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Posthatch Changes in Morphology and Function of the Small Intestines in Heavy- and Light-Strain Chicks
Posthatch Changes in Morphology and Function of the Small Intestines in Heavy- and Light-Strain Chicks ZEHAVA UNI, YAEL NOY, and DAVID SKLAN1 Faculty of Agriculture, Hebrew University, P.O. Box 12, Rehovot, Israel, 76-100
1995 Poultry Science 74:1622-1629
INTRODUCTION In the posthatch chick, metabolites are supplied at first from the yolk and development to exogenous nutrient uptake occurs in the first few days. During this period, feed intake increases many fold whereas pancreatic and bile secretions to the duodenum escalate to match these developments. Passage time through the small intestines decreases by one third during the 1st wk posthatch. Digestion of fatty acids and starch is essentially complete by Day 4 in broiler chicks, whereas N digestion increases from close to 80% on Day 4 to approximately 90% on Day 10 (Noy and Sklan, 1995). The relative weight of the intestines increases posthatch, reaching a maximum at 3 to 7 d, and declines slightly thereafter (Murakami et al., 1992; Pinchasov and
Noy, 1994). Although the chicks' intestinal system is anatomically complete in the embryonic stage (Overton and Shoup, 1964; Lim and Low, 1977; Chambers and Grey, 1979), the absorptive surface changes considerably posthatch and the rate of enterocyte proliferation increases (Cook and Bird, 1973; Moran, 1985). The rate of growth in heavy and light chick strains is very different and this is likely to be due to differences in the digestive and absorptive systems. The objective of this study was to determine changes from hatch in the morphology of the small intestines of heavy- and light-strain birds and in parallel measure secretion of enzymes to the duodenum, passage time, and digestion. MATERIALS AND METHODS Animals and Treatments
Received for publication November 28, 1994. Accepted for publication May 1, 1995. x To whom correspondence should be addressed.
Day-old Arbor Acres (AA) chicks and Lohman (L) chicks were obtained from
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ABSTRACT The morphology of the small intestines of heavy (Arbor Acres) and light (Lohman) chicks was determined posthatch in parallel with digestion, enzyme secretion, and passage time. Villus height and volume increased from 4 to 10 d, particularly in the jejunum and ileum. The number of enterocytes per villus increased with age, but enterocyte density was greater in jejunum than ileum. Villus volume and enterocyte density was greater in Arbor Acres than Lohman chicks from hatching and the rate of change with age was similar in both strains. Enzyme secretion to the duodenum was higher per gram of feed intake in heavy-strain birds on Day 4 after hatching but thereafter no differences were apparent. Passage time was 50% shorter in light-strain chicks on Day 4 but the difference between the strains was not significant from Day 10. No differences in fatty acid digestion were observed with age or between strains. Nitrogen digestion increased in both strains from approximately 70% on Day 4 to close to 90% on Day 14. Starch digestion was 90 to 95% from 4 to 14 d in Arbor Acres birds, but increased from approximately 80% on Day 4 in Lohman chicks to 93% on Day 14. Feed intake may determine the amount of uptake in posthatch chicks. (Key words: chick, intestine, morphology, absorption)
1623
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
commercial hatcheries and transferred to a battery brooder. Chicks were fed the experimental diet shown in Table 1, which were formulated according to requirements of the NRC (1994). Two hundred /tCi/kg 141 Ce was added as an unabsorbed reference substance (Sklan et ah, 1975a) to the chick diet for 4 d before slaughter. Five chicks from each treatment were killed with intracardiac overdose of sodium pentobarbital on Days 4, 7,10, and 14 after hatching.
Digesta were obtained by gentle manipulation from duodenum, jejunum, and lower ileum and the length and diameter of the segments measured. Digesta were weighed, homogenized with cold .9% NaCl, and aliquots of the homogenate taken for 141Ce determination, for lipase (Sklan et ah, 1975b), trypsin (Sklan and Halevy, 1985), and amylase activities (Pinchasov and Noy, 1994). Enzyme activities were defined as the amount of enzyme hydrolyzing 1 mmol of substrate/min under the specified conditions. The 141Ce was determined by scintillation counting (Sklan et al, 1975a), nitrogen by the Kjeldahl method [Association of Official Analytical Chemists (AOAC), 1990], starch as described by Rose et al. (1991), fatty acids after methylation by gas chromatography using an internal standard, and bile acids colorimetrically (Sklan et al, 1975b). Calcium, P, and fiber were determined by standard methods (AOAC, 1990). Morphology On Days 4, 7, and 10 posthatching, 1-cm segments from duodenum, jejunum, and ileum were removed from the following sites: 1) the apex of the duodenum, 2) 10 cm distal to the point of entry of the distal bile ducts, and 3) 10 cm proximal to the cecal junction. The intestine was flushed clean with PBS and samples were fixed in 10% neutral buffered formalin solution and routinely processed to 5 mm hematoxylineosin-stained sections and examined by
2
Applitec MSV-800 System, Tel Aviv, Israel.
