Physiological responses of pacu (Piaractus mesopotamicus) to intermittent cold exposure: A comprehensive analysis of stress, immunity, antioxidant, and metabolic adaptations

doi:10.21203/rs.3.rs-4419616/v1

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Physiological responses of pacu (Piaractus mesopotamicus) to intermittent cold exposure: A comprehensive analysis of stress, immunity, antioxidant, and metabolic adaptations

https://doi.org/10.21203/rs.3.rs-4419616/v1

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This study investigated the profile of energy-dependent physiological responses, such as those of the stress, innate immune and antioxidant systems, as well as some indicators of energy mobilization in pacu (Piaractus mesopotamicus) exposed to intermittent cold to observe the correlation between these responses. Juvenile fish were divided into two groups and exposed to alternating temperatures (16 ºC and 28 ºC). The fish were acclimated to 28°C, with half kept at 28°C (control), and the other half exposed to 16°C for two 24-h periods with a 5-day interval between them. The fish were sampled at six times: baseline (after acclimatization to 28°C), 24 h after the 1st exposure to 16°C, after 5 days of recovery at 28°C, 24 h after the 2nd exposure to 16°C, and after 24 and 48 h of recovery at 28°C. The reduction in temperature activated the stress response, the innate immune system and the antioxidative system as well as mobilized lipids from the visceral stores and preserved the circulating levels of triglycerides. The intermittent exposure of fish to cold increased plasma cortisol after both exposures, activated leukocyte respiratory activity after the 2nd exposure, and activated the compensatory lysozyme response after temperature recovery in both cold exposures in addition to increasing the number of circulating monocytes and granulocytes. The activity of the enzymes catalase (CAT) and superoxide dismutase (SOD) increased after the 1st and 2nd cold exposure, respectively. Glutathione peroxidase (GPx) was activated after the 2nd exposure compared to the control. The subtropical fish pacu was sensitive to intermittent exposure to cold and was able to display protective physiological responses.

Temperature change

cortisol

oxidative stress

antioxidant system

immune system

Water temperature is an important issue for fish, which are ectothermic organisms, whose metabolism is directly dependent on water temperature (Prosser and Nelson 1981; Handeland et al. 2008). Thus, thermal changes, which are usually sudden and repetitive, have a significant impact on fish physiology, and suboptimal temperatures can negatively impact fish health (Dan and Little 2000; Ma et al. 2015; Shi et al. 2013). The impact of temperature on biological systems is an important issue related to global climate changes. Extreme temperature events, such as unpredictable cold waves, have occurred with higher frequency and magnitudes due to increased climate variability (Tingley and Huybers 2013; IPCC 2012). According to the Climate Change Science Program (2008), the increase in global temperature makes the consequences of these changes more evident and the development of extreme weather events more common.

The temperature of the environment directly affects all biological processes, including metabolism (Pörtner et al. 2006). As temperature decreases, the mechanisms of the production and retention of metabolic heat in fish are insufficient to maintain body heat (Prosser and Nelson 1981). Temperature affects metabolic rate and sets the pace for virtually all biochemical reactions and physiological processes in fish (Van De Pol et al. 2017; Wen et al. 2017; Kovacevic 2019; Volkoff and Rønnestad 2020) affecting biological systems such as the immune system (Abram et al. 2017) and antioxidant defense system (Xu et al. 2019). The immune system has high cell turnover and is vulnerable to changes in metabolic rates induced by fluctuations in temperature. Low temperatures can suppress innate immune responses and decrease the ability of fish to fight pathogens during the winter or acute temperature decreases, negatively impacting their health (Abram et al. 2017; Zhou et al. 2019). Temperature modulates metabolic oxygen consumption and can cause an increase in the production of reactive oxygen species (ROS) (Lushchak and Bagnyukova 2006; Dalvi et al., 2017; Lushchak 2011; Welker et al. 2013). At low temperatures, there is greater availability of dissolved oxygen, which can contribute to an increase in the production of ROS and consequent oxidative damage to lipids in cell membranes (Bagnyukova et al. 2007; Vinagre et al. 2012). Under these conditions, the natural antioxidant system is not able to neutralize the excess of ROS produced Martínez-Álvarez et al. 2005). In view of the above, this study aimed to investigate the profile of physiological energy-dependent responses, such as those of the stress, innate immune and antioxidant systems as well as indicators of metabolic mobilization in pacu (Piaractus mesopotamicus) exposed to intermittent cold to observe the correlation between these responses. For this, the fish acclimated to 28 ºC were exposed repetitively to 16 ºC, since 16 ºC is a temperature out of the thermal comfort range for pacu (Pinto et al. 2020). Pacu was chosen as the target fish due to its economic value (Valladão et al. 2018) and its distribution in subtropical regions where temperature variations are common, strongly influenced by the entry of cold fronts that occur during autumn and winter (Sant’Anna Neto et al. 2015) being a concern in relation to fish health.

