e n l e v e l s i n human blood can be an i n d i c a t i o n of r e n a l impairment, hyperthyroidism, shock, haemorrhage, f e v e r , vomiting, d i a r r h e a , c a r d i a c decompen-s a t i o n or dehydration pregnancy and, s i m i l a r i l y , even should b l o o d chemistry r e v e a l the malfunction of some organ i n the f o w l , the breakdown i n t h i s s p e c i f i c p a r t of the body might be the m a n i f e s t a t i o n of a multitude of e n t i t i e s . The r o l e of c l i n i c a l chemistry i n the di a g n o s i s of human ailments cannot be d i r e c t l y a p p l i e d to p o u l t r y work. In human medicine the l a b o r a t o r y f i n d i n g s serve t o s u b s t a n t i a t e p h y s i c a l f i n d i n g s . In the l a t t e r the i n d i v i d u a l worth i s so valuable the l i m i t a t i o n s of blood chemistry f o r diagnosis and therapy are p a r t i a l l y overcome by an i n t e n s i v e study of many bl o o d c o n s t i t u e n t s and corroborated by p a t i e n t c o l l a b o r a t i o n . I n p o u l t r y disease c o n t r o l the i n d i v i d u a l , as such, does not warrant i n v e s t i g a t i o n ; that i s , unless a malady con-cerns l a r g e numbers where the best procedure may be t o do away w i t h the few s i c k b i r d s . For t h i s reason when an e n t i t y was being i n v e s t i g a t e d , numerous i n d i v i d u a l b l o o d assay r e -s u l t s would be a v a i l a b l e . This advantage would minimize some of the l i m i t a t i o n s of bl o o d chemistry f o r d i a g n o s t i c work i n p o u l t r y . The d i f f e r e n c e s i n the physiology of b i r d s and mam-mals changes the r e l a t i v e value of the "various b l o o d c o n s t i t u -ent determinations i n a i d i n g c l i n i c a l work. Review of the L i t e r a t u r e Numerous papers have been p u b l i s h e d r e p o r t i n g the chemical c o n s t i t u e n t l e v e l s i n chicken blood. Working on b i r d s l a b e l l e d 'normal* by the i n d i v i d u a l authors, the f o l l o w i n g con-t r i b u t e d the values f o r a number of c o n s t i t u e n t s : F o l i n and Denis (1913); Scheunert and-V. P e l c h r z i m (1923); Hayden and F i s h (192S); Hayden ( 1929); Hayden and Sampson (1931); Dyer and Roe ( l $ 3 k ) ; and Gonzaga (1935). 'Normal' g e n e r a l l y r e f e r s to a r b i t r a r y standards or co n d i t i o n s recognized by the worker concerned. Age, time of bl e e d i n g , d i e t , c y c l i c changes and so f o r t h , may or may not i n -5. fluence a b l o o d c o n s t i t u e n t ' s l e v e l . Some i n v e s t i g a t i o n s have been c a r r i e d out to determine the v a r i a t i o n , i f any, i n b l o o d values under a v a r i e t y of i n f l u e n c e s . Thompson and Oarr (1923) s t u d i e d the d e f i c i e n c y d i e t i n f l u e n c e s ; Thompson and Powers (1925) the e f f e c t of the moult-in g season; Braucher (1929) the i n f l u e n c e of the B i g L i v e r D i -sease; Horvath (1930) the e f f e c t of feeding c o m smut and sprouted soy beans; Gonzaga (1930) the v a r i a t i o n between venous and a r t e r i a l b l ood c o n s t i t u e n t l e v e l s ; Russel and Webber (193^) the e f f e c t of the p r o t e i n l e v e l of the r a t i o n upon c e r t a i n blood c o n s t i t u e n t s of the hen; Henry, Magee and R e i d (193*0 some e f f e c t s of f a s t i n g on the composition of the b l o o d and r e -s p i r a t o r y exchanges .<in f o w l s ; Shimer (1937) the v a r i a t i o n be-tween breeds, high and low p r o t e i n d i e t , time of drawing, i n -fluence of c e r t a i n p a t h o l o g i c a l e n t i t i e s ; H . i l l e r and P a r s e l l (1937) v a r i a t i o n ' i n b l o o d c o n s t i t u e n t s w i t h age, chemical composition of the blood of a hen d u r i n g i t s l i f e c y c l e . Considerable work has a l s o been done on s p e c i f i c p e riods i n the b i r d ' s l i f e c y c l e which cover one or two con-s t i t u e n t s . In s p i t e of a l l the work reported upon normal chicken blood values i t was necessary to e s t a b l i s h another set. This d e c i s i o n i s e x p l a i n e d by the f o l l o w i n g example which r e f l e c t s the d i s c r e p a n c i e s between the f i n d i n g s of v a r i o u s authors. Gonzaga (1933) found the normal l e v e l of nonprotein n i t r o g e n i n the fowl ranged from a maximum of 20 mgms.$ to a minimum of 12.7 mgms.$>. Thompson and Powers (1925) u s i n g the same p r o -cedure of F o l i n and Wu (1919), reported a maximum of 85 .7 rogms.$, and a minimum of 4 l . 4 mgms.$>. This example where the maximum l e v e l determined by one author i s l e s s than the minimum of another worker when done by the same, method i l l u s t r a t e s the d i f f i c u l t y of accepting a normal range from the l i t e r a t u r e . Shimer (1937) summarizes the work on normal l e v e l s up to that date and accepts the f a c t that "no w e l l - d e f i n e d l i m i t s have, as yet, been e s t a b l i s h e d f o r the c o n s t i t u e n t s of the blood of healthy normal a d u l t b i r d s " . He suggests the wide range noted depends upon a number of f a c t o r s t h a t have not been taken i n t o account. He i n c l u d e s amongst the f a c t o r s that i n f l u e n c e at l e a s t some c o n s t i t u e n t s , s t a t e of h e a l t h , egg production, food consumed, time of l a s t feeding, and the c y c l i c a c t i o n of the l i v e r . To t h i s l i s t f o r i n v e s t i g a t i o n could be added the f o l l o w i n g : shock and excitement; c o n d i t i o n under which b i r d s are h e l d , f o r example, caged, on range, e t c . p h y s i o l o g i c a l changes, f o r example, moulting, breeding; b l e e d i n g , f o r example, rate of blood replacement dependent upon i n t e r v a l and between b l o o d drawing and amount removed; geographic l o c a t i o n ; temperature; sex; and age. In view of the f a c t that work of t h i s nature has not been reported as having been done i n t h i s area, p r e l i m i n a r y t o the f u r t h e r use of c l i n i c a l blood chemistry, an experiment was designed to (a) Determine the normal range of nonprotein n i t r o -gen, urea n i t r o g e n , and c r e a t i n i n e i n the blood of s e v e n t y - f i v e healthy l a y i n g p u l l e t s ; (b) Complete a s i m i l a r i n v e s t i g a t i o n w i t h ten healthy breeding c o c k e r e l s . M a t e r i a l s and Methods The experimental work was conducted on two s e r i e s o f b i r d s as f o l l o w s -S e r i e s 1(a) - This group c o n s i s t e d of s e v e n t y - f i v e fourteen-month o l d White Leghorn p u l l e t s . These b i r d s were se-l e c t e d from pens each on a standard but d i f f e r e n t commercial l a y i n g mash. They were i n heavy egg pro d u c t i o n , and d i s p l a y e d no evidence of i l l h e a l t h at the time of bleedings. R e s u l t s were discarded where b i r d s were rechecked s e v e r a l weeks a f t e r b l e e d i n g and any i n d i c a t i o n of i l l h e a l t h was observed. Since o n l y a p o r t i o n of the b i r d s i n t h i s u n i v e r s i t y f l o c k were used, r i g i d s e l e c t i o n was p r a c t i s e d to o b t a i n only those i n good f l e s h and top general w e l l - b e i n g . S e r i e s 1(b) - The breeding c o c k e r e l s i n the S e r i e s 1(a) pens were used. In view of the l i m i t e d numbers of such b i r d s a v a i l a b l e the same r i g i d p r i n c i p l e s o f s e l e c t i o n as were ap-p l i e d to the p u l l e t s were impossible. These males were f o u r -teen months o l d and were mating p r i o r to and durin g the t e s t p e r i o d . Procedures The choice of method f o r the common b l o o d c o n s t i t u e n t s analyses i n v o l v e s s i m p l i c i t y and absolute accuracy. In c l i n i -c a l chemistry absolute values are not always e s s e n t i a l . For d i a g n o s t i c and/or p r o g n o s t i c purposes of t e n the degree of de-v i a t i o n from normal of a bl o o d c o n s t i t u e n t , and not i t s abso-l u t e value guides medical therapy. With regard to a s e l e c t i o n of procedures that would be p r a c t i c a l f o r l a r g e numbers, these methods were chosen. The method used f o r nonprotein n i t r o g e n determination was a f t e r F o l i n and Wu (1919)» ^ox urea n i t r o g e n Van Slyke and Oullen (1916) ( p r i n c i p l e ) ; and f o r c r e a t i n i n e F o l i n and Wu (1919). The nonprotein n i t r o g e n and c r e a t i n i n e determinations were completed on a Coleman spectrophotometer ( U n i v e r s a l l l ) . Many of the assays were repeated on the same sample, p a r t i c u l a r l y where r e s u l t s were at the extreme ends of the range. A l l r e s u l t s were not repeated, since i t c o u l d be as-sumed the l a r g e numbers done would cancel the small personal e r r o r , f o r example, t i t r a t i o n s f o r urea n i t r o g e n were done on s i n g l e samples. Minor changes i n procedure were introduced to take advantage of the optimum ranges on the spectrophotometer, f o r example, c r e a t i n i n e standard was added to the unknown because of the minute q u a n t i t i e s found i n chicken blood. Blood Samples The term "Normal 1 embodies an a r b i t r a r y set of con-d i t i o n s recognized by an author. The o b j e c t i v e when d e c i d i n g l e n g t h of f a s t p r i o r to b l e e d i n g , i n t e r v a l between b l e e d i n g s , feeds, e t c . , was to i n s t i t u t e measurable and reproducable u n i -form c o n d i t i o n s . The s e l e c t i o n of 'standard c o n d i t i o n s ' was such that would e l i m i n a t e or minimize a l l f a c t o r s that might a l t e r the normal blood c o n s t i t u e n t l e v e l s , save those a t t r i b u -t a b l e to st a t e of h e a l t h . B i r d s i n S e r i e s 1(a) were free i n pens, removed from the roosts a f t e r dusk to c r a t e s , and b l e d the f o l l o w i n g morn-in g . A f t e r b l e e d i n g they were returned to the pens. The cockerels i n S e r i e s 1(b) were free i n the pens p r i o r to the f i r s t t e s t but were r e t a i n e d i n b a t t e r y cages t h e r e a f t e r . A twelve-hour f a s t p r i o r to b l e e d i n g was standard f o r a l l b i r d s . This d u r a t i o n was adopted 6ince i t caused only a l i m i t e d disturbance to the b i r d s ' feeding h a b i t s ; but i t was long enough to a l l o w f o r emptying of the d i g e s t i v e t r a c t and was complementary to l a b o r a t o r y r o u t i n e . Shimer (1937) i n v e s -t i g a t e d the e f f e c t of time of b l e e d i n g on nonprotein n i t r o g e n l e v e l s but o f f e r s no conclusions. H© d i d observe that s t a r v i n g y i e l d s more uniform r e s u l t s . The twelve-hour f a s t i s rou t i n e i n human medical work. The b l o o d samples were a l l taken by c a r d i a c puncture a f t e r the method of Sloan and Wilgus (1930). Gonzaga (1935) re-por t e d on the v a r i a t i o n s i n l e v e l of some blood c o n s t i t u e n t l e v e l s between samples on venous and a r t e r i a l blood. I t was found that 2-inch s i z e 20 needles and larg e syringes were most s a t i s f a c t o r y . Andrews (1944) suggests an improvement i n method by the use of a cannula but success encountered w i t h an o r -dinary needle d i d not warrant p r o v i n g h i s method. The p o i n t of entry of the needle was approximately that suggested by Genest (19^6). Only one attempt was made to p i e r c e the heart. In 10. the few cases where blood was not obtained on the f i r s t t r y the operation on the b i r d was not repeated. This was done to av o i d any l o s s since the birds-were being used f o r other e x p e r i -mental work at the same time. An adjustable t a b l e , s i m i l a r t o a caponizing board, was used to h o l d the b i r d s during the operation (See F i g . 1, p. 11). I t was found s u i t a b l e f o r a l l s i z e d b i r d s , adequate weights could be used that would reduce the b i r d s ' s t r u g g l i n g , and the l e v e l was. adjustable t o s u i t the operator. The use of such a board permitted one i n d i v i d u a l to complete the operation. S l i g h t s u c t i o n was a p p l i e d through the syringe when drawing blood. A minimum of f i v e days was allowed to elapse between bleedings and g e n e r a l l y a week or more. Three to Twelve c.c.'s of bl o o d were removed at one time. Potassium oxalate was used e x c l u s i v e l y as the an t i - c o a g u l a n t . A f t e r the f i r s t few punctures, when speed was developed, only the rare sample c l o t t e d . Needles were not r i n s e d w i t h a n t i - c o a g u l a n t . The high r e s i s t a n c e of b i r d s to b a c t e r i a l i n f e c t i o n obviates the n e c e s s i t y of maintaining s t e r i l e c o n d i t i o n s . P r o t e i n - f r e e blood f i l t r a t e s were made i n a l l cases the same day as the b l o o d was drawn. Blood and f i l t r a t e s were kept i n the r e f r i g e r a t o r u n t i l examined. Blood analyses were completed i n a l l cases w i t h i n t h i r t y hours a f t e r drawing. 1 1 . Fig. 1. Bird secured for cardiac puncture Fig. 2. Point of entry for needle 12. Re s u l t s S e r i e s 1(a) A l l b i r d s were re-examined at the completion of the experiment. Table 1 in c l u d e s only r e s u l t s of b i r d s i n good h e a l t h at that time. NONPROTEIN NITROGEN (Whole Blood)  Summary on 6 6 B i r d s . RANGE: Maximum 4 6 . 5 mgms.%. Minimum 29 .0 mgms.$>. MEAN 37.1 mgms.$. FREQUENCY DISTRIBUTION Range-(Mgms.$>.) l e s s than 30 30-35 35.1-40 40.1-4-5 over 4-5 Number of b i r d s 1 13 4-1 5 1 Percentage o f To t a l UREA NITROGEN (Whole Blood) Summary on 23 B i r d s RANGE: Maximum 5 .6 mgms.fe. Minimum 3 .5 mgms.$>. MEAN 4.3 mgras.#. CREATININE (Plasma) Summary on 28 B i r d s RANGE: Maximum .31 mgms.fo. Minimum .4-8 mgms.$>. MEAN . 