Evaluation of Dietary Supplements of Lipase, Detergent, and Crude Porcine Pancreas on Fat Utilization by Young Broiler Chicks

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1 Evaluation of Dietary Supplements of Lipase, Detergent, and Crude Porcine Pancreas on Fat Utilization by Young Broiler Chicks W. AL-MARZOOQI 1 and S. LEESON 2 Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 ABSTRACT The purpose of Experiment 1 was to improve gain (P < 0.01) and a quadratic effect on apparent fat the digestibility of fat through the use of supple- mental lipase enzymes. A 2 3 factorial arrangement of treatments involving two levels of animal-vegetable blend fat (AV) (4 and 8%) and three enzyme treatments, digestibility (P < 0.05) and feed utilization (P < 0.01). Experiment 3 was designed to test the effect of Pancreatic enzyme at 0 or 1.339% in combination with two levels of detergent, namely 0 and 10% (with 4% added AV). The namely none; Pancreatic, 0.714%; and Pancreatin, detergent used consisted of a mixture of 95% Span %, were randomly allocated within a battery and 5% Tween 60. In general, there was no significant brooder. There was an increase in diet ME and apparent effect of detergent (P > 0.05). Experiment 4 was conducted to test the effect of supplementation of graded levels of fat digestibility when Pancreatic and Pancreatin enzymes were used (P < 0.01). However, both enzymes ground crude porcine pancreas at 0, 0.321, 0.535, 0.750, 0.964, 1.178, or 1.392% of the diet on performance of male caused lower feed intake and lower BW gain (P < 0.01). broiler chicks to confirm the anorexic effect caused by In Experiment 2, Pancreatic enzyme was used at graded supplementing with Pancreatic enzyme. In general, levels of 0, 0.214, 0.429, 0.643, 0.857, and 1.071%, involving there was no significant effect of feeding crude porcine 4% dietary AV fat. The ME values were greater as the pancreas on the performance of male broiler chicks (P enzyme level increased (P < 0.01). However, as found in > 0.05). In these studies, lipase enzymes improved fat Experiment 1 lower feed intake and BW gain were observed with all enzyme levels compared with the control group. There was a linear effect on feed intake and BW digestion, although it is suspected that associated reduced feed intake may be associated with contaminants such as cholecystokinin hormone. (Key words: lipase, detergent, metabolizable energy, fat digestion) 1999 Poultry Science 78: INTRODUCTION Young poultry do not utilize and absorb fats effectively, especially animal fats. Sell et al. (1986) reported that fat retention improved with age in poults, regardless of fat source or fat level. Fat retention ranged from 66.4 to 83.7% and from 90.8 to 96.5% at 2 and 8 wk of age, respectively. Wiseman and Salvador (1991) observed a marked reduction in overall AME of fats that linearly decreased with increasing free fatty acids; the effect was more pronounced with younger birds. Leeson and Atteh (1995) concluded that turkey poults, similar to chicks, have an age-related depression in fat utilization that is undoubtedly related to use of the saturated fatty acids found Received for publication November 30, Accepted for publication July 28, Current address: Department of Animal and Veterinary Sciences, College of Agriculture, Sultan Qaboos University, College of Agriculture, Muscat, Sultanate of Oman. 2 To whom correspondence should be addressed: SLEESON@aps.uoguelph.ca predominantly in animal fats. An additional problem with young birds is that they have a low level of natural lipase production. Krogdahl and Sell (1989) found that dietary tallow and animal-vegetable fat (AV) were not efficiently utilized until the time that lipase activity reached its maximum levels between 40 and 56 d of age. In addition, Noy and Sklan (1995) reported that net duodenal secretion of amylase, trypsin, and lipase were low at 4 d and increased 100-, 50-, and 20-fold, respectively, by 21 d of age in broiler chicks. The age-related effect on utilization of fats is also correlated with less efficient recirculation of bile salts in very young chicks (Serafin and Nesheim, 1970). Jackson et al. (1971) and Smallwood et al. (1970, 1972) attributed the inefficient bile salt recycling to the chick s small bile salt pool. Consequently, there is a low rate of bile salt synthesis and inefficient enterohepatic circulation of bile salts. Several studies have shown that supplementing the diet with bile salts improves the utilization of dietary fat by Abbreviation Key: AV = animal-vegetable fat; CCK = cholecystokinin; HLB = hydrophilic lipophilic balance. 1561

