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IntroductionThe immune system in birds may be regarded as being

intermediate in development between that of fish, amphi-bians and reptiles on the one hand, and of mammals on theother. Amongst the primitive features of the organization ofthe lymphoid system are the presence of collections of lym-phoid tissue scattered throughout the body, and the absenceof lymph nodes similar to those of mammals. The develop-ment of the immune system in birds is initiated duringembryogenesis but is not complete until weeks or monthsafter hatching. The primary immune organs, the thymus andbursa of Fabricius, are both present, and are populated bylymphoid tissue. The secondary immune organs, such as thespleen, caecal tonsils, Meckel's diverticulum, Harderian gland,and the diffuse lymphoid tissue of the gut and respiratory

systems are incomplete at hatching [33, 37]. The lack oflymph nodes in most avian species increases the relativeimportance of the spleen in disease resistance. In the pastdecade, the avian spleen has been frequently used in the studiesof avian ecology, parasitology and evolution to infer immunesystem strength in birds because functions of the embryonicand neonatal spleen are different [38]. Soon after the spleenprimordium emerges, it begins to function as a haemopoieticorgan [39]. After hatching, the spleen is a predominantlylymphocyte-producing organ [18, 22, 25, 37, 39]. The immunerole of the spleen in birds is similar to that of the mammals,but the contribution to tissue oxygen supply seems lessextensive; spleen storage of erythrocytes, for example, isunrecorded in birds [16]. Various studies related to themorphological structure and development of the spleen havebeen performed in some bird species, such as in chickens [1,

SUMMARY

The structure of the quail spleen at late embryonic stages and during thepost-hatching period was determined by light microscopy using differentstaining techniques. Whereas the spleen growth was limited compared to thebody growth in embryonic stages, the relative spleen weight increased 2.8fold during the first 4 weeks post-hatching in quail. At 10 days of egg incu-bation, erythropoiesis was predominant, but on the 11th day, granulocyteislets were formed around vessels and the ellipsoids appeared around thepenicilliform capillary on the 12th day. The capsule of Schweigger-Seidelsheath (CSSS) wrapped them on the 15th day and although they were notwell defined, the peri-arterial lymphatic sheath (PALS) and the peri-ellip-soidal white pulp (PWP) appeared on the 16th day. At hatching, the granulo-cyte islets disappeared and the white pulp was gradually colonized withlymphocytes according to the age. In parallel, the thickness of the CSSSincreased, especially after the 10th day post-hatching. In 30 day old quails,the PALS mainly consisted of small lymphocytes. The number of lympho-cytes was superior in the PALS areas than in the PWP in 60 days old quails.Plasma cells were found in the PWP and especially in the red pulp aroundpulp veins during the post-hatching period. In conclusion, the quail spleenundergoes structural changes throughout the pre- and post-hatching periods.

Keywords: Quail, spleen, hatching, ellipsoid, Schweigger-Seidel sheath, peri-arterial lymphatic sheath, peri-ellipsoidalwhite pulp.

RÉSUMÉ

Structure de la rate avant et après éclosion chez la caille (Coturnixcoturnix japonica)

La structure de la rate aux stades embryonnaires tardifs et après l’éclosion aété déterminée chez la caille par photomicroscopie en utilisant différentestechniques de coloration. Alors que la croissance de la rate par rapport àcelle du corps s’est avérée limitée dans les stades embryonnaires, le poidsrelatif de l’organe a été multiplié par 2.8 durant les 4 premières semainessuivant l’éclosion. A 10 jours d’incubation des œufs, l’érythropoïèse étaitprépondérante mais le 11ème jour, des îlots de granulocytes se sont formésautour des vaisseaux et les structures ellipsoïdes sont apparues autour descapillaires penicilliformes le 12ème jour. Au 15ème jour, elles étaient enveloppéespar une capsule, appelée la capsule de la gaine de Schweigger-Seidel(CGSS). Bien qu’encore peu clairement définies, les gaines lymphatiquespéri-artérielles (GLPA) et la pulpe blanche péri-ellipsoïdale (PBPE) étaientprésentes le 16ème jour. A l’éclosion, les îlots granulocytaires ont disparu etla pulpe blanche a été graduellement colonisée par des lymphocytes en fonctionde l’âge. En parallèle, l’épaisseur de la CGSS s’est accrue, essentiellementaprès le 10ème jour suivant l’éclosion. Chez les cailles de 30 jours, les GLPAétaient principalement constituées de petits lymphocytes. Les lymphocytesétaient plus nombreux dans les GLPA que dans les zones de pulpe blanchechez les cailles de 60 jours. Les plasmocytes ont été retrouvés dans la pulpeblanche mais surtout dans la pulpe rouge entourant les veinules durant lapériode après l’éclosion. En conclusion, la rate subit des modificationsstructurales avant et après l’éclosion chez la caille.

