Influence of Age, Dietary Cholic Acid, and Calcium Levels on Performance, Utilization of Free Fatty Acids, and Bone Mineralization in Broilers

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1 Influence of Age, Dietary Cholic Acid, and Calcium Levels on Performance, Utilization of Free Fatty Acids, and Bone Mineralization in Broilers J. O. ATTEH and S. LEESON Department of Animal and Poultry Science, University ofguelph, Guelph, Ontario Canada NIG 2W1 (Received for publication September 17, 1984) ABSTRACT The effects of age on the utilization of dietary palmitic or a 50/50 mixture of palmitic and oleic acid at the 8% inclusion level in the absence or presence of.2% cholic acid and also in the presence of low (.8%) or high (1.2%) calcium were investigated using broiler chicks from 1 to 56 days of age. Significant interactions (P<.01) were observed between the type of fatty acid supplemented and the presence or absence of cholic acid on weight gain and feed efficiency. Supplementing diets with a mixture of equal weights of palmitic and oleic acid, reduced feed intake relative to control diets and diets supplemented with palmitic acid alone. There was an interaction between the age of the bird and the type of fatty acid supplemented on fat retention and metabolizable energy (ME) of diets (P<.01). There was also a significant interaction between the type of fatty acid supplemented and the addition of cholic acid on fat retention and ME of diets. While cholic acid reduced soap formation during the process of digestion (P<.05), increasing dietary calcium level increased the proportion of the digesta fat that was present as soap (P<.01). The proportion of digesta and excreta fat, present as soap, depended on the type of fatty acid supplemented. The addition of free fatty acids to broiler diets resulted in a decrease in bone ash and bone calcium content relative to those birds fed the control diet. It is concluded that the ability of broilers to utilize dietary free fatty acids depends on the age at which they are fed, although in all cases supplemental cholic acid enhances fatty acid utilization. (Key words: age of broilers, palmitic acid, cholic acid, calcium, chick performance, bone minerals) INTRODUCTION Variation in fat utilization with the age of bird has been documented. Duckworth et al. (1950) observed that the ability of chicks to digest mutton fat increased with age up to 4 weeks. Fedde et al. (1960) also showed increases in digestion of beef tallow by chicks from 53% at 7-days of age to 80% at 12 weeks of age, while Renner and Hill (1960) reported that the 8-week-old bird was capable of utilizing tallow equally as well as the adult hen. There is an indication that the variation in fat utilization with age depends on the type of fat supplemented. Whitehead and Fisher (1975) reported that, although there was an improvement in utilization of tallow from 57% at 2 weeks of age to about 74% at 8 weeks of age, there was no change in utilization of corn oil or lard during the same period. Atten and Leeson (1984, 1985) observed a substantial difference between broiler chicks and laying hens in the proportion of digesta 1 A/S N. Foss Electric, Hiller^d, Denmark Poultry Science 64: fatty acids that were present as soap, which could partly explain differences in fatty acid utilization by young and adult birds. Also, while fatty acid utilization by broiler chicks depended on dietary calcium level, fatty acid utilization by laying hens was independent of the dietary calcium level. Carew et al. (1972) reported that the young chicken lacked the full physiological capacity for fat absorption. This lack of full physiological capacity for fat absorption is probably associated with producing enough bile acids to cope with fat digestion. Katongole and March (1980), showed that bile salt supplementation improved utilization of tallow by chicks. The study reported herein was undertaken to investigate the weekly changes in utilization of dietary free fatty acids and other nutrients in the presence or absence of supplementary cholic acid and whether such changes are influenced by the dietary calcium content. MATERIALS AND METHODS Four hundred and eighty day-old male broiler chicks were housed in electrically heated battery brooders and fed the experimental diets 1959

