EFFECTS OF TRANSPORT AND HOLDING STRESS ON PRUSSIAN CARP (Carassius gibelio, Bloch, 1782.) LEUKOGRAM PATTERN

Stress inevitably occurs during any ﬁ sh handling and manipulation in culturing, research, or clinical examination situations that require capture and removal of ﬁ sh from water. Different stress factors can affect the changes in the relative numbers and function of cells of the ﬁ sh immune system. Catching, transportation and over-crowding caused stress-induced changes in the total number of leukocytes and thrombocytes, as well as changes in the leukocyte formula in Prussian carp ( Carassius gibelio , Bloch, 1782) presented here. Cytochemical characterization of leukocyte cell types was performed by applying Myeloperoxidase (MPO), Periodic Acid Schiff (PAS) and Sudan Black B (SBB) staining of Prussian carp blood smears. Cytochemical characterization is a rapid and ef ﬁ cient method for white leukocyte differentiation and insight in their functional status. Comparison and analysis in Prussian carp hematological parameters from ﬁ sh with and without exposure to stressful conditions such as capture, manipulation, transport and holding, revealed signi ﬁ cant differences between stressed and non-stressed ﬁ sh. Signi ﬁ cant reduction in the total number of thrombocytes and lymphocytes and the increase in total neutrophil count were observed in stressed animals. However, differences in total leukocyte number and the number of monocytes were not observed. Deviations from the estimated reference intervals for Prussian carp hematological parameters clearly indicated the presence/absence of a stress reaction and to some extent its intensity. Estimated reference intervals and characterization of morphological and cytochemical appearance of blood cells form a solid basis for further research of the cellular immune function and hematology changes in Prussian carp.


INTRODUCTION
Intensive production technology is the dominant approach in modern aquaculture.However, together with industrial and communal wastewater, and runoff from agricultural areas, effl uent from intensive aquaculture can contribute to increases of pollution in open waters.Therefore, there is a need to consider the infl uence of the polluted environment as a stress factor on fi sh health.Conditions of acute or chronic stress can cause severe consequences, including decrease in production parameters and deteriorated health with increased mortalities.Fishes sensitivity and susceptibility to environmental stress is high compared to terrestrial animals due to the very close relationship with the water in which they live.It is known that in well-managed ponds mortalities due to acute stress are very rare.It is also known that chronic stress can be responsible for the increased susceptibility to diseases, metabolic changes and changes in energy utilization leading to reduced growth, suppression of the immune response, and inhibition of gonadal maturation and ovulation [1].
In European warm water pond aquaculture, the Prussian carp (Carassius gibelio, Bloch, 1782) is often present as an accompanying species.Their natural habitats are still waters and lowland rivers, and ecologically they are tolerant to pollution and low oxygen concentrations.Presently, it is the most dominant fi sh species in lentic and slow running aquatic habitats [2].Prussian carp has been shown to both have resilience and be responsive to different external factors that can lead to increased stress.Such a combination of eco-physiological characteristics indicates the potential for this species to be used as a model for environmental contaminant effect on fi sh populations [3].However, the availability of literature data on the values of hematological parameters, mechanisms of stress responses to various factors and their connections with immune responses of Prussian carp are limited [3].
Hematological methods are relatively simple and effective tools used in fi sh health assessment.Processing and analysis of blood samples can be routinely performed in veterinary and research laboratories.Analytical approaches in routine clinical evaluation of blood smears (cell morphology and count) do not require specialized equipment other than a staining station and a microscope.However, expert knowledge of the hematology of the given species, as well as at least some information on the base reference values of the observed parameters is mandatory.While leukograms can be used to indicate the presence of a disorder, in this case stress, there are defi nite limitations of the technique in the determination of the underlying cause of the observed abnormal fi ndings.The presence of acute, or sometimes chronic, stress can be detected in fi sh by monitoring changes in the values of hematological parameters.Acute stress in fi sh and other animals is known to cause neutrophilia and lymphopenia.Functional consequences of stress on leukocytes include inhibition of phagocytosis, reduction in the ability to synthetize antibodies and inhibition of immunological memory formation.The consequences of stress can be observed for a few days after the removal of stressors [4].
Differences in total white blood cell counts can be attributed to many factors, both biotic (such as age, gender, sexual maturity, pathogens) and abiotic (water temperature, pH, dissolved oxygen content, season).Technical issues with the slide preparation, staining methodology, and experience in counting can also interfere with the standardized evaluation of fi sh blood smears.Characterization of fi sh leukocytes, especially granulocytes, has not yet been systematized, and with interchangeable use of morphological versus functional characteristics to defi ne different cell types, it may lead to confusion.Furthermore, with signifi cant biological end evolutionary diversity of over 34,000 fi sh species [5], it is diffi cult to develop a classifi cation system to accommodate the variability in the shape of the leukocyte, forms and function.Some species have only neutrophilic granulocytes and others only heterophils, while in a few species both cell types can be present [6], reinforcing the need for experienced evaluator and use of cytochemical stains to further delineate clinically relevant morphological and functional characteristics of white blood cells.
Classifi cation of fi sh leukocytes was frequently performed using criteria related to mammalian leukocytes [7] and several studies indicated that many fi sh leukocytes have morphological and functional similarities to their mammalian counterparts [8][9][10].Within the above-mentioned limitations, leukograms are used in the clinical assessment of health status and stress in fi sh [11].As fi sh leukocytes show variable morphology [12], additional characterization is often required to support accurate classifi cation of blood cell types prior to clinical evaluation and determination of potential for function testing.In such cases, rapid cytochemical staining kits enable the precise identifi cation and differentiation of fi sh leukocytes based on the presence of certain enzymes in the granules and structural components of the organelle membranes [13].The purpose of the present study was to characterize the changes in leukogram of Prussian carp exposed to acute stress, using cytochemical stains to improve the accuracy of blood smear counts of selected cell types (granulocytes, lymphocytes and thrombocytes).

