Thermal adaptability and disease association in common carp (Cyprinus carpio communis) acclimated to different (four) temperatures

Journal of Thermal Biology 36 (2011) 492–497

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Thermal adaptability and disease association in common carp (Cyprinus carpio communis) acclimated to different (four) temperatures S.M. Ahmad a,n, F.A. Shah a, F.A. Bhat a, J.I.A. Bhat b, M.H. Balkhi a a b

Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology—Kashmir, Shuhama, Srinagar 19006, India Division of Environmental Sciences, Sher-e-Kashmir University of Agricultural Sciences and Technology—Kashmir, Shalimar, Srinagar 19006, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 June 2011 Accepted 30 August 2011 Available online 8 September 2011

The present study was designed to investigate the effect of temperature (20 1C, 24 1C, 28 1C and 32 1C) on the heamato–biochemical and histological alterations of Cyprinus carpio communis. Increase in the temperature showed significant decrease in the serum protein, while a reduced level of blood glucose at high temperature of 32 1C was observed leading to hypoglycemic conditions in the experimental fishes. A significant correlation (P o 0.01) was observed between cholesterol (Cho) and triglycerides (TG) for different temperature treatments. Elevated blood urea nitrogen (BUN) at high temperatures was a good indicator of gill osmoregulatory failure. A variation of 86.40% and 38.33%, respectively, was noticed in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 32 1C over minimum experimental temperature of 20 1C. The increase in red blood cell (RBC) and Heamoglobin (Hb) concentration is associated with the decrease of mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), could be the reason for observed poikilo-anisocytosis. Histological studies of different organs of experimental fishes showed accumulation of MMC’s (melanomacrophagic centers) and atrophy of the interrenal tissue on exposure to various levels of temperature. These changes were related to severity of thermal stress, being most marked when high temperature was prolonged during acclimatization. Some fishes were found infested by protozoan parasite at elevated temperature of 32 1C. Increased levels of certain biochemical and haemotological parameters studied were strongly correlated with disease in the Cyprinus carpio communis species. & 2011 Elsevier Ltd. All rights reserved.

Keywords: Temperature Acclimation Biochemistry Haemotology Poikilo-anisocytic anemia Histology Parasitic infection Cyprinus carpio communis

1. Introduction Physiological processes in fish are often carried out in a harsh aquatic environment, and fish face conditions and challenges that do not exist for terrestrial animals. The maintenance of internal homeostatic equilibrium is essential for the normal functioning of the animal and in case of any disturbance the fish will try to establish a new equilibrium. Any environmental disturbance can be considered a potential source of stress, as it prompts a number of responses in the animal to deal with the physiological changes triggered by exterior changes. However, the harmful effects of many of these stress-induced factors on the physiological condition significantly increases with even a moderate change in stress factors, such as a shift of a few degrees in water temperature or a reduction by a few mg per liter of dissolved oxygen (Pickering, 1993; Wedemeyer, 1997). Temperature affects virtually all biochemical and physiological activities of an animal. It should be

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0306-4565/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2011.08.007

viewed as an environmental factor, which evokes multiple effects on animals (Magnuson et al., 1997). A long-term change in temperature causes fishes to display acclimatory responses, which may include enzymatic changes to mitigate the effect of temperature on metabolism (Hazel and Prosser, 1974). Temperature is one of the important abiotic factors, which influences biochemical reactions and therefore has significant impact on the physiology and biochemistry of exothermic organisms (Woiwode and Adelman, 1991; Jobling, 1996; Person-Le Ruyet et al., 2004). Temperature beyond optimum limits of a particular species adversely affects the health of aquatic animals due to metabolic stress (Smith, 1989) hence increases susceptibility to diseases that have suppressive effects on growth, reproduction and immune capacity. Temperature of aquatic environment is important for ensuring survival, distribution and normal metabolism of fish, failure to adapt to temperature fluctuations is generally ascribed to the inability of fish to respond physiologically with resultant mortality, which is related to changes in the metabolic pathways (Forghally et al., 1973). According to Donaldson (1981) the physiological responses can be detected in fish and in other vertebrates in the form of changes in hormonal or substrate concentrations in the plasma or

