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Effects of Dietary Calcium and Phosphorus on the Utilization of Dietary Iron by the Chick1,2
Effects of Dietary Calcium and Phosphorus on the Utilization of Dietary Iron by the Chick1'2 D. G. WADDELL AND J. L. SELL Animal Science Department, University of Manitoba, Winnipeg, Canada (Received for publication April 20, 1964)
T
HE influence of various dietary factors on the utilization of dietary iron by animals has been reported by numerous investigators. Demulder (1958) listed dietary phosphorus as a factor which may decrease the utilization of dietary iron by monogastric animals. Previously, Hegsted et al. (1949) reported that the amount of iron deposited in the liver of the rat was inversely related to the phosphorus content of the diet. Buttner and Muhler (1959) also found that liver iron was reduced as the phosphorus level of rations fed to rats increased. Evidence presented by Kletzien (1940) and Chapman and Campbell (1957) demonstrated that excess dietary calcium also exerted an adverse effect on iron utilization by the rat. Chapman and Campbell (1957) observed that both the level and the relative solubility of dietary calcium salts were inversely related to the magnitude of hemoglobin regeneration by anemic rats, but were unable to detect an effect of excess dietary phosphorus on iron utilization. The data reported herein describe the effects of dietary calcium and phosphorus on the utilization of dietary iron by the chick. EXPERIMENTAL PROCEDURE
Male, broiler-type chicks obtained from "This study was supported, in part, by a grant-in-aid from the National Research Council of Canada. 2 A portion of a thesis submitted by the senior author in partial fulfillment of the requirements for a Master of Science degree.
a commercial hatchery were used in two experiments. The chicks were placed in electrically-heated, wire-floored batteries at one day of age. The feeders and heatingpad guards of the batteries were sprayed with an acrylic resin to reduce iron contamination and distilled water was supplied ad libitum in glass watering founts fitted with plastic bases. In experiment 1, a semi-purified ration (Table 1) containing 13 parts per million TABLE 1.—Composition of low-iron ration used for the one-week depletion period in experiments 1 and 21 Ingredient Corn starch Dried egg white Alphacel Sunflower oil Choline chloride Lysine Glycine Arginine Vitamin premix2 Mineral premix3 Calcium carbonate Ammonium phosphate
% 56.95 26.84 6.00 4.00 0.22 0.10 0.80 0.20 1.00 1.00 1.88 1.01 100.00
1 Dietary levels of calcium, phosphorus and iron were adjusted by inclusion or exclusion of calcium carbonate, ammonium phosphate and ferrous sulfate, respectively. 2 Vitamin premix supplied the following per pound of ration: vitamin A, 4,500 I.U.; vitamin D 3 , 396.0 I.C.U.; thiamine, 2.3 mg.; niacin 46.0 mg.; riboflavin 7.0 mg.; Ca pantothenate, 9.0 mg.; biotin 500.0 mg.; folic acid, 2.0 mg.; inositol, 46.0 mg.; P.A.B.A., 1.0 mg.; menadione, 1.0 mg.; vitamin E 10.0 I.U.; ascorbic acid, 115.0 mg.; vitamin B12, 9.0 mg.; vitamin B6, 3.0 mg. 3 Mineral premix supplied the following per pound of ration: sodium chloride, 0.30%; magnesium, 111.6 mg.; manganese 25.00 mg.; zinc 23.50 mg.; copper 2.30 mg.; iodine, 0.77 mg.; potassium, 0.20%.
1249
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D. G. WADDELL AND J. L. SELL
(p.p.m.) of iron was fed ad libitum for the first week to deplete the chicks of iron. At the end of the week, the chicks were individually weighed and allotted to the experimental pens in such a manner that average body weight of each group was approximately equal. Six chicks were used per experimental pen. A complete factorial arrangement of six ration treatments was used. Three levels of dietary iron were each tested in rations containing either 0.75% of calcium and 0.3% of phosphorus or 2.50% of calcium and 1.00% of phosphorus. Thus, a constant calcium to phosphorus ratio was maintained and the effect of calcium and phosphorus level of the ration on the utilization of dietary iron by the chick could be studied. Each ration treatment was assigned to three experimental pens according to a randomized block design. Ferrous sulfate, in weak acid solution, was added to the basal (13 p.p.m. of iron) ration to obtain levels of 26 and 39 p.p.m. of iron for each calcium-phosphorus level. Calcium and phosphorus levels were adjusted by varying the amounts of technical grade calcium carbonate and monobasic ammonium phosphate included in the rations. There was essentially no organic phosphorus in the rations. All rations were made isonitrogenous and isocaloric by adjusting the levels of corn starch and sunflower oil. Ingredients were handled and mixed in stainless steel or glass equipment that had been rinsed in weak hydrochloric acid and distilled water. Feed consumption data and individual chick weights were recorded weekly. Blood samples were collected weekly by heart probe using heparinized syringes and blood tubes. Immediately following collection of the blood, hemoglobin concentration was determined by the method of Schultze and Elvehjem (1934). Wintrobe hematocrit tubes were used to determine the percent
