0WSN 153(2) (2021) 181-191 EISSN 2392-2192
The histopathological condition of hepatopancreas
of the Prussian carp (Carassius gibelio (Bloch,
1782)) in the modern conditions of the Zaporizhian
(Dnipro) reservoir
V. O. Kurchenko*, T. S. Sharamok, O. V. Holub
Department of General Biology and Aquatic Biological Resources, Faculty of Biology and Ecology, Oles Honchar Dnipro National University, Gagarin Ave, 72, Dnipro, 49050, Ukraine
*E-mail address: kurchenko.viktoriia.3@gmail.com
ABSTRACT
The article presents data on the peculiarities of the histological structure of the hepatopancreas and cytometric parameters of the Prussian carp hepatocytes from the Zaporizhian (Dnipro) reservoir in the modern conditions. The morphological changes were not observed during the morphological examination of the hepatopancreas of the Prussian carp (Carassius gibelio (Bloch, 1782)). Cytometric examination revealed that the fish from the Samara Bay showed signs of typical hypertrophy. They also showed a decrease in the nuclear-cytoplasmic ratio. During histopathological examination of the hepatopancreas necrosis was revealed, the most common necrosis occurred in autumn in fish of the Samara Bay and was 8,1 ± 0.1% of the researched samples in females and 8.2 ± 0.2% in males. In the fish from the lower part, this figure was slightly lower. Adipose degeneration of the Prussian carp hepatocytes from the Samara Bay was more common compared to the fish from the lower part of the reservoir, namely the highest percentage of this pathology was recorded in fish in autumn and was 36 ± 0.4% in females and 34.3 ± 0.5% in males, respectively.Most histopathologies in the hepatopancreas are found in the Prussian carp from the Samara Bay, which may be due to the complex action of the external factors.
Keywords: hepatopancreas, Prussian carp, histopathology, necrosis, adipose degeneration, Zaporizhian (Dnipro) reservoir, Carassius gibelio
1. INTRODUCTION
Aquatic ecosystems play an important role in the agricultural sector of the economy of Ukraine. It is known that the disposal of pollutants in water has a detrimental effect on the health of fish and other aquatic organisms. Heavy metals damage the ecological balance, affecting various physiological, biochemical and cellular processes [1]. It is important to correctly interpret and understand the biological effects that occur under the influence of chemicals in the wild and are reflected in the physiological state of aquatic organisms, especially fish. Fish are ideal model objects, and their organs and tissues can serve as bioindicators (biomarkers) of the state of aquatic ecosystems. Biomarkers respond quickly to the presence and exposure to pollutants after their acute or chronic action [2-4].
Fish liver histopathology is an effective bioindicator and can be used to detect chemical contamination and exposure to stressors [5-7].
Liver is a target organ that comes into contact with absorbed nutrients and xenobiotics from the environment. In addition, liver is recognized as the main organ of detoxification and accumulates more toxicants than other tissues [8-11].
The aim of our research was to investigate the histological condition of the liver (hepatopancreas) of the Prussian carp in modern conditions of the Zaporizhian (Dnipro) reservoir.
2. MATERIALS AND METHODS
Figure 1. Prussian carp (Carassius gibelio (Bloch, 1782))
The subjects were four-year-old males and females of the Prussian carp (Carassius gibelio (Bloch, 1782)) (Figure 1).
Fishing was carried out in the waters of the Zaporizhian (Dnipro) reservoir with the help of gill nets in the summer-autumn period during 2018–2019 from two points of the reservoir, which differ in hydroecotoxicological conditions and have an important fishery value - the lower part of the reservoir. (near the village Viyskove), (48°22’30.75” N; 35° 20’80.05” E) and the Samara Bay (48°53’40.21” N; 35 ° 18’73.20” E) (Figure 2).
Figure 2. The scheme of the Zaporizhian (Dnipro) reservoir
The fish hepatopancreas were obtained for histological examination from freshly caught fish with anatomical dissection. Organ fragments 0.3–0.5 cm in size were selected for fixation.
Fixation of the material for histological examinations was performed in 10% aqueous solution of neutral formalin for 24 hours at room temperature. After fixation, the material was washed for 24 hours under running tap water. For pouring in paraffin, the washed material was dehydrated in alcohols, clarified in xylene at a temperature of 37 ° C. The residence time of the
tissue in each of the solvents ranged from 2 to 4 hours. Subsequently, the material was kept in paraffin-xylene (saturated solution) at a temperature of 54 °C for 1 hour, in two changes of paraffin - 2 hours. in each at the same temperature, and poured into paraffin blocks. The sections were 10 μm thick and made on a microtome MS-2 and stained with hematoxylin and eosin [12].
