Teleost fish have both innate and adaptive immune systems. The innate system is central to defense against disease manifestations in fish because the acquired immune response is slower and is rather inefficient. The simplicity and limited protection provided by the acquired immune system reflect the early evolutionary history and the poikilothermic nature of fish(Magnadóttir, 2006; Whyte, 2007). Previous studies have documented the important influence of external factors such as temperature, photoperiod, the presence of precipitation and suspended solids, oxygen content, sewage discharge, and other environmental conditions on the innate immune systems of fish(Bowden et al., 2007; Bowden, 2008; Al-Bahry et al., 2009).

For most teleost fish species, swimming is an important aspect of their life history in the aquatic environment. Fish often swim against water currents within a modest range of swimming speeds under natural conditions. However, the intensive culture systems often found in fish farms for commercial production have been shown to increase fish stress by limiting swimming space and increasing the possibility of body surface damage. This, in turn, greatly elevates the risk of disease being spread to a large number of fish(Iguchi et al., 2003). Swimming is intimately related to important biological processes taking place in fish, such as early development, somatic growth, gonadal growth, maturation and survival(Bjørnevik et al., 2003; Palstra and Planas, 2011) and it has also been suggested that sustained swimming may naturally stimulate the immune systems of fish(Palstra and Planas, 2011). Improved growth rates and flesh quality are important goals for the sale of fish in commercial markets. Previous studies have shown that swimming to exhaustion alters the blood biochemistry of fish and that sustained swimming improves growth rates and metabolic processes(Anttila et al., 2010; Ibarz et al., 2011; Li et al., 2011). Few reports however, have specifically studied the eff ect of sustained swimming on blood immune parameters in teleosts(Castro et al., 2011).

Barbonymus schwanenfeldii(Bleeker, 1854), a tropical fish native to Southeast Asia, was introduced to China several decades ago and rapidly became commercially important. Previous research on juvenile B. schwanenfeldii showed that their swimming performance changed and their rates of oxygen consumption markedly increased with increasing swimming intensity(Song et al., 2008; Li et al., 2010). This study investigated the eff ects of swimming on blood oxygen-carrying capacity and immune parameters of cultured juvenile B. schwanenfeldii. The following measurements were made at defined levels of swimming training: red blood cell(RBC)count, mean corpuscular volume(MCV), hematocrit(Hct), hemoglobin(Hb)concentration, mean corpuscular hemoglobin concentration(MCHC), white blood cell(WBC), count nitroblue-tetrazolium(NBT)-positive cells, lysozyme activity, antibacterial activity, alkaline phosphatase(ALP)activity, and superoxide dismutase(SOD)activity in the serum.

The trial was conducted at the Research Center of Hydrobiology, Jinan University in the Guangdong Province of China. B. schwanenfeldii were provided by the Jiahe Aquatic Research Institute(Baiyun District, Guangzhou City, China) and were acclimated to the experimental circulating aquarium system for one month prior to the experiment. Experiments were conducted in 21 circular fiberglass tanks(diameter: 86 cm; water depth: 40 cm)comprising three treatment groups each with seven replicates.

A total of 168 fish(body weight: 75.21±2.82 g, body length: 15.10±1.35 cm)were selected for the study and distributed r and omly to the 21 tanks with eight fish per tank. The fish were trained as described elsewhere(Ibarz et al., 2011). Water velocities were controlled by aquarium pumps(HX-6850; Guangdong Hailea Group Co., Ltd, Guangdong, China), as described by Ibarz et al.(2011). Similar tanks without pumps were used for the control group. Water velocities were measured at three tank depths(surface, mid-tank, and near the bottom)using an ultrasonic current meter(Starflow 6526c; Unidata, O’Connor, WA, Australia).

Water current speeds were chosen based on previous research in our laboratory indicating that swimming speeds of <2 bl/s(body length per second)had no adverse eff ects on the welfare or stress of tinfoil barb(Li et al., 2010). Current speeds in the three experimental groups were: control group 0.06± 0.01 bl/s; low-intensity training group 0.66±0.17 bl/s; high-intensity training group 1.92±0.45 bl/s.