red blood cells. Methods described by the Association of Official Agricultural Chemists (A.O.A.C.) (1960) were used to determine calcium, phosphorus and iron content of the rations. In experiment 2, calcium, phosphorus and iron were the independent ration variables. The levels of the ration variables used are shown in Table 2. The basal ration was similar to that used in experiment 1 (Table 1) except that chromic oxide was included as an index substance at a level of 0.3% of the ration. The sources of calcium, phosphorus and iron added to the ration were the same as in experiment 1. The fifteen ration treatments formed a three dimensional, central composite design (Box and Wilson, 1951). Treatments 1 through 8 formed a 23 factorial experiment and the additional treatments 9 through 15 formed a fractional 3 3 design. With this design, good estimates can be made of main effects, two-factor interactions and quadratic effects (Cragle et al., 1955). For a period of one week, all chicks were fed a ration, designed to be adequate in calcium and phosphorus (1.0% and 0.6%, respectively) but deficient in iron (16 p.p.m.). At one week of age, the chicks TABLE 2.—Levels of calcium, phosphorus and iron used in rations in experiment 2 Calcium
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
%
0.60 1.40 0.60 1.40 0.60 1.40 0.60 1.40 1.00 0.20 1.80 1.00 1.00 1.00 1.00
Phosphorus
% 0.35 0.35 0.85 0.85 0.35 0.35 0.85 0.85 0.60 0.60 0.60 0.10 1.10 0.60 0.60
Iron
1251
CALCIUM, PHOSPHORUS AND IRON
were individually weighed and allotted to the experimental pens as in experiment 1. Each ration treatment was assigned to 3 experimental pens according to a randomized block design. Feed and distilled water were offered ad libitum. Blood samples were collected at two and three weeks of age and analyzed as in experiment 1. In addition, red blood cell counts were determined, and mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated. At three weeks of age, three chicks per experimental pen were sacrificed and the iron content of the livers was determined by a modification of the A.O.A.C. (1960) procedure. Dry matter was also determined on the livers and the iron content was expressed as milligrams of iron per gram of dry liver. During the last ten days of the experiment, excrement was collected on waxed paper. Air-dried excrement samples were analyzed for iron content by the method described by A.O.A.C. (1960). All rations were analysed for iron, phosphorus and calcium by the methods of A.O.A.C. (1960). Chromic oxide content of the rations and excrement samples was determined by the method of Czarnocki et al. (1961). The retention of dietary iron was determined using the feed to excrement ratio of chromic oxide. The data of experiment 1 were subjected to analysis of variance procedures (Snedecor, 19S6). The sources of variation used for the subdivision of the sum of squares in experiment 2 are shown in Table 3. RESULTS
In experiment 1, treatment effects were very similar after 7 and 14 days on the experimental rations and so only the latter are reported (Table 4). Weight gains
TABLE 3.—The analysis of variance used for experiment 2 showing the subdivision of sums of squares Source of variation Blocks Treatments Linear X!
x x2
3 Interaction X1X2 X1X3 X2X3
Quadratic Residual Error Total
Degrees , ,
S u m of
Mean
squares
square
2 14 3 1 1 1 3 1 1 1 3 5 28 44
significantly (P < 0.01) increased with increasing levels of dietary iron, but the calcium-phosphorus level had little effect on weight gain. Hemoglobin concentration was significantly (P < 0.01) lower on the high calcium-phosphorus ration, but irrespective of calcium-phosphorus level, increasing levels of iron resulted in higher hemoglobin concentration in the blood. Hematocrit values varied in parallel with hemoglobin concentration of the blood. There were no interactions between iron and calcium-phosphorus levels. The fact that calcium-phosphorus level did not exert a main effect on weight gain but did adversely affect hemoglobin and hematocrits demonstrates that growth (weight gain) is not necessarily a valid criterion for assessing iron status of the chick in short term experiments. In contrast, circulating hemoglobin concentration appears to be a sensitive indicator of iron status, as is hematocrit. In experiment 2, increasing levels of dietary calcium significantly3 reduced circulating hemoglobin and hemotocrit of 33 The level of statistical significance referred to in experiment 2 is P<0.05.
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D. G. WADDELL AND J. L. SELL
TABLE 4.—Ejfect of dietary calcium-phosphorus and iron levels on weight gain, hemoglobin concentration and hematocrit of chicks—experiment 1
Ca+P %
Iron (p.p.m.) 13
26
Analysis of variance
39
X
Source
d.f.
Mean square
Iron Ca-P Ca-PXIron Error
2 1 2 10
23181 53 24 236
Iron Ca-P Ca-PXIron Error
2 1 2 10
32.41 33.li 0.3 0.3
Iron Ca-P Ca-PXIron Error
2 1 2 10
183.91 70.0i 7.4 4.0
Weight gain/chick (g.) 0.75+0.30
76
110
118
101
2.50+1.00
79
106
110
98
X
78
108
114
Hemoglobin (g./lOO cc.) 0.75+0.30
7.70
11.08
12.72
10.05
2.50+1.00
5.54
8.14
9.68
7.79
X
6.62
9.61
11.20
Hematocrit 0.75+0.30
20
27
29
25.3
2.50+1.00
15
22
28
21.7
X
17.5
24.5
28.5
Statistically significant at P < 0 . 0 1 .
week old chicks (Figure l). 4 This was most apparent when the rations contained 56 p.p.m. or less of iron. Increasing dietary phosphorus also exerted a depressing effect on hemoglobin concentration and hematocrit. Furthermore, in contrast to calcium, this effect was most pronounced when iron was present in the ration at 56 p.p.m. or more. Increasing the iron level of the ration consistently increased hemoglobin concentration regardless of calcium or phosphorus level. Hematocrit values also increased as the dietary iron level increased except when the ration contained 0.85% of phosphorus in combination with either 0.6% or 1.4% of calcium. There was a significant phosphorus X iron interaction which resulted in a greater depressing ef4
The data from experiment 2 are given in the 3-dimensional, spatial arrangement inherent in the experimental design used.