Ethical approval
The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.The experiment was approved by the Ethics Committee of the Faculty of Veterinary Medicine, University of Belgrade and the Ministry of Agriculture, Forestry and Water Management -Veterinary Administration of the Republic of Serbia, decision No. 323-07-01116 / 2023-05 / 1.

Fish and blood sampling
Prussian carps (Carassius gibelio, 20 individual fi sh) were caught by netting and angling for health examination by licensed veterinarians.Veterinary health inspections of the ponds and the behavior of the fi sh were carried out during sample collection.The fi sh were anesthetized using buffered solution of tricaine-methane-sulfonate (pH 7.4; MS-222, Sigma, St. Louis, Missouri, USA) at a concentration of 75 mg/L for 2 minutes.Blood samples were collected from all sampled and anesthetized fi sh by caudal vein puncture immediately after capture.Pasteur pipettes (Ø 6x150 mm, Sodalime-glass) with anticoagulant lithium heparin were used for blood collection from the caudal artery and vein.Blood smears were immediately prepared in triplicate for each type of staining, air-dried, stored in a box to prevent exposure to light and kept at 4 o C until staining (<72h).The procedure of blood sampling and making blood smears lasted <2 minutes per fi sh.Fish were transported live to the Laboratory for fi sh diseases, Faculty of Veterinary Medicine, University of Belgrade, basic morphological parameters of the examined specimens were measured (total length, weight) and fi sh placed in aquaria with strong aeration, simulating prolonged transport conditions.After 24 hours (next day), blood was again collected from the same individuals for the preparation of new blood smears as described above.Following anesthesia for blood collection, fi sh were killed by an overdose of anesthetic (1 g L -1 of buffered tricainemethyl-sulfonate solution, MS222, Sigma, Germany) followed by a sharp blow on the head [14].
Diff Quick ® staining was used to determine morphological characteristic of the leukocytes on the blood smears.The following cytochemical kits were used to characterize and differentiate the granulocytes of Prussian carp: myeloperoxidase (Sigma, kit N o 391A), Sudan Black B (Sigma, N o 380-B) and Periodic acid-Schiff (Sigma 395 B).Staining was performed as per manufacturer's instructions with modifi cations as per Palić et al. [13].Stained blood smears were examined using bright fi eld and differential interference contrast microscopy (400-1000 magnifi cation).Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of specifi c chromogens.Presence of lymphocytes, monocytes, thrombocytes and granulocytes was determined.

Absolute leucocyte number and leucocyte formula
The total number of white blood cells, the total number of thrombocytes and the leukocyte formula were determined on blood smears stained with the Diff-Quick ® method, in the areas of the blood smear where cells only touch each other, without overlap and blank spaces.The total number of leukocytes and thrombocytes was determined by bright fi eld microscopic examination of the blood smear under 400 X magnifi cation.Leukocytes and thrombocytes were counted in 10 fi elds of view.The total count was divided by total number of fi elds of view used and multiplied by 2000 [15].Determination of the leukocyte formula was performed on blood smears, observed under a microscope with a magnifi cation of 1000 X. Minimum of 200 leukocytes were counted on the smear, recording each type of leukocyte separately, with special attention to differentiate thrombocytes from small lymphocytes (Figure 1).Calculation of the absolute number of individual types of leukocytes was determined from the total number of leukocytes and their differential ratio in percentages using the following formula: total number of leukocytes /mL blood x % one type of leukocyte 100

Statistical analysis
Statistical analysis of the obtained data before and after stress was performed using paired t-test in GraphPad Prism version 8.00 (GraphPad Software, USA) and in MS Excel.