6 6 mgms.fo. 1-3-S e r i e s 1(b) Table 11 i n c l u d e s r e s u l t s of coc k e r e l s that appeared to be i n good h e a l t h at the time of bleeding. NONPROTEIN NITROGEN Summary of 10 B i r d s RANGE Maximum 4-6.5 mgras.$ Minimum 35.0 mgms.$. MEAN 42.4 mgms.fo. UREA NITROGEN Summary of & B i r d s RANGE Maximum 5.25 mgms.$>. Minimum 3.^5 mgms.$> MEAN 4. 53 mgms.$>. CREATININE Summary of 9 B i r d s RANGE Maximum .75 mgms.$. Minimum .40 mgms.$> MEAN .54 mgms.$. Table 1 S e r i e s 1(a) CHEMICAL BLOOD CONSTITUENT OF HEALTHY FOUR-TEEN MONTH OLD LAYING WHITE LEGHORN PULLETS DATE OF NPN MGMS.fo. BLEEDING BIRD NO. ' MGMS.$ UREA N. June 14 PEN 2. 1171 35.5 4.2 1175 43.00 4.9 1182 32.50 4.55 11515 3S.75 June IS PEN 3. I656 39.25 4.55 * 1S03 34.OO 4.20 1275 42.50 250 33.50 3.50 1274 35.50 3.99 416 29.00 June 20 PEN 4. 1315 33.25 1339 14§2 37.00 40.0 166 42.00 1326 3S .00 June 22 i4sq 36.5 PEN 5. 1S14 36.5 230 35.0 9S 33.0 1226 36.5 1406 37.75 3^.75 1450 35.75 34.25 1457 35.75 1SS5 37.00 1494 3^.25 395 37.00 Table 1 '(don't.-) 15. DATE OF NPN MGMS.f*. -BLEEDING BIRD NO. MGMS..% UREA N. CREATININE June 26 PEN 6 77 37.75 j+,13 1503® 40.00 35 • 39.25 4.55 em - 46.50 5.6 1506 39.25 .73 - 185 32.25 3.63 1560 39.00 .76 1525 36.00 - .62 1883 38.50 40.00 5.08 335* 3.68 1530 3.25 .60 703 June 30 PEN 8 J u l y 2 PEN 1A 1570 1531 1841 1788 3 1768 242 184-9 245 1705 278 297 1335 1710 114 90® 1085® 1051 1071 1019 4-31 3 f . 2 5 3*+. 50 35.00 33.50 37.50 • 37.25 ; 32.20* 39.75 ' 39.25 39.25 32.OO 4-1.75 32.50® 35-75-34.25® 30.00* 3.5 3.5 .60 .30 .54 .70 . 6 4 .60 . 5 4 .60 . 73 .70 .62 .43 .81 . 7 2 .79 . 6 3 J u l y 4 PEN 2A 191 1178 1107 33.25® .00® 00 5.43 4.55 3.S5 Table 1 (Oont.) DATE OF MGMS.% UREA N.^  BLEEDING BIRD NO. NPN IGMS.%.' CREATININE J u l y 4 34.25 PEN 2A 1S30 3.25 .54 (Cont.) 1174 37.00 4.20 1160 39.25 5.25 .74 1216 32.50 40.00 179 4.73 293 32.50 412 3^.75 1297 37.25 .5.6 1122 @ Average of two t e s t s . * Average of three t e s t s Table II Series 1(b) NONPROTEIN NITROGEN, UREA NITROGEN AND CREATININE BLOOD VALUES OF FOURTEEN MONTH OLD WHITE LEGHORN COCKERELS BIRD NO. NPN MGMS«% UREA N. MGMS.% CREATININE July 1 1 , 1947 461926 4 1 . 5 0 4 . 7 3 0 . 6 0 462744 3 7 . 2 5 4 . 9 0 0 . 5 5 461645 46.OO 4 . 5 5 0 . 4 0 58 4 2 . 0 0 .53 461218 4 3 . 2 5 4 . 9 0 460623 46>50 3 . 8 5 0 . 5 5 461695 4 . 2 0 9 . 7 5 1620 4 6 . 0 0 3 . 8 5 0 . 5 0 461254 3 5 . 0 0 461233 4 2 . 5 0 5 . 2 5 0 . 4 9 461917 4 3 . 7 5 0 . 5 0 Observations and Discussion Less than one per cent of birds that were bled d"ied from the operation. No noticeable setback in appearance or production was observed in birds that had been bled. The technique of cardiac puncture was developed with the f i r s t few attempts after which no d i f f i c u l t y was encountered in securing sufficient blood for analyses. In the course of the routine withdrawals of blood the clotting time was estimated to vary between three and five minutes. Rinsing the needles with anti-coagulant, as recommended in the literature, proved to be unnecessary. Series 1(a) The level of nonprotein nitrogen in the blood of sixty-six White Leghorn pullets varied from 29 to 46.5 mgms.#. These results compare favourably with Thompson and Carr (1923) who, on a series comprised of seven birds, reported the normal range as 34.4 to 45.3 mgms.^ j Scheunert and V. Pelchrzim (1923) who worked on three birds, reported a range of 26.2 to 46.2 mgms.^j and Dyer and Roe (1934), on thirteen birds, 28 to 47 m.gms.%. Protein-free f i l t r a t e s were made according to the Hayden (1923) modification of Folin and Wu (1919) method. Simmons and Gentzkow (1944) state that f i l t r a t e s prepared in this manner may contain as much as 10 per cent more nonprotein nitrogen than i f the original Folin-Wu procedure is followed. This fact could explain some of the discrepan-cies in results that were discussed in the introduction. It i s to be noted that 98.5 per cent of the values were 44 mgms.^ . or- less. This could serve as the upper arbitrary c r i t i c a l level for c l i n i c a l diagnosis. The urea nitrogen blood level was found to vary from 3 . 5 to 5*6 mgms.#. The Van Slyke Cullen ( 1916) aera-tion method was found to be practical for large numbers of tests being done simultaneously. The results are close to those reported by Hayden ( 1929) who, on the basis of ten birds, found the normal range to be 2 . 5 5 to 7.80 mgms.$; and a l i t t l e higher than those reported by Dyer and Roe (1934) of 2 . 1 to 5 . 4 mgms.# on the basis of four birds. Extreme normal values as high as 1 6 . 9 and as low as 2 . 0 7 mgmso^ , both arrived at by the Folin-Wu (1919) method, are listed in the literature. According to Clementi ( 1 9 3 2 ) , a l l the urea figures in the literature are too high. The normal plasma levels of creatinine found in this investigation are lower than those recorded in the literature for whole blood. A composite of ten authors' reported findings yields a maximum of 1.80 mgms.$ and a minimum of 1 . 0 0 mgms.#. The range obtained here was 0 . 4 0 to 0 , 8 5 mgms.$ which is close to the results of 0 . 6 0 to 0 . 8 9 mgms.# obtained by Hayden and Sampson (1931) working on two birds. Nearly a l l of the results in the literature, on creatinine levels in fowl, have been determined on whole blood. It i s recognized that the colorimetric determinations (Jaffe reaction) used here and also by the authors referred to in this manuscript, is by no means 20 specific. Chromogenic substances are present in the blood which can contribute to the fi n a l color. These substances are chiefly in the red blood cel l s , so that results on whole blood f i l t r a t e s probably are too high. The very low levels of creatinine found- in chicken blood coupled with the limits of accuracy of the spectrophotometer make diagnosis on the basis of small deviations from normal nearly impossible. Series 1(b) Severe crop impaction was observed in many of the cockerels after they had been confined to the cages for several days. Results are not included where this condition was clearly evident in a bird. The average nonprotein nitrogen level in the males was found to be higher than the normals established for females in series 1(a). The urea nitrogen and creatinine results were comparatively similar in both males and females. In view of the limited numbers of male birds reported on, the findings presented are subject to further confirmation. PART 1(B) - Blood Constituent L e v e l s In Some P o u l t r y Disease I n t r o d u c t i o n During the past decade i n the Province of B r i t i s h Co-lumbia, an i n c r e a s i n g percentage of fowl m o r t a l i t y has been r e -cognized on post mortem examination to involve the kidneys. The incidence of t h i s c o n d i t i o n , with i t s a s s o c i a t e d cost to the poultryman, has evoked considerable a t t e n t i o n . A review of post mortem repo r t s reveal that kidney malfunction of fowl has been a serious economic f a c t o r f o r some time. Beach (1927) of C a l i f o r n i a reviewed the diagnosis of 1694 post mortems and d i s c l o s e s the f a c t that 4$ of the f a t a l -i t i e s were gout and 2.5$ were l i s t e d as swollen kidneys. Hor-vath (1930) r e f e r r i n g to the United S t a t e s , revealed that "the occurrence of gout i n hens i s quite frequent". M y l r o i e (1942) i n a survey of p o u l t r y m o r t a l i t y at the U n i v e r s i t y of B r i t i s h Columbia p o u l t r y farm observed "kidney t r o u b l e c h a r a c t e r i z e d by accumulation of u r i c a c i d c r y s t a l s i n the kidneys and u r e t e r s accounted f o r 10$ of a l l deaths". The m o r t a l i t y w i t h kidney involvment observed here amongst b i r d s from the U n i v e r s i t y farm and specimens sent i n by p o u l t r y farmers can be d i v i d e d i n t o three age groups -(1) Baby c h i c k s , 2 - 3 weeks of age; (2) P u l l e t s s h o r t l y a f t e r commencing to l a y ; (3) Older hens approaching the end of the f i r s t year of l a y i n g . "Kidney c o n d i t i o n s " have not been l i m i t e d to these groups. Small numbers of males, and females not i n the above c l a s s i f i c a t i o n , have been examined that d i s p l a y e d s i m i l a r r e n a l l e s i o n s . Jungherr and Levine (1941) describe a disease of c h i c -kens "which manifests a d e f i n i t e syndrome" and i s known by the common name of "so c a l l e d P u l l e t s 1 Disease". I t a f f e o t 6 " r e -c e n t l y housed p u l l e t s " and i s c h a r a c t e r i z e d by a sudden drop i n egg production and food consumption, darkening of the comb, dehydration and di a r r h e a . They add that the disease may occur i n other age groups and s t a t e that i t has been seen i n chicks four weeks o l d , i n t y p i c a l form as to symptomatology and r e n a l gross l e s i o n s . They emphasize the great v a r i a b i l i t y and range of the syndrome i n p a t h o l o g i c a l manifestations but add "the r e s u l t s of the i n v e s t i g a t i o n s by microscopic methods tend t o show many b a s i c features i n common and would j u s t i f y the i n -c l u s i o n of the various manifestations under one e n t i t y " . The pathology of the syndrome i s c h a r a c t e r i z e d by hepato-nephrotic l e s i o n s a s s o c i a t e d at times with f i s h - f l e s h l i k e ( d y s t r o p h i c ) changes i n the s k e l e t a l musile and a chalky appearance of the pancreas. They observed that "the gross l e s i o n s of the kidneys presents a gradient beginning w i t h a b s o l u t e l y i n s i g n i f i c a n t change to marked enlargement and f i n a l l y the f a m i l i a r p i c t u r e of u r i c n e p h r i t i s known as v i s c e r a l gout". 2 3 . The broad description of symptoms of the "so called pullet disease" would encompass the "kidney condition" encountered here in British Columbia.. Jungherr and Matterson ( 1 9 4 4 ) , on the basis of their hemalological findings, propose* that "so called pullet disease" be called Avian Monocytosis. They report on i t s occurrence in Massachusettes, Connecticut, New York, New Jersey, Michigan, California, Ontario and most of the Atlantic seaboard States. Contributions to the literature on the subject would indicate that the entity i s even more widespread in both Canada and the United States. Veterinary history gives i t s age as an entity as nineteen years. Beaudette (1929) under the term X Disease, described a disorder of fowl which was characterized by cyanosis of the head, catarrhal enteritis and scattered haemorrhages on the internal organs. The disease resembles fowl cholera except no filterable agent or bacteria were demonstrable. The great variability of sumptomalogic and pathologic features has led to the condition being designated by numerous popular names in different areas. What appears to be a single condition, according to Jungherr and Matterson (1944) or two related entities Bullis (1944) has been labelled pullet disease, Tom Barron's, acute toxemia, x disease, xx, summer, housing, new wheat, blue comb and cholera-like disease, coli bacillosis, contagious indigestion, kidney disease, avian monocytosis and others. 24, Jungherr and Matterson (1944) thoroughly review a l l aspects of the disease. In their review of blood chemistry they suggest i f additional chemico-pathological work would confirm present findings i t would mark a physio-logical disturbance of the nitrogen and water metabolism, and the electrolyte balance. They confirm earlier findings on the blood of high average values for nonprotein nitrogen and uric acid, low calcium and normal values for phosphorus and magnesium. The serum potassium was reported to be low but the whole blood potassium high. The stricken birds had an increased haemoglobin and in a small number of severe cases that were available total chlorides were strikingly low. The diverse character of the symptoms of pullet disease syndrome lends the condition to diagnostic abuse. Further work on the blood chemistry as an aid to i t s accurate diagnosis appears warranted. With this object in mind the following work was planned. (1) To follow the blood constituent levels over a period of time to determine the history of the blood changes in "kidney conditions". ( 2 ) To compare the blood chemistry findings of a miscellaneous group of cull birds. Materials and Methods The experimental work was completed on two groups of birds. Series 2(§Q The cull birds were selected from the same pens as were the pullets that were used in series 1(a). No special criterion was exercised in selecting these birds except that they f e l l into the general classification of " c u l l " . A l l were sixteen month old White Leghorn pullets. The work was done between July 1 1 t h and 3 1 s t , 1947. Series 2(b) These birds were a miscellaneous group collected from the University farm and also from several commercial establishments in the Fraser Valley. Considerable diffi c u l t y was encountered in securing the desired number of birds displaying the typical external symptoms of kidney disease. For this reason, many of the birds chosen to study as kidney condition cases were, at the time of selection, doubtful. The birds were a l l females ranging in age from one to two years. They included White Leghorns, Rhode Island Reds, New Hampshires, Barred Rocks, Cambars and crosses. The work was done between August 2 0 t h and September 1 4 t h , 1 9 4 7 . The birds in series 2(a) were free in pens, placed in crates for fasting twelve hours prior to bleeding. The birds in series 2(b) were confined to laying cages throughout the experiment. A l l birds were given a commercial ration, supplemented with some whole wheat, and water ad libitum. The standard conditions adopted in establishing the normal blood values were applied. The only exception was the removal of a second blood sample from a bird without allowing the minimum five day interval. This was necessitated where death was imminent and a con-firmatory analysis was desired. Also in a few cases a small blood sample was taken to do a preliminary non-protein nitrogen test. Procedures The methods used for nonprotein nitrogen, urea nitrogen and creatinine determinations were as described" in part 1(a). The method of Brown (1945) was used for determining uric acid levels. This method proved practical for large numbers of tests and although'it i s not as widely used as some other procedures, Hawk, Oser and Summers (1947) describe i t as satisfactory and simpler than most methods. Chlorides were determined by the method of Schales and Schales (1941). Hawk, Oser and Summerson (1947) describe i t as "not quite as accurate as the common iodometric method but i s known to give satisfactory results". The procedure is very simple and especially suitable where large numbers of c l i n i c a l determinations are being done. 2 7 . Both the uric acid and the chloride determinations were done on plasma. The uric acids were completed on the Coleman spectrophotometer. Post Mortems Post mortem examinations were performed on birds that died during the course of the experiment. At the conclusion of the blood work a l l birds that exhibited any sign of i l l health were kil l e d and examined. Amongst those birds that appeared to be functionally normal and whose blood constituent levels were within normal limits only representative individuals were ki l l e d for internal examination. Results Table 111(a) includes the blood results of cull pullets in series 2(a) in which the post mortem examination revealed dormant ovaries but were otherwise negative. Birds 1794 and 1789 were poorly fleshed. Summary of Results in Table I l l ( a ) Number of birds bled 14 Number of blood samples 30 Nonprotein nitrogen (28 tests) Range - Maximum 4 5 . 