feet of increasing phosphorus levels on hemoglobin concentration and hematocrit at the 56 and 76 p.p.m. levels of iron than when the ration contained 36 p.p.m. of iron. Neither calcium or phosphorus exerted a significant main effect on MCH or MCHC (Fig. 2). However, MCH and MCHC were consistently increased by increasing the iron level of the ration. No interactions were observed. While neither calcium or phosphorus altered MCV significantly both of these dietary variables significantly decreased red blood cell number (Fig. 3). This effect was apparent at all levels of iron but it was most pronounced at 36 p.p.m. of iron. MCV was consistently increased by increasing the iron level of the ration. The significant influence of ration iron on red blood cell number appears to have been due to the marked increases in red blood
1253
CALCIUM, PHOSPHORUS AND IRON
(12)- Ration N°
, 13-64- Hb 31*5 -Hematocrit
0-10
.«40 25-5 ,0-35
,0-60 -§. o
,0-85
,6 10-43 240 16
36
56 p.p.m. Iron
110
76
96
FIG. 1. Effect of dietary calcium, phosphorus and iron on hemoglobin concentration (Hb—g./lOO cc.) and hematocrit—experiment 2.
cell counts found with increasing iron levels in rations containing 1.0% calcium and 0.6% phosphorus. As would be expected, increasing the level of dietary iron significantly increased liver iron concentration regardless of calcium and phosphorus levels (Fig. 4). On the other hand, increasing the ration calcium significantly reduced liver iron concentration. This effect of calcium was particularly noticeable at 56 p.p.m. of iron. Phosphorus did not consistently affect liver iron concentration, but significantly reduced retention of dietary iron with this effect being most pronounced at 36 p.p.m. of iron. Although the data shown in Figure 4 suggests that calcium interfered slightly with iron retention when the ration contained 56 p.p.m. or more of iron, this influence was not statistically significant.
Similarly, as the level of dietary iron increased, iron retention, on a percentage basis, decreased. A notable exception to this was when the iron level was increased from 16 to 96 p.p.m. in rations containing 1.0% of calcium and 0.6% of phosphorus. The low value of iron retention when chicks were fed the ration containing 16 p.p.m. of iron, 1.0% of calcium and 0.6% of phosphorus was unexpected since it is generally accepted that under conditions of inadequate iron intake, dietary iron is utilized more efficiently than under normal circumstances. Essentially all the iron in the 16 p.p.m. ration was supplied by ingredients such as cellulose, egg white and corn starch and it appears that this iron was of relatively low availability. It is also possible that at such a low level of dietary iron, phosphorus was present in sufficient quantity so as to markedly interfere with
1254
D. G. WADDELL AND J. L. SELL
510-MCH ' 43 4-MCHC
010
5»8 48-6
0-2
0-35
0-60
3 O
o 0-85 55-8 50-3 1.10 16
36 56 p.p.m Iron.
76
96
FIG. 2. Effect of dietary calcium, phosphorus and iron on mean corpuscular hemoglobin (MCH— u.u.g.) and mean corpuscular hemoglobin concentration (MCHC—g./lOO cc. of red blood cells)—experiment 2.
iron retention. There were no interactions apparent with regard to iron retention. A significant calcium X phosphorus interaction was observed in which increasing phosphorus levels in the ration augmented the depressing effect of ration calcium on liver iron. A significant phosphorus X iron interaction was also observed. It would appear that while ration phosphorus per se had no effect on liver iron, higher phosphorus levels enhanced the effect of increasing ration iron levels whereby liver iron concentration was increased. DISCUSSION
Using hemoglobin concentration as a criterion of iron status, the results of experiment 1 show that excess dietary calcium and phosphorus decreased the utilization of dietary iron by the chick. This was apparent even though the calcium to phosphorus ratio was constant. The data ob-
tained in experiment 2 demonstrate that both calcium and phosphorus, in increasing amounts in the ration, exert independent, adverse effects on hemoglobin concentration. Associated with the decrease in hemoglobin and hematocrit, due to increasing levels of dietary calcium in experiment 2, was a reduction in red blood cell number. Kletzieh (1940) and Chapman and Campbell (1957) reported that excess calcium adversely affected hemoglobin formation in the rat. Chapman and Campbell (1957) postulated that this effect of calcium was the result of a direct interference with the absorption of iron from the digestive tract. In contrast, the data reported herein show that calcium had little influence on the retention of dietary iron by the bird but do suggest that calcium interferes with iron metabolism. The reduction in red blood cell number indicates that calcium may
1255
CALCIUM, PHOSPHORUS AND IRON
2-693-RBC 117-2- MCV
010
2 078
1-993 127-0
25» 17-6
, P-35 u> 3
0-60
Q.
O
x:
0-85 1-950 111-0
1-975 113-2 2- 030 119-2 16
a.
36 56 p.p.m. Iron.
110
76
96
FIG. 3. Effect of dietary calcium, phosphorus and iron on red blood cell number (RBC—millions/ cmm.) and mean corpuscular volume (MCV—(x.3)—experiment 2.
45-8- Fe. ret n. ' 480-Liver Fe
16
36
56 p.p.m. Iron.
76
96
FIG. 4. Effect of dietary calcium, phosphorus and iron on retention of dietary iron (% of that ingested) and liver iron concentration (mg./lOO g. dry matter)—experiment 2.
