Chemical Changes in Nerves from Birds Affected by Fowl Paralysis

ACIDULATED SOAPSTOCKS Leibovitz, Z., 1961. Personal communication. Lipstein, B., and S. Bornstein, 1964. Studies with acidulated cottonseed oil soapstock. 1. Its use as a fat supplement in practical broiler rations. Poultry Sci. 43: 686-693. Lyman, C. M., 1953. Discussion to paper by Couch (1953). Pons, W. A., D. J. Mitcham, R. T. O'Connor and M. F. Stansbury, 1956. Improved method for determining gossypol in crude cottonseed oils.

701

J. Am. Oil Chem. Soc. 33:324-330. Schapiro, A., 1961. Personal communication. Stansbury, M. F., V. 0. Cirino and H. P. Pastor, 1957. Composition of acidulated cottonseed soapstocks as influenced by commercial methods of processing seed and oil. J. Am. Oil Chem. Soc. 34:539-544. Withers, W. A., and F. E. Carruth, 1918. Gossypol the toxic substance in cottonseed. J. Agric. Res. 12:83-101.

P. J. HEALD, H. G. BADMAN* AND B. F. FRTJNIVAL Twyford Laboratories Limited, Twyford Abbey Road, London, N.W.10, AND

P. A. L. WIGHT Agricultural Research Council, Poultry Research Centre, Edinburgh (Received for publication November 22, 1963)

A

LTHOUGH the histology and pa>• thology of affected nerves in fowl paralysis (neurolymphomatosis gallinarium, Marecks disease) has been well documented (see Campbell, 1961; Wight, 1962; Biggs, 1963), no chemical analysis of these tissues has been reported. Reasons for this could lie in the fact that the majority of affected nerves are found to be heavily infiltrated with inflammatory cells, thus making it extremely difficult or impossible to decide whether an analytical figure refers to the diseased tissue itself or to the infiltrating cell. It was found by Wight (1962) that nerves from cases of fowl paralysis in the flock at the Poultry Research Centre, Edinburgh, differed in microscopic appearance from those following the classical description. A large proportion of affected nerves showed comparatively little cel* Present address: Dept. of Physiological Chemistry, The University, Reading, Berkshire.

lular infiltration, the predominant histological changes being oedema, demyelination, and ultimately an appearance similar to that found in Wallerian degeneration. Accordingly it was considered desirable to examine such nerves to determine whether gross chemical changes took place similar to those described for Wallerian degeneration in mammalian sciatic nerve (Rossiter, 1962) and to correlate such changes with the histological picture observed. MATERIALS AND METHODS

Sciatic nerves were obtained from birds of the Brown Leghorn flock maintained at the Poultry Research Centre, Edinburgh. Nerves were removed as soon as possible after death, samples were taken for histological examination and the remainder of each nerve was packed in solid carbon dixoide and stored at —18° until required for analysis. The birds were generally aged 12-20 weeks with an oc-

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Chemical Changes in Nerves from Birds Affected by Fowl Paralysis

702

P. J. HEALD, H. G. BADMAN, B. F. FRUNIVAL AND P. A. L. WIGHT

casional older bird aged 52-124 weeks. All the cases of fowl paralysis were naturally occurring and were killed when the clinical condition became readily apparent. Where it could be assessed the period of clinical involvement ranged from 2 days to 2 weeks. Normal birds of the same age groups were killed to provide control material.

Because the primary requirement was the chemical analysis of the relatively small avian nerves, as small pieces as practicable were taken for histological examination. These were fixed in Susa, and paraffin sections of 5/u-thickness were cut and stained with haematoxylin and eosin. Chemical methods The general method of extraction of the nerves was based on the procedure of Johnson et al. (1949). The nerves were weighed and, after removing a segment for the determination of dry weight, the remainder was cut into small pieces with scissors and transferred to a small centrifuge tube containing 5 ml. of cold trichloroacetic acid (TCA 10% w./v.). The nerve was ground in the tube by means of a loosely fitting pestle, the tube being kept cold by frequent immersions in an ice/water mixture. The suspension was then centrifuged at 10,000 g. for 10 min. at 0° and the supernatant removed. The residue was extracted twice more with TCA (5% w./v.) and the combined supernatants taken for the determination of total acid-soluble phosphorus. Extraction of lipids. The residue was then extracted by grinding with 5 ml. of chloroform-methanol (2:1 v./v.) (Folch et al., 1951) and, after centrifuging, was extracted twice more with 2.5 ml. of chloroform-methanol. The combined

