Hydrolysis and Absorption in the Small Intestines of Posthatch Chicks

Hydrolysis and Absorption in the Small Intestines of Posthatch Chicks D. Sklan 1 and Y. Noy Faculty of Agriculture, Hebrew University, Rehovot, Israel 76-100 ABSTRACT In the immediate posthatch period, chicks activities, and these increased only after feed consumption. must transfer from metabolic dependence on yolk to utilization of exogenous feed. This study describes changes in intestinal luminal pancreatic enzyme activity and mucosal uptake posthatch as influenced by feed and Na intake. Chicks with access to feed increased in and small intestinal weight in the 48-h posthatch, whereas chicks without access to feed decreased in ; however, small intestinal weight increased during this period. Chicks ingesting feed showed increases in total intestinal trypsin, amylase and lipase activities that were correlated with intestinal weights and. Chicks without access to feed showed little change in trypsin and amylase Feeding a low-na diet did not significantly change the regression coefficient between pancreatic enzyme activity and. Mucosal uptake was estimated by measuring Na +,K + - adenosine triphosphatase (ATPase) activity in small intestinal segments. In fed birds this activity increased in relationship to growth, whereas in nonfed birds uptake increased only after access to feed. Low-Na diets allowed only minimal mucosal uptake in all intestinal segments. This study indicates that secretion of trypsin and amylase into the intestine was triggered by feed intake. In addition, Na plays a critical role in intestinal uptake in the immediate posthatch period. (Key words: chick, small intestines, posthatch, absorption) 2000 Poultry Science 79:1306 1310 INTRODUCTION In the initial posthatch period, the young bird must make the transition from metabolic dependence on endogenous lipid-rich yolk to exogenous carbohydrate- and protein-rich feed. This transition is a prerequisite for rapid growth and involves dramatic changes in the gastrointestinal tract, including secretion of digestive enzymes and the initiation of uptake of amino acids and hexoses (Uni et al., 1995). Hydrolysis of macromolecules in the small intestine is achieved to a large extent by pancreatic enzymes. Net enzyme secretion into the duodenum determined by steady state measurements with nonabsorbed markers from 4-d posthatch indicated that secretion occurs in increasing amounts with age, either due to increased feed intake or to organ size (Noy and Sklan, 1995, Uni et al., 1996). Such measurements cannot be made close to hatch because it is not possible to achieve a steady state, and thus changes in pancreatic enzyme secretions close to hatch have yet to be determined. Crane (1965) showed that sugar transport into mammalian intestinal epithelial cells is driven by Na-glucose co- Received for publication October 14, 1999. Accepted for publication May 8, 2000. 1 To whom correspondence should be addressed: Sklan@agri.huji. ac.il. 2 Yavne Hatcheries, Kibbutz Yavne, Israel 79233. transport. More recently, cdna-encoding, Na + -coupled glucose cotransporters located in the brush border have been cloned (Wright, 1993). In addition, several amino acid transporters with overlapping specificity in the brush border membrane are Na cotransporters (Nakanashi et al., 1994; Munck and Munck, 1999). Once within the cell, homeostasis is maintained by active exclusion of Na + across the basolateral cell membrane by the Na +,K + adenosine triphosphatase (ATPase) (Del Castillio and Robinson, 1985), whereas glucose and amino acids are transported out of the enterocyte by passive mechanisms. This transport can be determined by measuring the Na +,K + ATPase activity in the mucosa. Park et al. (1998) indicated that 31 to 37% of total jejunal O 2 uptake was used by the Na +,K + ATPase. Thus determining either intestinal O 2 uptake or Na +,K + ATPase mucosal activity provides an estimate of Na-dependent intestinal transport. This paper describes changes in intestinal activity of some pancreatic enzymes and of Na +,K + ATPase in the small intestines of chicks close to hatch as influenced by feed and Na intake. MATERIALS AND METHODS Animals and Treatments Male Ross Ross 2 broiler chicks were taken immediately following hatch, which was defined as the time Abbreviation Key: ATPase = adenosine triphosphatase. 1306