1256
D. G. WADDELL AND J. L. SELL
have inhibited erythropoietic activity and, in addition, calcium significantly decreased liver iron concentration even though iron retention was not affected appreciably. Increasing levels of dietary phosphorus significantly decreased circulating hemoglobin, hematocrit and red blood cell number. In contrast to calcium, phosphorus had little influence on liver iron concentration but significantly decreased retention of dietary iron. Assuming that urinary iron excretion is negligible and that the preponderance of fecal iron represents unabsorbed dietary iron (Underwood, 1962) the iron retention data would be indicative of iron absorption. On this basis, it is suggested that phosphorus exerted its deleterious effect on hemoglobin, hematocrit and red blood cell number by inhibiting absorption of dietary iron. Demulder (1958), Buttner and Muhler (1959) and Hegsted et al. (1949) reported that excess dietary phosphorus interfered with iron utilization. Demulder (1958) and Buttner and Muhler (1959) suggested that excess phosphorus forms an insoluble iron-phosphate complex in the gastrointestinal tract, thereby rendering the iron unavailable for absorption. Our data lend support to this hypothesis and also indicate that excess dietary phosphorus has little effect on iron metabolism following absorption. In view of the main effects of calcium and phosphorus on hemoglobin concentration, hematocrit and red blood cell number, and the absence of a calcium X phosphorus interaction in this regard suggests that calcium and phosphorus exert independent effects on iron utilization and also indicates that there is probably a fundamental difference in the mechanisms involved. The fact that increasing levels of dietary iron increased circulating hemoglobin, hematocrit, MCH, MCHC, MCV, red blood cell count and liver iron concentra-
tion would be anticipated considering the role of iron in the body. Worthy of consideration is that the data from experiment 2 strongly indicate that approximately 56 p.p.m. of iron is required in a chick ration containing 1% of calcium and 0.6% of phosphorus, for near maximum hemoglobin concentration. On this basis, it would appear that when hemoglobin concentration is the criterion, the National Research Council (1960) requirement for the chick of 9 milligrams of iron per pound of ration is too low. The data of Hill and Matrone (1961) and Davis et al. (1962) support this view. Although not included in the data presented herein, observations of poikilocytes as described by Hill and Matrone (1961) were made. The incidence of poikilocytes in blood of chicks fed the ration containing 16 p.p.m. of iron for 3 weeks was 11 percent of the total red blood cells. SUMMARY Two experiments were conducted using broiler-type chicks. In both experiments an iron-deficient, semi-purified diet was fed ad libitum for the first week to deplete the chicks of iron. The ration treatments were then fed ad libitum for the succeeding two weeks. The data from both experiments showed that weight gain to 3 weeks of age was a relatively poor criterion for iron status as compared to hemoglobin (Hb) concentration. Increasing levels of dietary calcium decreased Hb and hematocrit. In addition, increasing levels of calcium decreased red blood cell number and liver iron concentration but had little effect on mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) or retention of dietary iron. Increasing levels of dietary phosphorus decreased Hb, hematocrit, red blood cell number and retention of dietary iron but
CALCIUM, PHOSPHORUS AND IRON
did not affect MCH, MCV or liver iron concentration appreciably. The data of experiment 2 suggest that dietary calcium and phosphorus exert independent effects on iron utilization and also indicate that there is a fundamental difference in the mechanisms involved. The deleterious effects of dietary calcium and phosphorus on the iron status of the chick were alleviated by increasing the level of dietary iron. Using hemoglobin concentration as the criterion for iron status of the chick, the data reported herein indicate that approximately 56 p.p.m. of iron is required in a ration containing 1.0% of calcium and 0.6% of phosphorus. ACKNOWLEDGMENTS
The authors are grateful to Mr. S. Antonation, Mr. W. Guenter and Mr. J. McKirdy for their technical assistance. REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis. 9th ed. Box, G. E. P., and K. B. Wilson. 1951. The experimental attainment of optimum condition. J. Roy. Stat. Sco., Series B, 13 : 1. Buttner, W., and J. C. Muhler. 1959. Effect of dietary iron on phosphate metabolism. Proc. Soc. Exper. Biol. Med. 100: 440-442. Chapman, D. C , and J. A. Campbell. 1957. Effect of calcium and phosphorus salts on the utilization of iron by anemic rats. Brit. J. Nutr. 11: 127-133.
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Cragle, R. G., R. M. Myers, R. K. Waugh, J. S. Hunter and R. L. Anderson. 1955. The effects of various levels of sodium citrate, glycerol and equilibrium time on survival of bovine spermatozoa after storage at —79 °C. J. Dairy Sci. 38: 508-514. Czarnocki, J., I. R. Sibbald and E. V. Evans. 1961. The determination of chromic oxide in samples of feed and excreta by acid digestion and spectrophotometry. Can. J. Ani. Sci. 4 1 : 167-171. Davis, P. N., L. C. Norris and F. H. Kratzer, 1962. Iron deficiency studies in chicks using treated isolated protein diets. J. Nutr. 78: 445-453. Demulder, R. 1958. Iron metabolism, biochemistry and clinical pathological physiology—review of recent literature. Arch. Int. Med. 102: 254-301. Hegsted, D. M., C. A. Finch and T. D. Kinney. 1949. The influence of diet on iron absorption. 11. The interrelation of iron and phosphorus. J. Expt. Med. 90:137-156. Hill, C. H., and G. Matrone. 1961. Studies on copper and iron deficiencies in growing chickens. J. Nutr. 73 : 425-431. Kletzien, S. W. 1940. Iron metabolism. The role of calcium in iron assimilation. J. Nutr. 19 : 187-197. National Research Council, 1960. Nutrient requirements of domestic animals. Number 1. Nutrient requirements of poultry. Revised 1960. National Academy of Science—National Research Council. Publication 827. Schultze, M. O., and C. A. Elvehjem. 1934. An improved method for the determination of hemoglobin in chicken blood. J. Biol. Chem. 105: 253-257. Snedecor, G. W., 1956. Statistical Methods, 5th ed. The Iowa State College Press, Ames, Iowa. Underwood, E. J. 1962. Trace Elements in Human and Animal Nutrition. 2nd ed. Academic Press Inc. New York, N.Y.
NEWS AND NOTES (Continued from page 1219) foro Bonadonna. In its 46 chapters the author describes his observations and experiences on his missions around the World in the years 1962-1963, under the auspices of the Ministry of Foreign Affairs (Ministero degli Affari Esteri, Direzione Generale dei Rapporti Culturali) and the Ministry of Public Instruction (Ministero della Pubblica Istruzione, Direzione Generale dell' Istruzione Universitaria).
