Effect of Dietary Lipase Enzyme on Gut Morphology, Gastric Motility ...

METABOLISM AND NUTRITION Effect of Dietary Lipase Enzyme on Gut Morphology, Gastric Motility, and Long-Term Performance of Broiler Chicks W. Al-Marzooqi1 and S. Leeson2 Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 acteristics, especially feed intake. Only starter diets (0 to 21 d) were supplemented with 0, 0.375, 0.750, or 1.125% enzyme, and each diet was represented by three replicate pens of 25 male chicks each. Subsequent diets did not contain any enzyme. During the first 3 wk, increased dietary concentration of lipase enzyme caused a linear reduction of feed intake and body weight gain (P < 0.01). At 21 d the percentage weight of the liver was significantly greater with 1.125% enzyme (P < 0.01). However, added enzyme had no effect on 21 to 42 d or 1 to 42 d growth or feed intake (P > 0.05) or on the size of any internal organs examined at 42 d. Pancreatic威 enzyme has previously been shown to improve fat digestion and increase diet AMEn for young chicks fed animal-vegetable blended fats. These positive effects, however, are associated with marked anorexia, and from the present study, it seems that this effect was not related to physical changes in gut histology or in prolonged digesta transit time.

ABSTRACT Three experiments were conducted to test a previously described anorexic effect of graded dietary supplements of Pancreatic威 lipase enzyme on gut structure, gastric motility, and long-term performance of broiler chicks. In Experiment 1, dietary Pancreatic威 enzyme was used at graded levels of 0, 0.214, 0.429, 0.643, 0.857, and 1.071% to test the effect of this enzyme on gut structure, whereas Experiment 2 was designed to test its effect at 0, 0.268, 0.536, 0.804, 1.071, and 1.339% on gastric motility. The histological examination of the small intestine and a cineradiographic study of birds fed diets supplemented with lipase enzyme failed to detect any difference in gut structure, and there was no apparent adverse effect on gastric motility. Experiment 3 was conducted to test the effect of graded supplements of Pancreatic威 enzyme on performance of 300 male broiler chicks raised for 6 wk to determine whether the enzyme had any long-term effect on performance char-

(Key words: lipase, fat, intestinal physiology) 2000 Poultry Science 79:956–960

and Gentle (1980) reported that intravenous injection of cholecystokinin (CCK) depresses feed intake in chickens. Antin et al. (1975) observed that CCK not only reduces feed intake but also elicits satiety in rats. The Pancreatic威 enzyme used in these studies is extracted from the pancreas of pigs, and so could conceivably contain some CCK activity. The aim of the experiments reported here was to test the effect of Pancreatic威 enzyme on gut structure and gastric motility and to test whether this enzyme has any lasting effect on feed intake and performance of male broiler chicks.

INTRODUCTION Krogdahl and Sell (1989) found that pancreatic lipase activity was maximized 42 to 56 d after hatch. Over the same period there was a marked improvement in utilization of dietary tallow and animal-vegetable fat (AV) by young birds. Noy and Sklan (1995) reported that lipase activity increased at a slower rate than for most other digestive enzymes. Therefore, a low level of natural lipase production in young birds likely limits fat digestion. However, based on the results obtained in our previous studies (Al-Marzooqi, 1998), a clear pattern of reduced feed intake and growth rate caused by various supplemental lipase enzymes is cause for concern in any practical application of such supplements. Savory

MATERIALS AND METHODS Experiment 1 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

Received for publication November 1, 1999. Accepted for publication February 23, 2000. 1 Present address: Department Animal and Veterinary Sciences, College of Agriculture, Sultan Qaboos University, PO Box 34, Al-Khod 123, Muscat, Sultanate of Oman. 2 To whom correspondence should be addressed: SLEESON@aps. uoguelph.ca.

Abbreviation Key: AV = animal-vegetable fat; CCK = cholecystokinin.