2 1562 AL-MARZOOQI AND LEESON chicks (Edwards, 1962; Eyssen et al., 1965; Garlich and Nesheim, 1965; Katongole and March, 1980; Polin and Hussein, 1982; and Atteh and Leeson, 1985). Unfortunately, synthetic bile salts are very expensive, and so an alternative is to identify cheaper emulsifying agents or detergents that have the ability to transform a hydrophobic surface into a hydrophilic one. Such surfactants, detergents, and emulsifiers are commonly used in many industrial processes and are available in large quantities at more reasonable prices than bile salts. For example, Webling and Holdsworth (1965) used lauryl sulphate as a detergent to improve calcium utilization in the diet of broilers. Jones et al. (1992) showed that addition of emulsifiers increased digestibility of tallow (P < 0.01). Furthermore, other researchers (Augur et al., 1947; Polin, 1980) reported that the addition of lecithin, an emulsifier, increased digestibility of fats containing long-chain, saturated fatty acids. Experiments were conducted with the aim of improving digestibility of fat by young broilers through the use of supplemental lipase enzymes and detergents. Two lipase enzymes were chosen for study based on previous in vitro screening of those potentially available in commercial quantities (W. Al-Marzooqi and S. Leeson, unpublished data). Detergent selection was based on the system of hydrophilic/lipophilic balance as developed by ICI (1992). Experiment 1 MATERIALS AND METHODS Two hundred eighty-eight male broiler chicks of commercial strain were obtained from a local hatchery at 1 d of age. They were housed in electrically heated battery brooders, and 24 h of light were provided. Feed and water were provided for ad libitum consumption. The 2 3 factorial arrangement of treatments involved two levels of added blended AV (50% unsaturates at 4 or 8%), and three enzyme treatments: none, 0.714%, Pancreatic, and 0.714% Pancreatin (enzyme activity claimed to be 25 units USP/mg by manufacturer). There were six replicates for each of the six dietary treatments, and each replicate cage contained eight male broiler chicks. Treatment/replicate combinations were randomly allocated within the battery brooder. The control diet, containing only 4% AV (Table 1), was used to feed all chicks from 1 to 3 d of age to allow enough time to reduce any variation of nutrient absorption from yolk residue among the chicks. From each replicate cage, six chicks of eight within the same range of BW were introduced to experimental diets on Day 4. Body weight was determined for each replicate on Days 4 and 12, and feed was recorded over this period. A total feed intake/excreta collection proce- 3 Leco Corp., St. Joseph, MI Enzyme Development Corp., New York, NY (25 units USP/mg). TABLE 1. Percentage diet composition and calculated nutrient content Ingredient Diet 1 Diet 2 Corn Soybean meal, 48% Canola meal Limestone Calcium phosphate Animal-vegetable fat Iodized sodium chloride DL-methionine Vitamin-mineral premix Calculated nutrient content ME, kcal/kg 3,052 3,072 Crude protein, % Crude fat, % Calcium, % Available phosphorus, % Methionine, % Lysine, % Rothsay, Dundas, Ontario, Canada L9H 5G1. Contains 14:0, 2.1%; 16:0, 21.2%; 18:0, 14.5%; 16:1, 0.3%; 18:1, 31.5%; and 18:2, 24.7%. 2 Provided per kilogram of diet: vitamin A, 8,000 IU (retinyl palmitate); cholecalciferol, 40 mg; vitamin E, 11 IU (dl-α-tocopheryl acetate); riboflavin, 9.0 mg; biotin, 0.25 mg; pantothenic acid, 11.0 mg; vitamin B 12, 13 mg; niacin, 26 mg; choline, 900 mg; vitamin K, 1.5 mg; folic acid, 1.5 mg; ethoxyquin, 125 mg; manganese, 55 mg; zinc, 50 mg; copper, 5 mg; iron, 30 mg; and selenium, 0.1 mg. dure was undertaken from 6 to 9 d of age to measure diet AME n and fat digestion. Feed and excreta were dried for 48 h at 40 C. All samples of feed and excreta were ground prior to assay for determination of moisture content by oven drying at 135 C for 2 h and 20 min, for crude fat by petroleum ether extraction using a Soxhlet apparatus, for nitrogen content by the AOAC (1997) method of combustion #9903 FP428 using the model FP428 nitrogen analyzer, 3 and for gross energy content by adiabatic bomb calorimetry. To estimate the proportion of fatty acids in the excreta that was present as soap, the method of fat determination reported by Atteh and Leeson (1984) was used. These samples were subjected to two stages of petroleum ether extraction. The first extraction (1) was to remove neutral fat and free fatty acids. Thimbles containing the residue of the first extraction were placed in 25% hydrochloric acid for about 2 h at room temperature to liberate fatty acids that were present as soap. The samples were then freeze-dried, and the process of ether extraction was repeated (2). Data collected were subjected to analysis of variance; where significant differences were observed, means were further subjected to Tukey s test (ANOVA, SAS Institute, 1991). Experiment 2 Two hundred eighty-eight commercial strain male broiler chicks were obtained from a local hatchery at 1 d of age. They were housed in electrically heated battery brooders, and 24 h of light were provided. Feed and water were offered for ad libitum intake. Diet 1, as used in the previous experiment (Table 1), was used. The six dietary treatments consisted of six graded levels of Pancreatic enzyme, 4 namely 0, 0.214, 0.429, 0.643, 0.857, and 1.071%.

