- Email: [email protected]
Effect of different temperature regimes on metabolic and blood parameters of silver catfish Rhamdia quelen
Aquaculture 239 (2004) 497 – 507 www.elsevier.com/locate/aqua-online
Effect of different temperature regimes on metabolic and blood parameters of silver catfish Rhamdia quelen Carine Luı´sa Lermena, Rosiele Lappea, Ma´rcia Crestania, Vaˆnia Pimentel Vieiraa, Carolina Rosa Giodaa, Maria Rosa Chitolina Schetingera, Bernardo Baldisserottob, Gilberto Moraesc, Vera Maria Morscha,* a
Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, Campus Camobi P 18, 97105-900, Santa Maria, RS, Brazil b Departamento de Fisiologia, Centro de Cieˆncias da Sau´de, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil c Departamento de Gene´tica e Evoluc¸a˜o, UFScar, Rodovia Washington Luı´s Km 235, Caixa Postal 676, 13565-905, Sa˜o Carlos, SP, Brazil Received 13 August 2003; received in revised form 18 June 2004; accepted 18 June 2004
Abstract The silver catfish (Rhamdia quelen) is a teleost native to South America. It can resist cold winters and grow quickly in the summer and is an important species for aquaculture in both temperate and subtropical climates. The effect of different water temperatures (15, 23 and 31 8C) on hematological and metabolic parameters in blood, liver and white muscle was investigated in this species following chronic (21 days) and acute (12 h) exposure. In both experiments, hematocrit, hemoglobin and cortisol were unchanged, but plasma glucose levels increased at a temperature of 31 8C and decreased at 15 8C, when compared to control. Fatty acid levels decreased at 31 8C, and triacylglycerol levels increased at 15 8C in the 21-day experiment compared to control. One of the most interesting results of this study was the decrease of total protein in the liver and white muscle of fish exposed to 31 8C for 21 days. Following 21 days, liver glycogen levels decreased at 15 and 31 8C and glucose and
* Corresponding author. Tel.: +55 220 8665; fax: +55 220 8978. E-mail address: [email protected] (V.M. Morsch). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.06.021
498
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
lactate contents increased at 31 8C compared to control. In white muscle, glycogen and glucose levels increased at 15 and 31 8C, while lactate decreased at 31 8C. No alterations were observed in the liver or white muscle of fish exposed to the different temperature for 12 h. Taken together, these results suggest that temperature and time of exposure influence metabolic parameters in the plasma, liver and white muscle of silver catfish. D 2004 Elsevier B.V. All rights reserved. Keywords: Fish; Temperature; Metabolism; Cortisol; Blood; Silver catfish
1. Introduction Temperature is known to affect the functional and structural properties of proteins in ectothermic animals like fishes. Both the habitat and a change of temperature can influence the catalytic properties of enzymes (Klyachko and Ozernyuk, 1998). However, the adaptability of fishes and their ability to exhibit normal activity at extremes of temperature suggest that cellular processes may be maintained at appropriate levels following a period of thermal acclimation or adaptation (Gerlach et al., 1990). In ectothermic organisms, physiological rates can be adjusted to compensate for some changes in temperature. In fish, thermal acclimation is generally determined by metabolic changes, during which an initial period of thermal stress is followed by a gradual compensation. When a stable metabolic level that is consistent between the old and new thermal state is reached, the animal is considered to be fully acclimated (MaricondiMassari et al., 1998). Hematological parameters are increasingly used as indicators of the physiological stress response to endogenous or exogenous changes in fish (Adams, 1990; Santos and Pacheco, 1996; Cataldi et al., 1998). In addition, plasma cortisol levels and alterations in carbohydrate metabolism, such as plasma glucose and lactate concentrations, can be used as general stress indicators in fish (Santos and Pacheco, 1996). Fish are generally thought to have a limited ability to utilize carbohydrate and possess low metabolic rates, especially when compared with mammals. The clearance of glucose from plasma is sluggish, leading a number of authors to regard fish in general as bglucoseintolerantQ (Mommsen and Plisetskaya, 1991; Wilson, 1994; Gallego et al., 1995; Wright et al., 1998). Some authors consider that fish utilize dietary carbohydrate poorly (Walton and Cowey, 1982), thus protein is an important energy fuel in fish (Perago´n et al., 1999; Kikuchi, 1999). However, these animals have a high capacity for anaerobic metabolism, and their skeletal muscle (40–60% body mass) is mainly composed of poorly vascularized anaerobic white fibers (75–100% total muscle mass) (Bennett, 1978). In South America, there is interest in the aquaculture of fish species that can survive cold winters and then grow fast in the warm summer. The silver catfish (Rhamdia quelen) have such characteristics (Barcellos et al., 2001, 2002). However, questions on the metabolic traits of the species and its ability to cope with environmental stressors have received little attention. Moreover, data on comparison and selection of species based on their biochemical potential are still lacking. Effects of three different temperatures during a chronic and an acute treatment on the metabolic and serum parameters of the freshwater fish
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
499
R. quelen were studied to investigate the ability of this species to cope with temperature stress. The range of temperatures selected in these trials was based on the environmental temperatures which the silver catfish encounters in South America. The questions posed in this study were: (i) Is thermal shock equally effective as long-term exposures? (ii) Is cold shock as stressful as heat shock? (iii) Are the metabolic changes, observed under temperature variations, a relevant tool to evaluate stress? This is a primary study to assess the thermal biochemical responses of R. quelen for future biological comparisons.
2. Materials and methods 2.1. Fish Silver catfish R. quelen obtained from a local fish farm were maintained in 250-l tanks with constantly aerated water for 7 days at a constant temperature of 20F0.3 8C. The animals were fed commercial fish food (SUPRA) daily to avoid the accumulation of food residues in the tanks. 2.2. Experimental procedure 2.2.1. Experiment 1 Twenty-four silver catfish (body weight 96F10.6 g; length 22.5F1.0 cm; n=6, meanFS.E.M.) were distributed evenly among four 250-l continuously aerated tanks. These were static systems cleaned by suction daily, where approximately 10% of the water in the tanks was replaced daily. The temperature was then changed over a period of 6–7 days to the target temperature of 15, 23 or 31 8C, whereas the control group was kept at 20 8C. The fish were maintained at these four temperatures for 3 weeks and then sampled. These temperatures were chosen in agreement with a previous experiment carried out on silver catfish: for specimens adapted at 21 8C, the 100% survival range to rapid water temperature change was observed between 11 and 31 8C (Chippari-Gomes et al., 1999). Consequently, the chosen temperatures were nonlethal but close to the thermal limits of this species. 2.2.2. Experiment 2 Twenty-four silver catfish (body weight 129F11.6 g; length 23.7F0.6 cm; n=6, meanFS.E.M.) were distributed evenly among four holding tanks. The temperature was then changed to the target temperature of 15, 23, or 31 8C over a period of 12 h, whereas the control group was kept at 20 8C. Fish were maintained at these four temperatures for 12 h and then sampled. 2.3. Water quality DO2, pH and temperature were monitored daily. Hardness and alkalinity were measured weekly according to standard methods for analysis of waste water (Boyd and Tucker, 1992), and total ammonia nitrogen (TAN), un-ionized ammonia-N and nitrite was determined three times a week.
