Magnesium Requirement of the Chick*

CARBON DISULFIDE AND ROUNDWORMS

weight and higher toxicity was manifested by large weight losses. A higher efficacy was found among fasted birds than among non-fasted ones, thus emphasizing the importance of fasting chickens prior to administering CS2. The drug was not effective against any of the larval stages of A. galli at the dosage used in this study. ACKNOWLEDGMENTS

REFERENCES Bartlett, M. S., 1947. The use of transformations. Biometrics, 3: 39-51. Fryer, H. C , 1954. Elements of Statistics. John Wiley and Sons, Inc., New York, 262 pp. Gardiner, J. L., and E. E. Wehr, 1950. Selecting ex-

perimental groups of chicks by weight. Proc. Helm. Soc. Wash. 17: 25-26. Hansen, M. F., L. J. Olson and J. E. Ackert, 1954. Improved techniques for culturing and administering ascarid eggs to experimental chicks. Exptl. Parasit. 3: 464-473. Knapp, S. E., and M. F. Hansen, 1954. Observations on the anthelmintic action of carbon disulfide on the fowl ascarid, Ascaridia galli. J. Parasit. 40: 17-18. Knapp, S. E., M. F. Hansen, H. C. Moser and R. H. McFarland, 1960. Uptake of sulfur-35-labeled carbon disulfide by Ascaridia galli (Roundworm) and its chicken host. Exptl. Parasit. 9: 56-62. Kruskal, W. H., and W. A. Wallis, 1952. Use of ranks in one-criterion variance analysis. J. Amer. Stat. Assoc. 47: 583-621. Roberts, F . H. S., 1937. Studies on the biology and control of the large roundworm of fowls, Ascaridia galli (Schrank, 1788) Freeborn, 1923. Bui. No. 2, Animal Health Station, Yeerongpilly, Australia. 106 pp. Shumard, R. F., 1957. The toxicity to chickens and the anthelmintic effect of two forms of a piperazine-carbon disulfide complex on Ascaridia galli and Heterakis gallinae. Poultry Sci. 36: 613-618. Tugwell, R. L., and J. E. Ackert, 1952. On the tissue phase of the life cycle of the fowl nematode Ascaridia galli (Schrank). J. Parasit. 38: 277-288.

Magnesium Requirement of the Chick* E. E. GARDINER, J. C. ROGLER AND H. E. PARKER Purdue University, Lafayette, Ind. (Received for publication December 1, 1959)

ALMQUIST (1942), working with a -**• washed casein, cerelose-type diet containing 40 p.p.m. of magnesium, reported that the addition of 350 p.p.m. of supplemental magnesium produced as good growth rates as any higher level. The author concluded that 350 p.p.m. was a barely adequate supplementary level and stated that the requirement during the first weeks of life was approximately 400 p.p.m. The deficiency symptoms that * Journal paper no. 1539 of the Purdue University Agricultural Experiment Station.

were observed included convulsions, gasping and a coma which sometimes was fatal but usually ceased in a few minutes. Bird (1946) reported a cerebellum disorder in chicks being fed a washed casein, cerelose diet. Increasing the basal dietary level of MgS04 to supply 500 p.p.m. (calculated) of elemental magnesium corrected the condition. His description of the deficiency syndrome was very similar to that of Almquist's. Scott et al. (1956) supplemented a cerelose, Drackett-protein diet with 0,50,100,

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The authors wish to thank Dr. Stanley Wearden, Department of Statistics, Kansas State University for his assistance with the statistical analyses, and the United States Atomic Energy Commission for funds supporting the study under Contract At(ll-l)-308.

1111

1112

E. E. GARDINER, J. C. ROGLER AND H. E. PARKER

TABLE 1.—Composition of basal rations Trial number Item Cerelose Soy protein 220* Mineral mix 1 Vitamin premix 2 DL-methionine Glycine Corn oil (MgCa)4Mg(OH)rnH20 (A.R.)« 1

Basal magnesium (p.p.m.) Phosphorus (%) Calcium (%) Crude protein (%)