The photographs of histological specimens were taken using a SciencelabT500 5.17M digital camera connected to a Ulab XY-B2TLED microscope. The calculations were performed using the program "ScienceLabView7" 100 fields of view were viewed on the preparations at X 400 magnification. The following parameters were determined: large longitudinal and small transverse diameters of the hepatocyte, hepatocyte area, large longitudinal and small transverse diameters of the hepatocyte nucleus, hepatocyte nucleus area, nuclear-cytoplasmic ratio.
Statistical processing of the obtained data was carried out according to generally accepted methods using the program "Microsoft Excel 2010".
3. RESULTS AND DISCUSSIONS
According to our previous researches, using the ecological classification of surface water quality of the reservoir according to the degree of their purity (pollution), the water in the Samara Bay is characterized as "moderately polluted", and in the area of the village Viyskove - "slightly polluted". The concentration of almost all investigated heavy metals in the water of the Samara Bay is higher compared to the lower part of the Zaporizhian (Dnipro) reservoir.
There were statistically significant differences between the content of copper (by 35%), zinc (by 42%), nickel (by 65%), lead (by 75%) and cadmium (by 85%) in the two areas of the reservoir (p <0.05 ) [13, 14].
Figure 3. The hepatocytes of the Prussian carp from the Zaporizhian (Dnipro) reservoir
A characteristic feature of carp family is the union of the liver and pancreas into a single organ - the hepatopancreas. At the morphological research of a hepatopancreas of the Prussian crucian visual changes were not revealed. Hepatopancreas had a satisfactory structure and color, parasitic infestations were not detected.
The cytometric examination showed that hepatocytes of the hepatopancreas of the Prussian carp were stained in light purple, the walls of the vessels were stained in dark purple, red blood cells were seen in the vessels, the nuclei of hepatocytes were stained dark blue (Figure 3).
The morphometric analysis of hepatocytes showed that the fish of the Samara Bay showed signs of typical hypertrophy (Table 1).
Table 1. The cytometric parameters of hepatocytes of the Prussian carp from the Zaporizhian (Dnipro) reservoir
Indicators The lower part The Samara Bay
Summer Autumn Summer Autumn
♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂
Cell area, μm2 310.15 ±9.8* 302.6 ±9.8* 322.11 ±10.1* 311.10 ±10* 360.8 ±11.8* 359.8 ±11.7* 371.6 ±12.1* 368.1 ±12.01*
D of cells, μm 25.8 ±0.5 25.7 ±0.4 26.06 ±0.5 25.9 ±0.4 26.1 ±0.5 24.6 ±0.4 26.90 ±0.5 25.9 ±0.5
d of cells, μm 15.7 ±0.4 15.5 ±0.3 16.74 ±0.4 16.7 ±0.4 19.5 ±0.3 18.3 ±0.4 19.31 ±0.4 18.9 ±0.4
Nuclei area, μm2 26.3 ±0.9 26.1 ±0.9 30.5 ±0.9 30.1 ±0.8 26.9 ±0.8 26.7 ±0.7 27.74±1. 02 26.9 ±0.9
D of nuclei, μm 5.9 ±0.1 5.6 ±0,1 7.61 ±0.2 7.5 ±0.1 7.1 ±0.2 6.1 ±0.2 7.07 ±0.2 6.9 ±0.2
d of nuclei, μm 4.8 ±0.1 4.8 ±0,1 5.77 ±0.1 5.65 ±0.1 5.2 ±0.2 5.3 ±0.1 5.37 ±0.1 5.45 ±0.1
Nuclear- cytoplas
mic ratio 0.084 ±0.002* 0.086 ±0.002* 0.099 ±0.002* 0.096 ±0.002* 0.072 ±0.001* 0.074 ±0.001* 0.076 ±0.001* 0.073 ±0.001*
*- The difference between the indicators is statistically significant at the different parts of the reservoir, at p < 0,05
The area of hepatocytes in fish of the Samara Bay was larger than in the fish from the lower part, namely: in summer - in females by 16.33% in males by 18.9%; in autumn - 15.36 and 18.32%, respectively, the difference is statistically significant (at p <0.05).
The hypertrophy manifests itself in an increase in cell size, which may be due to the preparation of cells for mitosis or an increase in their ploidy. The increase in the size of the cytoplasm and nucleus without increasing the ploidy of cells is observed under conditions of increased functional activity of cells and their swelling in pathology [15].
Fish of both sexes from the Samara Bay also have a decrease in the nuclear-cytoplasmic ratio by 12.5% in summer and 22.2% in autumn, which may indicate inhibition of cell functional activity and cessation of synthetic processes [16].
During the histopathological research the centers of necrosis in the tissues of a hepatopancreas of the Prussian carp from the both sites were traced (Figure 4).
A B
C D
E F
Figure 4. Necrosis in the tissues of the hepatopancreas of the Prussian carp from the Zaporizhian (Dnipro) reservoir: A & B the lower part; C-F – the Samara Bay
It was found that the highest percentage of necrosis occurs in autumn in fish of the Samara Bay and is 8.1 ± 0.1% of the researched samples in females and 8.2 ± 0.2% in males. In the fish from the lower part, this indicator was slightly lower and was 7.8 ± 0.2% and 7.4 ± 0.5%, respectively. Necrosis in the hepatopancreas of fish is quite common. This is usually a manifestation of the effects of water pollution by toxic substances, such as heavy metals or the result of parasitic infestations [17, 18].