Mots clés : Caille, rate, éclosion, structure ellipsoïde,gaine de Schweigger-Seidel, gaine lymphatique péri-artérielle, pulpe blanche péri-ellipsoïdale.

Structure of the quail (Coturnix coturnixjaponica) spleen during pre-and post-hatchingperiods

N. LIMAN1*, G. K. BAYRAM1

1Department of Histology and Embryology, Faculty of Veterinary Medicine, University of Erciyes, 38 090, Kayseri, TURKEY.

*Corresponding author: narinliman@gmail.com or limann@erciyes.edu.tr

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26 LIMAN (N.) AND BAYRAM (G. K.)

6, 14-17, 19-21, 26-30, 35], ducks [10, 12], Amazon parrots[9], guinea fowls [11, 31, 32] and pigeons [24] and revealedstrong similarities but some originalities according to specieswere evidenced. The spleen is round to oval in gallinaceousbirds, ducks and psittacines but elongated in charadiiformesand Passeriformes. In proportion to the body weight, thespleen is smaller in birds than in mammals. The diameter ofa normal chicken spleen is approximately one quarter thelength of the proventriculus [13]. The trabeculae of the dove[24] and goose [12] spleens were poorly developed and thewhite and red pulps could not be distinguished from eachother as is also the case in the chicken spleen. The peri-arteriallymphoid tissue and the peri-venous lymphoid tissue havebeen referred to as the thymus-dependent element of whitepulp in chick spleen [15] while the duck peri-arterial lymphoidtissue has been designated as thymus-dependent, and theperi-venous lymphoid tissue as thymus- and bursa-independent,respectively [12]. Quails are more resistant to diseases thanchickens or other avian species, and the possibility of pro-duction without vaccination suggests that the quail’s immunesystem organs may exhibit some particularities.

The primary objective of the present study was to determinethe structural changes in the quail spleen throughout the pre- andpost-hatching periods. The second objective was to characterizethe growth of the spleen in quails after hatching, and the thirdobjective was to compare the structural characteristics of thequail spleen with chickens and other investigated avian species.

Materials and Methods

ANIMALS AND TISSUE PREPARATION

Fertilized eggs from quails (Coturnix coturnix japonica)were obtained from the Safiye Çikrikçioglu VocationalCollege, University of Erciyes (Kayseri), and incubated instandard incubators at 37.5 ± 0.3ºC, and in relative humidityof approximately 50-60%. Embryos were staged accordingto the number of incubation days (ED). Eggs were openedand embryos of different ages were collected from 10 to 16days of incubation. Ten embryos of each stage were weighedand decapitated and their spleens were immediately collec-ted and weighed. In addition, quails were obtained from theSafiye Çikrikçioglu Vocational College, University ofErciyes (Kayseri), on days 1–60 post hatching. Ten quails ateach of the following ages - 1, 2, 3, 5, 10, 30 and 60 days old- were killed under ether anaesthesia and their spleen werequickly removed and weighed. All the experiments describedfulfilled accepted national and international animal well-fareregulations. This study was approved by the EthicsCommittee of University of Erciyes (approval number 2010-7-65).

