Effect of Zinc Deficiency on Bone Mineralization and Plasma Proteins of Turkey Poults*

PERFORMANCE IN SINGLE VS. MULTIPLE CAGES

dividually. Whether the commercial egg producer should cage birds at a particular density may be dependent, in part, upon his ability to control cannibalism. REFERENCES Dunnett, C. W., 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. SO: 1096-1121. Dunnett, C. W., 1964. New tables for multiple comparisons with a control. Biometrics, 20: 482-491. Lowe, R. M., and B. W. Heywang, 1964. Performance of single and multiple caged White Leghorn layers. Poultry Sci. 43 : 801-805. Moore, B. W., R. Plumley and H. M. Hyre, 1965. A cage density study of laying hens. Poultry Sci. 44: 1339, and Mimeo Pub. with same authors and title, 14 pp. Schupe, W. D., and J. H. Quisenberry, 1961. Effect of certain rearing and laying house environments on performance of incross egg production type pullets. Poultry Sci. 40: 1165-1171. Snedecor, G. W., 1956. Statistical Methods, Iowa State College Press, Ames, Iowa.

Effect of Zinc Deficiency on Bone Mineralization and Plasma Proteins of Turkey Poults* Department

PRAN VOHRA AND F. H. KRATZER of Poultry Husbandry, University of California, Davis, California 95616 (Received for publication November 21, 1967)

A

DEFICIENCY of zinc depresses the Harrison, 1964), but not confirmed for growth of chicks (O'Dell et al., chicks (O'Dell et al., 1958; Morrison and 1958), poults (Supplee et al, 1958; Sarett, 1958; Rahman et al, 1961) and Kratzer et al., 1958) and Japanese quail turkey poults (Kratzer et al, 1958). Also, (Fox and Harrison, 1964). Reports of the a small increase in packed cell volume due effect of zinc deficiency on the tibia ash to a deficiency of zinc has been reported content are highly contradictory. A de- for chicks but the latter could be explained crease in tibia ash has been observed for in terms of hemoconcentration (O'Dell et chicks (Young et al., 1958), poults (Sulli- al, 1958; Rahman et al., 1961). No changes van, 1961) and Japanese quail (Fox and in hemoglobin or hematocrit were observed for quail (Fox and Harrison, 1964) with a * Supported by Grant Number AM-5334 from zinc deficiency but a marked difference in the U. S. Department of Health, Education and the plasma proteins was observed by disc Welfare, Public Health Service.

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five cage regimes were not significant although the calculated F ratios imply that the mortality differences among densities are not the same for each house. There were no great differences among the five treatment groups in average random sample body weight taken at 500 days of age or in 400th day average egg weight. When the hen-housed egg production is placed on the basis of providing 64 square inches of cage space per bird, the cage regime in which two birds are caged in an 8 inch cage had a 187 egg advantage over those caged individually (128 square inches); the three bird (12 inch) cage had a 195 egg advantage; the four bird cage (16 inch) had a 193 egg advantage and the six bird cage (24 square inch) had a 187 egg advantage over the single bird cages. The data obtained lead to the conclusion that income per unit of cage space can be maximized by caging layers in multiple cage units in preference to caging layers in-

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P . VOHRA AND F . H . KRATZER TABLE 1.—Zinc-dejlcieni basal diet