7 5 mgms. % - Minimum 3 0 . 2 5 mgms. % Frequency distribution Range (mgms. %) 3 0 - 3 5 35.1^-40 4 0 . 1 - 4 5 over 45 No. of samples 7 13 7 1 Percentage of total 2 5 . 0 4 6 . 4 2 5 . 0 3 . 6 28. Urea nitrogen (24 tests) Range - Maximum - Minimum 5.75 mgms. 3 . 2 5 mgms. % % Creatinine (19 test • ) Range - Maximum - Minimum 1 . 0 0 mgms. . 4 3 mgms. % • % TABLE III( a) Blood Constituent Levels of Cull Pullets - Disease Free Bird No. Blood sample s N.P.N.X Creatinine x Urea N. 1292 1 2 3 4 0 . 5 0 3 8 . 0 0 3 7 . 7 5 0 . 4 8 0 . 7 0 5 . 9 5 4 . 2 0 4 . 5 5 431 1 3 8 . 0 0 0 . 6 9 4 . 3 8 388 1 3 4 . 0 0 0.80 1539 1 3 6 . 0 0 0 . 4 8 4 . 5 5 1707 1 2 3 3 4 . 2 5 3 9 . 2 5 4 0 . 0 0 . 0 . 4 3 O.65 0 . 5 0 3 . 8 5 4 . 2 0 4 . 5 5 1673 1 3 8 . 0 0 4 . 9 0 1691 1 2 3 3 2 . 2 5 4 1 . 7 5 3 7 . 5 0 0 . 6 0 4 . 9 0 4 . 9 0 3 . 2 5 25 1 2 3 4 3 . 2 5 4 2 . 2 5 0 . 5 0 0 . 6 3 5 . 2 5 3 . 8 5 3 . 6 4 1332 l 3 0 . 2 5 3 - 8 5 1678 1 2 3 6 . 0 0 3 8 . 5 0 1732 1 2 3 3 5 . 0 0 3 5 . 0 0 0 . 5 6 0 . 6 8 0 . 5 8 4 . 5 5 3 . 5 7 x Results in mgms. % Table I l l ( a ) (Cont'd) Blood x Bird No. samples N.P.N. Creatinine x Urea N.x 1794 1 4 0 . 0 0 0 . 4 8 5 . 6 0 2 3 9 . 5 0 0 . 5 3 4 . 0 6 1789 1 4 0 . 2 5 2 4 3 . 2 5 0 . 4 8 4 . 9 0 3 4 2 . 2 5 3 . 6 4 1114 1 3 1 . 5 0 2 4 5 . 7 5 0 . 8 8 4 . 2 0 3 3 9 . 5 0 1 . 0 0 4 . 5 5 Table 111(b) covers the blood results of the birds in series 2(b) who, with the exception of A 1 0 , were not laying during the t r i a l period. These fowl appeared to be in good health at the completion of the experiment. Summary of Results in Table 111(b) Number of birds bled 7 Number of blood samples 23 Nonprotein nitrogen (23 tests) Range - maximum 4 6 . 2 5 mgms. % - minimum 3 1 . 2 5 mgms. % Frequency distribution Range (mgms. %) 3 0 - 3 5 3 5 . 1 - 4 0 4 0 . 1 - 4 5 over 45 Number of samples 7 11 4 1 Percentage of total 3 0 . 4 4 7 . 8 7 . 4 4 . 4 Urea nitrogen (5 tests) Range - Maximum 5 .6 . - Minimum 3 . 7 Creatinine (21 tests) Range - Maximum . 75 - Minimum . 4 3 x Results in mgms. % 3 0 . Uric acid (17 tests) Range - maximum 5.00 mgms. % - minimum 2.60 mgms. % Chlorides (18 tests) Range - maximum 128.9 " --minimum 114*0 " TABLE 111(b) Blood Constituent Levels of Cull Pullets - Disease Free Blood x x Urea Uric Chlorides* Bird No. samples N.P.N. Creatinine N. x Acid xTotal As NaCl CI , 1 35.00 . 5 0 3.70 4.00 120.2 703.2 2 3 4 . 7 5 . 5 4 5.25 3 . 6 8 117.5 687.4 B7 1 41.75 2 36.50 . 6 7 3 . 4 0 122.8 718.4 3 40.00 . 6 0 4.00 122.8 718.4 4 3 4 . 2 5 .60 2.60 117.5 687.4 B3 1 40.00 .71 127.3 744.7 2 3 3 . 2 5 . 6 5 4 .50 121.0 707.9 3 3 7 . 7 5 . 4 3 5.00 121.0 707.9 B8 1 42.50 2 40.00 .75 5 . 9 5 3 . 3 0 128.9 754.1 3 3 5 . 0 0 . 5 4 2 .90 121.9 713.1 B10 1 44.00 2 40.75 . 6 7 3.80 121.0 7 0 2 . 9 3 4 6 . 2 5 . 4 3 4 . 7 0 121.0 707.9 4 3 6 . 5 0 . 5 4 4 . 4 0 120.2 703.2 A10 1 38.75 4 .55 4.10 117.5 681.4 2 31.25 . 6 9 3 37.00 .71 4 .90 3 . 9 4 118.3 6 9 2 . 6 Bl 1 3 7 . 7 5 2 3 4 . 7 5 . 5 7 5.60 3 . 9 4 121.0 707.9 3 3 6 . 5 0 .60 3 . 9 0 118.4 6 9 2 . 6 4 38.50 . 5 7 3 .50 114.0 6 6 6 . 9 x Results in mgms. 3 1 . Table IV(a) includes the results of blood analyses of birds in series 2(a) who were afflicated with I r i t i s . Summary of Results in Table IV(a) Number of birds bled 6 Number of blood samples 12 Nonprotein nitrogen (12 tests) Range - maximum 4 2 . 0 mgms. % - minimum 3 0 . 0 mgms. % Frequency distribution Range (mgms. %) 3 0 - 3 5 3 5 . 1 - 4 0 4 0 . 1 - 4 5 Number of samples 2. 7 3 Percentage of total 1 6 . 7 5 8 . 3 25 Range - maximum - minimum 5 . 9 5 mgms. % 3 . 8 5 mgms. % Creatinine (8 tests) Range - maximum - minimum 0 . 5 3 mgms. % 0 . 7 5 mgms. % TABLE IV(a) Blood Constituent Levels of Cull Pullets - I r i t i s Bird No. Blood samples N.P.N. x Creatinine X Urea N. 1274 1 3 5 . 5 0 3 . 9 9 108 1 3 8 . 0 0 0 . 5 5 • 1422 1. 2 4 2 . 0 0 0 . 7 5 4 1 . 2 5 0 . 6 3 4 . 2 0 4 . 2 0 1344 1 2 4 0 . 5 0 0 . 6 5 3 8 . 7 5 0 . 5 4 5 . 9 5 3 . 8 5 X Results in mgms. % 3 2 . Table IV(a) (Cont'd) Blood B i r d No. samples 1785 1745 1 2 3 1 2 3 N.P.N. 3 9 . 0 0 3 5 . 0 0 3 0 . 0 0 3 8 . 0 0 3 6 . 5 0 3 8 . 2 5 C r e a t i n i n e * Urea N. 3.85 4 . 9 0 ' 0 . 7 0 0 . 4 9 0.53 Table IV(b) i n c l u d e s the r e s u l t s of blood analyses of b i r d s with fowl p a r a l y s i s . Summary of R e s u l t s i n Table IV(b) Number of b i r d s b l e d 2 Number o f blood samples 5 Nonprotein n i t r o g e n (5 t e s Range - maximum 39.25 - minimum 33-00 Urea n i t r o g e n (2 t e s t s ) Range - maximum 4.90 - minimum 4.90 C r e a t i n i n e (3 t e s t s ) • Range - maximum .54 - minimum .50 U r i c a c i d (3 t e s t s ) Range - maximum 4.16 - minimum 4.00 C h l o r i d e s (3 t e s t s ) Range - maximum 121.9' - minimum 115.8 x R e s u l t s i n mgms. % 33. TABLE IV(b) Blood Constituent Levels of Cull Pulleta - Fowl Paralysis Chloride s Blood ' • Creatin- Uric As Bird No. samples N.P.N. Urea N.x ine * acid Total NaCl C5 1 3 4 . 5 0 2 37.50 4 . 9 0 .50 4 . 0 0 1 2 1 . 9 7 1 3 . 1 3 3 9 . 2 5 4 . 1 0 1 2 1 . 9 7 1 3 . 1 A7 1 3 8 . 0 2 3 3 . 0 4 . 9 0 . 5 4 4 . 1 6 1 1 5 . 8 6 7 7 . 4 The blood constituent levels of pullets with "kidney condition" are reported in Table V(a). Condition of bird number 1409 was complicated by dropsy and a tumorous, ovary; bird number 1491 with an obstruction of the oviduct. Summary of Results in Table V(a) Number of birds bled 4 Number of blood samples 7 Nonprotein nitrogen (7 tests) Range - maximum 60.50 mgms. % - minimum 3 7 . 5 0 mgms. % Frequency distribution Range (mgms. %) below 45 4 5 . 1 - 5 0 5 0 . 1 - 5 5 55.1-60 over 60 Number of samples 1 1 2 1 2 Urea nitrogen (3 tests) Range - maximum 5 . 6 0 mgms. % - minimum 4 . 2 0 mgms. % Creatinine (4 tests) Range - maximum 1.00 mgms. % - minimum . 55 mgms. % x Results in mgms. % TABLE V(a) Blood Constituent Levels of Cull Pullets Kidney Condition (pullet disease) Blood x  Bird no. samples N.P.N. Creatinine Urea N. 279 1 6 0 . 5 0 lvOO 275 1 4 8 . 5 0 0 . 5 6 4 . 2 0 1409 1 6 0 . 5 0 2 5 6 . 7 5 0 . 6 3 1491 1 3 7 . 5 0 0 . 5 5 5 .14 2 5 4 . 0 0 5 . 6 0 3 5 2 . 2 5 Table V(b) includes the blood results of birds in series 2(b) who on post mortem were diagnosed as kidney condition cases. Summary of Results in Table V(b) Number of birds bled 3 Number of blood samples 4 Nonprotein nitrogen (4 tests) Range - maximum 82.75 mgms. % - minimum 51 .50 mgms. % Urea nitrogen ( 1 test) Value - 7 . 2 0 mgms. % Uric acid ( 2 tests) Range - maximum - over 25 mgms. % - minimum - over 15 mgms. % Chlorides (2 tests) Range - maximum 1 4 8 . 2 mgms. % - minimum 1 2 9 . 8 mgms. % x Results in mgms  % 3 5 . TABLE V(b) Blood Constituent Levels of Cull Pullets -Kidney Condition (pullet disease)  __ « TT • Chlorides Blood x Uric Bird no. samples N.P.N, Urea N. Acid Total As NaCl A2 1 5 1 . 5 0 2 66.00 7.70 15.00 129.8 759.3 C3 1 82.75 27.00 148.2 867.0 A5 1 51.75 Table VI(a) includes the blood results of pullets with lymphomatosis. The condition of number 1309 was complicated by blindness. A l l four birds were emaciated. Summary of Results in Table VI(a) Number of birds bled 4 Number of blood samples 10 Nonprotein nitrogen (9 tests) Range - maximum 5 6 . 7 5 mgms. % - minimum 3 2 . 7 5 mgms. % Frequency distribution over Range (mgms.50 to 35 3 5 . 1 - 4 0 4 0 . 1 - 4 5 4 5 . 1 - 5 0 5 0 . 1 - 5 5 55 No. of .samples 1 1 4 1 1 1 Urea nitrogen (9 tests) Maximum - 6 . 3 0 mgms. % Minimum - 4 . 4 1 mgms. % Creatinine (8 tests) Maximum - 0.80 mgms. % " Minimum - 0 .48 mgms. % x Results in mgms. % TABLE VI(a) Blood Constituent Levels of Cull Pullets - Lymphomatosis Blood x x x Bird no. samples N.P.N. Creatinine Urea N. 1628 1 4 0 . 0 0 O .55 5 . 2 5 2 56 .75 6 . 3 0 283 1 0 . 4 8 4 . 4 1 2 3 2 . 7 5 4 . 5 5 3 5 0 . 5 0 0 . 8 0 1309 1 4 5 . 0 0 O . 6 5 5 .46 2 4 5 . 0 0 O .65 5 .60 3 4 3 . 7 5 0 . 5 0 4 . 5 5 4 4 7 . 5 0 0 . 7 5 5 . 2 5 326 1 4 2 . 0 0 0 . 8 0 5 .6O Table Vl(b) includes the blood results of birds in series 2(b) whose malfunctioning was attributed to lymphomatosis on the basis of the post mortem findings. Summary of Results in Table Vl(b) Number of birds bled 2 v Number of blood samples 6 Nonprotein nitrogen (6 tests) Range - maximum 6 4 . 0 0 mgms. % - minimum 4 2 . 7 5 mgms. % Urea nitrogen (4 tests) Range r maximum 7 .87 mgms. % - minimum 4 . 2 0 mgms. % Creatinine (1 test) Value *81 mgms. % Uric acid ( l test) Value 4*60 mgms. % x Results in mgms. % 3 7 . Chlorides ( 4 tests) Range - maximum 1 2 7 . 3 mgms. %. - minimum 1 0 4 . 3 mgms. %. TABLE VI (b) Chlorides Blood x Crea- x Uric As Bird No. sample N.P.N. Urea N.x tinine acid Total NaCl A3 1 5 9 . 7 5 2 6 1 . 7 5 7 . 8 7 119.8 7 0 0 . 6 3 6 4 . 0 0 7 . 0 0 .81 1 0 4 . 3 6 0 5 . 4 A6 1 4 6 . 5 0 2 4 2 . 7 5 4 . 2 0 1 1 4 . 5 6 6 9 . 6 3 4 7 . 7 5 . 5 - 2 5 4 . 6 0 1 2 7 . 3 7 0 4 . 7 Table VII includes the blood results of pullets with oviduct obstruction. A l l the birds in this group exhibited severe emaciation. Summary of Results in Table Vll(a) Number of birds bled 3 Number of blood samples 5 Nonprotein nitrogen ( 5 tests) Range - maximum 5 2 . 5 0 mgms. % - minimum 4 3 . 0 0 mgms. % Frequency distribution Range (mgms.#) 4 0 - 4 5 4 5 . 1 - 5 0 5 0 . 1 - 5 5 Number of samples 3 1 1 Urea nitrogen ( 3 tests) Range - maximum 8 . 7 5 mgms. % - minimum 4 . 2 0 mgms. % Creatinine ( 3 tests) Range - maximum 0 . 7 5 mgms. % - minimum 0 . 6 0 mgms. % x Results in mgms. % 3 8 . TABLE VII(a) Blood Constituent Levels of Cull Pullets Oviduct Obstruction Blood x x x Bird No. samples N.P.N. Creatinine Urea N. 1 6 7 7 1 5 2 . 5 0 0 . 7 5 6 . 3 0 1 4 4 2 1 4 5 . 0 0 2 4 4 . 0 0 4 . 2 0 3 4 7 . 0 0 . 0 . 6 3 1 7 4 8 1 4 3 . 0 0 0 . 6 0 8 . 7 5 Table Vll(b) includes the blood results of one bird:- with an oviduct obstruction. Summary of Results in Table VII(b) Number of birds bled 1 Number of blood samples 5 Nonprotein nitrogen ( 5 tests) Range - maximum 5 2 . 5 0 mgms. % - minimum 4 2 . 5 0 mgms. % Urea nitrogen ( 3 tests) Range - maximum 5 * 6 0 mgms. % - minimum 4 . 5 5 mgms. % Creatinine ( 2 tests) Range - maximum . 8 5 mgms. % - minimum . 7 6 mgms. % Uric acid ( 2 tests) Range - maximum 7 * 4 0 mgms. % - minimum 7 . 0 0 mgms. % Chlorides ( 3 tests) Range - maximum 1 1 5 . 8 mgms. % - minimum 1 1 2 . 3 mgms. % x Results in mgms. % TABLE VII(b) Blood Constituent Oviduct Levels of Cull Obstruction Pullets •* Blood Crea- Uric Chlorides Bird no. sample N.P.N.X Urea N.x t i n i n e x acid Total As NaCl A9 1 5 2 . 2 5 1 1 4 . 0 6 6 6 . 9 2 4 5 . 0 0 4 . 5 5 3 4 2 . 5 0 4 . 9 0 7 . 0 0 1 1 5 . 8 6 7 7 . 4 4 4 4 . 0 0 . 8 5 • 5 5 2 . 5 0 5 . 6 0 . 7 6 7 . 4 0 1 1 2 . 3 6 5 7 . 0 Table VIII(a) includes results on birds whose blood analyses are essentially negative but the post mortem findings were positive. Bird number 1 0 4 0 had i r i t i s and an enlarged right ureter. Bird number 515 was emaciated, was suffering with an impacted crop and i t s kidneys were a yellowish colour but gave no suggestion of pullet disease. Number 1 4 1 0 had kidneys similar to 515 but was otherwise normal. TABLE VIII(a) Blood Constituent Levels of Cull Pullets - positive post mortems with normal blood pictures Bird No. Blood sample N.P.N. Creatinine Urea N. 1 0 4 0 1 3 8 . 5 0 0 . 5 0 6 . 2 0 2 3 9 . 7 5 0 . 6 3 4 . 2 0 3 3 9 . 2 5 0 . 5 0 4 . 2 0 515 1 1 . 0 0 2 4 0 . 5 0 4 . 9 0 3 3 2 . 0 0 4 . 2 0 1 4 1 0 1 3 8 . 0 0 2 3 8 . 7 5 X Results in mgms. % Table VIIl(b) l i s t s the results of birds whose blood constituent levels are within normal limits but where the post mortem examinations revealed the following conditions. ,Bird number AS was suggering from a severe infestation of worms. Bird number B4 had catarrhal enteri-t i s . Bird number C6 was extremely emaciated and i t s spleen, li v e r and kidneys were enlarged. The lungs and pancreas were abnormal. TABLE VIII(b) Blood Constituent Levels of Cull Pullets - positive post mortems with normal blood pictures  T>i , „ TT . Chlorides Blood Crea- Uric Bird No. sample N.'P.N. Urea N, tinine Acid Total As NaCl A8 1 40.00 4.2 114.5 6 6 9 . 6 2 34.15 4 .9 .75 117.5 687.4 B4 1 40.75 2 40.75 .71 3 . 6 0 121.9 713-1 3 35.75 . 6 5 3.10 121.0 707.9 4 34.25 .43 4 . 4 0 118.4 692.6 C6 1 44.00 2 40.00 4 . 9 0 .88 3 . 3 0 121.0 707.9 Table IX(a) includes the blood constituent levels of two birds whose malady could not be classified in any of the other groupings. Number 1411 was extremely emaciated and revealed on post mortem a massive caseation of the right lung. The kidneys were small, pale and atrophied but contained none of the urate deposits charac-t e r i s t i c of pullet disease. Bird number 1861 was thin and 41. when opened displayed severe inflammation of the caecum but the kidneys appeared normal. TABLE IX(a) Blood Constituent Levels of Cull Pullets -Miscellaneous Diseases  Bird No. Blood samples N.P.N. Creatinine Urea N.x 14H 1 6 0 . 