HISTOLOGICAL ANALYSIS

The spleen samples were fixed in formol-alcohol solutionfor 18 hours, dehydrated, cleared and embedded in paraffin.Serial sections, 6-µm thickness, were cut. After being depa-

raffinised and hydrated, the sections were stained with theCrossman’s triple technique to identify the general structureof spleen and to evaluate collagen synthesis [4], the Periodicacid Schiff (PAS), the James silver and the AldehydeFuchsine techniques for evidencing basement membranes,reticular and elastin fibres respectively [2], the Giemsa [8]and the Methyl Green-Pyronin [2] techniques for identifyingblood cells and particularly plasma cells. Tissue sectionsfrom different pre- and post-hatching days were examined byconventional light microscopy (BX51 Olympus, Tokyo,Japan).

IMMUNOHISTOCHEMICAL ANALYSIS

Furthermore, changes in B-cell and macrophage areaswithin the spleen during the post-hatching period were deter-mined by immunohistochemistry. Because specific antibodiestargeted to quail T- and B-cells and macrophages are notavailable, the CD79a/mb 1 antibody [Ab-1, (cloneHM47/A9, Thermo Fisher Scientific Lab VisionCorporation, Fremont, USA) at a dilution of 1:200] pre-viously recognized as a B-cell marker in humans, monkeys,cows, pigs, rabbits, guinea pigs, rats, mice and chickens [3]was used in this study with the hypothesis that it could alsobind to quail antigens. For the second antibody used in thepresent study, the macrophage marker CD68 [Ab-3 (CloneKP1, Thermo Fisher Scientific Lab Vision Corporation,Fremont, USA) at a dilution of 1:40] it is unknown if it isreactive for chicken or quail tissues.

Immunohistochemistry was performed at room temperatu-re according to the previously reported immunostainingprotocol of Thermo Fisher Scientific (Lab VisionCorporation, Fremont, CA, USA). Two slides were preparedfrom each sample, and each slide contained a minimum oftwo sections cut at 5 µm thickness. The tissue sections weremounted on glass slides coated with 3-aminopropyl-ethoxy-silane (APES) (Sigma- Aldrich Chemicals, St. Louis, MO,USA) and dried at 37°C. Briefly, sections were de-paraffinizedin xylene and rehydrated through a graded series of ethanol.To block any endogenous peroxidase activity or non-specificstaining, the sections were treated with 3% H2O2 in methanolfor 15 min at room temperature and washed with phosphate-buffered saline (PBS; pH 7.4) two times. Antigen retrievalwas performed in citrate buffer (pH 6) for 30 min at 95°Cwith cooling for 30 min before immunostaining. After that,the sections were washed in PBS (pH 7.4), incubated in bloc-king serum (Ultra V Block; TA-125UB; Thermo FisherScientific Lab Vision Corporation) for 5 min at room tempe-rature to block non-specific binding, and then left in the cor-responding primary antibody for 60 min. One series of slideswas incubated with a Mouse monoclonal antibody againstthe CD79a/mb 1 at a dilution of 1: 200. The second series ofslides was incubated with a mouse monoclonal antibodyagainst the macrophage marker CD68 at a dilution of 1:40.Thereafter, sections were washed in PBS, incubated withbiotinylated anti-mouse antiserum (Thermo Fisher ScientificLab Vision Corporation) for 20 min at room temperature, andwashed in PBS. Sections were incubated with streptavidinperoxidase for 20 min at room temperature, and washed with

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STRUCTURE OF THE QUAIL SPLEEN 27

PBS. The peroxidase activity was visualized with 3,3-diami-nobenzidine (DAB; Thermo Fisher Scientific Lab VisionCorporation) producing a brown colour. Sections were coun-terstained with Gill’s haematoxylin, dehydrated through analcohol series, cleared in xylene and finally embedded inEntellan® under a coverslip.