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LIPASE ENZYME AND GUT FUNCTION TABLE 1. Percentage diet composition and calculated nutrient content Ingredient Corn Soybean meal (48%) Canola meal Limestone Calcium phosphate Animal-vegetable fat Iodized salt DL-methionine Vitamin-mineral premix1 Calculated nutrient content ME, kcal/kg Crude protein, % Crude fat, % Calcium, % Available phosphorus, % Methionine, % Lysine, %

1 to 21 d Starter

21 to 42 d Grower

54.68 31.71 5.46 1.28 1.62 4.00 0.31 0.19 0.75

60.70 30.68 ... 1.50 1.50 4.50 0.31 0.06 0.75

3,052 22.08 6.33 0.93 0.45 0.56 1.24

3,144 19.96 6.95 0.95 0.49 0.40 1.10

1 Provided per kilogram of diet: vitamin A, 8,000 IU (retinyl palmitate); cholecalciferol, 40 mg; vitamin E, 11 IU (d1-α-tocopheryl acetate); riboflavin, 9.0 mg; biotin, 0.25 mg; pantothenic acid, 11.0 mg; vitamin B12, 13 µg; 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.

brooders, and 24 h of light was provided. Feed and water were provided ad libitum. Birds were fed a corn-soybean meal diet containing 4% animal-vegetable (AV) blended fat (Table 1). Six treatments consisted of graded levels of Pancreatic威 enzyme3 (25 units USP/mg), namely 0, 0.214, 0.429, 0.643, 0.857, and 1.071% of the diet. There were six replicates for each of the six treatments, and each replicate cage contained eight male broiler chicks. The unsupplemented diet was used to feed all chicks up to 3 d of age to allow enough time to reduce any variation due to nutrient absorption from yolk residue. From each replicate cage, six of the eight chicks, within the same range of body weight, were introduced to experimental diets on Day 4. A total feed intake per excreta collection procedure (Namkung and Leeson, 1999) was undertaken from 9 to 12 d, although these data are not reported in this paper. On Day 12, one bird per replicate was killed by cervical dislocation to examine the small intestine. A section of jejunum 2 cm from Meckel’s diverticulum was fixed in 10% buffered formalin, embedded in paraffin, sectioned at 6 µ, and stained with hematoxylin and eosin for observation using light microscopy.

Experiment 2 One hundred ninety-two 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 was provided. Feed and

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Enzyme Development Company, New York, NY 10121-0034. Phillips Diagnostics, Mississauga, Ontario, Canada N0B 2K0.

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water were provided ad libitum. Six dietary treatments consisted of graded levels of Pancreatic威 enzyme, namely 0, 0.268, 0.536, 0.804, 1.071, and 1.339% added to the basal diet shown in Table 1. In this experiment, the activity of Pancreatic威 enzyme was 20 units USP/ mg, and the concentration of this enzyme was adjusted to equate the same enzyme activity (25 units USP/mg), as used in Experiment 1. Feed intake was not recorded. There were four replicates for each of six dietary treatments, and each replicate cage contained eight male broiler chicks. At 12 d of age, six birds from each of the control group and the highest level of enzyme were subjected to a cineradiographic technique (Dzuik and Duke, 1972). The examination began after administration of 20 cc undiluted barium sulfate was given by gavage, and it involved lateral and ventrodorsal fluoroscopic examinations of birds placed in a restraint box, using a Phillips Diagnost 66 System,4 and was recorded on Super-VHS media for review. No sedative or anesthetic agents were administered. We recorded patterns of duodenal motility, refluxes per minute counted by the flow of barium sulfate passing from the muscular stomach to the duodenum and upper part of the ileum, and duodenal diameter. Wing tags attached to each bird from each treatment controlled internal measurements, and this procedure controlled any variation in radiographic magnification. At the end of the cineradiographic examination, three birds from each group were weighed and then killed by cervical dislocation. The heart, jejunum and ileum, and duodenal loop with pancreas were excised, dried with blotting paper, and weighed separately for each bird. Because no significant differences were observed, birds from other treatments were not tested. Data collected were subjected to ANOVA, and when 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 3 Three hundred, 1-d-old male broiler chicks of a commercial strain were weight-sorted, wing-banded, and randomly allocated to one of four diet treatment groups. Each pen was 2.44 × 1.83 m, and these were located in one of two identical rooms providing environmental control. The temperature was maintained at 32 C for 5 d and was subsequently reduced gradually with normal brooding practices. Lighting was at 80 lx for 23 h/d. Feed and water were provided for consumption ad libitum. Four starter diets were formulated to provide a similar nutrient profile (Table 1) with the exception of using four graded levels of Pancreatic威 enzyme, namely 0, 0.375, 0.750, or 1.125% of the diet. There were three replicates for each treatment, and each replicate pen contained 25 male broiler chicks. Experimental diets were started when the chicks were 1 d old and were replaced by a standard grower diet containing no enzyme, on Day 21 for all treatment groups (Table 1). Body weight gain, feed intake, and feed efficiency were determined