S.M. Ahmad et al. / Journal of Thermal Biology 36 (2011) 492–497

alterations in erythrocyte parameters, such as cell volume or enzyme activities. Measurement of serum biochemical parameters can be especially useful to help identify general health status of animals, and is advocated to provide early warning of potentially damaging changes in stressed organism (Folmar, 1993; Jacobson-Kram and Keller, 2001). Hematological values of fishes can be affected by environmental and biological factors such as age, weight, sex, food, bacteria, parasites and water quality parameters including water temperature, salinity, oxygen, availability and pH (Steinhagen et al., 1990 and Haider, 1973). Common carp (Cyprinus carpio communis) has been one of the oldest domesticated species of fish for food, dwells well in middle to lower reaches of rivers and shallow confined waters. Best growth is obtained at water temperature of 23–30 1C. The fish can survive cold winter periods, salinity up to about 5%, pH (6.5–9.0), low oxygen concentration (0.3–0.5 mg/1) as well as super saturation (Flajˇshans and Hulata, 2006). However, due to ongoing global warming conditions about one-fifth of all freshwater fishes are considered to be endangered (Heywood, 1995). Kashmir is the north most part of India and lies in north western part of greater Himalayas and is well known coldwater fisheries sector of the country. It is the temperate region where the air temperature fluctuates from 8 to 351C. Common carp (C. c. communis) is one of the major pond-culture fish of this region with high commercial value in domestic market. It is highly preferred fish of this region owing to its high nutritive value. Being commercially important and amenable to aquaculture, it is important to have knowledge of the acclimatory response of C. c. communis to thermal stress and this experiment is expected to provide better know-how for its aquacultural practices.

2. Materials and methods Fish samples were purchased from commercial fish ponds and were acclimatized in the laboratory for a week at 20 71 1C. Healthy fishes (6/aquarium) with a mean weight and length of 20375 g and 1973 cm, respectively, were subjected to experimental conditions. Fishes were stocked at 20 1C (control), 24 1C, 28 1C and 32 1C for a period of one month in 24 tanks (200 L capacity), six replicates to each treatment. Acclimation procedure followed in this experiment was based on Manush et al. (2004). Temperature in each treatment was maintained using temperature controller fitted with sensors (Selectron process controls Pvt. Ltd., Mumbai, India). Dissolved oxygen concentration was maintained at 670.5 mgl  1 by continuous aeration. Rearing experiments tanks were placed in an indoor experimental area and the light source was naturally enhanced with fluorescent light. Fishes were fed once in a day with practical diet (35% crude protein). Each day, dead fish (if occurred) was removed and in each tank half of the water volume was renewed every day with filtered water to assure water quality. The daily removal of water helped in maintaining the alkaline side of neutrality ranging from 7.5 to 8.00. Similarly the nitrate—nitrogen of water tanks remained in normal range between 480 mg and 720 mg (APHA, 1998). The supplemental aeration was also provided to maintain dissolved oxygen levels near saturation levels. The dissolved oxygen was measured by Winkler’s Modified method (APHA, 1998) and was maintained above 6 mgl  1. 2.1. Sample collection and analysis 2.1.1. Sampling Heamato–biochemical parameters were assessed after a period of one month rearing experimental fishes at these