2 1960 ATTEH AND LEESON as shown in Table 1. The basal diet contained 22.3% crude protein and provided 3000 kcal metabolizable energy (ME)/kg. The 12 experimental diets consisted of a 3 x 2 x 2 factorial combination of types of fatty acids supplemented, cholic acid, and calcium levels. The fatty acids supplemented at 8% inclusion level were palmitic (91%) or a 50/50 (w/w) mixture of oleic (90%) and palmitic acid (91%) (O/P mixture), each being substituted for alpha floe (cellulose) in the control diet. The fatty acids and cholic acid were obtained from US Biochemical Corporation, Cleveland, OH. All fatty acids were initially ascribed an ME value of zero, as reported ME and absorbability values of saturated fatty acids range from 0 to 80% (Renner and Hill, 1961; Hurwitz et al., 1973), and such values are affected by bird age (Renner and Hill, 1960) and diet inclusion level (Miller, 1974). It was presumed that this arbitrary decision would not influence most biochemical parameters measured, while those production measurements influenced by diet energy level could subsequently be discussed in relation to actual dietary ME values determined in the trial. Each of the fatty acid treatments was applied with 0 or.2% cholic acid (99.1%) in the diet while calcium levels were.8 or 1.2%. Each treatment was tested with 4 replicate cages of 10 chicks, with feed and water available ad libitum during a trial period that lasted 8 weeks. Performance of the chicks during the trial period was monitored. This included weighing the chicks in each replicate cage at 2 weeks of age and every week thereafter between 2 to 6 weeks of age and then at 8 weeks of age. Corresponding feed intake and feed efficiency during the same period was ascertained. Incidence of leg deformities was subjectively evaluated when the chicks were 5 weeks old. Chicks were either classified as having normal or deformed legs, the latter being valgus-varus deformity of the intertarsal joint or swollen hocks. Also at 5 weeks of age, half of the birds in each replicate cage were randomly selected and killed by dislocation of the cervical vertebrae. The body cavity was exposed and the contents of the small intestine (digesta) were 2 Parr Instrument Co., Moline, IL. 3 Techtron PTY Ltd., Melbourne, Australia. 4 Technicon Instruments Co., Chauncey, NY. flushed out with double distilled water. The digesta of all birds killed within a replicate were pooled and freeze-dried prior to grinding. Also, the left tibia of two of the killed birds per replicate were removed and cleaned of adhering flesh, dried at 100 C for 48 hr, subjected to petroleum ether extraction overnight using Soxhlet extraction apparatus, and dried again prior to dry ashing at 600 C overnight. A nutrient retention study using chromic oxide as a marker was undertaken during the trial. Excreta samples were collected over the last 48 hr of each week throughout the trial. Each weekly excreta collection was dried in a forced air oven at 70 C and ground prior to chemical analysis. Birds remaining at 8 weeks of age were sacrificed and subjected to the same procedures as described for 5 weeks of age. Chemical Analysis. Nitrogen in feed and excreta samples was determined by Kjeldahl procedure (Kjel-foss Automatic, Model 16310) 1 while gross energy was determined by adiabatic oxygen bomb calorimetry (Parr, Model 1241) 2. Samples of the feed, excreta, and bone were ashed at 600 C and ash weight taken for bone samples. The ash samples were then digested using the method of Association of Official Analytical Chemists (AOAC, 1980) for preparation of sample solutions of inorganic materials for atomic absorption spectrophotometry. The resulting solutions were transferred into 100 ml volumetric flasks and made up to volume with strontium chloride (1.5% w/v). Further dilutions were made where necesary before the solutions were analyzed for both calcium and magnesium, using a Techtron atomic absorption spectrophotometer (Model AA 4 ) 3, and for phosphorus, using a Technicon auto analyzer (Model AA 2 ) 4. Chromium in feed and excreta samples was determined using the method of Fenton and Fenton (1979). Total glycerides and fatty acids in feed, digesta, and excreta were determined by petroleum ether extraction using a Soxhlet apparatus. To estimate the proportion of digesta and excreta fat present as soap, the principle of fat determination in feces reported by Carroll (1958) was used. Digesta and excreta samples were subjected to two stages of ether extraction. The first extraction was to remove neutral and fatty acids that were not present as soap. Thimbles containing the residue of the first extraction were placed in 25% hydrochloric acid for about 2 hr at room temperature to liberate the fatty acids that were present as

3 Ingredient Basal 1, 91% Oleic acid, 90% Cholic acid, 99.1% Limestone Alpha-floc Total Analyzed nutrient content Crude protein, % Crude fat, % Calcium, % Total phosphorus, % Magnesium, % TABLE 1. Percentage diet composition Treatment at Penn State University (Paterno Lib) on May 12, 'Contains corn, 29.06%; corn gluten meal, 24.20%; corn starch, 25.91%; fish meal, 8.0%; diealcium phosphate, (.11% CoI 2 ),.25%; chromic oxide,.3%; mineral-vitamin premix,.75% (provides per kg of diet: vitamin A, 8000 IU, v 9.0 mg; pantothenic acid, 11 mg; vitamin B 12, 13 Mg; niacin, 25 mg; choline, 900 mg; vitamin K, 1.5 mg; biotin, mg; iron, 30 mg; copper, 5.0 mg; and seleninm,.1 mg).

