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The effect of dietary fat level on calcium utilization by the chick Salmon, Raymond Edward 1957

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THE EFFECT OF DIETARY FAT LEVEL ON CALCIUM UTILIZATION BY THE CHICK by Raymond Edward Salmon, B. S. A. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE IN THE DEPARTMENT of P O U L T R Y S C I E N C E We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE IN ACrRICtgLTURE Members of the Department of Poultry Science THE UNIVERSITY OF BRITISH COLUMBIA October, 1957 ABSTRACT THE EFFECT OF DIETARY FAT LEVEL ON CALCIUM UTILIZATION BY THE CHICK A series of three balance experiments were conducted to study the effect of the addition of animal fat to the diet on the utilization of calcium by growing chicks. In two experiments, the chicks were fed diets containing four levels of calcium, 0.75$, 1.0%, 1.25% and 1.50%, with a calcium: phosphorus ratio of 1.7:1- A f i f t h treatment was fed in which the top calcium level was adjusted to provide a calcium:phosphorus ratio of 2-5:1. A l l diets fed contained an excess of Vitamin D. Each of the five mineral treatments was fed as a control diet, to which no supplemental fat was added, and as a high fat diet, in which 8% of ground cellulose was replaced by 8% animal fat. In one experiment the diets contained 2^% protein. In the other, the protein level was reduced to 20.6%. In the third experiment, to clarify the effect of adjusting the calcium:phosphorus ratio in the diet, three levels of calcium were fed with calcium:phosphorus ratios of approximately 1.5:1 and 2:1. As in the previous experiments, the different mineral levels were fed as low fat diets, and as high fat diets containing 8% animal fat. The diets fed In this experiment contained 23.6% protein. Criteria of calcium utilization used in a l l these experiments were rate of growth, feed efficiency, calcium balance (% of calcium retained) and calcium retention (calcium retained per 100 grams of gain in body weight). In addition, in the first two experiments, the per-centage of bone ash was determined on a sample of chicks from each group at the conclusion of the experiment. It was found that: 1. The calcium requirement for growth and feed efficiency was not affected by the addition of 8$ animal fat to the diet. The calcium requirement for maximum growth when the diet contained 24$ protein appeared to be between 1.0 and 1.25$ of the diet. A l l levels of calcium fed provided equal growth with diets containing 20.7$ protein. 2. The addition of 8$ fat to the diet significantly impaired bone calcification when the diet contained 24$ protein. Wo impairment was evident at the 20.7$ protein level. Diets containing 1$ calcium provided calcification equal to that provided by diets containing 1.25$ or 1.5$ calcium. 3« Calcium balance ($ of calcium retained) was found to be in-fluenced excessively by differences in feed efficiency. A more useful index of calcium utilization was calcium retent-ion, expressed in terms of body weight. k. The addition of 8$ animal fat to the diet impaired calcium retention (expressed as calcium retained per unit gain in weight) when the diet contained 1$ to 1.5$ calcium. In the case of the low fat diets, calcium retention rose as the level of calcium in the diet was increased to 1.25$, and remained constant as the calcium level was increased further to 1.5$. When fat was added to the diet, calcium retention rose more slowly as the calcium level was increased, and failed to reach the maximum retention of the low fat diets. The degree to which calcium retention was reduced was not sufficient to affect growth, i n the presence of vitamin D above the usual allowance. The addition of 8$ animal fat to diets containing 2k<$> protein increased the rate of growth and improved feed efficiency. The addition of fat to diets containing 20-7$ protein did not affect: the rate of growth and had l i t t l e , i f any, effect on feed efficiency. Adjusting the calcium:phosphorus ratio of the diet within the limits tested did not affect growth, cal c i f i c a t i o n , or calcium retention. I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. ACKNOWLEDGMENT The author takes this opportunity to thank Professor Jacob Biely, Head of the Department of Poultry Science, University of British Columbia, for making this study possible, and for his interest and encouragement. He also wishes to express his thanks to Mrs. B. E. March of the Department of :Poultry Science for her supervis-ion of the planning and execution of these experiments, and for her guidance in the preparation of this essay. The author is above a l l grateful to his wife, Judy, for her assistance in caring for the chicks used in these experiments, and for her unfailing encouragement and tolerance during the course of this study. TABLE OF CONTENTS Page INTRODUCTION . A 1 REVIEW OF LITERATURE 3 EXPERIMENT 1 INTRODUCTION 6 EXPERIMENTAL 7 RESULTS AND DISCUSSION . . . . 8 TABLES . 17 FIGURES 26 EXPERIMENT 2 INTRODUCTION . . 28 EXPERIMENTAL. 29 RESULTS AND DISCUSSION 29 TABLES . 31* FIGURES 39 EXPERIMENT 3 INTRODUCTION . . hi EXPERIMENTAL 'hi RESULTS AND DISCUSSION k2 TABLES 9^ FIGURES 55 DISCUSSION 57 CONCLUSIONS 63 LITERATURE CITED 65 INTRODUCTION There i s evidence that the presence of large amounts of fat i n the diet may impair the u t i l i z a t i o n of dietary calcium by experimental animals. The degree of impairment has been shown to be related to the type of fat fed. High melting, relatively i n -digestible fats, which form relatively insoluble calcium soaps i n the digestive tract, cause heavy losses of calcium i n the feces• Lower melting fats, which form more soluble calcium soaps, cause less heavy losses of calcium. This impairment of calcium u t i l i z a t i o n could be of pract-i c a l importance i f losses are sufficient to reduce the calcium ab-sorption to below that required for maximum growth or bone c a l c i f i c a -tion, which would in effect increase the calcium requirement. In view of this p o s s i b i l i t y , i t seemed desirable to i n -vestigate the effect of a fat available for use i n poultry rations on the calcium, retention of growing chicks and on the calcium re-quirement for growth and bone cal c i f i c a t i o n . I t was also of interest to study the effect of a moderate calcium'.phosphorus imbalance on calcium u t i l i z a t i o n under the conditions of high protein and fat levels i n the diet. In the course of these investigations, the effect of the level of dietary protein on calcium u t i l i z a t i o n was studied. Each of two levels of fat in the diet was fed at two protein levels. It was therefore possible to study the relationship between protein and fat levels in the diet for maximum growth. - 3 -REVIEW OF LITERATURE The effect of dietary fat on calcium absorption by experi-mental animals was first studied by Givens (1917) who reported that fecal calcium excretion was increased when the absorption of fat was poor. He fed dogs rations containing 12$ lard, palmitic acid, or ethyl or glyceryl palmitate. Palmitic acid was utilized to the extent of 80$, while lard was 9**$ utilized. Ethyl palmitate was only 53$ utilized. Givens found that the fecal calcium excretion was inversely proportional to the utilization of the fat. He concluded that in "poor utilization of fat or fatty acids may increase the excretion of lime in the feces and prevent the storage of calcium even when the calcium intake is comparatively abundant." In 1918 Bosworth concluded that soap formation in the in-testine is determined by the presence of soluble ionised calcium, while fecal calcium excretion depends not only upon the amount of calcium soaps formed but also on their solubility, since calcium oleate is much more soluble in intestinal fluids than calcium palmitate or calcium stearate. Boyd and Lyman (1929-30) and Boyd, Crum and Lyman (1932) also showed that calcium soaps may be well utilized, calcium oleate being absorbed to the extent of 9°-91$» although calcium stearate was only 25-^ 5$ absorbed and palmitate was 38-65$ absorbed (depending on the amount fed). The digestibility of fats has been shown to depend, in part - k -at least, on the melting point of the fat. In general, fats with melting points below 50°C. are well utilized (Deuel, 1955). The digestibility of most fats melting above this critical temperature is inversely proportional to the melting point (Holmes and Deuel, 1920). Cheng et al (19^9) presented data which support this con-clusion and showed that the digestibility of higher melting fats is reduced in the presence of excessive calcium or magnesium. Their coefficients of digestibility for some fats in the presence and ab-sence of calcium were as follows: bland lard (m.p.47.8°C.) 92.