3 USE OF LIPASE AND DETERGENTS IN BROILER DIETS 1563 There were six replicates for each of six treatments, and each replicate cage contained eight male broiler chicks. The control diet devoid of enzyme was used to feed all chicks from 1 to3dofagetoallow enough time to reduce any variation of nutrient absorption from yolk residue among the chicks. From each replicate cage, six chicks of eight, within the same range of BW, were introduced to experimental diets on Day 4. A total feed intake/excreta collection procedure was undertaken from 9 to 12 d of age. Body weight was determined at Days 4 and 12, and feed intake was measured over this time. Other measurement criteria were diet AME n and fat digestion undertaken when birds were 9 to 12 d of age. Excreta collection and analyses were as described for Experiment 1. The data collected was subjected to regression analysis using the PROC REG function of SAS (SAS Institute, 1991). Experiment 3 Commercial strain male broiler chicks were obtained from a local hatchery at 1 d of age. They were housed in electrically heated battery brooders, and 24 h of light were provided. Diet 1, offered for ad libitum intake (Table 1), was used for all birds. The experimental design was a 2 2 factorial involving the same Pancreatic enzyme as used in Experiment 2, at 0 or 1.339% of the diet, and two levels of detergent, namely, 0 and 0.4%. In this experiment, the activity of Pancreatic enzyme was 20 units USP/mg. Treatment/replicate combinations were randomly allocated to cages within a battery brooder. The detergent used was a mixture of 95% sorbitan monosterate [Span 60 ; hydrophilic lipophilic balance (HLB) 4.7] and 5% polyoxyethylene sorbitan monosterate (Tween 60 ; HLB 14.9) to give a HLB value of 5, which is that appropriate for an oil-in-water emulsion involving lard (ICI, 1992). The detergent was premixed with the AV prior to diet incorporation. There were four replicates for each of the four treatments, and each replicate cage contained eight male broiler chicks. Other experimental details were as described in Experiment 1. Excreta samples were collected and ground prior to assay for moisture, crude fat, nitrogen, and gross energy content as described in Experiment 1. Data collected were subjected to analysis of variance, and, where significant differences were observed, means were further subjected to Tukey s test. Statistical analysis of all data was carried out by ANOVA (SAS Institute, 1991). Experiment 4 One hundred sixty-eight male broiler chicks of a commercial strain were obtained from a local hatchery at 1 d of age. They were housed in electrically heated battery brooders, and 24 h of light were provided. Feed and water were offered for ad libitum intake. Diet formulation was as Diet 1 (Table 1). The seven dietary treatments consisted of seven graded levels of ground dried crude porcine pancreas at 0, 0.321, 0.535, 0.750, 0.964, 1.178, and 1.392%. The porcine pancreases were collected from animals within 1 h of slaughter; separated from connective tissues and excessive fat layers; cut into small, thin pieces; and then freeze-dried to a constant weight over 3 d. There were three replicates for each of seven dietary treatments, and each replicate cage contained eight male broiler chicks. Other experimental details were as described in Experiment 1. Total feed intake and BW were determined for each replicate at 4 and 12 d of age. Data collected were subjected to ANOVA (SAS Institute, 1991). Experiment 1 RESULTS There was no significant difference in AME for diets containing 4 and 8% fat (Table 2); however, there was TABLE 2. Effects of supplemental lipase enzymes on performance of male broiler chicks from 4 to 12 d of age, Experiment 1 Dietary Fat Excreta 1 treatment Feed intake Weight gain Feed:gain digestibility soap AME n (g/b) (%) (kcal/kg) Enzyme level None a a 1.30 c 75.6 c 18.6 a 2,814 c Pancreatic c 86.0 c 1.51 a 88.1 a 13.0 b 3,113 a (0.714%) Pancreatin b b 1.41 b 83.5 b 12.2 b 2,974 b (0.714%) Significance * ** ** ** ** ** Fat level 4% ,001 8% ,932 Significance ** ** NS NS ** NS Enzyme fat NS ** ** NS NS NS MSE a c Values with the different superscript letters are different (P < 0.01). n = 48 birds per treatment. 1 Excreta fat as soap as proportion of total excreta fat. 2 MSE = mean standard error.