In its 520 pages, various information and data are interpreted and discussed, enabling the reader to keep up to-date with agricultural and animal husbandry economy in the countries visited, with the activities of the scientific and experimental institutes, and with various problems of production related to human social conditions. The book is illustrated with a number of original photographs and was printed by the "Edizione
(Continued on page 1262)
T
HE influence of various dietary factors on the utilization of dietary iron by animals has been reported by numerous investigators. Demulder (1958) listed dietary phosphorus as a factor which may decrease the utilization of dietary iron by monogastric animals. Previously, Hegsted et al. (1949) reported that the amount of iron deposited in the liver of the rat was inversely related to the phosphorus content of the diet. Buttner and Muhler (1959) also found that liver iron was reduced as the phosphorus level of rations fed to rats increased. Evidence presented by Kletzien (1940) and Chapman and Campbell (1957) demonstrated that excess dietary calcium also exerted an adverse effect on iron utilization by the rat. Chapman and Campbell (1957) observed that both the level and the relative solubility of dietary calcium salts were inversely related to the magnitude of hemoglobin regeneration by anemic rats, but were unable to detect an effect of excess dietary phosphorus on iron utilization. The data reported herein describe the effects of dietary calcium and phosphorus on the utilization of dietary iron by the chick. EXPERIMENTAL PROCEDURE
Male, broiler-type chicks obtained from "This study was supported, in part, by a grant-in-aid from the National Research Council of Canada. 2 A portion of a thesis submitted by the senior author in partial fulfillment of the requirements for a Master of Science degree.
a commercial hatchery were used in two experiments. The chicks were placed in electrically-heated, wire-floored batteries at one day of age. The feeders and heatingpad guards of the batteries were sprayed with an acrylic resin to reduce iron contamination and distilled water was supplied ad libitum in glass watering founts fitted with plastic bases. In experiment 1, a semi-purified ration (Table 1) containing 13 parts per million TABLE 1.—Composition of low-iron ration used for the one-week depletion period in experiments 1 and 21 Ingredient Corn starch Dried egg white Alphacel Sunflower oil Choline chloride Lysine Glycine Arginine Vitamin premix2 Mineral premix3 Calcium carbonate Ammonium phosphate
% 56.95 26.84 6.00 4.00 0.22 0.10 0.80 0.20 1.00 1.00 1.88 1.01 100.00
1 Dietary levels of calcium, phosphorus and iron were adjusted by inclusion or exclusion of calcium carbonate, ammonium phosphate and ferrous sulfate, respectively. 2 Vitamin premix supplied the following per pound of ration: vitamin A, 4,500 I.U.; vitamin D 3 , 396.0 I.C.U.; thiamine, 2.3 mg.; niacin 46.0 mg.; riboflavin 7.0 mg.; Ca pantothenate, 9.0 mg.; biotin 500.0 mg.; folic acid, 2.0 mg.; inositol, 46.0 mg.; P.A.B.A., 1.0 mg.; menadione, 1.0 mg.; vitamin E 10.0 I.U.; ascorbic acid, 115.0 mg.; vitamin B12, 9.0 mg.; vitamin B6, 3.0 mg. 3 Mineral premix supplied the following per pound of ration: sodium chloride, 0.30%; magnesium, 111.6 mg.; manganese 25.00 mg.; zinc 23.50 mg.; copper 2.30 mg.; iodine, 0.77 mg.; potassium, 0.20%.
1249
1250
D. G. WADDELL AND J. L. SELL
(p.p.m.) of iron was fed ad libitum for the first week to deplete the chicks of iron. At the end of the week, the chicks were individually weighed and allotted to the experimental pens in such a manner that average body weight of each group was approximately equal. Six chicks were used per experimental pen. A complete factorial arrangement of six ration treatments was used. Three levels of dietary iron were each tested in rations containing either 0.75% of calcium and 0.3% of phosphorus or 2.50% of calcium and 1.00% of phosphorus. Thus, a constant calcium to phosphorus ratio was maintained and the effect of calcium and phosphorus level of the ration on the utilization of dietary iron by the chick could be studied. Each ration treatment was assigned to three experimental pens according to a randomized block design. Ferrous sulfate, in weak acid solution, was added to the basal (13 p.p.m. of iron) ration to obtain levels of 26 and 39 p.p.m. of iron for each calcium-phosphorus level. Calcium and phosphorus levels were adjusted by varying the amounts of technical grade calcium carbonate and monobasic ammonium phosphate included in the rations. There was essentially no organic phosphorus in the rations. All rations were made isonitrogenous and isocaloric by adjusting the levels of corn starch and sunflower oil. Ingredients were handled and mixed in stainless steel or glass equipment that had been rinsed in weak hydrochloric acid and distilled water. Feed consumption data and individual chick weights were recorded weekly. Blood samples were collected weekly by heart probe using heparinized syringes and blood tubes. Immediately following collection of the blood, hemoglobin concentration was determined by the method of Schultze and Elvehjem (1934). Wintrobe hematocrit tubes were used to determine the percent
red blood cells. Methods described by the Association of Official Agricultural Chemists (A.O.A.C.) (1960) were used to determine calcium, phosphorus and iron content of the rations. In experiment 2, calcium, phosphorus and iron were the independent ration variables. The levels of the ration variables used are shown in Table 2. The basal ration was similar to that used in experiment 1 (Table 1) except that chromic oxide was included as an index substance at a level of 0.3% of the ration. The sources of calcium, phosphorus and iron added to the ration were the same as in experiment 1. The fifteen ration treatments formed a three dimensional, central composite design (Box and Wilson, 1951). Treatments 1 through 8 formed a 23 factorial experiment and the additional treatments 9 through 15 formed a fractional 3 3 design. With this design, good estimates can be made of main effects, two-factor interactions and quadratic effects (Cragle et al., 1955). For a period of one week, all chicks were fed a ration, designed to be adequate in calcium and phosphorus (1.0% and 0.6%, respectively) but deficient in iron (16 p.p.m.). At one week of age, the chicks TABLE 2.—Levels of calcium, phosphorus and iron used in rations in experiment 2 Calcium
