Introduction

Basic haematopoietic structures and mechanisms in fishes are similar to those operating in other vertebrates (Fijan 2002a, b). According to Kobayashi et al. (2007), in the ginbuna carp, and according to Chen and Zon (2004), in larval and adult zebrafish, all haematopoietic cell types were very similar to those of mammals. However, in mammals and other higher vertebrates all myeloid cells proliferate and mature in the bone marrow, while in teleosts there is more than one haematopoietic site (Ivanovski et al. 2009).

Homechaudhuri and Jah (2001) reported that in Cyprinus carpio and Oreochromis niloticus, the pronephric kidney (with spleen) functioned as main erythropoietic organ. The head (pronephros) (Stosik and Deptuła 2001) and trunk (mesonephros) kidney and the spleen are main lymphomyeloid organs, while the thymus gland and the gut-associated lymphoid tissue (GALT) are lymphoid sites (Stosik and Deptuła 1993). Liu et al. (2004) and Patel et al. (2009) suggested that the lymphoid organs in fish include thymus, spleen and head kidney. In some fishes, both haematopoietic organs function equally, whereas in others one is more active than the other. Catton (1951) found that in Salmo trutta, only the spleen showed haematopoietic activity, in Rutilus rutilus only the kidney, while in Perca fluviatilis both organs were active.

The head kidney is an organ in which erythrocytes and leucocytes, such as macrophages, granulocytes and B lymphocytes, develop and differentiate into teleosts Romano (1998). Esteban et al. (1989) reported that the head kidney of the sea bass is a source of erythropoietic and thrombopoietic cells (no significant lymphopoietic activity is found). Fijan (1961); Houston et al. (1996); Romano et al. (2002); Moritomo et al. (2004); Rombout et al. (2005) suggested that pronephros in fish is basic organ forming the blood elements, and it is also the reservoir of cells. Lange et al. (2000) noted that haemopoietic tissue containing blood precursor cells with a high proliferation rate was found in the following inner organs of sturgeon: pronephros, mesonephros, epicardium, spleen and the serosa of the middle part of the intestine.

However, little information is available about the haematopoietic stem or progenitor cells in kidney haematopoietic tissue (Kobayashi et al. 2006, 2007). There are several reports on histology and ultrastructure of head kidney tissue (Esteban et al. 1989; Diago et al. 1998; Meseguer et al. 1990; Romano et al. 2002; Liu et al. 2004; Travares-Dias 2006; Abdel-Aziz et al. 2010). There are also very few quantitative data on the proportions among blood cell lineages in haematopoietic organs of teleosts (Peters and Schwarzer 1985; Wlasow and Dabrowska 1989; Fijan 2002a, b).

It is known that the circulating blood cells are the good index of environmental impacts (e.g. toxic substances) or other stress. However, little data are available on the effects of environmental factors on haematopoietic tissue which should be particularly sensitive to various impacts due to its high rate of cell turnover. For that reason, the present study is aimed at identifying quantitative and qualitative blood cells in head kidney of common carp, an important aquaculture fish species.

Materials and methods

Ten juvenile healthy common carps (Cyprinus carpio) weighting 42.1 ± 10.2 g were used for the experiment in October 2008. Fish were transported from the hatchery of Inland Fisheries Institute in Zabieniec to the laboratory of Department of Animal Physiology in Siedlce for 2 h in plastic bags with pond water supplied with pure oxygen and allowed to acclimate to the laboratory flow-through tank, at the density of about 40 g/l, for over a month. Water temperature was 20–22°C, dissolved oxygen saturation level 70–80%, and pH 6.9. The fish were fed twice a day formulated Futura feed, at daily rate of 2% body weight.

The fish were killed by fast cutting the spinal chord just behind the head using sharp scissors. After opening of the abdominal cavity, head kidney was collected for the preparation of three smears from each of the organs. The surface of isolated fresh organs was blotted and then smeared gently on fat-free slides. After being dried for 24 h, smears were stained using May-Grünwald and Giemsa solutions. Then, the smears were viewed using light microscope (1,000× magnification), and the cellular structure of organs was determined. From each fish, 500 blood cells were identified, counted, and their relative abundance calculated. Cells were identified according to Fijan’s terminology (2002a). The results were expressed as percentages of the total number of blood cells present. Area of cells occupied by the nucleus was estimated visually. Fields with crowded, shrivelled, deformed, damaged or poorly stained cells were excluded. Cells of unclear characteristics were placed into the group of unidentifiable cells. Areas of kidney tissue with numerous erythrocytes (possibly blood vessel contents) were also ignored.