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AL-MARZOOQI AND LEESON TABLE 2. Effect of supplemental Pancreatic威 enzyme on gastric motility and organ weights (% body weight) of 12-d-old broiler chicks, Experiment 2 Dietary treatment

Duodenal refluxes per minute

Duodenal diameter

Heart

Jejunum and ileum

Pancreas and duodenum

0.87 0.86 NS 0.051

4.92 4.19 NS 0.352

2.37 2.19 NS 0.210

(mm) Enzyme (%) None Pancreatic威 (1.071%) Significance Error mean square

5.87 5.10 NS 1.13

1.61 2.27 * 0.25

*P < 0.05.

for each replicate at 21 and 42 d. On Day 21, four birds per replicate per treatment were weighed then killed by cervical dislocation. The heart, liver, jejunum and ileum, and duodenal loop with pancreas were excised, surface dried using blotting paper, and weighed separately for each bird. The liver only was removed at Day 42. The data collected were subjected to regression analysis using the PROC REG function of SAS (SAS Institute, 1991).

zyme supplementation. There was no significant difference in feed intake and body weight gain among treatment groups from 21 to 42 d (when no enzyme was fed) or when data were expressed over the 1 to 42 d grow-out period. However, birds did utilize feed more efficiently from 21 to 42 d when they had received prior (1 to 21 d) enzyme treatment at 1.125% (P < 0.05; Table 3) as

RESULTS AND DISCUSSION Experiment 1 There was a significant (P < 0.01) linear decline in feed intake from 4 to 12 d of age as lipase concentration increased [(Y = 243.6 (± 4.0) − 127.3 (± 6.2)x), where Y = grams feed intake per bird, and x = percentage inclusion lipase enzyme]. Histological examination of the small intestine from birds fed the highest dietary enzyme vs. the control birds essentially showed no difference in morphology, and there was no sign of villi effusion or cell death (Figure 1).

Experiment 2 The cineradiographic study results showed no significant differences in gastric refluxes per minute between the two groups (Table 2). The duodenal diameter of birds fed the diet supplemented with lipase enzyme was significantly larger compared to that of the control birds (P < 0.05; Table 3), even though these birds were expected to eat less feed (Experiment 3). There was no significant difference (P > 0.05) between the treatments in percentage weight of the heart, small intestine, or duodenal loop with pancreas (P > 0.05). These results minimize the likelihood that the Pancreatic威 enzyme used was contaminated with a polypeptide hormone, which has been shown to depress gastroduodenal motility (Duke et al., 1979; 1985).

Experiment 3 Body weight gain, feed intake, and feed efficiency of male broiler chicks are presented in Table 3 for 1 to 21, 21 to 42, and 1 to 42 d. From 1 to 21 d, there was a linear decrease in growth and feed intake associated with en-

FIGURE 1. Section of jejunum (×100) from 12-d-old broiler chicks fed the control diet (I) or the diet supplement with 1.071% dietary Pancreatic威 enzyme (II). a) Intestinal wall, b) crypts, c) goblet cells, and d) villi.

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LIPASE ENZYME AND GUT FUNCTION TABLE 3. Effect of supplemental Pancreatic威 enzyme fed from 1 to 21 d on performance of male broilers, Experiment 3 1 to 21 d Body weight gain/chick

Dietary treatment

21 to 42 d

Feed intake/chick

Feed:gain

Body weight gain/bird

(g) Enzyme (%) None 0.375 0.750 1.125 Linear regression Error mean square

600 588 561 473 ** 39.5

Feed intake/bird

1 to 42 d

Feed:gain

Body weight gain/bird

(g)

994 978 943 847 ** 32.5

1.65 1.67 1.68 1.78 NS 0.08

1,609 1,582 1,575 1,552 NS 104

Feed intake/bird

Feed:gain

4,055 3,892 3,823 3,626 NS 216

1.84 1.81 1.79 1.79 NS 0.04

(g) 3,061 2,914 2,880 2,779 NS 188

1.90 1.84 1.83 1.79 ** 0.39

2,209 2,170 2,136 2,025 NS 145

*P < 0.05. **P < 0.01.