500
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
2.4. Blood component analysis Blood samples were quickly collected from the caudal vein of serially netted and manually immobilized silver catfish without anesthesia. Blood was collected with heparinized syringes and plasma was obtained after centrifugation at 5400g for 10 min and stored at 20 8C for subsequent analysis. Plasma glucose (Labtest Kit), triacylglycerol (Chernecky et al., 1993) and fatty acids (Nova´k, 1965) were determined. For cortisol analysis, serum was collected with nonheparinized syringes, placed in a waterbath at 37 8C for 2 h, centrifuged at 5400g for 10 min and stored at 20 8C. Cortisol levels (ng/ml) were determined by chemiluminescence with an Immulite kit. The percentage of hematocrit and hemoglobin concentration (g/dl) was determined in whole blood. 2.5. Metabolite determination Liver and white muscle were dissected from freshly sacrificed animals and kept in a freezer at 20 8C until the time of use. Tissues were mechanically disrupted in an equal volume of 20% trichloracetic acid (TCA) using a motor-driven Teflon pestle. Acid homogenates were centrifuged at 10,000g for 10 min and the supernatants were used for metabolite determination. Glucose was estimated by the method of Duboie et al. (1956), lactate by the method of Harrower and Brown (1972), and protein by the method of Lowry et al. (1951). Tissue glycogen was determined after alkaline hydrolysis followed by ethanol precipitation as described by Bidinotto et al. (1997). 2.6. Statistical analysis All statistical analyses were carried out using the GraphPad Instat program (GraphPad software, version 2.05–1994). Data for water quality, hematocrit, hemoglobin, cortisol, triacylglycerol, fatty acid, glucose, lactate and glycogen obtained in each experiment were
Table 1 Hematological data for fish exposed to different temperatures for 21 days and 12 h Hematocrit (%)
Hemoglobin (g/dl)
Cortisol (ng/ml)
21 days Control 15 8C 23 8C 31 8C
35.33F2.75 31.83F3.74 34.16F1.30 26.00F2.22
10.16F0.79 8.50F0.50 10.00F0.51 8.00F0.68
25.20F6.28 36.40F2.42 24.40F6.69 23.20F5.93
12 h Control 15 8C 23 8C 31 8C
38.50F3.39 36.00F2.08 29.90F3.74 38.50F1.17
11.00F0.85 9.67F0.55 8.17F1.25 10.67F0.21
23.00F6.08 18.67F11.05 20.33F4.33 20.67F3.75
The values represent the meanFS.E.M., n=6.
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
501
Fig. 1. Plasma from fish exposed to different temperatures for 21 days. Data represent the meanFS.E.M. (n=6). The control values were: 169.00F9.12, 65.50F3.05 and 44.16F0.48 for fatty acid (nmol/ml of plasma), glucose (mg/dl) and triacylglycerol (mg/dl) levels, respectively. Different from the control group: *pb0.05; **pb0.001.
tested for significant differences by one-way analysis of variance followed by the Tukey– Kramer test. P values b0.05 were considered to be significant.
3. Results The water quality parameters (DO2, pH, hardness, alkalinity, total ammonia nitrogen, un-ionized ammonia-N and nitrite) were not changed by the different temperatures (data not shown). Hematocrit, hemoglobin and cortisol were also unchanged after both chronic and acute exposures (Table 1).
Fig. 2. Plasma from fish exposed to different temperatures for 12 h. Data represent the meanFS.E.M. (n=6). The control values were: 169.17F3.29, 58.50F1.17 and 42.17F1.28 for fatty acid (nmol/ml of plasma), glucose (mg/ dl) and triacylglycerol (mg/dl) levels, respectively. Different from the control group: **pb0.001.
502
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
Table 2 Total protein level in liver and white muscle from fish exposed to different temperatures during 21 days and 12 h Liver (mg protein/g tissue)
White muscle (mg protein/g tissue)
21 days Control 15 8C 23 8C 31 8C
16.17F0.87 20.00F1.34 15.33F0.76 8.17F0.79*
65.83F2.64 61.67F4.34 74.33F6.90 23.00F1.48*
12 h Control 15 8C 23 8C 31 8C
17.33F0.61 15.83F1.08 16.83F1.64 17.33F0.88
64.12F3.52 77.00F3.96 70.00F2.23 71.83F2.24
The values represent the meanFS.E.M., (n=6). *pb0.001 compared to control.
Plasma glucose levels increased in the 21-day experiment in silver catfish after exposure to 31 8C [ F(3.20)=25.61; pb0.05] and decreased after exposure to 15 8C [ F(3.20)=25.61; pb0.001]. Fatty acid level was decreased in plasma at 31 8C [ F(3.20)=7.70; pb0.05] compared to control. Triacylglycerol level decreased significantly in fish exposed to 15 8C [ F(3.20)=21.38; pb0.001] (Fig. 1). In the 12-h experiment, glucose levels decreased at 15 8C and increased at 31 8C [ F(3.20)=48.09; pb0.001], whereas fatty acid and triacylglycerol levels did not differ from control (Fig. 2).
Fig. 3. Liver from fish exposed to different temperatures during 21 days. Data represent the meanFS.E.M. (n=6). The control values were: 185.5F18.1, 45F1.5 and 20.50F2.62 for glycogen, glucose and lactate levels, respectively. The levels were expressed as umol/g of tissue. Different from the control group: *pb0.05; **pb0.001.
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
503
Table 3 Parameters for liver and white muscle from fish exposed to different temperatures for 12 h Glycogen (umol/g tissue) Liver Control 15 8C 23 8C 31 8C White muscle Control 15 8C 23 8C 31 8C
Glucose (umol/g tissue)
Lactate (umol/g tissue)
225F22.2 180F10.3 210F15.5 195F20.1
21.72F1.55 20.70F1.85 24.99F2.05 25.13F1.95
12.33F1.30 10.17F1.94 14.67F1.65 9.00F2.24
32.67F1.33 25.50F1.12 35.67F3.23 32.83F2.87
27.00F0.63 26.16F1.81 26.00F2.55 32.17F1.10
63.50F5.25 68.83F5.91 66.50F5.82 73.17F5.32
The values represent the meanFS.E.M., (n=6).
Protein content in the liver and white muscle of fish exposed to 31 8C decreased in the chronic experiment compared to control. In the 12-h experiment, no changes were observed (Table 2). In the liver of fish acclimated for 21 days, glycogen levels decreased at 15 and 31 8C [ F(3.20)=9.67; pb0.05] compared to control, and glucose content was only increased at 31 8C [ F(20.3)=7.23; pb0.001]. Lactate increased at 31 8C compared to control [ F(20.3)=4.08; pb0.05] (Fig. 3). No alterations were observed in the liver of fish exposed for 12 h (Table 3). In the 21-day experiment, white muscle showed an increase in glycogen content at 15 8C [ F(3.20)=8.01; pb0.05]. Glucose levels increased at the highest temperature [ F(3.20)=6.34; pb0.05], whereas lactate levels decreased at this same temperature
Fig. 4. White muscle from fish exposed to different temperatures for 21 days. Data represent the meanFS.E.M. (n=6). The control values were: 34.33F2.25, 26.67F0.84 and 54.00F3.44 for glycogen, glucose and lactate levels, respectively. The levels were expressed as umol/g of tissue. Different from the control group: *pb0.05; **pb0.001.
