Effects of dietary protein source on the digestive enzyme activities and electrolyte composition in the small intestinal fluid of chickens

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1 Effects of dietary protein source on the digestive enzyme activities and electrolyte composition in the small intestinal fluid of chickens L. Q. Ren,* F. Zhao,* 1 H. Z. Tan, J. T. Zhao, J. Z. Zhang,* and H. F. Zhang * * The State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing , China; and Wen s Foodstuffs Group Corporation Co. Ltd., Guangzhou , China INTRODUCTION 2012 Poultry Science Association Inc. Received December 7, Accepted March 16, Corresponding author: zsummit@163.com ABSTRACT An experiment was conducted to investigate the effects of dietary protein source on the digestive enzymes and electrolyte composition of digesta fluid in the duodenum, jejunum, and ileum of chickens. A 2 3 factorial and completely randomized design that used 2 types of diets that differed only in their protein source [a corn-soybean meal-miscellaneous meal diet (CSMD) and a corn-miscellaneous meal diet (CMD)] and 3 types of cannulated roosters (duodenal, jejunal, and ileal cannulations) was adopted. The experiments included 5 replicates for each of the 6 treatments, and each replicate involved 3 cannulated chickens. The digesta samples were collected for 1 h every 4 h between 09:30 and 18:30 h on d 31, 33, and 35 of the experiment. The amylase, lipase, trypsin, and chymotrypsin activities and the electrolyte composition in the duodenal, jejunal, and ileal fluid were determined. In general, no significant differences between the 2 dietary groups were observed for the mean of duodenal, jejuna, and ileal amylase, trypsin, chymotrypsin, and lipase, respectively. However, the duodenal amylase activity was lower in the CMD group than the CSMD group (P < 0.05), which was probably related to the lower duodenal ph value that was observed in this group (P < 0.01). A higher jejunal Mg 2+ concentration was observed in chickens that were fed the CMD (P < 0.05), whereas the differences in the Na +, K +, Cl, and Ca 2+ concentrations in the small intestine were not significant between the 2 diets (P > 0.05). In conclusion, the digestive enzymes and electrolytes in the small intestinal fluid of chickens adapted to the protein source of the diet, and each segment of the small intestine displayed different modifications. Key words: chicken, dietary protein source, digestive enzyme activity, electrolyte composition, small intestinal fluid 2012 Poultry Science 91 : The small intestine is the primary digestion and absorption site of dietary nutrients. The digestive enzymes and electrolytes in the intestinal fluid that are secreted by the pancreas, intestinal glands, and mucosal cells are responsible for the hydrolysis of dietary macronutrients and play vital roles in regulating products for transport. Several studies have shown that protease activities in the intestinal fluid are changed proportionately in response to the amounts of protein in the diet, whereas the amylase and lipase activities are dependent on their respective substrate carbohydrate and lipid contents (Corring, 1980; Valette et al., 1992; Yago et al., 1997; Zhao et al., 2007). Snook and Meyer (1964) reported that when rats received whole-egg protein, trypsin and chymotrypsin activities increased within intestinal contents when compared with a casein diet, which indicated that the types of ingested protein sources could affect digestive enzyme secretion. As a component of intestinal fluid, electrolytes in the intestinal tract are required for proper digestive enzyme functionality and the absorption of dietary hydrolysates (Clemens and Maloiy, 1978; Knarreborg et al., 2003). Sullivan et al. (1974) reported that magnesium and calcium could bind protein and were correlated with the amylase and protease activities in the pancreatic juice of pigs, which meant dietary composition could affect the ion concentration of intestinal fluid. In the practical feed formulation for Chinese yellow chickens, corn is the main source of cereal grain, whereas the proteinaceous ingredient varies greatly. To reduce the feed costs, soybean meal is usually substituted with relative low-quality protein feed, such as cottonseed meal, peanut meal, or rapeseed meal. However, the antinutritional ingredients and imbalanced digestible amino acids in the diet may have related influences on the activities of intestinal digestive enzymes and the concentration of ion in intestinal fluid. Few data were reported on the relationship 1641