compared to the control group. Organ weights expressed as a percentage of body weight at 21 and 42 d of age are shown in Table 4. The percentage weight of the liver, which was not different at 42 d, was significantly greater at 21 d with the 1.125% enzyme compared to most other treatments (P < 0.01; Table 4). All livers had a normal shape and color similar to those of the control birds. The increase in liver weight is perhaps due to the increase in metabolic activity related to increased fat utilization. The lipase enzyme seems to have no long-term effect on feed intake or growth because 21 to 42-d performance was unaffected. As stated previously, the clear pattern of reduced feed intake and growth rate might be due to the contamination of the lipase enzyme with products such as CCK, which influences satiety signals and so influences appetite (Antin et al., 1975; Savory and Gentle, 1980). Various methods have been suggested as a means of controlling growth and body weight gain in broilers and breeder flocks at various times. Based on the results obtained in Experiments 1 and 2, the histological examination of the small intestine and the cineradiographic study showed that the reduced feed intake was not associated with changes in gut structure or with reduced gastric motility. In addition, the grow-out study (Experiment 3) clearly showed that feeding the enzyme from 1 to 21 d of age does not have any long-term negative carry-over effects. Therefore, this enzyme could be used to control the body weight gain and growth rate in

broiler breeder pullets, because it can linearly reduce feed intake and body weight gain without apparently having any adverse effect on the physiology of the bird. Although early growth depression, as a means of obtaining low-weight broiler breeder hens, can be achieved by severe feed restriction or by feeding diets deficient in selected nutrients, few systems are as effective as the dose-related response shown in Table 3. Therefore, it may be useful to study the role of Pancreatic威 enzyme in inducing controlled levels of anorexia in both broilers and broiler breeders, because the Pancreatic威 enzyme seems to have some potential benefit in inducing a voluntary reduction in feed intake. In terms of improving fat utilization by young birds, it may be beneficial to identify microbial sources of lipase, which presumably would not be contaminated by any hormones active in the bird.

REFERENCES Al-Marzooqi, W. S., 1998. Use of supplemental lipase enzyme and detergent to improve fat digestion in poultry. M.Sc. Thesis. University of Guelph, Guelph, Ontario, Canada. Antin, J., J. Gibbs, J. Holt, R. C. Young, and G. P. Smith, 1975. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J. Comp. Physiol. Psychol. 89:784–790. Duke, G. E., J. R. Kimmel, H. P. Hunt, and H. G. Pollock, 1985. The influence of avian pancreatic polypeptide on gastric secretion and motility in laying hens. Poultry Sci. 64:1231–1235.

TABLE 4. Effect of supplemental Pancreatic威 enzyme fed from 1 to 21 d on organ weights of male broilers (% body weight), Experiment 3 21 d Dietary treatment

Heart

Jejunum and ileum

Enzyme (%) None 0.375 0.750 1.125 Linear regression Error mean square

0.68 0.67 0.69 0.68 NS 0.09

3.24 3.33 3.15 3.47 NS 0.35

**Significant at P < 0.01.

42 d Pancreas and duodenum

Liver

Liver

1.47 1.82 1.79 1.92 NS 0.24

2.87 3.06 3.02 3.34 ** 0.26

1.51 2.58 2.28 2.54 NS 0.34

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Duke, G. E., J. R. Kimmel, P. T. Redig, and H. G. Pollock, 1979. Influence of exogenous avian pancreatic polypeptide on gastrointestinal motility of domestic turkeys. Poultry Sci. 58:239–246. Dzuik, H. E., and G. E. Duke, 1972. Cineradiographic studies of gastric motility in turkeys. Am. J. Physiol. 222:159–166. Krogdahl, A., and J. L. Sell, 1989. Influence of age on lipase, amylase, and protease activities in pancreatic tissue and intestinal contents of young turkeys. Poultry Sci. 68:1561– 1568.

Namkung, H., and S. Leeson. 1999. Effect of phytase enzyme on dietary AMEn and ileal digestibility of nitrogen and amino acids in broiler chicks. Poultry Sci. 78:1317–1320. Noy, Y., and D. Sklan, 1995. Digestion and absorption in the young chick. Poultry Sci. 74:366–373. SAS Institute, 1991. SAS User’s Guide. SAS Institute Inc., Cary, NC. Savory, C. J., and M. J. Gentle, 1980. Intravenous injection of cholecystokinin and caerulin suppress food intake in fowls. Experientia (Basel) 36:1191–1197.