The specificity of immunohistochemical procedures waschecked by using negative and positive control sections. Aspositive controls, sections of human intestine, lymph nodeand spleen were incubated with primary antibodies. As negativecontrol, sections were incubated with PBS alone, without theprimary antibody. All samples were treated with exactly thesame protocol.

STATISTICAL ANALYSIS

Statistical means of the spleen weight and the spleenweight relative to the body weight were determined in eachage group. One-way ANOVA analysis was used to evaluatethe occurrence of differences between the age groups.Differences were considered significant at P < 0.05 unlessotherwise stated.

ResultsSpleen weights and the spleen weight / body weight ratios

in the pre-hatching (from the 10th to the 16th days of egg incu-bation) and post-hatching (from hatching to 60 days) periodswere determined. The average weights of spleen increasedwith the age (Table I) and differences between the pre- andpost-hatching stages were statistically significant (P < 0.05).Furthermore, the relative spleen weights (ratios ofspleen/body weights) in the foetal and the early post-hatchingperiods (1 to 2 days post-hatching) have greatly differedfrom the other age groups (P < 0.05) and they have signifi-cantly increased (P < 0.05) during the whole post-hatchingperiod.

PRE-HATCHING PERIOD

During the pre-hatching period, the spleen weight gradual-ly and weakly increased (P < 0.05) but in parallel the relati-ve spleen weight (spleen weight / body weight ratio) gra-dually declined according to time (Table I).

At 10 days of incubation, spleens were covered with meso-thelium. The sinuses were lined with flat endothelial cellsand blood vessels inserting into the capsule and arteries insi-de the parenchyma were rarely observed. The erythropoiesiswas more dominant than the granulopoiesis (figure 1).Thereafter, a relative increase in the granulopoiesis was obs-erved since the 11th day of incubation to the 14th day. The gra-nules of early granulocytes were stained with the aldehydefuchsine. The granulocytes circularly arranged around thevessels and formed islets (figure 2) which the number hasincreased until the day 14. Then, the number of granulocyteislets gradually declined to the 16th day.

On day 12 of incubation, the central artery and penicilliformcapillary were established. A single muscle layer was seenalong the entire length of the central artery and the endothelialcells rested on basal membrane which was stained with silverimpregnation and PAS stain. The penicilliform capillarieswere lined with cubic endothelial cells resting on a thin basementmembrane. The mesenchymal cells, which were ellipsoidalreticular cells, concentrically arranged around the penicilliformcapillary and they formed the ellipsoids or Schweigger-Seidelsheaths (SSS) (figure 3). It was observed that the number ofellipsoids increased with the embryonic age and their struc-ture was similar to those of adults. The large area of thespleen parenchyma was occupied with the ellipsoids at 16days of incubation. At 15th day of incubation, the wall of thetrabecular and central arteries were quite distinct: whereastrabecular arteries were bordered with numerous musclelayers, the wall of the central artery was constituted by a singlemuscle layer lined with flat endothelial cells on the basementmembrane. The basement membranes of both central arteryand penicilliform capillaries were stained with silver impre-gnation and PAS stain. Furthermore, some ellipsoids werewrapped by a novel discontinuous basement-membrane-like

Age (day) Spleen weight (mg) Spleen weight / body weight (%)11 0.612 ± 0.063 0.029 ± 0.00112 0.860 ± 0.102 0.028 ± 0.00213 0.888 ± 0.069 0.024 ± 0.00214 1.000 ± 0.100 0.021 ± 0.00215 -16 1.625 ± 0.070 0.019 ± 0.0011 2.220 ± 0.205 0.027 ± 0.0022 2.283 ± 0.205 0.027 ± 0.0025 8.844 ± 0.818 0.058 ± 0.00310 20.933 ± 7.938 0.059 ± 0.00430 99.866 ± 27.209 0.076 ± 0.00760 138.125 ± 30.073 0.067 ± 0.005

TABLE I: Spleen weights and the spleen weight / body weight ratios in the pre-hatching (from the 10th to the 16th days of egg incu-bation) and post-hatching (from hatching to 60 days) periods. Ten animals were sacrificed by day. Results are expressed as means± standard errors.