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The birds were housed in an electrically heated battery containing stainless steel gm./kg. cages, feeders and waterers. Duplicate Cornstarch 486.7 groups were studied on each treatment. Isolated soybean protein1 330.0 2 The composition of the zinc-deficient basal Cellulose, powdered 50.0 CaHP0 4 -2H 2 0 30.0 diet is given in Table 1. It contained about CaC0 3 25.0 17 mg./kg. zinc and was supplemented Mineral mixture 3 23.1 Vitamin mix4 10.0 with either 0.684 mmole ethylenediaSoybean oil 35.0 minetetraacetic acid (EDTA) or 15 mg. DL-Methionine 4.5 Choline Chloride, 44%' 5.7 zinc per kg. diet. 1 The poults were fed the same amount of Assay Protein C-l (Skidmore Enterprises, Cincinnati, Ohio) contained 0.95% P. EDTA or zinc-supplemented diets as was 2 Solka Floe, Brown Company, New Hampshire. 3 consumed over the past 24 hours by the Supplied the following minerals in gm-NaCl (uniodized), 9.9: MnSCvH 2 0, 0.297; FeSCv7H 2 0, zinc-deficient groups. The poults were 0.644; CuS04-5H 2 0, 0.079; cobalt acetate tetrahydrate, 0.02; Kl, 0.009; A12(S04)3-18H20, 0.25; weighed as a group twice each week and inMgS0 4 -7H 2 0, 3.97; KC1, 2.97; K 2 HP0 4 , 4.95; dividually at the end of the experiment on Na 2 Mo0 4 -2H 2 0, 0.009. 4 the 21st day. Supplied the following: (in mg.) riboflavin, 10; thiamine HCl, 10; pyridoxine HCl, 10; Ca pantoBlood samples were taken by heart thenate, 30; niacin, 120; folic acid, 5; menadione, 10; puncture for determination of packed cell biotin, 0.4; (in g.) BHT, 1; inositol, 1; vitamin A, 5,000 I.U.; vitamin D 3 , 4,500 I.C.U.; vitamin E, 88 volume and for study of protein compoI.U; vitamin Bi 2 , 10 ^g. 6 nents by disc electrophoresis on polyacryla44% in wheat middling carrier. mide gels (Ornstein, 1964) as well as by electrophoresis on polyacrylamide gels after cellulose acetate electrophoresis. starvation of zinc-deficient quail for 24 hours The ash, Ca and P determinations of the (Fox and Harrison, 1965). Without starva- left tibia of the dead poults were made by tion, the plasma protein patterns were sim- the A.O.A.C. method (1960). Zinc and ilar for deficient and normal quail (Fox and manganese contents of the ash, dissolved in Harrison, 1965) and chicks (Rahman et al., dilute HCl, were determined by the atomic 1961). absorption method (Perkin-Elmer, 1966). In the studies cited, the birds were fed The data were subjected to statistical ad libitum. The feed consumption of the analysis by Duncan's method (Cochran zinc deficient group was always less than and Cox, 1960). the zinc-supplemented groups. The present RESULTS AND DISCUSSION study was undertaken to investigate the plasma proteins and the mineral composiThe pair-fed, zinc-supplemented and tion of the tibia of turkey poults when the EDTA-supplemented turkey poults had zinc-deficient and the marginally supple- normal feathers and increased in weight mented (15 mg. Zn/kg.) diets were fed at slightly more than the zinc-deficient conan equal level per day. trols which had abnormal feathers (Table 2). The poults which received the suppleEXPERIMENTAL mented diets ad libitum were the heaviest. Poults of a small sized strain of Bronze These differences were significant at a 5% turkeys were fed a practical turkey diet for level only. At a level of 1%, the differences 3 days and a zinc-deficient purified diet for in gain between the pair fed groups and the 2 days before distribution into groups of 10 ad libitum deficient group were statistically birds each of approximately equal weight. non-significant.

1137

ZN, BONE AND PLASMA PROTEINS

TABLE 2.—Effect of pair feeding of zinc-deficient, and zinc or EDTA supplemented diets on gain in body weight, ash, Ca, P, Zn and Mn contents of tibia of turkey poults and their packed cell volume (P.C.V.)

Treatment

Feeding

Gain, gm. 1

Zn-deficient EDTA-supp. Zn-supp. EDTA-supp. Zn-supp. 1 2

Ab lib. Pair fed Pair fed Ad lib. Ad lib.