^% and 95.8%; blended lard (m.p. 55.2°C.) 66.2% and 80.0%; hydrogenated lard (m.p. 6l.0°C.) 17.3% and 38.0%; tristearin (m.p. 70°C.) 10.6% and 18.9%. The depressing effect of calcium on the utilization of high melting fats was confirmed by Rao and De ( l95l)• These in-vestigators fed rats cocoanut oil which had been fractionated on the basis of melting point. The presence of calcium salts in the diet caused no appreciable change in the digestibility of cocoanut oil (m.p.22°C), which was 98% digestible, or in the unsaturated (liquid) fraction. The digestibility of the saturated fraction (m.p. 52°C.), however, was reduced from 89.1% to 80.0% when calcium was included in the diet. While i t is true that these experiments were conducted primarily from the point of view of the effect of calcium and mag-nesium on the digestibility of fats, they show clearly that the presence of large quantities of high melting fats may be expected to - 5 -reduce calcium absorption. As Cheng pointed out, " i t is evident that a minimum calcium loss will occur when the dietary fats have low melt-ing points; however, when the melting point of the digested fat ex-o ceeds 50 C., one may expect a marked loss of calcium, proportional to the amount of such fat fed." - 6 -EXPERIMENT 1 INTRODUCTION In this experiment, a study has been made of the effect of calcium retention and on the calcium requirement for growth and bone calcification of the addition of fat to a diet containing 24.0% protein. The chicks were fed diets which consisted of the basal diet supplemented with ground limestone and tricalcium phosphate at the expense of ground cellulose to provide four levels of calcium, 0.76%, 1.03%, 1.21%, and 1.50%. The calcium:phosphorus ratio was adjusted to 1.7:1. except in the case of the low calcium diet in which the calcium: phosphorus ratio was 1.4:1. The effect of a cal-cium: phosphorus ratio imbalance on a high calcium diet was observed in chicks fed a diet containing 1.51% calcium with the calcium: phosphorus ratio adjusted to 2.5:1* The diets contained an excess of Vitamin D above the recommended allowance for normal diets. Each mineral treatment was tested with a control diet, to which no supplementary fat was added, and with a diet supplemented with 8% of animal fat, replacing 8% of ground cellulose in the control diet. The productive energy level of the basal diet was calculated to be 803 calories per pound of feed, on the basis of the productive energy values of poultry feedstuffs reported by Praps (19^6). The addition of 8% fat to the basal diet increased the energy level to 979 calories per pound. EXPERIMENTAL Day-old New Hampshire female chicks were distributed at random into 20 lots of approximately 17 chicks each. The chicks were wing-banded, vaccinated intraocularly against Newcastle dis-ease, and placed in electrically heated battery brooders, with free access to feed and water. At four weeks of age the chicks were moved to unheated wire batteries. The composition of the experimental diets is shown in Table 1:1. Each of the ten diets was fed to duplicate lots. The chicks were weighed individually at weekly intervals throughout the experiment. Feed consumption of each lot was recorded by weighing in feed daily and weighing back; the feed remaining at the end of the week. Calcium balance studies were carried out during the second and subsequent weeks of the experiment. During the early weeks of the experiment, an aliquot of each week's total production of feces was collected from each lot, and dried in an air oven at 100°C. The dried feces were ground in a pulverising mill and analysed for calcium by the official method of the Association of Official Agricultural Chemists (1955)• During the later weeks of the experiment, when i t became impractical to sample the f u l l week's accumulation of feces, the balance studies were conducted over a two day period of each week. At the conclusion of the experiment, when the chicks were six weeks of age, six birds from each lot, selected to be near the average weight for the lot, were killed and their left tibias removed for determination of ash content. The ash content was determined on - 8 -the pooled left tibias from each lot by the official method of the A.O.A.C. (1955). RESULTS AND DISCUSSION The average weights of the chicks at 3 and 6 weeks of age are summarized in Table 1:2. It will be seen from the table that on both low and high fat diets the heaviest chicks were those receiving 1.21% calcium. The average weights were analysed statistically by the method of Analysis of Variance (Table 1:3) • The average weight of the chicks which received 1.21% calcium was 2l6 gm. at 3 weeks. These chicks were significantly heavier than those fed 0.76% calcium, which aver-aged 195 gm. They were not, however, significantly heavier than the chicks receiving 1.03% calcium in the diet. The chicks fed 1.03% calcium were significantly heavier (209 gm«) than those on 0.76% calcium. Those receiving 1.51% calcium were significantly lighter (206 gm.) than those on 1.21% calcium. At 6 weeks, the average weight of the chicks consuming 1.21% calcium (551 gm.) was significantly greater than that of the chicks on the low calcium (0.76%) diet (498 gm.). The differences between these groups and the chicks on 1.03% and 1.50% calcium, which averaged 531 and 521 gm., respectively, were not statistically significant. It will be seen from Table 1:2 that the addition of 8% fat to the diet, which contained 24% protein, caused highly significant differences in weight at both 3 and 6 weeks. The boost in growth attributable to the addition of fat to the diet indicates that at the - 9 -24$ level of protein provided the energy content of the low fat diet did not provide sufficient caloric intake for maximum growth. Increasing the calcium:phosphorus ratio from 1.7 to 2.5 in diets containing 1.5$ calcium (diets k and 5, and 9 and 10) had l i t t l e effect on rate of growth, and did not affect the response to fat (Table 1:2). The feed efficiency of the birds on each diet at 3 and 6 weeks is shown in Table 4. The feed efficiency of the low fat groups was best in the case of the 1.21$ calcium diet (diet 3) at both 3 and 6 weeks, followed by diets 4, 5> and 1, which gave similar feed efficiency, 2.24 to 2.34 at 3 weeks and 2.64 to 2.73 at 6 weeks. Diet 2 (1.03$ Ca) gave the poorest feed efficiency (2.51 at 3 weeks, 2.74 at 6 weeks). The high values of feed efficiency shown by this group are difficult to explain in terms of the diets fed. Among the high fat diets, diet 8 (l .21$ calcium) gave the best feed efficiency at 3 weeks (1.75)> followed by diets 7 (l«03$ Calcium) and diet 9 (1.50$ calcium) with 1.8l in each case, diet 10 (l .51$ calcium) with 1.82, and diet 6 (0.76$ calcium) with 1.86. The high fat diets showed l i t t l e variation in feed efficiency at 6 weeks, ranging from 2.15 to 2.19. The results of the calcium balance studies have been expressed as "calcium balance" and as "calcium retention". The former term re-presents the percent of calcium ingested which was retained by the chicks in each group. "Calcium retention" indicates the calcium re-tained per hundred grams of gain in body weight. In the discussions - 10 -which follow, the terms "calcium balance" and "calcium retention" will be used as defined here. The values obtained for "calcium balance" are shown in Tables 5 and 6 and are presented graphically in Fig. 1:1. A study of the graphs in Fig. 1:1 reveals that the addit-ion of 8$ fat to the diet caused a reduction in calcium balance dur-ing the early weeks of growth, except in the case of the diet lowest in calcium. During the second week of the experiment, the chicks on diets 1 and 6, the low and high fat diets containing 0.76$ calcium, retained approximately 59$ of the calcium ingested. An increase in dietary cal-cium level to 1.03$ caused a drop in calcium balance of the high fat group to 55.4$. There was then a difference of approximately 13$ be-tween the calcium balance of the high and low fat groups. A further increase in calcium level to 1.21$ reduced the calcium balance of the high fat group to *i6$, 18$ below that of the chicks receiving the low fat diet of similar calcium content. The calcium balance of the high fat group receiving 1.50$ calcium was 38.3$> 5$ below that of the corresponding low fat group. The graphs of calcium balance for the later weeks of the ex-periment show that in general the calcium balance followed a similar pattern throughout the experiment. The calcium balance of the low fat, low calcium groups decreased to produce a flatter curve toward the end of the experiment. The calcium balance of the high fat groups decreased to a less marked degree so that during the final week of the experiment \ - 11 the calcium balance of the high fat group receiving 0.76% calcium was 51•6%» l6% above that of the corresponding low fat group. The calcium balance of the higher calcium groups was variable but tended to be higher in the case of the low fat groups receiving 1.03% and 1.21% calcium, early in the experiment. In later weeks, the difference between the high and low fat groups decreased until at the end of the experiment they showed similar calcium balance. The calcium balance of the groups receiving 1.50% calcium also tended to decrease more rapidly as the chicks became older in the case of the low fat groups than in the case of the high fat groups. As a result, during the final test period the low fat groups retained 35'5% of the calcium ingested, 8% less than the balance of the corres-ponding high fat groups. Increasing the calcium:phosphorus ratio- from 1.7:1 to 2.5:1 in the diet containing 1.5% calcium had no consistent effect on calcium balance. In the case of the low fat diets, the group lower in phos-phorus showed slightly lower calcium balance during the second and third weeks of the experiment, by 8% and 5%, respectively, and slightly higher balance during the third, fourth and f i f t h weeks by 10%, 4% and 8%, respectively, than the group receiving a normal