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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 t h i s t h e s i s as conforming t o the standard required from candidates f o r the degree o f MASTER OF SCIENCE IN ACrRICtgLTURE  Members o f the Department o f 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$, 1.0%, phosphorus ratio of 1.7:1-  1.25% and 1.50%, with a calcium:  A f i f t h treatment was fed i n which the  top calcium level was adjusted to provide a calcium:phosphorus ratio of 2-5:1.  A l l diets fed contained an excess of Vitamin D. Each of the five mineral treatments was fed as a control  diet, to which no supplemental fat was added, and as a high fat diet, in which 8% of ground cellulose was replaced by 8% animal fat. In one experiment the diets contained 2^% protein.  In the  other, the protein level was reduced to 20.6%. In the third experiment, to clarify the effect of adjusting the calcium:phosphorus ratio i n the diet, three levels of calcium were fed with calcium:phosphorus ratios of approximately 1.5:1  and 2:1.  As i n the previous experiments, the different mineral levels were fed as low fat diets, and as high fat diets containing 8% animal fat. The diets fed In this experiment contained 23.6% protein. Criteria of calcium utilization used i n a l l these experiments were rate of growth, feed efficiency, calcium balance (% of calcium retained) and calcium retention (calcium retained per 100 grams of gain  in body weight).  In addition, in the f i r s t two experiments, the per-  centage of bone ash was determined on a sample of chicks from each group at the conclusion of the experiment. It was found that: 1.  The calcium requirement for growth and feed efficiency was not affected by the addition of 8$ animal fat to the diet. The calcium requirement for maximum growth when the diet contained 24$ protein appeared to be between 1.0 and of the diet.  1.25$  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. impairment was evident at the 20.7$ protein level.  Wo  Diets  containing 1$ calcium provided calcification equal to that provided by diets containing 1.25$ 3«  or 1.5$  calcium.  Calcium balance ($ of calcium retained) was found to be i n fluenced excessively by differences i n feed efficiency.  A  more useful index of calcium utilization was calcium retention, expressed i n terms of body weight. k.  The addition of 8$ animal fat to the diet impaired calcium retention (expressed as calcium retained per unit gain i n 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$, and  remained constant as the calcium l e v e l was increased further to 1.5$.  When f a t was added to the d i e t , calcium retention  rose more slowly as the calcium l e v e l was increased, and f a i l e d t o reach the maximum retention of the low f a t d i e t s . The degree t o which calcium retention was reduced was not s u f f i c i e n t t o a f f e c t growth, i n the presence of vitamin D above the usual allowance. The addition o f 8$ animal f a t t o d i e t s containing 2k<$> protein increased the rate o f growth and improved feed e f f i c i e n c y . The addition o f f a t t o d i e t s containing 20-7$ p r o t e i n did not affect: the rate o f growth and had l i t t l e , i f any, e f f e c t on feed e f f i c i e n c y . Adjusting the calcium:phosphorus r a t i o o f the d i e t within the l i m i t s tested d i d not a f f e c t growth, c a l c i f i c a t i o n , or calcium retention.  In presenting the  this  r e q u i r e m e n t s f o r an  thesis in partial  advanced degree at the  of B r i t i s h Columbia, I agree that it  freely  agree t h a t for  available  the  f o r r e f e r e n c e and  permission for extensive  s c h o l a r l y p u r p o s e s may  D e p a r t m e n t o r by  fulfilment  be  s h a l l make  study.  I  the  gain  s h a l l not  Department  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a .  Columbia,  Head o f  thesis my  I t i s understood  copying or p u b l i c a t i o n of t h i s t h e s i s a l l o w e d w i t h o u t my  further  copying of t h i s  that  be  University  Library  g r a n t e d by  his representative.  of  for  written  financial  permission.  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 supervision of the planning and execution of these experiments, and for her guidance i n the preparation of this essay. The author i s above a l l grateful to his wife, Judy, for her assistance in caring for the chicks used i n these experiments, and for her unfailing encouragement and tolerance during the course of this study.  TABLE OF CONTENTS .  INTRODUCTION  Page 1  A  3  REVIEW OF LITERATURE EXPERIMENT  1  INTRODUCTION  6  EXPERIMENTAL  7  RESULTS AND DISCUSSION  . . . .  8  . 17  TABLES  26  FIGURES EXPERIMENT 2  . . 28  INTRODUCTION EXPERIMENTAL.  29  RESULTS AND DISCUSSION  29  TABLES FIGURES  .  3* 1  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 d i e t may  impair the u t i l i z a t i o n o f d i e t a r y calcium  experimental animals.  by  The degree of impairment has been shown to  be related to the type of f a t fed.  High melting, r e l a t i v e l y i n -  d i g e s t i b l e f a t s , which form r e l a t i v e l y insoluble calcium soaps i n the digestive t r a c t , cause heavy losses of calcium i n the feces• Lower melting f a t s , which form more soluble calcium soaps, cause l e s s heavy losses of  calcium.  This impairment of calcium u t i l i z a t i o n could be of practi c a l importance i f losses are s u f f i c i e n t to reduce the calcium  ab-  sorption to below that required f o r maximum growth or bone c a l c i f i c a t i o n , which would i n e f f e c t increase the calcium requirement. In view of t h i s p o s s i b i l i t y , i t seemed desirable to i n vestigate the e f f e c t of a f a t available f o r use i n poultry rations on the calcium, retention o f growing chicks and on the calcium r e quirement f o r growth and bone c a l c i f i c a t i o n .  