Animals and experimental design

We used 88 juvenile fish (115.84 ± 24.28 g) distributed into eight 100-L tanks, acclimated for seven days to the lab conditions, with running water at 28°C, and two treatments: temperature 28 ºC – control (temperature 28.11°C ± 0.25; dissolved oxygen 6.18 ± 0.23 mg l-1) and low temperature (16.1 ºC ± 0.18; dissolved oxygen 7.48 ± 0.11 mg l-1), with four tanks/treatment (Fig. 1). The temperature of 16 ºC was reached with a cooling equipment (Chiller Gelaqua® Split) by a reduction of 2 ºC/h. For this, in the experimental group, the water flow was closed, in a recirculating system, and when the temperature of 16 ºC was reached, 24 h was determined for each exposure (two) with an interval of five days between them (Fig. 1). The same procedure was carried out for the control fish with the temperature maintained at 28 ºC. During the entire experiment, after the 24 h exposure to 16°C, the recirculating system changed to a constant flow (CF), which promoted the return of the temperature to 28°C. Eight fish from each treatment, being 2 per tank, were sampled: before cooling (baseline), 24 h after the 1st exposure to 16°C (when 16 ºC was reached), 5 days after returning to 28°C (when 28 ºC was reached), 24 h after the 2nd exposure to 16°C, and 24 and 48 h after returning to 28°C (Fig. 1). The two groups of fish were fed (3% b.w.) during the period of 5 days at 28°C.

Samplings and analyses

The fish were anesthetized with MS222 (50 mg L-1), and blood was drawn from caudal vessels and added to tubes with and without anticoagulants. After the fish were euthanized with an overdose of anaesthetic, the liver and visceral fat were removed and kept at -80 ºC.

Biomarkers of stress

One blood sample with Glistab (Labtest code 29; https://labtest.com.br/reagentes/glistab/) was used to separate plasma to measure glucose and cortisol concentrations with commercial kits (Labtest code 133; https://labtest.com.br/wp-content/uploads/2016/09/Ref_133_Edi%C3%A7%C3%A3oDezembro2011_Ref050219_Port.pdf) and DRG Cortisol ELISA code 1887 (https://www.drg-diagnostics.de/files/eia-1887_ifu--cortisol_2019-05-02_endeitesfr.pdf), respectively). The intra-assay and inter-assay variations for cortisol were < 6% and < 8%, respectively.

Biomarkers of innate immunity

In a sample of blood containing heparin, the respiratory activity of leukocyte was measured according to protocol of Anderson and Siwicki (1995) adapted for pacu by Biller-Takahashi et al. (2013). The blood was processed to extract the serum to determine lysozyme concentrations and hemolytic activity of the complement system. The lysozyme concentrations were measured in serum according to protocol of Demers and Bayne (1997) adapted for pacu by Zanuzzo et al. (2015). The hemolytic activity of the complement system was measured in serum following protocol of Ferriani et al. (1990) and Polhill et al. (1978) adapted for pacu by Zanuzzo et al. (2017).

Biomarkers of the antioxidant system and oxidative stress

The liver samples were homogenized in a pH 7.0 phosphate buffer solution centrifuged, and the supernatants were used to determine the indicators of the antioxidant system and oxidative stress. The activity of superoxide dismutase (SOD) was determined following protocol of Mccord and Fridovich (1969); the activity of catalase (CAT) was determined according to Beutler et al. (1975); the activity of glutathione peroxidase (GPx) was measured according to Hopkins e Tudhope (1973) and the activity of glutathione transferase (GST) was measured following protocol of Gagné (2014). The concentrations of GSH were measured following Beutler et al. (1963) and the lipid peroxidation (LPO) was determined by thiobarbituric acid reactive substances (TBARS) assay (Camejo et al., 1998).

Metabolic biomarkers

The concentrations of triglycerides were measured in plasma with commercial kit (Labtest code 87; https://labtest.com.br/reagentes/triglicerides-liquiform/). The liver content of glycogen was determined according to Perry et al. (1998). The liver protein concentration was determined according to Bradford (1976). Liver and visceral fat were weighed to calculate the hepatosomatic index (HSI) and fat-visceral-somatic index (FVSI) [(tissue weight/body weight) x 100].

Biomarkers of blood cells

Total and differential leukocyte were counted in blood smears stained using Rosenfeld dye (Giemsa/May-Grunwald/Wright) by an indirect method (Mukherjee and Ghosh 2010). Leukocytes and thrombocytes were quantified every 2,000 erythrocytes (RBC), and in the differential leukocyte count the cells were quantified every 200 leukocytes and estimated for the total leukocytes. The erythrocytes were counted in whole blood with heparin in a Neubauer chamber.