Results and discussion

Haematopoiesis is a complex process in which haematopoietic stem cells (HSCs), the most immature elements of the haematopoietic hierarchy, proliferate and differentiate into various classes of haematopoietic progenitor cells (HPCs) (Kobayashi et al. 2008; Katakura et al. 2009). These progenitor cells further have been shown to be able to differentiate into mature blood cells: erythrocytes, lymphocytes, thrombocytes, granulocytes and monocytes (Moritomo et al. 2004; Kobayashi et al. 2006; Kobayashi et al. 2007; Katakura et al. 2009).

In the present study, in juvenile common carp pronephros smears 22 blood cell types can be easily identified and counted (Table 1). The haematopoiesis is formed by the following series: unidentified blast cells, erythroid, granuloid, lymphoid, monocytoid and thrombocytoid (Fig. 1).

Table 1 Frequency of various haematopoietic cells in the head kidney of common carp (n = 10, mean ± SD)
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Fig. 1
figure 1

Diagram on morphogenesis of head kidney haematopoietic cells from common carp. – – uncertainty about a pathway of development (Fijan 2002a—modified)

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In subsequent morphological description, they are listed together with synonyms used by other authors (Catton 1951; Fijan 1961; Peters and Schwarzer 1985; Wlasow and Dabrowska 1989; Fijan 2002a, b; Kobayashi et al. 2006; Abdel-Aziz et al. 2010).

Blast cells

In the present study, the percentage of early blast cells was 2.10 ± 0.53%. It was similar to the values reported by Wlasow and Dabrowska (1989) in common carp (2.54%) and by Fijan (2002b) in channel catfish (2.84%). Blast cells (Fig. 2.1) were larger than mature cells. Cell shape varied from round to oval, similarly as reported by Fijan (2002a). The blast cells showed a large (usually round) nucleus which filled over half of the cell, as observed by Peters and Schwarzer (1985), and intensely blue cytoplasm.

Fig. 2
figure 2

Blood cells in head kidney: Blast cells (1), Erythroid lineages: basophilic erythroblast (2), polychromatic erythroblast (3), orthochromic erythroblast (4), young erythrocyte (5), mature erythrocyte (6); Granuloid lineages: neutrophilic progranulocyte (7), neutrophilic metagranulocyte (8), band neutrophil (9), segmented neutrophil (10), basophilic metagranulocyte (11), young basophil (12), basophil (13), eosinophils (14); Lymphoid cells: prolymphocyte (15), lymphocyte (16), proplasmacyte (17); Large agranular cells: promonocyte (18), mature monocyte (19), thrombocyte (20)

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Erythroid cells

In the present study, the percentage of erythroid lineage cells (13.14 ± 1.00%) was similar to the results obtained by Fijan (2002b): 13.0 ± 5.1% for channel catfish and considerably lower in relation to the results obtained by Peters and Schwarzer (1985), near 45% for rainbow trout and Wlasow and Dabrowska (1989) near 37% for common carp. Number of observed erythroid developmental stages was higher than numbers of developmental stages of any other lineage. The erythroid stages were similar as in sea bass (Esteban et al. 1989), in gilthead sea bream (Zuasti and Ferrer 1989), in tilapia (Abdel-Aziz et al. 2010), in common carp and Nile tilapia (Homechaudhuri and Jah 2001) and in other teleosts (Fijan 1961, 2002a).

The basophilic erythroblast (Fig. 2.2) was characterized by more coarse and granular chromatin, smaller, peripheral nucleus, smaller cell size and darker blue cytoplasm than the early blast cells (Fijan 2002a).

In this study, the polychromatic erythroblast was the most frequent erythroid cell (8.28 ± 0.95%), similarly as observed by Wlasow and Dabrowska (1989): 15.9 ± 6.43% and Fijan (2002b): 4.61 ± 3.18%. The cytoplasm of the polychromatic erythroblast (Fig. 2.3) was grey. Nucleus occupied about half of the cell similarly as observed by Peters and Schwarzer (1985). Nuclear chromatin was coarsely granular. Some early and intermediate cells were oval in shape. Late cells of this stage were round, the smallest among maturing erythroid cells.

