Cerebrospinal Fluid Composition Studies in Calves on Varying Carotene Intakes1

1396

JOURNAL

OF D A I R Y S C I E N C E

6.0 2.0 i

1.0 g

o

(]o r~

J

A

iN

0.5

J:o /

2.0

//EF~LUENY.V VOLUME,VLITERS

FIG. 1. Effluent diagram of milk protein on DEAE-SF (1.3 × 33 era.) ; effluent collected in 5-ml. fractions. Buffers: I, 0.005 M Na phosphate, pit 7.0; II, gradient to 0.02 M Na phosphate, pH 6.0; III, gradient to 0.05 M NaH_~PO,; IV, gradient to 0.1 M NaC1---0.05 M Natt2PO4; V, gradient to 0.5 M N a C ~ 0 . 1 M NaH2PO,. REFERENCES (1) PETERSON, E. A., AND SOBER, I-I. A. Chromatography of Proteins. I. Cellulose IonExchange Adsorbents. J. A m . Chem. Sot., 78: 751. 1956. (2) SOBER,H. A., GUTTER,F. J-., WYCKOI~F,M. M., AND PETERSON, E. A. Chromatography of

Proteins. II. Fractionation of Serum Protein on Anion-Exchange Cellulose. J. A m . Chem. Soc., 78: 756. 1956. (3) SOBER, I-I. A., AND PETERSON, E. A. Chromatography of Proteins on Cellulose IonExchangers. J. A m . Chem. Soc., 76: 1711. 1954.

CEREBROSPINAL FLUID COMPOSITION STUDIES IN CALVES ON V A R Y I N G C A R O T E N E I N T A K E S 1 An increase in cerebrospinal fluid ( C S F ) pressure appears to be the first measurable change in vitamin A deficiency in the calf (8, 10). On this basis, it appeared plausible that concentration changes of certain coustituents in CSF might occur prior to this increased pressure. Twelve Guernsey and 12 Holstein 64-day old male calves were placed on a vitamin A depletion ration until their blood plasma vitamin A level had decreased to ---~12 ~/%. Thereafter, with restrictions as to breed, each animal received one of four carotene intakes, 30, 60, 120, or 240 y/lb live weight/day, from artificially dehydrated alfalfa. A more complete description of the depletion ration and the experimental 1 The author is indebted to H. D. Eaton for helpful suggestions in the preparation of this manuscript.

procedure used in this study has been reported by Eaton et at. (5). The 30-~/ intake level was chosen on the basis that it should result in an increased CSF pressure in the Guernsey calves, but be just adequate to prevent this in the Holsteins (9). The remaining levels of carotene were considered more than adequate to prevent all increased CSF pressure in both breeds. A f t er 12 wk. on their respective rations, C S F pressures were obtained by puncture of the spinal canal through the dorsal opening of the atlanto-occipital articulation and by measuring the height to which the CS F rose, in a rigid polyethylene tube attached to the hub of the needle (7, 11). A f t er measuring CSF pressures, approximately 25 ml. o£ fluid were withdrawn by means of a hypodermic syringe. Sodium, potassium, and calcium concentrations of CS F

TE CIcli~ICAL NOTES were determined flame photometrically (1, 4), and inorganic phosphorus was measured eolorimetrically (6). Analysis for free amino acids in C S F was carried out as follows: A p p r o x i mately 35 ml. of C S F were evaporated to dryness on the steam bath, to precipitate the proteins, the residue taken up in hot w a t e r and the solution filtered. The filtrate was then desalted electrolytically, evaporated to dryness again, and the residue taken up in a small volume of J_0% isopropyl alcohol. This solution was used f o r spotting the chromatograms. Two dimensional p a p e r chromatography was used to separate and identify the free amino acids; 80% p y r i d i n e and ethanol : butanol : formic acid : water (60 : 20 : 5:3-5) were the p r i m a r y solvents employed. Statistical procedures used f o r the analysis of sodimn, potassium, calcium, and phosphorus data were taken f r o m Cochran and Cox (3). Inspection of :the nfineral analyses results, substantiated by statistical analysis, revealed that there were inappreciable differences between carotene intake or breed in the sodium, potassium, inorganic phosphor-as, and caleimn concentrations of C S F (Table 1). Only two calves, both Guernseys on the 3() ~ carotene intake group, were f o u n d to have an increased terminal pressure (av. 125 ram. H~O). The present results thus would indicate that no changes in the concentration of these constituents occur p r i o r to an increased C S F pressure; however, the possibility that this balance is altered in a more severe or prolonged deficiency still exists. The only previous r e p o r t on bovine C S F found in the literature gave a mean calcimn value of 5.5 mg/100 ml. (2), which is in reasonable agreement with the present results. The free amino acids and their p e r cent occurrence, based on the analysis of C S F from eight Guernsey and two Holstein calves, are