calcium:phosphorus ratio. In the case of the high fat diets, the low phosphorus groups were lower in calcium balance by 1% during the fourth and fi f t h weeks, and higher by 7%« .6% and 13% during the second, third, and sixth weeks, respectively. The trend in this experiment was toward slightly higher - 12 -calcium balance in the groups low in phosphorus, but the differences were too small and inconsistent to be considered significant. The figures for percentage of dietary calcium retained (calcium balance) are of some value in showing the effect of the experimental treatments on calcium balance, but may have been in-fluenced by differences in feed-efficiency. For example, the groups receiving the diet containing 0.76$ calcium show a higher percentage of calcium retained in the case of the high fat diet than the low fat diet during the later weeks of the experiment. This difference seems to indicate that fat improves the utilization of calcium at this low level of dietary calcium. It is seen, however, from Table 1:4, that the feed efficiency of the high fat groups is some 20$ better than that of the low fat groups. The high fat groups therefore consumed less feed per unit of gain in weight, so that they consumed less feed, hence less calcium, than the low fat groupB. The higher calcium balance of the high fat groups indicates only that the birds retained a higher percentage of the calcium ingested. They may have actually retained no more calcium than the corresponding low fat groups. It is clear, then, that in order to assess the effect of the treatments on the retention of calcium by the chicks a measure of absolute calcium retention must be used. For this reason, to compensate for differences in feed efficiency and body weight between groups, "calcium retention" has been calculated; i.e. the calcium retained (grams) per 100 grams of gain in body weight. The values obtained for calcium retention are shown in - 13 -Tables 1:7 and 1:8, and are shown graphically in Fig. 1:2. A study of the graphs and tables reveals that the addi-tion of 8% fat to the diet markedly reduced the retention of calcium at a l l levels of dietary calcium fed, during the first five weeks of growth, and caused a less pronounced decrease in calcium retent-ion during the sixth week. The effect of fat in reducing calcium retention was most pronounced in the case of the diets containing 1.03% and 1.21% calcium, and less marked in the case of the diets containing 0.76% and 1.50% calcium. The graphs in Fig. 1:2, showing the weekly calcium retent-ion, show that this pattern holds, generally, during each week of the experiment, in spite of some variation in pattern from week to week, particularly in the case of the diets containing 1.50% cal-cium. The total calcium retention for the six-week experimental period was also calculated, and is shown in Tables 1:7 and 1:8 and graphed in Fig. 1:2. During the first two weeks of the experiment, calcium re-tention was measured only during the last three days of the two-week period. During each of the last two weeks, calcium retention was measured for two days of each week. In order to calculate calcium re-tention over each of these periods i t was necessary to assume that (for example, during the first two weeks) Ca retained (2 wk.)/ Ca retained (3 day test period) was equal to Feed consumed (2 wk.)/ Feed consumed (3 day test period). . - lk -An examination of the total calcium retention, in Fig. 1:2 and in Tables 1:7 and 1:8 reveals that the birds on the low fat diet containing 0.76$ calcium retained 0.97 gm. of calcium per hundred grams of total gain in weight. An increase in dietary calcium from 0.76$ to 1.03$ resulted in an increase in calcium retention from 0.97 gm- to I.36 gm. per 100 gm. of gain. This level of calcium appears to be sufficient for maximum calcium retention, since a further increase in dietary calcium, to 1.50$ of the diet caused no appreciable increase in calcium retention. In the case of the high fat diets, the birds receiving 0.76$ calcium in the diet retained 0.92 gm. of calcium per 100 gm. gain in weight, 5'2$ less than the birds on the corresponding low fat diet. As the calcium content of the diet was increased to 1.50$, I'the calcium retention rose steadily in proportion to the level of calcium fed. At the 1.03$ level, the birds retained 1.03 gm. per 100 gm. of gain, 24$ less than that retained by the birds on the low fat diet; at the 1.21$ level, they retained 1.11 gm. of calcium per 100 gm. of gain, 23$ less than that retained by the birds on the low fat diet. At the highest level of calcium fed, 1.50$, the birds on the high fat diet retained I.32 gm. of calcium per 100 gm. of gain, 9$ less than the retention of the birds on the corresponding low fat diet. The addition of 8$ fat to the diet impaired calcium retent-ion, with the greatest impairment occurring when fat was added to diets which permitted maximum calcium absorption in the absence of supplementary fat (l .03 to 1.21$ calcium). The addition of fat had - 15 -least effect on calcium retention when added to diets markedly de-f ic ient in calcium. The calcium retention of the low fat groups rose rapidly as the calcium leve l in the diet increased, reaching a maximum when the diet contained 1.21% calcium, and remained at that l eve l when the calcium leve l was increased further to 1.50%. In contrast, the calcium retention of the high fat groups rose less rapidly as the leve l in the diet increased, without reaching a maximum, and without reaching the leve l of calcium retention attained by the low fat groups. Increasing the calciumrphosphorus rat io from 1.7:1 to 2.5:1 i n the diet containing 1.5% calcium had l i t t l e effect on calcium re-tention. In the case of the low fat diets , the group lower in phos-phorus showed s l ight ly higher calcium retention, retaining a to ta l of 1.54 gm. of calcium per 100 gm. of gain, compared with 1.4-5 gm. per 100 gm. of gain, retained by the group receiving a normal leve l of phosphorus. In the case of the high fat d iets , the groups lower and higher i n phosphorus showed similar calcium retention, retaining a to ta l of 1.33 and I.32 gm. of calcium per 100 gm. of gain, respectively. The average values for the percentage of ash in the bones tested in each group are shown in Table 1:9. The s t a t i s t i c a l analysis of these data i s shown in Table 1:10. I t w i l l be seen from the Tables that the addition of fat to the diet s ignif icant ly reduced the ash con-tent in the bones. The percent ash in the bones of the chicks re-ceiving 0.76% calcium was s ignif icant ly lower than that of the groups receiving 1.21% or 1.5% calcium in the d ie t . There was no significant - 16'-difference in bone ash between the groups fed 0.76% and 1.03% calcium, or between those fed 1.03% and 1.21% calcium. Increasing the calcium:phosphorus ratio of the diets con-taining 1.5% calcium had no apparent effect on the bone ash of the chicks fed the high fat diets. Increasing the calcium:phosphorus ratio in the low fat diets from 1.7:1 to 2.5:1 resulted in an increase in bone ash from an average of 44.3% to 45%. It may be concluded from the results of this experiment that the addition of 8% animal fat to the diet impairs the retention of calcium by the chick. The addition of fat to the diet did not affect the calcium requirement for maximum growth which appeared to be 1.03% to 1.21% of the diet. The addition of 8% fat to the diet increased the calcium requirement for maximum retention to 1.5% or higher. Increasing the calcium:phosphorus ratio, in the presence of an excess of Vitamin D, from 1.7:1 to 2.5:1 did not affect growth or feed efficiency. - 17 -Table 1:1 Composition of diets, Experiment 1 A l l Diets Diet 1 Diet2 Diet 3 Diet 4 Diet 5 ingredient % % % % % % ground yellow corn 15-0 ground wheat 37.84 fishmeal (74% protein) 9*0 soybean o i l meal (44% protein) 1 8 . 3 dehydrated cereal gras6 2 .5 dried brewers yeast 3«0 dried skim milk 2 .5 feeding o i l (2250A-300D) 0 .25 salt (iodized) 0 .5 mineral mixture1 0.1 D P P D 0.0125 dl methionine 0.15 ground cellulose ^  0 ground limestone tricalcium phosphate protein content (%) calcium content (%) phosphorus content (%) Ca/p ratio 9.98 9 . 3 2 8.67 8 . 0 3 8 . 0 0 0 . 8 7 1 .18 1.09 O.98 2.50 -- 0 . 3 5 1 .09 1.84 0 . 3 5 24.01 2 3 . 8 6 24.15 24.03 24.10 O.76 1.03 1.21 1.50 1.51 0 . 5 3 0 . 6 0 . 7 5 0 . 9 0 . 6 1 .4:1 1 .7:1 1 .7 :1 1 .7:1 2 . 5 : 1 1. "Delamix", a trace mineral mixture containing: Mn 6.0%, I 0.12%, Fe 2.0%, Cu 0.2%, Zn 0.006%, Co 0.020%, Ca 27-0%. 2. Diphenyl - p - phenylenediamine. 