The experiment was conducted over 45 days, and the fish were fed to satiation twice daily(09:00 and 18:00)with commercial feed(crude protein ≥39%, crude fiber ≤5%, crude fat ≥3%, and ash content ≤15%). Water currents were stopped 1 h prior to feeding; uneaten feed and fecal matter were cleared 1 h after feeding before restarting the pumps. Thus, the actual swimming period lasted 20 h each day with two feeding breaks. The tanks were continuously aerated using commercial air-stones to ensure their dissolved oxygen(DO)concentrations remained at acceptable levels. The DO concentration, ammonia nitrogen concentration(NH₄-N), and pH of each tank were monitored twice weekly. Water temperatures in each tank were monitored daily. The average values of these parameters throughout the experiment were: DO>6.2 mg/L, NH ₄ -N<0.02 mg/L, pH=7.9-8.2, and temperature 28.0±1.0°C. The fish were exposed to indoor illumination(‘daylight’ lamp; 1 200±100 lx; 12 h:12 h photoperiod).

On day 1, prior to commencing training, one tank was r and omly selected from each group and three fish were r and omly sampled from each tank. On day 23, three tanks were r and omly chosen from the six remaining in each group and three fish were r and omly sampled from each tank. On day 45, three fish were r and omly sampled from each of the final three remaining tanks in each group; i.e., nine fish were sampled on each occasion. Sampled fish were anesthetized with a sub-lethal dose(100 mg/L)of tricaine methanesulphonate(MS-222) and dissected immediately.

Approximately 0.8-1.0 mL of blood was drawn from the caudal dorsal aorta using a 2-mL disposable syringe with internal surfaces and needles pre-soaked in 1% heparin sodium solution. Blood samples were dispensed into a 1.5-mL Eppendorf tubes treated with heparin sodium powder(70-100 U/mL)for the determination of RBCs, MCV, Hct, Hb, WBCs and NBT-positive cells. An additional 0.8-1.0-mL blood sample was centrifuged at 3 000 r/min(15 min at 4°C) and the plasma was collected and stored at -20°C for the determination of antibacterial activity and the activities of lysozyme, ALP and SOD.

RBCs, MCV, Hct, Hb, and WBCs were determined using a hematological analyzer(CELL-DYN3700, Abbott Laboratories, Abbot Park, IL USA) and ALP was determined using an ARCHITECT c16000 analyzer(Abbott Laboratories). Mean corpuscular hemoglobin concentration(MCHC)was calculated as Hb/Hct×100. The number of NBT-positive cells was determined using the method of Anderson et al.(1992). The dark blue positively staining cells were counted under a microscope. Five coverslips were examined for each fish and ten r and om fields were counted on each slide(magnification 40×10). The means and st and ard deviations of the 50 fields were calculated for each fish.

Serum lysozyme activity was determined as previously described(Hultmark et al., 1980; Wang et al., 1995). Bacteria Micrococcus lysodeikticus used as the substrate were provided by Nanjing Jiancheng Bioengineering Institute(Nanjing, China). Serum lysozyme activity was calculated as:

Click Browse formula

Antibacterial activity was determined using the bacterium Colibacillus as a substrate(Wang et al., 1995) and calculated according to the equation:

Click Browse formula

SOD activity was determined according to the original method described by Marklund and Marklund(1974) and Deng et al.(1991). In this method, the autoxidation of pyrogallol was determined at first by the following steps. 4.5 mL of dipotassium-hydrogenphosphate buff er(pH 8.3)was first mixed vigorously with the appropriate volume of pyrogallol(50 mmol/L) and the absorbance at 325 nm was measured photometrically at 30-s intervals until the rate of change due to autoxidation was less than 0.07 per minute. SOD activity was then determined after addition of plasma to the reaction system before pyrogallol was added.

The differences among the three groups on each sampling day were analyzed by one-way analysis of variance(ANOVA) and compared using post hoc tests(LSD)using SPSS17.0. Diff erences were considered significant at P <0.05. Values are presented as means±SD.

Table 1 shows the hematological and biochemical parameters of fish under diff erent intensities of training. The RBC count and Hct in the 0.66 bl/s and 1.92 bl/s groups were significantly higher than in the control group on both day 23 and on 45. No significant diff erence was observed between the two training groups on day 23 but these values in the 1.92 bl/s group were significantly higher than in the 0.66 bl/s group on day 45. No significant diff erence was observed in MCV among the three groups on day 23 but a significant increase was observed in the 1.92 bl/s group compared with that in the other groups on day 45. Furthermore, the Hb concentration in the 1.92 bl/s group was significantly higher than in the other two groups on days 23 and 45 but no statistical diff erence was observed between the control group and the 0.66 bl/s group. There were no significant diff erences in either MCHC or WBC counts among the control and two training groups on either day 23 or day 45.