Ingredients and composition
Percentage
Corn Soybean meal Soybean oil CaCO? Dicalcium phosphate NaCl Methionine Vitamins + minerals1 Analyzed nutrient composition Protein Fat Fiber Ca P
51.0 37.6 6.0 1.6 1.6 .4 .3 1.5 22.5 7.8 2.5 1.06 .70
1
The vitamin and mineral mix contained in milligrams per kilogram of diet: FeS04,2.5; CoCO^ .64; KI, 3.8; MnS04, 208; NajMoSO^ 3.5; C11SO4, 20; ZnSO^ 220; thiamin, 5; riboflavin, 3.5; nicotinic acid, 60; pantothenic acid, 15; pyridoxine, 5; folic acid, .5; biotin, 2; menaquinone, .5; cyclocobalamine .02, a-tocopherol acetate, 30; cholecalciferol, .125; retinol acetate, 35.
light microscopy. Morphometric indices were determined using a computer-aided light microscope image analysis 2 as described by Bird et al. (1994). Villi height was measured using the lamina propria as the base; crypt height was defined as the depth of the invagination between adjacent villi; villus perimeter as the distance from the base of one villus to the adjacent villus; number of enterocytes were measured per side of a longitudinal section through the villus; number of villi per area in a horizontal section through the base of the villi. Values are means ± SD from 10 adjacent villi or .1 cm2 areas. Calculations When an unabsorbed reference substance such as 141Ce is fed until a steady state is obtained with respect to inputoutput of the isotope, assuming no chemical changes occur, a decrease in ratio of any component (A) to 141Ce between two points in the intestine can be defined as absorption and any increase as secretion (Sklan et al, 1975a). Overall net absorption between feed and lower ileum can be calculated as follows: 100 [(A/i«Ce)f - (A/i4iCe)J/(A/i4iCe)f]
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 6, 2014
Analyses
TABLE 1. Composition of the experimental diets
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UNI ET AL.
where (A/i«Ce)f is the ratio of A to 141Ce in the feed and (A/141Ce); the ratio in the ileum. Net secretion of any component to the duodenum can be calculated by: (daily i«Ce intake)[(A/i4iCe)d - (A/i«Ce)f]
RESULTS The growth and feed intake of the two strains of chicks are shown in Figure 1, and the more rapid growth and higher feed intake of the AA chicks is clear. Sections from the different segments of the small intestine for both strains on Day 4 (Figure 2) show the differences in development of the duodenum, jejunum, and ileum. The villi are larger and the degree of organization greater in AA than in L chicks. Table 2 presents measurements for
200 -
$L
150 j -
' 0 ' 0
t^^^C^-—-*'^'^^^ i10 ' 5
' 10
' 0 15
Age, d
FIGURE 1. Body weight (circles) and feed intake (triangles) for Arbor Acres (open symbols) and Lohman (filled symbols) chicks from hatching to Day 14.
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where (A/141Ce)d is the ratio of A to 141Ce in the duodenum. Passage time through any segment can be calculated by dividing the mean amount of isotope recovered from the segment by the daily intake of 141Ce. Least squares means of results are presented after analysis of variance and linear regression using the General Linear Models procedures of SAS® (SAS Institute, 1986). Differences between means were tested using t tests and significance was P < .01 unless otherwise stated.
the different segments in both strains. Villus height and perimeters in all segments increased 25 to 100% between 4 and 10 d for both strains, yet villi in AA were consistently higher then in L. Greatest increases in villus height with age were in the jejunum and in L chicks. Crypt depth increased with age with the exception in the AA ileum. There were no changes in submucosa thickness between 4 and 10 d, although the AA had a thicker layer in jejunum and ileum. The number of enterocytes per longitudinal section of villus increased with age and the rate was more rapid in L chicks. The density of enterocytes in the different segments is shown in Figure 3 (bottom) and this did not change with age, but was higher in AA. As the villi grew larger with age, the number of villi per area decreased, particularly in the duodenum of both strains. Villus volume is shown in Figure 3 (top) and increased slightly with age in the duodenum, and more so in the jejunum and ileum. The AA villus volume was consistently greater then in L. Jejunal and ileal villus volume and were linearly correlated with feed intake (r = .77 and .67), whereas duodenal villus volume was not significantly correlated. Enterocyte density in the jejunum was also correlated with feed intake (r = .62). Passage time through the small intestine (Figure 4) decreased in the AA from 115 min on Day 4 to 74 min at Day 7, increasing at Day 10 and reaching approximately 122 min at Day 14. Passage time was shorter in L chicks; however, a similar pattern was observed, with passage time decreasing slightly between Days 4 and 7 and increasing on Day 10 and at Day 14. The daily enzyme secretion per gram feed in the duodenum is shown in Figure 5 for amylase, trypsin, and lipase. Daily secretion per gram of feed of these three enzymes was higher on Day 4 in AA than in L birds. Secretion of trypsin per gram of feed intake decreased between Days 4 to 7 in AA chicks, but no change with age was observed in L birds. Secretion of amylase per gram of feed increased in L chicks between Days 4 and 7, with no changes thereafter. Lipase increased in L chicks between Days 4 and 7 and decreased in both strains between Days 10 and 14.
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
1625
V
VI
FIGURE 2. Sections from 4-d-old Arbor Acres chicks (left) and Lohman chicks (right). The upper panel is from the duodenum (apex), the middle panel is the jejunum (10 cm distal to the bile duct) and the bottom panel the ileum (10 cm proximal to the cecal junction). Sections were stained with eosin-hematoxylin, magnification is 10-fold, and bars are 40 jtm.
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IV
IH
1626
UNI ET AL.
DISCUSSION
Villus volume Duodenum
Jejunum
Ileum
•1
X
1i 1 1 I i I
I
1
JL^
X
JL
JL
!
\
7
10
4
7
10
4
!
7
Age, d
FIGURE 3. Villus volume (top panel) and enterocyte density (bottom panel) in duodenum, jejunum, and ileum of Arbor Acres (open bars) and Lohman (hatched bars) chicks at 4, 7, and 10 d old. Samples were taken from sites as described in Figure 1. Results are mean ± SD from 10 adjacent villi. Villus volume was higher for Arbor Acres then Lohman chicks in all segments, whereas enterocyte density was higher in Arbor Acres chicks only in the jejunum and ileum.