52 59 60 180 211

Tibia ash,

% I

a ab b c d

40.2 a 43.1b 40.4 a 38.6 a 38.9 a

Bone Ca/P I

2.1 a 2.2 a 2.2 a 2.1a 2.1a

Mg./lOO gm. bone Zn 2

6.2a 9.8b 12.8 c 5.0 a 5.9a

Mn l

1.3 1.5 1.4 1.2 0.9

% P.C.V. j

a a a a a

23.3 a 27.5a 23.4 a

As expected, the restriction in growth of the zinc-deficient or pair-fed supplemented poults over the experimental period of 21 days was largely related to the decreased feed consumption. Addition of EDTA at a level of 0.684 mmole/kg. diet improved the availability of zinc already present in the diet, improved the weight gains and the feed consumption on an ad libitum basis. The growth could also be improved by increasing the level of dietary zinc and feeding ad libitum. The diets were still marginally deficient in zinc. The pair feeding data indicated that zinc might improve the utilization of the feed because the gains tended to be better for supplemented poults in comparison to zinc-deficient groups. The poults with very poor gain in body weight appeared to have higher tibia ash than the heavier poults which had free access to EDTA or Zn-supplemented feeds. There was, however, no difference in the calcium/phosphorus ratio of the tibia ash in any of the treatments. Instead of a decrease in tibia ash with zinc deficiency as reported for various avian species (Fox and Harrison, 1964; Young et al., 19S8; Sullivan, 1961), there was a decreased tibia ash when the poults were fed ad libitum diets supplemented with either EDTA or a marginal level of zinc in contrast to pair-fed diet supplemented with EDTA. However, this observation was statistically significant

at a level of 5% only and is also different from the previous observation when the bone ash was of the same order (Kratzer et al., 1958). The pair-fed zinc-supplemented group had the highest zinc in the tibia (12.8 mg./lOO gm. bone), the EDTA-supplemented pair-fed group had an intermediary level (9.8 mg./lOO gm. bone) but more than the ad libitum fed poults. Zinc is believed to be associated with the organic matrix of the bone and not its mineralization (Vohra and Kratzer, 1966). More zinc was available for association with the organic matrix from the EDTA- or Zn-supplemented diets when pair-fed with a zincdeficient diet to the poults. This is reflected in the increased zinc levels in the bone. However, when these diets were fed ad libitum, the total organic matrix synthesis was very high and the diets lacked zinc to maintain these high levels in the tibia bones of turkeys. Manganese, which is known to be involved in bone formation (Asling and Hurley, 1963) was not significantly different in the tibia of birds on any of the treatments. Our unpublished data indicate that manganese is associated with the organic matrix of the bone and not its mineralization in turkey poults. As there was no manganese deficiency in the diets, no differences in bone manganese were noticed in this experiment.

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Statistical significance at 5% level in a column is denoted by different letters. Statistical significance at 1% in a column indented by different letters.

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P . VOHRA AND F . H .

T A B L E 3.—Effect of starvation on packed cell volume of zinc-deficient poults

Diet

Starvation time, hours 0

Zinc-deficient 31.1+l.Oa' +15mg.Zn/kg. 36.0±0.5b»

24

48

32.1(5/6)2 32.1(9/10)'

33.1(2/6)! 34.4(9/10)2

1 Statistical; significance at 0.1% is indicated by Fisher's t test2 (Cochran and Cox, 1960) by different letters in a column. The number of poults surviving/poults started is indicated within parenthesis.

the heavy Broad Breasted Bronze strain and no restriction was put on feed consumption. The deficient diet was supplemented with 15 mg./kg. zinc. In this case, there was a marked decrease in the hematocrit value of poults fed the zinc-deficient diet (Table 3). Severe mortality was also observed on starvation for this group but not the zinc-supplemented groups. By withholding of feed, the difference in hematocrit values disappeared. The hematocrit value may be dependent upon the restrictions on feed consumption. Since many metals are associated with blood proteins, an attempt was made to measure the overall zinc carrying capacity of blood of deficient and supplemented poults. No significant differences were observed in the stability constants of blood plasma of zinc-deficient and zinc-supplemented groups as determined by the ion exchange method (Vohra et al, 1966). The stability constant (K t ) was calculated from the relationship: Kf = ( (K»d/Kd) - 1)/A n , where K»d and Kd are distribution coefficients for zinc between the exchanger and the solution in the absence and presence of plasma proteins, and An denotes the concentration of plasma protein in terms of moles of nitrogen per liter. The nitrogen content of the plasma in each case was about 5.3 mg./ ml. The values calculated for Kf were 3.34, 3.49 and 3.46 for plasma of poults on a zinc-deficient, EDTA-supplemented and zinc-supplemented (15 mg. zinc/kg.) diets. In an attempt to find any component of plasma which may combine preferentially with zinc, poults were fed Zn65 and the blood was drawn by heart puncture and the plasma was subjected to electrophoresis at a pH of 8.6 on cellulose acetate strips. About i of the strip was cut length-wise and stained with either Ponceau S or Nigrosin when at least six bands could be detected, some of which were so close that these could not be cut into separate seg-