I t was  also o f i n t e r e s t  to study the e f f e c t 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 l e v e l s i n the d i e t . In the course o f these investigations, the e f f e c t of the l e v e l of d i e t a r y 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 experimental animals was f i r s t 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$,  while lard was  utilized.  9**$  utilized.  Ethyl palmitate was only  53$  Givens found that the fecal calcium excretion was inversely  proportional to the utilization of the fat. He concluded that i n "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 i s comparatively abundant." In 1918  Bosworth concluded that soap formation i n the i n -  testine i s 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 i s much more soluble i n intestinal fluids than calcium palmitate or calcium stearate. Boyd and Lyman  (1932)  (1929-30)  and Boyd, Crum and Lyman  also showed that calcium soaps may be well utilized, calcium  9°-91$» although calcium palmitate was 38-65$ absorbed  oleate being absorbed to the extent of stearate was only  25-^5$ absorbed  and  (depending on the amount fed). The digestibility of fats has been shown to depend, i n 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 conclusion 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 absence 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 o i l 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 o i l (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 magnesium 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 i s evident that a minimum calcium loss w i l l occur when the dietary fats have low melting points; however, when the melting point of the digested fat exo 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 i n the case of the low calcium diet i n 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 i n 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 disease, and placed i n 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 i s shown i n 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 l o t was recorded by weighing i n 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 l o t , and dried i n an a i r oven at 100°C. The dried feces were ground i n a pulverising m i l l and analysed for calcium by the o f f i c i a l method of the Association of O f f i c i a l 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 l o t , selected to be near the average weight for the l o t , were killed and their l e f t tibias removed for determination of ash content. The ash content was determined on  - 8 -  the pooled l e f t tibias from each lot by the o f f i c i a l 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 i n Table 1:2. It w i l l 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 averaged 195 gm.  They were not, however, significantly heavier than  the chicks receiving 1.03% calcium i n 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 s t a t i s t i c a l l y significant. It w i l l 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 i n 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 i n 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 i s shown i n 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 d i f f i c u l t to explain i n 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 i n 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 i n 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 i n each group.  "Calcium retention" indicates the calcium re-  tained per hundred grams of gain i n body weight.  In the discussions  - 10 -  which follow, the terms "calcium balance" and "calcium retention" w i l l 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 addition of 8$ fat to the diet caused a reduction in calcium balance during the early weeks of growth, except i n 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 calcium level to 1.03$ group to 55.4$.  caused a drop in calcium balance of the high fat  There was then a difference of approximately 13$ be-  tween the calcium balance of the high and low fat groups. A further increase i n 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. fat group receiving 1.50$  The calcium balance of the high  calcium was 38.3$> 5$ below that of the  corresponding low fat group. The graphs of calcium balance for the later weeks of the experiment 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 f i n a l 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 i n the case of the low fat groups receiving 1.03% and 1.21% calcium, early i n 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 i n the  case of the low fat groups than i n 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 corresponding 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 i n 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 i n calcium balance by 1% during the fourth and f i f t h weeks, and higher by 7%« .6% and 13% during the second, third, and sixth weeks, respectively.  The trend i n this experiment was toward slightly higher  - 12 -  calcium balance i n 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 i n fluenced by differences in feed-efficiency.  For example, the groups  receiving the diet containing 0.76$ calcium show a higher percentage of calcium retained i n 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. 