2 1642 Ren et al. Table 1. Composition and nutrient content of the pre-experimental and experimental diets on DM basis Ingredient (%) Pre-experimental diet CSMD 1 CMD 1 between dietary nutrient content and digestive enzyme activities, ion concentration in intestinal fluid of duodenum, jejunum, and ileum of chicken, respectively. This information is important to understand how to improve the utilization of dietary nutrients formulated with lowquality protein ingredients. Recently, our laboratory has designed intestinal cannula to repeatedly collect a great volume of digesta from intestine of live birds for analyzing the composition of intestinal fluid (Zhao et al., 2007). With this method, the current study was conducted to determine the effects of dietary protein source on the digestive enzyme activities and electrolyte composition of digesta fluid in the duodenum, jejunum, and ileum of chickens. Birds and Diets Corn Soybean meal, 43% CP Cottonseed meal, 42% CP Peanut meal, 45.5% CP Rapeseed meal, 36% CP Corn gluten meal, 60% CP Soybean oil Wheat middings Wheat bran 2.80 Expanded soybean Calcium hydrophosphate Limestone Premix l-lys (65%) Sodium chloride dl-met (solid) Sodium bicarbonate l-thr Choline chloride (50%) Nutrient content ME 3 (kcal/kg) 2,900 3,150 3,150 CP 4 (%) Crude fat 4 (%) Crude fiber 4 (%) ADF 4 (%) NDF 4 (%) Starch 4 (%) Calcium (%) Total P (%) CSMD = corn-soybean meal-miscellaneous meal diet, CMD = corn-miscellaneous meal diet. 2 Supplied per kilogram of diet: vitamin A (retinyl acetate), 2,700 IU; vitamin D 3 (cholecalciferol), 400 IU; vitamin E (dl-α-tocopheryl acetate), 10 IU; vitamin K 3 activity, 0.5 mg; thiamine, 2.0 mg; riboflavin, 5.0 mg; pantothenic acid, 10.0 mg; niacin, 30 mg; pyridoxine, 3.0 mg; choline, 750 mg; folic acid, 0.5 mg; biotin, 120 μg; vitamin B 12, 10 μg; manganese (MnSO 4 H 2 O), 80 mg; zinc (ZnSO 4 ), 80 mg; copper (CuSO 4 5H 2 O), 8 mg; iron (FeSO 4 7H 2 O), 80 mg; iodine (KI), 0.7; and selenium (Na 2 SeO 3 ), 0.3 mg. 3 The values are calculated according to the AME values of feedstuffs for chickens (Ministry of Agriculture of China, 2004). 4 The values are determined values. ADF = acid detergent fiber; NDF = neutral detergent fiber. MATERIALS AND METHODS This experiment was conducted at Guangdong Wen s Foodstuffs Group Co. Ltd. and was approved according to the animal care and handling procedures at the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing. A 3 2 factorial, completely randomized design was used for the experiment. One hundred and twenty 15-wk-old Chinese yellow roosters were selected by weight (2.3 to 2.5 kg) and randomly divided into 3 groups with 40 birds per group. All of the birds were placed into individual cages (0.50 m length 0.42 m width 0.55 m height) in a temperaturecontrolled room (25 C) under 12 h of light per day. The birds from 1 group were fitted with cannulas in the duodenum, the remaining 2 groups were cannulated in the jejunum or ileum according to procedures described by Zhao et al. (2007). The duodenal cannula was fitted on the U-shaped corner of the duodenum, whereas the jejunal cannula was fitted 1.5 to 2.0 cm from Meckel s diverticulum of the jejunum, and the ileal cannula was placed parallel to the end of a pair of ceca. After the intestinal cannulation, feed was withdrawn and the birds were tube-fed 20 ml of a dextrose solution (20 g/100 g of water) 3 times daily for 3 d. Then, birds were fed a pre-experimental diet (Table 1) from d 4 to 15 of the experiment. On d 16, 30 of the 40 cannulated chickens in each group were randomly selected and divided into 2 subgroups. There were 5 replicates with 3 cannulated chickens each. Two subgroups of each cannulated chicken group were submitted to a 15-d adaptation period

3 Table 2. Analyzed amino acids composition of the experimental diets on a DM basis Item CSMD 1 CMD 1 Essential amino acid (%) Arg His Ile Leu Lys Met Phe Thr Val Nonessential amino acid (%) Ala Asp Cys Glu Gly Pro Ser Tyr Total amino acid CSMD = corn-soybean meal-miscellaneous meal diet; CMD = cornmiscellaneous meal diet. to one of 2 experimental diets that were isonitrogenous (165 g of CP/kg) and isoenergetic (3.15 Mcal of ME/ kg) but differed only in their protein sources (Table 1). Sample Collection and Chemical Assay PROTEIN SOURCE ON DIGESTIVE ENZYME ACTIVITIES Digesta were collected for 1 h every 4 h from 09:30 to 18:30 h on d 31, 33, and 35 of the experiment, and the collection method was similar to the procedure described by Zhao et al. (2007). The birds had free access to feed and water during the digesta collection. The small intestinal fluid was made by centrifuging digesta samples for 10 min at 1,250 g at 4 C according to the method that was described by Furuya et