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No significant differences in hematocrit values (packed cell volume) of blood of poults on any of the pair-fed diets were noticed and this is in agreement with a similar finding on quail (Fox and Harrison, 1964). When the blood plasma of five individual poults from various treatments was subjected to electrophoresis on cellulose acetate strips using various buffering systems based on tris-barbituric acid (pH 8.6), no differences in protein patterns were observed for any of the treatments or even after starving the poults either for 24 or 48 hours. There was heavy mortality on starvation of zinc-deficient poults even for 24 hours. Again, no differences in blood plasma proteins of poults starved for 24 hours or 48 hours were noticed even on disc electrophoresis on polyacrylamide gels. It appears that turkey poults behave differently from quail in which a difference in plasma protein patterns was observed with a deficiency of zinc after starvation for about 24 hours (Fox and Harrison, 1965). In contrast to the diets used by Fox and Harrison (1965), the diets used in the present study were still marginally deficient in zinc. No differences in plasma proteins of poults fed zinc-deficient diets and diets supplemented with EDTA- or 15 mg. Zn/kg. ad libitum, but starved for 24 hours, were observed. In another experiment, an attempt was made to study the changes in packed cell volume with starvation. The poults were of

K.RATZER

ZN, BONE AND PLASMA PROTEINS

SUMMARY The growth of turkey poults over 3 weeks was of the same order when fed zinc-deficient (1) purified diets ad libitum, and pair-fed diets supplemented with either 15 mg./kg. zinc (2) or 0.684 mmole EDTA/kg. (3). Growth was markedly improved by feeding diets 2 and 3 ad libitum. Tibia ash was greater on pair-fed diet 3 but was of the same order in poults subjected to the other treatments. The Ca/P ratio and the manganese content of the bones were unaffected. About 12 mg. and 10 mg. zinc/100 gm. bone was incorporated on pair fed diets 2 and 3, and about 6 mg. zinc on the rest of the treatments. No differences in plasma proteins by electrophoresis were observed on any of the treatments even after starvation for 24 hours or longer. The stability constant (K f ) of plasma protein for zinc was of the same order (3.34-3.49) on these treatments. ACKNOWLEDGMENT The technical help of Mr. G. D. Gott-

fredson and Mrs. L. F. Chio is gratefully acknowledged. REFERENCES Asling, C. W., and L. S. Hurley, 1963. The influence of trace elements on the skeleton. Clinical Ortho. 27: 213-264. Association of Official Agricultural Chemists, 1960. Official Methods of Analysis, A.O.A.C, Washington, D.C. Cochran, W. G., and G. M. Cox, 1960. Experimental Design, John Wiley and Sons, New York. Creger, C. R., M. N. A. Ansari, L. B. Colvin and J. R. Couch, 1967. Distribution of calcium and strontium in the blood of the laying hen. Proc Soc. Exp. Biol. Med. 124: 799-800. Fox, M. R. S., and B. N. Harrison, 1964. Use of Japanese quail for the study of zinc deficiency. Proc. Soc. Exp. Biol. Med.116: 256-259. Fox, M. R. S., and B. N. Harrison, 1965. Effect of zinc deficiency on plasma proteins of young Japanese quail. J. Nutrition, 86: 89-92. Kratzer, F. H., P. Vohra, J. B. Allred and P. N. Davis, 1958. Effect of zinc upon growth and incidence of perosis in turkey poults. Proc. Soc. Exp. Biol. Med. 98: 205-207. Morrison, A. M., and H. P. Sarett, 1958. Studies on zinc deficiency in the chick. J. Nutrition, 6 5 : 267-280. O'Dell, B. L., P. M. Newberne and J. E. Savage, 1958. Significance of dietary zinc for the growing chicken. J. Nutrition, 65: 503-523. Ornstein, L., 1964. Disc electrophoresis-1: background and theory. Ann. New York Acad, of Sci. 121: 321-349. Perkin-Elmer, 1966. Supplement to Analytical Methods for Atomic Absorption Spectrophotometry, Perkin-Elmer Corp., Norwalk, Conn. Rahman, M. M., R. E. Davies, C.W. Deyoe, B. L. Reid and J. R. Couch, 1961. The role of zinc in the nutrition of growing pullets. Poultry Sci. 40: 195-200. Sullivan, T. W., 1961. The zinc requirement of Broad Breasted Bronze poults. Poultry Sci. 40. 334-340. Supplee, W. C , G. F. Combs and D. L. Blamberg, 1958. Zinc and potassium effects on bone formation, feathering and growth of poults. Poultry Sci. 37: 63-67. Vohra, P., E. Krantz and F. H. Kratzer, 1966. Formation constants of certain zinc-complexes by ion-exchange methods. Proc. Soc. Exp. Biol. Med. 121: 422^25. Vohra, P., and F. H. Kratzer, 1966. Effect of var-