504
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
[ F(3.20)=15.65; pb0.001] compared to control (Fig. 4). No alterations were observed in the white muscle of fish exposed to the three different temperatures for 12 h (Table 3).
4. Discussion The mean water temperature in South Brazil, where the most extensive culture of silver catfish exists, is within the 15–30 8C range (Chippari-Gomes et al., 1999; Carvalho et al., 2003). Water temperature significantly affects some physiological fish processes such as growth and metabolism. Nonoptimal water temperature, insufficient food and low dietary protein have been found to inhibit fish growth (Fine et al., 1996). As shown in Table 1, hematocrit and hemoglobin concentrations were not altered by the different water temperatures. The values detected in our experiment are in agreement with those found by Tavares-Dias et al. (2002) who studied hematologic characteristics R. quelen at 25 8C. This fact attests good tolerance of the species to this range of temperature variation. Alterations of serum cortisol level are considered to be a primary indicator of the stress response (Roche and Boge´, 1996; Cataldi et al., 1998). In a study by Chen et al. (1995), plasma cortisol concentrations increased in common carp only after acute exposure to 4 8C, but in the chronic experiment, they returned to levels similar to those of the control group. The results obtained with Adriatic sturgeon (Acipenser naccarii) showed that colder temperatures decreased cortisol concentrations (Cataldi et al., 1998). Another experiment on common carp (Cyprinus carpio) showed a significant increase in plasma cortisol levels after single or multiple rapid temperature drops (Tanck et al., 2000). In our experiment, plasma cortisol levels were unchanged in both the acute and chronic treatments. This result plus the observed hematological responses indicate that the range of temperature employed in these experiments did not act as a stressor. A very interesting finding of this study is the decrease of total protein in the liver and white muscle only in the fish exposed at 31 8C for 21 days. This reduction cannot be explained solely by the effect of elevated temperature and implies that after 21 days of exposure to 31 8C, fish may be using protein as an energy source. In an experiment with Ictalurus melas, blood sugar levels were studied in relation to seasonal periods (spring and autumn) and were found not to differ significantly between seasons, although they decreased with increasing water temperature (Ottolenghi et al., 1995). Sun et al. (1992, 1995) observed a significant hyperglycemia in tilapia Oreochromis niloticus L. subjected to a 14–16 8C temperature, but this hyperglycemia only became visible at least 24 h after the beginning of the experiment. The glycemic response of R. quelen differs from the examples above; our results showed hyperglycemia in the fish exposed to 31 8C and hypoglycemia in the fish exposed to 15 8C in both the acute and chronic experiments. Interestingly, this metabolic response was not associated with the change in hematology or cortisol. It may be assumed that water temperature could modify the rate of lipolysis (fatty acid mobilization from triacylglycerol) or have an impact on fatty acid reesterification. Lipolysis always exceeds oxidative needs; therefore, a fraction of all the fatty acid released must be reesterified—simultaneous flux through lipolysis and reesterification from the triacylglycerol/fatty acid cycle, a substrate cycle known to play an important role in
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
505
adjusting fatty acid supply during exercise (Wolfe et al., 1990). In the present study, it was observed that triacylglycerol level decreased at 15 8C in the chronic experiment, with mobilization of fat stores probably occurring. At the temperature of 31 8C, there was a decrease of fatty acid, probably due to an increase on metabolic rate with fatty acid consumption by the tissue. The liver is a glucose-utilizing, glucose-producing and glucose-storing organ. As such, it acts as a glucostat in the vertebrate organism, regulating glucose levels of blood (Moon and Foster, 1995). The major part of intracellular glucose in teleost hepatocytes is channeled into glycogen (Pereira et al., 1995). In the present study, an increased glucose level in liver and white muscle after 21 days of exposure to 31 8C was observed. This fact, associated with the decrease of hepatic glycogen and protein concentrations, suggests glucose export from the liver to white muscle through a couple of associated mechanisms, gluconeogenesis and glycogenolysis. The decrease in muscle protein indicates that the high temperature (31 8C) was sufficiently stressful to extend proteolysis toward other tissues. Higher temperature demands protein as an energy source in addition to sugars. Glycogen levels in fish liver vary over a wide range of concentrations from 20 to 2000 Amol g 1 liver (Moon and Foster, 1995). Once formed, fish liver glycogen is remobilized with relative difficulty, whereas muscle glycogen is rapidly used to provide energy. After white muscle, liver glycogen represents the major carbohydrate store in the fish body. Hepatic glycogen is rapidly mobilized during stress by catecholamines (Ottolenghi et al., 1984; Janssens and Lowrey, 1987). Our results demonstrate that liver glycogen was decreased either at 15 or 31 8C. However, muscle displayed a different behavior to cope with cold and warm waters. Under cold conditions, the silver catfish spared glucose, resulting in gluconeogenesis, whereas in the warm water, it likely consumes glucose. Increase of liver and decrease of the muscular lactate in fish exposed to warm waters suggest aerobic catabolism of glucose with significant cooperation of the liver. The present results demonstrate that silver catfish do not show the classical stress response either to cold or heat shock. However, despite the lack of change in cortisol and hematological values with temperature, the metabolic responses were distinct. As should be expected, the chronic thermal exposures resulted in greater metabolic changes than acute exposure. Heat shock was more stressful to silver catfish than cold shock. The metabolic demand of the species was likely higher as fish exposed to warm water were metabolizing lipids, carbohydrates and even protein. In addition, the use of metabolism as an indicator of stress in the silver catfish was a good tool to evaluate long-term changes, and glucose was a better index than the classical tools usually applied. We can conclude that the present set of data can partially explain the ability of R. quelen to resist a wide range of environmental temperatures.
References Adams, S.M., 1990. Biological Indicators of Stress in Fish. American Fisheries Society, Bethesda, MD, pp. 1 – 8. Barcellos, L.J.G., Wassermann, G.F., Scott, A.P., Woehl, V.M., Quevedo, R.M., Ittze´s, I., Krieger, M.H., Lulhier, F., 2001. Steroid profiles in cultured female Jundia´, the siluridae Rhamdia quelen (Quoy and Gaimard, Pisces Teleostei) during the first reproductive cycle. General and Comparative Endocrinology 121, 325 – 332.