Statistical analysis

The experiment was set up in an entirely randomized design in a 2x1 factorial arrangement with six longitudinal factors (sampling times). Data were analyzed for normality (Cramer-von Mises) and homoscedasticity (Brown-Forsythe), and a two-way analysis of variance was used. When differences were significant (P < 0.05), the Tukey test was applied to compare means using the SAS software (version 9.3).

Biomarkers of stress

In the control fish exposed to 28 ºC, the cortisol concentration decreased after the 2nd exposure (p = 0.0445) and recovered the initial values after 24 h. In fish exposed to 16 ºC, cortisol increased after the 1st and 2nd exposure and reduced at 24 h after the reestablishment of the control temperature (p = 0.0018). In fish exposed to 16 ºC, cortisol values were higher compared to control fish after the 2nd exposure (p = 0.0008) (Fig. 2A). Plasma glucose concentrations in fish exposed to 16 ºC had lower values than the control fish at 24 h after returning to 28°C, in the 2nd exposure (p = 0.0092) (Fig. 2B).

Biomarkers of innate immunity

Respiratory activity of leukocyte (RAL) increased in both groups of fish after the 1st exposure and decreased 5 days after of the reestablishment of the control temperature (p < 0.0001). After the 2nd exposure, activation was observed only in fish exposed to 16 ºC (p < 0.0001). Then, the RAL of all fish decreased after the reestablishment of the control temperature (24 h and 48 h at 28°C) (p < 0.0001). In fish exposed to 16 ºC, the RAL was higher than in the control group at 24 h of recovery, after the 2nd exposure (p = 0.0034) (Fig. 3A).

The hemolytic activity of the complement system (HAC50) in the control group increased from the 2nd exposure (p = 0.0076), while fish exposed to 16 ºC showed no difference throughout the experiment (Fig. 3B).

Regarding lysozyme, an activation profile was observed in both groups of fish as the experiment proceeded, but control fish showed significantly higher concentrations 24 h after the reestablishment of the control temperature in the 2nd exposure (p = 0.0099), while this response was observed in fish exposed to 16 ºC in the last sampling (48 h) at 28 ºC (p = 0.0093). Comparing the groups, lysozyme concentrations were lower in fish at 16°C, 24 h after the temperature had returned to 28°C, and after the 2nd exposure (p = 0.0200) (Fig. 3C).

(B) and serum lysozyme concentration (C) in juvenile pacu (Piaractus mesopotamicus) exposed to intermittent drops of temperature. Samplings: baseline (28°C), 24 h (1st exposure to 16°C), 5 days of recovery (28°C), 24 h (2nd exposure to 16°C), 24 and 48 h of recovery (28°C). Different lowercase letters indicate differences between sampling times in the same treatment. Letters A and B indicate difference between treatments at the same sampling time (p < 0.05). Absence of symbols indicates absence of statistical difference.

Biomarkers of the antioxidant system and oxidative stress

SOD activity was reduced in control fish 5 days after the reestablishment of the control temperature and remained throughout the experiment, while in fish exposed to 16 ºC the values decreased in the same sampling (5 days) and returned to baseline values in the other sampling times (control, p = 0.0133; 16 ºC, p = 0.0190) (Fig. 4A).

CAT activity increased in fish exposed to 16 ºC from the 1st exposure (p = 0.0253), maintaining intermediary values until the experiment is over. The values were higher in fish exposed to 16 ºC than in the control group, after the 2nd exposure (p = 0.0253) and 24 h after the reestablishment of the control temperature (p = 0.0419) (Fig. 4B).

GPx activity did not change statistically in fish exposed to 16 ºC, but there was a trend for increased activity 24 h after the 2nd exposure and 24 h after reestablishment of the control temperature (p = 0.1890), when GPx activity was higher compared to the control fish (p = 0.0359). In the control group, the enzyme was activated after the 1st exposure and reduced 5 days after the reestablishment of the control temperature and 24 h after the 2nd exposure (p = 0.0560) (Fig. 4C).

GST activity reduced, in general, in both groups of fish 5 days after the reestablishment of the control temperature (control, p = 0.0040; 16 ºC, p = 0.0500) (Fig. 4D).

GSH concentrations increased in both groups of fish after the 1st exposure and decreased from 5 days of the reestablishment of the control temperature until the end of the experiment in control fish (p < 0.0001), while in fish exposed to 16°C the concentrations reduced in the last sampling after 48 h at 28°C (p = 0.0005) (Fig. 4E).

