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IntroductionThe avian pancreas is composed of the dorsal, ventral and

splenic lobes positioned between the ascending and descendingloops of the duodenum [12] and another lobe known as thethird lobe [14]. The pancreas is divided into 4 lobes in chickens[14] quails [20] and geese [10], whereas only 3 lobes areidentified in ducks and many other bird species [18].

The exocrine pancreas is composed of glandular epithelialcells named as acinus cells, and excretory ducts of small andlarge diameter. The cytoplasm of acinus cells is rich in roughendoplasmic reticulum (ER), mitochondria of the crystaltype, free ribosomes and secretory zymogene granules [17].Cells with euchromatic nuclei that are found in the lumen ofacini and named as centro-acinus cells do not exist in chickens,

but although infrequently, may exist in the starling [24] anddomestic goose [10]. Secretions of acinus cells are emptiedinto the duodenum via intercalated, intralobular, interlobular,and interlobar excretory ducts [7]. In birds, the main excretoryducts that open into the duodenum are reported to be the dorsaland ventral pancreatic ducts, and in certain other bird speciesanother third duct [3].

The endocrine pancreas of avian species is composed ofsmall and large islets that are named as alpha, beta and mixedislets [4, 24]. Alpha islets that are stained with silver are alsoknown as dark islets, whereas beta islets that are not stainedwith silver are also known as light islets [12, 16]. Alpha isletsthat are found in great numbers in the caput region of thesplenic lobe and third lobe [21] are composed of A, D and Bcells in chickens [12] and A, D and Type IV cells in quails

SSUUMMMMAARRYY

The structure and ultra structure of the pancreas was investigated in thisstudy in 20 healthy adult quails using histochemistry and electron microscopy.The quail pancreas inserted between the duodenal arms was divided into 4lobes (dorsal, ventral, third and splenic lobes) and was anatomically relied tothe ascendens duodenum by 3 main excretory ducts proceeding from theventral, dorsal and the third lobes respectively. The pyramidal secretory cellsexhibited a basal nucleus and a cytoplasm rich in rough endoplasmic reticulum(ER) and in zymogene granules and were regrouped into acini. Two cell types(dark and light acinus cells) were identified according to the profusion of ribo-somes and polysomes (dark type) and to the aspect of the reticulum cisterns(broadened vesicles in the light type or narrower structure in the dark type).The endocrine cells were amassed into α, β and mixed islets: the α islets weremainly constituted by A and D cells and by C and PP cells at a lesser extend,the β islets principally contained B cells and few D and A cells in periphery andthe mixed islets were characterized by the identification of all cellular types.The β islets were the most frequently found in the different lobes (from 53.23± 1.07% in the third lobe to 66.07 ± 0.93% in the ventral lobe), whereas theproportions of α- and mixed islets were weaker (from 26.20 ± 0.92% in thesplenic lobe to 35.00 ± 0.79% in the third lobe and from 5.03 ± 0.50% in theventral lobe to 11.77 ± 0.30% in the third lobe, respectively). However, α isletswere bigger and less abundant in the splenic lobe than in the other lobes.

Key-words: Quail, exocrine pancreas, endocrine panc-reas, electron microscopy.

RRÉÉSSUUMMÉÉ

Etudes en microscopies optique et électronique du pancréas de lacaille (Coturnix coturnix japonica)