The orthochromic erythroblast (Fig. 2.4) typically showed rosy-red cytoplasm and a round nucleus, more condensed than in the previous stage. The young erythrocyte (Fig. 2.5) was irregularly round to oval, larger than orthochromic erythroblast, with less condensed nucleus, and smaller than the mature erythrocyte, similarly as shown by Fijan (2002a). Mature erythrocyte (Fig. 2.6) was larger, more elongated and showed more intensely rosy-red cytoplasm, and more elongated nucleus when compared to the young erythrocyte.

During maturation (Fig. 1), the increasingly abundant cytoplasm gradually changed colour from blue to rosy red, and the relative nuclear size decreased, cell size gradually decrease to the stage of polychromatic erythroblast and then increased again, similarly as reported by Fijan (2002a).

Granuloid cells

Meseguer et al. (1990) and Berman et al. (2005) observed that the granulopoiesis in the head kidney of the sea bass was similar to that of higher vertebrates.

The neutrophilic progranulocyte (Fig. 2.7) was the most frequent granuloid cell (8.40 ± 0.97%), similarly as noted by Wlasow and Dabrowska (1989): 3.40 ± 1.62%, and by Fijan (2002b): 4.53 ± 1.53% (who counted together neutrophilic progranulocyte and neutrophilic metagranulocyte). It showed light bluish cytoplasm, a round and mostly eccentric nucleus which occupied about half of the cell. In the neutrophilic metagranulocyte (Fig. 2.8), the mostly eccentric nucleus was sometimes oval and occupied less than half of the cell Fijan (2002a). Small parts of the cytoplasm near the nucleus were sometimes still faintly bluish, but the rest was loaded with specific granulation. Some cells at this stage attained the largest size in the neutrophilic series. Neutrophilic progranulocyte and neutrophilic metagranulocyte stages matched the myelocyte and metamyelocyte as observed by Diago et al. (1998), and the promyelocyte and myelocyte reported by Wlasow and Dabrowska (1989). The neutrophil was characterized by ungranulated cytoplasm (Kobayashi et al. 2006) and a smaller, more compact nucleus. Its size decreased during ageing (Scharsack et al. 2003), and the nucleus could become band (Fig. 2.9) and then segmented (Fig. 2.10).

In the present study, the lineage of basophils was regularly present in kidneys (near 2%). Zuasti and Ferrer (1989) reported the cells of the basophilic series being very scarce. Concerning percentage of basophils, only Fijan (2002b) reported that they comprised near 1% of head kidney haematopoietic cells. Basophilic granulocytes showed a thin, barely distinguishable basophilic cytoplasmic mesh surrounded by unstained spaces and basophilic granulation. Nuclei of basophils were smaller and more condensed than those of neutrophils and located eccentrically. The rare basophilic young progranulocyte had foamy basophilic cytoplasm with scarce fine basophilic granulation. The slightly to completely eccentric nucleus of the basophilic progranulocyte occupied less than half of the cell and had a more condensed chromatin than the neutrophilic progranulocyte (Fijan 2002a). Basophilic metagranulocyte (Fig. 2.11) showed more elongated nucleus, darker foamy cytoplasm with more abundant granulation. Young basophils (Fig. 2.12) were the largest cells in the series and were characterized by the appearance of dense coarse basophilic granules, similar to azurophilic granules as observed by Abdel-Aziz et al. (2010). In the basophil which was the smallest cell of this lineage (Fig. 2.13), granulation occupied the whole cytoplasm. The nuclear membrane was not distinguishable, and the nuclear margin was often irregularly serrated due to superpositioned unstained granulation. According to Kobayashi et al. (2006), the basophil had a grey cytoplasmic mesh and a dense, small, round to oval or rarely bilobed nucleus. The size of basophils diminished during ageing, and the cytoplasmic mesh became less distinguishable.