DIGESTION AND ABSORPTION IN POSTHATCH CHICKS 1307 TABLE 1. Composition of the experimental diets Diet Item Control Low-Na (% of diet) Dehulled soybean meal 38.1 38.1 Corn 32.5 32.9 Wheat 19.3 19.3 Oil 6.8 6.8 CaCO 3 1.1 1.1 Dicalcium phosphate 1.6 1.6 Sodium choride 0.37... Vitamin and mineral mix 1 0.23 0.23 1 Provided vitamins and minerals as previously described (Uni et al., 1995). when birds completely cleared the shell. Some chicks were held for 48 h without access to feed, whereas other chicks immediately received a starter feed meeting or exceeding NRC (1994) requirements or a feed formulated similarly but without addition of NaCl (low Na) (Table 1). The control diet was analyzed to contain 0.15% Na, whereas the low-na diet contained 0.07% Na. Water was available ad libitum. At the specified times, yolk and intestines were removed. The contents of the duodenum (from the pylorus to the distal point of entry of the bile ducts), jejunum (Meckel s diverticulum marked the end point of the jejunum), and ileum (the ileocecal junction marked the end of the ileum) were washed quantitatively with a 50-mm Tris HCl ph 7.4 buffer containing 50 mm mannitol solution at 4 C for enzyme analysis. The intestine was homogenized with four volumes of 50-mm Tris HCl ph 7.4 containing 50 mm mannitol for Na +,K + ATPase determination. All procedures were approved by the Animal Care and Welfare Committee of our institute. RESULTS Chicks with access to feed increased in after hatch, whereas chicks without access to feed decreased in by 6 g in the 48 h after hatch, but once feed consumption began, they grew at a rate parallel with fed birds (Figure 1). Chicks fed the low-na diet increased in posthatch; however, after 48 h the rate of growth decreased. The rate of yolk utilization was more rapid in chicks with access to feed than either nonfed or low-na-fed chicks. The weight of the small intestine increased by two-fold in fed chicks and by about 60% in nonfed chicks in the 2 d posthatch. After feed ingestion, the rate of increase in intestinal weight was parallel to fed chicks. Chicks fed the low-na diet showed lower increases in small intestinal weight from Day 3. Weights of the individual segments of the small intestine (not shown) and total small intestinal weight were closely correlated with ( = 36.2 ± 1.8 + (6.9 ± 0.36)*small intestinal weight, r = 0.94). Total trypsin, lipase, and amylase activities were determined in the duodenum, jejunum, and ileum. However, because the treatment effects in each individual segment were similar, only total small intestinal activities are Analyses Lipase (Belfrage and Vaughan, 1969), trypsin (Sklan and Halevy, 1985), and amylase (Pinchasov and Noy, 1994) activities were determined in the washings from each intestinal segment. The Na +,K + ATPase activity was assayed as described by Del Castillio et al. (1991) in an incubation medium containing 50 mm Tris (ph 7.2), 5 mm MgCl 2, and 20 mm KCl or 20 mm KCl and 100 mm NaCl, as required, in the presence and absence of 5 mm ouabain. One unit of enzyme activity was defined as the amount of enzyme hydrolyzing 1 mmol of substrate/min under the specified conditions. Sodium was determined by flame photometry. Statistical Analysis Least squares means of results are presented after analysis of variance using the general linear models procedures of SAS. Differences between means were tested using t tests, and significance was P < 0.05 unless otherwise stated (SAS Institute, Inc., 1986). FIGURE 1. Changes in (A), yolk (B), and small intestine (C) weights with age in chicks with immediate access to feed (open triangles), held for 48 h without access to feed (circles), or fed a low-na diet (filled triangles). Results are means ± SD (where SD does not fall within the data symbol) of at least eight birds per data point. Body weights were significantly lower in non-fed chicks from Days 1 through 7 and in low-na-fed chicks from Days 3 through 7. The regression coefficient of the log of the yolk weight of control chicks with age was significantly different from that of nonfed chicks and low-na-fed chicks. Intestinal weights in nonfed chicks were significantly lower from Days 1 through 7 and in low-na-fed chicks from Days 3 through 7.

