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Table 1. Proximate composition of experimental diets

Proximate composition

Crude protein

Crude ash

Crude fat

Crude Fiber

Gross energy



wet weight








2.2. hematological and boichimical factor:

Blood was collected from five fish by venepuncture using syringes coated with heparin (Sigma-Aldrich, Basingstoke, Great Britain) and transferred immediately into 9 ml capacity lithium heparin vacuettes (Greiner, Stonehouse,Great Britain) on ice and the serum was separated by centrifugation. Haematocrit (PCV) value (% red blood cell) was determined in heparinised capillary tubes after centrifugation in a standard microhaematocrit centrifuge at 12.000 g for 10 minute and comparison of the capillary tube with a reference scale. The red blood cell (RBC) was determined using Neubauer Chamber, total leucocytes counts were determined by standard haematological procedures and Haemoglobin (Hb) level was determined by the cyanmethaemoglobin method (Dacie and Lewis, 1976). The mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated from hematological data. The concentration of serum protein, globulin, lysozyme, glucose were determined by the methods described by (Shimeno et al., 1990), while the albumin content was estimated spectrophotometrically using a standard kit (Glaxo, India). Serum cortisol concentration was determined by use of a commercially available enzyme-linked (ELISA) immunoassay kit (Alpha Diagnostic International, USA). All the calculation were made using the SPSS

program version 18.0.1 ( SPSS Inc., Chicago, IL, U.S.A) and t-test statictical method (Mela et al., 2007).


Summary of haematology and boichimical parameters indices were presented in Table 2 and 3.

Table 2: Average hematology parameters from Cammon carp (C.carpio) fingerlingmat the end of 60 days experiment._

Blood index Treatment

Total Leucocytes x104 No/mm3 (±SD)

R.B.Cx 104

No/mm3 (±SD)

Hct (%)(±SD)


(gl-1) (±SD)


(pg) (±SD)


(g/dl) (±SD)

Control group

2.3±0.14 a

1.42±0.71 c

37±02 c

8.2±0.7 a

67±0.4 a

21±1.3 b


2.6±0.08 a

1.64±0.07 c

40.2±01 c

8.8±0.8 b

72.9±0.7 b



3.8±1.32 b

2.43±0.11 b

47.1±01 b

9.1±0.1 b

75.4±0.8 b

29.7±0.8 c


3.96±0.40 b

2.64±0.04 b

49.3±01 b

9.9±0.2 c

81.1±0.5 b

34.4±0.4 a

Table 3. Effect of probiotic, Lactobacilus plantarium on total protein, albumin, globulin,glucose,cortisol and lysozyme ratio of common carp fingerling at the end of 60 days experiment._






Total protein(g/L)

42.38 ± 0.41 a

53.80 ± 7.77 b

57.60± 4.15 b

58.41±0.15 b


4.05 ± 1.32 a

4.19 ± 0.02 a

4.92 ± 0.74 a

4.92 ± 0.91 a


38.14 ± 4.02 a

42.28 ± 5.48 b

42.70 ± 0.07 b

46.01 ± 1.02 b

glucose (mg/dl)

72.54 ± 41 a

61.25± 80 b

54.25± 80 c

53 ± 69 c

Cortisol (ng mL-1)

191.42 ±2.52c

180.31±2.48 b

170.33±1.95 a


Lysozyme (IU/ml)

349 ±14.7 a

354±72.1 a

365±26.6 a

397±48.3 b

Means in the same row with different superscript were significantly different (P < 0.05).


Haematological and biochemical parameters have been acknowledged as valuable tools for monitoring fish health. The results indicated a positive effect represented by significant increase in RBCs count, total leucocytes,PVC%, Hb Conc., MCH and MCHC. These could be attributed to the fact that, the probiotics used increased the blood parameter values as a result of hemopoitic stimulation. These results supported the results of Sarma et al ( 2003). Serum albumin and globulin