3. In diets-6 - 10 inclusive, 8% of ground cellulose was replaced with 8% of stabilized animal fat ftsta - fat"). Otherwise the com-position of the diets was similar to that of.diets 1 to 5> res-pectively. "Sta - fat" contains not more than 0.02% butylated hydroxyanisole, 0.01% propyl gallate, and 0.004% citric acid. - 18 -Table 1:2 Average weights of chicks at 3 and 6 weeks, Experiment 1 low and high low fat diets high fat diets fat diets Ca in P in diet av wt av wt diet av wt av wt av wt av wt diet diet no. 3 wks 6 wks no. 3 wks 6 wks 3 wks 6 wks $ 1» (gm.) (gm.) (gm.) (gm.) (gm.) (gm.) O.76 0.53 1 1.77 499 6 203 552 1.83 46l 215_ 478 O o H^O 209 49o" 195 498 1.03 0.6 2 189 475 7 223 577 195 491 228 578 192 483 "22b" 5 ? S 209 531 1.21 0.75 3 199 503 8 226 584 204 522 233 202 513 230 506 216 551 1.50 0.9 4 197 512 9 219 544 186 484 218 541 192 H98" 219 54"3 206 521 Av 191 494 221 556 1.51 0.6 5 1.88 494 10 216 562 1.90 470 220 561 1TB9 482 2T5 502 -.19 -Table 1:3 Analysis of variance, average weights of chicks, Experiment 1 3 weeks Source of variation total calcium level fat level interaction error M. S. D. calcium level S.S. 4,619 924 3,451 219 6 weeks Source of variation S.S. total 25,780 calcium level 5,773 fat level 15,563 interaction 2,273 error 2,171 M. S. D. calcium level d.f. 15 3 1 4 variance 306 3,451 8 27.4 12 11.2** 126 ** d.f. 15 3 1 3 variance 1,924 15,563 758 271 7.10* 57.4 ** 2.80 - 20 -Table 1:4 Feed efficiency at 3 and 6 weeks, Experiment 1 low fat diet high fat diet $ Ca # P diet feed efficiency diet feed efficiency in in no 0-3 wks 0-6 wks no 0-3 wks 0-6 wks diet diet 0.76 0.53 1 2.34 2.64 6 1.86 2.19 1.03 0.6 2 2.51 2.74 7 I.81 2.16 1.21 0.75 3 2.02 2.54 8 1.75 2.17 1.50 0.9 4 2.28 2.65 9 1.81 2.19 1.51 0.6 5 2.24 2.73 10 1.82 2.15 - , . feed consumed feed efficiency - — : — 3 T - T - . " gain in weight - 21 -Table 1:5 Calcium balance, low fat diets, Experiment 1 Calcium balance, # 0 C a % P diet 11-14 15-21 22-28 33-35 to-42 0-42 in in no days days days days days days diet diet O.76 0.53 1 59-8 61.2 46.1 41.9 44.9 52.7 56.8 56.I 42.7 40.4 44.0 45.3 5BT3 5cT?f 4TT2 4T75 49T0 1.03 0.6 2 60.6 58.3 ^ . 0 37.9 43.1 46.5 66.7 62.2 46.3 46.1 45.9 51.7 S3T6 60.3 43Tl 42T0 44T5 4o\7 1.21 0.75 3 53.1 52.4 45.4 43.O 25.3 47.8 60.1 54.3 47.0 48.0 20.8^ 51.5 56T0" 53^ 5672 4 T ? 23.1 49T7 1.50 0.9 4 36.4 38.9 31-0 39.2 30.0 34.5 44.5 50.6 34.0 41.1 40.9 41.4 W% S O 32.5 1*oT2 35-5 3BT0 1.51 0.6 5 34.8 41.6 32.7 37-9 35-2 36.I 40.1 43.7 38.9 45.2 4 i .2 40.0 ypi 42T7 3T0 4i7eT 38^2 3S7T 1. Calcium balance - Ca ingested - Ca excreted x 1 0 0 % Ca ingested 2. Disregarded in calculating calcium balance (0-42 days) - 22 -Table 1:6 Calcium balance, high fat diets, Experiment 1 Calcium balance, # % Ca % p. diet 11-14 15-21 22-28 33-35 40-42 0-42 in in no days days days days days days diet diet O.76 0.53 6 61.2 64.3 54.3 44-3 52.6 53-5 58.9 59.8 59.0 57.4 50.5 56.3 59^6 6 l 3 50T6 50.9 51V6 54T9 1.03 0.6 7 55-0 60.O 42.7 50.9 kh.l2 46.6 55.8 57.0 44a i£j 25/f 55TT 58T5 4 T ¥ 5578 547i 37.6 47.4 39.0 38-8 40.0 30V3 46Tf 37.2 4o7T 38T5 1. Calcium balance - Ca i^ested^-^Ca^excreted x 1 0 0 £ 2. Disregarded in calculating calcium balance (0 - 42 days) 1.21 O.75 8 46.2 49.1 33.5 39-3 33-5 39-0 46.0 43.9 40. g 46.5 46.0 44-9 5o"7T 46T5 36*^ 9 3 9 3 Wfi 1.50 0.9 9 38.9 45.0 35.3 4l .3 36.9 38.9 40.0 39-5 1.51 0.6 10 42.8 48.6 3^.0 40.2 45-5 42.1 38.7 45.0 39-4 3^0 41^6 40.5 4o78 463 36T9 39Te7 4J7S 41T3 - 23 -Table 1:7 Calcium retention, low fat diets, Experiment 1 Calcium retention (gm. per ICO gm. gain) $ Ca # P diet 11-14 15-21 22-28 33-35 40-42 0-42 in in no days days days days days days diet diet 0.76 0.53 1 1.03 0.6 2 1.21 0.75 3 1.50 0.9 4 1.51 0.6 5 1. Calcium retention - Ca retained (grams) per 100 grams gain in body weight 2. Disregarded in calculating calcium retention (0-42 days) 1.16 O.96 1T00" 1.04 oTfcT 0.88 0.74 5TBT 1.00 0.92 OT90" 1.16 1.00 iToB" 1.47 1.72 175c" l . 4 i 1.42 m+2 1.17 1.14 1T10" 0.96 1.22 1.36 O f 1.27 1.44 1T30" I.38 1.34 1T30" 1.58 1^6 1.57 0.86 0.78, O 2 2 1.42 1.45 1754 1.16 1T31* 1.18 IT38" 1.01 i7o8 1.64 1.72 1. 1.30 2.06 1.27 1.62 I74"5 1.46 1.45 1. 1.16 1.33 1.77 1.89 1TB3 1.62 1.46 i 3 T 1.45 1.64 IT54-- 24 -Table 1:8 Calcium retention, high fat diets, Experiment 1 Calcium retention (gm. per 100 gm. gain) $ Ca # P diet 11-14 15-21 22-28 33-35 40-42 0-42 in in no days days days days days days diet diet 0. 76 O.53 6 1.03 0.6 7 1.21 0.75 8 1.50 0.9 9 1.51 0.6 10 1. Calcium retention - Ca retained (grams) per 100 grams gain in body weight 2. Disregarded in calculating calcium retention (0 - 42 days) 0.92 0.92 0.86 0.76 6TB9 oT8¥ 1,05 1.03 1..08 0.96 1.07 1.00 1.09 1.03 1.13 0.89 l . l l 0T90" 1.10 1.16 1.09 1.24 1.10 1.20 1.20 1.24 0.72 _ 0.87 1.02 * 0.93 0.72 O.9O 0.8 l O.9O 0.81 1.08 oTBT 0.99 0.69 1.18 0-97 1.40 0TB3 1.29 1.02 1 . 4 l 1.16 1.33 1.07 1.37 0.92 1.50 1-13 1-33 1.03 IT42 1.10 0.90 134 0.92 1.10 1.02 4.852 1.04 1.10 1.03 1.13 1.01 1.60 1.21 1.37 L U 1.89 1.31 1.72 1.32 ITST 1.32 1.73 1.33 1.81 1.33 1.77 1.33 - 25 -Table 1:9 Bone ash at 6 weeks, Experiment 1 high and low fat low fat diet high fat diet diets % Ca in diet % P in diet diet av bone no ash diet no av bone ash * av bone ash % O.76 0.53 1 43.9 6 42.2 42.5 WX 43.0 1.03 0.6 2 43.7 43.4 w% 7 43.4 ^3.3 43.5 1.21 0-75 3 44.2 43.6 53T9 8 43.1 43.7 5|T4 43.7 1.50 0.9 4 43.8 9 44.2 43.4 44.1 av diets 1-4 43.9 Av diets 6-9 43.3 1.51 0.6 5 44.2 45.7 45T0 10 43.5 43.2 43Tf Table 1:10 Analysis of variance, and 6 to 9> inclusive, bone ash at 6 weeks, diets 1 to 4, Experiment 1 Source of variation s . s . d.f. variance tt^ tt total calcium level fat level interaction error 5.66 2.08 1.56 0.76 15 3 1 -1 O.69 I.56 0.25 0.16 4.31* 9.75* 1.56; M. S. D. calcium treatments 0.7 11-1*4- days 15-21 days 22-2S days 75 50 - 25 CO c • H +> co fc cd u 75 CD CJ C as rH cd I 50 • H O r H CC o 25 33-35 days 0.75 i . o o 1.25 1.50 Calcium In diet {%)' kO-K2 days o„75 l . o o 1.25 1.50 control d i e t -I I i u 0-1+2 days x 0.75 1.90 1.25 1.50 x * fat added FIGURE 1:1 Calcium balance, Experiment 1, 1 ro l 11-14 days 1.5 1.0 0 . 5 -fc CD P . +3 CO bO C -H <H CO cd > CD C fc -H Cd C o -H — ' cd bo c o • +3 hO c coo T r-CD H fc 33-35 days _ _ X a •H o H cd o 1.0 0 . 5 -0.75 1.00 1.25 1.50 Calcium i n die t {%) 15-21 days _l ; l_ 40-42 days s-22-28 days — -1 i— 0-42 days 0.75 1.00 1.25 1.50 0.75 1.00 1.25 1.50 . . control diet x. _« &% fat added FIGURE 1:2 Calcium retention, Experiment 1. 1 ro I - 28 -EXPERIMENT 2 INTRODUCTION In experiment 2, a study was carried out with diets contain-ing 20.6% protein similar to the study which was conducted on 24% protein diets in Experiment 1. As in Experiment 1, four levels of calcium were fed, 0.74%, 1.01%, 1.22%, and 1.51%. The calcium:phosphorus ratio was from 1.5:1 to 1.7:1. A fifth diet containing 1.54% calcium, in which the calcium: phosphorus ratio was adjusted to 2.6:1$ was fed in order to determine i f there were an effect of calcium:phosphorus imbalance with the high-est level of calcium. As in the previous experiment, an excess of Vitamin D was provided. Each mineral treatment was tested with a control diet, to which no supplementary fat was added, and with a diet supplemented with 8% of animal fat replacing 8% of ground cellulose in control diet. The productive energy level of the basal diet was calculated to be 8l4 calories per pound of feed on the basis of the productive energy values of poultry feedstuffs reported by Fraps (1946). The addition of 8% fat to the basal diet increased the energy level to 990 calories per pound. - 29 -EXPERIMENTAL Day-old New Hampshire chicks were distributed into 10 lots of 25 chicks each. The chicks were wing banded, vaccinated intra-ocularly against Newcastle disease, and placed in electrically heated battery brooders. The chicks had free access to feed and water. The composition of the experimental diets is shown in Table 2:1. The chicks were weighed individually at weekly intervals throughout the experiment. Feed consumption of each lot was recorded weekly. Calcium balance studies were carried out during each week of the experiment as described in Experiment 1. The experiment was terminated when the chicks were 3 weeks of age. Six birds from each lot, selected to be near the average weight of the lot, were killed. The left tibia was removed from each bird for determination of ash content. The bone ash content was determined on the individual left tibias. RESULTS AND DISCUSSION The average weights and feed efficiency of chicks in Experi-ment 2 at 3 weeks of age are summarized in Table 2:2. It will be seen from the table' that the experimental treat-ments caused no significant difference in average weight of the chicks fed the various diets at 3 weeks. The average weight of chicks in a l l groups was 193 gm«> with no group deviating from this average by more than 7 gm. The fact that the addition of 8$ fat to the diet caused no appreciable increase in rate of growth indicates that, at the 20.7$ - 30 -level of protein, provided in this experiment, the optimum caloric intake for utilization of the available protein for growth was pro-vided by the low fat diet. The addition of fat to the diet resulted in some improve-ment in feed efficiency. The average feed efficiency was improved from an average of 2.07 gm. of feed consumed per gm. of gain in the case of the low fat diets to an average of 1.9% gm. of feed per gm. of gain in the case of the high fat groups. Increasing the calcium:phosphorus ratio from 1.7:1 to 2.6:1 in diets 5 and 10, containing 1.5$ calcium, had l i t t l e or no effect on body weight or feed efficiency. The results of the calcium balance studies have been ex-pressed, as in Experiment 1, as "calcium balance", i.e., the percent of ingested calcium retained, and as "calcium retention", the calcium retained per 100 gm. of gain in body weight. The values obtained for calcium balance are shown in Table 2:3 and are presented graphically in Fig. 2:1. A study of the graphs in Fig. 2:1 reveals that the addition of 8$ fat to the diet had l i t t l e effect on the percent of calcium re-tained by the chicks. The table and graph of calcium balance for the 21-day period of the experiment indicate that at the 0.74$ and 1.22$ levels of calcium the chicks receiving 