Cette étude a visé à déterminer la structure et l’ultra structure du pancréaschez la caille (20 adultes cliniquement sains) par histochimie et microscopieélectronique. Leur pancréas, inséré entre les branches duodénales, est diviséen 4 lobes (lobes dorsal et ventral, 3e lobe, lobe splénique) et est anatomi-quement relié au duodénum ascendant par 3 principaux canaux d’excrétionissus respectivement des lobes ventral, dorsal et du 3e lobe. Les cellulessécrétoires de forme pyramidale présentent un noyau en position basaleainsi qu’un cytoplasme riche en réticulum endoplasmique granuleux (RER)et en granules de zymogène et sont organisées en acini. Deux types cellulaires(le type sombre et le clair) ont été identifiés en fonction de l’abondance desribosomes et des polysomes (cas du type sombre) et de l’aspect des citernesdu réticulum (dilatées dans le cas du type clair). Les cellules endocrines sontregroupées en îlots α, β et mixtes : les îlots a sont constitués principalementde cellules A et D, mais aussi de cellules C et PP ; les îlots β contiennent descellules B essentiellement et quelques cellules D et A en périphérie, et lesîlots mixtes sont caractérisés par la présence de tous les types cellulaires.Les îlots β ont été les plus abondants dans les différents lobes (de 53.23 ±1.07% dans le 3e lobe à 66.07 ± 0.93% dans le lobe ventral) tandis que lesproportions des îlots α et mixtes ont été plus faibles : respectivement, de26.20 ± 0.92% dans le lobe splénique à 35.00 ± 0.79% dans le 3ème lobe etde 5.03 ± 0.50% dans le lobe ventral à 11.77 ± 0.30% dans le 3ème lobe.Cependant, les îlots α ont été plus grands et moins abondants dans le lobesplénique que dans les autres lobes.

Mots-clés : Caille, pancréas exocrine, pancréas endocrine,microscopie électronique.

N. SIMSEK1* and B. ALABAY2

1 Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Atatürk, 25240, Ilıca-Erzurum, TURKEY.2 Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Ankara, 06110 Dıskapı-Ankara, TURKEY.

*Corresponding author: E-mail: nsimsek58@gmail.com

Light and electron microscopic examinationsof the pancreas in quails (Coturnix coturnixjaponica)

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199PANCREAS STRUCTURE IN QUAIL

FIGURE 1a and 1b: Anatomical connections of quail pancreas with duodenum. Descendens duodeni (DD), ascendensduodeni (AD), dorsal lobe (DL), third lobe (TL), ventral lobe (VL), splenic lobe (arrow), spleen (S), third lobeduct (1), ventral duct (2), dorsal duct (3), hepatic duct (4), bile duct (5). 1c-1d: Light micrograph ofpancreatic ducts. Intralobular duct (id), interlobular duct (ild), interlobar duct (ilbd), Triple stain. X 325.

FIGURE 2: Ultra-structures of acinus cells. 2a: nucleus (N), zymogene granules (Z), mitochondrion (m), granules ofmitochondrial matrix (arrow head), cistern of rough endoplasmic reticulum (bold arrows) X 20000. 2b: lumen ofacinus (L), centro-acinar cells (CAC), microvillus (arrows) X 4200.

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SIMSEK (N.) AND ALABAY (B.)200

FIGURE 3: Ultra-structures of light and dark acinus cell. Light acinus cell (LAC), dark acinus cell (DAC), cistern ofrough endoplasmic reticulum (arrow heads). 3a: X 3800. 3b: X 15400.

FIGURE 4: Identification of endocrine islets of the endocrine pancreas in quails by histochemistry. α islet (A), mixed islet(M), A cells (arrows), B cells (arrow heads). 4a: Triple stain. X 325. 4b: Aldehyde fuchsine stain. X 325.

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FIGURE 5: Identification of α and β islets by toluidine blue stain. 5a: The α islet. A cell (arrow heads), toluidine bluenegative cells (arrows). X 1000. 5b: The β islet. B cells (B), A cells (arrow heads). X 1100.

FIGURE 6: Ultra-structural analysis of the α islet of the endocrine pancreas in quails. 6a: The α islet. A cells (A), D cells(D), PP cell (PP), not granulated cell (C). X 3375. 6b: A cell. Nucleus (N), secretory granules (g), mitochondrion(m), endoplasmic reticulum (arrow heads), immature granule (bold arrows). X 16000.

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202 SIMSEK (N.) AND ALABAY (B.)