values in fish treated with different immunostimulants were always higher than the control (Choudhury et al., 2005). The results indicated higher (P < 0.05) total protein and globulin concentrations of Common carp serum were obtained in T3 compared with control in the present study. This was in agreement with the previous studies which showed that serum protein and globulin in fish treated with different immunostimulants, i.e. P-glucan and yeast RNA, were always higher than the control (Choudhury et al., 2005). As in other vertebrates, fish experiencing stress show a number of physiological changes that are expressed through a number of particular indicators (Wendelaar Bonga, 1997). It is well known that cortisol plays an adaptive function against stressors since it regulates metabolic energy, hydro-mineral balance, oxygen uptake, and immune competence (Wendelaar Bonga, 1997). In the present study, serum cortisol was measured as an indicator of primary stress response and serum glucose as an indicator of secondary stress response. Generally, the analysis of serim cortisol and glucose levels carried out in this study gave the evidence that the groups fed with L. plantarium showed a better tolerance to captive rearing conditions. This was evidenced by the lower cortisol and glucose levels detected in the treated experimental groups with respect to control. Lysozyme, being an enzyme with antibacterial activity, can split peptidoglycan in bacterial cell walls especially of the gram positive species and can cause lysis of the cells (Chen et al., 1996). Lysozyme concentrations in fish have been reported to increase after injection of a bacterial product and in response to bacterial infection (Chen et al., 1996). Fletcher and White (1976) reported increased values of lysozyme with activation of immune system. Modulation of lysozyme activity in fish related to a probiont has not been reported yet. Apart from serum lysozyme content, probiotics can also enhance the lysozyme level in skin mucosa of fish (Taoka et al., 2006). Taoka et al (2006) reported significantly high lysozyme level in skin mucosa by supplementing commercial probiotics through water in comparison to oral supplementation in O. niloticus. In the present study LAB fed groups showed elevated level of lysozyme activity, but the group receiving the highest density (T3) was observed to have

significantly higher lysozyme activity compared to that of the others (control,T1,T2) indicating activation of the immune system. Based on these results, use of (65/6 LogCFU/100g) supplement of probiotics in common carp diet was recommended to stimulate productive performance.


Aly SM, Abd-El-Rahman AM, John G, Mohamed MF (2008).Characterization of some bacteria isolated from Oreochromis niloticus and their potential use as probiotics. Aquaculture, 277: 1-6.

Aymerich T, Martin B, Garriga M, Hugas M: Microbial quality and direct PCR identification of lactic acid bacteria and nonpathogenic staphylococci from artisanal low-acid sausages. Appl Environ Microbiol 2003, 69(8):4583-4594.

Carnevali, O., Zamponi, M.C., Sulpizio, R., Rollo, A., Nardi, M., Orpianesi, C., Silvi, S.Caggiano, M., Polzonetti, A.M., Cresci, A., 2004. Administration of probiotic strain to improve sea bream wellness during development. Aquac. Int. 12, 377­386.

Chen, S.C., Yoshida, T., Adams, A., Thompson, K.D., Richards, R.H., 1996. Immune response of rainbow trout to extracellular products of Mycobacterium sp. J. Aquat. Anim. Health. 8, 216- 222. Chipman, D.M., Sharon, N., 1969. Mechanism of lysozyme action. Science 165, 454—465.

Choudhury, D., Pal, A.K., Sahu, N.P., Kumar, S., Das, S.S., Mukherjee, S.C., 2005.

Dietary yeast RNA supplementation reduces mortality by Aeromonas hydrophila in rohu (Labeo rohita L.) juiveniles. Fish & Shellfish Immunology, Vol.19. No.3,


Fletcher, T., 1976. The lysozyme of plaice (Pleuronectes platessa L.). Experientia 29,


Irianto, A.and B. Austin,2002. Probiotics in aquaculture.j. Fish Dis., 25: 633-642.

Kim DH, Austin B (2008). Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine. Lett. Appl. Microbiol. 47(3): 141-147.

Kumar, R., S.C. Mukherjee, R. Ranjan and S.K. Nayak, 2008. Enhanced innate immune parameters in Labeo rohita (Ham.) following oral administration of Bacillus subtilis. Fish Shellfish Immune., 24: 168-172.

Sarma, M., D. Sapcto, S. Sarma and A.K. Gohain, 2003. Herbal growth promoters on hematobiochemical constituents in broilers. Indian. Ver. J., 80: 946-948.

Taoka Y, Maeda H, Jo JY, Kim SM, Park S, Yoshikawa T, et al. Use of live and dead probiotic cells in tilapia Oreochromis niloticus. Fisher Sci 2006; 72:755e66.