8$ fat in the diet retained 52-55$ of the calcium ingested, about 4$ more than the groups receiving the corresponding low-fat diets. At the 1.01$ and 1.51$ levels of calcium the high fat groups retained only 46$ of the calcium ingested, or 22$ less than the corresponding low-fat groups. The weekly graphs - 31 -of calcium balance show considerable variation in pattern. There i s , in the graphs for 0-7 and 8-l4 days, a depression of calcium balance from 2-20% when 8% fat is added to the diet. This effect is not evident in the graph of the date for 15-21 days, or the graph of total calcium balance (0*21 days). The graphs reveal no effect on calcium balance of increas-ing the level of calcium in the diet. It will be seen from the graph showing calcium balance for the 21-day period of the experiment, and from Table 2:3> that the chicks in a l l groups retained between If5.8% and 57.8% of the calcium ingested. Increasing the calcium:phosphorus ratio of the high calcium diet resulted in a drop in calcium balance of the chicks fed the low fat diets from 54.6% to 48.1% of the calcium consumed. The calcium balance of the chicks fed the high fat diets increased from 45.9% to 47.2% when the calcium:phosphorus ratio was increased. Calcium retention (calcium retained per 100 gm. of gain in body weight) is shown in Table 2:4 and graphed in Fig. 2:2. A study of the graphs and the table reveals that the addit-ion of 8% fat to the rations reduced the retention of calcium at a l l levels of dietary calcium fed, except the 0.74% level, at which level the addition of tallow did not appear to affect calcium retention. It is seen from Table 2:4 that increasing the level of cal-cium from 0.74% to 1.51% resulted in an increase in total (0-21 days) calcium retention from 0.77 to 1.79 in the case of the chicks fed the low fat diets- With the chicks fed the high fat diets the range in cal-- 32 -cium retention was only from O.87 to 1.35 when the calcium level in the diet was increased from 0.7%$ to 1.51$« The fluctuations in the calcium retention curves due to experimental error are not sufficient to obscure the differences attri-butable to the experimental diets. The average percentage of bone ash in each group is shown in Table 2:5. These data are analyzed statistically in Table 2:6. It will be seen from the table that the addition of fat to the diets did not significantly affect calcification. There was, however, a highly significant increase in ash content of the bones when the calcium level was increased from 0.74$ to 1.01$. Further increases in diet-ary calcium level did not significantly affect calcification. An increase in the calcium:phosphorus ratio of the 1.5$ calcium group from 1.69:1 to 2.57:1 caused l i t t l e or no difference in calcification. In the case of the high fat groups, the high ratio group showed calcification of 43-9$ ash, compared with 44.0$ in the group receiving the more normal calcium:phosphorus ratio. In the case of the chicks fed the low fat diets, the values for ash content were again not affected by the modification of calcium:phosphorus ratio. It is concluded from the results of this experiment that the addition of 8$ animal fat to a diet containing 20.7$ protein did not affect the growth rate to 3 weeks of age, but resulted in some improvement in feed efficiency. Increasing the level of calcium in the diet from 0.74$ to 1.51$ caused no difference in average weight at 3 weeks. - 33 -The addition of fat to the diet had l i t t l e or no effect on calcium balance (percentage, of calcium retained) but reduced calcium retention (calcium retained per 100 gm. of gain in body weight) at a l l levels of calcium except the 0.74% level. Increasing the level of calcium in the diet did not affect calcium balance but there was an increase in calcium retention as the calcium level was increased. Bone calcification was not influenced by the addition of fat to the diet. Calcification was improved by increasing the diet-ary calcium level from 0.74% to 1.01%. Increases in calcium level above 1.01% did not affect calcification. Increasing the calcium:phosphorus ratio at the high calcium level in the presence of an excess of Vitamin D did not affect growth, calcium balance,: calcium retention or bone calcification. - 34 -Table 2:1 Composition of diets, Experiment 2 ingredient A l l Diets Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 ground wheat 6l.91 fishmeal (74$ protein) 6.0 soybean o i l meal (44$ protein) 12.0 dehydrated cereal grass 2.5 dried brewers' yeast 3.0 dried skim milk 2.5 feeding o i l (2250A-300D) 0.25 salt (iodized) 0.5 mineral mixture1 0.1 D P P D 2 0.0125 dl methionine 0.15 ground cellulose^ ground limestone tricalcium phosphate protein content ($) calcium content ($) phosphorus content ($) Ca/P ratio 1. "Delamix", a trace mineral mixture containing: Mn 6.0$, I 0.12$, Fe 2.0$, Cu 0.2$, Zn 0.006$,Co 0.020$, Ca 27-0$. 2. Diphenyl - p - phenylenediamine. 3. In diets 6 - 1 0 inclusive, 8$ of ground cellulose was replaced with 8$ of stabilized animal fat ("Sta - fat"). Otherwise the composition of the diets was similar to that of diets 1 to 5, respectively. "Sta - fat" contains not more than 0.02$ butylated hydroxyanisole, 0.01$ propyl gallate, and 0.004$ citric acid. 9.0 8.68 8.35 8.04 8.00 0.57 0.59 0.54 0.48 1.27 0.30 0.68 1.05 0.30 20.67 20.35 20.52 20.84 20.62 0.74 1.01 1.22 1.51 1.54 0.48 0.6 0.75 0.9 0.6 1.5:1 1.7:1 1.6:1 1.7:1 2.6:1 - 35 -Table 2:2 Average weight of chicks and feed efficiency at 3 weeks, Experiment 2 low fat diets nigh fat diets % Ca % P diet av wt feed diet av wt feed in in no 3 wks gain no 3 wks gain diet diet (gm) 0-3 wks (gm) 0-3 wks 0.74 0.48 1 196 2.12 6 193 1-99 1.01 0.6 2 190 2.17 7 198 1.93 1.22 0.75 3 193 1.92 8 195 1.89 1.51 0.9 k 186 2.07 9 198 1.89 1.54 0.6 5 190 2.07 10 186 2.01 „ , „„. , feed consumed feed efficiency - — ; — . . . . J gain in weight - 36 -Table 2:3 Calcium balance, Experiment 2 Calcium balance, $ $ Ca in diet *P in diet diet no 0-7 days 8-14 days 15-21 days 0-21 days low fat diets 0.74 0.48 1 32.2 62.5 49.2 52.8 1.01 0.6 2 61.3 56.1 54.3 57.5 1.22 0.75 3 64.2 53-0 43.6 50.4 1.51 0.9 4 67.8 60.0 46.3 54.6 1.54 0.6 5 57-2 50.1 41.6 48.1 nigh fat diets 0.74 0.48 6 52.4 57.7 54.0 55-0 1.01 0.6 7 60.3 51.1 35-6 45.8 1.22 0.75 8 61.6 51.9 50.5 52.1 1.51 0.9 9 55.8 48.1 40.5 45.9 1.5% 0.6 10 51.9 50.3 43.3 47.2 Ca ingested - Ca excreted Calcium balance - Ca ingested - 37 -Table 2:4 Calcium retention, Experiment 2 Calcium retention (gm. per 100 gm. gain) f> Ca <$> P in in diet 0-7 8-14 15-21 0-21 diet diet no days days days days low fat diets 0.74 0.48 1 O.96 1.12 O.69 0.77 1.01 0.6 2 1.54 1.34 1.18 1.30 1.22 0.75 3 1.84 1.39 1.05 1.29 1.51 0.9 4 2.27 2.17 1.38 1.79 1.54 0.6 5 1.84 1.71 I.36 1-59 high fat diets 0.74 0.48 6 O.96 0.95 0.79 O.87 1.01 0.6 7 1.27 0.97 0.62 0.85 1.22 0.75 8 1.64 1.47 1.01 1.23 1.51 0.9 9 1.72 1.46 1.14 1.35 1.54 0.6 10 1.63 1.54 1.33 1.46 Calcium retention - Ca retained (grams) per 100 grams gain in body weight - 38 -Table 2:5 Bone ash at 3 weeks, Experiment 2 high and low fat low fat diets high fat diets diets % Ca % P av bone av bone av bone in in diet ash diet ash ash diet diet no % no % % 0.74 0.48 1 40.4 6 41.6 41.0 1.01 0.6 2. 43.4 7 43.0 43.2 1.22 0.75 3 44.4 8 43.1 43.8 1-51 0.9 4 44.3 9 44.0 44.2 av 1-4 43.1 av 6-9 42.9 1.54 5 43.4 10 ^3.9 Table 2:6 Analysis of variance, bone ash at 3 weeks, diets 1 to 4 and 6 to 9} inclusive, Experiment 2 Source of variation s.s. d.f. variance It£>tt total 147.88 47 calcium level 71.72 3 23.9 14.6** fat level 0.42 1 0.42 interaction error 10.19 65.55 3.40 1.60 2.07 M. S. D. Calcium treatments 1.1 0-7 days &-lk days 75 -i i i 1 — _. _ I—i 1 1 1 — 0.75 L o o 1.25 1-50 0.75 1.00 1.25 1.50 Calcium i n diet {%) . „ control diet x- -* f a t added r FIGURE 2:1 Calcium balance, Experiment 2. + 3 + 3 © X! fc hO I i • 1 1 I « 1 1 L cd co • c I I 1 1 I L 1 1 1 0.75 i . o o 1.25 1-50 0.75 1.00 1.25 1.50 Calcium i n di e t (%) . . control diet x- 6% fat added FIGURE 2:2 Calcium retention, Experiment 2. - kl -EXPERIMENT 3 INTRODUCTION In this experiment, further study was made of the calcium requirement for maximum growth and feed efficiency on a relatively high protein diet (23.6% protein). Three calcium levels were provided ranging from 0.95% calcium to 1.35% calcium, thus covering the range in which the calcium requirement was shown to f a l l in Experiment 1. The possible effect of an imbalance in calcium:phosphorus ratio on calcium utilization was not conclusively established in the earlier experiments. In this experiment, two levels of phosphorus in the diet were provided at each of the 3 levels of calcium fed. Each level of calcium was fed in a diet in which the calcium:phosphorus ratio was approximately 1.6:1, and a diet in which the calcium phos-phorus ratio was adjusted to approximately 2 :1. The calcium:phosphorus ratio was adjusted by varying the levels of tricalcium phosphate and ground limestone in the diet. Each mineral treatment was tested with a control diet, to which no supplementary fat was added, and with a diet supplemented with 8% animal fat, replacing 8% of ground cellulose in the control diet. The diets were similar in productive energy to those of Experi-ment 1. The basal diet contained 803 calories per pound, calculated from the productive energy values of Fraps (l9k6), and the high fat diet, 979 calories per pound. EXPERIMENTAL Day-old New Hampshire