Posthatch Changes in Morphology and Function of the Small Intestines in Heavy- and Light-Strain Chicks

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1 Posthatch Changes n Morphology and Functon of the Small Intestnes n Heavy- and Lght-Stran Chcks ZEHAVA UNI, YAEL NOY, and DAVID SKLAN Faculty of Agrculture, Hebrew Unversty, P.O. Box 2, Rehovot, Israel, ABSTRACT The morphology of the small ntestnes of heavy (Arbor Acres) and lght (Lohman) chcks was determned posthatch n parallel wth dgeston, enzyme secreton, and passage tme. Vllus heght and volume ncreased from 4 to 0 d, partcularly n the jejunum and leum. The number of enterocytes per vllus ncreased wth age, but enterocyte densty was greater n jejunum than leum. Vllus volume and enterocyte densty was greater n Arbor Acres than Lohman chcks from hatchng and the rate of change wth age was smlar n both strans. Enzyme secreton to the duodenum was hgher per gram of feed ntake n heavy-stran brds on Day 4 after hatchng but thereafter no dfferences were apparent. Passage tme was 50% shorter n lght-stran chcks on Day 4 but the dfference between the strans was not sgnfcant from Day 0. No dfferences n fatty acd dgeston were observed wth age or between strans. Ntrogen dgeston ncreased n both strans from approxmately 70% on Day 4 to close to 90% on Day 4. Starch dgeston was 90 to 95% from 4 to 4 d n Arbor Acres brds, but ncreased from approxmately 80% on Day 4 n Lohman chcks to 93% on Day 4. Feed ntake may determne the amount of uptake n posthatch chcks. (Key words: chck, ntestne, morphology, absorpton) INTRODUCTION In the posthatch chck, metaboltes are suppled at frst from the yolk and development to exogenous nutrent uptake occurs n the frst few days. Durng ths perod, feed ntake ncreases many fold whereas pancreatc and ble secretons to the duodenum escalate to match these developments. Passage tme through the small ntestnes decreases by one thrd durng the st wk posthatch. Dgeston of fatty acds and starch s essentally complete by Day 4 n broler chcks, whereas N dgeston ncreases from close to 80% on Day 4 to approxmately 90% on Day 0 (Noy and Sklan, 995). The relatve weght of the ntestnes ncreases posthatch, reachng a maxmum at 3 to 7 d, and declnes slghtly thereafter (Murakam et al., 992; Pnchasov and 995 Poultry Scence 74: Noy, 994). Although the chcks' ntestnal system s anatomcally complete n the embryonc stage (Overton and Shoup, 964; Lm and Low, 977; Chambers and Grey, 979), the absorptve surface changes consderably posthatch and the rate of enterocyte prolferaton ncreases (Cook and Brd, 973; Moran, 985). The rate of growth n heavy and lght chck strans s very dfferent and ths s lkely to be due to dfferences n the dgestve and absorptve systems. The objectve of ths study was to determne changes from hatch n the morphology of the small ntestnes of heavy- and lght-stran brds and n parallel measure secreton of enzymes to the duodenum, passage tme, and dgeston. MATERIALS AND METHODS Receved for publcaton November 28, 994. Accepted for publcaton May, 995. x To whom correspondence should be addressed. 622 Anmals and Treatments Day-old Arbor Acres (AA) chcks and Lohman (L) chcks were obtaned from