Wendelaar Bonga SE. The stress response in fish. Physiol Rev 1997; 77: 591-625.

The effects of heavy metal (cadmium chloride) on the haematological and biochemical parameters in sturgeon

fish (Huso huso)

Mohsen pourabasali*1, mitra esmaili2, Mohammad Gholizade1, Nazanin Peyvandi1

*1 Department of Fishery, Islamic azad University, babol branch, Iran

2 Department of Fishery, Islamic azad University, azadshahran branch, Iran


The aim of the present study was to determine the effect of heavy metal cadmium in aquatic system on strugeon fish (huso huso) by using a set of haematological and biochemical parameters. Cadmium chloride LC50 was found out for 96 h (28mg/L) (Sprague, 1971) and 1/15th, 1/10th and 1/5th of the LC50 values were 1.93, 3.11 and 5.78mg/L respectively taken as sublethal concentrations for this study. The results indicated that the values of the Leucocytes were in the range of 13.74+0.42 to 40.64 + 2.01 (x103cell /mm3) (p<0.05) and the RBC was in the range of 4.31+0.35 to 2.28+0.35 (p<0.05). Concentrations of M.C.H (pg), M.C.H.C (g/ld), Haematocrit and haemoglobin were significantly decreased (p<0.05). The exposed groups showed a marked decline in serum total protein, albumin and globulin (p<0.05). Conversely an increase in serum glucose and Cholesterol comparison to control was observed (p<0.05). The study suggested that the cadmium has strong influence on the hematological and biochimical parameters in H.huso.

Keywords: heavy metal, cadmium, huso huso., haematological, biochimical

Todays well understanded that environmental problems have increased exponentially mainly because of quick growth in human population and industrial growth is an important part of the evolution of human civilization and is essential for the development of any society. However, industries also often prove hazardous to aquatic life when their toxic effluents are discharged into water. Amidst the aquatic animals, fishes are chiefly utilized as food resources.Major component of inorganic contaminates are heavy metals. They have some different problems than organic contaminants (Dreibach and Robertson 1987; Gad and Saad, 2008).Toxic metals can enter human organism through inhalation, ingestion and in skin contact. The most frequent effects are hemotoxic nephrotoxic, effects on respiratory and reproduction system (Dreibach and Robertson, 1987). The presence of metals varies between fish species; depend on age, developmental stage and other physiological factors. Pollutants generally produce relatively rapid changes in blood characteristics of fish (Ezzat et al., 1998). Therefore haematological and biochimical data can provide useful information in evaluating the health of fish and in monitoring stress responses.The haematological parameters like hemoglobin, haematocrit, blood cell counts, glycemia and ion concentrations can be used to find physiological res-ponse of contaminated environment (Dethloff et al., 2001). Heavy metals become toxic when they are not metabolized by the body and accumulate in the soft tissues. Cadmium belongs to the group of highly toxic heavy metals. The toxicity of Cd is ascribed to its ability to generate reactive oxygen species that may act as signaling molecules in the induction of gene expression and apoptosis (Waisberg et al., 2003), deplete endogenous radical scavengers, and also damage a variety of transport proteins including the Na+ / K+ - ATPase. Several studies show their bioaccumulation in different fish tissues skin, gill, brain, liver, muscle, kidney, intestine, gonads etc. (Vinodini and Narayanan, 2008). Cadmium may also affect the blood cells (Witeska, 1998). Cadmium accumulation in these organs appears to be related to the presence of cadmium-binding