0 0 0 . 7 5 1 3 . 3 0 1861 1 5 0 . 5 0 2 40 .00 0 . 3 6 1 0 . 3 6 3 4 7 . 5 0 4 4 2 . 0 0 0.54 3.85 Table IX(b) includes the blood results of birds with a variety of ailments. One or more blood constituent levels of every bird was found to be abnormal. A brief description of individual post mortem findings was as follows. Bird number Description Al Severe hemorrhage in pericardial-sac A4 Emaciated; gall bladder to four or five times the normal size B2 Thin but otherwise appeared normal B9 Emaciated; growth along the ureters B12 Cecitis; worms C2' Pale, yellowish kidneys, but did not suggest pullet disease C4 Pale, yellowish kidneys, but did not suggest pullet disease x Results in mgms. % TABLE IX(b) Blood Constituent Levels of Cull Pullets -Miscellaneous Diseases  Chloride s Blood Crea- Uric Bird No.samples N.P.N.X Urea N.x tinine x Acid Total As NaCl Al 1 4 0 . 0 0 2 4 4 . 2 5 > . 6 4 9 . 0 0 1 1 4 . 9 6 7 2 . 2 A4 1 5 1 . 2 5 2 5 1 . 5 0 4 . 9 0 1 0 1 0 7 . 5 6 2 8 . 6 B9 1 4 9 . 7 5 2 4 4 . 2 5 4 . 9 0 . 8 6 9 . 0 0 1 3 1 . 6 7 6 9 . 8 3 4 4 . 2 5 5 . 2 5 . 7 9 1 4 . 0 0 1 2 9 . 8 7 2 9 . 8 B 1 2 1 4 4 . 0 0 1 1 4 . 0 6 6 6 . 9 2 4 1 . 7 5 . 5 0 3 . 1 0 1 0 5 . 3 6 1 6 . 0 C4 1 ' 4 2 . 2 5 1 1 . 4 118.4 6 9 2 . 6 C2 1 4 4 . 2 5 B2 1 4 4 . 0 0 . 6 7 9 . 0 0 1 2 0 . 2 7 0 3 . 2 2 4 0 . 5 0 . 5 0 1 2 2 . 8 7 1 8 . 4 3 4 4 . 0 0 . 5 7 3 . 0 0 1 2 0 . 2 7 0 3 . 2 Observations and Discussion Theoretically every disease is associated with a chemical blood picture. Some maladies are readily dis-tinguished by a single or a combination of abnormal blood constituent levels; others are characterized by the fact they do not alter the chemical component levels of the blood. The former is exemplified by the increased.con-centration of glucose with Diabetes Mellitus, or the decrease in sodium and chlorine with an associated increase in the potassium level as i s observed in Addison's disease. Uncomplicated protozoan infestations leaves the blood unchanged. The change in level of a specific blood con-stituent, or constituents, the combination of abnormal levels, and the degree of change a l l assist in the i d e n t i f i -cation of an entity. For reasons already discussed in the introduction, blood chemical test results cannot always be interpreted as specific entities. The range of abnormals reported here for the same pathological condition illustrates this d i f f i c u l t y . However, when blood findings are based upon a large number of similar cases the importance of results that do not conform to the expected characteristic pattern i s minimized. In the course of the blood determinations, no special consideration was given to the possible direct or indirect effect of the repeated bleeding on the blood constituent levels of the birds. The mortality due to cardiac puncture, at the time of the operation, was found to be less than one per cent. The post mortem examinations revealed that several times this percentage were l e f t with clots of varying sizes within the pericardial sac. Although no definite detrimental effects on the health of the birds could be related to these clots the possibility must be considered. The range of blood constituent levels as found in series 1 have to be amended for diagnostic purposes. The results of the c u l l birds that were reported on as 44. disease f r e e were found to f a l l roughly i n t o the normal range e s t a b l i s h e d i n s e r i e s 1 ( a ) . However, t a k i n g i n t o c o n s i d e r a t i o n the r e s u l t s of the p a t h o l o g i c a l cases the f o l l o w i n g changes are suggested. Although the nonp r o t e i n n i t r o g e n l e v e l was found to be as high as 46.5 mgms. % i n a h e a l t h y b i r d , l e v e l s above 42.0 mgms. per cent should be c l a s s i f i e d as d o u b t f u l and should warrant f u r t h e r i n -v e s t i g a t i o n . I t should be noted that o n l y a 98.5 per-centage o f the r e s u l t s or normals were 44*0 mgms. or l e s s . S i m i l a r l y the normal range f o r urea n i t r o g e n was found to be 3*5 to 5.6 mgms. per cent, t h i s could be broadened to 6.0 mgms. % with v a l u e s over 5.0 considered f o r f u r t h e r i n v e s t i g a t i o n . The c r e a t i n i n e normal range was found to be .48 to .81 mgms. % but the work i n s e r i e s 2 suggest t h i s range be enlarged from..0..3 to 1.0 mgms. %. Th i s l a s t change i s n e c e s s i t a t e d by the l i m i t s i n accuracy of the procedure used. The frequency d i s t r i b u t i o n o f nonprotein n i t r o g e n l e v e l s i n the blood of h e a l t h y c u l l b i r d s with dormant o v a r i e s r e v e a l e d a g r e a t e r percentage i n the higher v a l u e s than was found i n the normals. Some of these b i r d s may have been s u f f e r i n g from s u b - c l i n i c a l c o n d i t i o n s which would account f o r t h i s s l i g h t s h i f t upwards. The r e s u l t s i n any event a l l f e l l w i t h i n the normal range e s t a b l i s h e d i n s e r i e s 1 ( a ) . Marked d e v i a t i o n s from normal appeared i n v a r i o u s c l i n i c a l e n t i t i e s and these have been summarized i n Table X. TABLE X Blood Constituent Levels in Various Conditions Crea- Uric No. N.P.N. Urea N. tinine Acid Chlorides Casi Kidney X HN to x WNL X X condition ( 1 1 ) ( 4 ) ( 4 ) ( 2 ) ( 2 ) 7 Lymphomatosis x HN to x WNL WNL Inconsis-( 1 5 ) ( 1 3 ) ( 9 ) ( 1 ) tent ( 4 ) 6 I r i t i s WNL WNL WNL ( 1 2 ) ( 7 ) ( 8 ) 6 Fowl paraly- WNL WNL WNL WNL WNL sis ( 5 ) ( 2 ) ( 3 ) ( 3 ) ( 3 ) 2 Oviduct HN to x HN to x WNL X - to LN obstruction ( 1 0 ) ( 6 ) ( 5 ) ( 2 ) ( 3 ) 4 Dormant WNL WNL ovary ( 4 8 ) (28) Worm WNL WNL infestation (2) (2) Catarrhal WNL enteritis ( 4 ) Cecitis H ( 2 ) Enlarged x WNL gall bladder (2) ' (l) WNL WNL WNL ( 3 8 ) ( 1 5 ) ( 1 6 ) 20 WNL. WNL ( 1 ) ( 2 ) 1 WNL WNL WNL ( 3 ) ( 3 ) ( 3 ) 1 WNL WNL 1 to LN ( 1 ) ( 1 ) ( 2 ) 2 x ( 1 ) ( 1 ) 1 WNL within normal limits HN high normal LN low normal x above normal below normal ( ) number of tests The urea nitrogen values did deviate from the normal in several diseases, but the value of this deter-mination for the diagnosis of diseases encountered was . limited. Birds produce a very limited amount of creatinine as an end product of their waste metabolism. For the entities investigated, creatinine level determinations appeared to be of no value as a c l i n i c a l blood test. The uric acid levels of birds with dormant ovaries were found to vary between 2 . 9 0 and 5 . 0 0 mgms. %. This range should be confirmed on the basis of a large number of normal healthy pullets. Subject to further work the upper normal limit of 6 . 0 mgms. per cent would be justified. The uric acid level appears toe be the most valuable chemico-pathological test for diagnostic purposes. The method used here proved suitable for large numbers. It was found necessary to do several standards with every group determination even where work was being done on the same or successive days. Horvath ( 1 9 3 0 ) working on 10 birds found the normal range to be 2 . 7 6 to 7 . 2 7 mgms. per cent. Results by other authors varied from a minimum of 1 . 3 5 mgms. % to a maximum of 1 1 . 6 6 mgms. %. The method used for chloride determinations of chicken blood plasma appeared reliable. Results obtained were consistent both when repeated on the same sample and upon successive samples of the same bird. Although the healthy cull birds exhibited a range of 1 1 4 . 0 mgms. to 47. 128.9 mgms. the t e n t a t i v e normal range was accepted as 114 to 127 mgms. %. The r e s u l t s i n the l i t e r a t u r e f o r c h l o r i d e s i n fowl blood have a l l been determined on whole blood and are c o n s i d e r a b l y lower than those obtained here. The very high l e v e l s of u r i c a c i d i n kidney c o n d i t i o n confirms the f i n d i n g s o f Jungherr and Matterson (1944), but contra r y to t h e i r whole blood c h l o r i d e r e s u l t s the two cases reported here had high plasma c h l o r i d e l e v e l s . Hawk, Oser and Summerson (1947) s t a t e that high c h l o r i d e l e v e l s are a s s o c i a t e d with n e p h r i t i s . I f the plasma c h l o r i d e r e s u l t s would be confirmed i t would be a most v a l u a b l e d i a g n o s t i c t e s t since none of the other e n t i t i e s i n v e s t i g a t e d e x h i b i t e d a p a r a l l e l i n f l u e n c e on t h i s blood c o n s t i t u e n t . The blood r e s u l t s of b i r d 1491 i n Table V suggested t h a t the onset of kidney c o n d i t i o n i s r a p i d . In an i n t e r v a l of one week the nonprotein n i t r o g e n i n -creased from 3 7 * 5 0 to 54.00 mgms. %. Conc l u s i o n 243 blood samples dravm from 137 b i r d s were used i n 215 nonprotein n i t r o g e n , 108 urea n i t r o g e n , 86 c r e a t i n -i n e , 39 u r i c a c i d and 49 c h l o r i d e d e t e r m i n a t i o n s . On the b a s i s of over 800 i n d i v i d u a l blood analyses the f o l l o w i n g chemical blood p i c t u r e s were o f f e r e d . 48. 1. The normal range of nonprotein nitrogen in blood of healthy 14 month old White Leghorn pullets and cockerels was found to be 89.0 to 46.50 mgms. %. The levels for non-laying pullets, older fowl and a variety of breeds fell within the same limits. 2. The range of urea nitrogen in the blood of the same groups of birds was found to be 3.25 to 5.95 mgms. 3. The range of creatinine in the blood plasma of the same groups of birds was found to be 0.43 to 1.00 mgms. % 4. The range of uric acid in the blood plasma of non-laying female birds of a variety of breeds was 2.60 to 5.00 mgms. 5. The range of total chlorides in the blood plasma of non-laying female birds of a variety of breeds was 114.0 to 128.9 mgms. %. 6. Kidney condition, lymphomatosis and gall bladder trouble was observed to increase the nonprotein nitrogen level above the normal limits. The level with cecitis or oviduct obstruction was observed to be high noma! or beyond the upper arbitrary limits. Dormant ovaries, Iritis, fowl paralysis, worm infestation and catarrhal enteritis in the birds investigated, failed to abnormally elevate or depress the level of this constituent in fowl blood. .7. Urea nitrogen level determinations of fowl blood was found to be of little diagnostic value. 8. Creatinine level determinations on the blood plasma of chickens, by the method employed in this experiment, was of no diagnostic value in the bird disease studied. 49. 9. Uric acid level determinations on the plasma of fowl blood appeared to be a valuable chemi co-diagnostic test. In. kidney condition the increase in level was several hundred per:cent of normal whereas in the limited.numbers done in.other.con-ditions where i t did rise the response was not as great* 10. The chloride level of plasma in two cases of kidney condition was above notmal. Should further work confirm thi s finding thi s test would serve to simplify clinical differential diagnosis. PART II - A STUDY OF THE EFFECTS OF A MINERAL STRESS Introduction "So called pullet disease* (kidney condition) is characterized by. symptoms of a diverse nature but related by common pathology. In the literature various reports have been presented by different authors to explain the cause of kidney condition, and different treatments have been suggested to alleviate the disease. The diversity of opinions is illustrated in the following review. Weisner (1940) claimed the entity is caused by certain strains . of Escherichia coli. No other bacterial agent has been consistently t associated with i t . Waller (1942) reported the isolation and cultivation of a filterable virus. No investigator to date has confirmed this finding. Faeces of birds i l l with the disease have been purposely placed in the pens of healthy birds with no resultant occurrence of the condition. Selye (1943) reported the probability of this disease being due to accidental sodium chloride intoxication. Quigley (1944) suggested the disease may be related to certain unidentified principles in wheat. 0»Neil and Rae (1944) suggested that at least several varie-ties of wheat do not produce this condition. Jungherr and Matterson (1944) ruled out vitamin A as a cause of the entity. 