In the present study, percentage of eosinophil cell lineage (Table 1) was similar to that reported by Fijan (2002b) for channel catfish and equalled only 0.20%. It was significantly lower compared to the data reported by Wlasow and Dabrowska (1989) for common carp (almost 40%). That eosinophilic granulocytes were the dominant granulopoietic series in the haematopoietic tissue of tilapia (Abdel-Aziz et al. 2010). Mature eosinophils (Fig. 2.14) were larger than neutrophils. The eccentrically located elliptical or lobed nucleus was small. The light blue to colourless cytoplasm presented relatively large rosy-red granules similar to eosinophilic granules as observed by Travares-Dias (2006). In the present study, precursors of this granulocyte were not encountered.

Abdel-Aziz et al. (2010) observed that granulopoietic series consisted of cells with variable shape and size at different stages of maturity from myeloblasts to mature granulocytes in the kidney of tilapia, Oreochromis niloticus. Maturation of granulocytes was formed by a reduction in cell and nuclear size, nuclear condensation and the appearance of secondary granules with a gradual increase in their number in mature granulocyte.

Lymphoid cells

In this study, lymphoid cells were the most abundant among all observed haemopoietic cell lineages (39.00 ± 1.86%) (Table 1). Data obtained by various authors on the percentage of lymphoid cells in the head kidney of the same fish species often differ dramatically. In the present study, the frequency of these cells in common carp was similar to the percentage of lymphoid cells in the head kidney of common carp reported by Fijan (1961) and by Fijan (2002b) for channel catfish, but much higher than that reported in the same organ and species by Wlasow and Dabrowska (1989): only 8.5%.

The prolymphocyte (Fig. 2.15) was the small cell, but larger than the lymphocyte (Fig. 2.16). It had pale blue cytoplasm. The amount of lymphocytes as well as their size and the colour of cytoplasm varied considerably. Similarly as reported by Peters and Schwarzer (1985), nucleus filled over three-fourths of the cell. The form of the nuclei ranged from round to deeply invaginated (Kobayashi et al. 2006). Lymphocytes observed in the present study were similar to those of four species described by Travares-Dias (2006): variously sized spherical cells, with basophilic cytoplasm and without granulations. In this study, the lymphocyte was the most frequent cell of the lymphoid lineage in the kidney (35.5 ± 2.26%), similarly to the findings by Wlasow and Dabrowska (1989), Quentel and Obcach (1992), Fijan (2002b) and Liu et al. (2004).

The proplasmacyte (Fig. 2.17) and plasmacyte had an eccentrically located nucleus and deep blue cytoplasm with small peripheral pale vacuoles surrounded by darker cytoplasm. The nucleus of the plasmacyte occupied about one-fourth of the cell (Fijan 2002a).

Monocytoid cells

The frequency of present monocytoid cells in the head kidneys (0.98 ± 0.45%) was similar to that reported by Fijan (2002b) for channel catfish: 0.91 ± 0.82.

Two types of large agranular cells were discernible. Promonocyte (Fig. 2.18) was slightly bigger then neutrophilic progranulocyte and showed medium blue cytoplasm. The nucleus occupied over half of the cell, similarly as observed by Fijan (2002a). The mature monocyte (Fig. 2.19) had a more abundant light blue cytoplasm and eccentric, a more compact, reniform or irregularly oval nucleus. Travares-Dias (2006) occasionally observed also horseshoe-shaped monocyte nuclei. Some monocytes showed different sizes and contained many vacuoles in the cytoplasm, which confirms the findings by Peters and Schwarzer (1985), Zuasti and Ferrer (1989) and Abdel-Aziz et al. (2010).

Thrombocyte

In the present study, less thrombocytes were found in the head kidney compared to channel catfish (Fijan 2002b). The thrombocytes (Fig. 2.20) were oval or irregularly round, predominantly smaller than the lymphocytes, with a central compact nucleus and a minimum of or no cytoplasm (Fänge 1994). In the present study, developmental stages were not identified.

Unclassified cells

Cells not matching any of the above-described lineage stages or not recognizable due to damage were categorized as unclassified.

The results of the present study and their comparison with data obtained by other authors indicate that cellular composition of head kidney haematopoietic tissue is quite similar in various fish species if the same cell classification criteria are applied. This is an advantage that makes possible interspecific comparisons and use of haematopoietic system as an indicator of environmental impacts. On the other hand, the differences may occur even within the same species if different methodologies and cell nomenclature are used. Such discrepancies indicate the need for the unification of nomenclature and the cell differentiation criteria in the study of haematopoietic organs.