molecules called metallothioneins (Carpene and VaSik, 1989). The lethal and sub-lethal concentration, cadmium has a cumulative polluting effect and could cause serious disorders in fish metabolism such as locomotor anomalies and (Cicik and Engin, 2005). Most cadmium contamination comes from metal foundries, the dye industry, production of plastics and of accumulators. This exposure results in pathological changes in water ecosystems, mostly demonstrated in fishes, which are affected by heavy metals through the respiratory and digestive systems and through the skin. For example, Baker and Montgomery (2001) showed that cadmium ions were responsible for impaired olfactory function and altered rheotaxis behaviour associated with damage to the lateral line system in freshwater fish. Daei et al.(2009) with study on (Chalcalburnus chalcoides) showed that cadmium with ratio (P<0.05 ) replaced with ferritin (Fe) over the time in the fish blood but metal Pb couldn,t so. Those results indicated that by increasing in lead density, in fishes this metal was absorbed by other tissues Daei et al.(2009). Strugeon fish (huso huso), is one of the vulnerable species of Caspian Sea sturgeons. The adults live in the Caspian Sea (10-13 ppt) but spawning occurs in freshwater Rivers, fry live in rivers for two months and then they return back to the Caspian Sea. The Caspian Sea is an enclosed water body that is fed from several freshwater rivers, many of them carrying different land source pollutants, such as heavy metals, etc cause h.huso, has listed as threatened and endangered throughout their ranges (Moghim et al. 2002). In this context, the present research has been designed to study the haematological and biochimical effect resulting from the exposure of the Caspian Sea sturgeon (huso huso) exposed to sublethal concentrations of cadmium chloride.

Material and method

Experimental design

Total of sixty fish ( 70 - 100 g ) were acclimatized to laboratory conditions for two weeks before use. Cadmium chloride LC50 was found out for 96 h (28mg/L) (Sprague, 1971) and 1/15th, 1/10th and 1/5th of

the LC50 values were 1.93, 3.11 and 5.78mg/L respectively taken as sublethal concentrations for this study. Sixty fish were selected and divided into 4 groups of 15 each. The first group was maintained in free from cadmium chloride and served as the control. The other 3 groups were exposed to sub lethal concentration of cadmium chloride in 100 litre capacity aquaria. During the rearing period, pH and dissolved oxygen were 7.1 + 0.5 and 8 mg/l, respectively. The temperature in each aquarium was maintained at 27+1°C by means of thermostats. The photoperiod was 12 hrs light-length/days. During the experiment the fish were fed 3% of their body weight per day (twice a day). Siphoning three quarters aquariums was done every day for waste removal and replacing it by an equal volume of water containing the same concentration of Cd. Dead fish were removed and recorded daily. The formulation and proximate composition of diet is shown in Table1.

Table 1:Experimental diet formulation and proximate composition

Ingredients (%)


Fish meal


Wheat meal


Fish oil


Soybean oil




Vitamin mixture


Mineral mixture


Anti oxidane


proximate composition


Dry mater(%)








Digestible energy(kj g -1 diet)


Gross energy(kj g -1 diet)


Blood and hematological factor

Blood was collected by venepuncture using syringes coated with heparin (Sigma-Aldrich, Basingstoke, Great Britain) and transferred

immediately into 9 ml capacity lithium heparin vacuettes (Greiner, Stonehouse,Great Britain) on ice and the serum was separated by centrifugation. Haematocrit (PCV) value (% red blood cell) was determined in heparinised capillary tubes after centrifugation in a standard microhaematocrit centrifuge at 12.000 g for 10 minute and comparison of the capillary tube with a reference scale. The total leucocytes counts were counted by standard haematological procedures and Hemoglobin (Hb) level was determined colorimetrically by measuring the formation of cyanomthaemoglobin using a commercial kit. The mean corpuscular hemoglobin (MCH, pg = (Haemoglobin [g/dL] x 10 / (RBC count [in millions/lL])) and mean corpuscular hemoglobin concentration (MCHC,g/dl= Haemoglobin [g/dL] / Hematocrit [%]) were calculated from hematological data.


The concentration of serum total protein, globulin, glucose were determined by the methods described by (Shimeno et al., 1990), while the albumin content was estimated spectrophotometrically using a standard kit (Glaxo, India). Total Serum cholesterol was determined by the method of Allain (1974).

Statistical analysis

Data were analysed by one-way analysis of variance (ANOVA) and Duncan's comparison of means. Percentage data weret transformed to square-root arcsine values to homogenize variance.All statistical tests were performed using SPSS software (SPSS, Release 14.0, SPSS, Chicago, IL.). Differences were considered statistically significant when P < 0.05.

Result and Discussion

Summary of haematological and biochimical parameters indices were presented in Tables 1, 2.

Table 1. Effects of cadmium exposure on hematological parameters of Huso huso.