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temperatures. Individual animals were anaesthetized before sampling with clove oil (150 ml/L) in water. Once the fish lost equilibrium, the blood samples were drawn by cardiac puncture, the 25-gage needle of disposable syringes were passed through the posterior wall of the branchial chamber directly into the lumen of the bulbus arteriosus. The ammonium salt of heparin, used in combination with ethylenediaminetetraacetic acid (di-sodium EDTA) was found adequate to prevent coagulation. 2.1.2. Blood analysis For the serum biochemical analysis, blood samples were prepared using the method described by Bernet et al. (2001) with slight modification. Blood was allowed to coagulate at room temperature for 2 h. Serum was obtained by centrifugation of blood samples at 1500  g (for 10 min at 4 1C) and then stored at  80 1C for several weeks until analysis. The concentration of glucose, total protein, cholesterol, triglyceride, blood urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by using a Semi-automatic blood chemistry analyzer (model—ERBA CHEM—PRO.) and test kits from Accurex Biomedical Ltd. Immediately after sampling, the red blood cell (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC), lymphocytes and granulocytes were determined with the help of Automated Hematology Analyzer Hema Screen—18 (Hopitex Diagnostics, Italy). 2.2. Histology After 30 days of exposure, six fishes from the experimental and control groups were euthanized with overdose of MS222 and then the second gill arch, liver, spleen, trunk kidney and head kidney were carefully removed and preserved in 10% neutral-buffered formalin (NBF) for 48 h. Organs were rinsed in 4 changes of 70% ethanol (ETOH), and stored in 70% ETOH until further processing. They were dehydrated in isopropanol, cleared in xylene, infiltrated in paraffin and sectioned at a thickness of 5 mm. Sections were stained with hematoxylin and eosin and examined with a light microscope. Sections were stained with hematoxylin and eosin (Luna, 1968), and examined with a light microscope. 2.3. Data analysis Data was analyzed using one way analysis of variance (ANOVA) as described by Gomez and Gomez (1984) and Tukeys’s multiple range test was carried out for post hoc comparison of means (Po0.05), if they were significantly different.

3. Results 3.1. Haemato–biochemical profile Data pertaining to biochemical and hematological parameters of C. c. communis at 20 1C (control), 24 1C (T24), 28 1C (T28) and 32 1C (T32) are presented in Tables 1 and 2. The result based on biochemical parameters showed that with the increase in temperature the blood glucose of the experimental fishes increased from 84.8375.4 mg% (20 1C) to 97.14 73.1 mg% (28 1C) and decreased to 59 76.6 mg% at 32 1C. The decline in the concentration of total serum protein at elevated acclimation temperatures was observed. The mean total serum protein concentration (1.1670.26 g/dl) at the highest temperature employed (33 1C), is 66.47% lower than that of the lowest temperature group (20 1C,

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Table 1 Effect of acclimation temperatures on biochemical parameters in the Cyprinus carpio communis (mean 7S.E). Temperature

20 1C (Control)

24 1C

28 1C

32 1C

Glucose (mg%) Protein (gram/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) Blood urea nitrogen (mg/dl) Alanine amino transferase (ALT, mkat l  1) Aspartate amino transferase (AST, mkat l  1)

84.83 7 2.24c 3.46 7 0.16c 142.877 5.88c 108.50 7 1.52c 1.11 7 0.01d 0.147 0.05c 1.93 7 0.09b

88.76 7 1.81c 3.137 0.17c 144.537 2.66c 112.227 2.34c 1.387 0c 0.157 0.09c 1.987 0.17b

97.14 7 1.28b 2.48 7 0.16b 167.287 3.40b 125.927 3.15b 2.14 7 0.05b 0.357 0.18b 2.34 7 0.44b

59.66 7 2.57a 1.167 0.49a 194.50 7 5.09a 149.677 2.74a 2.987 0.15a 1.037 0.25a 3.137 0.67a

*Different superscripts (a, b, c, d) within the row differ significantly (P o 0.05). Means were compared using ANOVA and Tukeys’s multiple range test for post hoc comparison.