51. Jungherr and Levine (1941) attempted to artificially produce the condition by innoculating both filtered and unfiltered extracts of tissue and intestinal contents. The results were negative. They considered the possibility of its being a metabolic disorder and they fed various precursors of uric acid, or substances with specific neph-rotoxic action as urea, ammonium carbonate, lactic acid, adenine sulfate, potassium nitrate, arsenic acid, mercuric chloride, lead acetate and sodium tartrate, but a l l failed to produce the histo-pathology of "so called pullet disease" of chickens. Potassium dichramate, injected daily for four days in doses of 10-20 mgms. per kilo body weight, caused a gross pathological picture indistinguishable from renal gout. The dichromate radical was demonstrated to exert the nephrotoxic effect. Oral administration of the potassium dichromate did not have the same effect. Up to the present no wholly successful method of treatment or control can be offered. Reports of some success in ameliorating the severity and reducing the mortality of the disease have been published. Jungherr (1943) lists some of the medicinal measures used; amongst them are: 1:2000 copper sulfate or 1:4000 potassium, dichromate in the drinking water, or about 40 per cent black strap molasses in a wet mash composed half-and half of bran and oats. Biely and Gasperdone (1948) using the copper sulfate on half of a 1400 hen flock found no significant differences in incidence of kidney conditions between treated and untreated birds. Scott, Jungherr and Matterson (1944) reported on the protective action of potassium chloride (as muriate of potash) in experimental 52. uric nephrosis produced by sodium citrate. According to Correl (1941) they suggest that feeding extra potassium may be B e n e f i c i a l in ameliorating the after effects of "so called pullet disease". Van>;NesB (1947) studied the effects of high intakes of sodium chloride, calcium carbonate, potassium chloride and combinations of these compounds. Ee suggests further consideration of the rate of effect of sodium and calcium. The possibility of interference with electrolyte metabolism being a cause of kidney condition, must be considered. The pathologi-cal fact of pancreas injury raises the question of organic metabolism interference. The pancreatic trouble, of course, could be primary or secondary to irregularity in protein, fat, vitamin or hormone metabolism. Post mortems yielding a "kidney condition" diagnosis have often been complicated'by other diseases. These diseases have been accused of being the primary cause. The specific pathology of these conditions differ from that of pullet disease, but that any of these diseases can act as the mediate prodromal cause of the syndrome of "so called pullet disease" and this lead to a terminal uremia cannot be denied. In spite of all-the possible causes of the disease offered, experimental procedures to induce the condition have never produced the identical pathological picture. Commercial rations naturally have on occasion been blamed for producing the disease. Follovlng complaints, idiere high mortality with associated renal impairment was encountered, investigations were carried out. Biely (1947) was unable to induce an abnormally high incidence of kidney conditions in the University of British Columbia birds by the feeding of any specific commercial ration. The .. occurrence of the disease has been observed in flocks being fed a i-multitude of different rations. Wakely (1946) fed rations high in protein in an attempt to produce the entity but obtained negative results. In view of the increased usage of fish meal as a protein supplement during the past decade attempts were made to induce "kidney condition" by the use of diets where fish meal was the sole protein supplement. No evidence to indict any-of alnumber of commercial fish meals as the causative agent of "kidney condition" was obtained. The entity has.been observed here in all commercial breeds but there is no statistical survey available to reveal the relative breed susceptibility. Post mortem statistics in different localities have listed greater percentages of deaths in some breeds than in others but the relation to the local breed population was not provided. The condition ie widespread (see Part IB.). Biely (1947) found the incidence of mortality with involvement of the kidneys has varied in British Columbia from year to year. Jungherr and Matterson (1944) report the disease may occur throughout the year but there is a definite concentration of occurrences during the warm season. It has been observed here that amongst a flock of Identical age, and under parallel conditions of housing and feeding, a varying percentage of the birds will prove susceptible. Further there is no 54, evidence to suggest the productivity of the visibly unaffected birds is reduced* The divergent nature of reports on "so called pullet disease" could in itself be significant. Thus far, however, for all practical purposes, the question of cause and control is unanswered. The information available would suggest that in at least some birds an interference with electrolyte metabolism could be the causative agent. This does not in any way preclude the possibility of other causes.being associated with production of pullet disease. It was with this attitude that the following line of in-vestigation was adopted: An experiment was designed to investigate: 1. If the stress of a diverse group of minerals fed in excess over a long period of time would induce "kidney condition" in pullets. 2. Some blood constituent changes, i f any, of the afore-mentioned birds throughout the experiment. 55 Materials and Methods  Experimental birds Thirty-six four month old single comb White Leg-horn pullets, selected from a flock of 600 were used as the experimental birds. They were chosen for uniformity in development, and were a l l in good health. They ranged in weight from 1435 grams to 1588 grams. Their weights were recorded at eight additional periods in the course of the experiment. The pullets were confined to laying cages (figs. 3 and 4) throughout the duration of the experiment. The cages were in a separate building away from other birds. Although the room had considerable window area a r t i f i c i a l light was provided from 8 a.m. to 8 p.m. when the season warranted i t . The birds were divided into groups of four which shared a common water trough and two feed troughs. Diet The birds were transferred to the laying cages on October 11, 19.47>. and were fed.the basal mash until November 8, 1 9 4 7 « This was done to allow the birds to accustom themselves to their new environment. The same basal mash was used in preparing the experimental diets. Fig. 3. Experimental arrangement of birda Fig. 4. Upper birds receiving excess manganese Lower birds receiving excess phosphorus The basal mash consisted of: wheat oats bran fishmeal meat meal soyabean meal alfa l f a lime stone bone meal B.Y. cod liver o i l 512 lbs, 2 0 0 100 4 0 50 20 50 10 5 10 3 On November 8 , 1 9 4 7 , the birds were started on the experimental rations indicated in Table I. The mineral supplement was added in excess to the extent of two to three times the generally recommended ration level. The review by Branion ( 1 9 3 7 ) of minerals in poultry nutrition was used as a guide. The mash and the water was offered ad libitum. The diet was supplemented by small quantities of wheat which was given to a l l birds in equal quantities on alter-nate days. Since the mash was prepared at one time for the whole experiment additional quantities of fish o i l were given to a l l birds after the second month. Oyster shell was added to the diet when birds were in heavy lay in quantities sufficient to avoid soft shells in control birds. The experiment was continued for six months and a careful record was maintained of the feed and water consumption. Evaporation was accounted for but no allowance for waste of either water or feed was made. In the case of the water particularly this was a formidable amount because TABLE I Special Mineral Diets Diet Number Al A2 A3 Bl B2 B3 CI C2 C3 Supplement Ammonium chlo ride Iron Nil Calcium carbonate Magne sium oxide Potassium acetate Mangane se dioxide Ammonium phosphate (dibasic) Sodium bicarbonate Mineral Percentage in of mash Supplement 3 . 0 2 1 . 0 0 N i l 1 2 . 4 9 3 . 3 2 5 . 0 2 0 . 1 6 8 . 5 2 5 . 1 0 chlorine iron Nil calcium magnesium potassium manganese phosphorus sodium Percentage of mash 2 . 0 1 . 0 N i l 5 . 0 2 . 0 2 . 0 0 . 1 2 . 0 1 . 5 of the design of the water trough. The waste of feed was observed to vary considerably between the different diets. The egg production for the individual birds was recorded throughout their laying periods. Blood samples The blood samples were removed from the birds in the manner described in Part 1(a). Standard conditions as established for previous work were observed. A variety of.' anti-coagulants. were used depending upon the nature of the respective c l i n i c a l tests to be carried out on the blood, A maximum of about 85 cc. of blood was taken from an individual bird in single collections of up to 1 2 cc. at one time. The f i r s t was taken when the birds were f i r s t placed in the laying cages and the remainder were at least widely spaced over the experimental period. Procedures Nonprotein nitrogen, urea nitrogen, creatinine, uric acid and chloride determinations were completed by the methods described in Part I. Duplicate tests were done for uric acid and total chlorides on most of the blood-sample s. The glucose level was determined on whole blood by the method of Shaffer and Hartman ( 1 9 2 1 ) as modified by Somogyi ( 1 9 2 6 ) . The sugar determinations were done in duplicate. The whole blood and plasma specific gravities, the hemoglobin, and the plasma protein determinations were computed with the use of the "shock k i t " devised by Philips et a l ( 1 9 4 3 ) . Post mortems Birds that died early in the experiment were replaced since i t was concluded that the deaths were not directly related to the mineral stress applied through the diet. Post mortem examinations were made of a l l birds 60. that died in the course of the experiment. Results The feed, mineral and water consumption of a l l groups are given in table II. The egg production for each individual bird i s summarized in table III. Sick birds were retained in their cages u n t i l they died of their i l l n e s s . When only two or three of the birds in each group were being bled the selection was limited as much as possible to birds that appeared to be healthy. The results for whole blood nonprotein nitrogen and urea nitrogen are given in table IV. Whole blood glucose levels are reported in table V. Plasma creatinine, chlorides and uric acid levels are given respectively in tables VI, VII and VIII. The progressive weights of the individual pullets are given in table IX. The whole blood specific gravities, the plasma specific gravities, the haemoglobin and the plasma proteins are reported in table X. Tables IX and X are in Appendix I. 61, TABLE II Feed and Water Consumption Feed x Hen Group Water cc's. Total Mash-_No_j_ (Evap.deducted) m a s h minerals Grain days 96.98$ CI Al 219,155 39,400 38,210 7,580 632 99.00$ Fe / A2 83,155 47,015 46,545 6,835 595 Con- ,100.00$ t r o l A3 149,340 69,745 69,745 7,700 645 8 7 . 5 2 $ Ca Bl 120,570 60,695 53,120 8,920 732 9 6 . 6 8 $ Mg B2 248 ,655 70,625 68 ,280 9,120 732 97.49$ K B3 229,345 53,250 51,913 8,145 663 9 9 . 8 4 $ Mn CI 139,240 67,785 67,677 7,745 641 89.34$ P C2 302,050 40,775 36,428 8,370 684 9 4 . 9 $ Na C3 304,445 74,190 70,406 9,000 732 x Results in grams TABLE III Egg Production Chart Bird Nov .8- Dec.7- Jan . 4 - Feb.l- Feb.29- Mar.28- Apr. 25 - ' Re-Diet No. Dec.6 Jan .3 Jan.31 Feb.28 Mar.27 Apr .24 May 8 Total mark! Ammonium 2 9 8 0 3 11 2 4 7 5 0 3 2 1 ss chloride 2 3 0 7 0 0 2 2 0 4 2 10 2 6 6 0 0 0 0 0 2 3 0 6 0 0 0 0 0 0 0 0 Iron 40 3 1 0 0 0 2 5 3 1 8 Q 1 0 1 1 12 2 3 1 0 0 11 0 7 0 0 1 19 4 5 8 6 0 2 Control 9 3 6 10 17 22 20 19 20 10 118 2 ss 2988 10 14 0 2 9 9 3 n\ 9 19 2 2 1 9 2 1 10 91 2 ss 7 5 ( 1 ) 1 0 15 19 1 5 20 22 11 112 Calcium carbonate L 2 4 4 0 1 13 11 13 17 9 64 584 0 0 0 0 1 3 11 5 5 0 1 ( 2 ) 1 2 6 3 6 3 2 2 3 20 4 1 12 6 5,, 14 8 50 Magnesium 2 9 5 3 1 6 0 4 3 9 0 23 oxide 561 0 0 0 1 4 9 1 15 3 0 5 9 10 3 8 8 12 ' 6 56 1 ss 3 1 0 7 0 2 2 3 4 3 . 0 1 4 (1) Started as No. 2 9 3 7 ; replaced Nov. 27 ( 2 ) Started as No. 2 9 1 3 ; replaced Dec. 10 ss - soft shell TABLE III  Egg Production Chart Bird Nov.8- Dec.7- Jan.4- Feb.l- Feb.29- Mar.28- Apr.25- Re-Diet No. Dec.6 Jan .3 Jan.31 Feb.28 Mar .27 Apr .24 May 8 Total marks Potassium 781(3) 3 10 2 3 -acetate 6 7 8 27 23 9 23 ..17 22 12 1 3 3 10 ss 2 9 9 5 ( 4 ) 6 10 17 5 1 8 17 8 71 2 ss 3 1 4 4 6 8 2 0 13 18 * 14 9 9 1 4 ss Manganese 518 9 10 10 8 16 11 2 66 69 ss dioxide 2 9 0 4 4 15 20 20 19 22 9 109 3 S3 4 6 9 11 0 3 1 1 13 18 2 1 2 1 18 20 7 118 Ammonium 3 1 8 0 0 0 2 0 1 3 6 1 ss pho sphate i 2984 0 0 0 0 0 0 3 2 1 0 0 .1 0 0 0 0 1 2 9 2 5 0 0 0 0 0 0 0 0 Sodium 7 0 1 5 16 0 11 18 15 0 65 3 ss bicar- 417 0 10 6 7 0 17 1 0 50 1 ss bonate 3 0 9 16 19 11 15 1 4 0 0 75 2 9 1 4 5 9 10 3 7 15 6 . 54 3 ss ( 3 ) Started as No. 4 6 4 ; replaced Jan. 26th ( 4 ) Started as No. 8 8 3 J replaced Dec. 20th indicates death and removal ss-softshell TABLE IV  Nonprotein Nitrogen (Results in mgms.#) Urea Nitrogen Diet Bird No. Oct. 24th May 9th Oct.24th May 9th Al 2980 41.0 31.0 4.90 4 . 9 0 2307 49.3 28.1 4.20 3 . 3 3 2 6 6 43.0 4.90 2306 45.0 "• 38.1 4 . 9 0 4 . 5 0 A2 40 42.8 4.20 2 5 3 42.5 34.8 4 . 7 3 4.20 2 3 1 0 4 4 . 5 47.5 4.20 4 . 9 0 45 4 0 . 5 4 . 5 5 A3 9 3 6 45.0 33.8 3.85 4.20 2988 56.0 7 5 ( 1 ) 33.8 4 . 0 3 2993 4 3 . 3 3 0 . 6 4.20 4 . 9 0 Bl 2 4 4 42.5 37.5 4.20 4.90 584 35.0 38.8 3.85 4 . 5 5 501(2) 43.5 ; 3 2 . 5 3.85 4 . 7 3 20 40.0 3 3 . 1 3.85 4 . 5 5 B2 2953 40.5 3 4 . 4 5.25 561 3 7 . 5 48.8 4.20 4.20 3 0 5 38.0 35.0 5 . 2 5 3107 38.0 33.8 3.85 4.20 B3 781(3) 39.0 4.20 678- 36.0 33.8 3.85 4.20 883 45.0 3.85 2 9 9 5 38.8 3 . 5 0 3 1 4 4 . 41.0 3 5 . 3 3.85 3 . 6 5 CI 518 42 .5 37 .5 3.85 4 . 5 5 2904 4 1 . 3 39.8 3.85 3-85 4 6 4 4 . 3 311 33.8 3.85 3 . 5 0 C2 318 40.0 30.0 4 . 0 3 4.20 2984 38.3 3 . 6 8 3 2 1 3 6 . 3 3.85 2 9 2 5 44.5 3 9 . 4 3.85 4.38' C3 7 0 1 417 37 . 3 3.85 3 0 9 3 7 . 5 3 6 . 3 3.8$ 2 9 4 41.8 4 . 3 8 ( 1 ) original bird 2937; (2) 2913 ( 3 ) 4 6 4 65 TABLE V BLOOD GLUCOSE (Results in mgms. %) Bird Diet No. Oct. 31/47 Nov.28/47 Dec.19/47 Feb.27/48 May 4/48' Al 2980 182 188 182 175 2307 186 217 195 191 266 186 205 2306 189' 20 5 209 A2 40 186 182 183 253 183 171 161 187 2310 183 190 164 182 45 199 183 A3 936 189 197 177 174 2988 174 215 75(1) 180 191 169 186 2993 189 184 181 Bl 244 180 180 180 584 184 185 182 2913 177 501 188 20 189 185 192 185 B2 2953 177 180 185 561 183 186 208 305 186 180 188 202 3107 192 B3 781(2) 180 183 678 185 167 160 156 883 189 2995 193 173 3144 189 195 CI 518 183 183 171 2904 193 197 170 199 46 189 221 311 185 195 167 168 C2 318 180 197 182 174 2984 183 ' 205 321 213 202 200 2925 177 193 186 C3 701 196 172 417 186 191 204 205 186 309 167 188 162 * . 294 192 188 (1) Started as bird 2937; (2) 464 66. TABLE VI Plasma Creatinine (Results in mgms. %) Bird -No. Oct. 2 4 / 4 7 Nov. 28/47 Dec.19/47 Feb.27/48 May 4 / 4 8 2980 L 5 0 2 3 0 7 0 . 4 5 . 4 0 2 6 6 0 . 4 8 . 4 2 2 3 0 6 40 .42 . 4 8 253 . 5 0 . 4 2 2 3 1 0 . 4 8 . 5 7 45 . 5 0 9 3 6 . 4 6 . 4 0 2 9 8 8 . 4 0 2937 .. 75 . 5 0 . 5 5 . 3 8 2 9 9 3 0 . 4 5 1 . 4 0 2 4 4 . 6 0 .45 . 4 0 5 8 4 . 4 4 . 4 5 5 0 1 ( 1 ) . 7 1 . 6 0 20 . 4 8 . 5 0 2 9 5 3 . 4 6 . 3 8 561 . 5 0 . 4 3 3 0 5 . 5 2 1.45 . 5 0 .80 3 1 0 7 . 5 5 781(2) . 4 6 . 4 6 6 7 8 . 4 8 .45 . 4 0 8 8 3 .66 . 5 0 2 9 9 5 3 1 4 4 1 . 0 0 . 7 5 5 1 8 . 4 0 . 4 0 2 9 0 4 . 5 0 . 5 0 . 4 5 46 . 4 4 3 1 1 . 4 0 . 5 3 . 4 4 3 1 8 . 5 0 . 7 5 . 4 0 2 9 8 4 .55 321 . 5 0 . 4 5 . 4 5 2 9 2 5 . 4 1 . 4 5 7 0 1 . 5 8 4 1 7 .65 . 4 1 309 . 4 1 . 4 0 2 9 4 . 