506
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
Barcellos, L.J.G., Wassermann, G.F., Scott, A.P., Woehl, V.M., Quevedo, R.M., Ittze´s, I., Krieger, M.H., Lulhier, F., 2002. Plasma steroid concentrations in relation to the reproductive cycle of cultured male Rhamdia quelen. Journal of Fish Biology 61, 751 – 763. Bennett, A.F., 1978. Activity metabolism of lower vertebrates. Annual Review of Physiology 400, 447 – 469. Bidinotto, P.M., Moraes, G., Souza, R.H.S., 1997. Hepatic glycogen and glucose in eight tropical freshwater teleost fish: a procedure for field determinations of micro samples. Boletim Te´cnico CEPTA. Pirassununga 10, 53 – 60. Boyd, C.E., Tucker, C.S., 1992. Water Quality and Pond Soil Analyses for Aquaculture. Alabama Agricultural Experiment Station, Auburn University, Alabama, USA. 183 pp. Carvalho, P., Thomaz, S.M., Bini, L.M., 2003. Effects of water levels, abiotic and abiotic factors on bacterioplankton abundance in lagoons of a tropical floodplain (Parana´ River, Brazil). Hydrobiologia 510, 67 – 74. Cataldi, E., Di Marco, P., Mandich, A., Cataudella, S., 1998. Serum parameters of Adriatic sturgeon Acipenser naccarii (Pisces: Acipenseriformes); effects of temperature and stress. Comparative Biochemistry and Physiology 121 A, 351 – 354. Chen, G.R., Sun, L.T., Lee, Y.H., Chang, C.F., 1995. Characteristics of blood in common carp, Cyprinus carpio, exposed to low temperatures. Journal of Applied Aquaculture 5 (3), 21 – 31. Chernecky, C.C., Krech, R.L., Berger, B.J., 1993. Laboratory Tests and Diagnostic Procedures. WB, Saunders, Philadelphia, pp. 932 – 933. Chippari-Gomes, A.R., Gomes, L.C., Baldisserotto, B., 1999. Lethal temperatures for Silver Catfish, Rhamdia quelen, fingerlings. Journal of Applied Aquaculture 9 (4), 11 – 21. Duboie, M., Gilles, K.A., Hamilton, J.K., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350 – 358. Fine, M., Zilberg, D., Cohen, Z., Degani, G., Moav, B., Gertler, A., 1996. The effect of dietary protein level, water temperature and growth hormone administration on growth and metabolism in the common carp (Cyprinus carpio). Comparative Biochemistry and Physiology 114 A, 35 – 42. Gallego, M.G., Bazoco, J., Sua´rez, M.D., Sanz, A., 1995. Utilization of dietary carbohydrates by fish: a comparative study in eel and trout. Animal Science 61, 427 – 436. Gerlach, G.F., Turay, L., Malik, K.T.A., Lida, J., Scutt, A., Goldspink, G., 1990. Mechanisms of temperature acclimation in carp: a molecular biology approach. American Journal of Physiology 259, R237 – R244. Harrower, J.R., Brown, C.H., 1972. Blood lactic acid. A micromethod adaptable to field collection of microliter samples. Journal of Applied Physiology 32 (5), 224 – 228. Janssens, P.A., Lowrey, P., 1987. Hormonal regulation of hepatic glycogenolysis in the carp, Cyprinus carpio. American Journal of Physiology 252, R653 – R660. Kikuchi, K., 1999. Use of defatted soybean meal as a substitute for fish meal in diets of Japanese flounder Paralichthys olivaceus. Aquaculture 179, 3 – 11. Klyachko, O.S., Ozernyuk, N.D., 1998. Functional and structural properties of lactate dehydrogenase from embryos of different fishes. Comparative Biochemistry and Physiology 119B, 77 – 80. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 – 275. Maricondi-Massari, M., Kalinin, A.L., Glass, M.L., Rantin, F.T., 1998. The effects of temperature on oxygen uptake, gill ventilation and ECG waveforms in the Nile tilapia, Oreochromis niloticus. Journal of Thermal Biology 23, 283 – 290. Mommsen, T.P., Plisetskaya, E.M., 1991. Insulin in fishes and agnathans: history, structure and metabolic regulation. Review of Aquaculture Science 4, 225 – 259. Moon, T.W., Foster, G.D., 1995. Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences. In: Hochachka, P.W., Mommsen, T.O. (Eds.), Biochemistry and Molecular Biology of Fish, Metabolic Biochemistry, vol. 4. Elsevier Sciences Publishers, New York, pp. 65 – 100. Nova´k, M., 1965. Colorimetric ultramicro method for the determination of free fatty acids. Journal of Lipid Research 6, 431 – 433. Ottolenghi, C., Puviani, A.C., Baruffaldi, A., Brighenti, L., 1984. Effect of insulin on glycogen metabolism in isolated catfish hepatocytes. Comparative Biochemistry and Physiology 78 A, 705 – 710.
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
507
Ottolenghi, C., Puviani, A.C., Ricci, D., Morsiani, E., 1995. The effect of high temperature on blood glucose level in two teleost fish (Ictalurus melas and Ictalurus punctatus). Comparative Biochemistry and Physiology 111 A, 229 – 235. Perago´n, J., Barroso, J.B., Garcia-Salguero, L., Letı´cia, B., De La Higuera, M., Lupian˜ez, J.A., 1999. Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout (Oncorhynchus mykiss). Aquaculture 179 (3–4), 425 – 437. Pereira, C., Vijayan, M.M., Storey, K.B., Jones, R.A., Moon, T.W., 1995. Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes. Journal of Comparative Physiology 165, 62 – 70. Roche, H., Boge´, G., 1996. Fish blood parameters as a potential tool for identification of stress caused by environmental factors and chemical intoxication. Marine Environmental Research 41, 27 – 43. Santos, M.A., Pacheco, M., 1996. Anguilla anguilla L. Stress biomarkers recovery in clean water and secondarytreated pulp mill effluent. Ecotoxicology and Environmental Safety 35, 96 – 100. Sun, L.T., Chen, G.R., Chang, C.F., 1992. The physiological responses of tilapia exposed to low temperatures. Journal of Thermal Biology 17, 149 – 153. Sun, L.T., Chen, G.R., Chang, C.F., 1995. Acute responses of blood parameters and comatose effects in saltacclimated tilapias exposed to low temperatures. Journal of Thermal Biology 20, 299 – 306. Tanck, M.W.T., Booms, G.H.R., Eding, E.H., Bonga, S.E.W., Komen, J., 2000. Cold shocks: a stressor for common carp. Journal of Fish Biology 57, 881 – 894. Tavares-Dias, M., Melo, J.F.B., Moraes, G., Moraes, F.R., 2002. Caracterı´sticas hematolo´gicas de teleo´steos brasileiros: IV. Varia´veis do jundia´ Rhamdia quelen (Pimelodidae). Cieˆncia Rural 32, 693 – 698. Walton, M.J., Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish. Comparative Biochemistry and Physiology 73B, 59 – 79. Wilson, R.P., 1994. Utilization of dietary carbohydrates in fish. Aquaculture 124, 67 – 80. Wolfe, R.R., Klein, S., Carraro, F., Weber, J.M., 1990. Role of triglyceride–fatty acid cycle in controlling fat metabolism in humans during and after exercise. American Journal of Physiology 258, E382 – E389. Wright, J.R., O’Hali, W., Yang, H., Han, X., Bonen, A., 1998. GLUT-4 deficiency and absolute peripheral resistance to insulin in the teleost fish tilapia. General and Comparative Endocrinology 111 (1), 20 – 27.