Table 2 Effect of acclimation temperatures on heamtological parameters in the Cyprinus carpio communis (mean7 S.E). Temperature

20 1C (Control)

WBC (  103/mm3) Lymphocytes (  103/mm3) Granulocytes (  103/mm3) RBC (  106/mm3) HCT (%) Hb (g/dl) MCV (fl) MCH (Pg) MCHC (gdl  1)

26.31 7 0.99b 41.12 7 0.90d 0.54 7 0.02c 1.32 7 0.03b 26.65 7 0.15c 4.37 7 0.14b 198.767 1.54a 43.67 7 1.21a 24.16 7 0.60c

c

24 1C

28 1C

32 1C

26.98 7 1.02c 38.56 7 0.75c 0.51 7 0.16c 1.35 7 0.05b 27.04 7 0.39c 5.83 7 0.20a 175.437 2.13c 42.88 7 1.82a 22.73 7 0.76bc

23.63 7 0.58b 33.56 7 1.02b 0.367 0.14b 1.61 7 0.05a 30.67 7 0.60b 6.057 0.16a 188.547 2.50b 42.16 7 0.90a 21.44 7 0.79b

16.71 7 1.15a 25.12 7 0.70a 0.237 0.08a 1.777 0.04a 34.71 7 0.17a 6.087 0.10a 201.77 7 1.43a 39.62 7 1.60a 19.32 7 0.53a

*Different superscripts (a, b, c, d) within the row differ significantly (P o 0.05). Means were compared using ANOVA and Tukeys’s multiple range test for post hoc comparison.

Fig. 1. Peripheral blood film from C. c. communis at high temperature (32 oC) for a period of 1 month revealing poiklo-anisocytic, hypochromic RBC’s, (a)  40 and (b)  100 (Giemsa stain).

3.4670.11 g/dl). Except 20 1C and 24 1C a significant decrease (Po0.05) existed among different temperature treatments for protein levels. Unlike serum protein the concentrations of both these enzymes (ALT and AST) used in this study increased with the increase in temperature. The concentration of ALT at temperature 32 1C increased 8–9 times (0.14 70.01 mkat l  1 to 1.0370.03 mkat l  1), while as the concentration of AST increased 2–3 times (1.9370.09 mkat l  1 to 3.13 70.67 mkat l  1) over the control. A significant increase at the 0.05 level of probability was found for both the enzymes at 28 1C and 32 1C temperature. As might be anticipated, cholesterol varies in the same fashion as triglycerides. The highest values for both these parameters were observed at 32 1C. A significant correlation (Po0.01) was observed between cholesterol and triglycerides for different temperature treatments. Blood urea nitrogen (BUN) concentrations at higher temperature exposure exhibited higher values than those of the control group. The significant increase (P o0.05)

of BUN was found throughout all the temperature treatments. The difference between the lowest (BUN, 20 1C; 1.11 70.41 mg/d) and highest blood urea nitrogen (BUN, 32 1C; 2.98 71.43 mg/d) was 62.75%. The hematological data of C. c. communis (Table 2), showed decrease in leukocytes (WBC) with the rise in acclimation temperature. A decrease of 36.48% was noticed at 32 1C over an ambient temperature of 20 1C. The lymphocytes and granulocyte number of the experimental animals decreased significantly (Po 0.05) with the rise in temperature. The peripheral blood film of thermally stressed fishes revealed poikilo-anisocytosis and hypochromia (Fig. 1). This type of anemia was only found at protracted exposure of fishes at 32 1C. The results of the study upon Hb, RBC and Hct are evident in Table 2, showed a common trend of increase with respect to the increase in temperature. Although the highest mean Hb level was that encountered at 32 1C (6.08 70.46 g/dl), this was not greatly different from that of