Pre

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28 LIMAN (N.) AND BAYRAM (G. K.)

structure, heavily stained with silver impregnation but notwith PAS stain (figure 4), corresponding to the capsule ofSchweigger-Seidel sheath (CSSS). Smooth muscle cells in asingle line were observed in the capsule on day 14.

On day 16, the peri-arterial lymphatic sheaths (PALS) andthe peri-ellipsoidal white pulp (PWP) were progressively

organized (figure 5). During the prenatal period abundantmitotic figures were seen in the spleen parenchyma. The redpulp with its numerous sinusoids was not distinct. Silverimpregnation revealed some reticular fibres in spleen, parti-cularly around the vessels at day 10 to 11 of incubation (figure6a), whilst the network of reticular fibres was determined onthe 12th day of incubation (figure 6b).

POST-HATCHING PERIOD

In the post-hatching period, the spleen weight dramaticallyincreased particularly at the 10th day until the 60th day (P < 0.05)leading to a marked increase of the relative spleen weight onday 30 (P < 0.05) (Table I).

At hatching, the capsule was composed of one or twolayers of smooth muscle cells. Trabecular arteries, veins andcentral arteries were quite obvious. With aldehyde fuchsinestain, the laminae elastica interna and the adventitia of thetrabecular arteries but not of the central arteries containedelastin fibres in 2-3 rows. The pale coloured ellipsoids werespread throughout the spleen; therefore, the small area of thespleen parenchyma was occupied with the PALS and PWP.The distribution of red pulp was sparse when compared with

FIGURE 2: Granulocyte islets (square), erythropoietic area (arrowheads)and sinuses (s) at the 14th days of incubation. Crossman’s triple stain.Bar = 100 µm.

FIGURE 1: Erythropoietic area (arrowheads) and granulocytes (arrows) atthe 10th days of incubation. Crossman’s triple stain. Bar = 50 µm.

FIGURE 4: The capsule of Schweigger-Seidel sheath (arrows) and thebasement membrane of penicilliform capillary (arrowheads) at the15th days of incubation. James silver stain. Bar = 20 µm.

FIGURE 3: Ellipsoid (circle), penicilliform capillary (Pc), mesenchymalcells (arrowheads) at the 12th days of incubation. PAS stain. Bar = 20 µm.

FIGURE 5: The peri-ellipsoidal white pulp (PE) and ellipsoids (circles) atthe 16th days of incubation. Crossman’s triple stain. Bar = 100 µm.

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STRUCTURE OF THE QUAIL SPLEEN 29

white pulp, however, the sharply distinguished areas of redand white pulp were not observed at hatching (figure 7). In1-2 days old quails, ellipsoids were similar to structures obs-erved at the end of the pre-hatching period. The granulocyteislets have disappeared and erythrocytes, less abundant, werenow located with some heterophils within the blood vesselsand sinuses. Mitotic figures were still present. Moreover, thespleen capsule contained some collagen and elastin fibres.

In 3-day-old quails, the ellipsoid matured and emerged atthe beginning of the penicilliform capillaries from the centralartery, including the branching area. Furthermore, the CSSScovered the entire length of the penicilliform capillary andended at the edge of the peri-ellipsoidal white pulp (figure 8)and the PWP exhibited a pale stained area which surroundsellipsoids. A weak increase in the number of lymphocytes inthe white pulp was seen. With silver impregnation it wasnoted that the content of reticular fibres was lower in thePWP than in the red pulp. Five days after hatching, sinusesopened to the pulp veins which opened to the trabecularveins and the white pulp increased compared to the red pulp.In 10 day old quails, the one or two ellipsoids, which may beconfluent with one another, were embedded in the PWP(figure 9) and the red pulp separated various groups of ellip-soids.