4 1962 ATTEH AND LEESON soap. The samples were then freeze dried and the process of ether extraction repeated. This second ether extract was considered to represent the fatty acids that were previously present as soap. Samples of the feed were subjected to similar two-stage ether extractions to act as standards. The fat and fatty acids, extracted from the feed and from each of the two extractions from the digesta and excreta, were esterified with.2 N methanolic trimethyammonium hydroxide, and the methyl esters were analyzed for component fatty acids by gas/liquid chromatography (Varian, Model 2100) using the method of AOAC (1980). A 5' X.08 in column of 5% DEGS PS on 100/120 mesh Supelcoport was used in the chromatograph. Data were analyzed statistically by both regression analysis and analysis of variance using the Statistical Analysis Systems (SAS) (1982). Significant differences among treatments were determined using the protected least significant difference (LSD) (Steel and Torrie, 1980). RESULTS Chick Performance. The overall performance of the chicks at 8 weeks of age is shown in Table 2. There were no significant effects of cholic acid supplementation on feed intake, incidence of leg deformities, or mortality. There was also no significant effect of increases in dietary calcium level on any of the performance parameters measured, with the notable exception of a decrease in incidence of leg deformities with increase in dietary calcium level. Birds on diets supplemented with the O/P mixture consumed less feed compared to birds offered the control and palmitic acid diets. There was also an increase in leg deformities with fatty acid supplementation. Significant interactions were, observed between the types of fatty acid supplemented and the presence or absence of supplemental cholic acid for both weight gain and feed efficiency (P<.01), details of which are shown in Table 3. In the absence of bile acids, there was no significant difference in weight gain between birds on the control diets and those on diets TABLE 2. Effect of dietary fatty, cholic acids, calcium levels on broiler performance to 8 weeks of age Dietary treatments Source of fatty acid (Fa) O/P mixture 2 Cholic acid (Ba, %).0.2 Calcium (Ca, %) Fa X Ba FaX Ca BaX Ca Fa X Ba X Ca Standard deviation Feed intake 0 8 weeks * 4215 b 4202b 4092* Body weight gain 0 8 weeks vg/dlld; 1771 a 1894 b 2242 c * 1937 a 2001 b Feed intake: weight gain 0 8 weeks 2.38 c 2.22b 1.83 a * 2.18 b 2.11 a Leg deformities 1 * 3.8 a 6.9 b 8.8b b 5.0 a 1.2 Mortality ' ' Within main treatment categories, means within columns followed by different superscripts are significantly different (*P<.05; P<.01). 1 Chicks with leg deformities expressed as a percentage of chicks in each treatment. 2 Oleic and palmitic acid. 3 = No significant difference (P>.05).