Clinical and pathomorphological examination of fi sh
During the clinical examination carried out during fi sh collection non-specifi c symptoms of disease such as gathering of fi sh at the water inlet/outlet, lethargy, swimming in unnatural positions, resting at the pond bottom or rapid fatigue, were not observed.According to the anamnestic data collected, the fi sh had a good appetite and were within expected production parameters.Mean total length was 172 ± 33 mm and mean weight 118 ± 71 g.All captured fi sh survived the transport to the laboratory.External examination of fi sh arriving at the laboratory revealed no obvious signs of illness (wounds, visible secondary bacterial and fungal infections, and cachexia).Necropsy of the specimens after second blood drawing did not reveal any gross signs of disease.Based on the collected information fi sh were healthy and in good condition.

Cytology of blood leukocytes observed with Diff-Quick staining
On smears stained with the Diff-Quick staining method, it was possible to distinguish white blood cells based on the shape and size of the cells, their color, relative size ratio of the cytoplasm and nucleus, and the presence/absence of cytoplasmic granules.
Prussian carp neutrophils describe as round and relatively large (9-12 μM) with moderate nuclear to cytoplasm ratio and cytoplasm containing fi ne neutrophilic granules.The nucleus is kidney-shaped, sometimes segmented with a moderately coarse nuclear chromatin.Monocytes are round in shape, with basophilic cytoplasm that sometimes contains vacuoles.The nucleus is large, euchromatic in appearance, usually round and placed eccentrically.Lymphocytes in Prussian carp are round and relatively small (7-9 μM) cells with basophilic cytoplasm.The nucleus is round and occupies almost the entire cytoplasm.Thrombocytes are predominantly spindle-shaped, but it was also possible to observe some round-shaped cells with a nucleus that follows the shape of the cell (Figure 1).

Cytological characteristics of leukocytes subjected to specifi c staining
Examination of blood smears stained with MPO and PAS method and Sudan Black B dye under 1000 X magnifi cation with the use of immersion oil revealed the following cytochemical characteristics of Prussian carp white blood cells:

Myeloperoxidase method
Neutrophilic leukocytes stained with the MPO method show brown-black granulation because they have the enzyme myeloperoxidase in their cytoplasmic granules.Monocytes stained by this procedure stain weakly positive.Lymphocytes and thrombocytes did not show myeloperoxidase activity as evidenced by the negative MPO staining (Figure 1).

Periodic acid Schiff method
PAS staining indicated the presence of glycogen in neutrophil leukocytes.The color of their cytoplasm ranged from purple to red, while the nucleus is colored blue.Prussian carp monocytes contain a signifi cantly smaller number of glycogen granules in their cytoplasm as suggested by the weakly positive reaction during PAS staining.Prussian carp lymphocytes do not contain glycogen granules, supported by negative reaction during PAS staining.Prussian carp thrombocytes contain a signifi cantly smaller number of glycogen granules in their cytoplasm, giving weakly positive reaction during PAS staining (Figure 1).

Sudan Black B method
Sudan Black B staining reveals that membrane of neutrophil granules, and neutrophil leukocytes show blue-black intracellular granulation.Monocytes, lymphocytes and thrombocytes of Prussian carp do not stain with Sudan Black B dye (Figure 1).

Stress leukogram in Prussian carp
Total differential leukocyte counts were analyzed with paired t-test comparing leukocyte counts before and after stress.Total number of leukocytes between Prussian carp that were not exposed to stress factors compared to those exposed to stress was not signifi cantly different (p>0.05), which indicated that the leukopenia (reduction of the total number of leukocytes, not including thrombocytes) in Prussian carp that were exposed to stress factors is not readily observable.However, contrary to this fi nding, the same test proved a statistically signifi cant decrease in the total number of thrombocytes (signifi cant thrombocytopenia) in Prussian carp who were exposed to stress, compared to those who were not exposed to stress factors (p < 0.01) (Figure 2).
The absolute number of neutrophil leukocytes in Prussian carps that were not exposed to stress was signifi cantly lower compared to stressed fi sh (p < 0.01), indicating acute neutrophilia in Prussian carps exposed to stress factors.Concurrently, statistically signifi cant lymphopenia in Prussian carp was observed in stressed fi sh (p < 0.01).Monocytes showed no statistically signifi cant change between Prussian carp that were not exposed to stress factors compared to those exposed to stress factors (p>0.05)(Table 1).