After Day 7, no differences were observed between strains. Digestion between feed and ileum is shown in Figure 6 for nitrogen, fatty acids, and starch for AA and L birds. Small intestine nitrogen digestion increased in AA from 78% on Day 4 to close to 90% on Day 14. The L nitrogen digestion changed similarly with age and increased from 67% at Day 4 to 89% at Day 14. Starch digestion in AA changed little during this same period and ranged from 90 to 95%. In L birds, starch digestion increased during this period from 80% on Day 4 to 94% on Day 14. Small intestine fatty acid digestion did not change between 4 and 21 d for either AA or L, ranging from 80 to 86%.
150 -
4
7
10
14
Age, d
FIGURE 4. Passage time through the small intestines in Arbor Acres (open bars) and Lohman (shaded bars) chicks. Passage time was shorter in Lohman chicks at 4 and 7 d. Results are the mean ± SD for five chicks.
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Enterocyte density
In this study, the development of the absorptive structure of the intestine has been examined close to hatching together with changes in digestion and absorption of feed. Villus growth in the young chick is stimulated by presence of feed and involves generation of cells in the crypt that attain maturity while ascending the shaft (Moran, 1985). Baranylova and Holman (1976) measured morphological changes in the intestines of fed and feeddeprived chicks through 1st wk of life. Access to feed was observed to markedly increase villus height, whereas feed deprivation prevented villi growth and reduced epithelial cell turnover time. The expansion of surface area that occurs with villus growth has been used to explain the increased absorptive capacity. (Moran, 1985). The duodenum had the greatest absorptive surface along the intestine as indicated by the higher and denser villi as compared with the jejunum, which in turn had higher and denser villi then the ileum. The enterocyte density was similar throughout the intestine, thus higher villi had more enterocytes. In the immediate posthatch period from 4 to 10 d of age, villi increase in size and number, with a greater absorptive surface per unit intestine. Bayer et al. (1975) showed micrographs qualitatively indicating increased villus surface area and
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
Trypsin
Amylase
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Lipase
i
Age, d
FIGURE 5. Net daily secretion per gram feed intake of trypsin, amylase, and lipase to the duodenum of Arbor Acres (open bars) and Lohman (hatched bars) chicks at different ages. Enzyme activities were defined as the amount of enzyme hydrolyzing 1 /unol of substrate/min under the specified conditions. Bars are means ± SD from five chicks. Secretion was higher in AA chicks on Day 4.
N
Starch
FA
100
7
10
14
i1]|1 4
7
10
14
4
7
10
14
Age, d
FIGURE 6. Apparent digestion between feed and lower ileum of nitrogen, starch, and fatty acids from 4 to 14 d posthatch in Arbor Acres (open bars) and Lohman (hatched bars) chicks. Bars are means ± SD from five chicks. Digestion of N and starch was higher in Arbor Acres chicks on Day 4.
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1111i I i i
1628
UNI ET AL.
at three sites in the intestine indicated that AA chicks had larger villi and more enterocytes then L chicks. The rate of growth of the villi with age was generally similar in AA and L chicks. However, it should be noted that the overall length of the small intestines in L chicks was some 70% of the AA chicks. With the methods used in this study we are not able to quantify in detail the convolutions of the mucosa, which are pronounced in the proximal small intestine, and thus we cannot evaluate precisely overall intestinal absorptive capacity. However, using computerized imaging techniques we have compared the structure and volume in the different intestinal regions and the differences in structure parallel the large differences in feed uptake between the two strains. In this study we also examined enzyme secretion, digestion, and small intestinal
TABLE 2. Morphological measurements in the duodenum, jejunum, and ileum of Arbor Acres (AA) and Lohman (L) chicks AA Measurement Duodenum Villi height,1 /tm perimeter,1 /an Crypt depth, 1 /tin Submucosa, /tm Enterocytes/villus 2 Villa/.l cm2<3 Jejunum Villi height,1 /tm perimeter, /tm Crypt depth, /tm Submucosa, /tm Enterocytes/villus Villa/.l cm* Ileum Villi height, /tm perimeter, /tm Crypt depth, /tm Submucosa, /tm Enterocytes/villus Villa/.l cm*
P
L
4 d
7 d
10 d
4 d
7 d
10 d
SEM
Age
Strain
511 1121 98 76 119 12
661 1422 110 75 146 8
710 1548 139 77 164 6
360 751 88 81 59 21
410 849 102 82 62 13
483 1003 112 82 95 11
7 21 7 2 21 2
.001 .004 .03 .64 .01 .01
.001 .001 .02 .04 .001 .02
310 646 65 82 71 16
383 836 103 83 93 13
432 948 112 83 101 14
156 332 54 75 34 25
242 531 87 73 51 17
324 707 112 75 67 17
32 41 7 2 6 1
.006 .001 .004 .86 .004 .18
.001 .001 .001 .04 .001 .05
221 474 98 81 66 19
251 503 100 80 61 17
281 621 101 82 67 18
147 262 85 73 28 27
183 373 96 76 36 19
213 405 99 76 44 20
39 31 4 1 13 1
.005 .001 .08 .15 .11 .19
.02 .006 .05 .05 .04 .06
Willi height was measured using the lamina propria as the base, crypt height as the depth of the invagination between adjacent villi, and the perimeter is the distance from the base of one villus to the adjacent villus. Values are means ± SD from 10 adjacent villi. 2 Number of enterocytes were measured per side of a longitudinal section through the villus. Values are means ± SD from 10 adjacent villi. 3 Number of villi/.l cm2 in a horizontal section through base of the villi of the area under examination. Values are means ± SD from 10 adjacent .1-cm2 areas.