1397

given in Table 2. Differences in the p e r ~ n t occurrence of the free amino acids a p p e a r e d not to be related to carotene intake or elevated C S F pressure; however, because of the limited numbel,s (calves) and unequal distribution aznong carotene intakes and breeds, no definite conclusions could be made f r o m these data. The amino acids repo~¢ed in Table 2 represent those conTABLE 2 Free amino acids present in cerebrospinal fluid of eight Guernsey and two Holstein calves Amino Acid

Occurrence

(%) Alanine Arginine-Lysine Aspartie Acid Cysteine-Cystine Glutamic Acid Glutamine Glycine Leueines Serine Threonine Valine

100 50 40 80 100 100 100 ~0 lOO

100 30

stituents which were identified; however, several unknown ninhydrin-positive spots were obselwed in some of the C S F samples. On the basis of visual color density and area of spots, alanine, serine, and glutamine were the amino acids consistently present in the highest concentration. B. A. DE~Om~Y ~ Storrs (Connecticut) Agricultural E x p e r i m e n t Station -"Present address: Department of Animal Science, Ohio Agricultural Experiment Station, Woos.ter, Ohio.

TABLE 1 Concentrations of sodium, potassium, inorganic phosphorus, and calcium in eerebrospinal fluid of Guernsey and Holstein calves Carotene intake S 30 7

60 3~

120 3'

240 7

Mean

Standard deviation per calf

(mg/lO0 ~l.) Sodium Gb 335 H~ 3'39c Potassium G 12.0 H 12.4 e Inorganic phosphorus G 1.66 H 1.83 c Calcium G 4.9 H 5.1 ¢

333 338 12.5 12.5

337 343 12.3 12.8

334 338 12.7 12.4

337 12.4

4 4 0.3 0.3

1.79 1.92

1.66 1.66

1.75 1.65

1.74

0.16 0.16

5.1 5.2

5.1 5.0

4.9 5.1

5.1

0.4 0.4

a 3,/lb live weight/day. b G, Guernseys; H, Holsteins. The value under each intake represents the mean concentration obtained from the eerebrospina] fluid of three calves. Cerebrospinal fluid sample not obtained from one calf, missing value calculated (3).

]398

JOURNAL OF DAIRY SCIENCE

REFERENCES (1) B.~IRD ASS~CZ~_TF~S,I~C. Manual for Flame Photometer. (2) C.~A~H~CHA~L~J., ANt) JO~E~S, E. R. Cerebrospinal Fluid in the Bovine~Its Composition and Properties in Health and Disease with l~eference to Turning Sickness. J. Comp. Pathol. Therap., 52: 222. 1939. (3) COCHa~, W. G., A~]) Cox, G. M. Experimenta~ Designs. John Wiley and Sons, Inc., New York. 1957. (4) D~Ho~I~Y, B. A. Determination of Bovine Serum Calcium with a Simple Flame Photometer. J. Dairy Sci., 42: 872. 1959. (5) E ~ , H. D., M : ~ s , G. S., d~., DIC~S, MAI~TI~_ W., D]~;HOaITY, B. A., GRII~O, A. P., JI~., TE~ICHMAN, R., HHHF~LMBOLDT~ C. F., J u s t i c e s , E. L., A~D Gossrm~, D. G. Conversion of Carotene from Alfalfa to ~ritamin A by Guernsey ~nd Holstein Calves. J. Dairy Sci., 42: 642. 1959. (6) FISKE,, C. H., A~) S c ~ t { o w , Y. The Colorlmetric Determination of Phosphorus. J. Biol. Chem., 66: 375. 1925.