5 0 ( 1 ) Started as bird no. 2 9 1 3 ( 2 ) 4 6 4 TABLE VII Plasma C h l o r i d e s ( R e s u l t s i n mgms. %) B i r d Oct.24/47 Nov.28/47 p e c.19/47 D i e t No. CI NaCl CI NaCl CI NaCl A l 2980 113.2 662 .2 119.3 697.9 2307 120.2 703.2 118.9 695.3 266 115.8 677.4 2306 118 . 4 692.6 A2 40 119.3 697.9 115 .8 677.4 253 114 .0 666;9 117.5 687.4 2310 112.3 657.0 116.3 680 .1 45 118.4 692.6 116.7 682 .7 A3 936 117.5 678.4 122.8 718.4 2988 115 .8 677.4 117 .1 685.6 (1) 75 116.7 682.7 116.3 680 .1 2993 121.0 707.9 112.3 657.0 B l 244 124.6 728.6 118.9 695.3 584 115.8 677.4 122.8 718.4 2913 H 9 . 3 697.9 501 ' ' 120.2 703.2 20 122.8 718.4 125.4 733.6 B2 2953 120.2 703.2 112.3 657.0 561 125 . 4 733.6 115.4 674.8 305 119.3 697.9 111.8 6 5 4 . 4 3107 120.2 703.2 118 .0 690.0 (1) S t a r t e d as b i r d No. 2937 Feb.27/48 CI NaCl May 4/48 CI NaCl 121.5 12 5.0 710.5 731.3 119.8 124.6 700.6 728 .9 133.8" 782.5 115.8 116.5 117.5 677.4 680 .1 687.4 117.5 114.5 687.4 669.6 119.8 700.6 118 .4 692.6 118 .4 124.2 692.6 726.3 125.9 736.8 116.3 121.0 680.1 707.9 114.5 669.6 119.3 697.9 115.4 674.8 107.9 114.9 111.8 631.2 672.2 654.4 111.8 654.4 O N - J TABLE VII Plasma Chlorides (Results in mgms. %) Oct.24/47 Nov.28/47 Dec. 19 /47 Feb.27/4^ May 4 / 4 § Bird Diet No. CI- NaCl CI NaCl CI NaCl CI NaCl CI NaCl B3 (2 )781 119 .3 697 . 9 116 .3 680 .1 678 120.2 703.2 1 1 8 . 4 692 . 6 117.1 6 8 5 . 6 116 .3 680 .1 883 119 .3 697 . 9 122.8 718 . 4 2995 121.0 7 0 7 . 9 123 .7 723 . 6 3144 115.8 677 . 4 121 .9 713.1 CI . 518 115.8 677.4 112.8 659.6 117.1 6 8 5 . 6 2904 116 .7 682 .7 112 .3 657.0 122.8 718 . 4 117.1 6 8 5 . 6 46 122.8 7 1 8 . 4 119.8 7 0 0 . 6 311 1 2 0 . 2 703.2 116 .3 680 .1 1 2 0 . 6 705 .6 1 1 7 . 5 687.4 C2 318 120.2 703.2 115 .8 677.4 119.8 7 0 0 . 6 116 .7 682 .7 2984 1 1 7 . 5 6 8 7 . 4 114 .5 6 6 9 . 6 321 121 .9 713.1 121 .9 713.1 110.1 643.8 108 .8 636 .5 2925 117 .5 687.4 116 .7 682 .7 114.0 666 . 9 C3 701 120.2 7 0 3 .2 111.0 6 4 9 . 6 417 120.2 703.2 119 .3 697 . 9 I O 9 . 6 641.2 111 .4 651 .7 117.1 6 8 5 . 6 309 117 .5 6 8 7 . 4 113.2 6 6 2 .2 1 1 8 . 9 695 .3 294 1 1 8 . 4 6 9 2 . 6 107 .9 631o2 (2) Started as bird no. 464 Os 03 69. TABLE VIIT Plasma Uric Acid (Results in mgms. %) Bird no . Oct.24/47 Nov.28/47 Dec.19/47 Feb.27/48 May U/l 2980 2 .60 1.67 •- 1.85 2.62 2307 3 .30 2.00 2.10 266 3 .90 2306 2.85 6.20 40 3.02 2.76 4.84 253 2 .50 2.66 2.60 2.84 2310 2.05 2 .90 3.36 2.94 45 2 .05 1.70 936 6 .40 1.91 ' '1.60 2.60 2988 3.00 2 .07 755(1) 2.65 2.00 2.75 2.34 2993 2 .05 2.47 1.60 244 2 .30 2.99 5.89 2.58 584 2.99 3.68 2913(2) 2.12 3 .04 20 2.42 2.85 2.80 3.44 2953 2.84 4.48 3.76 561 1.88 3 .15 305 2.10 3.59 1 .90 2.62 3107 2.32 4.80 781(3) 2.00 . 2.40 . 678 1.80 2.56 2.53 3.60 883 1.80 2.96 2995 1.41 '3.54 3144 1.70 3 .70 518 2 .30 2.06 1.72 2904 2.22 2.16 1.95 2.38 46 1.95 3.01 311 3 .40 2 .23 3.56 3.58 318 4.20 2.19 • - 1.69 4.22 2984 3.00 3.66 321 2.60 4.59 3.34 2.38 2925 1.72 2.3 5 .04 701 5 .05 . 4.46. . 11 .40 417 2 .50 7.46 6 .05 6.00 309 3.00 5.09 3.08 294 5.60 (1) Started as bird no. 2 9 3 7 ; (2) 5 0 1 ; (3) 464 Post Mortems: Bird lumber Diet Date of death Post mortem findings 266 A l Feb. 4, 1948 cause of death undetermined 45 A2 Feb. 14, 1948 friable, abnormal liver 40 A2. March' i 14, 1948 friable, abnormal l i v e r j pancreas degeneration 2937 A3 Nov. 26, 1947 osteomalacosis 2988 A3 Feb. 10, 1948 lymphomatosis; 2913 Bl Dec. 10, 1947 pullet disease 883 B3 Dec. 20, 1947 accidental death due to haemorrhage in pericardial sac 464 B3 Dec. 2 6 , 1947 lymphomato si s 781 B3 Feb. 28, 1948 lymphomato si s 46 CI Feb. 16, 1948 cause of death undetermined 2984 C3 March 23 , 1948 pullet disease 309 C3 May 8, 1948 pullet disease Normal Ranges On the basis; of over 97$ of the analyses completed for plasma uric acid, on the pullets while they were s t i l l on the basal ration, a range of 1 . 7 0 to 4 . 2 0 mgms % was observed. A single very high result was eliminated. Even should extremes of this nature be normal but rare, i t s exclusion from a range to be used for c l i n i c a l diagnosis i s justifiable. The ranges obtained by the shock k i t , on the f i r s t blood samples were as follows: whole blood specific gravity 1 . 0 4 7 0 to 1 . 0 5 2 0 plasma specific gravity 1 . 0 2 0 0 to 1 .0220 plasma proteins 4 . 4 5 to 5 .12 gms % haemoglobin 1 1 . 7 0 to 1 4 . 1 0 gms % Observations and Discussion Branion (1937) stated " i f excessive amounts of certain minerals are continuously fed, the safety mechanisms break down, the bird is no longer able to main-tain the normal composition of body fluids and tissues, and injury ensues." In this experiment the excess minerals were considered in the nature of a stress upon the body mechanism rather.than their individual effect upon metabolism. Minerals .of a diverse character were purposely selected so as to vary the stress. The 'general adaptation syndromte1 theory presented by Selye (-1947) suggests there i s an endocrine pathway over which the body adapts i t s e l f to any specific sublethal stress. The protective adaptation can fatigue after a period of time and 'diseases of adaptatio are encountered. One group of these involves a disturbance in the electrolyte metabolism. This theory could explain the variety of causes, reported in the literature, of pullet disease. The mortality encountered in this experiment i s greater than would normally be anticipated on ordinary rations. The small number of birds in the experiment questions the r e l i a b i l i t y of this s t a t i s t i c . Further the mortality could be partially due to abnormal conditions other than those created by the experimental diet. Numerous blood samples were taken from the birds for the reported analyses and also for ashing and spectrograph!c analyses. The birds were handled unnumerable times in the course of weighing, bleeding and general examination. Regardless of the diet some birds in every group were able to maintain good health. With the possible ex-ception of the birds in group A2 who were receiving an excess of iron no deaths could be directly attributed to the respective diets. There is no evidence that would suggest the isolated cases of kidney condition encountered are related to the diet the birds were receiving. Some of the diets appeared to exert effects which were common to birds in an individual group. The friable l i v e r in the two birds of group A2 may have been related to' the high iron content of the diet. The haemoglobin level of two birds in this group exhibited a marked abnormal increase towards the end of the fourth month. The plasma protein levels of the birds receiving sodium bicarbonate and those with magnesium oxide in their diet were high. The haemoglobin levels of the three remaining birds on the diet supplemented by potassium acetate were comparatively low. 73. The plasma chloride levels of the birds in group Al receiving ammonium chloride were relatively high but not abnormal. On the other hand the chloride level of the birds whose diets were supplemented with magnesium oxide, ammonium phosphate or sodium bicarbonate were low. No distinct group changes in the blood levels of the other constituents studied were observed. The nomograph employed with the shock kit in this experiment was devised for mammalian blood. The plasma protein results obtained with i t were slightly higher than the average of 4 , 2 5 9 gms. % obtained by Hayden (1928). Hayden reported on ten birds and his results were cal-culated from the refractive index of the blood. The haemoglobin results obtained by the shock kit method were in the general range of results reviewed by Olson (1937). Conclusion Diets containing different mineral supplements at levels considered to constitute a stress were fed to eight experimental lots of four White Leghorn pullets over a period of six months. Some of the blood chemical con-stituent levels of the birds were determined at regular intervals and post mortem investigations on fa t a l i t i e s were carried out to determine i f the diets could e l i c i t a condition parallel to pujlet disease. 1. Ammonium chloride, iron, calcium carbonate, magnesium oxide, potassium acetate, manganese 7 4 . dioxide, ammonium phosphate and sodium carbonate individually included in the mash of a ration failed to produce the c l i n i c a l entity known as pullet disease. 2 . The birds were able to adapt themselves to the abnormal diets, and healthy individuals were observed in a l l of the lots. However, egg production was inhibited to varying degrees in the birds on some of the diets. 3 . The shock kit was found to give consistent and reliable plasma protein and haemoglobin results. The level of haemoglobin in the blood of four month old White Leghorn pullets was found to range between 1 1 . 7 0 and 1 4 . 1 0 grams per cent. The plasma protein range on the same birds was 4 . 4 5 to 5 .12 grams per cent. 4« The blood level of glucose in the thirty-six White Leghorn pullets varied from a minimum of 167 mgms. per cent to a maximum of 199 mgms. per cent. R E F E R E N C E S Andrews,. F. N., Nov. 1944. An improved method of obtaining blood, from the chicken heart. Poultry Science, vol. XXIII, no. 6. Beach, J. R., 1927. Survey of postmortems at University of California. Delivered to World's Poultry Congress. Beaudette, F. R., 1929. X disease. Poultry Pathology Notes, New Jersey Experimental Station, 1, 6-7. Biely, J., and H. Gasperdon, 1948. Unpublished data. Biely, J., 1947. Unpublished data. Branion, H. D., 1938. Minerals in poultry nutrition. Sci. Agric. 18, 5; Braucher, P. F., 1929. Studies in the composition of chicken blood as influenced by the "Big Liver" disease. Master* s thesis, Pennsylvania State College. Bronkhoist, J. J., and G. 0. Hall. The physiology and chemi3try of high and low hatching eggs. Cornell University, Ithaca, N. Y. Brown, Herman, 1945. The determination of uric acid in human blood. J. Biol. Chem. 158, 601. Bullis, K. L., 1940. Unknown disease. Proc. 13th Annual Conference Lab. Work, in Pullorum Disease Control. Mass. Agric. Expt. Sta. Mim. Report, Amherst, Mass. Bullis, K. L., 1944. Symposium on pullets* disease - discussion. Proc. 16th Ann. Conf. Lab. work, in Pullorum Disease Control. Univ. Conn., Sloirs, Conn., Mim. Report. Clementi, A., 1932. Urea Content of the blood and liver of chickens. Chem. Abstracts 27 , 238. Correil, J. T., 1941. The biological response of chickens to certain organic acids and salts with particular reference to their effect on ossification. J. of Nutrition 21, 515-525. Dyer, H. M. and J. H. Roe, 1934. The chemistry of the blood of normal chickens. J. Nutrition 7, no. 6, 623-6. Foling, Um. H., 1919. A system of blood analysis. J. Biol. Chem. 38, 81-110. Genest, P. A., Jan. 1946. Technique de Prete vement de Sang chez la Poule par la Fonction Cardiaque, Can. Jour, of Comp. Med., vol. 10, no. 1. - 2 -Gonzaga, A. C, 1935. A contribution to the chemical study of chicken blood. Report of N..Y. State Vet. Coll., 1933-34, 53-57. Hawk, Oser and Summerson, 1937. Practical Physiological Chemistry. The Blakiston Co., Toronto. Hayden, C. E. and P. A. Fish, 1928. The normal blood of same domesticated animals. The Cornell Veterinarian, vol. 18, no. 2, 197-203. . Hayden, C. E., 1929. The physiology of the blood of chickens. Report. of New York State Vet. Coll., 1927-28, 213-220. Hayden, C. 1. and 35. L. Brunett, 1930. A study of the influence of bacterial pullorum upon some organic and inorganic constituents of the blood.of S. C. \White Leghorns. Report of the New York state Veterinary Coll. 1928-1929, 135-142. Hayden, C. E. and J. Sampson, 1931. A study of some organic and inorganic constituents of the wing and throat blood of laying and non-laying R.I.R. hens. Report of N. Y. State Vet. Coll., 1929-30, 174-182. > Heller, V. G. and L. Parsell, 1937. Chemical composition of the blood of a hen during its life cycle. Jour, of Biochem., vol. 118, p 549. Henry, K. M., H. E. Magee and E. Ried, 1934. Some effects of fasting on the composition of the blood and respiratory exchanges in fowl. J. 3Sxpt. Bio., vol. 118, p 58-72. Horvath, A. A., 1930. Blood composition of animals under pathological conditions - feeding hens corn smut. Poultry Sci. 9313-9. Horvath, A. A., 1930. Changes in hen's blood produced by a diet of sprouted soybeans. Amer. Jour. Physics 94, 65-68. Jungherr, E. and L. D. Matterson, 1944. Avian monocytosis; so-called pullets' disease. Reprinted from Proc. of the Forty-eighth Ann. Meeting of the U. S. Livestock Sanitary Ass'n. Jungherr, E., 1943. H. E. Biester and 1. Devries - Diseases of Poultry. Iowa State College Press, p 511-522. Jungherr, E. and J. M. Levine, 1941. The pathology of so-called pullets' disease. Am. J. Vet. Res. 2, 261-271. Mylrose, R. L., 1943. Adult poultry mortality in the University of British Columbia's poultry flock. Bachelor's thesis, University of British Columbia. - 3 -Olson, C, 1937. Variations in the cells and hemoglobin content in the blood of the normal domestic chicken. Cornell Vet. 3, 235-263. O'Neil, J. B. and W. J. Rae, 1944. Is the type of wheat fed a factor in "New Wheat Disease"? Poultry Science, vol. XXIII, no. 6 , 548-49. Quigley, G. D., 1944. The effect of wheat upon the incidence of pullets' disease or blue comb. Poultry Science, vol. XXIII, no. 5. Russel, Walter C. and Albert L. Weber, Nov. 1934. The effect of the protein level of the ration upon certain blood constituents of the hen. Poultry Science, vol. XIII, no. 6. Schales, Otto and Selma S. Schales, 1941. A simple and accurate method for the determination of chlorides in biological fluids. J. Biol. Chera. 