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Histological methods

supernatants were brought to 10 ml. with chloroform-methanol and washed, first with 0.2 vol. of 0.03 M NaCl and three times with 0.2 vol. of the "pure solvents upper phase" [prepared by mixing 5 parts of chloroform-methanol (2:1 v./v.) with 1 part of 0.03 M NaCl]. The washings were discarded. A slight interfacial layer (insoluble on addition of ethanol) was obtained during this treatment and was removed either by filtration or centrifugation. Extraction of the residue. The residue was washed twice with 5 ml. ethanol and once with 5 ml. of TCA (5% v./v.). The residue was then extracted at 90° for 15 min. with 4 ml. 5% TCA (Schnieder, 1945) and, after cooling, the supernatant was removed for the determination of DNA and RNA phosphorus. Separation of lipid fractions, (a) Quantitative. Lipid extracts were prepared for chromatography as described by Long and Staples (1961). The extract was evaporated to dryness in a stream of nitrogen using a rotary evaporator. The residue was dissolved in 10 ml. chloroform-methanol-water (64:32:4 v./v.) and again taken to dryness. A solution of the residue, in chloroform-methanol (98:2 v./v.), was filtered through sintered glass (porosity 3) and a Whatman No. 50 filter paper previously washed with the solvent. The filtrate was evaporated to dryness and the residue suspended in 5 ml. of chloroform-methanol (98:2 v./v.). A sample was taken for the determination of lipid weight and phosphorus and the remainder evaporated to dryness. It was finally dissolved in a volume of chloroformmethanol to give a lipid concentration of 10-12 mg./ml.; 1.0 ml. of this solution was loaded onto a silic acid column. Columns were prepared from 100 mesh silicic acid. (Mallinkrodt Chemical Works, St. Louis, Missouri), which was first sieved with a

CHEMICAL CHANGES IN FOWL PARALYSIS

(b) Qualitative analysis. Samples of the eluates were examined qualitatively by thin-layer chromatography. Neutral lipids were separated in petroleum ether (b.p. 60-80)/diethyl ether (9:1 v./v.), and phospholipids in di-isobutylketone/acetic acid/water (57:36:7), (Kates et al., 1960). Plates were first examined in ultraviolet light and were then developed using iodine vapour, spraying with sulphuric acid (20iV) and heating at 100°. Analytical methods Inorganic phosphorus was determined by the method of Berenblum and Chain (1938) as modified by Long (1943) for use in a single tube. Total phosphorus was determined in a similar manner after digestion with perchloric acid as described by King (1932). DNA-P. This was determined in trichloroacetic acid extracts by the method of Burton (1956). Two specimens of highly purified DNA (supplied by Dr. M.F.H.

Wilkins and Dr. K. V. Shooter) were used as standards. RNA-P. This was determined by measuring the extinction at 268.5 mju of the TCA extracts as described by Logan et al. (1952a) and subtracting the values obtained for DNA-P as estimated directly. Acyl esters. These were determined by the method of Stern and Shapiro (1952). Cholesterol and cholesterol esters. These were determined as described by Pearson etal. (1953). Cerebrosides. These were determined as described by Long and Staples (1961). Materials. Lecithin and cephalin were obtained from the Californian Corporation for Biochemical Research and were of Grade A quality. Triglycerides and cholesterol were obtained from British Drug Houses Ltd., Poole, Dorset, England. A specimen of galactocerebroside was given by Dr. C. Long and one of sphingomyelin by Dr. M. G. McFarlane. Both specimens were obtained from ox brain. RESULTS