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
%
0.60 1.40 0.60 1.40 0.60 1.40 0.60 1.40 1.00 0.20 1.80 1.00 1.00 1.00 1.00
Phosphorus
% 0.35 0.35 0.85 0.85 0.35 0.35 0.85 0.85 0.60 0.60 0.60 0.10 1.10 0.60 0.60
Iron
1251
CALCIUM, PHOSPHORUS AND IRON
were individually weighed and allotted to the experimental pens as in experiment 1. Each ration treatment was assigned to 3 experimental pens according to a randomized block design. Feed and distilled water were offered ad libitum. Blood samples were collected at two and three weeks of age and analyzed as in experiment 1. In addition, red blood cell counts were determined, and mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated. At three weeks of age, three chicks per experimental pen were sacrificed and the iron content of the livers was determined by a modification of the A.O.A.C. (1960) procedure. Dry matter was also determined on the livers and the iron content was expressed as milligrams of iron per gram of dry liver. During the last ten days of the experiment, excrement was collected on waxed paper. Air-dried excrement samples were analyzed for iron content by the method described by A.O.A.C. (1960). All rations were analysed for iron, phosphorus and calcium by the methods of A.O.A.C. (1960). Chromic oxide content of the rations and excrement samples was determined by the method of Czarnocki et al. (1961). The retention of dietary iron was determined using the feed to excrement ratio of chromic oxide. The data of experiment 1 were subjected to analysis of variance procedures (Snedecor, 19S6). The sources of variation used for the subdivision of the sum of squares in experiment 2 are shown in Table 3. RESULTS
In experiment 1, treatment effects were very similar after 7 and 14 days on the experimental rations and so only the latter are reported (Table 4). Weight gains
TABLE 3.—The analysis of variance used for experiment 2 showing the subdivision of sums of squares Source of variation Blocks Treatments Linear X!
x x2
3 Interaction X1X2 X1X3 X2X3
Quadratic Residual Error Total
Degrees , ,
S u m of
Mean
squares
square
2 14 3 1 1 1 3 1 1 1 3 5 28 44
significantly (P < 0.01) increased with increasing levels of dietary iron, but the calcium-phosphorus level had little effect on weight gain. Hemoglobin concentration was significantly (P < 0.01) lower on the high calcium-phosphorus ration, but irrespective of calcium-phosphorus level, increasing levels of iron resulted in higher hemoglobin concentration in the blood. Hematocrit values varied in parallel with hemoglobin concentration of the blood. There were no interactions between iron and calcium-phosphorus levels. The fact that calcium-phosphorus level did not exert a main effect on weight gain but did adversely affect hemoglobin and hematocrits demonstrates that growth (weight gain) is not necessarily a valid criterion for assessing iron status of the chick in short term experiments. In contrast, circulating hemoglobin concentration appears to be a sensitive indicator of iron status, as is hematocrit. In experiment 2, increasing levels of dietary calcium significantly3 reduced circulating hemoglobin and hemotocrit of 33 The level of statistical significance referred to in experiment 2 is P<0.05.
1252
D. G. WADDELL AND J. L. SELL
TABLE 4.—Ejfect of dietary calcium-phosphorus and iron levels on weight gain, hemoglobin concentration and hematocrit of chicks—experiment 1
Ca+P %
Iron (p.p.m.) 13
26
Analysis of variance
39
X
Source
d.f.
Mean square
Iron Ca-P Ca-PXIron Error
2 1 2 10
23181 53 24 236
Iron Ca-P Ca-PXIron Error
2 1 2 10
32.41 33.li 0.3 0.3
Iron Ca-P Ca-PXIron Error
2 1 2 10
183.91 70.0i 7.4 4.0
Weight gain/chick (g.) 0.75+0.30
76
110
118
101
2.50+1.00
79
106
110
98
X
78
108
114
Hemoglobin (g./lOO cc.) 0.75+0.30
7.70
11.08
12.72
10.05
2.50+1.00
5.54
8.14
9.68
7.79
X
6.62
9.61
11.20
Hematocrit 0.75+0.30
20
27
29
25.3
2.50+1.00
15
22
28
21.7
X
17.5
24.5
28.5
Statistically significant at P < 0 . 0 1 .
week old chicks (Figure l). 4 This was most apparent when the rations contained 56 p.p.m. or less of iron. Increasing dietary phosphorus also exerted a depressing effect on hemoglobin concentration and hematocrit. Furthermore, in contrast to calcium, this effect was most pronounced when iron was present in the ration at 56 p.p.m. or more. Increasing the iron level of the ration consistently increased hemoglobin concentration regardless of calcium or phosphorus level. Hematocrit values also increased as the dietary iron level increased except when the ration contained 0.85% of phosphorus in combination with either 0.6% or 1.4% of calcium. There was a significant phosphorus X iron interaction which resulted in a greater depressing ef4
The data from experiment 2 are given in the 3-dimensional, spatial arrangement inherent in the experimental design used.
feet of increasing phosphorus levels on hemoglobin concentration and hematocrit at the 56 and 76 p.p.m. levels of iron than when the ration contained 36 p.p.m. of iron. Neither calcium or phosphorus exerted a significant main effect on MCH or MCHC (Fig. 2). However, MCH and MCHC were consistently increased by increasing the iron level of the ration. No interactions were observed. While neither calcium or phosphorus altered MCV significantly both of these dietary variables significantly decreased red blood cell number (Fig. 3). This effect was apparent at all levels of iron but it was most pronounced at 36 p.p.m. of iron. MCV was consistently increased by increasing the iron level of the ration. The significant influence of ration iron on red blood cell number appears to have been due to the marked increases in red blood
1253
CALCIUM, PHOSPHORUS AND IRON
(12)- Ration N°
, 13-64- Hb 31*5 -Hematocrit
0-10
.«40 25-5 ,0-35
,0-60 -§. o
,0-85
,6 10-43 240 16
36
56 p.p.m. Iron
110
76
96
FIG. 1. Effect of dietary calcium, phosphorus and iron on hemoglobin concentration (Hb—g./lOO cc.) and hematocrit—experiment 2.