140, 879. Scheunert, A. and H. Von pelchrizm, 1923. Uber den gehalt des Blutes vers chiedener Fierarten an Zucker, Rest-N, Harastoff-N. Kreatlnkorpern und Harrisauer nach den Folinschen Methoden. Biochem. Zeitschrift, 1929, 17-29. Scott, H. M., Jungherr, E. and L. D. Matterson, 1944. Possible role of potassium in pullet disease. Proc. Soc. Expt. Biol* and Med., 57, p 7-10. Selye, H., 1944. production of nephro sclerosis in the fowl by sodium chloride. Jour. After. Vet. Med. Ass., 706, p 140-143. Selye, H., 1947. Textbook of Endocrinology. Acta Endocrinologica, University of Montreal, Montreal. Shaffer, P. A. and A. F. Hartman, 1921. II. Methods for the deter-mination of reducing sugars in blood, urine, milk and other solutions. J. Biol. Chem. 45, 365-390. Shimer, S. R., Aug. 1937. Chemical studies on chicken blood. Univ. of New Hampshire Expt. Sta., Durham, N. H., Tech^ Bull. 69. Simmons, J. S. and C. J. Bentzkow, 1944. The laboratory methods of the United States Army. Lee and Febiger, Philadelphia, p 199-200. Sloan, H. J. and H. S. Wilgus, Nov. 1930. Heart probe - a method for obtaining blood samples from chickens. Poultry Sci., vol. X, no. 1. Somazyi, M., 1926. Notes on sugar determination. J. Biol. Chem. 70, p 599-612. Thompson, T. J. and H. H. Powers, 1925. The variation of certain blood constituents of chickens during the moulting season. Poultry Sci. 4, 186-8. .. - 4 -Thompson, T. J. and I. L. Carr, 1923. The relation of certain blood constituents to a deficiency diet. Biochem. Jour. 17, 373-5. Van Ness, G., 1947. The production of so-called "pullets* disease", Poultry Sci. 8, 3, 304-5. Wakely, W. 1947. Essay submitted in the Faculty of Agriculture, Department of Poultry Husbandry, University of British Columbia. Waller, E . F., 1942. Isolation of a filterable virus from chickens affected with Blue Comb Disease. Science 95, 560-561. Weisner, E . s., 1940. How the veterinarian can develop a poultry practise, Mich. State Coll. Vet-. 10-13. WS7G//T C7//IRT i i , s i i i pcT86,mr. 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THE TOXICITY OF SODIUM CHLORIDE AND ITS RELATION TO WATER INTAKE IN BABY CHICKS by Morley R. Kare A Thesis Submitted in Partial Fulfilment of The Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE In the Department of POULTRY HUSBANDRY The University of British Columbia June, 1 9 4 8 . ABSTRACT Two hundred and fifty-two New Hampshire chicks divided into 21 lots of 12 chicks were used to investigate the toxicity of salt, i t s influence on water intake and i t s relation to feed utilization efficiency. A l l lots receiving mash containing over 4 per cent salt showed characteristic edematous condition in some of the chicks. The chicks exhibited individual differences in tolerance for sodium chloride. The comparative toxicity of sodium chloride in mash and drinking water was studied and toxic levels established in each case. The water intake per gram of feed consumed increased progressively with the salt percentage of the mash diet. Withholding water from chicks on a high salt diet was found to make no difference,with reference to total water consumed, to the supplying of water at a l l times. There is no significant variation in efficiency of feed utilization on diets containing 1.08 to 3.IB per cent sodium chloride. TABLE Off CGNTEOTS Introduction. Experimental Resulte Discussion Summary References Page 1 4 7 11 18 \ II. THE TOXICITY OF SODIUM CHLORIDE AND ITS RELATION TO WATER INTAKE IN BABY CHICKS Introduction The amount of sodium chloride required for normal growth and production of the fowl is subject to considerable variations. Sjollema (1935) reviewed a feeding experiment in which day old chicks fed basal rations containing .033 and ,086 per cent sodium chloride failed to grow satisfac-t o r i l y . Prentice (1933) reported that 0.5 per cent of common salt in the mash supplied to young chicks is essen-t i a l for satisfactory growth, resistance to disease, economical utilization of feed, and early maturity in the pullets. The amount of salt added to a mash depends upon the variable amount of salt found in such feeds as meat meal, fish meal and dried milk. Halpin,.Holmes, and Hart (1934) reported that 0.5 per cent added salt w i l l meet a l l needs for growth or production and maintenance. The chicken can tolerate considerably more salt than i t needs. Mitchell, Card, and Carman (1926) reported that chickens 9 to 21 weeks old were reared on rations containing 8 per cent salt with no apparent detrimental effects. They also concluded that i f salt is administered directly into the crop in doses twice daily, the minimum lethal dose is close to 4 grams per kgm. body weight. This dosage agrees with that determined by Suffran (1909). Quigley and Waite (1932) found that young chicks were able to endure salt levels as high as 30 per cent of the total ration, for short periods; and they confirmed the minimum lethal dose as stated above. Selye (1943) tested the tolerance of baby chicks for salt by replacing the drinking water with various concentrations of NaCl; 2 per cent salt caused 100 per cent mortality among two day old chicks within a few days. When 19 day old chicks were given 0,9 per cent NaCl instead of drinking water many survived up to 20 days; 0.3 per cent NaCl solutions were not toxic for 4 weeks' old chicks. Selye States that comparatively weak NaCl solutions such as 0.9 per cent are highly toxic for chicks. Based upon his observations of "NaCl intoxication" and the "striking similarity" of so-called pullet disease (Jungherr and Levine, 1941) Selye concludes that " i t is probable that this disease of fowl is also due to accidental NaCl intoxi-cation". He suggests.that a normal sodium chloride intake is more important for birds than for mammals, Blaxland (1946) found that no mortality could be attributed to sodium chloride poisoning when mashes 3. containing up to 20 per cent of salt were fed to mature fowl over a period of four weeks. On the other hand, he found 5 and 10 per cent salt in mash fed to baby chicks to be highly toxic within the f i r s t week. Confirming Selye 1s observations on the toxicity of sodium chloride solutions, he reported 100 per cent mortality in baby chicks within five days when 0,9 or 2.0 per cent sodium in solution was given instead of drinking water, A pertinent discrepancy arose between the findings of the two author's post-mortem examinations in the^chicks that died on the 2 per cent NaCl solution. Selye observed tissue edema whereas Blaxland clearly pointed out the absence of such edema, Gericke (1945) recommends that "a mash ration containing 5 to 7 per cent fish meal should not be supple-mented with common salt". Computing the possible per-centages of salt added to such rations, in terms of the sodium chloride content of Gericke 1s fish meal samples, the range is 0,13 to 0.39 per cent of the total mash ration. In view of the contradictory evidence in the literature, an experiment was designed to investigate: 1. The toxicity of sodium chloride incorporated in the mash as compared with an equivalent quantity consumed in the drinking water; 2. The water-feed ratio in relation to salt intake of baby chicks; 3. The possibility that inadequate water provision would enhance the toxicity of sodium chloride for baby chicks; 4 . The variation, i f any, in feed utilization efficiency with varying sodium chloride levels. EXPERIMENTAL The experimental study was conducted with 2 5 2 : two day old New Hampshire chicks consisting of 21 lots of 12 chicks each. At the start of the experiment the. total weights of the lots ranged from 554 grams to 5 7 0 grams. Most of the chicks were female. The chicks were reared in a special air-con-ditioned roomj four identical battery brooders containing, a total of 3 2 compartments were used. The chicks were reared in the top three levels. The basal ration consisted of the following: Wheat 1 0 0 pounds Oats 2 0 " Bran 20 " Middlings 2 0 " Fish meal (herring) 16 " Meat meal 14 " Alfalfa 10 " B. Y. 2 1 1 Fortified fish oil; 1/3 " Limestone 2 " Manganese ( 4 oz. per ton) The feed was before the chicks at a l l timesj the water or salt solutions were offered as described in Table 1 . The experiment was continued for 29 days. Variations in room temperature, relative humidity in the brooder room and water temperature in each of the troughs were a l l recorded. The evaporation from the individual troughs was carefully measured. 5.-. In the lots receiving salt in their water, the troughs were emptied, washed, and replaced with fresh solutions every 4 8 hours. The salt intake in these lots was corrected for increased concentration by adjusting the percentage in accordance with the data for evaporation over a 24-hour period. The water measurements were made at the same times as the feed measurements in a l l lots, except Lots 1 1 , 1 2 , 1 3 , 1 4 , and 1 5 . Lots 11 to 13 were measured every 48 hours, 14 and 15 every 24 hours. In Lots 17 to 2 1 , from which water was withheld, consump-tion was measured every day with due* allowances in evaporation calculations for periods when troughs were empty. Additional measurements were made when the troughs were emptied for cleaning in accordance with standard sanitary precautions. The chicks were weighed at the i n i t i a l grouping and at eight additional dates during the experiment. The feed consumption was measured at the end of the f i r s t 15 days and again at the end of each of the two succeeding weeks. Waste was carefully estimated from pans placed beneath the feed trough. The contents of the pans were screened,and taken into consideration in the calculations. Sodium chloride, C. P., was used throughout the experiment. The chlorides in the basal ration were 6 TABLE I PERCENTAGE ADDED -SALT IN FEED AND WATER Lot Per cent salt Per cent salt Water No. added in feed added to water offered 1 0.3 2 0.9 3 1.4 4 2.0 5 3 .0 6 n i l 7 4.0 8 5.0 9 6.5 10 8.0 11 n i l 12 n i l 13 n i l 14 n i l 15 n i l 16 n i l 17 1.0 18 2.0 19 .. 3 .0 20 4.0 21 n i l n i l ad libitum ii II ii it II it II n n n it ti it it II ti ti n 0.3 u 0.9 it 2.0 ti . 9 X tt 1 . 8 X it nil) tt ti \ deprived of ti S water for 4-6 ti \ hours during it \ 12 hours of it ) light provided Salt solution as indicated, and pure water on alternate days. 7. determined by the method of Fraps and Brown (1947); expressed as sodium chloride, they amounted to 0.182 per cent of the basal ration. Since the chloride content of the tap water was negligible i t was not considered in total salt intake data. Post-mortem examinations were completed on a l l chicks that died during the experiment and also upon representative chicks at the completion of the t r i a l s . RESULTS A summary of feed, salt, and water intake, gains and their relationship is included in Table II and a review of mortality is presented in Table III,. Post-mortem examination of chicks in Lots 7 to 11 which died during the course of the experiment offered a f a i r l y typical picture. With the exception of one chick in Lot 8, for which no cause of death could be determined, a l l others displayed a l l or most of .the following patho-logical abnormalities: 1. Edema - from localized to generalized. Water in the body cavities was common. 2. Fluid in the pericardial sac. 3 . Varying degrees of heart enlargement with large clots in the auricles. 4. Gelatinous covering of the body. 5 . Gonad enlargement which proved to be due to accumulation of a watery f l u i d . TABLE II SALT, FEED, WATER, GAINS, AND THEIR RELATIONSHIP Salt (grams) Gain grams per 1 0 0 grams feed K Water Salt : per gram feed Lot No. Added to feed In feed Added to water Total cc. in-take per gram feed at 1 2 0 . 1 1 2 . 2 - 3 2 . 3 4 0 . 3 4 1 . 8 5 0 . 4 8 2 7 8 . 5 1 5 . 9 - 9 4 . 4 4 1 . 8 8 1 . 9 2 1.08 3 1 1 6 . 5 1 5 . 1 - 1 3 1 . 6 4 1 . 6 7 2 . 3 4 1 . 5 8 4 1 5 6 . 6 1 4 . 3 - 1 7 0 . 9 41 .84 2 . 7 3 2.18 5 2 0 6 . 0 1 2 . 5 - 218.5 4 1 . 5 5 3.81 3.18 6 - 1 3 . 6 - 1 3 . 6 4 0 . 1 2 1 . 7 4 0.18 7 2 3 1 * 2 1 0 . 5 - 2 4 1 . 7 X 4 . 6 9 4.18 8 2 2 3 . 2 8 . 1 - 2 3 1 . 3 X 5 . 4 8 5.18 9 1 8 7 . 6 5 . 2 mm 1 9 2 . 8 X 7 . 2 9 6 . 6 8 10 2 4 2 . 5 5 . 5 - 2 4 8 . 0 X 8 ; 7 1 8.18 11 - 1 3 . 7 4 6 . 4 6 0 . 1 4 0 . 2 7 . 1 . 9 6 0.80 12 - 9 . 0 199.6 208.6 X 4 . 3 5 4 . 3 1 13 - 0 . 4 4 . 7 5 . 1 X 1 . 2 1 2 . 2 7 14 - 1 2 . 2 7 8 . 7 9 0 . 9 X 2 . 2 3 1 . 3 6 15 - 1 1 . 5 5 9 . 9 7 1 . 4 3 9 . 7 9 1.82 1 . 1 4 16 - 1 3 . 7 - 1 3 . 7 3 9 . 3 2 1 . 6 6 0.18 17 6 9 . 4 1 2 . 7 - 82.1 40.08 2 . 2 7 1.18 18 144.1 13.1 - 1 5 7 . 2 4 0 . 4 1 2 . 6 3 2.18 19 2 0 1 . 6 1 2 . 2 - 2 1 3 . 8 X 3.80 3.18 20 . 2 5 2 . 9 1 1 . 5 - 2 6 4 . 4 X 4 . 6 0 4.18 2 1 — 1 3 . 8 — 1 3 . 8 3 9 . 5 3 1 . 8 9 0.18 k Feed consumed minus salt content x Not computed because of mortality 9 TABLE III SUMMARY OF MORTALITY Number of deaths during experiment Lot No. lst-8th day 9th-15th l6th-22nd 23rd-29th Total 1,2,3, 4,5 Nil - • Nil Nil •- Nil N i l 6,16,21" Nil • •• Nil N i l Nil Nil 7 Nil ' 1 1 Nil 2 8 2 3 Nil 1 6 9 Nil 4 4 Nil 8 10 Nil 1 5 Nil 6 11 Nil Nil Nil Nil Nil 12 1 Nil Nil 3. 4 13 12 Nil N i l Nil 12 14 Nil • 1 Nil Nil 1 15 Nil ' Nil Nil Nil Nil 17,18 Nil Nil Nil Nil Nil 19 1 Nil Nil Nil 1 20 Nil . 1 Nil Nil 1 10. 6. Lung congestion in varying degrees of severity. 7. Kidney swelling or engorging with blood. 8. Livers engorged with blood. 9. Ureters f i l l e d with chalky material. The four deaths in Lot 12 on 0.9 per cent salt, included only one with most of the above symptoms. One chick in the lot died from undetermined causes. Two chicks in this group displayed no abnormalities of internal organs except slight enlargement of the kidneys. The absence of generalized tissue edema was con-spicuous in Lot 13, although some chicks had flu i d in their body cavities. Most of the post-mortems revealed slight heart enlargements and lung congestion., The chalky material f i l l i n g the ureters was common to nearly a l l of them but on gross inspection only half of the chicks showed enlarged kidneys. The individual deaths in Lots 14, 19, and 20 could a l l be classed pathologically with Groups 7 to 10. Upon completion of the t r i a l s post-mortem examinations were carried out on representative chicks in a l l of the groups. Some of the chicks remaining (Lots 9 and 10) on the high salt intakes were stunted but their internal organs revealed no abnormalities. One chick died in Lot 17 the day after the experiment concluded and two chicks in both Lots 9 and 10 suffered from severe edema. 1 1 , Table IV shows the percentage total solids of chicks selected at the end of the experiment and evaporated to dryness . TABLE IV PERCENTAGE TOTAL SOLIDS OF CHICKS 31 DAYS OLD I n i t i a l Lot Percentage weight No. Diet total solids 0,3% salt added 2 7 . 5 3 3 0 . 9 8 U.0% " " 2 6 . 8 3 28 .15 8 . 0 $ » " 2 5 . 1 4 2 2 . 