5 AGE, CHOLIC ACID, AND CALCIUM IN BROILERS 1963 TABLE 3. Interaction of fatty acid with presence (+ Ba) or absence ( Ba).2% of cbolic acid on body weight gain and feed efficiency (0 to 8 weeks) Fatty acids Body weight gain, g/bird Palmitic O/P mixture 1 Feed intake: weight gain Palmitic O/P mixture -Ba 1807 a 1828 a 2175 c 2.34 de 2.29 d 1.91 b + Ba 1735 a 1959 b 2308 d 2.42«2.14 c 1.76 a For both body weight gain and feed efficiency, means followed by different superscripts are significantly different (P<.01). 1 Oleic and palmitic acid. supplemented with palmitic acid, while birds on diets with added O/P mixture gained more than those on other diets. However, in the presence of cholic acid supplementation, birds on diets supplemented with palmitic acid gained more weight than those on the control diets (P<.01), while birds consuming the O/P mixture outperformed all others. Observed from another perspective, supplementation of the diets with cholic acid improved weight gain of birds only when diets contained fatty acids. The efficiency with which birds utilized the diets followed a trend similar to that observed with body weight gain. These same interactions were also apparent at both 4 and 6 weeks of age. Nutrient Retention. Nutrient retention as influenced by dietary treatments is shown in Table 4. There was no significant effect of fatty acid supplementation on either phosphorus or magnesium retention. However, there was variation in crude protein retention with fatty acid supplementation (P<.01). Thus, protein retention was improved in the presence of O/P mixture compared to the control diet, while there was no significant difference in protein retention between birds on the control diet or those diets supplemented with palmitic acid. Supplemental cholic acid had no effect on protein, calcium, phosphorus, or magnesium retention (P>.05). Similarly, calcium level in the diet had no effect on protein or phosphorus retention. However, increasing the dietary calcium level resulted in a decrease in both percentage calcium and magnesium retention (P<.01). Also, birds on the control diets retained a larger percentage of dietary calcium than birds on diets with added palmitic acid (P<.01), although there was no significant difference between those on the control diets and those on diets supplemented with the O/P mixture. There were significant interactions (P<.01) between the type of fatty acid supplemented and the use of cholic acid on fat retention, the ME of diets, and the proportion of excreta fat that was present as soap; details are shown in Table 5. In all cases, addition of cholic acid to the diets improved fat retention while reducing soap formation. The ME of diets supplemented with free fatty acids was improved with cholic acid supplementation, while addition of cholic acid to the control diets had no effect on their ME. There were also interactions (P<.01) between the types of fatty acids added and dietary calcium levels on fat retention, dietary ME, and soap formation (Table 6). Although there was no change in fat retention with an increase in dietary calcium level for the control diets, increasing the dietary calcium content in the presence of diets with supplemental free fatty acids resulted in a significant decrease in fat retention; this trend was also seen with diet energy levels. Increasing dietary calcium level in all diets also increased the proportion of unabsorbed fat that was present as soap. Regression analyses of age on retention of individual nutrients are presented in Figures 1 to 6. The corresponding regression equations and correlation coefficients are shown in Table 7. The regression analyses for protein and calcium retention was based on data collected from 2 to 8 weeks of age because the values at 1 week of age were low relative to other values. Without the Week 1 data, the decreases in protein and calcium retention with increasing age were not significant (P>.05). Although there were significant increases in fat retention with age for all diets, the rate of increase differed significantly with diet (P<.01). Increase was least for the control diet, followed by diets supplemented with palmitic acid, while that of the birds on diets supplemented with the O/P mixture was highest (Fig. 2, Table 7). There was no increase in ME of the control diet with an increase in the age of the chicks (Fig. 3). However, for diets supplemented with palmitic acid or O/P mixture, dietary ME values increased with increase in the age of the bird (P<.01, Fig. 3). With older birds, there was a