1:4,  It i s seen, however, from Table  that the feed efficiency of the high fat groups i s 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 i s clear, then, that i n 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 i n body weight. The values obtained for calcium retention are shown i n  - 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 addition of 8% fat to the diet markedly reduced the retention of calcium at a l l levels of dietary calcium fed, during the f i r s t five weeks of growth, and caused a less pronounced decrease i n calcium retention during the sixth week. The effect of fat in reducing calcium retention was most pronounced i n the case of the diets containing 1.03% and 1.21% calcium, and less marked i n 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, i n 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 i s shown i n Tables 1:7 and 1:8  and  graphed i n Fig. 1:2. During the f i r s t two weeks of the experiment, calcium retention 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 retention over each of these periods i t was necessary to assume that (for example, during the f i r s t 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, i n Fig. 1:2 and i n 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 i n an increase i n 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 i n calcium retention. In the case of the high fat diets, the birds receiving 0.76$ calcium i n the diet retained 0.92 gm. of calcium per 100 gm. gain i n 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 retention, with the greatest impairment occurring when fat was added to diets which permitted maximum calcium absorption i n the absence of supplementary fat ( l . 0 3 to 1.21$ calcium).  The addition of fat had  - 15 -  l e a s t effect on calcium retention when added to d i e t s markedly def i c i e n t i n calcium.  The calcium retention of the low fat groups rose  rapidly as the calcium l e v e l i n the d i e t increased, reaching a maximum when the d i e t contained 1.21% calcium, and remained at that l e v e l when the calcium l e v e l was increased further to  1.50%.  In contrast,  the  calcium retention of the high fat groups rose l e s s r a p i d l y as the l e v e l i n the d i e t increased, without reaching a maximum, and without reaching the l e v e l of calcium retention attained by the low fat groups. Increasing the calciumrphosphorus r a t i o from 1.7:1 to  2.5:1  i n the d i e t containing 1.5% calcium had l i t t l e effect on calcium r e tention.  In the case of the low fat d i e t s , the group lower i n phos-  phorus showed s l i g h t l y higher calcium retention, retaining a t o t a l of 1.54 gm. of calcium per 100 gm. of g a i n , compared with 1.4-5 gm. per 100 gm. of g a i n , retained by the group receiving a normal l e v e l of phosphorus.  In the case of the high fat d i e t s ,  the groups lower and  higher i n phosphorus showed s i m i l a r calcium retention, retaining a t o t a l of 1.33 and I.32 gm. of calcium per 100 gm. of g a i n ,  respectively.  The average values for the percentage of ash i n the bones tested i n each group are shown i n Table 1:9. of these data i s shown i n Table 1:10.  The s t a t i s t i c a l  analysis  I t w i l l be seen from the Tables  that the addition of fat to the d i e t s i g n i f i c a n t l y reduced the ash content i n the bones.  The percent ash i n the bones of the chicks r e -  ceiving 0.76% calcium was s i g n i f i c a n t l y lower than that of the groups receiving 1.21% or 1.5% calcium i n the d i e t .  There was no s i g n i f i c a n t  - 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 containing 1.5% calcium had no apparent effect on the bone ash of the chicks fed the high fat diets.  Increasing the calcium:phosphorus  ratio i n the low fat diets from 1.7:1 to 2.5:1 resulted i n 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, i n 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 %  ingredient  ground yellow corn ground wheat fishmeal (74% protein) soybean o i l meal (44% protein) dehydrated cereal gras6 dried brewers yeast dried skim milk feeding o i l (2250A-300D) salt (iodized) mineral mixture 1  DPPD  d l methionine ground cellulose ^ ground limestone tricalcium phosphate protein content (%) calcium content (%) phosphorus content (%) Ca/p ratio 1.  Diet 1 Diet2 Diet 3 Diet 4 Diet 5 % % % % %  15-0 37.84 9*0 18.3 2.5 3«0 2.5 0.25 0.5 0.1  0.0125  0.15 0  9.98 0.87  9.32 1.18 0.35  8.67 1.09 1.09  8.03 O.98  8.00 2.50 0.35  24.01  23.86 1.03 0.6 1.7:1  24.15  24.03  24.10 1.51 0.6 2.5:1  --  O.76 0.53 1.4:1  1.21 0.75 1.7:1  1.84  1.50 0.9 1.7:1  "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 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% c i t r i c acid.  - 18 -  Table 1:2  Average weights of chicks at 3 and 6 weeks, Experiment 1  low fat diets Ca i n P i n diet diet  $  1»  O.76  0.53  1.03  1.21  1.50  0.6  0.75  0.9  diet no.  1  2  3  4  Av  1.51  0.6  5  av wt 3 wks (gm.)  av wt 6 wks (gm.)  1.77 1.83  499 46l  high fat diets diet av wt av wt no. 3 wks 6 wks (gm.) (gm.) 6  203 215_ 209  552 478 49o"  223 228 "22b"  577 578  226 233 230  584  Oo  H^O  189 195 192  475 491 483  7  199 204 202  503 522 513  8  197 186 192  512  9  219 218 219  544  484 H98"  191  494  221  556  216 220 2T5  562 561 502  1.88  1.90 1TB9  494 470 482  10  5?S  506  541  54"3  low and high fat diets av wt 3 wks (gm.)  av wt 6 wks (gm.)  195  498  209  531  216  551  206  521  -.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.  d.f.  4,619 924 3,451 219  15 3 1  4  variance  306 3,451 8 27.4 12  11.2** 126 **  6 weeks Source of variation total calcium level fat level interaction error M. S. D. calcium level  S.S.  d.f.  25,780 5,773 15,563 2,273  15 3 1  2,171  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 in diet  # P in diet  diet no  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 efficiency "  feed efficiency  0-3 wks 0-6 wks  feed consumed - — : — 3 T-T-. gain i n weight  diet no  feed efficiency  0-3 wks 0-6 wks  - 21 -  Table 1:5  Calcium balance, low fat diets, Experiment 1  Calcium balance, # 0 in diet  % P in diet  diet no  11-14 days  15-21 days  22-28 days  33-35 days  to-42 days  0-42 days  O.76  0.53  1  59-8 56.8 5BT3  61.2 56.I 5cT?f  46.1 42.7  41.9 40.4 4TT2  44.9 44.0 4T75  52.7 45.3 49T0  1.03  0.6  2  60.6 66.7 S3T6  58.3 62.2 60.3  ^.0 46.3  43Tl  37.9 46.1 42T0  43.1 45.9 44T5  46.5 51.7 4o\7  53.1 60.1 56T0"  52.4 54.3 53^  45.4 47.0 5672  43.O 48.0 4T?  25.3 20.8^ 23.1  47.8 51.5 49T7  36.4 44.5 W%  38.9 50.6 S O  31-0 34.0 32.5  39.2 41.1 *oT2  30.0 40.9 35-5  34.5 41.4 3BT0  34.8 40.1 ypi  41.6 43.7 42T7  32.7 38.9 3T0  37-9 45.2 4i7eT  35-2 4i.2 38^2  36.I 40.0 3S7T  C  a  1.21  1.50  1.51  0.75  0.9  0.6  3  4  5  1  1.  