female chicks were distributed into 1 - k2 -2k lots of 25. The chicks were wing banded, vaccinated intraocularly against Newcastle disease. They were reared in electrically heated battery brooders with free access to feed and water. The composition of the experimental diets is shown in Table 3:1. The chicks were weighed individually at weekly intervals throughout the experiment. Feed consumption of each lot was recorded weekly. Calcium balance studies were carried out each week as des-cribed in Experiment 1. The values were determined for the entire week during each of the first four weeks and for a one day period during each of the final weeks of the experiment. The experiment was terminated when the chicks were six weeks old. RESULTS AND DISCUSSION The average weights of the chicks at 3 and 5 weeks of age are shown in Table 3:2. The addition of 8$ animal fat to the basal diet signific-antly increased the average weight at 3 and 5 weeks. The chicks fed the high fat diets averaged 217 gm. At 5 weeks, the chicks fed the low and high fat diets averaged 363 and 423 gm., respectively. The various modifications in calcium and phosphorus levels of the diets did not significantly affect growth to 3 weeks of age. At 5 weeks, however, there were significant differences in weight be-tween the groups fed the various calcium and phosphorus levels. There - 43 -was also a significant interaction between the mineral and fat treat-ments . In the case of the groups fed diets containing both a normal and an adjusted calcium:phosphorus ratio the chicks fed 1.10$ calcium in the diet were lighter in average weight at 5 veeks (379-384 gm.) than those fed 0.95$ or 1.34$ calcium, which ranged in weight from 396-1402 gm. (average of low and high fat diets). The difference in weight betweens the lots fed 1.10$ and 1.34$ calcium was statistically significant. The significant interaction between the mineral and fat treatments resulted largely from the difference in response of lots 1 and It compared to the response of the other lots to the addition of 8$ fat to the basal diet. Lots 1 and 7 were fed 0.95$ calcium with a normal calcium:phosphorus ratio. The chicks fed diet 1, the low fat diet, weighed 394 gm. at 5 weeks in contrast to the chicks fed the other low fat diets, which ranged in weight from 347 to 365 gm. When fat was added to the diet, the chicks fed diet 7' averaged 399 gm. at 5 weeks, only 5 gm. heavier than the chicks fed the corresponding low fat diet. In contrast, the chicks fed the other high fat diets ranged in weight from 410 to 446 gm., 62-89 gm« heavier than the chicks fed the corresponding low fat diets. If the weights of the chick fed diets 1 and 7 are not con-sidered, i t is seen that the diets containing 1.34$ calcium resulted in significantly greater growth than the diets containing 1.10$ calcium. There was no significant difference in weight between the chicks fed 0.95$ and 1.10$ calcium in the diet. It is therefore concluded that - 44 -increasing the level of calcium in the diet from 0.95% to 1.34% resulted in an increase in growth rate. Adjusting the calcium:phosphorus ratio in the diet did not affect the growth rate. There were no significant differences in weight between the treatments corresponding in calcium level but differing in calcium:phosphorus ratio. The feed efficiency of the birds on each diet at 3 and 5 weeks is shown in Table 3:4. The addition of fat to the diets im-proved feed efficiency, from an average of 2. l6 lb. of feed per lb. of gain at 3 weeks to an average of 1.79• At 5 weeks feed efficiency was improved from an average of 2.47 to an average of I.98 by the addition of fat to the diet. The results of the calcium balance studies have been ex-pressed, as in Experiment 1, as "calcium balance", i.e., the percent of ingested calcium retained, and as "calcium retention", the cal-cium retained per 100 gm. of gain in body weight. The values obtained for "calcium balance" are shown in Table 3:5 and are presented graphically in Fig. 3:1« A study of Table' 2:1 and the graphs in Fig. 5 indicates that the addition of 8% fat to the diet improved calcium balance. This is shown most clearly in the graph of calcium balance for the 0-35 day experimental period. This graph and Table 3:5 show that in both the groups receiving a normal calcium:phosphorus ratio and the groups receiving an increased ratio of calcium to phosphorus, the addition of 8% fat to the diet increased calcium balance by 4% to 29% at a l l levels of calcium fed. The values of calcium balance - 4 5 -of the chicks fed the low fat diets ranged from 36.8$ to 47.6$. On the high fat diets the range in calcium balance was from 43.8$ to 55.4$. The graphs show clearly that calcium balance was higher in the case of the low calcium diet (0.95$ calcium) than in the case of the diets containing 1.10$ or 1.34$ calcium. During the f u l l period of the experiment, the chicks receiving the low fat diet with a normal calcium:phosphorus ratio retained 47.6$ of the calcium consumed, when the diet contained 0.95$ calcium. When the calcium level was increased to 1.10$ the calcium balance f e l l to 39.2$. As the calcium level was increased further to 1.35$, the calcium balance f e l l less sharply to 36.8$. The other diets, which contained supple-mentary fat and/or an increased calcium:phosphorus ratio, resulted in similar patterns of calcium balance. Adjusting the calcium:phosphorus ratio from approximately 1.5:1 to 1.9:1 - 2.25:1 had l i t t l e effect on calcium balance. In the case of the low fat diets, calcium balance was reduced by 10$ by increasing the calcium:phosphorus ratio when.the diet contained 0.95$ calcium, by 3$ when the calcium level was increased to 1.10$, and was increased by 5$ when the calcium level was increased to 2.35$ of the diet. In the case of the diets containing 8$ fat, calcium balance was increased by 12$ by increasing the calcium:phosphorus ratio when the diet contained 0.95$ calcium, by 11$ when the calcium level was increased to 1.10$, and reduced by 2$ when the calcium level was increased to 2.35$ of the diet, reversing the pattern shown by the - k6 -low fat diets. In view of the absence of any consistent effect of adjusting the calcium:phosphorus ratio in either the low or high fat diets, i t is concluded that the differences in calcium balance which appeared between the calcium:phosphorus ratio treatments were not due to the diets fed. The values obtained for "calcium retention" (calcium retained per 100 gm. of gain in weight) are shown in Table 3:6 and are pres-ented graphically in Fig. 3:2. A study of the graph of calcium retention for the 35 - day experimental period may be expected to give the most accurate informa-tion on the effect of the experimental diets on calcium retention in this experiment. The addition of 8$ fat to the diet caused some impair-ment in calcium retention. The average calcium retention of the groups receiving the low fat diets at each of the three levels of calcium provided was O.96, O.95, and 1.15 gm. of calcium per 100 gm. of gain for the diets containing 0.95, 1.10 and 1.35$ calcium, respectively. Similar diets, with 8$ fat added, resulted in retention of 0.93, 0.86, and 1.02 gm. of calcium per 100 gm. of gain. The effect of the level of calcium in the diet on calcium retention i 6 not readily apparent from the graph. The graph reveals a tendency for calcium retention to remain approximately level or to drop as much as 0.10 gm. per 100 gm. of gain (from O.91 gm. per 100 gm. of gain) as the calcium level was increased from 0.95$ to 1.10$ of the diet, then to increase by 0.10 to 0.28 gm. when the calcium level was increased further to 1.35$. This behavior does not follow the - kl-expected pattern. Calcium retention might be expected to rise as the level of calcium in the diet is increased up to a point of maximum calcium retention, followed by l i t t l e or no further increase in re-tention as the level of calcium In the diet is increased further, within reasonable limits. No explanation is apparent for the f a i l -ure of the calcium retention values shown to follow the expected pattern. No effect on calcium retention of adjusting the calcium: phosphorus ratio within the limits included in this experiment is apparent. Increasing the calcium:phosphorus ratio of the low fat diets appeared to result in a reduction in calcium retention at the two lower levels of calcium fed, and an increase at the high level of calcium. Increasing the calcium:phosphorus ratio of the high fat diets appeared to reverse this pattern. Calcium retention was in-creased at the two lower levels of calcium and decreased at the high level of dietary calcium. It is concluded that adjusting the calcium: phosphorus ratio had l i t t l e or no effect on calcium balance. It is concluded from the results of this experiment that the addition of 8% animal fat to a diet containing 23.6% protein signi-ficantly increased the growth rate and feed efficiency, but did not affect the calcium requirement for maximum growth. Increasing the level of calcium in the diet from 0.95% to 1.35% resulted in an increase in growth rate. The addition of fat to the diet increased calcium balance (per cent of calcium retained)at a l l levels of calcium provided, but - 48 -appeared to have l i t t l e or no effect on calcium retention, expressed in terms of calcium retained per unit of gain in "body weight. The effect of the level of calcium in the diet on calcium "balance and calcium retention is not clearly indicated by the data presented. Increasing the calcium:phosphorus ratio from 1.46 - 1.58:1 to 1.90 - 2.25:1 in the presence of an excess of Vitamin D, did not affect the rate of growth, and appeared to have l i t t l e or no effect on calcium utilization by the chick. - 49 -Table 3:1 Composition of diets, Experiment 3 ingredient A l l Diets Diets Diets Diets $ 1 & 4 2 & 5 3 & 6 ground yellow corn 15-0 ground wheat 3T-8i*-fishmeal (74$ protein) 9.0 soybean o i l meal (44$ protein) 18.3 dehydrated cereal grass 2.5 dried brewers' yeast 3'0 dried skim milk 2.5 feeding o i l (2250A - 300D) 0.25 salt (iodized) 0.5 mine ral mixture1 0.1 D P P D 0.0125 dl methionine , 0.15 ground cellulose"5 ground limestone tricalcium phosphate protein content ($) calcium content ($) ' phosphorus content diets 1-3 ($) phosphorus content diets 4-6 ($) Ca/Pratio, diets 1-3 Ca/Pratio, diets 4-6 9.32 8.67 8.03 1.18 1.09 O.98 0.35 1.09 1.84 23.54 23.71 23.57 0.95 1.10 1.34 0.6 0.75 0.9 0.5 0.55 0.6 l ; 5 8:l 1.46:1 1.48:1 1.90:1 2.00:1 2.25:1 1. "Delamix", a trace mineral mixture containing: Mn 6.0$, I 0.12$, Fe 2.0$, Cu 0.2$, Zn 0.006$, Co 0.020$, Ca 27.0$. 