2 MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE 623 commercal hatcheres and transferred to a battery brooder. Chcks were fed the expermental det shown n Table, whch were formulated accordng to requrements of the NRC (994). Two hundred /tc/kg 4 Ce was added as an unabsorbed reference substance (Sklan et ah, 975a) to the chck det for 4 d before slaughter. Fve chcks from each treatment were klled wth ntracardac overdose of sodum pentobarbtal on Days 4, 7,0, and 4 after hatchng. Analyses Dgesta were obtaned by gentle manpulaton from duodenum, jejunum, and lower leum and the length and dameter of the segments measured. Dgesta were weghed, homogenzed wth cold.9% NaCl, and alquots of the homogenate taken for 4 Ce determnaton, for lpase (Sklan et ah, 975b), trypsn (Sklan and Halevy, 985), and amylase actvtes (Pnchasov and Noy, 994). Enzyme actvtes were defned as the amount of enzyme hydrolyzng mmol of substrate/mn under the specfed condtons. The 4 Ce was determned by scntllaton countng (Sklan et al, 975a), ntrogen by the Kjeldahl method [Assocaton of Offcal Analytcal Chemsts (AOAC), 990], starch as descrbed by Rose et al. (99), fatty acds after methylaton by gas chromatography usng an nternal standard, and ble acds colormetrcally (Sklan et al, 975b). Calcum, P, and fber were determned by standard methods (AOAC, 990). Morphology On Days 4, 7, and 0 posthatchng, -cm segments from duodenum, jejunum, and leum were removed from the followng stes: ) the apex of the duodenum, 2) 0 cm dstal to the pont of entry of the dstal ble ducts, and 3) 0 cm proxmal to the cecal juncton. The ntestne was flushed clean wth PBS and samples were fxed n 0% neutral buffered formaln soluton and routnely processed to 5 mm hematoxylneosn-staned sectons and examned by TABLE. Composton of the expermental dets Ingredents and composton Percentage Corn 5.0 Soybean meal 37.6 Soybean ol CaCO? Dcalcum phosphate.6 NaCl.4 Methonne.3 Vtamns + mnerals.5 Analyzed nutrent composton Proten 22.5 Fat 7.8 Fber 2.5 Ca.06 P.70 The vtamn and mneral mx contaned n mllgrams per klogram of det: FeS0 4,2.5; CoCO^.64; KI, 3.8; MnS0 4, 208; NajMoSO^ 3.5; CSO4, 20; ZnSO^ 220; thamn, 5; rboflavn, 3.5; ncotnc acd, 60; pantothenc acd, 5; pyrdoxne, 5; folc acd,.5; botn, 2; menaqunone,.5; cyclocobalamne.02, a-tocopherol acetate, 30; cholecalcferol,.25; retnol acetate, 35. lght mcroscopy. Morphometrc ndces were determned usng a computer-aded lght mcroscope mage analyss 2 as descrbed by Brd et al. (994). Vll heght was measured usng the lamna propra as the base; crypt heght was defned as the depth of the nvagnaton between adjacent vll; vllus permeter as the dstance from the base of one vllus to the adjacent vllus; number of enterocytes were measured per sde of a longtudnal secton through the vllus; number of vll per area n a horzontal secton through the base of the vll. Values are means ± SD from 0 adjacent vll or. cm 2 areas. Calculatons When an unabsorbed reference substance such as 4 Ce s fed untl a steady state s obtaned wth respect to nputoutput of the sotope, assumng no chemcal changes occur, a decrease n rato of any component (A) to 4 Ce between two ponts n the ntestne can be defned as absorpton and any ncrease as secreton (Sklan et al, 975a). Overall net absorpton between feed and lower leum can be calculated as follows: 2 Appltec MSV-800 System, Tel Avv, Israel. 00 [(A/«Ce) f - (A/4Ce)J/(A/4Ce) f ]

3 624 UNI ET AL. where (A/«Ce)f s the rato of A to 4 Ce n the feed and (A/ 4 Ce) ; the rato n the leum. Net secreton of any component to the duodenum can be calculated by: (daly «Ce ntake)[(a/4ce) d - (A/«Ce) f ] where (A/ 4 Ce) d s the rato of A to 4 Ce n the duodenum. Passage tme through any segment can be calculated by dvdng the mean amount of sotope recovered from the segment by the daly ntake of 4 Ce. Least squares means of results are presented after analyss of varance and lnear regresson usng the General Lnear Models procedures of SAS (SAS Insttute, 986). Dfferences between means were tested usng t tests and sgnfcance was P <.0 unless otherwse stated. RESULTS The growth and feed ntake of the two strans of chcks are shown n Fgure, and the more rapd growth and hgher feed ntake of the AA chcks s clear. Sectons from the dfferent segments of the small ntestne for both strans on Day 4 (Fgure 2) show the dfferences n development of the duodenum, jejunum, and leum. The vll are larger and the degree of organzaton greater n AA than n L chcks. Table 2 presents measurements for $L 50 j- ' t^^^c^- -*'^'^^^ 0 0 ' ' ' ' FIGURE. Body weght (crcles) and feed ntake (trangles) for Arbor Acres (open symbols) and Lohman (flled symbols) chcks from hatchng to Day 4. the dfferent segments n both strans. Vllus heght and permeters n all segments ncreased 25 to 00% between 4 and 0 d for both strans, yet vll n AA were consstently hgher then n L. Greatest ncreases n vllus heght wth age were n the jejunum and n L chcks. Crypt depth ncreased wth age wth the excepton n the AA leum. There were no changes n submucosa thckness between 4 and 0 d, although the AA had a thcker layer n jejunum and leum. The number of enterocytes per longtudnal secton of vllus ncreased wth age and the rate was more rapd n L chcks. The densty of enterocytes n the dfferent segments s shown n Fgure 3 (bottom) and ths dd not change wth age, but was hgher n AA. As the vll grew larger wth age, the number of vll per area decreased, partcularly n the duodenum of both strans. Vllus volume s shown n Fgure 3 (top) and ncreased slghtly wth age n the duodenum, and more so n the jejunum and leum. The AA vllus volume was consstently greater then n L. Jejunal and leal vllus volume and were lnearly correlated wth feed ntake (r =.77 and.67), whereas duodenal vllus volume was not sgnfcantly correlated. Enterocyte densty n the jejunum was also correlated wth feed ntake (r =.62). Passage tme through the small ntestne (Fgure 4) decreased n the AA from 5 mn on Day 4 to 74 mn at Day 7, ncreasng at Day 0 and reachng approxmately 22 mn at Day 4. Passage tme was shorter n L chcks; however, a smlar pattern was observed, wth passage tme decreasng slghtly between Days 4 and 7 and ncreasng on Day 0 and at Day 4. The daly enzyme secreton per gram feed n the duodenum s shown n Fgure 5 for amylase, trypsn, and lpase. Daly secreton per gram of feed of these three enzymes was hgher on Day 4 n AA than n L brds. Secreton of trypsn per gram of feed ntake decreased between Days 4 to 7 n AA chcks, but no change wth age was observed n L brds. Secreton of amylase per gram of feed ncreased n L chcks between Days 4 and 7, wth no changes thereafter. Lpase ncreased n L chcks between Days 4 and 7 and decreased n both strans between Days 0 and 4.