FIGURE 7: Ultra-structural analysis of the D and PP cells in α islet. 7a: D cell. A cell (A), D cell (D), round granules(arrows), rod-shape granules (arrow heads), heterochromatic area adhered to the nucleolus with a ring gem form(bold arrow). X 9500. 7b: PP cell. A cell (A), D cell (D), PP cells (PP), round granules (arrow), rod-shape granules(arrow head). X 9375.

FIGURE 8: Ultra-structural analysis of the β islet of the endocrine pancreas in quails. 8a: The β islet. B cells (B), D cell (D),A cell (A), empty granules of B cells (arrow heads). X 2400. 8b: B cell. Nucleus (N), mitochondrion (m), high elec-tron-dense granules (a) low electron-dense granules (b), electron-lucent cores granules (c), crystalline structures gra-nules (d). X 16500.

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PANCREAS STRUCTURE IN QUAIL 203

LLooccaalliissaattiioonn IIsslleett ttyyppeessαα ββ mmiixxeedd

Overall proportion 30.06 ± 3.68b 60.98 ± 5.71a 8.96 ± 2.82c

Repartition per lobeDorsal lobe 30.16 ± 1.03B 60.24 ± 0.76B 9.60 ± 0.55B

Ventral lobe 28.90 ± 0.84AB 66.07 ± 0.93C 5.03 ± 0.50A

Third lobe 35.00 ± 0.79C 53.23 ± 1.07A 11.77 ± 0.30C

Splenic lobe 26.20 ± 0.92A 64.37 ± 0.86BC 9.43 ± 0.85B

TABLE 1: Identification and repartition (in %) of the α, β and mixed islets of the endocrine pancreas according to the different lobesin 10 healthy adult quails. Results are expressed as mean ± standard deviation.

Different superscripts a,b,c in the same line indicate significant differences between proportions of the different islet types in the same pancreatic lobe.Different superscripts A,B,C in the same column indicate significant differences between proportions of the same islet type in the different pancreatic lobes.

[20]. Beta islets that are numerous in all lobes [21] are com-posed of BB and DD cells in chickens [16] and quails [20], andof A, B and D cells in geese [10].

Previous studies [3, 4, 18, 23] have reported the embryonicprogression and immunohistochemical, and electron micro-scopic structures of the alpha and beta cells in the fowl pancreas(generally, in chicken). The aim of this study was to analyzethe histological structure of the exocrine and endocrine pancreasby light and electron microscopy studies in adult quails andto determine the repartition of endocrine cells throughout thedifferent pancreatic lobes.

Material and Methods1. ANIMALS AND PANCREAS SAMPLES

Twenty healthy adult (6 month old) quails supplied frombreeding farms are used in this study. The quails wereslaughtered by exsanguinations under profound ether anaes-thesia and their pancreases were divided into the dorsal, ventral,third lobes and the splenic lobe. For light microscopic exa-mination, tissue samples taken from the caput, corpus andcaudal of each lobe of 10 birds were fixed in Bouin’s fixativefluid for 18 hours. Tissue samples taken for electron micro-scopic examination from the splenic lobe of pancreas of theother 10 birds were first subjected to pre-fixation in glutaral-dehyde-paraformaldehyde (pH 7.4) for 24 hours in accor-dance with the method described by KARNOVSKY [13],rinsed a first time with cacodylate buffer for three hours anda second time with a 1 % osmic acid solution for two hours.

2. HISTOLOGICAL ANALYSIS

The tissue samples fixed for light microscopic examinationwere passed through a standard alcohol dehydration-xylenesequence and embedded in paraffin, and 5µm thick serialsections cut from tissue blocks. The sections were stained bythe CROSSMON’s modified triple stain [5] for the examinationof the general histological structure of the organ and with thealdehyde fuchsine stain for demonstration of alpha, beta andmixed islets as well as of the A and B cells [11]. The locali-sation of the different islet types in the whole pancreas was

semi-quantitatively determined from five serial sections.Each islet type was counted in randomly 10 microscopicfields for each serial section (using a 10 X objective) then thearithmetic mean was calculated.