4 1564 AL-MARZOOQI AND LEESON TABLE 3. Details of enzyme fat level interaction, Experiment 1 Fat Enzyme level BWG 1 Feed:gain (%) (g) 4 None Pancreatic (0.714%) Pancreatin (0.714%) None Pancreatic (0.714%) Pancreatin (0.714%) BWG = body weight gain. more soap excreta with the 8% fat diet (P < 0.01, Table 3). Increased AME n values were recorded for diets when both Pancreatic and Pancreatin enzymes were used (P < 0.01; Table 2). Although both enzymes caused reduced feed intake, BW gain, and fecal soap formation (P < 0.01), apparent fat digestibility was improved (P < 0.01; Table 2). Poor feed utilization was observed with birds fed diets supplemented with both enzymes compared with the control group (P < 0.01; Table 3). The enzyme fat interactions were significant for BW gain and feed efficiency (P < 0.01; Table 3). Experiment 2 Pancreatic enzyme level was linearly related to improvements in diet AME n (P < 0.01). Again, enzyme addition caused a linear reduction of feed intake, BW gain, and fecal soap formation. The inefficient utilization of feed, for Pancreatic enzyme treatment compared with the control group, was associated with lower feed intake and BW gain (Table 4). A linear (P < 0.01) and a quadratic effect (P < 0.05) of dietary Pancreatic enzyme on apparent fat digestibility was observed (Table 4). Experiment 3 Detergent had no effect on diet AME feed intake, BW gain, feed efficiency, apparent fat digestibility or excreta soap formation. Adding enzyme to the diet improved AME (P < 0.01; Table 5), increased apparent fat digestibility, and reduced excreta soap formation (P < 0.01; Table 5); however, this result was accompanied by reduced feed intake and BW gain (Table 5). There was no significant interaction between enzyme and detergent for any of the parameters measured (P > 0.05). Experiment 4 Crude porcine pancreas had no effect on performance of male broiler chicks (P > 0.05). DISCUSSION In Experiment 1, the 4 and 8% AV content diets were formulated to have the same energy content (Table 1), and, therefore, no significant difference in ME value of the diet was expected. However, as the fat inclusion increased, there was greater fecal soap formation (Table 3). These findings agree with the report of Atteh and Leeson (1984), who showed that fat level and fat saturation influence the level of soap formation. Many researchers have found that the ability of young poultry to utilize and absorb fats, especially animal fats, improves as the chick ages (Katongole and March 1980; Sell et al., 1986). Others have attributed the poor digestibility of dietary fat to the limited secretion of lipase enzyme (Noy and Sklan, 1995) and bile salts (Serafin and Nesheim, 1970). Supplemental enzymes did improve fat digestion and reduce the formation of fat present in the excreta as soap. It is shown in Tables 2 and 3 that the supplemental enzymes caused an increase in fat digestion, a consequence of which is attributed to increased diet AME n. These results are somewhat contrary to those of Polin et al. (1980), who reported that supplemental lipase in the diet was not as effective as cholic acid in increasing tallow digestibility in chicks. Although both enzymes improved diet AME n and apparent fat digestibility, these effects were associated with reduced feed intake, BW gain, and less efficient feed utilization (Table 3). TABLE 4. Effect of supplemental Pancreatic enzyme on performance of male broiler chicks from 4 to 12 d of age, Experiment 2 (n = 48 birds per treatment) Pancreatic Feed Weight Fat Excreta 1 enzyme level intake gain Feed:gain digestibility soap AME n (%) (g/b) (%) (kcal/kg) , , , , , ,899 Linear ** ** NS ** ** ** Quadratic NS NS ** * NS NS R-square CV Excreta fat as soap as proportion of total excreta fat. *P < **P < 0.01.