4 MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE 625 IH V IV VI FIGURE 2. Sectons from 4-d-old Arbor Acres chcks (left) and Lohman chcks (rght). The upper panel s from the duodenum (apex), the mddle panel s the jejunum (0 cm dstal to the ble duct) and the bottom panel the leum (0 cm proxmal to the cecal juncton). Sectons were staned wth eosn-hematoxyln, magnfcaton s 0-fold, and bars are 40 jtm.

$626 UNI ET AL. Vllus volume Duodenum Jejunum Ileum Enterocyte densty JL I X I JL^ JL I \! 7 0 4 7 0 4 7 FIGURE 3.$

5 626 UNI ET AL. Vllus volume Duodenum Jejunum Ileum Enterocyte densty JL I X I JL^ JL I \! FIGURE 3. Vllus volume (top panel) and enterocyte densty (bottom panel) n duodenum, jejunum, and leum of Arbor Acres (open bars) and Lohman (hatched bars) chcks at 4, 7, and 0 d old. Samples were taken from stes as descrbed n Fgure. Results are mean ± SD from 0 adjacent vll. Vllus volume was hgher for Arbor Acres then Lohman chcks n all segments, whereas enterocyte densty was hgher n Arbor Acres chcks only n the jejunum and leum. After Day 7, no dfferences were observed between strans. Dgeston between feed and leum s shown n Fgure 6 for ntrogen, fatty acds, and starch for AA and L brds. Small ntestne ntrogen dgeston ncreased n AA from 78% on Day 4 to close to 90% on Day 4. The L ntrogen dgeston changed smlarly wth age and ncreased from 67% at Day 4 to 89% at Day 4. Starch dgeston n AA changed lttle durng ths same perod and ranged from 90 to 95%. In L brds, starch dgeston ncreased durng ths perod from 80% on Day 4 to 94% on Day 4. Small ntestne fatty acd dgeston dd not change between 4 and 2 d for ether AA or L, rangng from 80 to 86%. X! DISCUSSION In ths study, the development of the absorptve structure of the ntestne has been examned close to hatchng together wth changes n dgeston and absorpton of feed. Vllus growth n the young chck s stmulated by presence of feed and nvolves generaton of cells n the crypt that attan maturty whle ascendng the shaft (Moran, 985). Baranylova and Holman (976) measured morphologcal changes n the ntestnes of fed and feeddeprved chcks through st wk of lfe. Access to feed was observed to markedly ncrease vllus heght, whereas feed deprvaton prevented vll growth and reduced epthelal cell turnover tme. The expanson of surface area that occurs wth vllus growth has been used to explan the ncreased absorptve capacty. (Moran, 985). The duodenum had the greatest absorptve surface along the ntestne as ndcated by the hgher and denser vll as compared wth the jejunum, whch n turn had hgher and denser vll then the leum. The enterocyte densty was smlar throughout the ntestne, thus hgher vll had more enterocytes. In the mmedate posthatch perod from 4 to 0 d of age, vll ncrease n sze and number, wth a greater absorptve surface per unt ntestne. Bayer et al. (975) showed mcrographs qualtatvely ndcatng ncreased vllus surface area and FIGURE 4. Passage tme through the small ntestnes n Arbor Acres (open bars) and Lohman (shaded bars) chcks. Passage tme was shorter n Lohman chcks at 4 and 7 d. Results are the mean ± SD for fve chcks.