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microvillus development throughout the intestine with age. Crypt depth also increased with age throughout the intestine, which indicates increased activity (Moran, 1985), whereas the thickness of the submucosal layer did not change. Similar increases in villi area and crypt depth have been reported in the first 7 d after hatching in the duodenum of Leghorn chicks (Cook and Bird, 1973). The increase in volume of villi with age was greater in the jejunum and ileum than in the duodenum, and in this intestinal region a large quantitative increase in absorption occurs at 7 to 14 d posthatching (Noy and Sklan, 1995). The changes in villus height and enterocyte density in this region were also correlated with feed intake as suggested by Moran (1985) and Murakami et al. (1992). Comparing the intestinal structure of AA and L chicks, villus absorptive surface
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
REFERENCES Association of Official Analytical Chemists, 1990. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA. Baranylova, E., and J. Holman, 1976. Morphological changes in the intestinal wall in fed and fasted chickens in the first week after hatching. Acta Vet. Brno 45:151-158. Bayer, R. R., C. B. Chawan, F. H. Bird, and S. D. Musgrave, 1975. Characteristics of the absorptive surface of the small intestine of the chicken from 1 day to 14 weeks of age. Poultry Sci. 54: 155-169. Bird, A. R., W. J. Croom, Y. K. Fan, L. R. Daniel, B. L. Black, B. W. McBride, E. J. Eisen, L. S. Bull, and I. L. Taylor, 1994. Jejunal glucose absorption is enhanced by epidermal growth factor in mice. J. Nutr. 124:231-240. Chambers, C, and R. D. Grey, 1979. Development of the structural components of the brush border in absorptive cells of the chick intestine. Cell Tissue Res. 204:387-405. Cook, R. H., and F. H. Bird. 1973. Duodenal villus area and epithelial cellular migration in conventional and germ-free chicks. Poultry Sci. 52: 2276-2280. Lim, S. S., and F. N. Low, 1977. Scanning electron microscopy of the developing alimentary canal in the chick. Am. J. Anat. 150:149-174. Moran, E. T., 1985. Digestion and absorption of carbohydrates in fowl and events through perinatal development J. Nutr. 115:665-674. Murakami, H., Y. Akiba, and M. Horiguchi, 1992. Growth and utilization of nutrients in newly hatched chicks with or without removal of residual yolk. Growth Dev. Aging 56:75-84. National Research Council, 1994. Nutrient Requirements for Poultry. 9th. rev. ed. National Academy Press, Washington, DC. Noy Y., and D. Sklan, 1995. Digestion and absorption in the young chick. Poultry Sci. 74:366-373. Overton, J., and J. Shoup, 1964. Fine structure of cell surface specializations in the maturing duodenal mucosa of the chick. J. Cell Biol. 21:75-85. Pinchasov, Y., and Y. Noy, 1994. Early postnatal amylolysis in the gastrointestinal tract of turkey poults (Meleagris gallopavo). Comp. Biochem. Physiol. 106:221-225. Rose, R., C. L. Rose, S. K. Omi, K. R. Forry, D. M. Durall, and W. L. Bigg, 1991. Starch determination by perchloric acid vs enzymes: evaluating the accuracy and precision of six colorimetric methods. J. Agric. Food Chem. 39:2-11. SAS Institute, 1986. SAS® User's Guide. Version 6 Edition. Cary, NC. Sklan, D., and O. Halevy, 1985. Protein digestion and absorption along the ovine gastrointestinal tract. J. Dairy Sci. 68:1676-1681. Sklan, D., D. Dubrov, U. Eisner, and S. Hurwitz, 1975a. 51Cr-EDTA, 91Y and 141Ce as nonabsorbed reference substances in the gastrointestinal tract of the chicken. J. Nutr. 105: 1549-1552. Sklan, D., S. Hurwitz, P. Budowski, and I. Ascarelli, 1975b. Fat digestion and absorption in chicks fed raw or heated soybean meal. J. Nutr. 105: 57-63.
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 6, 2014
passage time in the two strains of chicks. We have recently shown that digestion of fat and starch changed little between 4 and 21 d in broiler chicks fed diets with 6% added fat, whereas N digestion increased during the 1st wk (Noy and Sklan, 1995). Similar results were observed here for N and fatty acid digestion in both breeds from 4 to 14 d posthatch. Secretion of some pancreatic enzymes to the duodenum per gram of feed intake was high at 4 d after hatch in AA chicks and values, which were similar to those found in L chicks from 7 to 14 d. Starch digestion was over 90% in AA chicks on 4 d, whereas L chicks digested approximately 80% starch at 4 and 7 d, and this increased to 90% and above on Days 10 and 14. On the basis of the amounts of amylase secreted into the duodenum in L chicks, this difference in starch digestion was not surprising. Small intestinal passage time was very different in the two strains, with digesta in L chicks having a 50% shorter stay in the intestines at 4 d of age. It is also possible in this early stage of development that this was too short a time for complete starch digestion. Passage time or absorption could not be directly correlated with the morphological changes observed. It would be expected that increased absorptive surface area would be at the expense of lumen volume. However, measurement of length and external diameter of the intestine do not reflect the route of the digesta through the intervillus spaces. Thus, whereas the intestine increased in length by 20 to 30% between 4 and 14 d and by 5 to 10% in external diameter, the throughput of feed increased some five- to eightfold. Passage time changed by a much smaller magnitude, thus the digesta must have been forced into more intimate contact with the absorptive surfaces, as, even accounting for the increased passage time, two- to threefold more nutrients were digested on Day 14. The absorption of starch and N in the light strains was lower on Day 4 then in the heavy birds but was similar from Day 7. Thus, slower growth would be expected close to hatching. Subsequently, however, with similar percentage digestion in both strains, it appears that the absolute uptake of nutrients was determined by feed intake.