Table 1 Eff ects of sustained training on the hematological and biochemical parameters of tinfoil barb

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On day 23 and day 45, the numbers of NBTpositive cells in the fish that were trained at 1.92 bl/s were significantly higher(P <0.05)than the numbers in the 0.66 bl/s group and the values in both training groups were significantly higher than in the corresponding control group(Fig. 1).

Fig. 1 Eff ects of sustained training on the number of NBTpositive cells in tinfoil barb Different letters denote signifi cant diff erences among the three groups on the same sample day (one-way ANOVA, P <0.05).

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A similar trend of significantly increased plasma lysozyme activities was observed with increasing training intensity. The values in the 1.92 bl/s group on days 23 and 45 were significantly higher than those in the 0.66 bl/s group and the values in both training groups were significantly higher than in the corresponding control group(Fig. 2).

Fig. 2 Eff ects of sustained training on lysozyme activities in the plasma of tinfoil barb Different letters denote signifi cant diff erences among the three groups on the same sample day (one-way ANOVA, P <0.05).

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On day 23, the plasma antibacterial activities in the training groups were significantly higher than in the control group but no statistical diff erence was observed between the two training groups(Fig. 3). No significant diff erence existed in plasma antibacterial activities between the control group and the two training groups on day 45 although the value in the 1.92 bl/s group was significantly higher than that in 0.66 bl/s group.

Fig. 3 Eff ects of sustained training on antibacterial activities in the plasma of tinfoil barb Different letters denote signifi cant diff erences among the three groups on the same sample day (one-way ANOVA, P <0.05).

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There were no significant diff erences in plasma ALP activities among the three groups on day 23. However, the plasma ALP activity of the group trained at 1.92 bl/s was significantly higher than in the other two groups on day 45; there was no significant between the control and 0.66 bl/s groups on day 45(Fig. 4).

Fig. 4 Eff ects of sustained training on ALP activities in the plasma of tinfoil barb Diff erent letters denote signifi cant diff erences among the three groups on the same sample day (one-way ANOVA, P <0.05).

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The SOD activities in the plasma were progressively higher with increasing training intensity on days 23 and 45; i.e., the values in the 1.92 bl/s group on each day were significantly higher than in the other two groups and the value in both the 0.66 bl/s and 1.92 bl/s groups were significantly higher than in the corresponding control group(Fig. 5).

Fig. 5 Eff ects of SOD activities in the plasma of tinfoil barb Diff erent letters denote signifi cant diff erences among the three groups on the same sample day (one-way ANOVA, P <0.05).

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The physiological changes resulted from changes in RBC counts and Hct were reflected in the swimming performance. It has been reported that the enhanced swimming capability continues to benefit over time from increased RBCs counts, Hb concentrations and Hct(Gallaugher et al., 1995). A 10% increase in Hct was shown to be associated with an increase in swimming performance of about 10% and there was a positive relationship between these factors(Buick et al., 1980). The present study showed significantly increased RBC count and Hct in post-swimming individuals, which has also been observed in humans(Buick et al., 1980). Furthermore, the concentration of Hb is a major determinant of oxygen uptake(V _{O 2})in vertebrates, which reaches a peak(V_O2 max)as Hb increases(Celsing et al., 1987; Jones and Lindstedt, 1993). An increased concentration of Hb was also observed in the training groups in the present study, suggesting that sustained training potentially enhances the swimming ability of fish. The reduced MCV induced by swimming provides a larger specific surface area of red cells, favoring increased uptake of O₂ into the red cells. These observations suggest that sustained training improves the oxygen-carrying capacity of the blood, which could result in enhanced swimming performance of the fish.

NBT-positive cells include phagocytes, neutrophils, and macrophages are the important components of the immune system that have critical roles in natural disease resistance in fish(Hazlett and Wu, 2011). The number of NBT-positive cells increased in postswimming individuals, demonstrating that this eff ect can be achieved naturally through training and might provide a viable means of increasing the disease resistance of fish in commercial aquaculture systems.Similar results have been documented in other species. Training improved the innate immune functions of the blood of mammals, with increased numbers of macrophages and elevated phagocytic function of neutrophils(Kizaki et al., 2008; García- Torres et al., 2011). Additionally, the number of neutrophils was markedly increased with increasing training time in humans(Nieman et al., 1995). These studies indicated that lymphocytes in the plasma were stimulated by training and this is consistent with observations in the current study of increased numbers of NBT-positive cells in the training groups. However, no significant diff erence was observed in the overall WBC counts in these groups. Possibly, up-regulation of the numbers of NBT cells occurred concurrently with down-regulation of the numbers of other blood lymphocytes, providing a potential explanation for the relative constancy of the overall WBC counts observed in the present study.