6 MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE 627 Trypsn Amylase Lpase I FIGURE 5. Net daly secreton per gram feed ntake of trypsn, amylase, and lpase to the duodenum of Arbor Acres (open bars) and Lohman (hatched bars) chcks at dfferent ages. Enzyme actvtes were defned as the amount of enzyme hydrolyzng /unol of substrate/mn under the specfed condtons. Bars are means ± SD from fve chcks. Secreton was hgher n AA chcks on Day N Starch FA ] FIGURE 6. Apparent dgeston between feed and lower leum of ntrogen, starch, and fatty acds from 4 to 4 d posthatch n Arbor Acres (open bars) and Lohman (hatched bars) chcks. Bars are means ± SD from fve chcks. Dgeston of N and starch was hgher n Arbor Acres chcks on Day 4.

7 628 UNI ET AL. mcrovllus development throughout the ntestne wth age. Crypt depth also ncreased wth age throughout the ntestne, whch ndcates ncreased actvty (Moran, 985), whereas the thckness of the submucosal layer dd not change. Smlar ncreases n vll area and crypt depth have been reported n the frst 7 d after hatchng n the duodenum of Leghorn chcks (Cook and Brd, 973). The ncrease n volume of vll wth age was greater n the jejunum and leum than n the duodenum, and n ths ntestnal regon a large quanttatve ncrease n absorpton occurs at 7 to 4 d posthatchng (Noy and Sklan, 995). The changes n vllus heght and enterocyte densty n ths regon were also correlated wth feed ntake as suggested by Moran (985) and Murakam et al. (992). Comparng the ntestnal structure of AA and L chcks, vllus absorptve surface Measurement Duodenum Vll heght, /tm permeter, /an Crypt depth, /tn Submucosa, /tm Enterocytes/vllus 2 Vlla/.l cm2<3 Jejunum Vll heght, /tm permeter, /tm Crypt depth, /tm Submucosa, /tm Enterocytes/vllus Vlla/.l cm* Ileum Vll heght, /tm permeter, /tm Crypt depth, /tm Submucosa, /tm Enterocytes/vllus Vlla/.l cm* at three stes n the ntestne ndcated that AA chcks had larger vll and more enterocytes then L chcks. The rate of growth of the vll wth age was generally smlar n AA and L chcks. However, t should be noted that the overall length of the small ntestnes n L chcks was some 70% of the AA chcks. Wth the methods used n ths study we are not able to quantfy n detal the convolutons of the mucosa, whch are pronounced n the proxmal small ntestne, and thus we cannot evaluate precsely overall ntestnal absorptve capacty. However, usng computerzed magng technques we have compared the structure and volume n the dfferent ntestnal regons and the dfferences n structure parallel the large dfferences n feed uptake between the two strans. In ths study we also examned enzyme secreton, dgeston, and small ntestnal TABLE 2. Morphologcal measurements n the duodenum, jejunum, and leum of Arbor Acres (AA) and Lohman (L) chcks 4 d d AA d d d L d SEM Age P Stran Wll heght was measured usng the lamna propra as the base, crypt heght as the depth of the nvagnaton between adjacent vll, and the permeter s the dstance from the base of one vllus to the adjacent vllus. Values are means ± SD from 0 adjacent vll. 2 Number of enterocytes were measured per sde of a longtudnal secton through the vllus. Values are means ± SD from 0 adjacent vll. 3 Number of vll/.l cm 2 n a horzontal secton through base of the vll of the area under examnaton. Values are means ± SD from 0 adjacent.-cm 2 areas