1308 SKLAN AND NOY Total activity of Na +,K + ATPase and the ouabain-sensitive Na +,K + ATPase activity were determined in the duodenum, jejunum, and ileum. The Na +,K + ATPase activity comprised 60 to 75% of total ATPase activity. The activity patterns and effects of the treatments were parallel in the different intestinal segments, and thus only total small intestinal activities are shown (Figure 3). Total small intestinal Na +,K + ATPase activity was highest in fed chicks from Days 1 through 4. Nonfed chicks showed low activity before feed ingestion, and then an increase in activity parallel to fed chicks. In contrast, chicks fed the low-na diet had low activity that increased only slightly with age. Table 2 indicates the relationships between intestinal enzymatic activities and. The pancreatic enzyme activities and the Na +,K + ATPase activities were significantly correlated with both and intestinal weight (not shown) over 7 d. The regression coefficients were significantly lower for Na +,K + ATPase activities in chicks fed the low-na diet. DISCUSSION As in previous studies, holding birds without food for 48 h decreased initial growth until after feed ingestion. However, during the 48 h posthatch, the small intestines increased in weight in all birds; this increase was approximately two-fold in fed birds and about 60% in nonfed birds. Yolk was utilized exponentially during this period at a slightly greater rate in fed than non-fed chicks. These results are similar to those of our previous studies (Noy et al., 1996; Noy and Sklan, 1999) and indicate that in nonfed birds, protein for synthetic and metabolic activities originates from yolk. Energy for maintenance is supplied by the yolk prior to use of exogenous nutrients, which FIGURE 2. Small intestinal trypsin (A), amylase (B), and lipase (C) activity with age in chicks with immediate access to feed (open triangles), held for 48 h without access to feed (circles) or fed a low-na diet (filled triangles). Results are means ± SD of at least six birds per data point. Nonfed chicks had significantly lower trypsin activity than controls on Days 1 through 4, and low-na-fed chicks had lower activity on Days 4 and 7. Non-fed chicks had significantly lower amylase activity than controls on Days 1 through 3, and low-na-fed chicks had lower activity on Days 4 and 7. Nonfed chicks had significantly lower lipase activity than controls from Day 1 through Day 7, and low-na chicks had lower activity on Days 3 through 7. shown (Figure 2). In nonfed chicks, small intestinal trypsin and amylase activities were lower prior to feed intake than in fed chicks. Once feeding commenced, activities increased and by Day 7 activities were not different from fed chicks. In contrast, low-na-fed chicks had similar trypsin and amylase activity to fed birds until 4 d posthatch, after which activity increased more slowly. Lipase activity was similar in all birds until Day 2, after which higher activity was found in fed than nonfed birds. In chicks fed the low-na diet, lipase activity was lower from Day 3 and remained lower until 7 d. FIGURE 3. Small intestine Na +,K + adenosine triphosphatase (ATPase) activity with age in chicks with immediate access to feed (open triangles), held for 48 h without access to feed (circles), or fed a low-na diet (filled triangles). Results are means ± SD of at least six birds per data point. Non-fed chicks had significantly lower activity than controls on Days 1 through 4, and low-na-fed chicks had lower activity on Days 1 and 7.