2

59.09 to 60.63 28.00 5.06 0.50 0.50 0.30 5.00 Variable

60.01 to 60.28 28.00 5.41 0.50 0.50 0.30 5.00 Variable

1,500

—. 850—

1,500

520 500 140 50 5 2 0.9 0.1 0.003 0.00065 122 0.84 1.20 25.0

—. 1,250—

2,100

850 520 500 140 50 5 2 0.9 0.1 0.003 0.00065 77 0.83 1.20 25.0

2 Vitamin premix used in both trials, supplied in amounts per 100 gms.: riboflavin, 882 meg.; Ca pantothenate, 1,764 meg.; niacin, 3,968 meg.; inositol, 24 mg.; menadione Na bisulfite, 0.074 mgsj vitamin E, 551 I.U.: pyridoxine HCI, 1.34 mgs.j thiamine HCI, 0.79 mgs.; folic acid, 0.37 mgs.; biotin, 0.03 mgs.; vitamin B12, 0.099 mgs.; penicillin, 0.66 mgs.; vitamin A, 1,000 I.U.; vitamin Ds, 150 I.C.U.; and choline^chloride, 200 mgs. 3 Gravimetric analysis showed that this compound contained 25.53% magnesium. * Purchased from Archer-Daniels-Midland Company, Minneapolis, Minnesota.

200, 400, and 600 p.p.m. magnesium. Maximal growth was obtained with 100 p.p.m. of supplemental magnesium. Higher levels did not depress growth. EXPERIMENTAL PROCEDURE

VantressX Hubbard male chicks purchased from a local hatchery were confined in wire floor battery brooders. Four replications of 10 birds each were used in both trials. Feed and water were supplied ad libitum. The water used in both trials had passed through an ion exchange water softener, which lowered the magnesium content from approximately 28 p.p.m. to 0.26 p.p.m. Feed consumption records were kept on a chick-day basis. The chicks were weighed individually at weekly intervals. The statistical analyses of chick weights,

Blood was obtained by heart puncture at the end of the four week periods with the use of heparin as an anticoagulant. The plasma obtained by centrifugation was frozen in plastic centrifuge tubes until magnesium and cholesterol assays could be conducted. The cerelose, isolated soy-protein basal rations are presented in Table 1. The important difference between the basal rations is that basal number 1 contained precipitated CaCC>3 and Ca3(PC>4)2; whereas basal number 2 contained analytical reagent grade CaC0 3 and CaHP04. This change lowered the basal magnesium level from 122 to 77 p.p.m.

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Mineral mix (mg.%): CaCOs (ppt.) CaCOs (A.R.) Caj(PO<)! (ppt.) CaHPOi (A.R.) KsHPOi (A.R.) N a 2 H P 0 4 (A.R.) N a C l (A.R.) Fe(C6Hs07)-6H20 M n S 0 4 (A.R.) ZnSOi-HiO CuS0 4 -5H20 H1BO3 CoCl-6H 2 0 Na 2 -Mo04-2H20 KI

1

plasma cholesterol and magnesium content of the plasma were made by the method of Newman (1939) as modified by Keuls (1952). Plasma cholesterol was determined by the Sperry and Webb method (1950). Magnesium content of the plasma was determined by the method of Young and Gill (1951). A modification of the Young and Gill method was used to determine the magnesium content of the basal diets. This modification involved removal of the calcium by double precipitation with ammonium oxalate, followed by adsorption of the magnesium from an aliquot of the "calcium-free" solution on a cation exchange column containing Amberlite CG-120 type 2 resin on the bottom of the column and Amberlite 1RC-50 resin on the top. The purpose of the column was to get solutions relatively free of phosphorus, which passes through the column, since phosphorus interferes drastically with the results. The magnesium was then eluted from the column with IN HCI and the procedure of Young and Gill (1951) followed for the color reaction. The magnesium content of the supplemental (MgC0 3 )4Mg(OH) 2 .nH 2 0 was determined by a slight modification of the A.O.A.C. (1955) gravimetric method.

1113

MAGNESIUM REQUIREMENT TABLE 2.—Results of Trial 1 Lot number Item 2

1

7

8

9

122

—

122 36 158

122 136 258

122 236 358

122 336 458

122 436 558

122 536 658

122 936 1,058

122 1,936 2,058

275 42.5

358 22.5

493 12.5

534 10.0

488 10.0

500 5.0

499 10.0

Basal Mg (p.p.m.) Added Mg (p.p.m.) Total Mg (p.p.m.)