Bright unstained vacuoles of various sizes were also visible in the cytoplasm of hepatocytes. There were large areas of cells without nuclei, which in some places merged and formed the so-called "fat cysts". General pathological signs of morphofunctional disorders of the hepatopancreas were morphologically represented by vacuolation of individual hepatocytes.
In summer, the fish from the Samara Bay had adipose degeneration (Figure 5), the percentage of this pathology was higher than in the fish from the lower part of the reservoir and was 33.7 ± 0.3% of samples in females and 33.1 ± 0.1% samples in males.
A B
C D
E F
Figure 5. Adipose dystrophy of the Prussian carp hepatocytes from the Zaporizhian (Dnipro) reservoir: A & B - the lower part; C-F- the Samara Bay
Table 2. The histopathological changes of the hepatopancreas of the Prussian carp.
Pathology
The lower part The Samara Bay
Summer Autumn Summer Autumn
♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂
Necrosis,
(%) 3.1±0.1 3±0.1 3.9±0.2 3.8±0.2 7.8±0.2 7.4±0.5 8.1±0.1 8.2±0.2
Adipose degenerati
on, % 18.7±0.3 18.4±0.1 20.3±0.3 20±0.3 33.7±0.3 33.1±0.1 36±0.4 34.3±0.5
The same trend is observed in autumn where the percentage of pathology increases slightly and is 36 ± 0.4 and 34.3 ± 0.5%, respectively (Table 2).
During the cytometric examination, the area of hepatocytes in the fish from the Samara Bay was larger than in thr fish from the lower part of the reservoir. An increase in the size of the cytoplasm without increasing the ploidy of the cell nucleus is observed under conditions of increased functional activity of cells and swelling of cells in conditions of pathology. Also in the Prussian carp from the Samara Bay there is a decrease in the nuclear-cytoplasmic ratio, which may indicate the suppression of synthetic processes [17].
Histological examination of the hepatopancreas of the Prussian carp from the Zaporizhian (Dnipro) reservoir revealed the following pathologies: necrosis and adipose dystrophy. The percentage of identified pathologies was higher in fish from the Samara Bay. It is in conditions of increased anthropogenic impact. High mineralization of water and high content of heavy metals in the complex can adversely affect the histological structure of the hepatopancreas of the Prussian carp, which manifests itself in the form of identified histopathologies.
The hepatocyte necrosis is the most severe type of pathology, it is manifested in the fact that hepatocytes lose their structure as a result of destruction of the cell membrane and internal structures. Such areas stand out against the background of healthy tissue spots with an amorphous structure. The presence of this type of pathology is evidence of a strong negative external influence on the individual [19]. In the fish from the Samara Bay necrosis is more common than in fish from the lower part of the reservoir.
Hepatocyte dystrophy should be regarded as a pathological process that occurs, as a rule, in metabolic disorders in cells and is characterized by the presence in hepatocytes of large vacuoles filled with cytoplasmic fluid (balloon dystrophy). This form of dystrophy is irreversible [20]. In pathological conditions, the functions of the hepatopancreas are impaired, and the morphological sign of this is adipose degeneration. In the Prussian carp, this pathology mostly appears in autumn in both parts of the Zaporizhian (Dnipro) reservoir. It can also be associated with seasonal changes before winter (the accumulation of fat by liver cells increases).
Thus, most histopathologies in the hepatopancreas are found in the Prussian carp from the Samara Bay, which may be associated with more intense anthropogenic load.
4. CONCLUSION
The cytometric researches of hepatocytes of the Prussian carp from the Samara Bay showed signs of hypertrophy against the background of a decrease in the nuclear-cytoplasmic ratio. The area of hepatocytes in the fish from the Samara Bay was larger than in the fish from the lower part by an average of 17.6% in summer and 16.8% in autumn. The nuclear- cytoplasmic ratio, by contrast, was lower by 12.5% in summer and 22.2% in autumn.
The research of the histological structure of the hepatopancreas of the Prussian carp from the Zaporizhian (Dnipro) reservoir revealed histological changes, manifested in the form of necrosis (3-8.2%) and adipose dystrophy (18.4-36%).
Most histopathologies in the hepatopancreas are found in the Prussian carp from the Samara Bay. This may be due to the more intense complex anthropogenic load on the bay.
Given the data obtained, the Prussian carp can be used as a bioindicator of the aquatic environment of the Zaporizhian (Dnipro) reservoir, and its hepatopancreas as a biomarker of the body. The obtained data is necessary for monitoring the physiological state of the population
of the experimental species. They deepen modern ideas about the structural organization of the hepatopancreas of the carp fish.
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