3. ULTRA-STRUCTURAL ANALYSIS

The tissue samples fixed for ultra-structural analysis, storedin a 0.5 % uranyl acetate for two hours, were passed throughgraded alcohols, propylene oxide, and embedded in aralditeM. Semi-thin sections slides were stained with toluidineblue-pyronine and sections with a thickness of 300-400 Åwere prepared and contrasted according to the method ofVENEABLE and COGGESHALL [22] then examined with theCarl Zeiss EM 9 S-2 model transmission electron microscope.

4. STATISTICAL ANALYSIS

The results of the repartition of the different islet typesthroughout the different pancreatic lobes were expressed asmeans ± standard deviations and were compared with the tstudent test. Differences were considered as significant whenp values were less than 0.05.

ResultsThe quail pancreas which is located between the ascending

and descending loops of the duodenum was determined to becomposed of the dorsal, ventral, third and splenic lobes(figures 1a -1b). The exocrine pancreas was demonstrated tocontain intercalated ducts lined with squamous epithelium,intralobular ducts lined with cubical epithelium, and interlo-bular and interlobar ducts lined with columnar epitheliumthat display one or two layers of muscle and a broad layer ofconnective tissue (figure 1c-1d). At the site where bile isemptied into the duodenum, in addition to the ducts originatingfrom the liver and the gall bladder, 3 main excretory ductsoriginating from the pancreas were determined to open sepa-rately into the duodenum (figure 1a). Glandular epithelialcells that compose the acini were demonstrated to be rich inhigh electron-dense zymogene granules with a diameter of0.2-1.2 µm, mitochondria of the crystal type and rough ER.

Furthermore, 2-3 small electron-dense granules were detectedin the matrix of the mitochondria (figure 2a). Centro-acinarcells that are infrequently found in quails were determined tobe flattened and to have irregular nuclei and microvillus inelectron microscopic examinations (figure 2b). Pyramidalacinus cells were determined to consist of light and dark acinuscells in quails (figure 3a). The ER of the dark acinus cellswas rich in ribosomes and their cytoplasm was rich in freeribosomes and polysomes. Moreover, the cisterns of the ERwere demonstrated to display a narrower structure. Bycontrast, in the light acinus cells, the ER was poor in ribosomesand cisterns displayed broadened vesicles (figure 3b).

Identification and repartition (in %) of the α, β and mixedislets of the endocrine pancreas according to the different lobesare shown in table I. The quail endocrine pancreas was mainlycomposed by beta islets (60.98 ± 5.71 %), then by alpha andmixed islets (30.06 ± 3.68% and 8.96 ± 2.82%, respectively).The endocrine islets were more frequently found in the splenicand the third lobes, whereas they were more scarcely observedin the ventral and the dorsal lobes (frequencies of α, β andmixed endocrine islets in different lobes: splenic lobe > thirdlobe > dorsal lobe > ventral lobe). Furthermore, they weremore abundant in the caput region of all pancreatic lobes thanin the corpus and tail (caudal) region. The β islets were alwaysmore numerous than the other types of islets in all pancreaticlobes (p < 0.05), their proportions ranging from 53.23 ± 1.07%in the third lobe to 66.07 ± 0.93% in the ventral lobe. Themixed islets were more scarcely found in the endocrine panc-reas, particularly in the ventral lobe (they represented only 5.03± 0.50% of the total islets) whereas they became more abundantin the third lobe (11.77 ± 0.30%) (p < 0.05). The proportions ofthe α type islet were intermediate, comprised between 26.20 ±0.92% in the splenic lobe and 35.00 ± 0.79% in the third lobe.The α islets were always bigger and less abundant in the sple-nic lobe than in the other pancreatic lobes, whereas they foun-ded more abundant in the third lobe (p < 0.05). The α isletswere stained in purple or in blue with the triple stain (figure 4a),in yellowish-green with the aldehyde fuchsine (figure 4b) andin blue with the toluidine blue (figure 5a). The β islets werestained in pink with the triple stain (figure 4a) and in purple-violet with the aldehyde fuchsine stain(figure 4b), whereas theywere not stained with the toluidine blue (figure 5b). The elec-tron microscopic examination revealed that A, D, PP and Ccells composed the a islets (figure 6a).