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1995 Poultry Science 74:1622-1629
INTRODUCTION In the posthatch chick, metabolites are supplied at first from the yolk and development to exogenous nutrient uptake occurs in the first few days. During this period, feed intake increases many fold whereas pancreatic and bile secretions to the duodenum escalate to match these developments. Passage time through the small intestines decreases by one third during the 1st wk posthatch. Digestion of fatty acids and starch is essentially complete by Day 4 in broiler chicks, whereas N digestion increases from close to 80% on Day 4 to approximately 90% on Day 10 (Noy and Sklan, 1995). The relative weight of the intestines increases posthatch, reaching a maximum at 3 to 7 d, and declines slightly thereafter (Murakami et al., 1992; Pinchasov and
Noy, 1994). Although the chicks' intestinal system is anatomically complete in the embryonic stage (Overton and Shoup, 1964; Lim and Low, 1977; Chambers and Grey, 1979), the absorptive surface changes considerably posthatch and the rate of enterocyte proliferation increases (Cook and Bird, 1973; Moran, 1985). The rate of growth in heavy and light chick strains is very different and this is likely to be due to differences in the digestive and absorptive systems. The objective of this study was to determine changes from hatch in the morphology of the small intestines of heavy- and light-strain birds and in parallel measure secretion of enzymes to the duodenum, passage time, and digestion. MATERIALS AND METHODS Animals and Treatments
Received for publication November 28, 1994. Accepted for publication May 1, 1995. x To whom correspondence should be addressed.
Day-old Arbor Acres (AA) chicks and Lohman (L) chicks were obtained from
1622
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 6, 2014
ABSTRACT The morphology of the small intestines of heavy (Arbor Acres) and light (Lohman) chicks was determined posthatch in parallel with digestion, enzyme secretion, and passage time. Villus height and volume increased from 4 to 10 d, particularly in the jejunum and ileum. The number of enterocytes per villus increased with age, but enterocyte density was greater in jejunum than ileum. Villus volume and enterocyte density was greater in Arbor Acres than Lohman chicks from hatching and the rate of change with age was similar in both strains. Enzyme secretion to the duodenum was higher per gram of feed intake in heavy-strain birds on Day 4 after hatching but thereafter no differences were apparent. Passage time was 50% shorter in light-strain chicks on Day 4 but the difference between the strains was not significant from Day 10. No differences in fatty acid digestion were observed with age or between strains. Nitrogen digestion increased in both strains from approximately 70% on Day 4 to close to 90% on Day 14. Starch digestion was 90 to 95% from 4 to 14 d in Arbor Acres birds, but increased from approximately 80% on Day 4 in Lohman chicks to 93% on Day 14. Feed intake may determine the amount of uptake in posthatch chicks. (Key words: chick, intestine, morphology, absorption)
1623
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
commercial hatcheries and transferred to a battery brooder. Chicks were fed the experimental diet shown in Table 1, which were formulated according to requirements of the NRC (1994). Two hundred /tCi/kg 141 Ce was added as an unabsorbed reference substance (Sklan et ah, 1975a) to the chick diet for 4 d before slaughter. Five chicks from each treatment were killed with intracardiac overdose of sodium pentobarbital on Days 4, 7,10, and 14 after hatching.
Digesta were obtained by gentle manipulation from duodenum, jejunum, and lower ileum and the length and diameter of the segments measured. Digesta were weighed, homogenized with cold .9% NaCl, and aliquots of the homogenate taken for 141Ce determination, for lipase (Sklan et ah, 1975b), trypsin (Sklan and Halevy, 1985), and amylase activities (Pinchasov and Noy, 1994). Enzyme activities were defined as the amount of enzyme hydrolyzing 1 mmol of substrate/min under the specified conditions. The 141Ce was determined by scintillation counting (Sklan et al, 1975a), nitrogen by the Kjeldahl method [Association of Official Analytical Chemists (AOAC), 1990], starch as described by Rose et al. (1991), fatty acids after methylation by gas chromatography using an internal standard, and bile acids colorimetrically (Sklan et al, 1975b). Calcium, P, and fiber were determined by standard methods (AOAC, 1990). Morphology On Days 4, 7, and 10 posthatching, 1-cm segments from duodenum, jejunum, and ileum were removed from the following sites: 1) the apex of the duodenum, 2) 10 cm distal to the point of entry of the distal bile ducts, and 3) 10 cm proximal to the cecal junction. The intestine was flushed clean with PBS and samples were fixed in 10% neutral buffered formalin solution and routinely processed to 5 mm hematoxylineosin-stained sections and examined by
2
Applitec MSV-800 System, Tel Aviv, Israel.
Ingredients and composition
Percentage
Corn Soybean meal Soybean oil CaCO? Dicalcium phosphate NaCl Methionine Vitamins + minerals1 Analyzed nutrient composition Protein Fat Fiber Ca P
51.0 37.6 6.0 1.6 1.6 .4 .3 1.5 22.5 7.8 2.5 1.06 .70
1
The vitamin and mineral mix contained in milligrams per kilogram of diet: FeS04,2.5; CoCO^ .64; KI, 3.8; MnS04, 208; NajMoSO^ 3.5; C11SO4, 20; ZnSO^ 220; thiamin, 5; riboflavin, 3.5; nicotinic acid, 60; pantothenic acid, 15; pyridoxine, 5; folic acid, .5; biotin, 2; menaquinone, .5; cyclocobalamine .02, a-tocopherol acetate, 30; cholecalciferol, .125; retinol acetate, 35.