Effect of different temperature regimes on metabolic and blood parameters of silver catfish Rhamdia quelen Carine Luı´sa Lermena, Rosiele Lappea, Ma´rcia Crestania, Vaˆnia Pimentel Vieiraa, Carolina Rosa Giodaa, Maria Rosa Chitolina Schetingera, Bernardo Baldisserottob, Gilberto Moraesc, Vera Maria Morscha,* a
Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, Campus Camobi P 18, 97105-900, Santa Maria, RS, Brazil b Departamento de Fisiologia, Centro de Cieˆncias da Sau´de, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil c Departamento de Gene´tica e Evoluc¸a˜o, UFScar, Rodovia Washington Luı´s Km 235, Caixa Postal 676, 13565-905, Sa˜o Carlos, SP, Brazil Received 13 August 2003; received in revised form 18 June 2004; accepted 18 June 2004
Abstract The silver catfish (Rhamdia quelen) is a teleost native to South America. It can resist cold winters and grow quickly in the summer and is an important species for aquaculture in both temperate and subtropical climates. The effect of different water temperatures (15, 23 and 31 8C) on hematological and metabolic parameters in blood, liver and white muscle was investigated in this species following chronic (21 days) and acute (12 h) exposure. In both experiments, hematocrit, hemoglobin and cortisol were unchanged, but plasma glucose levels increased at a temperature of 31 8C and decreased at 15 8C, when compared to control. Fatty acid levels decreased at 31 8C, and triacylglycerol levels increased at 15 8C in the 21-day experiment compared to control. One of the most interesting results of this study was the decrease of total protein in the liver and white muscle of fish exposed to 31 8C for 21 days. Following 21 days, liver glycogen levels decreased at 15 and 31 8C and glucose and
* Corresponding author. Tel.: +55 220 8665; fax: +55 220 8978. E-mail address: [email protected] (V.M. Morsch). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.06.021
498
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
lactate contents increased at 31 8C compared to control. In white muscle, glycogen and glucose levels increased at 15 and 31 8C, while lactate decreased at 31 8C. No alterations were observed in the liver or white muscle of fish exposed to the different temperature for 12 h. Taken together, these results suggest that temperature and time of exposure influence metabolic parameters in the plasma, liver and white muscle of silver catfish. D 2004 Elsevier B.V. All rights reserved. Keywords: Fish; Temperature; Metabolism; Cortisol; Blood; Silver catfish
1. Introduction Temperature is known to affect the functional and structural properties of proteins in ectothermic animals like fishes. Both the habitat and a change of temperature can influence the catalytic properties of enzymes (Klyachko and Ozernyuk, 1998). However, the adaptability of fishes and their ability to exhibit normal activity at extremes of temperature suggest that cellular processes may be maintained at appropriate levels following a period of thermal acclimation or adaptation (Gerlach et al., 1990). In ectothermic organisms, physiological rates can be adjusted to compensate for some changes in temperature. In fish, thermal acclimation is generally determined by metabolic changes, during which an initial period of thermal stress is followed by a gradual compensation. When a stable metabolic level that is consistent between the old and new thermal state is reached, the animal is considered to be fully acclimated (MaricondiMassari et al., 1998). Hematological parameters are increasingly used as indicators of the physiological stress response to endogenous or exogenous changes in fish (Adams, 1990; Santos and Pacheco, 1996; Cataldi et al., 1998). In addition, plasma cortisol levels and alterations in carbohydrate metabolism, such as plasma glucose and lactate concentrations, can be used as general stress indicators in fish (Santos and Pacheco, 1996). Fish are generally thought to have a limited ability to utilize carbohydrate and possess low metabolic rates, especially when compared with mammals. The clearance of glucose from plasma is sluggish, leading a number of authors to regard fish in general as bglucoseintolerantQ (Mommsen and Plisetskaya, 1991; Wilson, 1994; Gallego et al., 1995; Wright et al., 1998). Some authors consider that fish utilize dietary carbohydrate poorly (Walton and Cowey, 1982), thus protein is an important energy fuel in fish (Perago´n et al., 1999; Kikuchi, 1999). However, these animals have a high capacity for anaerobic metabolism, and their skeletal muscle (40–60% body mass) is mainly composed of poorly vascularized anaerobic white fibers (75–100% total muscle mass) (Bennett, 1978). In South America, there is interest in the aquaculture of fish species that can survive cold winters and then grow fast in the warm summer. The silver catfish (Rhamdia quelen) have such characteristics (Barcellos et al., 2001, 2002). However, questions on the metabolic traits of the species and its ability to cope with environmental stressors have received little attention. Moreover, data on comparison and selection of species based on their biochemical potential are still lacking. Effects of three different temperatures during a chronic and an acute treatment on the metabolic and serum parameters of the freshwater fish
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
499
R. quelen were studied to investigate the ability of this species to cope with temperature stress. The range of temperatures selected in these trials was based on the environmental temperatures which the silver catfish encounters in South America. The questions posed in this study were: (i) Is thermal shock equally effective as long-term exposures? (ii) Is cold shock as stressful as heat shock? (iii) Are the metabolic changes, observed under temperature variations, a relevant tool to evaluate stress? This is a primary study to assess the thermal biochemical responses of R. quelen for future biological comparisons.
2. Materials and methods 2.1. Fish Silver catfish R. quelen obtained from a local fish farm were maintained in 250-l tanks with constantly aerated water for 7 days at a constant temperature of 20F0.3 8C. The animals were fed commercial fish food (SUPRA) daily to avoid the accumulation of food residues in the tanks. 2.2. Experimental procedure 2.2.1. Experiment 1 Twenty-four silver catfish (body weight 96F10.6 g; length 22.5F1.0 cm; n=6, meanFS.E.M.) were distributed evenly among four 250-l continuously aerated tanks. These were static systems cleaned by suction daily, where approximately 10% of the water in the tanks was replaced daily. The temperature was then changed over a period of 6–7 days to the target temperature of 15, 23 or 31 8C, whereas the control group was kept at 20 8C. The fish were maintained at these four temperatures for 3 weeks and then sampled. These temperatures were chosen in agreement with a previous experiment carried out on silver catfish: for specimens adapted at 21 8C, the 100% survival range to rapid water temperature change was observed between 11 and 31 8C (Chippari-Gomes et al., 1999). Consequently, the chosen temperatures were nonlethal but close to the thermal limits of this species. 2.2.2. Experiment 2 Twenty-four silver catfish (body weight 129F11.6 g; length 23.7F0.6 cm; n=6, meanFS.E.M.) were distributed evenly among four holding tanks. The temperature was then changed to the target temperature of 15, 23, or 31 8C over a period of 12 h, whereas the control group was kept at 20 8C. Fish were maintained at these four temperatures for 12 h and then sampled. 2.3. Water quality DO2, pH and temperature were monitored daily. Hardness and alkalinity were measured weekly according to standard methods for analysis of waste water (Boyd and Tucker, 1992), and total ammonia nitrogen (TAN), un-ionized ammonia-N and nitrite was determined three times a week.