By contrast, 30 days after hatching, the expansion of theboth red and white pulp regions induced separation of ellip-soids. The PALS surrounding the central artery was mainlyconsisted by small lymphocytes, but few several medium-sized and large lymphocytes were also found. At this date,the amount of lymphocytes in the PALS was increased com-pared to 10 days after hatching. Sixty days after hatching, theamount of lymphocytes markedly increased in the PALSwhich consequently appeared darker. During the period of30-60 days post hatching, red pulp areas became more spa-cious and the sinuses of the red pulp were lined with flat andirregular endothelial cells. Plasma cells were observed in thePWP, but also and mainly in the red pulp around veins, andtheir number have gradually increased according to the quailage. On the 60th day post-hatching, the penicilliform capillarieswithin the ellipsoids were lined with 5-6 endothelial cellsthat bulged deeply into the capillary lumen. The ellipsoidalreticular cells were round or oval in shape and possessed a

euchromatic nucleus containing a nucleolus like the endo-thelial cells. During this period, the CSSS showed anilineblue reactivity similar to the basement membrane with theCrossman’s triple staining procedure, indicating the presen-ce of collagen (figure 10a). The ellipsoid extracellular matrixwas stained with PAS (figure 10b) and the endothelial base-ment membranes, covering the penicilliform capillaries,increased in thickness and also showed an intense PAS reac-tivity (figure 10c). The main structural changes in the tissueelements of quail spleen according to morphogenesis aresummarized in Table II.

Unfortunately, the results of immunohistochemical analysisin spleen of quails were negative. Therefore, the results forB-cells and macrophage could not be interpreted.

DiscussionThe present study revealed that the average weights of

spleen increased with the age (Table I) and differences betweenthe pre- and post-hatching stages were statistically significant(P < 0.05). The spleen weight increased 2.8 fold during thefirst 4 weeks of life in quail as reported in chicken [33].Furthermore, the relative spleen weights (ratios of spleen/body weights) in the foetal and the early post-hatching periods(1 to 2 days post-hatching) have greatly differed than in otherage groups (P < 0.05) and they have significantly increased(P < 0.05) during the whole post-hatching period. Theseresults indicated that the spleen growth continued after hat-ching in quails.

In contrast to chicken [26], erythropoiesis in quail spleenwas seen to begin before the 10th day of incubation [25].Although it has been reported that the granulopoiesis beganin the red pulp at 12 to 13th days of incubation in chicken [33,34] and that the arteries appeared during this period [34], thegranulocytes appeared around the arteries during an earlierperiod in quails than in chickens [33, 34] and they even formedislets surrounding arteries on the 10th day of incubation.According to the staining properties of granulocytes withaldehyde fuchsine [5] and Giemsa technique [8], these cellswere thought to be heterophils as reported for chicken

FIGURE 6: a. Reticular fibres (arrows) around the vessels (v) and erythrocytes (arrowheads) at the 11th days of incubation. b. Thenetwork of reticular fibres (arrows) around the vessels at the 12th days of incubation. James silver stain. Bar = 50 µm.

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30 LIMAN (N.) AND BAYRAM (G. K.)

FIGURE 8: The capsule of Schweigger-Seidel sheath (arrows) covered thepenicilliform capillary (Pc) and ended at the edge (arrowheads) of theperi-ellipsoidal white pulp (PE) on day 3 post-hatching. Ca: centralarteriole. James silver stain. Bar = 20 µm.

FIGURE 10: a. In 30 day old quails, aniline blue reactivity of the capsuleof Schweigger-Seidel sheath (arrowheads), of the basement mem-brane of ellipsoid (black arrows) and of the basement membrane ofcentral artery (white arrows). PA: peri-arterial lymphatic sheath, PE:peri-ellipsoidal white pulp. Crossman’s triple stain. Bar = 100 µm

FIGURE 7: Ellipsoids (circles), the peri-ellipsoidal white pulp (PE), theperi-arterial lymphatic sheaths (PA), central arteriole (Ca), trabecularartery (Ta) at hatching. Giemsa stain. Bar = 100 µm.