The A cells displaying euchromatic nuclei containednumerous round high electron-dense granules of varying size(with a diameter ranging between 400-550 and 150-250 nm),but a very scarce ER and few crystal type mitochondria inthe cytoplasm (figure 6b). The C cells, either containing 1-2granules or even not granulated, were generally found in theperiphery of the α islets (figure 6a). The D cells were mainlylocated in both the centre and the periphery of a islets andexhibited a very few number of granules. Their cytoplasmextensions, displaying no nucleus and located mostly in theround of capillary vessels were filled with oval (80-375 nm)and rod-shaped (150 x 400 nm) granules of large diameter(figure 7a). Moreover, a heterochromatic area with an electronlight centre that was adhered to the nucleolus in a formresembling to the gem of a ring was frequently observed intheir nucleus (figure 7a). Among cells that compose the α islets,

the PP cells presented an irregular nucleus and contained thesmallest granules mostly rod-shaped (with a diameter of 100 X250 nm) (figure 7b) and oval electron-dense secretory granules.

The β islets were composed by numerous B, a few D cellsand rare A cells located in the periphery of the islets (figure8a). Four or Five different forms of secretory granules witha diameter ranging between 250-450 nm (high electron-dense, low electron-dense, electron-lucent cores, crystallinestructures (figure 8b), and empty granules (figure 8a)) wereobserved in the cytoplasm of B cells. Generally, mixed isletswere consisted of connected α and β islets. Sometimes, asmall β islet has been settle dawn in the big α islet.

DiscussionThe quail pancreas is composed by 4 lobes (dorsal, ventral

and splenic lobes and a third lobe) like in chickens [18] andin geese [10], as previously described by RAWDON [18]. Inthe goose and in many other avian species, apart from thedorsal and ventral pancreatic ducts [9], a third excretory ductis also identified [3, 18] and glands are found in the laminapropria of the large excretory ducts in geese [9] and in cats[7]. In the present investigation, 2 main ducts originatingfrom the liver and the gall bladder respectively and 3 mainexcretory ducts originating from the pancreas were found toopen separately into the duodenum at the site where bile is emp-tied but the gland-like structures and Goblet cells previouslyreported were not be evidenced in the columnar epithelium andin the connective tissue of the excretory ducts in quails.

The acinus cells are rich in rough ER, free ribosomes, incrystal type mitochondria and in electron-dense zymogenegranules with a diameter ranging between 0.3 - 1.5 µm [15,17]. Cisterns of the ER, observed to be flattened in acinuscells in the resting period with electron microscopic exami-nation, are reported to become broadened in active acinuscells [19]. In this study, 2 types of acinus glandular cellswere identified in quails: light and dark types. Compared tothe light type, the dark acinus cells exhibited a heterochro-matin rich nucleus, a profuse rough ER, and numerous poly-somes in their cytoplasm. Moreover, the ER cisterns werecharacterized by a narrower structure. Consequently, in referencewith the studies of SAGLAM [19], the light acinus cellswere considered as functional cells whereas the dark acinuscells were quiescent cells.

According to histochemical staining procedures, A cellsare identified to be easily stained with acidophilic stains(phosphotungstic acid haematoxylin, Gomori's chrome alumhaematoxylin-phloxine stain), whereas B cells are well stainedwith aldehyde fuchsine, and D cells with aniline blue andwith light green [6]. In agreement with them, the β isletswere stained in purple with the aldehyde fuchsine in the presentstudy. In contrast with the findings of CULLING et al. [6],the A cells were also stained in blue and B cells in pink withthe triple stain.