500
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
2.4. Blood component analysis Blood samples were quickly collected from the caudal vein of serially netted and manually immobilized silver catfish without anesthesia. Blood was collected with heparinized syringes and plasma was obtained after centrifugation at 5400g for 10 min and stored at 20 8C for subsequent analysis. Plasma glucose (Labtest Kit), triacylglycerol (Chernecky et al., 1993) and fatty acids (Nova´k, 1965) were determined. For cortisol analysis, serum was collected with nonheparinized syringes, placed in a waterbath at 37 8C for 2 h, centrifuged at 5400g for 10 min and stored at 20 8C. Cortisol levels (ng/ml) were determined by chemiluminescence with an Immulite kit. The percentage of hematocrit and hemoglobin concentration (g/dl) was determined in whole blood. 2.5. Metabolite determination Liver and white muscle were dissected from freshly sacrificed animals and kept in a freezer at 20 8C until the time of use. Tissues were mechanically disrupted in an equal volume of 20% trichloracetic acid (TCA) using a motor-driven Teflon pestle. Acid homogenates were centrifuged at 10,000g for 10 min and the supernatants were used for metabolite determination. Glucose was estimated by the method of Duboie et al. (1956), lactate by the method of Harrower and Brown (1972), and protein by the method of Lowry et al. (1951). Tissue glycogen was determined after alkaline hydrolysis followed by ethanol precipitation as described by Bidinotto et al. (1997). 2.6. Statistical analysis All statistical analyses were carried out using the GraphPad Instat program (GraphPad software, version 2.05–1994). Data for water quality, hematocrit, hemoglobin, cortisol, triacylglycerol, fatty acid, glucose, lactate and glycogen obtained in each experiment were
Table 1 Hematological data for fish exposed to different temperatures for 21 days and 12 h Hematocrit (%)
Hemoglobin (g/dl)
Cortisol (ng/ml)
21 days Control 15 8C 23 8C 31 8C
35.33F2.75 31.83F3.74 34.16F1.30 26.00F2.22
10.16F0.79 8.50F0.50 10.00F0.51 8.00F0.68
25.20F6.28 36.40F2.42 24.40F6.69 23.20F5.93
12 h Control 15 8C 23 8C 31 8C
38.50F3.39 36.00F2.08 29.90F3.74 38.50F1.17
11.00F0.85 9.67F0.55 8.17F1.25 10.67F0.21
23.00F6.08 18.67F11.05 20.33F4.33 20.67F3.75
The values represent the meanFS.E.M., n=6.
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
501
Fig. 1. Plasma from fish exposed to different temperatures for 21 days. Data represent the meanFS.E.M. (n=6). The control values were: 169.00F9.12, 65.50F3.05 and 44.16F0.48 for fatty acid (nmol/ml of plasma), glucose (mg/dl) and triacylglycerol (mg/dl) levels, respectively. Different from the control group: *pb0.05; **pb0.001.
tested for significant differences by one-way analysis of variance followed by the Tukey– Kramer test. P values b0.05 were considered to be significant.
3. Results The water quality parameters (DO2, pH, hardness, alkalinity, total ammonia nitrogen, un-ionized ammonia-N and nitrite) were not changed by the different temperatures (data not shown). Hematocrit, hemoglobin and cortisol were also unchanged after both chronic and acute exposures (Table 1).
Fig. 2. Plasma from fish exposed to different temperatures for 12 h. Data represent the meanFS.E.M. (n=6). The control values were: 169.17F3.29, 58.50F1.17 and 42.17F1.28 for fatty acid (nmol/ml of plasma), glucose (mg/ dl) and triacylglycerol (mg/dl) levels, respectively. Different from the control group: **pb0.001.
502
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
Table 2 Total protein level in liver and white muscle from fish exposed to different temperatures during 21 days and 12 h Liver (mg protein/g tissue)
White muscle (mg protein/g tissue)
21 days Control 15 8C 23 8C 31 8C
16.17F0.87 20.00F1.34 15.33F0.76 8.17F0.79*
65.83F2.64 61.67F4.34 74.33F6.90 23.00F1.48*
12 h Control 15 8C 23 8C 31 8C
17.33F0.61 15.83F1.08 16.83F1.64 17.33F0.88
64.12F3.52 77.00F3.96 70.00F2.23 71.83F2.24
The values represent the meanFS.E.M., (n=6). *pb0.001 compared to control.
Plasma glucose levels increased in the 21-day experiment in silver catfish after exposure to 31 8C [ F(3.20)=25.61; pb0.05] and decreased after exposure to 15 8C [ F(3.20)=25.61; pb0.001]. Fatty acid level was decreased in plasma at 31 8C [ F(3.20)=7.70; pb0.05] compared to control. Triacylglycerol level decreased significantly in fish exposed to 15 8C [ F(3.20)=21.38; pb0.001] (Fig. 1). In the 12-h experiment, glucose levels decreased at 15 8C and increased at 31 8C [ F(3.20)=48.09; pb0.001], whereas fatty acid and triacylglycerol levels did not differ from control (Fig. 2).
Fig. 3. Liver from fish exposed to different temperatures during 21 days. Data represent the meanFS.E.M. (n=6). The control values were: 185.5F18.1, 45F1.5 and 20.50F2.62 for glycogen, glucose and lactate levels, respectively. The levels were expressed as umol/g of tissue. Different from the control group: *pb0.05; **pb0.001.
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
503
Table 3 Parameters for liver and white muscle from fish exposed to different temperatures for 12 h Glycogen (umol/g tissue) Liver Control 15 8C 23 8C 31 8C White muscle Control 15 8C 23 8C 31 8C
Glucose (umol/g tissue)
Lactate (umol/g tissue)
225F22.2 180F10.3 210F15.5 195F20.1
21.72F1.55 20.70F1.85 24.99F2.05 25.13F1.95
12.33F1.30 10.17F1.94 14.67F1.65 9.00F2.24
32.67F1.33 25.50F1.12 35.67F3.23 32.83F2.87
27.00F0.63 26.16F1.81 26.00F2.55 32.17F1.10
63.50F5.25 68.83F5.91 66.50F5.82 73.17F5.32
The values represent the meanFS.E.M., (n=6).
Protein content in the liver and white muscle of fish exposed to 31 8C decreased in the chronic experiment compared to control. In the 12-h experiment, no changes were observed (Table 2). In the liver of fish acclimated for 21 days, glycogen levels decreased at 15 and 31 8C [ F(3.20)=9.67; pb0.05] compared to control, and glucose content was only increased at 31 8C [ F(20.3)=7.23; pb0.001]. Lactate increased at 31 8C compared to control [ F(20.3)=4.08; pb0.05] (Fig. 3). No alterations were observed in the liver of fish exposed for 12 h (Table 3). In the 21-day experiment, white muscle showed an increase in glycogen content at 15 8C [ F(3.20)=8.01; pb0.05]. Glucose levels increased at the highest temperature [ F(3.20)=6.34; pb0.05], whereas lactate levels decreased at this same temperature
Fig. 4. White muscle from fish exposed to different temperatures for 21 days. Data represent the meanFS.E.M. (n=6). The control values were: 34.33F2.25, 26.67F0.84 and 54.00F3.44 for glycogen, glucose and lactate levels, respectively. The levels were expressed as umol/g of tissue. Different from the control group: *pb0.05; **pb0.001.