Calcium balance - Ca ingested - Ca excreted Ca ingested  2.  Disregarded i n calculating calcium balance ( 0 - 4 2 days)  x  1 0 0  %  - 22 -  Table 1:6  Calcium balance, high fat diets, Experiment 1  Calcium balance, # % Ca in diet  % p. in diet  O.76  0.53  1.03  0.6  diet no  6  7  11-14 days  15-21 days  22-28 days  33-35 days  40-42 days  0-42 days  61.2 58.9  64.3 59.8  54.3 59.0  44-3 57.4  52.6 50.5  53-5 56.3  59^6  6l3  50T6  50.9  51V6  54T9  55-0 55.8 55TT  60.O 57.0 58T5  42.7  44a  4T¥  i£j  25/f  kh.l  50.9  2  5578  46.6  547i  1.21  O.75  8  46.2 46.0 5o"7T  49.1 43.9 46T5  33.5 40. g 36*^9  39-3 46.5  33-5 46.0 393  39-0 44-9 Wfi  1.50  0.9  9  38.9 37.6 30V3  45.0 47.4  46Tf  35.3 39.0 37.2  4l.3 38-8 4o7T  36.9 40.0 38T5  38.9 40.0 39-5  42.8 38.7 4o78  48.6 45.0 463  3^.0 39-4 36T9  40.2 3^0 39Te7  45-5 41^6 4J7S  42.1 40.5 41T3  1.51  0.6  10  1.  Calcium balance  2.  Disregarded i n calculating calcium balance (0 - 42 days)  - Ca i^ested^-^Ca^excreted  x  1 0 0  £  - 23 -  Table 1:7  Calcium retention, low fat diets, Experiment 1 Calcium retention (gm. per ICO gm. gain)  $ Ca in diet  0.76  1.03  1.21  #P in diet  0.53  0.6  0.75  diet no  1  2  11-14 days  15-21 days  22-28 days  33-35 days  40-42 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  l.4i 1.42  1.17 1.14 1T10"  0.96  1.36  1.22  O f  1.58 1^6 1.57  0.86 0.78,2  O2  1.42 1.45 1754  1.64 1.72 1.  1.30 2.06  1.27 1.62  1.77 1.89 1TB3  1.62 1.46 i3T  175c"  I.38 1.34 1T30"  3  1.50  0.9  4  1.51  0.6  5  m+2  1.16  1.18  1.01  1T3 *  IT38"  i7o8  1.46 1.45 1.  1.16  1  1.33  0-42 days  1.27 1.44 1T30"  I74"5 1.45 1.64  IT54-  1. Calcium retention - Ca retained (grams) per 100 grams gain i n body weight 2. Disregarded i n calculating calcium retention (0-42 days)  - 24 -  Table 1:8  Calcium retention, high fat diets, Experiment 1 Calcium retention (gm. per 100 gm. gain)  $ Ca in diet  0. 76  1.03  1.21  # P in diet  O.53  0.6  0.75  11-14 days  15-21 days  0.92  6  0.92 0.86 6TB9  7  diet no  8  33-35 days  40-42 days  0.76  0.87 0.93 O.9O  1.10  0.90  oT8¥  0.72 _ 1.02 * 0.72  134  0.92  1,05 1..08 1.07  1.03 0.96 1.00  0.8l 0.81 oTBT  O.9O 1.08 0.99  1.10 1.10  1.03  1.09  1.03 0.89 0T90"  0.69 0-97 0TB3  1.18  1.13  1.40  1.29  1.13 1.60 1.37  1.01 1.21 L U  1.24  1.16  1.02 1.16 1.07  1.4l 1.33 1.37  1.89  1.72  1.31 1.32  ITST  1.32  1.24  0.92 1-13  1.50 1-33  1.73  1.33 1.33 1.33  l.ll  1.50  0.9  9  1.10 1.09 1.10  1.51  0.6  10  1.20  1.20  22-28 days  1.03  IT42  4.85  0-42 days  1.02 2  1.81  1.77  1.04  1.  Calcium retention - Ca retained (grams) per 100 grams gain i n body weight  2.  Disregarded i n calculating calcium retention (0 - 42 days)  - 25 -  Table 1:9  Bone ash at 6 weeks, Experiment 1  low fat diet % Ca in diet O.76  % P in diet  diet no  av bone ash  1  0.53  43.9  high fat diet diet no  6  av bone ash  *  0.6  2  43.7 43.4  7  w% 1.21  1.50  0-75  3  4  0.9  av diets 1-4  1.51  0.6  Table 1:10  5  8  43.8  9  44.2 45.7 45T0  %  42.5  43.0  43.4 ^3.3 43.5  44.2 43.6 53T9  43.9  av bone ash  42.2  WX 1.03  high and low fat diets  Av diets 6-9  10  43.1 43.7 5|T4 44.2 43.4  43.7  44.1  43.3 43.5 43.2 43Tf  Analysis of variance, bone ash at 6 weeks, diets 1 to 4, and 6 to 9> inclusive, Experiment 1  Source of variation  s.s.  d.f.  total calcium level fat level interaction error  5.66 2.08 1.56 0.76  15 3 1  M. S. D. calcium treatments  0.7  -1  variance  O.69 I.56 0.25 0.16  tt^tt  4.31* 9.75* 1.56 ;  11-1*4- days  15-21  22-2S days  days  75 50 -  CO  25  c  •H  -I  I  i  u  +>  co fc  cd u  1 33-35  CD CJ  C as  rH  cd  75  I  50  •H O rH CC  o  kO-K2 days  days  0-1+2 days  x  25 0.75 i . o o 1.25 1.50 Calcium In d i e t  {%)'  FIGURE 1:1  o„75 l . o o 1.25 1.50 x  *  0.75 1.90 1.25 1.50  control diet f a t added  Calcium balance, Experiment  1,  ro l  1 5 - 2 1 days  11-14 days  22-28 days  1.5 fc CD P.  1.0 —  +3  0.5-  CO bO C -H <H CO  _l  cd >  ;  l_  -1  i—  CD C fc -H Cd C o -H — ' cd  c  1  bo  o •  3 3 - 3 5 days  40-42 days  0-42 days  +3 hO  c  coo  T  r-  CD H  _ _ X  fc  a •H  o H  cd  o  s-  1.0 0.5-  0 . 7 5 1.00 1.25 1.50 Calcium i n d i e t  {%)  0 . 7 5 1.00 1.25 1.50 . .  x  FIGURE 1:2  0 . 7 5 1.00 1.25 1.50  . control diet _« &% f a t added  Calcium r e t e n t i o n ,  Experiment 1.  ro  I  - 28 -  EXPERIMENT  2  INTRODUCTION  In experiment 2, a study was carried out with diets containing 20.6% protein similar to the study which was conducted on 24% protein diets i n Experiment 1. As i n Experiment 1, four levels of calcium were fed, 0.74%, 1.01%, 1.22%, and 1.51%. The calcium:phosphorus ratio was from to 1.7:1.  1.5:1  A f i f t h diet containing 1.54% calcium, in which the calcium:  phosphorus ratio was adjusted to 2.6:1$ was fed i n order to determine i f there were an effect of calcium:phosphorus imbalance with the highest level of calcium. As i n 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 i n 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 intraocularly against Newcastle disease, and placed i n electrically heated battery brooders.  The chicks had free access to feed and water.  The composition of the experimental diets i s shown i n Table 2 : 1 . The chicks were weighed individually at weekly intervals throughout the experiment. weekly.  Feed consumption of each l o t was recorded  Calcium balance studies were carried out during each week of  the experiment as described i n Experiment 1. The experiment was terminated when the chicks were 3 weeks of age. Six birds from each l o t , selected to be near the average weight of the l o t , were k i l l e d .  