6 Dietary treatments Source of fatty acids (Fa) Palmitic O/P mixture 3 Cholic acid, (Ba, 0.2 Calcium, (Ca, %) FaX Ba FaX Ca Ba X Ca Fa X Ba X Ca Standard deviation %) TABLE 4. Effect of dietary fatty, cholic acids, calcium levels on nutrient utilization by broil Crude protein retention (%) 64. l a 64.8 ab 65.2 b Metabolizable energy (kcal/g) a 3.170b C a b b a.055 Excreta soaps 1 Fat Ca 11.0 a 47.6 C 23.3b 30.4b 24.2a 23.l a 31.5b C 36.8 a 60.6 b 54.0 a 62.9 b 60.6 b 56ja ' ' Within main treatment categories, means within columns followed by different superscripts are significantly di 1 Excreta ether Extract two as proportion of extracts one plus two. 2 = No significant difference (P>.05). 3 Oleic and palmitic acid. 4.5 ttp://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 12,

7 AGE, CHOLIC ACID, AND CALCIUM IN BROILERS 1965 TABLE 5. Interaction of fatty acid with presence (+ Ba) or absence ( Ba) of cholic acid on fat retention, dietary metabolizable energy (ME), and soap formation Fatty acids Fat retention, % O/P mixture 1 ME, kcal/g O/P mixture Excreta soap, % O/P mixture -Ba 77.0 e 32.3 a 52.7 C a 3.124b d 12.l b 51.8 f 27.4 d + Ba 78.8 f 41.3 b 68.6 d a c e 10.0 a 43.4 e 19. l c a-f For fat retention, ME, or excreta soap, means followed by different superscripts are significantly different (P<.01). 1 Oleic and palmitic acid. significant decrease in the proportion of the excreta fat present as soap regardless of the type of fat that was added to the diet (Fig. 4). Although there was no significant effect of age of broiler on magnesium retention, there was improved retention of phosphorus (P<.05) with older birds (Fig. 6). Table 8 indicates the effect of dietary fatty acid supplementation on soap formation. A substantial proportion of the digesta fat was present as soap in all cases. However, there were changes in these proportions as the soap progressed through the digestive system as evidenced by differences in digesta vs. excreta soap concentrations. was responsible for a large proportion of soap fat even in birds fed the O/P mixture. Digesta Soap Formation and Bone Minerals. There was a decrease (P<.01) in proportion of digesta fat that was present as soap in birds at 8 vs. 5 weeks of age (Table 9). While addition of cholic acid reduced soap formation during the process of digestion (P<.01), increasing the dietary calcium content increased soap formation (P<.01). There was also variation in digesta soap formation, depending on the fatty acid supplemented, with birds fed diets containing palmitic acid recording the highest level (P<.01) followed by those on diets with O/P mixture, whereas the least amount of soap was formed by birds offered the control diet. Although there was no difference in bone ash for birds at 5 and 8 weeks of age, bone calcium and phosphorus contents of birds killed at 8 weeks of age were higher (P<.01), whereas the bone magnesium content was lower (P<.01) than those of birds sampled at 5 weeks of age (Table 9). Bone ash and bone calcium content of birds fed the control diet were higher (P<.01) than those on diets with supplemented fatty acids. Increasing the calcium content of the diets reduced bone magnesium content. There were no significant effects of the calcium and cholic acid treatments on bone ash, bone calcium, or phosphorus content. There were also no significant interactions between treatments for any of the parameters measured. DISCUSSION Variation in overall feed intake can be explained on the basis of energy content of the diets. Although the fatty acids were initially assigned a zero energy value, the assumption was valid only during the first week of growth for palmitic acid, while the O/P mixture contributed substantial additional energy from the first week of age. Thus, the increase in diet energy with fatty acid supplementation (especially O/P mixture) resulted in a decrease in feed intake. However, the authors have no explanation for the fact that although there was a TABLE 6. Interaction of fatty acid with calcium levels on fat retention, dietary metabolizable energy (ME), and soap formation in broiler chicks Fatty acids Fat retention, % O/P mixture 1 Diet ME, kcal/g O/P mixture Excreta soap, % O/P mixture.8% Calcium 77.9 e 40.lb 63.7 d a c e 8.5a 40.5 e 20.2C 1.2% Calcium 77.9 e 33.5a 57.6 C 3.009a 3.147b d 13.5b 54.7 f 26.3 d a-f For fat retention, ME of diets, or excreta soap, means followed by different superscripts are significantly different (P<.01). 1 Oleic and palmitic acid.