504
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
[ F(3.20)=15.65; pb0.001] compared to control (Fig. 4). No alterations were observed in the white muscle of fish exposed to the three different temperatures for 12 h (Table 3).
4. Discussion The mean water temperature in South Brazil, where the most extensive culture of silver catfish exists, is within the 15–30 8C range (Chippari-Gomes et al., 1999; Carvalho et al., 2003). Water temperature significantly affects some physiological fish processes such as growth and metabolism. Nonoptimal water temperature, insufficient food and low dietary protein have been found to inhibit fish growth (Fine et al., 1996). As shown in Table 1, hematocrit and hemoglobin concentrations were not altered by the different water temperatures. The values detected in our experiment are in agreement with those found by Tavares-Dias et al. (2002) who studied hematologic characteristics R. quelen at 25 8C. This fact attests good tolerance of the species to this range of temperature variation. Alterations of serum cortisol level are considered to be a primary indicator of the stress response (Roche and Boge´, 1996; Cataldi et al., 1998). In a study by Chen et al. (1995), plasma cortisol concentrations increased in common carp only after acute exposure to 4 8C, but in the chronic experiment, they returned to levels similar to those of the control group. The results obtained with Adriatic sturgeon (Acipenser naccarii) showed that colder temperatures decreased cortisol concentrations (Cataldi et al., 1998). Another experiment on common carp (Cyprinus carpio) showed a significant increase in plasma cortisol levels after single or multiple rapid temperature drops (Tanck et al., 2000). In our experiment, plasma cortisol levels were unchanged in both the acute and chronic treatments. This result plus the observed hematological responses indicate that the range of temperature employed in these experiments did not act as a stressor. A very interesting finding of this study is the decrease of total protein in the liver and white muscle only in the fish exposed at 31 8C for 21 days. This reduction cannot be explained solely by the effect of elevated temperature and implies that after 21 days of exposure to 31 8C, fish may be using protein as an energy source. In an experiment with Ictalurus melas, blood sugar levels were studied in relation to seasonal periods (spring and autumn) and were found not to differ significantly between seasons, although they decreased with increasing water temperature (Ottolenghi et al., 1995). Sun et al. (1992, 1995) observed a significant hyperglycemia in tilapia Oreochromis niloticus L. subjected to a 14–16 8C temperature, but this hyperglycemia only became visible at least 24 h after the beginning of the experiment. The glycemic response of R. quelen differs from the examples above; our results showed hyperglycemia in the fish exposed to 31 8C and hypoglycemia in the fish exposed to 15 8C in both the acute and chronic experiments. Interestingly, this metabolic response was not associated with the change in hematology or cortisol. It may be assumed that water temperature could modify the rate of lipolysis (fatty acid mobilization from triacylglycerol) or have an impact on fatty acid reesterification. Lipolysis always exceeds oxidative needs; therefore, a fraction of all the fatty acid released must be reesterified—simultaneous flux through lipolysis and reesterification from the triacylglycerol/fatty acid cycle, a substrate cycle known to play an important role in
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
505
adjusting fatty acid supply during exercise (Wolfe et al., 1990). In the present study, it was observed that triacylglycerol level decreased at 15 8C in the chronic experiment, with mobilization of fat stores probably occurring. At the temperature of 31 8C, there was a decrease of fatty acid, probably due to an increase on metabolic rate with fatty acid consumption by the tissue. The liver is a glucose-utilizing, glucose-producing and glucose-storing organ. As such, it acts as a glucostat in the vertebrate organism, regulating glucose levels of blood (Moon and Foster, 1995). The major part of intracellular glucose in teleost hepatocytes is channeled into glycogen (Pereira et al., 1995). In the present study, an increased glucose level in liver and white muscle after 21 days of exposure to 31 8C was observed. This fact, associated with the decrease of hepatic glycogen and protein concentrations, suggests glucose export from the liver to white muscle through a couple of associated mechanisms, gluconeogenesis and glycogenolysis. The decrease in muscle protein indicates that the high temperature (31 8C) was sufficiently stressful to extend proteolysis toward other tissues. Higher temperature demands protein as an energy source in addition to sugars. Glycogen levels in fish liver vary over a wide range of concentrations from 20 to 2000 Amol g 1 liver (Moon and Foster, 1995). Once formed, fish liver glycogen is remobilized with relative difficulty, whereas muscle glycogen is rapidly used to provide energy. After white muscle, liver glycogen represents the major carbohydrate store in the fish body. Hepatic glycogen is rapidly mobilized during stress by catecholamines (Ottolenghi et al., 1984; Janssens and Lowrey, 1987). Our results demonstrate that liver glycogen was decreased either at 15 or 31 8C. However, muscle displayed a different behavior to cope with cold and warm waters. Under cold conditions, the silver catfish spared glucose, resulting in gluconeogenesis, whereas in the warm water, it likely consumes glucose. Increase of liver and decrease of the muscular lactate in fish exposed to warm waters suggest aerobic catabolism of glucose with significant cooperation of the liver. The present results demonstrate that silver catfish do not show the classical stress response either to cold or heat shock. However, despite the lack of change in cortisol and hematological values with temperature, the metabolic responses were distinct. As should be expected, the chronic thermal exposures resulted in greater metabolic changes than acute exposure. Heat shock was more stressful to silver catfish than cold shock. The metabolic demand of the species was likely higher as fish exposed to warm water were metabolizing lipids, carbohydrates and even protein. In addition, the use of metabolism as an indicator of stress in the silver catfish was a good tool to evaluate long-term changes, and glucose was a better index than the classical tools usually applied. We can conclude that the present set of data can partially explain the ability of R. quelen to resist a wide range of environmental temperatures.
References Adams, S.M., 1990. Biological Indicators of Stress in Fish. American Fisheries Society, Bethesda, MD, pp. 1 – 8. Barcellos, L.J.G., Wassermann, G.F., Scott, A.P., Woehl, V.M., Quevedo, R.M., Ittze´s, I., Krieger, M.H., Lulhier, F., 2001. Steroid profiles in cultured female Jundia´, the siluridae Rhamdia quelen (Quoy and Gaimard, Pisces Teleostei) during the first reproductive cycle. General and Comparative Endocrinology 121, 325 – 332.