2. Diphenyl - p - phenylenediamine. 3. In diets 4 - 6 inclusive, 8$ of ground cellulose was replaced with 8$ of stabilized animal fat ("Sta - fat"). Otherwise the composition of the diets was similar to that of diets 1 to 5> respectively. "Sta - fat" contains not more than 0.02$ butylated hydroxyanisole, 0.01$ propyl gallate, and 0.004$ citric acid. - 50 -Table 3:2 Average weight of chicks at 3 and 5 weeks, Experiment 3 low and high fat low fat diets high fat diets diets Av wt Av wt Av wt Av wt diet 3 wks 5 wks 3 wks 5 wks no (gm.) (gm.) (gm.) (gm.) % Ca % P Av wt Av wt in in diet 3 wks 5 wks diet diet no (gm.) (gm.) 0.95 0.6 1 210 394 1.10 0.75 2 199 347 1.34 0.9 3 I83 357 0.95 0.5 4 189 361 1.10 0.55 5 176 353 1.34 0.6 6 193 365 Av 192 363 7 205 399 208 397 8 205 410 202 379 9 230 446 207 402 10 225 430 207 396 11 216 415 196 384 12 222 ^37 208 401 Av 217 423 M. S. D. mineral treatments (5 weeks) - 51 -Table 3:3 Analysis of variance, average weights of chicks, Experiment 3 3 weeks Source of variation S.S. d.f. variance total mineral treatments fat level interaction error 612,253 5,425 49,592 26,183 531,053 296 5 1 285 1,085 49,592 5,237 1,863 26.6** 2.81* 5 weeks Source of variation S.S. d.f. variance f total 891,640 295 • mineral treatments 21,793 5 S359 2.24* fat level 267,748 1 267,748 138.** interaction 49,407 5 9,881 5-08** error 552,692 284 1,946 S. D. mineral treatments ^ i -L 1 7 - 52 -Table 3:4 Feed efficiency * at 3 and 5 weeks, Experiment 3 low fat diets h i S n * a t diets $ Ca % p feed/ feed/ feed/ feed/ in in diet gain gain diet gain gain diet diet no 0-3 wks 0-5 wks no 0-3 wks 0-5 wks 0.95 0.6 1 2.05 2.35 7 1.86 2.05 1.10 0.75 2 2.09 2.49 8 1.83 2.05 1.34 0.9 3 2.23 2.46 9 I.83 I.87 0.95 0.5 4 2.18 2.46 10 1.76 2.00 1.10 0.55 5 2.23 2.50 11 1.72 1.95 1.34 0.6 6 2.15 2.54 12 I.76 1.94 Av 2.16 2.47 Av 1.79 1.98 ^ . . feed consumed * Feed efficiency - g a l n ^ v e i g h t - 53 -Table 3:5 Calcium balance, Experiment 3 Calcium balance, % # Ca in diet % p in diet . diet no 8-14 days 15-21 days 26-28 days 33-35 days 0-35 days low fat diets 0.95 0.6 1 53-1 46.8 52.0 39-0 47.6 1.10 0.75 2 44.9 44.2 40.3 27.5 39.2 1.34 0.9 3 36.7 31.0 46.5 31-3 36.8 0.95 0.5 4 50.9 53.0 41.4 30.4 43.0 1.10 0.55 5 44.3 41.2 41.8 27.5 38.2 1.34 0.6 6 31.6 43.2 46.1 34.8 38.8 high fat diets 0.95 0.6 7 48.3 59.^  47.8 46.0 49.7 1.10 0.75 8 42.3 44.1 46.4 28.7 40.8 1.34 0.9 9 41.1 53-2 46.6 38.2 44.6 0.95 0.5 10 53.8 46.8 57.1 61.3 55.^ 1.10 0.55 11 45.2 40.6 53 A 40.2 45.6 1.34 0.6 12 45.2 40.0 47.2 41.7 43.6 Calcium balance - Ca ingested - Ca excreted Ca Ingested - 54 -Table 3:6 Calcium retention, Experiment 3 Calcium retention i. per ICO gm. gain) $ Ca in diet $ p in diet diet no 8-14 days 15-21 days 26-28 days 33-35 days 0-35 days low fat diets 0.95 0.6 1 1.01 1.00 1.24 0.86 1.04 1.10 0.75 2 0.97 1.05 1.16 O.78 1.00 1.34 0.9 3 0.97 1.06 I.56 O.85 1.10 0.95 0.5 4 0.88 1.09 O.89 O.63 0.88 1.10 0.55 5 0.95 1.11 O.98 0.59 0.89 1.34 0.6 6 O.QO 1.32 1.60 1.01 1.20 high fat diets 0.95 0.6 7 0.79 1.14 0.93 0.80 0.91 1.10 0.75 8 0.79 0.95 0.94 0.54 0.81 1.34 0.9 9 0.99 1.44 1.25 0.84 1.09 0.95 0.5 10 O.83 O.78 1.12 0.99 0.94 1.10 0.55 11 0.81 0.80 1.26 0.74 0.91 1.34 0.6 12 0.97 0.91 1.17 O.96 0.99 Calcium retention - Ca retained (grams) per 100 grams gain in body weight 75 50 25 CO c «H CO CD U CO o CD O e cO H 75 co S 50 «H O iH CO 25 O g-ll+ days 26-2S days 0.75 1.00 !v 25 1.50 Calcium in diet {%) 15-21 days 33-35 days 0.75 1.00 1.25 1.50 , control diet, normal Ca:P ratio fat added, nominal" Ca :P' t»atio A + control diet, high Ca:P-ratio + + fat added, high Ca:P ratio 0-35 d a ys 0.75 1.00 1.25 1.50 VJ1 VJl 1 FIGURE 3:1. Calcium balance, Experiment 3. 1.5 l . o 0.5L. S-lif daya 15-21 days +• +-• • ' control diet, normal Ca:P r a t i o J 1 i_ x x 07b fat added, normal Ca:P r a t i o control d i e t , high Ca:P r a t i o +• + fat added, high Ca:P r a t i o 1.5 1.0 0.51-26-28 daya 0.75 1.00 1.25 1.50 Calcium i n di e t (%) 33-35 days -J 1 1 i_ 0.75 1.00 1.25 1.50 0-35 clays -J 1 1 i_ 0.75 1.00 1.25 1.50 I VJl o\ 1 FIGURE 3:2 Calcium retention, Experiment 3. - 5 7 -DISCUSSION The addition of 8% animal fat to the diet of growing chicles did not affect the calcium requirement for growth and feed efficiency. When the diet contained 20.7% protein, there was no difference in growth rate of chicks fed different calcium levels, which ranged from 0.75% to 1.50% of the diet. In one of the two experiments in which the diets contained 24% protein, levels of calcium ranging from 0.75% to 1.50% were provided. Significant differences in average weight of chicks were observed between calcium levels, with 1.21% calcium provid-ing the most rapid growth in both the low fat diets and those contain-ing 8% supplementary fat. In the other high protein experiment, in which only 3 levels of calcium were fed, l i t t l e increase in growth re-sulted from an increase in calcium level from 0.95% to 1.35% of the diet. The failure of the chicks to show a consistent growth re-sponse to differences in calcium level may be explained by the fact that the diets fed in these experiments contained an excess of Vitamin D of 2.5 to 3 times the recommended level for a diet containing 20% protein (A.O.A.C. Nutrient Requirements for Poultry, 1954). The pre-sence of an excess of Vitamin D above the required level reduced the effect of differences in calcium level by promoting the absorption of calcium from the digestive tract. The apparent growth response in Experiment 1 which resulted from an increase in dietary calcium level from the recommended level, 1%, to 1.21% of the diet, suggests that at the higher protein level an increase in dietary calcium above the - 58 -present recommended level may be desirable. Further experiments, in which graded levels of calcium are fed with lower levels of Vitamin D than were used in these experiments, will be required to establish clearly the requirement of the growing chick for calcium for maximum rate of growth with diets containing high levels of protein. The failure of 8$ fat to affect the growth response of chicks to calcium at either level of protein shows that the fat did not seriously impair calcium absorption. It is possible that the high level of Vitamin D in the diets, by promoting the absorption of calcium, overcame a tendency of the fat to increase fecal calcium excretion. A more likely explanation for the failure of fat to affect the calcium requirement is indicated by the work of Cheng et al (1949) and Rao and De (1951) with rats, which was reviewed earlier. The results of these groups of workers suggest that although the in-gestion of high melting fats might be expected to lead to heavy losses of ingested calcium in the feces, low melting fats (m.p. below o V 50 C) may cause relatively l i t t l e increase in fecal calcium excretion. Under some conditions, in fact, with diets high in cereals which are high in phytin phosphorus, the presence of fat was shown by McDougall (1938) with rats, to promote calcium absorption by the formation of soluble calcium soaps with fatty acids, instead of the formation of an insoluble compound with the phytin in the diet. Since the fat used in the present experiments was a low-melting product which would lead to the formation of largely soluble calcium soaps in the digestive tract, i t should cause relatively l i t t l e impairment of calcium absorp-tion. - 59 -The results of bone ash analyses carried out in Experiments 1 and 2 showed that in both low and high protein diets, whether or not fat was added to the diet, the level of calcification produced by diets containing 1% calcium was equal to that produced by higher levels of calcium. Calcification at the 0.75% level of calcium was significantly inferior to calcification produced by 1% calcium at both the 20.7% and 24% levels of protein. At the 24% protein level the addition of 8% fat to the diet significantly depressed bone calcification. The addition of fat to the 20.7% protein diet did not depress calcification. The data on bone calcification suggest that the level of calcium required for maximum calcification may be lower than that required for maximum growth rate, at least in the presence of sufficient Vitamin