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ments. The unstained portion of the strips was divided into 7 segments, of about equal length and covered the 6 bands and both the ends of the stained zones. These segments were counted for zinc-65. The radioactivity was distributed along the whole strip but maximum activity was associated with the section just ahead of the albumen. That the activity of Zn65 is associated with the protein components of the plasma has been further confirmed by the Gel filtration technique on a Sephadex-2 5 column as described for Ca and Sr by Creger et al. (1967). During the staining of the cellulose acetate strips, all the radioactivity was lost indicating that the binding must be loose enough to be completely disassociated by dilute acetic acid present in staining solutions. Further work is needed to investigate the nature of this zinc and plasma protein binding.

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P . VOHRA AND F . H . KRATZER

ious phosphates on zinc and vitamin Da requirements of turkey poults. Proc. of the 7th International Nutrition Congress, Hamburg (in press).

Young, R. J., H. M. Edwards, Jr., and M. B. Gillis, 1958. Studies on zinc in poultry nutrition. 2. Zinc requirement and deficiency symptoms of chicks. Poultry Sci. 37: 1100-1107.

The Effect of Steam Pelleting Feed Ingredients on Chick Performance: Effect on Phosphorus Availability, Metabolizable Energy Value and Carcass Composition

(Received for publication November 18, 1967)

T

HE performance of growing chicks on a diet containing no added inorganic phosphate (42% corn and 40% soybean meal) was markedly increased when the diet was steam pelleted. The ash content of the bones of chicks receiving the diet in the mash form was low but when the ration had been steam pelleted the ash content of the bones was normal (Cisneros, 1966). It would appear that in a corn, soybean meal diet the availability of the organic phosphorus to the young chick is increased by steam pelleting. Summers et al. (1967) found that this was also true for rations containing wheat bran. In the present study the effect of steam pelleting the ingredients of a corn soybean meal diet, separately and in combination, on the response of growing chicks to supplemental inorganic phosphorus, was investigated. An experiment designed to show the effects of exploiting increased nutrient availability as a result of steam pelleting is also described. EXPERIMENTAL

The rations and ingredients were pelleted using a 60 H.P. California 'Hy-Flo' pellet machine in which the feed passed through a conditioning chamber where it

was subjected to steam, injected at a pressure of 55 pounds per square inch (150°C). Feed remained in the conditioning chamber between three and five seconds and the rate of steam addition was regulated to heat the feed to approximately 90°C. The feed was extruded through % 6 inch dies and the pellets were cooled with a forced air draught. Groups of twenty hybrid meat type chicks were assigned to cages in electricallyheated tiered brooders. Female chicks were used in experiment 1 and males in experiment 2. The chicks received the experimental diets from day of age for 23 days. Feed intake and excreta produced in two consecutive two day periods between the 18th and 22nd days were recorded and the excreta was freeze-dried and ground for gross energy determinations. Total feed consumption and gain were recorded and at the end of the experiment all the birds were killed with chloroform. One leg was removed from ten randomly selected birds in each pen and the remaining birds were frozen for carcass analysis. Bone ash determinations were made on the dry, fat free tibiae by the A.O.A.C. (1960) method. The frozen carcasses were ground in a meat grinder, freeze-dried and ground fine with

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H. S. BAYLEY, J. D. SUMMERS AND S. J. SLINGER Departments of Nutrition and Poultry Science, Ontario Agriculture College, University of Guelph, Guelph, Ont., Canada