cell counts found with increasing iron levels in rations containing 1.0% calcium and 0.6% phosphorus. As would be expected, increasing the level of dietary iron significantly increased liver iron concentration regardless of calcium and phosphorus levels (Fig. 4). On the other hand, increasing the ration calcium significantly reduced liver iron concentration. This effect of calcium was particularly noticeable at 56 p.p.m. of iron. Phosphorus did not consistently affect liver iron concentration, but significantly reduced retention of dietary iron with this effect being most pronounced at 36 p.p.m. of iron. Although the data shown in Figure 4 suggests that calcium interfered slightly with iron retention when the ration contained 56 p.p.m. or more of iron, this influence was not statistically significant.
Similarly, as the level of dietary iron increased, iron retention, on a percentage basis, decreased. A notable exception to this was when the iron level was increased from 16 to 96 p.p.m. in rations containing 1.0% of calcium and 0.6% of phosphorus. The low value of iron retention when chicks were fed the ration containing 16 p.p.m. of iron, 1.0% of calcium and 0.6% of phosphorus was unexpected since it is generally accepted that under conditions of inadequate iron intake, dietary iron is utilized more efficiently than under normal circumstances. Essentially all the iron in the 16 p.p.m. ration was supplied by ingredients such as cellulose, egg white and corn starch and it appears that this iron was of relatively low availability. It is also possible that at such a low level of dietary iron, phosphorus was present in sufficient quantity so as to markedly interfere with
1254
D. G. WADDELL AND J. L. SELL
510-MCH ' 43 4-MCHC
010
5»8 48-6
0-2
0-35
0-60
3 O
o 0-85 55-8 50-3 1.10 16
36 56 p.p.m Iron.
76
96
FIG. 2. Effect of dietary calcium, phosphorus and iron on mean corpuscular hemoglobin (MCH— u.u.g.) and mean corpuscular hemoglobin concentration (MCHC—g./lOO cc. of red blood cells)—experiment 2.
iron retention. There were no interactions apparent with regard to iron retention. A significant calcium X phosphorus interaction was observed in which increasing phosphorus levels in the ration augmented the depressing effect of ration calcium on liver iron. A significant phosphorus X iron interaction was also observed. It would appear that while ration phosphorus per se had no effect on liver iron, higher phosphorus levels enhanced the effect of increasing ration iron levels whereby liver iron concentration was increased. DISCUSSION
Using hemoglobin concentration as a criterion of iron status, the results of experiment 1 show that excess dietary calcium and phosphorus decreased the utilization of dietary iron by the chick. This was apparent even though the calcium to phosphorus ratio was constant. The data ob-
tained in experiment 2 demonstrate that both calcium and phosphorus, in increasing amounts in the ration, exert independent, adverse effects on hemoglobin concentration. Associated with the decrease in hemoglobin and hematocrit, due to increasing levels of dietary calcium in experiment 2, was a reduction in red blood cell number. Kletzieh (1940) and Chapman and Campbell (1957) reported that excess calcium adversely affected hemoglobin formation in the rat. Chapman and Campbell (1957) postulated that this effect of calcium was the result of a direct interference with the absorption of iron from the digestive tract. In contrast, the data reported herein show that calcium had little influence on the retention of dietary iron by the bird but do suggest that calcium interferes with iron metabolism. The reduction in red blood cell number indicates that calcium may
1255
CALCIUM, PHOSPHORUS AND IRON
2-693-RBC 117-2- MCV
010
2 078
1-993 127-0
25» 17-6
, P-35 u> 3
0-60
Q.
O
x:
0-85 1-950 111-0
1-975 113-2 2- 030 119-2 16
a.
36 56 p.p.m. Iron.
110
76
96
FIG. 3. Effect of dietary calcium, phosphorus and iron on red blood cell number (RBC—millions/ cmm.) and mean corpuscular volume (MCV—(x.3)—experiment 2.
45-8- Fe. ret n. ' 480-Liver Fe
16
36
56 p.p.m. Iron.
76
96
FIG. 4. Effect of dietary calcium, phosphorus and iron on retention of dietary iron (% of that ingested) and liver iron concentration (mg./lOO g. dry matter)—experiment 2.