FIGURE 9: In 10 day old quails, the one or two confluent ellipsoids(arrows) were embedded in the peri-ellipsoidal white pulp (PE). PA:peri-arterial lymphatic sheaths, Ca: Central arteriole, Arrowheads:the edge of the peri-ellipsoidal white pulp. James silver stain. Bar =100 µm.

FIGURE 10: b. Sixty days post hatching, the penicilliform capillaries (Pc) in the ellipsoids, ellipsoidal reticular cells (EAC), the capsule ofSchweigger-Seidel sheath (arrows), basal membrane (arrowheads), s: sinus. Crossman’s triple stain. Bar = 20 µm. c. In 60 day oldquails, PAS reactivity of the basement membrane of endothelial cells (arrowheads), covering the penicilliform capillaries (Pc), of theCSSS (arrows) and of the extracellular matrix of ellipsoid. EAC: Ellipsoidal reticular cells, PE: Peri-ellipsoidal white pulp. PAS stain.Bar = 20 µm.

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STRUCTURE OF THE QUAIL SPLEEN 31

embryos [22]. In quails, the granulopoiesis was not onlyreduced but also completely ceased by day 1 after hatchingand this result is in agreement with findings of Onyeanusi[32]. Therefore, the spleen in the quail embryos would bealso considered as a haemopoietic organ, having erythro-poietic and granulopoietic roles as reported in other birds[18, 22, 25, 32, 34, 39].

The typical characteristic of the avian spleen is the well-developed ellipsoids known as Schweigger-Seidel sheath.Ontogenic studies have shown that the ellipsoids appeared inthe chicken spleen at 18 days of incubation [17, 26]. In thepresent study, the ellipsoids appeared in the quail spleen withthe accumulation of the mesenchymal cells around the peni-cilliform capillary at 12 days of incubation and the capsuleof the Schweigger-Seidel sheath was formed around theellipsoid on the 15th day of incubation. Ellipsoids containedtwo histologically similar cell layers as reported in chickenand guinea fowl [11, 19, 27, 30, 31], but the ellipsoid-asso-ciated cells could not be identified. No information about theappearance of the CSSS in spleen of any avian species hasbeen available. Therefore, this study appears to be the firstdescription of the presence of the CSSS in the spleen ofquails during the pre-hatching period. The presence of theCSSS could indicate that the ellipsoids matured in the pre-hatching period. This capsule, a fenestrated external laminaof collagenous and reticular fibres, is defined as the externallamina of the Schweigger-Seidel sheaths in hens byMIYAMOTO et al. [23] and this structure was also found inother species, such as Guinea fowl [11, 31]. The Schweigger-Seidel sheaths (SSS) appeared grouped in the spleen of quailas in Guinea fowl [11, 31] but these groups were separatedby a narrow red pulp zone. In quails, the penicilliformcapillaries were lined with cubic endothelial cells [36] reston a basement membrane, which the thickness increasedwith the age. Furthermore, the surface of SSS was sharplyoutlined like in guinea fowl, but not in chicken [31]. In thisstudy, the silver impregnation revealed that SSS emerged atthe beginning of the penicilliform capillary and ended atedge of the PWP. In addition, the basement membrane andthe CSSS were differently stained with the silver impregnation

and PAS techniques, indicating specific chemical and probablyfunctional properties of these structures, as previously des-cribed in guinea fowl [11]. Ten days after hatching, CSSSwas stained in blue like the basement membrane of penicilli-form capillaries with the Crossman’s triple technique, thereactivity to the aniline blue evidencing the presence of col-lagen I. GUMATI et al. [11] have also previously demons-trated the presence of collagen I in the guinea fowl spleen.The occurrence of collagen I may indicate postnatal maturationof ellipsoids and/or functional activity of the ellipsoid cells;indeed, it may be suggested that some ellipsoid cells producedcollagen during the post-hatching period. From the 10th dayof egg incubation to the 60th day post-hatching, the reticularfibre network in the quail spleen was similar to that observedin the chicken [7]. The silver impregnation has revealed thepresence of a dense reticular fibre network in the red pulpwhile this network was absent in PWP surrounding the ellip-soids and was poorly developed in the PALS areas, as pre-viously reported in guinea fowl [11].