D. It is also apparent that the addition of animal fat to the diet caused greater impairment of calcium utilization, measured by the level of bone calcification, when the diet contained 24% pro-tein than at lower levels of protein. The results of the calcium balance studies, which were carried out in each of these experiments, showed that the addition of fat to the diet was associated with a reduction in calcium retention. Calcium balance, expressed as the per cent of ingested cal-cium which was retained by the chick, is a less valuable index of calcium utilization than calcium retention, expressed as calcium re-tained per hundred grams of gain in weight, in view of the differences in feed efficiency which resulted from the addition of fat to the diet. - 60 -In Experiments 1 and 3> in which the diets contained 24% protein, the addition of fat to the diet tended to improve calcium balance. The effect was most marked in the low calcium diets of Ex-periment 1. In Experiment 2, in which the diets contained 20.6% pro-tein, fat had l i t t l e effect on calcium balance. Since in Experiments 1 and 3. feed efficiency was markedly improved by the addition of fat to the diet, the improvement in calcium balance may merely reflect the decreased calcium intake per unit of body weight, rather than an in-crease in absolute calcium retention. This difficulty has been over-come by expressing calcium retention in terms of body weight or gain in weight from week to week. Differences in body composition may, of course, introduce an error in values for calcium retention expressed in terms of body weight. In a l l experiments, calcium retention (grams per hundred grams gain in weight) was impaired by the addition of fat to the diet. The effect was less marked at the low level of calcium (0.75%) in Experiments 1 and 2 than at the higher levels fed. In view of the growth and bone ash data, however, which reveal no difference in cal-cium requirement for growth or calcification when fat was added to the diet, i t Is concluded that the extent to which calcium retention was lowered by the addition of 8% fat to the diet was not of practical importance, in spite of the slight depression of calcification when fat was added to diets containing 24% protein. It remains possible, though, that with a lower level of Vitamin D in the diet, the impair-ment of calcium retention might be of greater significance. It is - 6i -possible, also, that the reduced calcium retention found on addition of fat to the diet may be due less to an impairment of calcium absorp-tion by the chick than to a relatively smaller size of skeleton in the more rapidly growing chicks on the high fat diets, in view of the fact that l i t t l e difference in calcification was found between the groups on high and low fat diets. Further experiments, which should include analyses of body composition and calcium distribution in chicks receiving diets of these types, will be required to test this hypothesis. The addition of 8$ animal fat to diets containing 24$ pro-tein in Experiments 1 and 3 caused a highly significant increase in growth rate and a marked improvement in feed efficiency. In Experi-ment 2, in which the diets contained 20.7$ protein, the addition of 8$ fat to the diet caused no appreciable improvement in rate of growth and l i t t l e change in feed efficiency from that shown by the groups re-ceiving the low fat diets. The fact that the addition of fat to a 20.7$ protein diet failed to improve growth or feed efficiency, while markedly improving both growth and feed efficiency in a 24$ protein diet, is in agreement with the findings of Biely and March (1954). It was reported that the addition of fat to a 10$ protein diet actually depressed growth and feed efficiency, while the addition of fat to a 24$ or 28$ protein diet either stimulated or failed to affect growth, and improved feed efficiency. Adjusting the calciumrphosphorus ratio in the diet within the limits tested in these experiments, in the presence of an excess of Vitamin D, did not affect growth or calcification and had no effect - 62 -on calcium balance or calcium retention in these experiments. This conclusion is in agreement with the findings of Gershoff and Hegsted (1956) that in the presence of Vitamin D the calcium: phosphorus ratio (varied from 4:1 to 1:2) had no significant effect on calcium absorption in the gastrointestinal tract of chicks, while in the absence of Vitamin D the calcium:phosphorus ratio in-fluenced calcium absorption. The high calcium:phosphorus ratio of approximately 2.5=1 provided in these experiments was regarded by Wilgus (1931) as borderline when fed in a ration containing 16.5$ protein. It is therefore possible that a similar calcium: phosphorus ratio, in diets containing the higher levels of pro-tein used in these experiments, but with a limited level of Vitamin D, might seriously interfere with growth and normal bone calcifica-tion. The results of the present experiments suggest that when the diet contains a moderate excess of Vitamin D the calcium-.phos-phorus ratio may be varied considerably without adverse effect on growth and bone formation. - 63 -CONCLUSIONS 1. The calcium requirement of growing chicks for growth and feed efficiency was not affected by the addition of 8% animal fat to the diet, in the presence of an excess of Vitamin D. The calcium re-quirement for maximum growth when the diet contained 24% protein appeared to be between 1.0 and 1.25% of the diet. All levels of cal-cium fed provided equal growth with diets containing 20.7% protein. 2. The addition of 8% animal fat to the diet significantly:im-paired bone calcification when the diet contained 24% protein. No impairment was evident at the 20.7% protein level. Diets containing 1% calcium resulted in calcification equal to that obtained with diets higher in calcium. 3« Calcium balance (% of calcium retained) was found to be in-fluenced excessively by differences in feed efficiency. A more useful index of calcium utilization was calcium retention, expressed in terms of body weight. 4. The addition of fat to the diet impaired calcium retention (expressed as calcium retained per unit gain in weight). In the case of the low fat diets, calcium retention rose as the level of calcium in the diet was increased to 1.25%, and remained constant as the cal-cium level was increased further to 1.5%. When fat was added to the diet, calcium retention rose more slowly as the calcium level was in-creased, and failed to reach the maximum retention of the low fat diets. The degree to which calcium retention was impaired was not sufficient to affect growth, in the presence of an excess of Vitamin D above the - 6k -usual allowance. 5. The addition of 8$ animal fat to diets containing 24$ pro-tein increased the rate of growth and improved feed efficiency. The addition of fat to diets containing 20.7$ protein did not affect the rate of growth and had l i t t l e , i f any, effect on feed efficiency. 6. Adjusting the calcium:phosphorus ratio of the diet within the limits tested did not affect growth, calcification, of calcium retention. 1 - 65 -LITERATURE CITED A.O.A.C., 1955. Methods of Analysis, 8th ed. Association of Of f i c -i a l Agricultural Chemists, Washington. Biely, J., and B. E. March, 1954. Fat studies i n poultry 2. Fat supplements i n chick and poultry rations. Poul. Sci.33: 1220-1227. Bosworth, A.W., H.I. Bowditch, and L.A. Giblin, 1918. The digestion and absorption of fats 1. Calcium i n i t s relation to the absorption of fatty acids. Am. J . Dis. Child. 15:397-407-Boyd, Oscar F-, Carlos L. Crum, and J.F. Lyman, 1932. The absorption of calcium soaps and the relation of dietary fat to calcium u t i l i z a t i o n i n the white rat. J . Bi o l . Chem. 95:29-41. Boyd, O.F., and J.F. Lyman, 1930. Utilization of calcium soaps by the white rat. Proc. Soc. Exp. B i o l . Med. 27:871-873. Cheng, Amber L.S., Margaret G. Morehouse, and Harry J . Deuel, J r . , 1949. The effect of the level of dietary calcium and mag-nesium on the d i g e s t i b i l i t y of fatty acids, simple t r i g l y -cerides, and some natural and hydrogenated fats. J . Nut. 37:237-250. Deuel, Harry J . J r . , 1955. The l i p i d s , their chemistry and biochem-is t r y , Vol. 11. Interscience, New York. Fraps, G. S., 1946. Composition and productive energy of poultry feeds and rations. B u l l . Texas Agric. Expt. Sta. #678. Gershoff, S.N., and D.M. Hegsted, 1956. Effect of Vitamin D and Ca:P ratios on chick gastrointestinal tract. Am. J . Physiol 187:203-206. Givens, Maurice H., 1917 • Studies i n calcium and magnesium meta-bolism III. The effect of fat and fatty acid derivatives. J . B i o l . Chem. 31:441-444. Holmes, A.D., and H.J. Deuel J r . , 1920. D i g e s t i b i l i t y of certain miscellaneous vegetable fats. J . B i o l . Chem. 41:227-235. McDougall, E.J., 1938. The counteraction by fat of the anti-calcify-ing action of cereals. Bioch. J . 32:194-202. N. R. C , 1954. Nutrient requirements for poultry. National Research Council Publication #301. - 66 -Rao, M. Narayana, and S.S. De, 1951. The effect of the level of dietary calcium on the digestibility of the different fatty-acid fractions of coco-n;ut o i l . Ind. Jour. Med. Res. 39:457-464. Wilgus, H.S., Jr., 1931' The quantitative requirement of the grow-ing chick for calcium and phosphorus. Poul. Sci. 10:107-117' 

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