light microscopy. Morphometric indices were determined using a computer-aided light microscope image analysis 2 as described by Bird et al. (1994). Villi height was measured using the lamina propria as the base; crypt height was defined as the depth of the invagination between adjacent villi; villus perimeter as the distance from the base of one villus to the adjacent villus; number of enterocytes were measured per side of a longitudinal section through the villus; number of villi per area in a horizontal section through the base of the villi. Values are means ± SD from 10 adjacent villi or .1 cm2 areas. Calculations When an unabsorbed reference substance such as 141Ce is fed until a steady state is obtained with respect to inputoutput of the isotope, assuming no chemical changes occur, a decrease in ratio of any component (A) to 141Ce between two points in the intestine can be defined as absorption and any increase as secretion (Sklan et al, 1975a). Overall net absorption between feed and lower ileum can be calculated as follows: 100 [(A/i«Ce)f - (A/i4iCe)J/(A/i4iCe)f]
Downloaded from http://ps.oxfordjournals.org/ at Columbia University Libraries on November 6, 2014
Analyses
TABLE 1. Composition of the experimental diets
1624
UNI ET AL.
where (A/i«Ce)f is the ratio of A to 141Ce in the feed and (A/141Ce); the ratio in the ileum. Net secretion of any component to the duodenum can be calculated by: (daily i«Ce intake)[(A/i4iCe)d - (A/i«Ce)f]
RESULTS The growth and feed intake of the two strains of chicks are shown in Figure 1, and the more rapid growth and higher feed intake of the AA chicks is clear. Sections from the different segments of the small intestine for both strains on Day 4 (Figure 2) show the differences in development of the duodenum, jejunum, and ileum. The villi are larger and the degree of organization greater in AA than in L chicks. Table 2 presents measurements for
200 -
$L
150 j -
' 0 ' 0
t^^^C^-—-*'^'^^^ i10 ' 5
' 10
' 0 15
Age, d
FIGURE 1. Body weight (circles) and feed intake (triangles) for Arbor Acres (open symbols) and Lohman (filled symbols) chicks from hatching to Day 14.
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where (A/141Ce)d is the ratio of A to 141Ce in the duodenum. Passage time through any segment can be calculated by dividing the mean amount of isotope recovered from the segment by the daily intake of 141Ce. Least squares means of results are presented after analysis of variance and linear regression using the General Linear Models procedures of SAS® (SAS Institute, 1986). Differences between means were tested using t tests and significance was P < .01 unless otherwise stated.
the different segments in both strains. Villus height and perimeters in all segments increased 25 to 100% between 4 and 10 d for both strains, yet villi in AA were consistently higher then in L. Greatest increases in villus height with age were in the jejunum and in L chicks. Crypt depth increased with age with the exception in the AA ileum. There were no changes in submucosa thickness between 4 and 10 d, although the AA had a thicker layer in jejunum and ileum. The number of enterocytes per longitudinal section of villus increased with age and the rate was more rapid in L chicks. The density of enterocytes in the different segments is shown in Figure 3 (bottom) and this did not change with age, but was higher in AA. As the villi grew larger with age, the number of villi per area decreased, particularly in the duodenum of both strains. Villus volume is shown in Figure 3 (top) and increased slightly with age in the duodenum, and more so in the jejunum and ileum. The AA villus volume was consistently greater then in L. Jejunal and ileal villus volume and were linearly correlated with feed intake (r = .77 and .67), whereas duodenal villus volume was not significantly correlated. Enterocyte density in the jejunum was also correlated with feed intake (r = .62). Passage time through the small intestine (Figure 4) decreased in the AA from 115 min on Day 4 to 74 min at Day 7, increasing at Day 10 and reaching approximately 122 min at Day 14. Passage time was shorter in L chicks; however, a similar pattern was observed, with passage time decreasing slightly between Days 4 and 7 and increasing on Day 10 and at Day 14. The daily enzyme secretion per gram feed in the duodenum is shown in Figure 5 for amylase, trypsin, and lipase. Daily secretion per gram of feed of these three enzymes was higher on Day 4 in AA than in L birds. Secretion of trypsin per gram of feed intake decreased between Days 4 to 7 in AA chicks, but no change with age was observed in L birds. Secretion of amylase per gram of feed increased in L chicks between Days 4 and 7, with no changes thereafter. Lipase increased in L chicks between Days 4 and 7 and decreased in both strains between Days 10 and 14.
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
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V
VI
FIGURE 2. Sections from 4-d-old Arbor Acres chicks (left) and Lohman chicks (right). The upper panel is from the duodenum (apex), the middle panel is the jejunum (10 cm distal to the bile duct) and the bottom panel the ileum (10 cm proximal to the cecal junction). Sections were stained with eosin-hematoxylin, magnification is 10-fold, and bars are 40 jtm.
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IV
IH
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UNI ET AL.
DISCUSSION
Villus volume Duodenum
Jejunum
Ileum
•1
X
1i 1 1 I i I
I
1
JL^
X
JL
JL
!
\
7
10
4
7
10
4
!
7
Age, d
FIGURE 3. Villus volume (top panel) and enterocyte density (bottom panel) in duodenum, jejunum, and ileum of Arbor Acres (open bars) and Lohman (hatched bars) chicks at 4, 7, and 10 d old. Samples were taken from sites as described in Figure 1. Results are mean ± SD from 10 adjacent villi. Villus volume was higher for Arbor Acres then Lohman chicks in all segments, whereas enterocyte density was higher in Arbor Acres chicks only in the jejunum and ileum.