1256
D. G. WADDELL AND J. L. SELL
have inhibited erythropoietic activity and, in addition, calcium significantly decreased liver iron concentration even though iron retention was not affected appreciably. Increasing levels of dietary phosphorus significantly decreased circulating hemoglobin, hematocrit and red blood cell number. In contrast to calcium, phosphorus had little influence on liver iron concentration but significantly decreased retention of dietary iron. Assuming that urinary iron excretion is negligible and that the preponderance of fecal iron represents unabsorbed dietary iron (Underwood, 1962) the iron retention data would be indicative of iron absorption. On this basis, it is suggested that phosphorus exerted its deleterious effect on hemoglobin, hematocrit and red blood cell number by inhibiting absorption of dietary iron. Demulder (1958), Buttner and Muhler (1959) and Hegsted et al. (1949) reported that excess dietary phosphorus interfered with iron utilization. Demulder (1958) and Buttner and Muhler (1959) suggested that excess phosphorus forms an insoluble iron-phosphate complex in the gastrointestinal tract, thereby rendering the iron unavailable for absorption. Our data lend support to this hypothesis and also indicate that excess dietary phosphorus has little effect on iron metabolism following absorption. In view of the main effects of calcium and phosphorus on hemoglobin concentration, hematocrit and red blood cell number, and the absence of a calcium X phosphorus interaction in this regard suggests that calcium and phosphorus exert independent effects on iron utilization and also indicates that there is probably a fundamental difference in the mechanisms involved. The fact that increasing levels of dietary iron increased circulating hemoglobin, hematocrit, MCH, MCHC, MCV, red blood cell count and liver iron concentra-
tion would be anticipated considering the role of iron in the body. Worthy of consideration is that the data from experiment 2 strongly indicate that approximately 56 p.p.m. of iron is required in a chick ration containing 1% of calcium and 0.6% of phosphorus, for near maximum hemoglobin concentration. On this basis, it would appear that when hemoglobin concentration is the criterion, the National Research Council (1960) requirement for the chick of 9 milligrams of iron per pound of ration is too low. The data of Hill and Matrone (1961) and Davis et al. (1962) support this view. Although not included in the data presented herein, observations of poikilocytes as described by Hill and Matrone (1961) were made. The incidence of poikilocytes in blood of chicks fed the ration containing 16 p.p.m. of iron for 3 weeks was 11 percent of the total red blood cells. SUMMARY Two experiments were conducted using broiler-type chicks. In both experiments an iron-deficient, semi-purified diet was fed ad libitum for the first week to deplete the chicks of iron. The ration treatments were then fed ad libitum for the succeeding two weeks. The data from both experiments showed that weight gain to 3 weeks of age was a relatively poor criterion for iron status as compared to hemoglobin (Hb) concentration. Increasing levels of dietary calcium decreased Hb and hematocrit. In addition, increasing levels of calcium decreased red blood cell number and liver iron concentration but had little effect on mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) or retention of dietary iron. Increasing levels of dietary phosphorus decreased Hb, hematocrit, red blood cell number and retention of dietary iron but
CALCIUM, PHOSPHORUS AND IRON
did not affect MCH, MCV or liver iron concentration appreciably. The data of experiment 2 suggest that dietary calcium and phosphorus exert independent effects on iron utilization and also indicate that there is a fundamental difference in the mechanisms involved. The deleterious effects of dietary calcium and phosphorus on the iron status of the chick were alleviated by increasing the level of dietary iron. Using hemoglobin concentration as the criterion for iron status of the chick, the data reported herein indicate that approximately 56 p.p.m. of iron is required in a ration containing 1.0% of calcium and 0.6% of phosphorus. ACKNOWLEDGMENTS
The authors are grateful to Mr. S. Antonation, Mr. W. Guenter and Mr. J. McKirdy for their technical assistance. REFERENCES Association of Official Agricultural Chemists, 1960. Official Methods of Analysis. 9th ed. Box, G. E. P., and K. B. Wilson. 1951. The experimental attainment of optimum condition. J. Roy. Stat. Sco., Series B, 13 : 1. Buttner, W., and J. C. Muhler. 1959. Effect of dietary iron on phosphate metabolism. Proc. Soc. Exper. Biol. Med. 100: 440-442. Chapman, D. C , and J. A. Campbell. 1957. Effect of calcium and phosphorus salts on the utilization of iron by anemic rats. Brit. J. Nutr. 11: 127-133.
1257
Cragle, R. G., R. M. Myers, R. K. Waugh, J. S. Hunter and R. L. Anderson. 1955. The effects of various levels of sodium citrate, glycerol and equilibrium time on survival of bovine spermatozoa after storage at —79 °C. J. Dairy Sci. 38: 508-514. Czarnocki, J., I. R. Sibbald and E. V. Evans. 1961. The determination of chromic oxide in samples of feed and excreta by acid digestion and spectrophotometry. Can. J. Ani. Sci. 4 1 : 167-171. Davis, P. N., L. C. Norris and F. H. Kratzer, 1962. Iron deficiency studies in chicks using treated isolated protein diets. J. Nutr. 78: 445-453. Demulder, R. 1958. Iron metabolism, biochemistry and clinical pathological physiology—review of recent literature. Arch. Int. Med. 102: 254-301. Hegsted, D. M., C. A. Finch and T. D. Kinney. 1949. The influence of diet on iron absorption. 11. The interrelation of iron and phosphorus. J. Expt. Med. 90:137-156. Hill, C. H., and G. Matrone. 1961. Studies on copper and iron deficiencies in growing chickens. J. Nutr. 73 : 425-431. Kletzien, S. W. 1940. Iron metabolism. The role of calcium in iron assimilation. J. Nutr. 19 : 187-197. National Research Council, 1960. Nutrient requirements of domestic animals. Number 1. Nutrient requirements of poultry. Revised 1960. National Academy of Science—National Research Council. Publication 827. Schultze, M. O., and C. A. Elvehjem. 1934. An improved method for the determination of hemoglobin in chicken blood. J. Biol. Chem. 105: 253-257. Snedecor, G. W., 1956. Statistical Methods, 5th ed. The Iowa State College Press, Ames, Iowa. Underwood, E. J. 1962. Trace Elements in Human and Animal Nutrition. 2nd ed. Academic Press Inc. New York, N.Y.
NEWS AND NOTES (Continued from page 1219) foro Bonadonna. In its 46 chapters the author describes his observations and experiences on his missions around the World in the years 1962-1963, under the auspices of the Ministry of Foreign Affairs (Ministero degli Affari Esteri, Direzione Generale dei Rapporti Culturali) and the Ministry of Public Instruction (Ministero della Pubblica Istruzione, Direzione Generale dell' Istruzione Universitaria).
In its 520 pages, various information and data are interpreted and discussed, enabling the reader to keep up to-date with agricultural and animal husbandry economy in the countries visited, with the activities of the scientific and experimental institutes, and with various problems of production related to human social conditions. The book is illustrated with a number of original photographs and was printed by the "Edizione
(Continued on page 1262)