Lysozyme activities, ALP and antibacterial activities in plasma play a key role in the non-specific immune response in fish; ALP is also an important enzyme that regulates a number of essential functions in all living organisms(Liu et al., 2013). We observed increased lysozyme, ALP and antibacterial activities in the plasma in post-training groups consistent with the positive eff ects of training on the innate immune parameters of fish. Lysozyme is usually identified histochemically in monocytes and neutrophils, which are the key sources of this enzyme in plasma(Ellis, 1999; Morozov et al., 2003). In fish, lysozyme is also mainly found in neutrophils and monocytes, with lower activities also present in macrophages(Saurabh and Sahoo, 2008). In the present study, the lysozyme activities markedly increased with increasing training intensity. Similarly, lysozyme levels in mucus and plasma were significantly increased after physical exercise in mammals(Morozov et al., 2003; Cripps et al., 2010). A positive correlation between antibacterial activities and training intensity has been documented in human subjects, demonstrating that the number of circulating neutrophils and overall antibacterial activities in the plasma increase markedly after exercise(Inoue et al., 2004). Furthermore, the concentration of lactoferrin, which is present in the specific granules of neutrophils, was also significantly increased immediately after exercise. Lactoferrin might play an important role in antibacterial defense by depriving microorganisms of iron and thereby inhibiting their growth(Bullen et al., 1991; Weinberg, 1992; Inoue et al., 2004). These observations are consistent with the increases in NBT-positive cell numbers and lysozyme activities found in the present study and further suggest that similar physiological mechanisms are responsible for the increase in lysozyme and antibacterial activities in fish. However, additional studies are required to assess the optimal level of training intensity in fish because it is possible that training at extremely high levels might actually cause decreased immunity or mechanical damage.

Increasing SOD activity has been previously associated with a response to environmental toxins and to excessive levels of reactive-oxygen species(ROS)(Campa-Córdova et al., 2009). It has also been shown that short-term stimulation of swimming enhanced the transcriptional activity of antioxidant defense genes that produce antioxidant enzymes, including Cu/Zn SOD, catalase, and phospholipid hydroperoxide and glutathione peroxidase in Atlantic cod(Gadus morhua)(Caipang et al., 2008). In the current study, SOD activities were significantly increased concurrently with the increase in training intensity, suggesting that training improved the antioxidant functions of fish, possibly because more ROS are produced in skeletal muscle during exercise.

Exercise of moderate intensity and duration is beneficial to the human body and is associated with direct and indirect improvements in cardiovascular function linked to nitric oxide-mediated adaptation, enhanced concentrations of neurotrophins, and improved modulation of redox homeostasis(Radak et al., 2008). The consensus from human medicine is that regular exercise reduces low-grade systemic inflammation. This eff ect is driven by cytokines that are released by active skeletal muscles and play a central metabolic and anti-inflammatory role at both local and systemic levels(Pedersen et al., 2007; Br and t and Pedersen, 2010). Fish skeletal muscle has also been shown to undergo structural, morphometric and biochemical changes in response to exercise(Johnston and Moon, 1980; Davison, 1997). Similarly, aerobic training might improve survival rates and strengthen disease resistance in Atlantic salmon(Salmo salar)by reducing cardiac transcription levels of the inflammatory cytokines TNF-α, IL-1β and IL- 6. Such training might also aid in regulating gene expression involved in immune responsiveness and in other processes of disease resistance(Castro et al., 2011). Our observations likewise suggest that the active skeletal muscle in the training groups likely enhanced the release of cytokines and gene expression, which play a key role in the stimulation of immune parameters in the plasma.

We have demonstrated that sustained aerobic training markedly enhances the blood oxygen-carrying capability and immune parameters of juvenile tinfoil barbs though mechanisms that are still to be determined. Although there are promising indications of the ability of swimming training to naturally enhance the innate immune system of fish, further studies will be required to fully characterize the mechanisms by which its various elements interact to control invading pathogens in fish. Further study of the feasibility of naturally improving fish immunity by sustained aerobic training in environmentally friendly aquaculture systems would be extremely worthwhile.