122

4 wk. wts. (gms.) 1 Mortality (%) Feed-gain ratios (0-4 wks.) Plasma M g (mg.%)* Plasma cholesterol (mg.%)» 1

1.74 0.47 115

4

3

1.72 0.53 163

5

6

1.55 0.72

1.57 0.86

1.63 1.14

1.60 1.30

—

—

—'

—

540 7.5

1.60

— —

1.57 —

10

509 2.5 1.66

—

•

—

163

122 3,936 4,058 502 7.5 1.67 2.40 161

Significant differences by Newman-Keuls test—4 week wts. Lot number

8 265** 182**

4 259** 176**

10

9 234" 151**

6

7

225** 142**

227** 144**

224** 141**

3 218** 135**

5 213** 130**

2 83**

2 Significant differences by Newman-Eeuls test—plasma magnesium mg.% (individual determinations of five birds from each group where averages are given).

Lot number

10

1 2 3 4 5 6

6

1.93** 1.87** 1.68** 1.54** 1.26** 1.10**

0.83* 0.78*

a Significant differences by Newman-Keuls test—plasma cholesterol mg.% (ndividual determinations of eight birds from each mp where averages are given). group Lot number

2 48*

6

10 46*

* Significant at the 5 percent level. ** Significant at the 1 percent level.

RESULTS AND DISCUSSION

The results of Trial 1 are presented in Table 2. Supplemental magnesium carbonate was added to give rations containing 122 to 4,058 p.p.m. of magnesium. The growth rates of the birds receiving the basal ration (122 p.p.m.) and the next higher level of magnesium (158 p.p.m.) were significantly lower than all other lots. There were no significant differences in four week weights between any of the lots receiving from 258 to 4,058 p.p.m. of magnesium. There were no indications of toxicity on the higher levels of magnesium. Deficiency symptoms were observed five days after the birds were placed on treatment. These symptoms were most noticeable on the basal ration. The deficiency symptoms included slow growth, nervous tremors, gasping and convulsions.

When the birds were excited some of them would go into a coma, which sometimes was fatal, but more often they recovered. Deaths began to occur on the 6th day of treatment and were fairly evenly distributed throughout the remainder of the experimental period. The results of the plasma magnesium determinations showed a positive linear relationship between the amount of dietary magnesium and the amount of magnesium in the plasma. Lot 10 (4,058 p.p.m.) was significantly higher than all the other groups tested while Lot 6 (558 p.p.m.) was significantly higher than lots 1 (122 p.p.m.) and 2 (158 p.p.m.). A significant reduction in plasma cholesterol was observed on the lowest level of magnesium (122 p.p.m.) as compared to the other levels studied. A possible

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1 2

1114

E. E. GARDINER, J. C. ROGLER AND H. E. PARKER TABLE 3

—Results of Trial 2 Lot number

Item Basal Mg (p.p.m.) Added Mg (p.p.m.) Total Mg (p.p.m.) 1

4 wk. wts. (gms.) Mortality (%) Feed-gain ratios (0-4 wks.) Plasma Mg (mg.%) 2 Plasma cholesterol (mg.%) 3 1

2

77

77 25 102

77 75 152

77 125 202

190 87.5

322 50.0 1.51 0.85 156

427 22.5 1.56

77 99 97.5

6

7

77 175 252

77 225 302

77 250 327

77 425 502

467 0.0 1.64 1.04 189

494 2.5 1.59

481 10.0 1.63

472 2.5 1.65 2.03 234

5

4

1

3

8

Lot number

3

4

6 7 8 5 4

172** 158** 149** 144** 104**

68** 54* 45* 40*

2 Significant differences by Newman-Keuls test—plasma magnesium (mg.%). (Individual determinations of five birds from each group where averages are given.)

Lot number 1.18** 0.99**

0.19*

3 Significant differences by Newman-Keuls test—plasma cholesterol (mg.%). (Individual determinations of 10 birds from each group where averages are given.)