8 1966 ATTEH AND LEESON 70-; oep E 8»- ACE IN WEEKS FIG. 1. Effect of age on crude protein retention. AGE IN WEEKS STAR-CONTROL DIET. DIAMOND-DIETS WITH SUPPLEMENTAL PALMITIC ACID. TRIANGLE-DIETS WITH SUPPLEMENTAL 0/P MIXTURE. FIG. 2. Effect of age and types of fatty acid on fat retention. ACE IN VEEKS STAR-CONTROL DIET. DIAMOND-DIETS WITH SUPPLEMENTAL PALMITIC ACID. TRIANGLE-DIETS WITH SUPPLEMENTAL O/P MIXTURE. FIG. 3. Effect of age and types of fatty acid on metabolizable energy of diets (kcal/g).

9 AGE, CHOLIC ACID, AND CALCIUM IN BROILERS 1967 ACE IN WEEKS FIG. 4. Effect of age on the proportion of excreta fat present as soap. S OO- FIG. 5. Effect of age on calcium retention. ft 27- > E T 24- y.iir...! I I I t.t I! i ]...i, 1.0 l.s AGE IN WEEKS STAR-PHOSPHORUS RETENTION. DIAMOND-MAGNESIUM RETENTION. FIG. 6. Effect of age on phosphorus and magnesium retention.

10 1968 ATTEH AND LEESON TABLE 7. Regression equations and correlation coefficients of the effect of age on nutrient in broiler chicks utilization Nutrient Regression Correlation equations 1 SEE 2 coefficient(r) Crude protein, % Fat, % diet Diets with added palmitic acid Diets with added O/P mixture 3 Metabolizable energy, kcal/g diet Diets with added palmitic acid Diets with added O/P mixture Excreta soap Calcium Phosphorus Magnesium Y = x 1 Y = Nutrient utilization, x = Age in weeks. 2 Standard error of estimate. Y = x Y = x Y = x Y = x Y = x Y = x Y= x Y= x Y= x Y = x 'Probability (* = P<.05; * = P<.01, = P>.05). "Oleic and palmitic acid. significant improvement in palmitic acid utilization, with associated increase in diet energy and with increase in the age of the chicks, there was only a marginal decrease in feed intake during the same period. In the absence of cholic acid supplementation, weight gain and feed efficiency of birds, on the control diet or those with additional palmitic acid or the O/P mixture, followed a trend similar to that of feed intake. However, in the presence of cholic acid supplementation, birds on diets with supplemental palmitic acid gained more weight and utilized their feed more efficiently than those fed the control diet. While the presence of cholic acid had no beneficial effect on utilization of the control TABLE 8. Relationship of dietary fatty acids to soap formation and fatty acid composition of soap fat in broiler chickens Proportion of total fatty acids in diet fat, % Fat in digesta existing as soap as proportion of total fat in digesta, % Fatty acid make-up of soap fat in digesta, % Excreta fat existing as soap as a proportion of total fat in excreta, % Fatty acid make-up of soap fat in excreta, % Crude fat retention using first excreta ether extraction alone, % Crude fat retention using first and second excreta ether extraction, % ME 5 contribution to control diet, kcal/g Diets with supplements Palmitic acid O/P mixture Oleic and palmitic acid. 2 Made up of 42.2 oleic palmitic acid. 3 Made up of 13.3 oleic palmitic acid. 4 Made up of 9.0 oleic palmitic acid. 5 Metabolizable energy.