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28 1C (6.05 70.88 g/dl) group. The RBCs at 32 1C (1.7770.05  106/mm3) were 25.42% higher than lowest value recorded (RBC 20 1C¼1.32 70.05  106/mm3). Effect of increasing temperature on Hct showed significant difference at 0.05 level of probability between different groups. Values derived for MCV showed wide variation with the rise in temperature. Compared to 24 1C and 28 1C, the variation of only 1.51% was observed between 20 1C (198.7675.99) and 32 1C (201.77 719.95). The values for both MCH and MCHC tend to decline at higher acclimation temperatures. The value for MCH at 20 1C ¼43.67 72.26 showed 99.21% and 98.49% similarity with 24 1C¼42.88 72.47 and 28 1C¼ 42.16 71.72, while as 9.2% variation was observed with 32 1C¼ 39.62 76.68. The level of variation between different values of MCHC however, ranged from 5.91% to 20%. 3.2. Histological examination of fish tissues No specific histological alterations were found in the organs of the fishes exposed to control (20), 24, and 28 1C. Most of the histopathological manifestations were marked after the prolonged exposure of 32 1C. In the present study, exposure of carp to thermal stress resulted interrenal cell hypertrophy and melanomacrophage centers were scattered throughout kidney and spleen. The kidney of the fish often showed vacuolar degeneration of tubular epithelium associated with lysis and loss of tubular structure. In some cases organization of necrosed tubules was observed (Fig. 2). Trunk kidney of fish showed enlarged sinusoids and decreased amount of hematopoietic tissue. Vacuolar change was the only lesion observed in liver. Gills revealed separation of epithelium from lamellae, lamellar fusion and swelling of the epithelial cells. Further histopathological examination of their gills revealed that the fishes were seen infected with protozoan parasite, which was identified as Trichodina (Fig. 3). The highest infestation of the parasite was seen in the branchial cavity. The parasitic infection was associated with excessive mucus production, which resulted in respiratory problems. Due to this problem the fish piped most of the time on the surface of the water tank followed by rhythmic flashing and bouts of fringed activity.

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temperature C. c. communis got stress, which affected all biochemical and physiological activities. High stress conditions were observed at a temperature of 32 1C. At this temperature the fish showed less appetite for feed, irregular behavior of swimming and always gulped for air. However, the adaptive capability of C. c. communis enabled it to survive through stressful temperature conditions. Measurement of serum biochemical parameters gave general health status, and provided early warnings of damaged changes in stressed fishes. The results depicted in Table 1 showed increase in the blood glucose up to 28 1C, a sharp decrease was noticed at 32 1C. A prolonged exposure of high temperature of 32 1C showed negative effect on blood glucose of C. c. communis. Blood glucose does not show significant correlation with other biochemical parameters studied. The highest blood glucose level at low temperatures is indicative of retarded carbohydrate metabolism, and is also an index of sub-lethal stress (Smith et al., 1976; Connors et al., 1978; Best et al., 2001). Further decreased serum glucose concentrations could be the result of undernourishment as well as impaired hormonal control. A continuous decrease in total serum protein with increase in temperature was observed in our studies. As shown in results 66.47% decrease of total serum protein was observed at 32 1C over the control temperature of 20 1C. Decreased concentrations of serum protein are common in many disease status and may result from impaired synthesis (liver disease), reduced absorption or protein loss (Bernet et al., 2001). As reported by Walsh et al. (2003) the liver is the primary organ for urea production and gills appearing to be the main organ of excretion. A significant effect of temperature on BUN was

4. Discussion Temperature beyond optimum limits for a particular species, adversely affects the health of aquatic animals due to metabolic stress hence increases susceptibility to diseases that have suppressive effects on growth, reproduction and immune capacity (Cnaani, 2006). Our results clearly showed that with increase in

Fig. 3. Gill photomicrography of C. c. communis revealing presence of Trichodina and degenerative changes in lamellae at 321C for a period of one month (bar¼ 100 mm).

Fig. 2. Histology section of trunk and portion of head kidney from thermally stressed C. c. communis at 32 oC for a period of 1 month revealing (a) aggregation of melanomacrophagic centers (MMC’s, H and E, bar ¼200 mm) (b) hypertrophy of renal tubules following thermal stress (bar¼ 100 mm).