9 4 DISCUSSION Best and Taylor (1945), in discussing the question of water and salt retention, state that even in the nephrotic type of glomerule-nephritis the a b i l i t y of the kidney to excrete chloride is l i t t l e , i f at a l l , reduced. They state that "the influence upon the produc-tion of edema is exerted by the sodium rather than by the chloride ion". Biely and Kare (1948) found that 1.5 per cent sodium incorporated in the mash in the form of sodium bicarbonate fed over a period of six months exerted no apparent detrimental effects upon well-being and only slightly depressed egg production of pullets; 2 . 0 per cent 354.9 2 219.5 3 2 2 . 4 7 268.9 199.3 10 218 12. c h l o r i d e i n c o r p o r a t e d i n the mash i n the form of ammonium c h l o r i d e depressed the weight of p u l l e t s and g r e a t l y i n -h i b i t e d egg p r o d u c t i o n . In Lots 1 to 10, where the sodium c h l o r i d e was i n c o r p o r a t e d i n the mash and pure water was giv e n ad l i b i t u m , no m o r t a l i t y occurred u n t i l the l e v e l of s a l t was 4.18 per cent of the mash i n t a k e . At l e v e l s as high as 6.68 and 8.18 per cent s a l t ( L o t s 9 and 10) some of the chic k s were a l i v e a f t e r 29 days. A l l the s u r v i v i n g c h i c k s i n these l o t s weighed markedly l e s s than the average i n the l o t s with no m o r t a l i t y . On the other hand, n e a r l y a l l of the s u r v i v i n g c h i c k s i n Lots 7 and 8 on l e v e l s of 4.18 and 5.18 per cent s a l t , d i s p l a y e d gains and w e l l -being e q u i v a l e n t to the best c h i c k s on the lower s a l t d i e t s . I n d i v i d u a l c h i c k s have an inhe r e n t c a p a c i t y to t o l e r a t e high l e v e l s of s a l t as demonstrated by Lot 8, f o r example, where h a l f the chi c k s died and h a l f ex-hibited.-normal gains and h e a l t h . The water intake per gram of feed consumed (Table II) i n c r e a s e d from 1.70 c c . on b a s a l d i e t con-t a i n i n g ,182 per cent s a l t to 8.71 cc. on a d i e t with 8 per cent added s a l t . The c h i c k s i n Lot 12 drank 21,076 cc, of 0,9 per cent sodium c h l o r i d e . The s a l t content, c o r r e c t e d f o r i n c r e a s e d c o n c e n t r a t i o n due to eva p o r a t i o n , was 199.6 grams. This was e q u i v a l e n t to 4.13 per cent of the mash consumed 13 o or a total of 4 . . 3 I per cent i f the salt in the mash is included. Lot 7 was fed mash containing 4.18 per cent salt and pure water was offered ad libitum. The mortality (Table III) in Lot 12 was lower than that of Lot 7 at the end of the third week. However, at the end of the experiment there were four f a t a l i t i e s in Lot 12 as compared to two in Lot 7 . The average weight of surviving chicks in Lot 12 was 2 6 7 . 8 grams with a range of 174 to 3 6 4 grams. The average weight of surviving, chicks in L o t V 7 was 2 8 3 , 2 grams with a range of 2 3 8 to 372 . grams. The feed-water ratio in the two lots was 4 . 6 9 cc, of water per gram feed consumed for Lot 7 as compared to 4 . 3 5 cc. per gram feed in Lot 1 2 . The data indicated that at a concentration of 0 . 9 per cent salt in the drinking water the toxicity of the salt was approximately equivalent to the mash containing 4 per cent added salt. Both Selye and Blaxland reported greater mortality with 0 . 9 per cent sodium chloride in the drinking water than was observed in this experiment. Both of these investigators used commercial chick mashes which contained possibly 0 . 5 per cent added salt. Additional salt in the ration could stimulate greater thirst and, of course, increased water consumption would further increase actual salt intake. The discrepancy might be due to the 14. difference in control of the increased concentration resulting from evaporation. The comparison of mortality should be based upon actual salt intake, but neither Selye or Blaxland provide this information. Lot 11 with 0 .3 per cent salt solution as drinking water was consuming the equivalent of 0.80 per cent salt in the mash. The chicks in this group appear-ed normal in a l l respects. A l l the chicks in Lot 13 which received 2 per cent sodium chioride as drinking water were dead within a week. The percentage salt in terms of mash eaten was 2 . 2 7 . The water consumption per gram feed was 1.21 cc. as compared to 2 .73 per cc. with chicks receiving 2.18 per cent salt in the mash. The salt intake of Lot.13 has been demonstrated in Lots 4 and 5 to be non-toxic when consumed incorporated in the mash. Lots 7 and 12 indicated that salt solutions were roughly as toxic as an equivalent amount of salt incorporated in the mash. Post-mortem examinations of the chicks in Lot 13 failed to reveal a single edematous condition characteristic of lethal salt, levels. On the evidence of the marked reduction of water consumed in relation to the feed and salt intake a diagnosis of water starvation appears jus t i f i a b l e . Lots 14 and 15 were set up to see how freely chicks drink salt water and whether the level of salt or the total salt intake was responsible for the toxicity. 15 • Lot 14 consumed 8,297 cc. of 0.9 per cent salt water in the 15 alternate days as compared to 6 , 5 7 2 cc. of pure water in 14 days. The 24-hour periods were approximate. Evaporation was taken into account but no allowance was made for waste. Perhaps the salt water was distasteful but was balanced by the increased t h i r s t . One death was reported. Lot 15 consumed 3,182 cc. of 1.8 per cent salt water in 15 days as compared to 11,450 cc. of pure water on the alternate 14 days. There was no mortality. 1.8 per cent salt water was obviously distasteful. The small quantity consumed, i f interpreted as salt intake, would be well below the minimum lethal dosage* If salt water is before the birds at a l l times i t is futi l e for them to attempt to adjust their water-salt ratio. Over short periods 0.9 and 1.8 per cent sodium chloride in the drinking water is not toxic. In the latter half of the experiment, particularly, the chicks in Lot 15 reduced their intake of salt water and com-pensated by large intakes on the alternate days when fresh water was provided. Chicks which were deprived of water four to six hours a day compensated by drinking more when the water was before them. Comparing the water intake per gram feed consumed (Table II) of chicks on the same salt levels, the chicks with water ad libitum and the chicks deprived of water during part of the day displayed approximately 16. the same feed-water ratios. One death occurred with the typical post-mortem picture (Lots.7-10) on 3.18 per cent salt in Lot 19 and only one in Lot 20 on 4.18 per cent salt. Lots 2 to 5 revealed a negligible difference in gain per hundred grams feed consumed. The range was from 41.55 to 41.88 grams and the average gain,was 41.74 grams in terms of one hundred grams feed after the salt content had been deducted. The chicks on basal rations,containing .182 per cent salt (Lots 6 and 1 6 ) made an average gain of 39.72 grams per 100 grams feed. Campbell (1946), working on rats, concluded that the presence or absence of sodium chloride in diets consisting of whole wheat and dried whole milk appeared to make no difference in growth or well-being. Campbell's work was based upon three diets using 1 . 3 2 , 2.59 and 5 . 0 6 per. cent sodium chloride. The data in Table II would suggest: that between a minimum level and the toxic upper limits the efficiency of food utilization is not influenced by sodium chloride intake. The evaporation to dryness<of chicks tabulated in Table IV offers two observations: The heavier birds in a l l lots had a greater percentage water content than the smaller birds in the same l o t . The increased percentage of water content of birds on higher salt diets.offers the opportunity to confirm this finding upon the basis of a large number of chicks. 1 7 . Since Lots 7 and 10 were known to produce edema, i t is a matter of conjecture whether salt content of mashes in ranges below the minimum edematous dose could influence the water content of the tissues. A l l chicks evaporated to dryness were, from external appearances, in good health. Gericke (1945).computed the average live weight of Rhode Island Red chicks and the average quantity of feed consumed per chick per week. Using this data as a guide to determine feed intake in relation to body weight, Gericke's table reveals that in the second week a chick eats i t s own weight in feed. This relationship changes progressively and by the sixth week the chicks consume .81 of their own weight, the 12th week.517, the 18th week, , 4 9 7 , and the 24th week, ,409. .This would explain why, as Blaxland ( 1 9 4 6 ) demonstrated, older chickens could tolerate much higher percentages of salt in their mash than baby chicks. It also could be used as a method of approaching inter-species comparisons of salt toxicity, Selye's ( 1 9 4 3 ) observation that the fowl is extremely sensitive to small doses of sodium chloride in the drinking water is misleading. Results in this paper have indicated that salt injeeted in water or in the feed is about equally toxic. Further, i t was demonstrated that 3.18 per cent salt in the feed caused no detrimental effect to baby chicks. Since chicks consume their own •18. weight in feed during their second week, evidently they can tolerate over that time 3»18 per cent of their own weight in salt. A 65-kilogram mammal on an equivalent basis would have to consume more than 2 kilograms of salt in a week. Both Mitchell et a l . (1926) and Blaxland (1946). have demonstrated that older birds can tolerate an even greater percentage of salt in terms of body weight. The evidence would justify the conclusion that fowl can tolerate considerable salt per kilogram body weight.. What Selye terms a "comparatively weak NaCl solution" in actuality results in an abnormally high salt intake. Further, on the basis of intake of feed and water in relation to body weight, his data on chicks were not directly applicable to older.birds. In view of the fowl's strong distaste for lethal levels of salt water, and the unlikelihood of feeding a mash with a toxic salt level, the probability of "so-called pullet disease" being due to accidental sodium chloride intoxication is open to question, SUMMARY Two hundred and fifty-two New Hampshire chicks divided into 21 lots of 12 chicks were used to investigate the toxicity of salt, its influence on water intake and its relation to feed uti l i z a t i o n efficiency. (l) A l l lots receiving mash containing over 4 per cent salt showed characteristic edematous condition in some of the chicks. 19. The chicks exhibited individual differences in tolerance for sodium chloride. For. some chicks 5.18 per cent salt in the mash was fatal; for others i t was apparently harmless. The toxicity of sodium chloride in low concentrations given in the drinking water was roughly that of an approximately equivalent intake of salt i n-corporated in the mash. The salt intake when 0.9 per cent sodium chloride,,was given as drinking water was 4.31 per cent in terms of mash eaten (0.18 per cent from the mash). The chicks exhibited a strong distaste for water containing 1.8 per cent sodium chloride. Chicks offered a 2 per cent solution instead of drinking water drank small quantities and died within a week, but their total salt intake was con-siderably less than a toxic amount. The 1.8 per cent sodium chloride solution in place of drinking water was not toxic when birds were periodically given access to pure water. The water intake per gram feed consumed increased progressively with the salt percentage of the mash diet. Chicks on diets containing 0.18 to 4•18 per cent salt, deprived of water from 4 to 6 hours per day, consumed practically the same amount of water per gram feed as chicks on the same diet but with water ad libitum. There is no significant variation in efficiency of feed u t i l i z a t i o n on diets containing 1,08 to 3.18 per cent sodium chloride. The average gain over 29 days was 41.74 grams per hundred grams feed. The gain per hundred grams feed on a basal diet containing 0.182 per cent sodium chloride was 39 .72 grams over 29 days under parallel conditions. R E F E R E N C E S Best, C. H. and N. B. Taylor, 1945 . The physiological basis of medical practice. The Williams & Wilkins Co., Baltimore, p 407-8 Biely, J. and M. R. Kare, 1 9 4 8 . Unpublished data. Blaxland, J. D., 1 9 4 6 . The toxicity of sodium chloride for fowls. Vet. Jour. 102 : 1 5 7 - 1 7 3 . Campbell, H. L., 1946 . Sodium chloride as an adjunct to a diet of whole wheat and whole milk. A. Jour, of Physiol. 1 4 7 : 3 4 0 - 3 4 2 . Fraps, G. S. and E..E. Brown, 1 9 4 7 . Determination of chlorides in commercial feeding stuffs. Jour, of the Ass. of O f f i c i a l Agr. Chemists, 3 0 : 3 7 0 - 3 7 2 . Gericke, A. M., 1 9 4 5 . The nutrition of poultry. Union of South Africa Dept. of A g r i c , Bull. 260 , 52 pp. Halpin, J. G., C. E. Holmes, and E. B. Hart, 1 9 3 6 . Salt requirements of poultry. Poultry Sci. 1 5 : 9 9 - 1 0 3 . Jungherr, E., and J. M. Levine, 1 9 4 1 . The pathology of so-called pullet disease. A. Jour, of Vet. Res. 2 : 2 6 1 - 2 7 1 . Mitchell, H. H., L. E. Card, and G. G. Carman, 1926 . The toxicity of salt for chickens. University of 1 1 1 . , Agr. Exp. Sta. Bull. 2 7 9 : 1 3 5 - 1 5 6 . Prentice, J. H., 1 9 3 3 . The role of salt in poultry nutrition. The Journal of the Ministry of Agriculture of Northern Ireland, p 7 2 : Abstract in International Review of Poultry Sci. 6: 6 5 - 6 6 . Quigley, G. D., and R. H. Waite, 1 9 3 2 . Salt tolerance of baby chicks. Maryland Agri. Exp. Sta. Bull. 340 : 3 4 3 - 3 7 0 . Selye, H., 1 9 4 3 . Production of nephro sclerosis in the fowl by sodium chloride. Jour, of the A. Vet. Med, Ass. 103 : 1 4 0 - 1 4 3 . - 2 ~ Sjollema, B. 1935. Studies on the sodium requirement of chickens and on the consequences of a diet almost free of sodium. Tierernahrung 7 : 1 8 4 » Suffran, F. 1909. Poisoning of poultry by common salt. Revue Generale De Med. Vet. 1 3 : 698-705; 1910 Abstract in -Journal of Comparative Pathology and Therapeutics 2 3 : 71-74. 

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