Biological indicators of stress in pacu ( Piaractus mesopotamicus ) after capture

The effects of capture (chasing, netting and air exposure) on cortisol, glucose, chloride, sodium, potassium and calcium concentrations, osmolality, hematocrit, hemoglobin concentration, red blood cells count (RBC) and mean corpuscular volume (MCV) were investigated in pacu (Piaractus mesopotamicus). A total of 132 fish (49.7 +/- 11.7 g) were subjected to capture and 3 minutes air exposure and capture and 5 minutes air exposure. Nine fish at each treatment were sampled at 5, 15, 30, 60 minutes and 24 hours after the procedure. Nine undisturbed fish were sacrificed before the handling and used as controls. Capture resulted in a rise in blood cortisol and glucose 30 and 5 minutes, respectively, after both air exposures. Both indicators returned to resting levels 24 hours after capture. In both fish groups, plasma chloride decreased 60 minutes after capture, not recovering the resting levels within 24 hours after, and serum sodium rose at 15 and 30 minutes and recovered the resting levels 24 hours later. There were no significant changes neither in potassium, calcium and osmolality nor in hematocrit, hemoglobin, RBC and MCV as a consequence of capture. The sequential stressors imposed to pacu during capture activated the brain-pituitary-interrenal axis (cortisol and glucose responses) but the activation of the brain-sympathetic-chromaffin cell axis was apparently moderate (ionic and hematological responses).

(4 fish per box), with constant water and air flow, where they were kept for 15 days to acclimate to the experimental conditions.Feeding was stopped 24 hours before the capture that consisted in chasing, netting and exposed the fish to the air.Fish were submitted to the conditions: T1: undisturbed fish (control) (3 boxes), T2: chasing and 3 minutes of air exposure (15 boxes), T3: chasing and 5 minutes of air exposure (15 boxes).Nine fish of T2 and T3 (3 fish/3 boxes) were sampled at each sampling time (5, 15, 30, 60 minutes and 24 hours after handling).The control fish (n = 9) were sampled before handling.Fish were anesthetized (benzocaine, 66 mg.L -1 ) and bled by caudal vessels puncture and serum and plasma were separated.The four fish of each box were simultaneously anesthetized but only 3 were sampled.

Statistical analysis
A completely randomized design (CRD) was employed and results were analyzed by a two-way analysis of variance (ANOVA), with 2 treatments (capture with 3 or 5 minutes air exposure) and 5 sampling times (5, 15, 30 and 60 minutes and 24 hours after capture) as the factors plus the resting condition (control fish).Data were expressed in means ± standard deviation of the mean.Means were compared by Tukey test (p < 0.05).

Results
No mortality was observed in any group during the experiment.There were hormonal and metabolic changes after the handling imposed on the fish.Serum cortisol concentrations in air-exposed fish either for 3 minutes (T2) or 5 minutes (T3) rose within 30 minutes (p = 0.0109) and then dropped until 24 hours later.The rise was higher in fish of treatment 3 (p = 0.0074) regardless of the sampling (Figure 1).Blood glucose in both air-exposed fish (T2 and T3) increased within

Introduction
There are many potential applications of the stress response.Experimental biologists need to know the baseline from which to assess whatever response they are studying.It is also important to know whether fish under intensive aquaculture are in or out of a stressed state.Aquaculture development depends on the establishment of appropriate management practices.Thus the features of the physiological stress responses can serve this purpose.
Various stressors, including fish capture (Mugnier et al., 1998;Arends et al., 1999;Barcellos et al., 2001;Rocha et al., 2004;Morales et al., 2005), are necessary components of modern intensive aquaculture (Wendelaar Bonga, 1997).The fish response to such stressors involves a series of physiological changes in an attempt to compensate for the challenge imposed upon it and, thereby cope with the stress.Such changes have been broadly categorized into primary, secondary and tertiary responses including hormonal, metabolic, osmoionic and hematological disturbances and have been used to characterize the degree of stress fish experienced (McDonald and Milligan, 1997;Wendelaar Bonga, 1997;Wojtaszek et al., 2002).

Fish and experimental procedure
Juveniles of pacu (132 fish, 49.7 ± 11.7 g) were randomly distributed in thirty three 100 L boxes