9.98± 1.54

9.94+ 0.52

9.11+ 0.17

8.46+ 1.42*


31.00+ 0.13

29.54+ 1.46

28.71+ 0.78




28.43+ 1.04*


40.64 + 2.01*

M.C.H (pg)

66.01+ 2.04



38.58+ 2.19*

M.C.H.C (g/ld)

35.25 ± 0.06

35.34 +1.84

35.14+ 3.61

30.77 +2.17*






*Significant difference with control (P<0.05). Values are mean + standard error.

Table 2. Effects of cadmium exposure on biochimical parameters of Huso huso.






Total protein(g/L)

45.19+ 2.35


33.25+ 1.08*

28.71+ 2.06*


5.18+ 1.47

4.27+ 1.48

3.35+ 3.46*




31.19+ 1.28*


25.34 + 0.01*

glucose (mg/dl)

56.31+ 0.02



75.05+ 0.19*

Cholesterol (mg/dl)

150.12 + 0.41

158.45 +0.25

178.10+ 0.36*

186.24 +1.43*

*Significant difference with control (P<0.05). Values are mean + standard error.

The toxic effects of heavy metal on fish are multidirectional and manifested by numerous changes in the physiological and chemical processes of their body systems (Dimitrova et al., 1994). Regarding hematological parameters, cadmium exposure for 45 days significantly diminished RBC count, HCT, MCHC , MCH and haemoglobin concentration in h.huso in comparison with control. In the present study, the mean value of PCV was 31.00+ 0.13 in the control group, which decreased progressively (29.54, 28.71, 27.16) in groups T1, T2 and T3. A decrease in the percent of haematocrit indicate the worsening of an organism state and developing anaemia. The reduction of these parameters in h.huso at sub-lethal levels of cadmium might be due to the destruction of mature RBCs and the inhibition of erythrocyte production

due to reduction of haemsynthesis that affected by pollutants (Wintrobe, 1978). Also, the decrease in RBCs count may be attributed to haematopathology that results in sever anemia in most vertebrates including fish species exposed to different environmental pollutants (Khangarot & Tripathi, 1991). According to Pamila et al. (1991), the reduction in haemoglobin content in fish exposed to toxicant could also be due to the inhibitory effect of the toxic substance on the enzyme system responsible for synthesis of haemoglobin. Also Gill & Epple (1993) found a significant reduction in the RBCs, Hb and HCT in American eel (Anguilla rostrata) after exposure to 150 ug Cd L-1. Karuppasamy et al. (2005) found a significant decrease in total erythrocyte count, haemoglobin content, haematocrit value and mean corpuscular haemoglobin concentration in air breathing fish, Channa punctatus after exposure to sub-lethal dose of Cd (29 mg Cd L-1). Leucocytes counts were found increased following cadmium exposure as shown in Table 1. Similar findings were also documented significantly higher in fish exposed to increased copper concentration (Nath and Banerjee, 1995). Mishra and Srivastava (1980) also reported an increase in leucocytes count when they exposed fishes to heavy metals. Respecting the serum protein, cadmium exposure for 45 days significantly decreased the levels of total protein, albumin and globuline ratio comparing with control. Decrease in serum protein may indicate some liver dysfunction. When exposed to stressor, the gills become leaky to water and ions, often resulting in osmoregulatory imbalances (30). So the decline in serum total protein, albumin and globuline may be also due to a degree of haemodulation under the stress of pollution. Heavy metals increase the glucose content in serum, because of intensive glycogenolysis and the synthesis of glucose from extra hepatic tissue proteins and aminoacids (Almeida et al., 2001). Many other workers reported hypoglycemic condition in fishes due to contaminants (Kurde1990, Sastry 1984). This may be to cope with high-energy demand in stress situations. Cholesterol is the most important sterol occurring in animal fats. It is equally distributed between plasma and red blood cells, but in adrenal cortex, it occurs in the esterified form. The

cholesterol occurs as white (or) faintly yellow almost odorless granules. In the current study, the serum cholesterol level was significantly (p<0.05) increased in heavy metal exposed experimental groups (Table 2). After the above discussion it had been concluded that cadmium chloride causes deleterious effects on fishes and much altars the biochemical characteristic of blood and serum. Contamination of aquatic environment by heavy metals whether as a consequence of acute or chronic events constitutes additional source of stress for aquatic organisms. Sublethal concentrations of toxicants in the aquatic environment will not necessarily result in outright mortality of aquatic organisms.