Whereas no accumulation of lymphocytes was observed inthe embryonic chicken spleen [26], lymphocytes wereregrouped and accumulated for forming peri-ellipsoidalwhite pulp (PWP) and peri-arterial lymphatic sheaths(PALS) on the 16th day of incubation in the quail spleen.Onyeanusi [32] has reported that the red and white pulps for-med on day 19 in the guinea fowl, but he did not indicate thelocalization of PALS and PWP. Furthermore, Mast andGoddeeris [21] have informed that the PALS, the ellipsoidsand their macrophage ring appeared on the 20th day of incu-bation in chicken. On the other hand, OGATA et al. [26] hasstated that the PALS and PWP were constituted in 4 and 6days old chickens, respectively. In the present study, PALSand PWP emerged on the 16th day of incubation but thesestructures were not completely achieved at this date, and the-reafter they gradually matured in the first week of hatchingas reported in chicken [21].

In 1 day old quails, as ellipsoids were spread throughoutthe spleen, the distribution in the spleen parenchyma of thePALS, PWP and the red pulp was reduced. In 30 day old

Pre-hatching days Post-hatching days10 11 12 13 14 15 16 1 2 3 5 10 30 60

Erythropoiesis + + + + + + + - - - - - - -Granulopoiesis + + + + + + + - - - - - - -Central a. / Penicilliform cp. - - + + + + + + + + + + + +Ellipsoid - - + + + + + + + + + + + +CSSS - - - - - + + + + + + + + +PALS - - - - - - ± + + + + + + +PWP - - - - - - ± + + + + + + +Plasma cells - - - - - - - + + + + + + +Red pulp - - - - - - ± + + + + + + +

TABLE II: Histological changes in the quail spleen during the pre-hatching (from the 10th to the 16th days of egg incubation) and post-hatching (from hatching to 60 days) periods.

a.: artery; cp: capillary; CSSS: capsule of Schweigger-Seidel sheath; PALS: Peri-arterial Lymphatic Sheaths; PWP: Peri-ellipsoidal White Pulp; -: absent; ±: vaguely.

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32 LIMAN (N.) AND BAYRAM (G. K.)

quails, the white pulp was more abundant than the red pulp,indicating therefore that the spleen growth was not yet com-plete. In 60 days old quails, the white and red pulp regionswere observed in equal proportions as reported in the chickenspleen [13, 33]. These findings showed that the quail spleenis a lymphocyte producing organ at the post-hatching stages.As evidenced by the Methyl Green Pyronin and Giemsa stainingtechniques, lymphocytes more accumulated in the PALSareas than in the PWP areas. Furthermore, the plasma cellswere observed in the PWP and in the red pulp especiallyaround the pulp veins. These results confirmed the possibilitythat the PWP is functionally identical to the mammalianmarginal zone (MZ) which is considered as a distinct B-dependent region [21, 27]. Immunohistochemistry would berequired to identify cells in the PALPS and PWP areas and inthe red pulp and to investigate changes in the B cells andmacrophages localisation during the post-hatching period.Unfortunately, there is no available specific primary antibo-dies targeting quail antigens and preliminary assays using theCD79a/mb1 antibody recognized as a B-cell marker inhumans, monkeys, cows, pigs, rabbits, guinea pigs, rats,mice and chickens [3] and the macrophage marker CD68gave negative results.

In conclusion, the most significant features of the quailspleen development during the pre-hatching and post-hatchingperiods are the presence of well-developed Schweigger-Seidelsheath and the formation of the inherent capsule. Moreover,the structural differentiation of the spleen continues duringthe post-hatching period, which is the growing period andthe structural rearrangements during this period were similarto those observed in guinea fowl.

AcknowledgementsThis resarch (Project No: 00-50-11) was financially sup-

ported by Scientific Research Council of Erciyes University.

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