After Day 7, no differences were observed between strains. Digestion between feed and ileum is shown in Figure 6 for nitrogen, fatty acids, and starch for AA and L birds. Small intestine nitrogen digestion increased in AA from 78% on Day 4 to close to 90% on Day 14. The L nitrogen digestion changed similarly with age and increased from 67% at Day 4 to 89% at Day 14. Starch digestion in AA changed little during this same period and ranged from 90 to 95%. In L birds, starch digestion increased during this period from 80% on Day 4 to 94% on Day 14. Small intestine fatty acid digestion did not change between 4 and 21 d for either AA or L, ranging from 80 to 86%.
150 -
4
7
10
14
Age, d
FIGURE 4. Passage time through the small intestines in Arbor Acres (open bars) and Lohman (shaded bars) chicks. Passage time was shorter in Lohman chicks at 4 and 7 d. Results are the mean ± SD for five chicks.
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Enterocyte density
In this study, the development of the absorptive structure of the intestine has been examined close to hatching together with changes in digestion and absorption of feed. Villus growth in the young chick is stimulated by presence of feed and involves generation of cells in the crypt that attain maturity while ascending the shaft (Moran, 1985). Baranylova and Holman (1976) measured morphological changes in the intestines of fed and feeddeprived chicks through 1st wk of life. Access to feed was observed to markedly increase villus height, whereas feed deprivation prevented villi growth and reduced epithelial cell turnover time. The expansion of surface area that occurs with villus growth has been used to explain the increased absorptive capacity. (Moran, 1985). The duodenum had the greatest absorptive surface along the intestine as indicated by the higher and denser villi as compared with the jejunum, which in turn had higher and denser villi then the ileum. The enterocyte density was similar throughout the intestine, thus higher villi had more enterocytes. In the immediate posthatch period from 4 to 10 d of age, villi increase in size and number, with a greater absorptive surface per unit intestine. Bayer et al. (1975) showed micrographs qualitatively indicating increased villus surface area and
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
Trypsin
Amylase
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Lipase
i
Age, d
FIGURE 5. Net daily secretion per gram feed intake of trypsin, amylase, and lipase to the duodenum of Arbor Acres (open bars) and Lohman (hatched bars) chicks at different ages. Enzyme activities were defined as the amount of enzyme hydrolyzing 1 /unol of substrate/min under the specified conditions. Bars are means ± SD from five chicks. Secretion was higher in AA chicks on Day 4.
N
Starch
FA
100
7
10
14
i1]|1 4
7
10
14
4
7
10
14
Age, d
FIGURE 6. Apparent digestion between feed and lower ileum of nitrogen, starch, and fatty acids from 4 to 14 d posthatch in Arbor Acres (open bars) and Lohman (hatched bars) chicks. Bars are means ± SD from five chicks. Digestion of N and starch was higher in Arbor Acres chicks on Day 4.
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1111i I i i
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UNI ET AL.
at three sites in the intestine indicated that AA chicks had larger villi and more enterocytes then L chicks. The rate of growth of the villi with age was generally similar in AA and L chicks. However, it should be noted that the overall length of the small intestines in L chicks was some 70% of the AA chicks. With the methods used in this study we are not able to quantify in detail the convolutions of the mucosa, which are pronounced in the proximal small intestine, and thus we cannot evaluate precisely overall intestinal absorptive capacity. However, using computerized imaging techniques we have compared the structure and volume in the different intestinal regions and the differences in structure parallel the large differences in feed uptake between the two strains. In this study we also examined enzyme secretion, digestion, and small intestinal
TABLE 2. Morphological measurements in the duodenum, jejunum, and ileum of Arbor Acres (AA) and Lohman (L) chicks AA Measurement Duodenum Villi height,1 /tm perimeter,1 /an Crypt depth, 1 /tin Submucosa, /tm Enterocytes/villus 2 Villa/.l cm2<3 Jejunum Villi height,1 /tm perimeter, /tm Crypt depth, /tm Submucosa, /tm Enterocytes/villus Villa/.l cm* Ileum Villi height, /tm perimeter, /tm Crypt depth, /tm Submucosa, /tm Enterocytes/villus Villa/.l cm*
P
L
4 d
7 d
10 d
4 d
7 d
10 d
SEM
Age
Strain
511 1121 98 76 119 12
661 1422 110 75 146 8
710 1548 139 77 164 6
360 751 88 81 59 21
410 849 102 82 62 13
483 1003 112 82 95 11
7 21 7 2 21 2
.001 .004 .03 .64 .01 .01
.001 .001 .02 .04 .001 .02
310 646 65 82 71 16
383 836 103 83 93 13
432 948 112 83 101 14
156 332 54 75 34 25
242 531 87 73 51 17
324 707 112 75 67 17
32 41 7 2 6 1
.006 .001 .004 .86 .004 .18
.001 .001 .001 .04 .001 .05
221 474 98 81 66 19
251 503 100 80 61 17
281 621 101 82 67 18
147 262 85 73 28 27
183 373 96 76 36 19
213 405 99 76 44 20
39 31 4 1 13 1
.005 .001 .08 .15 .11 .19
.02 .006 .05 .05 .04 .06
Willi height was measured using the lamina propria as the base, crypt height as the depth of the invagination between adjacent villi, and the perimeter is the distance from the base of one villus to the adjacent villus. Values are means ± SD from 10 adjacent villi. 2 Number of enterocytes were measured per side of a longitudinal section through the villus. Values are means ± SD from 10 adjacent villi. 3 Number of villi/.l cm2 in a horizontal section through base of the villi of the area under examination. Values are means ± SD from 10 adjacent .1-cm2 areas.