(7) MOOR~,, L. A., B ~ Y , M. It., A~I) SYKeS, J. F. Carotene l~equirements for the Maintenance of a Normal Spinal Fluid Pressure in Dairy Calves. J. Nutrition, 26: 649. 1943. (8) Moo~, L. A., AND SYKes, J. F. Cerebrospinal Fluid Pressure and Vitamin A De~ fieiency. Am. J. Physiol., 130: 684. 1949. (9) Moomc, L. A., SYKes, J. F., JAcoBso~, W. C., _a~l) WISE~AN, H. G. Carotene Requiremerits for Guernsey and Jersey Calves as Deternfined by Spinal Fluid Pressure. J. Dairy Sci., 31 : 53'3. 1948. (1O) ROUSSEAU,J. E., JR., EATON, It. D., HE~]~ BOLDT, ~. :F., JUNGItE~I~, E~ L., ROt~I~ISK, S. A., B s ~ , G., A~D Moo~:~, L. A. Rela~ tire Value of Carotene from Alfalfa and Vitamin A from a Dry Carrier Fed at Minimum Levels to Holstein Calves. J. Dairy Sci., 37 : 8~9. 1954. (11) SYKES, J. F., AND MOOl~, L. A. The Normal Cerebrospiaal Fluid Pressure and a Method for Its Determination in Cattle. Am. J. Vet. Research, 3 : 364. 1942.

EFFECT OF DURATION OF EXPERIMENT ON E X P E R I M E N T A L ERRORS IN CALF NUTRITION GROWTH STUDIES 1 Estimates of experimental errors of growth axe of importance for efficient planning and adequate interpretation of calf nutrition studies. F o r example, they are of value in estimating the number of calves required per treatment to obtain a specified statistically significant difference in growth between treatments (2, 4). As early as 1913, Mitchell and Grindley (5) demonstrated that the size of the experimental errors for increases in live weight, expressed as percentages of mean gains, of beef cattle, sheep, poultry, aad swine, decreased with increasing time on experiment. The rates of change of these decreases were most rapid during the early parts of the experfinents and, thereafter~ were at considerably slower rates or had a tendency to approach constant values. Somewhat similar findings have been reported (3) for increases in several linear growth measurements as well as live weight of dairy calves fed a single ration, ~niformity data, and for increases in live weight of dairy heifers (1). The present study was undertaken to determine the effect of duration of experiment on the experimental errors of increases in growth of dairy calves to which various dietary treatmenr~s This study was supported in part with funds provided by Wirthmore Feeds Inc., Waltham, Mass. The authors are grateful to Mrs. Mac Miller, Mrs. Elaine Trantum, R. T. Chatterton, Jr., A. P. Grifo, Jr., H. A. Kemmerer, Jr., and ~. Teichman, for assistance in computations. We are especially indebted to Dr. R. L. Anderson for discussing the statistical aspects of this study.

had been imposed, in contrast to the aforementioned estimates obtained from uniformity data (3). Data used in this study were from seven randomized block and one nonreplicated factorial experiment conducted at the Storrs Station from 1949 through 1956. As they became available, one-day-old calves from Connecticut State institution and private herds were assigned to treatment according to previous raudom allotment. In all but one experiment (a randomized block), breed and sex were included as treatment, so that the existence of possible interaction between these and other treatment effects might be ascertained. The number of calves per experiment ranged from 15 to 36. The number of degrees of freedom for the error mean squares ranged from four to 21. All calves were maintained in individual tiestalls throughout the course of their respective experimental periods. A f t er nursing their dam or receiving colostrum from a frozen colostrmn bank, calves in all but one experiment were fed a limited amount of whole milk, 168 to 182 lb., to the 35th day of age~ a limited amount of staz~er, 2.5 to 4.0 lb. per calf per day, and ad libitmn amounts of hay or ad libitum amounts of various mixtures of starter and hay. In the one experiment, one-half of the calves received milk as above, and one-half received milk at a daily rate o£ 10% of live weight up to. a daily maximum of 10 lb. per calf per day to the 63rd day of age. The nutritional treatments include