Analysis of lipids. Under the conditions employed, lipid standards containing predominantly the material stated were eluted in the order listed in Table 1. This table also shows the quantitative recoveries of known lipids added to each column together with a description of the major components of each fraction when obtained from extracts of sciatic nerves. Sphingomyelin appeared in at least two fractions. Determination of this material when distributed in this way proved extremely difficult when extracts of nerve were used owing to the small quantities present in each fraction, and was finally abandoned. Examination, by thin-layer chromatography, of the fractions eluted from extracts of sciatic nerves from normal birds showed that materials chro-

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British Standards Sieve 240 mesh and the dust discarded. The remainder was washed five times with methanol to remove small particles and finally with diethyl ether. It was activated by heating at 120° for 24 hr. and was stored under vacuum. A suspension of 4.5 g. in petroleum ether (b.p. 60-60) was added to a column 10 cms. X1-0 cms.2, half filled with petroleum ether. After settling, the petroleum ether was carefully drained until the column was free of excess fluid. The lipid sample was carefully added, taking care not to disturb the surface of the column and the column again allowed to drain. Lipids were eluted in a stepwise manner using 100 ml. of freshly prepared solvent for each step. Each eluate was evaporated to dryness at less than 45° in a stream of nitrogen and stored under nitrogen until required for analysis.

703

704

P. J. HEALD, H. G. BADMAN, B. F. FRTJNIVAL AND P. A. L. WIGHT

TABLE 1.—Elution of lipid fractions from silic acid columns and recovery of known samples of lipids added Samples were prepared and fractionated as described in the Materials and Methods section. The identification of the lipids present in extracts of sciatic nerves was made on the basis of elution characteristics when compared with known materials by means of thin layer chromatography. Fraction No. 1 2

4 5 6 7 8

Ethyl ether in light petroleum (b.p. 60-80°) (1:99 v./v.) Ethyl ether/light petroleum (4:96 v./v.) Ethyl ether/light petroleum (10:90 v./v.) Ethyl ether/light petroleum (25:75 v./v.) Ethyl ether Chloroform-methanol (4:1 v./v.) Chloroform-methanol (3:2 v./v.) Chloroform-methanol (1:4 v./v.)

%

Range

Cholesterol esters

108.6

96-127

Triglycerides

102.0

83-115

Cholesterol

106.0

95-115

Diglycerides

—

Lipids eluted

Monoglycerides Cephalins and Cerebrosides Lecithins and Sphingomyelin Sphingomyelin

Recovery

—

72.4*

—

53- 91*

—

Cephalins only.

matographically similar to those of the standards were being eluted in the appropriate fractions. Various unknown spots, which appeared consistently in fractions from all nerves, were observed in small amounts or traces. Though the overall quantities of some of the lipids changed according to the degree of degeneration of the nerves, with one exception no new "spots" indicating the possible presence of a new type of lipid was observed. The exception was the fraction containing cholesterol esters, which was found to contain two esters when derived from the nerves which were severely degenerated. Treatment of data. The development of the paralytic condition as regards sciatic nerves is usually accompanied by a marked swelling and oedema, and histologically, by a loss of myelin lipids. Since swelling without a change in the quantity of a component should not affect the total amount present, when calculated on a dry weight basis, such comparisons were made between nerves in the different histological groups. Comparisons were

also made in relation to the quantities of DNA-P since in those instances in which cellular proliferation was minimal this relationship permits description of the change on a cellular basis. Neither of these comparisons, however, is likely to be strictly accurate as the degeneration progresses since much of the dry weight of the nerve is represented by lipid. Thus a small decrease in a single lipid type could be obscured when nerves are compared on a dry weight basis since such a calculation would tend to correct for the loss. Similarly comparison on a DNA-P basis becomes less meaningful when the degree of cellularity increases. A third analysis was therefore performed to which these objections do not apply: the correlations between the individual components within nerves were calculated. The results, analysed on a dry weight basis, are presented in Table 2. They have been grouped according to the classification of Wight (1962). Thus group 1 includes nerves which were mildly affected and show a moderate leucocytic infiltra-

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CHEMICAL CHANGES IN FOWL PARALYSIS 1)