Whereas the α endocrine islets are abundant in all pancreaticlobes in ducks [18], they are mainly located in the splenicand in the third lobes in chickens [21], quails [20], and geese

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[10]. In the present experiment, the proportions of α islets(small or big) ranged between 26 and 35% in all pancreaticlobes: the highest counts were effectively obtained in the thirdlobe but in the splenic lobe, the α islets were bigger and lessabundant than in the other lobes. By contrast, β islets werealways in majority whatever the pancreatic lobes in adultquails (from 53.23 ± 1.07% in the third lobe to 66.07 ±0.93% in the ventral lobe and 60.98 ± 5.71% for the wholeendocrine pancreas). Numerous A cells, few D [4] and scarceB cells [23] were reported to compose the α islets in birds.In the present investigation, they were constituted by A, D,PP and C cells and B cells were found to be totally absent.While spaces have been reported to exist between the granularmembrane and granular content in A cells from chickens [1],granules were completely filled with secretory material inquails, and immature granules contained a structure similar tothat referred in the findings of ALUMENTS et al. [1]. The Dcells which are more frequently encountered in birds than inmammals [16] are located in the periphery of the β islets andscattered throughout the α islets in geese [10] and in quails[20]. The present study was in agreement with that since theywere found in the β islets (periphery) and within the whole αislets (center and periphery). Although D cells are mainlyabundant in the ventral lobe, these cells are least count in thesplenic lobe of pancreas in pigeons and in quails [20].Nevertheless, GÜLMEZ et al. [10] have reported that D cellsare the preponderant cellular type throughout the pancreas ingeese; the relative frequency of D cells in the quail pancreaswas inferior to the A and B cell frequencies in the presentstudy. The last type of cells found in the a islets cellular type,PP cells, were firstly named as X or F cells [8], then as TypeIV cells in quails [20], and finally as pancreatic polypeptide(PP) cells according to their hormonal secretions [2, 8].These PP cells which are the more prevalent endocrine celltype in pancreatic parenchyma of chickens contained smallerand round granules compared to those of the D cells [2]. Inquails, they differ from the D cells because they mostlycontained rod-shaped and concave granules [20]. In the presentexperiment, the cellular characteristics of the PP cells were inagreement with these previous reports, but PP cells were notobserved in the pancreatic parenchyma of the adult quails.

In the avian species [4, 16], a majority of B cells with fewD cells composed the b endocrine islets, although SMITH[20] in quails and GÜLMEZ et al. [10] in geese have reportedthe presence of some A cells in their periphery. In compliancewith SMITH [20] and GÜLMEZ et al. [10], some A cellswere also identified in the periphery of the β islets with Dcells in the present study. Pin-like crystalloid granules androd-shaped granules (diameter: 315 nm) were evidenced in Bcells of chickens [1, 23]. Furthermore, high electron-dense,electron-lucent cores and crystalline structures granules werealso observed in the B cell cytoplasm in quails [20]. Theultra structural analysis presented here reveals that B cellswere particularly rich in various secretory granules, probablycorresponding to the different steps of the maturation andpackaging of the insulin. Whereas the mammalian endocrinepancreas is essentially constituted of mixed islets (A and PPcells in the periphery, B cells in the centre, and D cells), 3types of islets (α, β and mixed) were clearly identified in theendocrine pancreas of birds [14]. Although GÜLMEZ et al.

[10] have not detected mixed islets in the geese, the presentlight microscopic examinations revealed their presence inthe endocrine pancreas of the adult quails.

In conclusion, the quail pancreas is divided into 4 lobes(the dorsal, ventral, third and splenic lobes) and 3 excretoryducts opening into the duodenum proceed directly from thedorsal, ventral and third lobes, respectively. The exocrinepancreas is composed by dark and light acinus cellscorresponding to quiescent and functional cells respectively.The endocrine pancreas consists in α, β and mixed islets,whose the cellular composition is relatively specific: the αislets are mainly composed by A, D, PP, and C cells; B cellsin majority with some peripherical D and A cells form the βislets whereas in the mixed type, all cell types is identified.Although the α islets are easily stained by the acidophilicstains included in the triple stain, their purple to blue stainingwould be probably related to the great prevalence of D cellsthat have affinity to the aniline blue.

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