11 AGE, CHOLIC ACID, AND CALCIUM IN BROILERS 1969 TABLE 9. Effect of age, dietary fatty and cholic acids, and calcium levels on digesta, soap, bone asb, and bone minerals in broiler chicks 1 Treatments Soap as % digesta fat Ash Calcium Bone Phosphorus Magnesium Age, weeks 5 8 Source of fatty acid O/P mixture 2 Cholic acid % 0.2 Calcium, % Interactions Standard deviation 31.lb 28.5 a 22.1* 40.7= 26.5 b 30.8b 28.7* 28,l a 31.5 b b 45.0 a 46.iab a 36.1 b 37.4b 33.9a 34.2a a 18.lb ' ' Within main treatment categories, means within column followed by different superscripts are significantly different (*P<.05; P<.01). 1 There was no significant interaction between any of the treatments on any of these parameters. 2 Oleic and palmitic acid. 3 = No significant difference (P>.05). diet, it did improve utilization of diets with supplemental fatty acids. There is evidence to suggest that this beneficial effect of cholic acid was mediated via a decrease in soap formation with corresponding increase in fat retention and energy utilization. The observed increase in weight gain and feed efficiency seem to contradict earlier reports (Edwards, 1962; Gomez and Polin, 1976; Polin and Hussein, 1982) that showed no significant improvement in weight gain and feed efficiency with bile acid supplementation. The improvement in weight gain with fatty acid supplementation was accompanied by an increase in incidence of leg deformities, confirming earlier observations of Atteh and Leeson (1984). Although this may be associated with reduced calcium retention, increase in weight gain with fatty acid supplementation could have contributed to the problem, since Andrews et al. (1975) reported that weight of broiler influenced the incidence of leg deformities. As discussed earlier, the positive effect of supplemental cholic acid on weight gain and b.60 a *.63b.60 a.04 feed efficiency of birds fed supplemental fatty acid was mediated via a decrease in soap formation and subsequent increase in fat retention and ME of diets. This observation agrees with the earlier reports (Edwards, 1962; Gomez and Polin, 1974, Katongole and March, 1980; Kussaibati et al, 1982) showing these positive effects of dietary bile acid supplementation. Although supplemental cholic acid reduced soap formation and increased fat retention of the control diet, the 2% fat content of these diets was not high enough to substantially influence ME. The role of bile acids in digestion is to emulsify fats by lying at the lipid-water interface (Moran, 1982), and this action probably reduces contact between fatty acid component of fat micelle and cations in the digesta, thus reducing the possibility of soap formation. However, there is evidence to indicate that chicks may not be able to meet their bile acid requirement with high fat diets. Serafin and Nesheim (1967) reported that young chicks are unable to replenish bile salts lost by excretion as readily as do older birds. Hence, the mode of action of cholic acid in this particular study was

12 1970 ATTEH AND LEESON likely one of direct supplementation to the inadequate innate production in relation to the high levels of fatty acids used in these diets. The effects of increasing dietary calcium level appear antagonistic to that of cholic acid, although the mechanisms are probably different. Soap formation between the free fatty acids and calcium is believed to be responsible for the decrease in fat retention and dietary ME, and this difference is accentuated with increases in dietary calcium levels. Hakansson (1974), Sibbald and Price, (1977), and Atteh and Leeson (1984) observed similar decreases in fat retention and ME of diets that were high in both fat and calcium concentrations. There does not seem to be any effect of supplemental cholic acid or calcium level on the type of fatty acid involved in soap formation, although there is some evidence to indicate that palmitic acid forms soaps more easily than does oleic acid. This is probably due to the fact that palmitic acid exhibits low micellar solubility in aqueous solutions containing bile salts (Garrett and Young, 1975). Thus, in diets supplemented with O/P mixture, 26.5% of the digesta fat was present as soap, 73.7% of which was accounted for by palmitic acid as against 13.3% by oleic acid. Evidence of absorption of soaps of oleic acid was manifested by the decrease in proportion of the soap fat in the excreta that was accounted for by oleic acid relative to what was observed in the digesta. Boyd et al. (1932) and Atteh and Leeson (1984) have