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tion. Group 2 includes all nerves which exhibit oedema with little or no infiltration whilst group 3 represents all nerves which were severely oedematous and showed massive leucocytic infiltration. Nerves in group 4 were histologically unaffected, but were derived from birds in which neurolymphomatosis was diagnosed by examination of the brachial plexus. By far the largest number of nerves fell into group 2, only five appearing in group 3 and three in group 1. This distribution is typical for birds derived from this experimental flock (Wight, 1962). Nerves in groups 1, 2 and 3 contained significantly less dry matter (i.e. more fluid) than normal nerves. Nerves in groups 2 and 3 contained significantly less cerebrosides than normal, while significant decreases in phospholipids and increases in DNA-P were noted only in nerves of group 3. No differences from normal were detectable in the nerves of group 4. For all nerves the triglycerides, cholesterol esters, cholesterol and lipid phosphorus was not significantly correlated with the dry weight; on the other hand cerebrosides, DNA-P and RNA-P were highly correlated, the correlation coefficients being r = 0A5 (P<0.01), r = - 0 . 6 3 (P<0.001), and r = - 0 . 3 9 (P<0.05) respectively. When calculated in terms of DNA-P (Table 3) the significances of the changes between the various groups of nerves resembled those which had been calculated in terms of dry weight. Here phospholipid and cerebrosides in nerves of groups 1 and 3 now showed significant changes from normal. Correlations of the individual fractions with one another are presented in Table 4. Over all the nerves cholesterol was positively correlated with cholesterol esters and RNA-P whereas cholesterol esters were negatively correlated with phospholipids and cerebrosides and posi-

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I- g "Stts S

705

706

P. J. HEALD, H. G. BADMAN, B. F. FRUNIVAL AND P. A. L. WIGHT

TABLE 3.—Comparison of quantities of lipids from sciatic nerves of birds suffering from fowl paralysis Data included in Table 2 was recalculated and expressed in terms of units jimoles of DNA-P. The classification of the nerves into normal, groups 1, 2 and 3 was made according to the system of Wight (1962). N.S. = not significant at the 5 % level. Group 1 Lipid

nerves

Triglyceride Cholesterol Ester Cholesterol Phospholipid Cerebroside

8.98 0.96 10.70 23.43 7.17

Mean

Significance of diff. from normal

1.85 3.12 10.98 3.4 0.86

P<0.05 N.S. N.S. P<0.05 P<0.01

Group 2 Mean

Significance of diff. from normal

9.77 1.91 9.16 16.18 3.37

N.S. N.S. N.S. N.S. P<0.001

DISCUSSION

The results can be discussed under two headings first in relation to the histological findings and secondly in relation to the progressive biochemical changes. Further, grouped according to the histological classification of Wight (1962), the increasing severity of the disease is accompanied by a highly significant increase in oedema (Table 2). The progression of the disease is also marked by a loss of total cerebrosides and phospholipids and a significant increase in the quantities of DNA-P. Little or no change occurred in the triglycerides, cholesterol or RNA-P. Indeed, the major feature of comparisons made on the dry weight basis is the apparent absence of significant changes with the exception of the dry matter, until the histological findings showed severe damage. It would thus seem that the swelling of the tissue is probably one of the earliest significant changes occurring during the onset of fowl paralysis, which can be detected in this manner. I t is also of importance to note that nerves of group 4, which, though derived from birds

Group 4

Mean

Significance of diff. from normal

Mean

Significance of diff. from normal

2.54 0.49 3.07 3.64 1.25

N.S. N.S. P<0.05 P<0.001 P<0.001

13.58 1.99 18.75 37.18 9.98

N.S. N.S. N.S. N.S. N.S.

suffering from fowl paralysis, did not themselves reveal anything but a normal histology, and were also apparently normal in respect to dry matter and the other components examined. A more significant change was noted in nerves of group 1 when the components were expressed in terms of DNA-P, i.e. on a cellular basis. Thus triglycerides, phospholipids and cerebrosides decreased significantly. Nerves of this group, however, show a generally increased degree of cellular proliferation and some slight infiltration of lymphocytes. This is more marked in nerves of group 3 where highly significant changes on a cellular basis were found in cholesterol, phospholipids and cerebrosides. However, since proliferation of Schwann cells and infiltration of lymphocytes automatically increases the DNA and RNA content of the tissue such results for nerves of the type placed in group 3 are to be expected. In nerves of group 2 where little infiltration occurred and only a mild proliferation of Schwann cells took place no significant change in DNA-P was found and only the cerebrosides decreased significantly when calculated on a cellular basis. It must be concluded that analyses of this type into broad groups of materials was not able to detect a change in a component before tissue damage could be detected histologically. In terms of the biochemical events dur-