DISCUSSION
Cytochemical staining kits often target the activity of intracellular/intragranular enzymes or membrane building components such as glycolipids or lipids, therefore staining results can provide an insight into the components of blood cells and give us evidence of their function status and differentiation stages, which is especially vital in fi sh immunology research [16].
Myeloperoxidase is a crucial component in the catalytic reaction involved in the production of ROI (reactive oxygen intermediates), specifi cally hydrogen peroxide from superoxide, which plays an important role in early innate clearance of bacterial pathogens [17].The examination of peroxidase reactivity of leukocytes was carried out in different fi sh species [4,18,19].Among myeloperoxidase (MPO) detection in blood cells of these fi sh species, most neutrophils primary granules were MPO positive, supporting their primary phagocytic and killing role in innate immune responses.The cytochemical fi ndings in the study of phagocytes implied that upon phagocytosis, the peroxidase in the phagosomes was part of a microbicidal system operating as a host defense mechanism against bacterial infection.Researchers believe that the presence of peroxidase indicates the potential for measuring oxidative bursts and degranulation in neutrophil primary granules, such as those in Arctic grayling, cutthroat trout and June sucker [4].
The positive fi nding of myeloperoxidase staining of neutrophil leukocytes in our research confi rms fi ndings in other fi sh species [9,12,13,20,21] and demonstrates very similar cytochemical characteristics to mammalian neutrophils.
A positive fi nding of myeloperoxidase staining is not exclusively related to neutrophil leukocytes, but can also be seen in monocytes, although it is signifi cantly less intense.
Monocytes can have a higher level of peroxidase activity [22], when the number of neutrophils is also increased [23].In our research, Prussian carp monocytes were not or barely visible positive on MPO staining.
Periodic acid-Schiff (PAS) and Sudan Black B (SBB) staining are an indication of glycogen in the cellular organelle membranes (e.g. in granules).In fact, except glycogen, glycoproteins and polysaccharides are also observed by PAS reaction.Glycogen in cells is believed to be an energy source for both aggregatory and phagocytic abilities [24,25].
Neutrophils in human and mammals are mainly involved in the phagocytosis and degradation of invading microorganisms [16,26,39].Strongly positive reactions for SBB and PAS indicated that the neutrophils of Prussian carp are similar to those of mammals, which have strong phagocytic and bactericidal abilities.
Monocytes of tilapia [22], Murray cod [25], S. prenanti [28], and piebald naked carp [29], were positive for PAS but negative for SBB staining, indicating that fi sh monocytes have phagocytic and antigen-presenting functions.In our study monocytes were only weakly positive and also SBB negative (Figure 1).
Thrombocytes are described as the most abundant blood cells after erythrocytes, and the thrombocyte count is frequently used as an indicator of health status because they are mainly involved in forming defensive barriers [33].Fish thrombocytes participate in hemostatic processes and in the immune response [34,35].There are no clear tendencies of changes in the total number of thrombocytes in fi sh exposed to different stress factors.Under the infl uence of various stress factors, blood coagulation is often accelerated, but this is not always accompanied by an increase in the total number of thrombocytes.The decrease in the total number of thrombocytes in Prussian carp exposed to stress is observed [23].Contrary to this fi nding, different stress factors signifi cantly affect the increase in the number of thrombocytes in the peripheral blood of channel catfi sh [24].
Studies have investigated whether thrombocytes in some fi sh have phagocytic abilities [24,25,29,32,36].However, the mechanism for killing pathogens after phagocytosis remains unknown [37].Some researchers believe that fi sh thrombocytes have a nonenzymatic mechanical phagocytic ability because no enzyme activity was detected in thrombocytes in Murray cod [25].The presence of MPO was closely related to the ability of cells to produce respiratory bursts.
Glycogen in thrombocytes represents a source of energy for them and those thrombocytes play a role in phagocytosis of foreign substances.This process requires the consumption of energy, both from endogenous and exogenous sources [38].
In thrombocytes of channel catfi sh and goldfi sh, the reaction of blood cells in PAS staining was weakly positive [24], while in rainbow trout and Pacifi c salmon it was negative [10].
We have demonstrated a weakly positive reaction of thrombocytes in PAS staining in the Prussian carp, which is in accordance with the fi ndings in the goldfi sh.
There are differences in the fi ndings of PAS and MPO staining even in fi sh species belonging to the same family, thrombocytes in grass carp were positive in both the PAS reaction and MPO, and the crucian carp's thrombocytes were positive for the PAS reaction but were negative for MPO activity [39].The fi nding in Prussian carp is the same as in crucian carp.
Positive Sudan Black B staining was observed in thrombocytes while in our study thrombocytes were SBB negative [40].
The fi nding of basophilic and eosinophilic leukocytes in the blood of fi sh is quite rare [12,41] and our research confi rms this claim.
Stress-induced hematological changes depend on type of stressor, its magnitude and time of action [42].Leukocyte count (WBC) is an important parameter in the assessment of the immune status in vertebrates [43].Stress may affect white blood parameters.Short-term stress sometimes results in an increase in WBC but chronic and/or strong stress usually causes leukopenia [44].In the course of our research, we found out that the total number of leukocytes in the peripheral blood of Prussian carp exposed to stress factors was only slightly lower compared to the values of the group of fi sh that were not exposed to stress.A tendency to increase the total number of leukocytes may indicate an increased protective function of blood in the body of the Prussian carp under the stress as action of chemical toxicants [45].
Differential leukocyte counts belong to important characteristics of the health state of fi sh and in many cases they are also helpful in evaluating the immune system and variations in the proportion of these cells are considered normal [9].The neutrophils and lymphocytes numbers are affected by stress in opposite directions [46].Generally, a fi sh stress leukogram refl ects leukopenia with lymphopenia and granulocytosis.Such changes may persist for several days after the stressor is removed [11].
Teleost neutrophils are phagocytes and typically the fi rst leukocytes recruited to an infl ammatory site by chemokines [47] and are capable of eliminating pathogens through multiple complementary mechanisms.Activated neutrophils become powerful killers, utilizing toxic intracellular granules, the production of reactive oxygen species (ROS) [48], and secrete neutrophil extracellular traps (NETs) which consist of antimicrobial granular proteins that prevent the dissemination of invading pathogens [49,50].
The characteristic mark of the stress-leukogram in fi sh is lymphopenia and neutrophilia [51,52].The results obtained in our research are in accordance with the results of these authors, and are in the contrary to studies claiming that t the numbers of circulating lymphocytes and neutrophils remain unchanged after exposure to stress [30].
Evaluation of the leukocyte formula cannot be equated with measuring the degree of immune response per se, but can be determined more directly using other methods [53].Changes in the total number of circulating leukocytes, their redistribution and quantitative shifts within the leukocyte formula are the most frequently observed effects in fi sh exposed to stress.