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found throughout all the treatments. Except for blood glucose, BUN showed significant correlation with all other biochemical parameters studied. Mensinger et al., 2005 reported that diseased fish showed significantly elevated BUN levels prior to dying, likely due to osmoregulatory failure. In our studies a variation of 28% in BUN was observed between 28 1C and 32 1C is an indicator of gill osmoregulatory problems. The elevated BUN is more likely associated with gill or liver disease and probably not indicative of renal disease (Mensinger et al., 2005). Osmoregulatory failure has often been demonstrated to be an important contributor to death in fish (Wood et al., 2003). Increased concentration of cholesterol and triglycerides with the rise in water temperature was observed in our study. This indicates disorders of lipid and lipoprotein metabolism and especially liver disease. The results suggest that the cells are able to adjust membrane fluidity by varying the concentration of cholesterol. These results are compatible with the concept of homeoviscous adaption: that fish strive to maintain an optimal level of membrane fluidity and when grown at different temperature will alter lipid composition in order to maintain this level. These results are consistent with that of Robertson and Hazel, 1995. Further, increased serum cholesterol concentrations can result from damage of liver or nephritic syndrome (Yamawaki et al., 1986; Seyit et al., 2000). Bernet et al., 2001, however reported that high concentrations of triglycerides may occur with nephritic syndrome or glycogen storage disease. Among the most sensitive and widely used liver enzymes are the aminotransferases. They include alanine aminotransferase (ALT or SGPT) and aspartate aminotransferase (AST or SGOT). These enzymes are normally contained within liver cells. The increased level of these enzymes in blood at high temperature in our study may be due to liver injury or hepatic metabolism hyperactivity of the experimental animals. The increased SGOT and SGPT values in fishes reveal enzymes exporting from liver into bloodstream (Yang and Chen, 2003; Perez-Rostro et al., 2004). According to Chen et al., 2004 increased level of SGOT and SGPT in Tilapia is associated with hepatic injury, acute injuries in trunk kidney, bacterial infection and myocardial infarction. Barcellos et al., 2003 suggested that acute stress condition of Rhamdia quelen Quoy and Gaimard Pimelodidae resulted in increased SGOT and SGPT due to hyper liver metabolism. Hematological profiles have often been used as stress indicators (Dobˇsikova et al., 2009). Major shifts in the haemogram are found in fish exposed to acute or chronic stress. The statistical analysis showed non-significant increase of RBC and Hb with the increase in acclimation temperature. These results are in agreement with those found by Zarejabad et al. (2009) who studied the effects of rearing temperature on hematological and biochemical parameters of great sturgeon and Carine Luı´sa et al. (2004) who studied the effect of different temperature regimes on metabolic and blood parameters of silver catfish. Naidu et al. (1989) reported that due to stress, fish tried to cope with adverse conditions by enhancing their respiratory capabilities through evaluated RBC and Hb synthesis. Our results also showed significant increase in Hct concentration over the control. In stressed fish an increase in RBC, hemoglobin and hematocrit concentration is often observed (Svobodova et al., 1994). Elevated hemoglobin and hematocrit increase the oxygen carrying capacity of blood, and thus the oxygen supply to major organs, in response to higher metabolic demand is the manifestation of stress (Ruane et al., 1999). However, prolonged exposure of stress may pose a great threat to their survival. The other hematological parameters affected by temperature fluctuations include MCH and MCHC. The results showed drop in MCH and MCHC is associated with the decrease in RBC and Hb. While examining the peripheral blood film after period of one month at 32 1C revealed poikilo-anisocytic