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tively correlated with RNA-P. Phospholipids were positively correlated with cerebrosides and DNA-P and negatively with RNA-P. The acid soluble phosphorus was positively correlated with DNA-P and RNA-P. The significance of these correlations is discussed below.

Group 3

—

—

—

—

—

—

Triglyceride

Phospholipid

Cerebroside

DNA-P

RNA-P

r = 0.745(28) P<0.001

^^ter*01

Cholesterol ester

Cholesterol

Substance

r=

—

—

—

—

—

0.0672(28) N.S.

r= - 0 . 1 3 9 6 (30) N.S.

Triglyceride 0.194 (30) N.S.

—

—

—

—

r= - 0 . 2 5 2 7 (30) N.S.

r=-0.3704(28) P<0.05

r=

Phospholipid 0.0499(30) N.S.

r=

r=

—

—

—

0.539 (31) P<0.001

0.1042(30) N.S.

r= - 0 . 3 4 8 1 (28) P = 0.05

r=

Cerebroside

0.3 P<0

0. N.

0.1 N.

0.1 N.

—

—

r= - 0 . 3 P<0

r=

r=

»•=

r=

DN

TABLE 4.—Correlation coefficients of the changes in lipids and nucleic acids from sciatic nerves of The values given are the correlation coefficients followed in parenthesis by the number of nerves teste

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708

P. J. HEALD, H. G. BADMAN, B. F. FRUNIVAL AND P. A. L. WIGHT

Nevertheless differences appear to exist Thus, increases in cholesterol esters were also accompanied by an increase in free cholesterol while both DNA-P and RNAP appeared to increase together and not RNA before DNA (Logan et al., 1952b). Such differences may in part be due to the presence of small numbers of infiltrating lymphocytes carrying DNA and possibly cholesterol and phospholipids, since avian blood cells, or erythrocytes alone, contain free cholesterol, cholesterol esters and phospholipids (Kates and James, 1961; Levielle et al., 1962). This interpretation is supported by a positive correlation between RNA-P and cholesterol ester. Also, because clinical diagnosis of naturally occurring poultry disease is inadequate the time which the nerve has been affected and the sequence of relative chemical changes cannot be exactly assessed.

Increases in the quantity of cholesterol esters have been considered to be reliable signs of active demyelination (Cummings, 1953, 1961), the cholesterol esters previously having been described incorrectly as "neutral fat" on the basis of staining reactions. In the present experiments cholesterol esters increased and in severely affected nerves at least two esters were present, but no significant change was noted in the quantities of triglycerides. In cat sciatic nerve undergoing Wallerian degeneration (Johnson et al., 1949) neutral fat decreased in the first few days following nerve section and then regained the original values. It was noted that qualitative analysis of the various lipid fractions showed them to be of the groups listed in Table 1, and, with the exception of the cholesterol esters mentioned above, no additional lipid appeared in any of the nerves examined. Together with the quantitative analyses these results lead to the conclusion that the onset of fowl paralysis in the flock examined is accompanied by a generalized demyelination, not by a lipidosis, and that oedema is one of the earliest changes detectable by the methods used here. This conclusion is in complete agreement with the histological findings of Wight (1962). It seems unlikely at present that additional information of the above type, obtained from clinical material, would add much further to an understanding of the nature and causation of the changes taking place and studies in a different connotation are envisaged. SUMMARY

1. Sciatic nerves from Brown Leghorns affected by fowl paralysis have been analysed for changes in various lipid groups, and nucleic acids. 2. When correlated on a dry weight basis the progression of the disease was marked by a continuing loss of dry mat-