reported previously on the ability of the chick to utilize the soaps of oleic acid. There is no evidence to suggest absorption of soaps of palmitic acid, since its proportion of soap in the excreta increased relative to that observed in the digesta. The proportion of excreta fatty acid, that was present as soap, decreased with increase in the age of the chick, regardless of fatty acid type or level of cholic acid and calcium. Associated with decreased soap formation by the older bird was a concomitant increase in both fat retention and ME of diets when fatty acids were supplemented. Fedde et al. (1960), Renner and Hill, (1960), and Whitehead and Fisher (1975) observed similar increases in fat retention. Carew et al. (1972), and Polin and Hussein (1982) showed that the young chick does not have full physiological capacity for fat absorption. Serafin and Nesheim (1967) also reported that young chicks are unable to replenish bile salts lost by excretion as readily as do older birds. Gomez and Polin, (1976) suggested that the maturation of the mechanism involved in the absorption of saturated fatty acids in chickens appeared to involve the availability of bile acids. Thus, the decreases in soap formation and increases in fat retention and ME of diets with supplemented fatty acids with increases in age of the chicks would suggest a possible increase in bile production that enabled the chicks to utilize the fatty acids better as they grew older. The increase in phosphorus retention occurring with increases in the age of the bird, was probably associated with bone growth (Scott et al, 1982). Holmes et al. (1931) also reported that calcium and phosphorus are deposited in a fairly constant ratio regardless of the completeness of bone development. In contrast to this, there does not seem to be any significant increase in magnesium requirement with increase in age of the chicken. The fact that magnesium requirements of laying hens, as suggested by the National Research Council (NRC, 1977), is lower than that of the chick would suggest that the broiler chick does not have to increase its magnesium retention substantially to meet its requirements, which was the case observed in this study. The difference in bone minerals between chicks sampled at 5 to 8 weeks agrees with earlier observations of mineral retention. Thus, the increase in bone calcium content with age was accompanied by an increase in bone phosphorus content, possibly to maintain a fairly constant calcium:phosphorus ratio. The decrease in bone ash and bone calcium content with fatty acid supplementation was probably due to a reduction in the amount of calcium that was available for bone calcification due to soap formation. Gardiner and Whitehead (1976) and Atteh and Leeson (1983b) observed similar decreases in bone ash and bone calcium content with fatty acid supplementation. The decrease in bone magnesium with increases in dietary calcium level agrees with earlier findings (Hakansson, 1975; Atteh and Leeson, 1983a) showing negative magnesium balance at high calcium levels. Although there was a decrease in percentage calcium retention with increases in dietary calcium levels, there was no difference in bone calcium content between birds on the diets with the two levels of calcium, since both groups retained identical absolute quantities of calcium. It is concluded that the ability of broilers to utilize fatty acids depends not only on the type of fatty acid involved, but also on bird age.

13 AGE, CHOLIC ACID, AND CALCIUM IN BROILERS 1971 Supplemental cholic acid results in enhanced utilization of free fatty acids through a process that reduces soap formation during the process of digestion, and as evidenced by the dramatic improvement in feed utilization, such supplementation warrants futher investigation. REFERENCES Andrews, L. D., G. S. Nelson, G. C. Harris, and T. L. Godwin, Performance of five strains of broilers in four tier cage systems with plastic mat floors. Poultry Sci. 54: Association of Official Analytical Chemists, Pages 21 and 447 in Official Methods of Analysis, 13th ed. Assoc. Offic. Anal. Chem., Washington, DC. Atteh, J. O., and S. Leeson, 1983a. Effects of excess dietary calcium, magnesium and phosphorus on performance and mineral metabolism of broiler chicks. Nut. Rep. Int. 28(3): Atteh, J. O., and S. Leeson, 1983b. Effects of dietary fatty acids and calcium levels on performance and mineral metabolism of broiler chickens. Poultry Sci. 62: Atteh, J. 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