Lot number

78* ' Significant at the 5 percent level. ! * Significant at the 1 percent level.

explanation for these results is afforded by Man and Gildea (1936) who reported a hypocholesterolemia in emaciated human patients suffering from prolonged malnutrition. Since the birds receiving the basal diet were severely deficient and feed consumption was very low, perhaps the birds could be considered in a state of malnutrition from a standpoint of energyproducing materials as well as magnesium, which might explain the reduced plasma cholesterol. It would also appear that a magnesium deficiency would reduce the utilization of energy-producing materials

because of the importance of this element in a wide variety of enzyme systems. The results of Trial 2 are presented in Table 3. The two lowest levels of magnesium were not included in the statistical analysis of the weights because of the high mortality. The birds receiving the diets containing 152 and 202 p.p.m. grew at a significantly slower rate than those receiving higher levels. There were no significant differences in four week weights of any of the groups receiving 252 p.p.m. of magnesium or more. These results are consistent with Trial 1 where 250 p.p.m.

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Significant differences by Newman-Keuls test—4 week wts. Not including treatment 1 and 2.

1115

MAGNESIUM REQUIREMENT

SUMMARY

Two experiments were conducted to determine the magnesium requirements of

chicks. Weight data and mortality indicated that the chicks' dietary magnesium requirement is approximately 250 p.p.m. No adverse effects were noted with dietary magnesium levels as high as 4,058 p.p.m. Deficiency symptoms included poor growth, nervous tremors, gasping and convulsions. Deficiency symptoms and mortality occurred Very quickly when day-old chicks were fed diets extremely deficient in magnesium. A direct relationship was observed between the level of dietary magnesium and the level of plasma magnesium. The plasma cholesterol levels of birds on magnesium deficient diets were significantly lower than the plasma cholesterol levels on diets adequate in magnesium. REFERENCES Almquist, H. J., 1942. Magnesium requirement of the chick. Proc. Soc. Expt. Biol. Med. 49:544-545. Association of Official Agricultural Chemists, 1955. Official Methods of Analysis, 8th Ed. Bird, F. H., 1946. Magnesium supplementation in relation to a previously reported cerebellar disorder in chicks. Poultry Sci. 25: 396-397. Keuls, M., 1952. The use of the studentized range in connection with an analysis of variance. Euphytica, 1: 112-122. Man, E. B., and E. F. Gildea, 1936. Serum lipoids in malnutrition. J. Clin. Invest. 15: 203-214. Newman, D., 1939. The distribution of the range in samples from a normal population, expressed in terms of an independent estimate of standard deviation. Biometrika, 31: 20-30. Scott, H. M., M. W. Moeller and S. W. Hinners, 1956. Studies on purified diets. 1. Supplemental magnesium levels. Poultry Sci. 35: 1169. Sperry, W. M., and M. Webb, 1950. A revision of the Schoenheimer-Sperry method for cholesterol determination. J. Biol. Chem. 187: 97-106. Young, H. Y., and R. F. Gill, 1951. Determination of magnesium in plant tissues with thiazole yellow. Analytical Chem. 23: 751-758.

OCTOBER 4-6. NEPPCO EXPOSITION AND CONVENTION, ONONDAGA WAR MEMORIAL AUDITORIUM, SYRACUSE, N. Y.

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also appeared to adequately supply the magnesium needs of the chicks. Deficiency symptoms were observed after the birds had been on treatment three days and were the same as those described for Trial 1. Approximately 75 percent of the mortality occurred between the third and tenth day of treatment. Mortality was extremely high on the two lowest levels of magnesium. The differences in mortality between this trial and that in Trial 1 can probably be attributed to the difference in the magnesium content of the basal diets. As in Trial 1, a direct relationship was observed between the amount of dietary magnesium and the plasma magnesium. The means given are all significantly different from each other. The observation noted in the first experiment on the reduction of plasma cholesterol on a low magnesium diet was confirmed in this trial. Electrophoretic patterns of plasma proteins were conducted and albumin/globulin ratios calculated using individual samples from eight birds each from Lots 3 (152 p.p.m.) and 8 (502 p.p.m.). Although the difference in the A/G was not significant, some individual patterns from Lot 3 showed decreased A/G ratios. (Method used is that outlined in "Model R Paper Electrophoresis System Instruction Manual RIM-5"). It is possible that a secondary infection could account for a rise in globulins, while a state of semi-starvation might lower the albumin (Man and Gildea, 1936).