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ing the course of the disease a more meaningful pattern is provided by correlating changes in one component with that in another (Table 4). Thus cholesterol and cholesterol esters increased together as did the level of cholesterol and RNA-P. Cholesterol esters increased as phospholipids and cerebrosides decreased and also as the level of RNA-P increased. Phospholipids and cerebrosides decreased simultaneously and both decreased as DNA-P and RNA-P increased. The acid soluble phosphorus increased as the DNAP and RNA-P increased. These changes are similar in many respects to those found in the demyelination accompanying Wallerian degeneration in the cat or rat and in multiple sclerosis or diffuse sclerosis in man (Bodian and Dziewiatkowski, 1950; Johnson et ah, 1949; Mannel, 1952; Poser, 1962). Thus there is a loss of phospholipids, cholesterol and cerebrosides, and an increase in cholesterol esters, DNA and RNA (Logan et al., 1952b; Causey and Stratman, 1956).

CHEMICAL CHANGES IN FOWL PARALYSIS

ACKNOWLEDGEMENTS

It is a pleasure to thank Miss S. V. Cunliffe for the statistical analyses, Dr. C. Long, Dr. G. R. Webster and Dr. M. G. McFarlane for gifts of lipids and for helpful discussions and Dr. M. F. H. Wilkins and Dr. K. V. Shooter for gifts of highly purified DNA. REFERENCES Berenblum, I., and E. B. Chain, 1938. An improved method for the colorimetric determination of phosphate. Biochem. J. 48:295-298. Biggs, P. M., 1961. A discussion on the classification of the avian leucosis complex and fowl paralysis. Brit. Vet. J. 117:326-334. Bodian, D., and D. Dziewiatkowski, 1950. The disposition of radioactive phosphorus in normal as compared with regenerating and degenerating nervous tissue. J. Cell. Comp. Physiol. 35: 155— 175. Burton, K., 1956. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315-323. Campbell, J. G., 1961. A proposed classification of the leucosis complex and fowl paralysis. Brit. Vet. J. 117:316-325. Causey, G., and C. J. Stratman, 1956. The nucleic acid content of peripheral nerve in degeneration and activity. Proc. Roy. Soc. (B) 144: 520-527.

Cummings, J. N., 1953. The cerebral lipids in disseminated sclerosis and in amaurotic family idiocy. Brain, 76: 551-562. Cummings, J. N., 1961. The National Hospital and the growth of neurochemistry. World Neurol. 2: 247-253. Folch, J., I. Ascoli., M. Lees, J. A. Meath and F. N. LeBaron, 1951. Preparation of lipid extracts from brain tissue. J. Biol. Chem. 191: 833-841. Johnson. A. C , A. R. McNabb and R. J. Rossiter, 1949. Chemical studies of peripheral nerve during Wallerian degeneration. I. Lipids. Biochem. J. 45:500-507. Kates, M., and A. T. James, 1961. Phosphatide components of fowl blood cells. Biochim. Biophys. Acta, 50:478-485. Kates, M., G. V. Marinetti., J. Erbland and J. Kochen, 1957. Quantitative chromatography of phosphatides. Fed. Proc. 16: 837-844. King, E. J., 1932. The colorimetric determination of phosphorus. Biochem. J. 26: 292-297. Levielle, G. A., J. W. Shockley and H. E. Sauberlich, 1962. Lipid content of chick erythrocytes and plasma. Proc. Soc. Exptl. Biol. Med. 109: 345-347. Logan, J. E., W. A. Mannel and R. J. Rossiter, 1952a. A note on the determination of deoxypentose nucleic acid and pentose nucleic acid in tissues from the nervous system by ultraviolet absorption. Biochem. J. 51:480-482. Logan, J. E., W. A. Mannel and R. J. Rossiter, 1952b. Chemical studies of peripheral nerve during Wallerian degeneration. 3. Nucleic acids and other protein bound phosphorus components. Biochem. J. 51:482-487. Long, C , 1943. The in vitro oxidation* of pyruvate and a-ketobutyric acids by ground preparations of pigeon brain: the effect of inorganic phosphate and adenine nucleotide. Biochem. J. 37: 215-225. Long, C , and D. A. Staples, 1961. Chromatographic separation of brain lipids: cerebroside and sulphatide. Biochem. J. 78:179-185. Mannel, W. A., 1952. Wallerian degeneration in the rat: chemical study. Canad. J. Med. Sci. 30: 173-179. Pearson, S., S. Stern and T. H. McGavack, 1953. A rapid accurate method for the determination of total cholesterol in serum. Anal. Chem. 25: 814820. Poser, C. M., 1962. In Cerebral Spingolipidoses. Ed. by Aronson, S. M., and B. W. Volk, Academic Press., N. Y., p. 141-153. Rossiter, R. J., 1962. The Chemistry of Wallerian Degeneration in Chemical Pathology of the Nervous System. Ed. by Folch-Pi, J., Pergamon Press, N. Y., p. 207-224.