CONCLUSIONS
Rapid cytochemical characterization of fi sh blood cell types with human-based staining kits proved to be an effective method for differentiating Prussian carp leukocytes.The presented information about morphological appearance and leukocyte counts provide a basis for further research into the function and changes of blood cells of Prussian carp exposed to variable environmental stress factors.

Figure 1 .
Figure 1.Cytochemical staining of peripheral blood cells in the Prussian carp.Lymphocytes were negative in all cytochemical staining applied (MPO, PAS, SBB).Monocytes were barely visibly positive for MPO, weakly positive for PAS, while negative for SBB.Neutrophils were positive for all cytochemical stains applied (MPO, PAS, SBB).Platelets were negative for MPO and SBB, while weakly positive for PAS.Scale bar = 10 µm.Magnifi cation is 1,000 ×.

Figure 2 .
Figure 2. Relative abundance of leukocytes in (A) non-stressed and (B) stressed fi sh; (C) Changes in relative abundance of leukocytes in stressed fi sh compared to non-stressed control.Lymphocytes are the dominant cell group, followed by neutrophils and leukocytes.The least represented cells are monocytes (A); Neutrophil leukocytes dominate, followed by lymphocytes.The percentage of monocytes in Prussian carp exposed to stress is also signifi cantly lower than the percentage of neutrophil leukocytes and lymphocytes (B); A statistically signifi cant increase in the percentage of neutrophil leukocytes (p<0.01)can be observed (*), as well as a statistically signifi cant (p<0.05)decrease in the percentage of lymphocytes and thrombocytes in Prussian carp exposed to stress compared to Prussian carp that were not exposed to stress (**).No statistically signifi cant difference was found in the percentage of monocytes (p>0.05)(C).

Table 1 .
Comparative analysis of leukograms in non-stressed and stressed Prussian carp with estimations of respective reference values