Discussion
Stress is a biological response of adaptation to adverse conditions and fish respond by activating responses such as increased circulating cortisol and glucose (Wendelaar Bonga, 1997), changes in blood ionic balance (McDonald and Milligan, 1997) and hematological profile (Wojtaszek et al., 2002).Although laboratory studies can be criticized for lack of realism, they allow for a systematic determination of general behavioral and physiological principles of stress response that is not possible in the field.
Interactions of sequential stressors, as those to which the pacu was exposed in the present study, often cause increased stress in fish.Capture, for instance, includes 15 and 30 minutes in both air-exposed fish (T2 and T3) and returned to resting levels 24 hours later (Figure 2).Potassium concentration (p = 0.2901) and osmolality (p = 0.5332) did not change after air exposure (Table 1).In fish of treatments 2 and 3, calcium levels did not differ significantly until 60 minutes after handling and decreased after 24 hours (p = 0.0001) compared to the other samplings but not to the control condition (Table 1).Excepting for MCV, which rose 30 minutes after capture and returned to resting levels 24 hours later (p = 0.0440), the hematocrit (Table 1), red blood cells count (RBC) and hemoglobin (Figure 3) were not affected by capture (p = 0.8382; p = 0.2980 and p = 0.5463, respectively).the first data on plasma catecholamines level in tropical fish (Perry et al., 2004) has shown that plasma catecholamines levels remained constant in pacu exposed to acute hypoxia, suggesting an inoperative or absent humoral adrenergic stress response in this species.
The results of the hematological assessement of stressed pacu confirmed the low degree of activation of the brain-sympathetic-chromaffin cell axis.Peripherical blood analysis has been used as a diagnosis to assess healthy state in fish and the effect of several stressors on them (Wojtaszek et al., 2002).The most significant effect of catechoalmines release during stress is to enhance blood O 2 transport by increasing the carrying capacity (Wells and Weber, 1990) and by enhancing Hb-O 2 binding affinity (Cossins and Richardson, 1985).No significant changes were found in the hematological parameters tested in pacu after the sequence of stressors during the capture.Concluding, the results of this work indicate that the submission of sequential stressors on pacu during capture activated the brain-pituitary-interrenal axis (cortisol and glucose responses) but the activation of the brain-sympathetic-chromaffin cell axis was apparently moderate (ionic and hematological responses).
the chasing and swimming of fish, physical injury provoked by the contact among fish and with the net and anoxia by air exposure (Mugnier et al., 1998;Arends et al., 1999;Ross and Ross, 1999;Barcellos et al., 2001;Morales et al., 2005).Confirming the literature regarding fish stress, elevated circulating cortisol and glucose of pacu were short-term responses in both air-exposed fish after capture and the recovery occurred within 24 hours.However, the magnitude of glucose responses was low (from 60 to 94 mg.dL -1 ) compared to values found previously in pacu in stressed state (Krieger et al., 1989;Martins et al., 2000).Air exposure of Sparus aurata for 3 minutes resulted, within 30 minutes, in an increase in plasma concentrations of cortisol and glucose.After 2 hours, plasma cortisol and after 12 hours plasma glucose had returned to control concentrations (Arends et al., 1999).On the other hand, netting and exposure of juvenile turbot to air for 1-4 minutes had no immediate effect on plasma cortisol concentrations (Mugnier et al., 1998).The stress response of pacu involved activation of the brain-pituitary-interrenal axis, as indicated by the cortisol and glucose elevations and it was probably elicited by the combined stimuli of fish swimming when chased, netting and the hypoxic condition provoked by air exposure.
Many stressors affect the ionic balance in fish (Wendelaar Bonga, 1997) facilitated by cathecolamineinduced increase of gill permeability responsible for chloride and sodium exchange with the environment (McDonald and Milligan, 1997).Decrease in plasma concentration of chloride and sodium were found in other stressed freshwater fish as in Brycon cephalus after transport (Carneiro and Urbinati, 2001;Urbinati et al., 2004).In our experiment, a partial and moderate disturbance of the ionic balance was found in both air exposed fish.In stressed pacu, chloride decreased 60 minutes after capture whereas sodium concentration increased transiently between 15 and 60 minutes after capture.Additionally, calcium and potassium levels and osmolality were not affected by the sequential stressors during capture.Apparently only a moderate and rather specific loss of permeability control occurred as consequence of capture.
The low magnitude of glucose responses, mild chloride and sodium disturbances, and the lacking potassium, calcium and osmolality responses indicated low activation of the brain-sympathetic-chromaffin cell axis, and hence a low release of catecholamines, which seemed though to occur to a higher extent at severe stressors (Mazeaud and Mazeaud, 1981).Several studies are known to initiate catecholamine secretion in fish including physical disturbance (Ristori and Laurent, 1985) and hypoxia (Ristori and Laurent, 1989).However, the degree of hypoxia required to initiate the responses is highly variable.Studies have shown that there exist hypoxia-tolerant species such as carp (Vianen et al., 2001) in contrast to hypoxia-intolerant species such as rainbow trout (Boutilier et al., 1988).A recent study that provided

Figure 1 .Figure 2 .
Figure 1.Blood cortisol and glucose of pacu undisturbed and air exposed for 3 and 5 minutes.Different capital letters indicate differences among sampling times (all air exposure times are represented in the same bar) and small letters between treatments (all samplings are represented in the same bar).Bars represent means of treatments or samplings.Vertical bars represent SEM.

Figure 3 .
Figure 3. Number of erythrocytes, mean corpuscular volume (MCV) and hemoglobin concentration of pacu undisturbed and air exposed for 3 and 5 minutes.Different capital letters indicate differences among sampling times (all air exposure times are represented in the same bar) and small letters between treatments (all samplings are represented in the same bar).Bars represent means of treatments or samplings.Vertical bars represent SEM.

Table 1 .
Hematocrit, serum potassium and calcium and osmolality of pacu undisturbed and air exposed for 3 and 5 minutes.Different capital letters indicate differences among samplings and small letters between treatments.