The l e f t t i b i a was removed from each  bird for determination of ash content. The bone ash content was determined on the individual l e f t tibias. RESULTS AND DISCUSSION The average weights and feed efficiency of chicks i n Experiment 2 at 3 weeks of age are summarized i n Table 2:2. It w i l l be seen from the table' that the experimental treatments caused no significant difference i n average weight of the chicks fed the various diets at 3 weeks. The average weight of chicks i n 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 i n 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 provided by the low fat diet. The addition of fat to the diet resulted i n some improvement 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 in diets 5 and 10, containing 1.5$  to  2.6:1  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 expressed, 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 i n 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 retained 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 i n 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 i n pattern.  There  i s , i n the graphs for 0-7 and 8-l4 days, a depression of calcium balance from 2-20% when 8% fat i s added to the diet.  This effect  is not evident i n 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 increasing the level of calcium i n the diet.  It w i l l be seen from the graph  showing calcium balance for the 21-day period of the experiment, and from Table 2:3> that the chicks i n 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 i n a drop i n 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 i n body weight) i s shown i n Table 2:4 and graphed i n Fig. 2:2. A study of the graphs and the table reveals that the addition 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 i s seen from Table 2:4 that increasing the level of calcium from 0.74% to 1.51% resulted i n an increase i n total (0-21 days) calcium retention from 0.77 to 1.79 i n the case of the chicks fed the low fat diets- With the chicks fed the high fat diets the range i n cal-  - 32 -  cium retention was only from O.87 to 1.35 when the calcium level i n the diet was increased from 0.7%$ to 1.51$« The fluctuations i n the calcium retention curves due to experimental error are not sufficient to obscure the differences a t t r i butable to the experimental diets. The average percentage of bone ash i n each group i s shown i n Table 2:5.  These data are analyzed s t a t i s t i c a l l y i n Table 2:6. I t  w i l l be seen from the table that the addition of fat to the diets did not significantly affect calcification.  There was, however, a highly  significant increase i n ash content of the bones when the calcium level was increased from 0.74$ to 1.01$.  Further increases i n diet-  ary calcium level did not significantly affect calcification. An increase i n the calcium:phosphorus ratio of the 1.5$ calcium group from 1.69:1 to 2.57:1 caused l i t t l e or no difference i n calcification.  In the case of the high fat groups, the high ratio group  showed calcification of 43-9$ s h , compared with 44.0$ i n the group a  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 i s 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 i n some improvement i n feed efficiency. Increasing the level of calcium i n the diet from 0.74$ to 1.51$ caused no difference i n 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 i n body weight) at a l l levels of calcium except the 0.74% level. Increasing the level of calcium i n the diet did not affect calcium balance but there was an increase i n 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  A l l Diets Diet 1 Diet 2  ingredient  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 mixture 0.1 DPPD 0.0125 d l methionine 0.15 ground cellulose^ ground limestone tricalcium phosphate  Diet 3 Diet 4 Diet 5  1  2  protein content ($) calcium content ($) phosphorus content ($) Ca/P ratio  1.  9.0 0.57  8.68 0.59 0.30  8.35 0.54 0.68  8.04 0.48 1.05  8.00 1.27 0.30  20.67 0.74 0.48 1.5:1  20.35 1.01 0.6 1.7:1  20.52 1.22 0.75 1.6:1  20.84 1.51 0.9 1.7:1  20.62 1.54 0.6 2.6:1  "Delamix", a trace mineral mixture containing:  Fe 2.0$, Cu 0.2$, Zn 0.006$,Co 0.020$, Ca 27-0$.  Mn 6.0$, I 0.12$,  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$ c i t r i c acid.  - 35 -  Table 2:2  Average weight of chicks and feed efficiency at 3 weeks, Experiment 2 low fat diets  % Ca in diet  % P in diet  diet no  nigh fat diets  av wt feed 3 wks gain (gm) 0-3 wks  diet no  av wt 3 wks (gm)  feed gain 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 efficiency J  feed consumed - —;—. .. . gain i n weight  - 36 -  Table 2:3  Calcium balance, Experiment 2  Calcium balance, $  low fat diets  nigh fat diets  $ Ca in diet  *P in diet  0.74  0.48  1.01  days  0-7  8-14 days  15-21  0-21  days  days  1  32.2  62.5  49.2  52.8  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  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  Calcium balance -  diet no  Ca ingested - Ca excreted Ca ingested  - 37 -  Table 2:4  Calcium retention, Experiment 2 Calcium retention (gm. per 100 gm. gain)  f> Ca  low fat diets  high fat diets  in diet  <$> P in diet  0.74  0.48  1.01  0-7  days  8-14 days  15-21 days  0-21 days  1  O.96  1.12  O.69  0.77  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  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  diet no  Calcium retention - Ca retained (grams) per 100 grams gain i n body weight  - 38 -  Table 2:5  Bone ash at 3 weeks, Experiment 2  low fat diets  high fat diets  high and low fat diets  % Ca in diet  % P in diet  diet 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  5  43.4  10  ^3.9  1.54  Table 2:6  av bone ash  %  diet no  av bone ash  %  av bone ash  %  Analysis of variance, bone ash at 3 weeks, diets 1 to 4 and 6 to 9} inclusive, Experiment 2  Source of variation total calcium level fat level interaction error M. S. D. Calcium treatments  s.s. 147.88 71.72 0.42 10.19 65.55 1.1  d.f.  47 3 1  variance  It£>tt  23.9 0.42 3.40 1.60  14.6** 2.07  0-7  days  i  i  &-lk days  75 -  i  0.75  Loo  1 —  1.25 1-50  Calcium i n d i e t  _.  _ I—i  1  1  0.75 1.00 1.25 1.50  {%)  . x-  „ control diet -* f a t added  r  FIGURE 2:1  1 —  Calcium balance, Experiment  2.  +3+3  © X! fc hO cd co  I  i  •  1  1  I  «  1  L  1  • c  I  0.75  I  i.oo  1  1  1.25 1-50  Calcium i n d i e t  (%)  I  L  Calcium r e t e n t i o n ,  1  1  0.75 1.00 1.25 1.50 . x-  FIGURE 2:2  1  . control  diet  6% f a t added  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 i n Experiment 1. The possible effect of an imbalance i n calcium:phosphorus ratio on calcium utilization was not conclusively established  i n 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 i n a diet i n which the calcium:phosphorus ratio was approximately 1.6:1, and a diet i n which the calcium phosphorus 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 i n the control diet. ment 1.  The diets were similar i n productive energy to those of ExperiThe 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 i n electrically heated  battery brooders with free access to feed and water. The composition of the experimental diets i s shown i n Table 3:1. The chicks were weighed individually at weekly intervals throughout the experiment.  Feed consumption of each l o t was recorded  weekly. Calcium balance studies were carried out each week as described i n Experiment 1.  The values were determined for the entire  week during each of the f i r s t four weeks and for a one day period during each of the f i n a l 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 i n Table 3:2. The addition of 8$ animal fat to the basal diet s i g n i f i c 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 i n 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 i n weight between the groups fed the various calcium and phosphorus levels.  There  - 43 -  was also a significant interaction between the mineral and fat treatments . In the case of the groups fed diets containing both a normal and an adjusted calcium:phosphorus ratio the chicks fed 1.10$ calcium i n the diet were lighter i n average weight at 5 veeks (379-384 gm.) than those fed 0.95$ or 1.34$ calcium, which ranged i n weight from 396-1402 gm. (average of low and high fat diets).  The difference i n  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 i n 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 i n contrast to the chicks fed the other low fat diets, which ranged i n 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 considered, i t i s 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 i n weight between the chicks fed 0.95$ and 1.10$ calcium i n the diet.  I t i s therefore concluded that  - 44 -  increasing the level of calcium i n the diet from 0.95% to 1.34% resulted i n an increase i n growth rate. Adjusting the calcium:phosphorus ratio i n the diet did not affect the growth rate.  There were no significant differences  in weight between the treatments corresponding i n calcium level but differing i n calcium:phosphorus ratio. The feed efficiency of the birds on each diet at 3 and 5 weeks i s shown in Table 3:4.  The addition of fat to the diets im-  proved feed efficiency, from an average of 2 . l 6 l b . of feed per l b . 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 expressed, as i n 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 i n body weight. The values obtained for "calcium balance" are shown i n Table 3:5 and are presented graphically i n Fig. 3:1« A study of Table' 2:1 and the graphs i n Fig. 5 indicates that the addition of 8% fat to the diet improved calcium balance. This i s shown most clearly i n 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  -45 -  of the chicks fed the low fat diets ranged from 36.8$ to 47.6$. On the high fat diets the range i n calcium balance was from 43.8$  to 55.4$. The graphs show clearly that calcium balance was higher in the case of the low calcium diet (0.95$ calcium) than i n 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 i s 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 i n weight) are shown i n Table 3:6 and are presented 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 information on the effect of the experimental diets on calcium retention in this experiment. The addition of 8$ fat to the diet caused some impairment 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  for the diets containing 0.95,  gm. of calcium per 100 gm. of gain  1.10  and 1.35$  calcium, respectively.  Similar diets, with 8$ fat added, resulted i n retention of 0.93, and 1.02  0.86,  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 of gain) as the calcium level was increased from 0.95$ to 1.10$  gm.  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 i n the diet i s increased up to a point of maximum calcium retention, followed by l i t t l e or no further increase in retention as the level of calcium In the diet i s increased further, within reasonable limits.  No explanation i s 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 i n this experiment i s apparent.  Increasing the calcium:phosphorus ratio of the low fat  diets appeared to result in a reduction i n 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 i n creased at the two lower levels of calcium and decreased at the high level of dietary calcium.  I t i s 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 significantly 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 i n 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 i n "body weight.  