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Baker, C.F., Montgomery, J.C., 2001. Sensory deficit induced by cadmium in banded kokopu,

Galaxias fasciatus, juveniles. Environ. Biol. Fish. 62, 455-464.

Carpene, E. and V asak, M., 1989. Hepatic metallothioneins from gold fish, Carassius auratus. Camp. Biochem. Physiol., 92: 463-468.

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in the liver and muscle tissues of Cyprinus carpio L. (1758)". Turk. J. Vet. Anim. Sci. 2005; 29: 113 - 117.

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Dethloff GM, Bailey HC, Maier KJ (2001). Effect of dissolved copper on selected haematological, biochemical and immunological parameters of wild rainbow trout (Oncorhynchus mykiss). Archi. Environ. Conta. Toxicol. 40: 371-380.

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Reproductive Biology of kura barbel (Barbus lacerta) in Babolrood River, Northern Iran

Mohsen Pourabasali, Nazanin Peyvandi Mohammad Gholi Zadeh,

Department of Fisheries, Islamic Azad University, Babol branch,IRAN


This study was carried out to examine reproduction characteristics of Kura Barbel, Barbus lacerta from August 2010 to July 2011, in the Babolrood River (Northern Iran) using electro-shocker with the voltage of 200-300 volts. A Total 206 specimens were caught. The total length and weight of females and males were 109.65 ± 1.33 (SD) mm and 17.87 ± 1.03 g, and 103.46 ± 1.78 (SD) mm and 16.06 ± 1.9 g respectively. Age readings showed that age composed of four age groups of (0+, 1+, 2+, and 3+), the most of the specimens belonged to the age group 1+. Sex ratio was 1.1 to 1, no significance from deviation from parity. The mean of ova diameters was 0.57 ± 1.03 mm (SD). Absolute fecundity ranged from4360 ± 21.96 (SD) and relative fecundity varied between 140.44 ± 18.45 (SD). The mean GSI for females and males was 2.45 ± 0.55 (SD) and 2.61 ± 0.14 (SD) respectively. mean condition factor (CF) was estimated 1.17 ± 0.12 (SD) for the females and 1.12 ± 0.09 (SD)for the males.

Key words: Reproduction, B. lacerta, Babolrood, Caspian Sea, Iran


The application of an accurate management over the repertoires of aquatic reserves and the development of aquaculture are successful when the genetic reserves of the native species are studied. The first step in this

way is the correct identification of species, populations or races. Such a matter is highly important regarding fishery management and protection planning of species (Coad 1980). The main composition of the inland waters consists of carps among which some of the species of the genus Barbus are the most important ones (Ramin 1999). Kura barbel B. lacerta belonging to the family Cyprinidae, lives in upper levels of rivers with sandy and stone beds rich in macrobenthic communities (often together with brown trout in the Chalus and Tajan rivers). They are usually found in fast, cold and highly currents of water with high levels of oxygen although (Abdoli 1999), including rivers of Tigris and Euphrates and of the southern Caspian basin (Aras, Sefid-Rud, Shahrud, Ghareh Soo) (www.briancoad.com). The existence of dark spots on its body is regarded as the key characteristic of the species, which are seen on the irregular brown back, the dorsal fin, and its tail. The food remaining components of plants, crustaceans such as amphipoda, and insects such as chironomidae and damselfly's larva were found in the fish's intestine. In addition, the consumption of plecoptera, ephemeroptera, and chironomidae were reported for this species (Abdoli 1999). Others reported the consumption of algae (Bogutskaya and Banarescu 2003). Spawning may occur two or three times a season, which have been identified by the mature eggs in the ovary. The species spawns from the late April to August. Noticeably, the temperature is effective on spawning that a water's temperature higher than 20° C and lower than 14° C inhibits the process of spawning (Bogutskaya and Banarescu 2003). The species is found in freshwater and does not belong to migrators. The present study aims to examine basic reproduction characteristics of this species including age composition, spawning period, GSI, sex ratio, absolute and relative fecundities, size range and mean diameter of eggs.

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