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microvillus development throughout the intestine with age. Crypt depth also increased with age throughout the intestine, which indicates increased activity (Moran, 1985), whereas the thickness of the submucosal layer did not change. Similar increases in villi area and crypt depth have been reported in the first 7 d after hatching in the duodenum of Leghorn chicks (Cook and Bird, 1973). The increase in volume of villi with age was greater in the jejunum and ileum than in the duodenum, and in this intestinal region a large quantitative increase in absorption occurs at 7 to 14 d posthatching (Noy and Sklan, 1995). The changes in villus height and enterocyte density in this region were also correlated with feed intake as suggested by Moran (1985) and Murakami et al. (1992). Comparing the intestinal structure of AA and L chicks, villus absorptive surface
MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE
REFERENCES Association of Official Analytical Chemists, 1990. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA. Baranylova, E., and J. Holman, 1976. Morphological changes in the intestinal wall in fed and fasted chickens in the first week after hatching. Acta Vet. Brno 45:151-158. Bayer, R. R., C. B. Chawan, F. H. Bird, and S. D. Musgrave, 1975. Characteristics of the absorptive surface of the small intestine of the chicken from 1 day to 14 weeks of age. Poultry Sci. 54: 155-169. Bird, A. R., W. J. Croom, Y. K. Fan, L. R. Daniel, B. L. Black, B. W. McBride, E. J. Eisen, L. S. Bull, and I. L. Taylor, 1994. Jejunal glucose absorption is enhanced by epidermal growth factor in mice. J. Nutr. 124:231-240. Chambers, C, and R. D. Grey, 1979. Development of the structural components of the brush border in absorptive cells of the chick intestine. Cell Tissue Res. 204:387-405. Cook, R. H., and F. H. Bird. 1973. Duodenal villus area and epithelial cellular migration in conventional and germ-free chicks. Poultry Sci. 52: 2276-2280. Lim, S. S., and F. N. Low, 1977. Scanning electron microscopy of the developing alimentary canal in the chick. Am. J. Anat. 150:149-174. Moran, E. T., 1985. Digestion and absorption of carbohydrates in fowl and events through perinatal development J. Nutr. 115:665-674. Murakami, H., Y. Akiba, and M. Horiguchi, 1992. Growth and utilization of nutrients in newly hatched chicks with or without removal of residual yolk. Growth Dev. Aging 56:75-84. National Research Council, 1994. Nutrient Requirements for Poultry. 9th. rev. ed. National Academy Press, Washington, DC. Noy Y., and D. Sklan, 1995. Digestion and absorption in the young chick. Poultry Sci. 74:366-373. Overton, J., and J. Shoup, 1964. Fine structure of cell surface specializations in the maturing duodenal mucosa of the chick. J. Cell Biol. 21:75-85. Pinchasov, Y., and Y. Noy, 1994. Early postnatal amylolysis in the gastrointestinal tract of turkey poults (Meleagris gallopavo). Comp. Biochem. Physiol. 106:221-225. Rose, R., C. L. Rose, S. K. Omi, K. R. Forry, D. M. Durall, and W. L. Bigg, 1991. Starch determination by perchloric acid vs enzymes: evaluating the accuracy and precision of six colorimetric methods. J. Agric. Food Chem. 39:2-11. SAS Institute, 1986. SAS® User's Guide. Version 6 Edition. Cary, NC. Sklan, D., and O. Halevy, 1985. Protein digestion and absorption along the ovine gastrointestinal tract. J. Dairy Sci. 68:1676-1681. Sklan, D., D. Dubrov, U. Eisner, and S. Hurwitz, 1975a. 51Cr-EDTA, 91Y and 141Ce as nonabsorbed reference substances in the gastrointestinal tract of the chicken. J. Nutr. 105: 1549-1552. Sklan, D., S. Hurwitz, P. Budowski, and I. Ascarelli, 1975b. Fat digestion and absorption in chicks fed raw or heated soybean meal. J. Nutr. 105: 57-63.
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passage time in the two strains of chicks. We have recently shown that digestion of fat and starch changed little between 4 and 21 d in broiler chicks fed diets with 6% added fat, whereas N digestion increased during the 1st wk (Noy and Sklan, 1995). Similar results were observed here for N and fatty acid digestion in both breeds from 4 to 14 d posthatch. Secretion of some pancreatic enzymes to the duodenum per gram of feed intake was high at 4 d after hatch in AA chicks and values, which were similar to those found in L chicks from 7 to 14 d. Starch digestion was over 90% in AA chicks on 4 d, whereas L chicks digested approximately 80% starch at 4 and 7 d, and this increased to 90% and above on Days 10 and 14. On the basis of the amounts of amylase secreted into the duodenum in L chicks, this difference in starch digestion was not surprising. Small intestinal passage time was very different in the two strains, with digesta in L chicks having a 50% shorter stay in the intestines at 4 d of age. It is also possible in this early stage of development that this was too short a time for complete starch digestion. Passage time or absorption could not be directly correlated with the morphological changes observed. It would be expected that increased absorptive surface area would be at the expense of lumen volume. However, measurement of length and external diameter of the intestine do not reflect the route of the digesta through the intervillus spaces. Thus, whereas the intestine increased in length by 20 to 30% between 4 and 14 d and by 5 to 10% in external diameter, the throughput of feed increased some five- to eightfold. Passage time changed by a much smaller magnitude, thus the digesta must have been forced into more intimate contact with the absorptive surfaces, as, even accounting for the increased passage time, two- to threefold more nutrients were digested on Day 14. The absorption of starch and N in the light strains was lower on Day 4 then in the heavy birds but was similar from Day 7. Thus, slower growth would be expected close to hatching. Subsequently, however, with similar percentage digestion in both strains, it appears that the absolute uptake of nutrients was determined by feed intake.
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