and hypochromic RBCs. These results clearly indicate stress and undernourishment (due to stopping of feeding in elevated temperatures) leading to anisocytic hypochromic anemia of experimental animals at highest temperature. This further suggests that C. c. communis has an adaptive ability upto certain limit of temperature. Alteration of hemoglobin leads to loss of hemoglobin solubility, resulting in structural damage to erythrocytes and it can subsequently cause rapid lyses of the erythrocytes (Bloom and Brandt, 2001; Everse and Hsia, 1997). As shown in Table 2, the results obtained for MCV are temperature-independent as it tends to stabilize at high temperatures. This also shows thermal adaptabity of experimental fishes to varied temperature conditions. The values of MCV, MCH and MCHC of the present study correspond to the results of Adeyemo et al. (2003) and Zarejabad et al. (2009). In present study the organs examined showed marked changes, which correlate well with the haemato–biochemical observation. Kidney (Bucher and Hofer, 1993), liver (ICES, 1997) and Gills (Poleksic and Mitrovic-Tutundzic, 1994) have been considered as suitable organs for histological examination in order to determine the effect of stress. The histopathological results of kidney showed vacuolar degeneration of tubular epithelium, loss of tubular structure and necrosis of the renal tubules. The present results are in agreement with those observed in Labeo rohita (Hamilton) acclimated to various temperatures (Dash et al., 2011). The liver plays a key role in the metabolism and biochemical transformations, which inevitably reflects on its integrity by creating lesions and other histopathological alterations of the liver parenchyma or the bile duct (Roberts, 1978). We found vacuolar change was the only lesion observed in liver. As discussed above the increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) may be due to hepatic pathological alterations of thermally stress fishes. Increased AST and ALT activities have been associated with hepatic pathology in fishes (Casillas et al., 1983). High temperatures can injure gills, thus reducing the oxygen consumption and disrupting the osmoregulatory function of fish (Ghate and Mulherkar, 1979; Saravana Bhavan and Geraldine, 2000). The present results showed degenerative changes of gills. These results are in congruence with the findings of earlier workers (Sunitha and Sahai, 1983; Roy and Munshi, 1991; Saravana Bhavan and Geraldine, 2000; Dash et al., 2011). The observation of parasitic infestation of gills in present study indicated the increased susceptibility of the thermally stressed fishes to such infections. Stress has been recognized as one of the important factors favouring parasitic infestation in fishes (Mcintyre, 1996). The pathological alterations observed in the gills of affected fishes may be ascribed partly to high parasitic load. The heavy load of parasite in branchial cavity associated with excess mucus production may be one of the factors causing osmoregulatory disturbance. Fish with severe gill infections of trichodina will have respiratory and osmoregulatory difficulty and may ‘‘pipe’’ as well as ‘‘flash’’ if there is cutaneous involvement (http://ag.ansc.purdue.edu/courses/aq448/diseases/para sites.htm). In summary, knowledge of the hematological and biochemical values is important tool that can be used as an effective and sensitive index to monitor physiological and pathological changes in a particular fish. Further, this study also supports the fact that fishes have a remarkable resilience to altered temperature especially with a prolonged time course and physiological changes during acclimation, which may help common carp of cold waters to accommodate climatic change. However, the study indicated the upper thermal limit as 32 1C for common carp as they couldn’t survive hotter conditions (above 32 1C) for more than a few hours.

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Acknowledgments We greatly appreciate all the help and support provided by Dr. M.H Balkhi, Dean Faculty of Fisheries SKUAST-K, Shalimar. The help of Dr. Masood Saleem Mir, for some valuable suggestions regarding histopathology is greatly acknowledged. We also thank an anonymous reviewer for very helpful comments. This work is the part of RCM, supported by Sher-e-Kashmir University of Agricultural sciences and Technology of Kashmir, India. References Adeyemo, O.K., Agbede, S.A., Olaniyan, A.O., Shoaga, O.A., 2003. The haematological response of Clarias gariepinus to changes in acclimation temperature. Afr. J. Biochem. Res. 6, 105–108. APHA/AWWA/WEF, 1998. Standard Methods for the Examination of Water and Wastewatertwentieth ed. American Public Health Association/American Water Works Association/Water Environment Federation, Washington, DC. 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