al. (1979), and then the supernatant was stored at 20 C. After the digesta collection, the frozen sample was thawed in 4 C. One milliliter of digesta fluid from each of the 3 collection times and 3 collection days was pooled and vortexed for the analysis of enzyme activities and electrolyte composition. Amylase activity was measured with soluble starch as a substrate at ph 6.9 and using the method that was described by Dahlqvist (1962). Trypsin activity was assayed with Na-p-toluolsulfonyl-l-arginine methyl ester hydrochloride (TAME) as a substrate at ph 8.1 according to procedures described by Wirnt (1974a,b). A similar method was used for the chymotrypsin determination, but TAME replaced N-benzoyl-l-tyrosine ethyl ester (BTEE) at ph 7.8; the TAME and BTEE were obtained from Sigma-Aldrich (St. Louis, MO). Lipase activity was determined using a Diasys reagent box (Lipase DC FS, DiaSys Diagnostic Systems GmbH, Holzheim, Germany). All of the digestive enzyme activities were expressed in of the intestinal fluid. The ph value of the intestinal fluid was measured with a ph meter at 39 C (PB-10, Sartorius, Germany). The Na +, K +, and Cl concentrations were determined using an electrolyte analyzer (Medica Easylyte, Bedford, UK). The Ca 2+ and Mg 2+ concentrations were assayed by the method described by Barnett et al. (1973) and Mann and Yoe (1957), respectively. All of the ion concentrations were expressed as millimoles per milliliter () of intestinal fluid. The DM contents of the diets were determined by oven drying at 105 C for 5 h. The nitrogen determination was according to the combustion method, using an FP2000 nitrogen analyzer (Leco Corp., St. Joseph, MI). The diets were also analyzed for crude fiber (method ) and ether extract (method ) according to the procedures of the Association of Official Analytical Chemists (AOAC, 1990). The acid detergent fiber and neutral detergent fiber contents were determined according to the procedure described by Van Soest et al. (1991). Starch content was analyzed by the enzyme digestion method (method ; AOAC, 2002). Amino acid concentrations in the experimental diets were determined by HPLC and shown in Table 2 (L-8900 Amino Acid Analyzer, Hitachi, Tokyo, Japan). The samples were hydrolyzed for 24 h at 110 C with 6 N HCl before analysis. Sulfur-containing amino acids were analyzed after cold performic acid oxidation for 16 h before acid hydrolysis. All analyses were performed in duplicate. Statistical Analysis The data were tested for homogeneity of variance using the Levene test (Milliken and Johnson, 1984), data were then analyzed by ANOVA according to a completely randomized design with treatments arranged in a 2 3 factorial arrangement using the GLM procedures of SAS version 8 (SAS Institute, 1990). An interaction was included in the statistical model as Y = diet effect + intestinal effect + diet intestine effect + error. Treatment means were separated using orthogonal contrasts with P < 0.05 considered significant. RESULTS AND DISCUSSION 1643 The effect of dietary protein source on the digestive enzyme activities of the small intestine are shown in Table 3. No differences between the 2 dietary groups were observed for the mean of duodenal, jejuna, and ileal amylase, trypsin, chymotrypsin, and lipase, respectively. However the duodenal amylase activity was lower in the corn-miscellaneous meal diet (CMD) group than the corn-soybean meal-miscellaneous meal diet (CSMD) group (P < 0.05). These results showed that the protease activity in the small intestinal fluid did not change in response to the type of dietary protein consumed. Similar results were obtained by Low (1982) and Partridge et al. (1982) in studies with pigs that were fitted with cannulas in the pancreatic ducts. However, Snook and Meyer (1964) and Valette et al. (1992) demonstrated that the consumption of diets with different protein sources significantly modified trypsin and chymotrypsin activity in the pancreatic juice of pigs and