5 USE OF LIPASE AND DETERGENTS IN BROILER DIETS 1565 TABLE 5. Effects of supplemental Pancreatic enzyme and detergent on performance of male broiler chicks from 4 to 12 d of age, Experiment 3 (n = 48 birds per treatment) Dietary Weight Fat Excreta 1 treatment Feed intake gain Feed:gain digestibility soap AME n (g/b) (%) (kcal/kg) Enzyme , ,107 Significance ** ** ** ** ** ** Detergent , ,964 Significance NS NS NS NS NS NS Enzyme NS NS NS NS NS NS detergent MSE Excreta fat as soap as proportion of total excreta fat. 2 0 vs 1.339% of the diet. 3 0 vs 0.4% of the diet. 4 MSE = mean standard error. **P < Supplemental crude porcine pancreas did not result in improved fat utilization and had no anorexic effect on the chicks. Enzyme levels were not assayed in this extract, and it is possible that inclusion levels were insufficient to mimic lipase supplements used previously, or that handling and drying conditions were not conducive to optimum enzyme activity. The reduced feed intake and growth rate associated with feeding lipase enzyme might be related to contamination with cholecystokinin (CCK), which is a polypeptide hormone released into the duodenum and jejunum in response to the entrance of digesta. Dockray (1977, 1979) reported that this hormone occurs in the brain and gut of birds, whereas Savory and Gentle (1980) showed that intravenous injection of CCK in two strains of domestic fowl resulted in a dose-related reduction in feed intake. Similar results were found in rats and rabbits (Gibbs et al., 1973; Houpt et al., 1978). Antin et al. (1975) argued that the action of CCK reflects the satiety signal in rats. It is concluded that Pancreatic enzyme has a role to play in improving fat digestion in young chicks. However, the associated problem of reduced feed intake must be resolved. TABLE 6. Effect of feeding crude porcine pancreas from 4 to 12 d on male broiler performance (n = 24 birds per treatment) Body weight Dietary treatment Feed intake gain Feed efficiency (%) (g/bird) (feed:gain) Significance NS NS NS MSE P < MSE = mean standard error. REFERENCES Antin, J., J. Gibbs, J. Holt, R. C. Young, and G. P. Smith, Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J. Comp. Physiol. Psychol. 89: AOAC, Official Methods of Analysis. AOAC, Arlington, VA. Atteh, J. O., and S. Leeson, Effects of dietary saturated and unsaturated fatty acids and calcium levels on performance and mineral metabolism of broiler chicks. Poultry Sci. 63: Atteh, J. O., and S. Leeson, Influence of age, cholic acid and calcium levels on performance, utilization of FFA s and bone mineralization in broilers. Poultry Sci. 64: Augur, H. S., H. S. Rollman, and H. J. Deuel, The effect of crude lecithin on the coefficient of digestibility and the rate of absorption of fat. J. Nutr. 33: Dockray, G. J., Progress in gastroenterology. Molecular evolution of gut hormones: Application of comparative studies on the regulation of digestion. Gastroentology 72: Dockray, G. J., Cholecystokinin-like peptides in avian brain and gut. Experientia 35: Edwards, H. M., Observation on feeding cholic acid to broilers. Poultry Sci. 41: Eyssen, H., M. Vandeputle, and E. Evard, Effect of various dietary bile acids on nutrient absorption and on liver size in chicks. Arch. Int. Pharmocodyn. 158: Garlich, J. D., and M. C. Nesheim, Effect of sodium taurocholate on fat malabsorption induced by feeding unheated soybean proteins. Proc. Soc. Exp. Biol. Med. 118: Gibbs, J., J. Holt, R. C. Young, and G. P. Smith, Cholecystokinin decreases food intake in rats. J. Comp. Physiol. Psychol. 84: Houpt, T. R., S. M. Anika, and N. C. Wolff, Satiety effects of cholecystokinin and caerulein in rabbits. Am. J. Physiol. 235:R23. I.C.I The HLB System. A Time Saving Guide to Emulsifier Selection. I.C.I. Surfactants, Wilmington, DE. Jackson, B. T., R. A. Smallwood, and G. J. Piasecki, Fetal bile salt metabolism. 1. The metabolism of sodium cholate 14 C in the fetal dog. J. Clin. Invest. 50: Jones, D. B., J. D. Hancock, D. L. Harmon, and C. E. Walker, Effects of exogenous emulsifiers and fat sources on nutrient digestibility, serum lipids, and growth performance in weanling pigs. J. Anim. Sci. 70:

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