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ter, cerebrosides and phospholipids. Similar changes were noted when correlations were made in terms of DNA-P. 3. The most significant correlations were obtained when each component was compared with another. The changes then resemble closely the pattern found in demyelination accompanying Wallerian degeneration. 4. Qualitative examination of the lipids by thin-layer chromatography failed to show the presence of any new lipid fraction other than a cholesterol ester in any of the affected nerves. 5. It is concluded that the onset of fowl paralysis is accompanied by a generalized demyelination and not by a lipidosis. This conclusion agrees precisely with the observed histological pattern.

709

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P. J. HEALD, H. G. BADMAN, B. F. FRTJNIVAL AND P. A. L. WIGHT

Schnieder, W. C , 1945. Phosphorus compounds in animal tissues. I. Extraction and estimation of desoxypentose nucleic acid. J. Biol. Chem. 161: 293-303. Stern, I., and E. Shapiro., 1953. A rapid and simple method for the determination of esterified fatty

acids and for total fatty acids in blood. J. Clin. Pathol. 6:158-160. Wight, P. A. L., 1962. Variations in peripheral nerve histopathology in fowl paralysis. J. Comp. Path. 72:40-48.

PAUL GRIMINGER AND HANS LTTTZ Nutrition Laboratory, Department of Animal Sciences, Rutgers, The Stale University, New Brunswick, New Jersey Received for publication November 22, 1963)

T

HE calcium needs of the laying hen can be met by furnishing the required calcium in the feed, by free-choice offering of a calcium supplement, or by a combination of both. The calcium requirement of a laying hen is largely determined by the fraction of dietary calcium retained and by the amount of egg shell secreted (Hurwitz and Griminger, 1962). Since the latter depends upon rate of egg production, size of eggs, and thickness of shell, considerable variability is to be expected among hens. When the calcium requirement is expressed as a percent of the ration, feed intake, and therefore caloric density of the ration, will constitute an additional factor (Griminger, 1961). In view of these variables adjustment of the calcium intake by a freechoice offering of calcium supplements appears to offer a logical solution to the problem of supplying the right amount of calcium for each hen, provided, of course, that laying hens are able to adjust their

intake of calcium supplement to their needs. It was the purpose of the observations recorded here to study the voluntary intake of calcium supplements by the laying hen. EXPERIMENTAL METHODS AND DESIGN

The intake of two sources of supplemental calcium (crushed oyster shell and calcite grit) was observed for individually caged hens and for small groups of birds kept on litter. The hens in the individual cages received a laying diet containing either an adequate calcium level (3%) in the form of mash or pellets, or diets with a reduced calcium content (1 and 2%). Each diet was fed for 3 weeks with calcite grit as the supplement, and for another 3 weeks with oyster shell, with both the diet and the supplement available to the hens at all times. Ten hens were used throughout the entire experiment (twelve 3-week periods) and feed intake, calcium supplement intake, and 1 Paper of the Journal Series, New Jersey egg production were recorded daily. The Agricultural Experiment Station, New Brunswick, rations are shown in Table 1, and the New Jersey. Supported in part by a grant-in-aid from physical arrangements in Figure 1. The the Central Jersey Farmers Cooperative, Highsexperimental animals were S.C.W. Legtown, New Jersey.

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Observations on the Voluntary Intake of Calcium Supplements by the Laying Hen 1