DIGESTION AND ABSORPTION IN POSTHATCH CHICKS 1309 TABLE 2. Regressions between total intestinal enzymatic activities and in posthatch chicks Dependent Independent Intercept 1 Slope 1 R 2 Lipase Fed 917 ± 315 21.2 ± 4.2 0.48 Non-fed 759 ± 180 18.4 ± 3.2 0.52 Low-Na 808 ± 278 19.0 ± 4.1 0.49 Trypsin Fed 7,255 ± 1,056 198 ± 14 0.78 Non-fed 10,002 ± 1,152 226 ± 21 0.80 Low-Na 10,076 ± 1,719 221 ± 31 0.77 Amylase Fed 3,788 ± 1,704 172 ± 23 0.61 Non-fed 4,985 ± 1,709 179 ± 32 0.51 Low-Na 5,766 ± 2,154 216 ± 57 0.58 Na-ATPase Fed 2.79 ± 1.78 0.29 ± 0.03 a 0.68 Non-fed 4.54 ± 3.30 0.29 ± 0.05 a 0.43 Low-Na 4.15 ± 3.23 0.19 ± 0.04 b 0.48 1 Regressions were calculated from data from chicks sampled at 0, 1, 2, 3, 4, and 7 d posthatch; intercepts and slopes are given together with SD. a,b Values without a common superscript differ (P < 0.05). are available after hydrolysis of feed macromolecules by pancreatic enzymes in the intestine. Activities of the three pancreactic enzymes examined in this study increased with age, although in nonfed chicks trypsin and amylase activities changed little before ingestion of feed. Similar effects of delayed placement on the pancreatic amylase and trypsin activities have been reported in poults by Corless and Sell (1999). Lipase activity in the intestine is required even before feed ingestion for hydrolysis of yolk triglycerides, and the changes following feed intake observed in the activity of this enzyme in this and previous studies (Uni et al., 1995, 1996) were less dramatic than those observed for trypsin and amylase. Intestinal pancreatic enzymatic activities were correlated with and intestinal weight. In the absence of feed intake and, thus, limited availability of amino acids, synthesis of intestinal tissue has priority over secretion of digestive enzymes. In chicks fed the low-na diet, increase was slower after 48 h, probably due to a decrease in feed intake accompanied by lower secretion of pancreatic enzymes. In this study, we determined total intestinal activity of pancreatic enzymes by quantitatively removing all of the intestinal contents. Previous determinations of secretion of pancreatic enzymes used steady-state nonabsorbed marker methodology from 4 d posthatch and indicated increases in secretion with age (Noy and Sklan, 1995), but not per gram feed intake (Uni et al., 1995). Marchaim and Kulka (1967) indicated that pancreatic enzymes are present in the small intestines in the late embryonic stages; however, the findings of this study suggest that feed intake triggers enhanced secretion of trypsin and amylase, which are then secreted at relatively constant amounts per feed intake as the chick grows. Because glucose and a large proportion of amino acids are absorbed by co-transport with Na, which is then removed from the enterocyte by active transport, we examined the Na +,K + ATPase activity in different intestinal segments. This mucosal activity was correlated with growth in all intestinal segments, although the slopes differed with intestinal site. Such a relationship is expected between intestinal uptake of major nutrients and growth. In nonfed birds, activity of Na +,K + ATPase was low before feed ingestion, then increased parallel to fed chicks. In contrast, in chicks fed the low Na diet, Na +,K + ATPase activity was strongly depressed, probably due to lack of the Na required for the Na +,K + ATPase activity. This result confirms that a sufficient Na supply is essential for nutrient uptake. Thus it appears that intestinal Na +,K + ATPase activity is a useful parameter for measuring intestinal absorptive activity. The low in situ uptake of glucose and methionine from yolk previously found close to hatch (Noy and Sklan, 1999) was probably due both to the hydrophobic nature of the substrate and to the low concentration of Na. Recently, additional Na-dependent transporters have been reported in small intestine brush border membranes, including: L-ascorbic acid (Tsukaguchi et al., 1999), phosphate (Field et al., 1999), multivitamin (Prasad et al., 1999), and taurocholate (Coleto et al., 1998) transporters. Thus Na appears to have a central role in nutrient uptake in the immediate posthatch period and may limit nutrient uptake when Na intake is low. REFERENCES Belfrage, P., and M. Vaughan, 1969. Simple liquid-liquid partition system for isolation of labelled oleic acid from mixtures of triglycerides. J. Lipid Res. 10:341 344. Coleto, R., J. Bolufer, and C. M. Vasquez, 1998. Taurocholate transport by brush border membrane vesicles from different regions of chicken intestine. Poultry Sci. 77:594 599. Corless, A. B., and J. L. Sell, 1999. The effects of delayed access to feed and water on the physical and functional development of the digestive system of young turkeys. Poultry Sci. 78:1158 1169. Crane, R. K., 1965. Na + dependent transport in the intestine and other animal tissues. Fed. Proc. 24:1000 1004.

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