506
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
Barcellos, L.J.G., Wassermann, G.F., Scott, A.P., Woehl, V.M., Quevedo, R.M., Ittze´s, I., Krieger, M.H., Lulhier, F., 2002. Plasma steroid concentrations in relation to the reproductive cycle of cultured male Rhamdia quelen. Journal of Fish Biology 61, 751 – 763. Bennett, A.F., 1978. Activity metabolism of lower vertebrates. Annual Review of Physiology 400, 447 – 469. Bidinotto, P.M., Moraes, G., Souza, R.H.S., 1997. Hepatic glycogen and glucose in eight tropical freshwater teleost fish: a procedure for field determinations of micro samples. Boletim Te´cnico CEPTA. Pirassununga 10, 53 – 60. Boyd, C.E., Tucker, C.S., 1992. Water Quality and Pond Soil Analyses for Aquaculture. Alabama Agricultural Experiment Station, Auburn University, Alabama, USA. 183 pp. Carvalho, P., Thomaz, S.M., Bini, L.M., 2003. Effects of water levels, abiotic and abiotic factors on bacterioplankton abundance in lagoons of a tropical floodplain (Parana´ River, Brazil). Hydrobiologia 510, 67 – 74. Cataldi, E., Di Marco, P., Mandich, A., Cataudella, S., 1998. Serum parameters of Adriatic sturgeon Acipenser naccarii (Pisces: Acipenseriformes); effects of temperature and stress. Comparative Biochemistry and Physiology 121 A, 351 – 354. Chen, G.R., Sun, L.T., Lee, Y.H., Chang, C.F., 1995. Characteristics of blood in common carp, Cyprinus carpio, exposed to low temperatures. Journal of Applied Aquaculture 5 (3), 21 – 31. Chernecky, C.C., Krech, R.L., Berger, B.J., 1993. Laboratory Tests and Diagnostic Procedures. WB, Saunders, Philadelphia, pp. 932 – 933. Chippari-Gomes, A.R., Gomes, L.C., Baldisserotto, B., 1999. Lethal temperatures for Silver Catfish, Rhamdia quelen, fingerlings. Journal of Applied Aquaculture 9 (4), 11 – 21. Duboie, M., Gilles, K.A., Hamilton, J.K., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350 – 358. Fine, M., Zilberg, D., Cohen, Z., Degani, G., Moav, B., Gertler, A., 1996. The effect of dietary protein level, water temperature and growth hormone administration on growth and metabolism in the common carp (Cyprinus carpio). Comparative Biochemistry and Physiology 114 A, 35 – 42. Gallego, M.G., Bazoco, J., Sua´rez, M.D., Sanz, A., 1995. Utilization of dietary carbohydrates by fish: a comparative study in eel and trout. Animal Science 61, 427 – 436. Gerlach, G.F., Turay, L., Malik, K.T.A., Lida, J., Scutt, A., Goldspink, G., 1990. Mechanisms of temperature acclimation in carp: a molecular biology approach. American Journal of Physiology 259, R237 – R244. Harrower, J.R., Brown, C.H., 1972. Blood lactic acid. A micromethod adaptable to field collection of microliter samples. Journal of Applied Physiology 32 (5), 224 – 228. Janssens, P.A., Lowrey, P., 1987. Hormonal regulation of hepatic glycogenolysis in the carp, Cyprinus carpio. American Journal of Physiology 252, R653 – R660. Kikuchi, K., 1999. Use of defatted soybean meal as a substitute for fish meal in diets of Japanese flounder Paralichthys olivaceus. Aquaculture 179, 3 – 11. Klyachko, O.S., Ozernyuk, N.D., 1998. Functional and structural properties of lactate dehydrogenase from embryos of different fishes. Comparative Biochemistry and Physiology 119B, 77 – 80. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265 – 275. Maricondi-Massari, M., Kalinin, A.L., Glass, M.L., Rantin, F.T., 1998. The effects of temperature on oxygen uptake, gill ventilation and ECG waveforms in the Nile tilapia, Oreochromis niloticus. Journal of Thermal Biology 23, 283 – 290. Mommsen, T.P., Plisetskaya, E.M., 1991. Insulin in fishes and agnathans: history, structure and metabolic regulation. Review of Aquaculture Science 4, 225 – 259. Moon, T.W., Foster, G.D., 1995. Tissue carbohydrate metabolism, gluconeogenesis and hormonal and environmental influences. In: Hochachka, P.W., Mommsen, T.O. (Eds.), Biochemistry and Molecular Biology of Fish, Metabolic Biochemistry, vol. 4. Elsevier Sciences Publishers, New York, pp. 65 – 100. Nova´k, M., 1965. Colorimetric ultramicro method for the determination of free fatty acids. Journal of Lipid Research 6, 431 – 433. Ottolenghi, C., Puviani, A.C., Baruffaldi, A., Brighenti, L., 1984. Effect of insulin on glycogen metabolism in isolated catfish hepatocytes. Comparative Biochemistry and Physiology 78 A, 705 – 710.
C.L. Lermen et al. / Aquaculture 239 (2004) 497–507
507
Ottolenghi, C., Puviani, A.C., Ricci, D., Morsiani, E., 1995. The effect of high temperature on blood glucose level in two teleost fish (Ictalurus melas and Ictalurus punctatus). Comparative Biochemistry and Physiology 111 A, 229 – 235. Perago´n, J., Barroso, J.B., Garcia-Salguero, L., Letı´cia, B., De La Higuera, M., Lupian˜ez, J.A., 1999. Carbohydrates affect protein-turnover rates, growth, and nucleic acid content in the white muscle of rainbow trout (Oncorhynchus mykiss). Aquaculture 179 (3–4), 425 – 437. Pereira, C., Vijayan, M.M., Storey, K.B., Jones, R.A., Moon, T.W., 1995. Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes. Journal of Comparative Physiology 165, 62 – 70. Roche, H., Boge´, G., 1996. Fish blood parameters as a potential tool for identification of stress caused by environmental factors and chemical intoxication. Marine Environmental Research 41, 27 – 43. Santos, M.A., Pacheco, M., 1996. Anguilla anguilla L. Stress biomarkers recovery in clean water and secondarytreated pulp mill effluent. Ecotoxicology and Environmental Safety 35, 96 – 100. Sun, L.T., Chen, G.R., Chang, C.F., 1992. The physiological responses of tilapia exposed to low temperatures. Journal of Thermal Biology 17, 149 – 153. Sun, L.T., Chen, G.R., Chang, C.F., 1995. Acute responses of blood parameters and comatose effects in saltacclimated tilapias exposed to low temperatures. Journal of Thermal Biology 20, 299 – 306. Tanck, M.W.T., Booms, G.H.R., Eding, E.H., Bonga, S.E.W., Komen, J., 2000. Cold shocks: a stressor for common carp. Journal of Fish Biology 57, 881 – 894. Tavares-Dias, M., Melo, J.F.B., Moraes, G., Moraes, F.R., 2002. Caracterı´sticas hematolo´gicas de teleo´steos brasileiros: IV. Varia´veis do jundia´ Rhamdia quelen (Pimelodidae). Cieˆncia Rural 32, 693 – 698. Walton, M.J., Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish. Comparative Biochemistry and Physiology 73B, 59 – 79. Wilson, R.P., 1994. Utilization of dietary carbohydrates in fish. Aquaculture 124, 67 – 80. Wolfe, R.R., Klein, S., Carraro, F., Weber, J.M., 1990. Role of triglyceride–fatty acid cycle in controlling fat metabolism in humans during and after exercise. American Journal of Physiology 258, E382 – E389. Wright, J.R., O’Hali, W., Yang, H., Han, X., Bonen, A., 1998. GLUT-4 deficiency and absolute peripheral resistance to insulin in the teleost fish tilapia. General and Comparative Endocrinology 111 (1), 20 – 27.