4 1644 Ren et al. Table 3. Effect of dietary protein quality on digestive enzymes in intestinal fluid of chickens Item Intestinal fluid the intestinal fluid of rats, respectively. It is difficult to compare the present work to previous studies because our study used 2 practical diets; in comparison, Snook and Meyer (1964) examined 2 semipurified diets that contained casein or rapeseed as protein sources, and Valette et al. (1992) investigated 2 purified diets that contained 150 g of casein or whole-egg protein per kilogram of diet. Because the substrate that was used was crucial in the determination of enzyme activity (Nicholson et al., 1974; Corring, 1980; Swanson et al., 2000), these inconsistencies may have been caused because the physical or chemical differences in our 2 experimental diets were too small to be practically significant (Tables 1 and 2). In the present study, duodenal amylase activity was less in the CMD diet group. The percentage of starch, acid detergent fiber, or neutral detergent fiber in the 2 experimental diets were similar (Table 1); therefore, the amylase activity modification may just slightly be influenced by dietary ingredients. Previous studies have shown that amylase activity is sensitive to the ph value of digesta, and the optimum ph of pancreatic amylase in chickens was 7.5 (Osman, 1982; Ao et al., 2008). In the current study, the duodenal ph value of the CMD group was lower than that of the CSMD group (4.78 vs. 5.17; Table 3); correspondingly, the amylase activity of the CMD group was much lower than that of the CSMD group (5.73 vs ). These results indicated that the lower duodenal ph in chickens in the CMD group may be another factor that was responsible for the decrease in duodenal amylase activity. In the current study, the effects of the dietary protein source on lipase activities in the small intestinal fluid were not significant. Amylase, 1 Lipase, 1 Trypsin, 1 Chymotrypsin, 1 Diet 2 CSMD Duodenum CMD Duodenum CSMD Jejunum CMD Jejunum CSMD Ileum CMD Ileum SEM Main effect Intestinal section Duodenum 55.9 c 1.40 a 13.8 b 7.48 b Jejunum a 0.41 b 50.2 a 13.7 a Ileum b 0.05 c 19.9 b 4.58 c Diet CSMD CMD Source of variation, P-value Intestinal section <0.001 <0.001 <0.001 <0.001 Diet Intestinal section diet CSMD vs. CMD Duodenum CSMD vs. CMD Jejunum CSMD vs. CMD Ileum a c Means within a column with no common superscripts differ significantly (P < 0.05). 1 Mean represents 5 observations of 9 pooled samples collected for 1 h every 4 h in 12 h in d 31, 33, and 35 of the experiment. 2 CSMD = corn-soybean meal-miscellaneous meal diet; CMD = corn-miscellaneous meal diet. The proper ph value of the intestinal tract is critical for proper digestive enzyme functionality, and ph values that are outside the normal ranges can result in decreased digestion and absorption, which can eventually reduce growth performance (Shih and Hsu, 2006). In the present study, the lower duodenal ph value in the CMD group indicated that it had a weaker buffer capacity than the CSMD group. This result was supported by Berot and Briffaud (1983) and O Hare et al. (1984), who demonstrated that the buffer capacity varied from one protein feed to another. The ph value varied over a large range in the duodenum, but it varied very little in the jejunum and ileum. The mean variation coefficients of the ph value in duodenum, jejunum, and ileum were 4.72, 1.27, and 0.80%, respectively (Table 4). Braude et al. (1976) also found that the ph value in the duodenum was the highest after feeding, and it decreased with increased time after feeding; however, the ph varied over a much smaller range in the jejunum and ileum, which indicated that the digesta were more effectively buffered by intestinal secretions in the jejunum and ileum. In our study, a large flow-rate in the duodenum was observed; it was approximately 3- and 4-fold the amount of the flows in the jejunum and ileum, respectively. Because the strong acid chymus flowing from the gizzard stayed such a short time in the duodenum for buffering, the duodenal fluid was more sensitive to changes in dietary macronutrients than the fluid in the jejunum or ileum. In the current study, an increasing distance from the stomach was correlated with an increase in the ph values; the mean ph values of the duodenum, jejunum, and ileum were 4.98, 8.12, and 8.77, respectively. Pang and Applegate (2007) ob-

5 PROTEIN SOURCE ON DIGESTIVE ENZYME ACTIVITIES 1645 Table 4. Effect of dietary protein quality on the electrolyte composition in intestinal fluid of chickens Item Intestinal fluid ph, 1 mean (CV, %) served a mean ph of 6.22 in the duodenum-jejunum and a ph of 6.26 in the ileum in a slaughter trial with 3-wk-old broilers. The same method was used by Heller (1936), who observed that the ph values of the duodenum, upper ileum, and lower ileum of 8-wk-old chickens were 6.52, 6.47, and 8.29, respectively. Although the age and species of the animals and the diets used in the previously mentioned studies may have contributed to the variation in the measurements, the digesta collection methods may primarily account for the differences between our study and the aforementioned reports. Intestinal electrolytes are primarily derived from the secretions of the bile, pancreatic, and intestinal glands, and they play an important role in digestive enzyme