8 MORPHOLOGY AND ABSORPTION IN CHICK INTESTINE 629 passage tme n the two strans of chcks. We have recently shown that dgeston of fat and starch changed lttle between 4 and 2 d n broler chcks fed dets wth 6% added fat, whereas N dgeston ncreased durng the st wk (Noy and Sklan, 995). Smlar results were observed here for N and fatty acd dgeston n both breeds from 4 to 4 d posthatch. Secreton of some pancreatc enzymes to the duodenum per gram of feed ntake was hgh at 4 d after hatch n AA chcks and values, whch were smlar to those found n L chcks from 7 to 4 d. Starch dgeston was over 90% n AA chcks on 4 d, whereas L chcks dgested approxmately 80% starch at 4 and 7 d, and ths ncreased to 90% and above on Days 0 and 4. On the bass of the amounts of amylase secreted nto the duodenum n L chcks, ths dfference n starch dgeston was not surprsng. Small ntestnal passage tme was very dfferent n the two strans, wth dgesta n L chcks havng a 50% shorter stay n the ntestnes at 4 d of age. It s also possble n ths early stage of development that ths was too short a tme for complete starch dgeston. Passage tme or absorpton could not be drectly correlated wth the morphologcal changes observed. It would be expected that ncreased absorptve surface area would be at the expense of lumen volume. However, measurement of length and external dameter of the ntestne do not reflect the route of the dgesta through the ntervllus spaces. Thus, whereas the ntestne ncreased n length by 20 to 30% between 4 and 4 d and by 5 to 0% n external dameter, the throughput of feed ncreased some fve- to eghtfold. Passage tme changed by a much smaller magntude, thus the dgesta must have been forced nto more ntmate contact wth the absorptve surfaces, as, even accountng for the ncreased passage tme, two- to threefold more nutrents were dgested on Day 4. The absorpton of starch and N n the lght strans was lower on Day 4 then n the heavy brds but was smlar from Day 7. Thus, slower growth would be expected close to hatchng. Subsequently, however, wth smlar percentage dgeston n both strans, t appears that the absolute uptake of nutrents was determned by feed ntake. REFERENCES Assocaton of Offcal Analytcal Chemsts, 990. Offcal Methods of Analyss. 5th ed. AOAC, Arlngton, VA. Baranylova, E., and J. Holman, 976. Morphologcal changes n the ntestnal wall n fed and fasted chckens n the frst week after hatchng. Acta Vet. Brno 45:5-58. Bayer, R. R., C. B. Chawan, F. H. Brd, and S. D. Musgrave, 975. Characterstcs of the absorptve surface of the small ntestne of the chcken from day to 4 weeks of age. Poultry Sc. 54: Brd, A. R., W. J. Croom, Y. K. Fan, L. R. Danel, B. L. Black, B. W. McBrde, E. J. Esen, L. S. Bull, and I. L. Taylor, 994. Jejunal glucose absorpton s enhanced by epdermal growth factor n mce. J. Nutr. 24: Chambers, C, and R. D. Grey, 979. Development of the structural components of the brush border n absorptve cells of the chck ntestne. Cell Tssue Res. 204: Cook, R. H., and F. H. Brd Duodenal vllus area and epthelal cellular mgraton n conventonal and germ-free chcks. Poultry Sc. 52: Lm, S. S., and F. N. Low, 977. Scannng electron mcroscopy of the developng almentary canal n the chck. Am. J. Anat. 50: Moran, E. T., 985. Dgeston and absorpton of carbohydrates n fowl and events through pernatal development J. Nutr. 5: Murakam, H., Y. Akba, and M. Horguch, 992. Growth and utlzaton of nutrents n newly hatched chcks wth or wthout removal of resdual yolk. Growth Dev. Agng 56: Natonal Research Councl, 994. Nutrent Requrements for Poultry. 9th. rev. ed. Natonal Academy Press, Washngton, DC. Noy Y., and D. Sklan, 995. Dgeston and absorpton n the young chck. Poultry Sc. 74: Overton, J., and J. Shoup, 964. Fne structure of cell surface specalzatons n the maturng duodenal mucosa of the chck. J. Cell Bol. 2: Pnchasov, Y., and Y. Noy, 994. Early postnatal amylolyss n the gastrontestnal tract of turkey poults (Meleagrs gallopavo). Comp. Bochem. Physol. 06: Rose, R., C. L. Rose, S. K. Om, K. R. Forry, D. M. Durall, and W. L. Bgg, 99. Starch determnaton by perchlorc acd vs enzymes: evaluatng the accuracy and precson of sx colormetrc methods. J. Agrc. Food Chem. 39:2-. SAS Insttute, 986. SAS User's Gude. Verson 6 Edton. Cary, NC. Sklan, D., and O. Halevy, 985. Proten dgeston and absorpton along the ovne gastrontestnal tract. J. Dary Sc. 68: Sklan, D., D. Dubrov, U. Esner, and S. Hurwtz, 975a. 5 Cr-EDTA, 9 Y and 4 Ce as nonabsorbed reference substances n the gastrontestnal tract of the chcken. J. Nutr. 05: Sklan, D., S. Hurwtz, P. Budowsk, and I. Ascarell, 975b. Fat dgeston and absorpton n chcks fed raw or heated soybean meal. J. Nutr. 05:

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