The  effect of the level of calcium i n the diet on calcium "balance and calcium retention i s not clearly indicated by the data presented. Increasing the calcium:phosphorus ratio from 1.46 - 1.58:1 to 1.90 - 2.25:1 i n 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  A l l Diets Diets  ingredient  $  ground yellow corn ground wheat fishmeal (74$ protein) soybean o i l meal (44$ protein) dehydrated cereal grass dried brewers' yeast dried skim milk feeding o i l (2250A - 300D) salt (iodized) mine r a l mixture 1  Diets  Diets  8.67 1.09 1.09  8.03 O.98 1.84  2 & 5  3 & 6  15-0 3T-8i*9.0 18.3 2.5 3'0 2.5 0.25 0.5 0.1  D P P D  0.0125 0.15  d l methionine , ground cellulose" ground limestone tricalcium phosphate 5  protein content ($) calcium content ($) ' phosphorus content diets 1-3 phosphorus content diets 4-6 Ca/Pratio, diets 1-3 Ca/Pratio, diets 4-6  1 & 4  ($) ($)  9.32 1.18 0.35  23.54 0.95 0.6 0.5  23.71 23.57 1.10 1.34 0.9 0.75 0.6 0.55 l;58: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$ c i t r i c acid.  - 50 -  Table 3:2  Average weight of chicks at 3 and 5 weeks, Experiment 3  low fat diets % Ca % P in in diet diet  diet no  0.95  0.6  1.10  high fat diets  low and high fat diets  Av wt 3 wks (gm.)  Av wt 5 wks (gm.)  1  210  394  7  205  399  208  397  0.75  2  199  347  8  205  410  202  379  1.34  0.9  3  I83  357  9  230  446  207  402  0.95  0.5  4  189  361  10  225  430  207  396  1.10  0.55  5  176  353  11  216  415  196  384  1.34  0.6  6  193  365  12  222  ^37  208  401  192  363  217  423  Av  Av wt diet 3 wks no (gm.)  Av  M. S. D. mineral treatments (5 weeks)  Av wt 5 wks (gm.)  Av wt 3 wks (gm.)  Av wt 5 wks (gm.)  - 51 -  Table 3:3  Analysis of variance, average weights of chicks, Experiment 3  3 weeks Source of variation  S.S.  d.f.  total mineral treatments fat level interaction error  612,253 5,425 49,592 26,183 531,053  296 5 1  variance  285  1,085 49,592 5,237 1,863  d.f.  variance  26.6** 2.81*  5 weeks Source of variation  S.S.  total mineral treatments fat level interaction error  891,640 21,793 267,748 49,407 552,692  L  S. D. mineral treatments  ^i1 7  295 5 1 5 284  • S359 267,748 9,881 1,946  f 2.24* 138.** 5-08**  - 52 -  Table 3:4  Feed efficiency * at 3 and 5 weeks, Experiment 3  low fat diets $ Ca in diet  % p in diet  0.95  0.6  1.10  h i  S  n  *  a t  diets  feed/ gain 0-3 wks  feed/ gain 0-5 wks  feed/ gain 0-3 wks  feed/ gain 0-5 wks  1  2.05  2.35  7  1.86  2.05  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  *  diet no  ^ . . Feed efficiency -  feed consumed ^  g a l n  v e i g h t  diet no  - 53 -  Table 3:5  Calcium balance, Experiment 3  Calcium balance, % # Ca  in diet low fat diets  % p in diet diet . no  8-14 15-21 26-28 33-35 days days days days  0-35 days  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  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  high fat diets 0.95  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  0.95  0.6  1.10  diet no  8-14 15-21 26-28 33-35  0-35  days  days  days  days  1  1.01  1.00  1.24  0.86  1.04  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  low fat diets  days  Calcium retention - Ca retained (grams) per 100 grams gain i n body weight  g-ll+ days  15-21 days  , control diet, normal Ca:P ratio  75  f a t added, nominal" Ca :P' t»atio  50 A  CO  25  +  c  «H CO  + control diet, high Ca:P-ratio + fat added, high Ca:P ratio  CD  U  CO  o  26-2S days  CD O  33-35 days  0-35  d a  ys  e  cO H co  S «H O iH CO O  75 50 25 0.75 1.00 !v 5 1.50 2  0.75 1.00 1.25 1.50  0.75 1.00 1.25 1.50  Calcium in diet {%) FIGURE 3:1. Calcium balance, Experiment 3.  VJ1 VJl  1  S-lif daya  15-21  days  control diet, normal Ca:P r a t i o  1.5 x  l.o  x 07b f a t added, normal Ca:P r a t i o control diet, h i g h Ca:P r a t i o  +-• • '  +•  0.5L. +• J  26-28 daya  1  33-35  i_  +  f a t added, h i g h Ca:P r a t i o  I  days  0-35  VJl  clays  o\  1  1.5 1.0 0.51-  0.75 1.00 1.25 1.50 Calcium i n d i e t  -J  1  1  i_  0.75 1.00 1.25 1.50  -J  Calcium  r e t e n t i o n , Experiment  1  i_  0.75 1.00 1.25 1.50  (%)  FIGURE 3:2  1  3.  -57 -  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 i n 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 i n which  the diets contained 24% protein, levels of calcium ranging from 0.75% to 1.50% were provided.  Significant differences i n average weight of  chicks were observed between calcium levels, with 1.21% calcium providing the most rapid growth i n both the low fat diets and those containing 8% supplementary fat. In the other high protein experiment, i n which only 3 levels of calcium were fed, l i t t l e increase i n growth resulted from an increase i n calcium level from 0.95% to 1.35% of the diet. The failure of the chicks to show a consistent growth response to differences i n calcium level may be explained by the fact that the diets fed i n 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 i n calcium level by promoting the absorption of calcium from the digestive tract.  The apparent growth response i n  Experiment 1 which resulted from an increase i n dietary calcium level from the recommended level, 1%, to 1.21% of the diet, suggests that at the higher protein level an increase i n 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, w i l l 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.  I t i s possible that the  high level of Vitamin D i n the diets, by promoting the absorption of calcium, overcame a tendency of the fat to increase fecal calcium excretion.  A more l i k e l y explanation for the failure of fat to  affect the calcium requirement i s indicated by the work of Cheng et a l (1949) and Rao and De (1951) with rats, which was reviewed earlier. The results of these groups of workers suggest that although the i n 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 i n fecal calcium excretion. Under some conditions, i n fact, with diets high i n 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 absorption.  - 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 i n the presence of sufficient Vitamin D. I t i s 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% protein 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 i n calcium retention. Calcium balance, expressed as the per cent of ingested calcium which was retained by the chick, is a less valuable index of calcium utilization than calcium retention, expressed as calcium retained per hundred grams of gain i n 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> i n 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, i n which the diets contained 20.6% pro-  tein, fat had l i t t l e effect on calcium balance.  Since i n Experiments  1 and 3. feed efficiency was markedly improved by the addition of fat to the diet, the improvement i n calcium balance may merely reflect the decreased calcium intake per unit of body weight, rather than an i n crease in absolute calcium retention.  This d i f f i c u l t y 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 i n 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 i n calcium 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, i n spite of the slight depression of calcification when fat was added to diets containing 24% protein.  I t remains possible,  though, that with a lower level of Vitamin D in the diet, the impairment 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 absorption by the chick than to a relatively smaller size of skeleton i n the more rapidly growing chicks on the high fat diets, i n view of the fact that l i t t l e difference i n calcification was the groups on high and low fat diets.  found between  Further experiments, which  should include analyses of body composition and calcium distribution in chicks receiving diets of these types, w i l l be required to test this hypothesis. The addition of 8$ animal fat to diets containing 24$ protein in Experiments 1 and 3 caused a highly significant increase i n growth rate and a marked improvement in feed efficiency.  In Experi-  ment 2, i n which the diets contained 20.7$ protein, the addition of 8$ fat to the diet caused no appreciable improvement i n rate of growth and l i t t l e change i n feed efficiency from that shown by the groups receiving 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 i n the diet within the limits tested i n 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 i n these experiments. This conclusion i s i n agreement with the findings of Gershoff and Hegsted (1956) that i n the presence of Vitamin D the calcium: phosphorus ratio (varied from 4:1 to 1:2) had no significant effect on calcium absorption i n the gastrointestinal tract of chicks, while i n the absence of Vitamin D the calcium:phosphorus fluenced calcium absorption. The high calcium:phosphorus  ratio i n ratio  of approximately 2.5=1 provided i n these experiments was regarded by Wilgus (1931) as borderline when fed i n a ration containing 16.5$ protein.  I t i s therefore possible that a similar calcium:  phosphorus ratio, i n diets containing the higher levels of protein used i n these experiments, but with a limited level of Vitamin D, might seriously interfere with growth and normal bone c a l c i f i c a tion. The results of the present experiments suggest that when the diet contains a moderate excess of Vitamin D the calcium-.phosphorus 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. A l l levels of calcium 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 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 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 o f Analysis, 8th ed. Association o f O f f i c i a l A g r i c u l t u r a l Chemists, Washington. B i e l y , J . , and B. E. March, 1954. Fat studies i n poultry 2 . Fat supplements i n chick and poultry r a t i o n s . Poul. Sci.33:  1220-1227.  Bosworth, A.W., H.I. Bowditch, and L.A. G i b l i n , 1918. The digestion and absorption o f f a t s 1. Calcium i n i t s r e l a t i o n t o the absorption o f f a t t y acids. Am. J . D i s . C h i l d . 15:397-407Boyd, Oscar F-, Carlos L. Crum, and J.F. Lyman, 1932. The absorption of calcium soaps and the r e l a t i o n o f d i e t a r y f a t t o calcium u t i l i z a t i o n i n the white r a t . J . B i o l . Chem. 95:29-41. Boyd, O.F., and J.F. Lyman, 1930. U t i l i z a t i o n o f calcium soaps by the white r a t . 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 e f f e c t o f the l e v e l o f d i e t a r y calcium and magnesium on the d i g e s t i b i l i t y o f f a t t y acids, simple t r i g l y cerides, and some natural and hydrogenated f a t s . J . Nut.  37:237-250.  Deuel, Harry J . J r . , 1955. The l i p i d s , t h e i r chemistry and biochemi s t r y , V o l . 11. Interscience, New York. Fraps, G. S., 1946. Composition and productive energy o f poultry feeds and r a t i o n s . B u l l . Texas Agric. Expt. Sta. #678. Gershoff, S.N., and D.M. Hegsted, 1956. E f f e c t o f Vitamin D and Ca:P r a t i o s on chick g a s t r o i n t e s t i n a l t r a c t . Am. J . Physiol  187:203-206.  Givens, Maurice H., 1917 • Studies i n calcium and magnesium metabolism I I I . The e f f e c t o f f a t and f a t t y acid d e r i v a t i v e s . J . B i o l . Chem. 31:441-444. Holmes, A.D., and H.J. Deuel J r . , 1920. miscellaneous vegetable f a t s .  D i g e s t i b i l i t y of c e r t a i n J . B i o l . Chem. 41:227-235.  McDougall, E.J., 1938. The counteraction by f a t o f the a n t i - c a l c i f y ing action o f cereals. Bioch. J . 32:194-202. N. R. C , 1954. Nutrient requirements f o r poultry. Council Publication #301.  National Research  - 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., J r . , 1931' The quantitative requirement of the growing chick for calcium and phosphorus. Poul. S c i . 10:107-117'  

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