activities and dietary hydrolysate transportation (Pinheiro et al., 2004). In the current study, the CMD decreased the jejunal Mg 2+ concentration, but it did not significantly influence the Na +, K +, Cl, and Ca 2+ concentrations in each segment of the small intestine (Table 4). This result was partially in agreement with the study by Hendrix and Bayless(1970), who found that the concentrations of major ions in the intestine were not easily affected by dietary nutrients because the major ions could be rapidly absorbed by enterocytes. The Mg 2+ ion can inhibit the net water absorption in the jejunum and influence the flow rate of digesta in the intestinal tract (Malagelada et al., 1978; Partridge et al., 1982). As a result, the higher jejunal Mg 2+ concentration in the CMD group might be correlated with a greater digestive water requirement in this group. The mechanism of the intestinal Mg 2+ concentration regulation is not very clear, and further investigation is needed. The current study showed that Na + was the major cation in the small intestine tract, whereas Cl Na +, 1 K +, 1 Cl, 1 Ca 2+, 1 Mg 2+, 1 Diet 2 CSMD Duodenum 5.17 (3.84) CMD Duodenum 4.78 (5.60) CSMD Jejunum 8.11 (1.15) CMD Jejunum 8.12 (1.39) CSMD Ileum 8.74 (0.90) CMD Ileum 8.79 (0.70) SEM Main effect Intestinal section Duodenum 4.98 c 14.2 c 13.3 b a 14.8 a 8.78 c Jejunum 8.12 b 35.9 b 11.5 c b 12.5 a 19.4 a Ileum 8.77 a 56.7 a 14.6 a 15.8 c 4.90 b 15.4 b Diet CSMD b CMD a Source of variation, P-value Intestinal section <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Diet Intestinal section diet CSMD vs. CMD Duodenum CSMD vs. CMD Jejunum CSMD vs. CMD Ileum a c Means within a column with no common superscripts differ significantly (P < 0.05). 1 Mean represents 5 observations of 9 pooled samples collected for 1 h every 4 h in 12 h in d 31, 33, and 35 of the experiment. 2 CSMD = corn-soybean meal-miscellaneous meal diet; CMD = corn-miscellaneous meal diet. was the major anion in the duodenum and jejunum. The apparent large anion deficit in the ileum may be partially explained by the unmeasured bicarbonate ions. The observed ph values in the ileum, which were as high as 8.77, strongly suggest that bicarbonate was the major anion that increased in concentration as the chloride concentration decreased (Fordtran and Locklear, 1966; Clemens and Maloiy, 1978). The Ca 2+ and Mg 2+ ions only slightly contributed to the total cation concentration; the same phenomenon was observed by Partridge et al. (1982) in pig. The length of the small intestine in adult chickens is approximately 1.1 to 1.2 m. Because the intestinal morphology, chyme retention time, and the influences of the pancreatic, bile, or intestinal gland secretions on each intestinal segment are diverse, the digestive enzymes and electrolyte composition of digesta from each intestinal segment may be quite different. In the present study, amylase, trypsin, and chymotrypsin activity in the jejunal fluid were significantly higher than in the duodenal and ileal fluid (P < 0.01), whereas the lipase activity in the duodenal fluid was higher than in that of the other 2 intestinal segments (P < 0.05). This result was confirmed by the report that dietary lipids are primarily digested in the duodenum (Krogdahl, 1985), while carbohydrates and some polypeptides were mainly hydrolyzed and absorbed in the jejunum (Sklan and Hurwitz, 1980; Osman, 1982; Wiseman, 2006). The electrolyte composition of the duodenal, jejunal, and ileal fluid were significantly different, which was likely because the electrolyte composition of the duodenal fluid was primarily influenced by pancreatic and bile secretions, whereas that of the jejunal and ileal fluid were mainly subject to intestinal gland secretions.

6 1646 Ren et al. In conclusion, the CMD may have influenced amylase activity by changing the ph value of digesta fluid, but it did not significantly affect protease activity. With the exception of magnesium in the hindgut, the electrolytes in the small intestinal fluid were not easily affected by dietary nutrients. The digestive enzymes and electrolyte composition differed among each segment of the small intestine to maintain proper physiological function. ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China ( ), State Innovation Method Project (2009IM033100), and GWFG for their financial support. We also thank S. J. Liu, Y. G. Yin, Z. K. Liu, J. Z. Tan, B. M. Mi, and F. Yan (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing) for the cannulation operations and sample collection. REFERENCES Ao, T., A. H. Cantor, A. J. Pescatore, and J. L. 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