The LPO values increased after the 1st exposure, and in the control fish there was a reduction 5 days after the reestablishment of the control temperature (p = 0.0040), while in fish exposed to 16°C the values did not differ among the sampling times (p = 0.2922) (Fig. 4F).

Metabolic biomarkers

The concentration of triglycerides increased 5 days after the reestablishment of the control temperature in both groups of fish and reduced after the 2nd exposure in the control group and 24 h after the reestablishment of the control temperature in the 2nd exposure to 16°C (control, p < 0.0001; 16°C, p < 0.0001) (Fig. 5A).

In both groups of fish, the lowest amount of liver glycogen was observed in the last sampling, 48 h after the reestablishment of the control temperature in the 2nd exposure to 16°C (control, p = 0.0026; 16 ºC, p = 0.0426). After the 2nd exposure, glycogen was lower in fish exposed to 16 ºC than in control fish (p = 0.0388) (Fig. 5B).

The FVSI changed in fish exposed to 16 ºC, with the lowest values observed after the 2nd exposure with recovery of the baseline values at the last sampling, 48 h after the reestablishment of the control temperature (p = 0.0139) (Fig. 5C). The highest values of HSI in control fish were observed after the 2nd exposure (p < 0.0001), when they were higher than in fish exposed to 16 ºC (p = 0.0001) in a profile that resembles that of liver glycogen (Fig. 5D).

at the same sampling time (p < 0.05). Absence of symbols indicates absence of statistical difference.

Blood cell markers

RBC values were higher in fish exposed to 16 ºC after the 2nd exposure compared to the control group (p = 0.0246). Throughout the experiment, the control fish showed a reduction after the 2nd exposure (p = 0.0195) (Fig. 6A). Fish exposed to 16 ºC showed an increase in the circulating leukocytes after the 2nd exposure, followed by reduction 24 h after the reestablishment of the control temperature and recovery of the initial values at 48 h (p = 0.0332). After the 2nd exposure, the number of leukocytes of fish exposed to 16°C was higher than those of the control group (p = 0.0480), and 24 h after returning to 28°C the values decreased, becoming lower than in the control group (p = 0.0387) (Fig. 6B).

The number of lymphocytes in both groups of fish decreased after the 2nd exposure when the temperature returned to 28 ºC, at 24 and 48 h in the control group (p = 0.0002), and at 48 h in fish exposed to 16 ºC (p = 0.0351). After 5 days of recovery at 28°C, the fish exposed to 16°C had fewer circulating lymphocytes compared to the control group (p = 0.0034) (Fig. 6C).

The number of neutrophils did not change in fish exposed to 16°C, but the variability between values was very high. In the control fish, there was a reduction in both samplings after returning to 28°C in the 1st and 2nd exposures (at 5 days and at 24 h at 28°C) compared to the values observed after the 2nd exposure (p = 0.0179) (Fig. 6B). The number of monocytes increased in the control fish after the 2nd exposure, at 24 and 48 h of recovery (p < 0.0001), and at 48 h in fish exposed to 16°C (p < 0.0001) (Fig. 6D).

The number of eosinophils fluctuated in control fish after 48 h of recovery in the 2nd exposure (p = 0.1580), while fish exposed to 16°C showed lower values than the control group after the 1st exposure (p = 0.0374) and at 5 days after the reestablishment of the control temperature in the 1st exposure (p = 0.0247) (Fig. 6E). The number of granulocytes increased in fish exposed to 16 ºC after the 1st exposure and remained high in the next samplings (p = 0.0097), being higher than the control 5 days after the reestablishment of the control temperature in the 1st exposure (p = 0.0051) and after the 2nd exposure at 24 h of recovery (p = 0.0117) (Fig. 6F).

Temperature changes in subtropical regions and southern Brazil are influenced by the entry of cold fronts, frequent during autumn and winter (Sant’Anna Neto et al. 2015). Sudden and repetitive thermal changes have a negative impact on fish, with consequences for the immune and antioxidant systems (Abram et al. 2017; Xu et al. 2019; Zhou 2019). To assess the impact of temperature reduction in the pacu, found in regions where temperature drops are common, we submitted juveniles to two temperature reductions (from 28 ºC to 16 ºC, for 24 h, with 5 days of recovery between exposures). The reduction in temperature, which took six hours, and the maintenance of fish in this condition for 24 h, activated the stress response, the innate immune system and the antioxidative system. We found that the intermittent exposure of fish to cold increased the cortisol response in both exposures, in particular in the 2nd one, after 5 days of the reestablishment of the control condition, indicating that lowering the temperature activated a stress response (Wendelaar Bonga 1997). Increased blood cortisol in after changes in water temperature has been described in other fish, such as Dicentrarchus labrax (Samaras et al. 2018), Oreochromis niloticus (Liu et al. 2011), Sebastes schlegelii (Zhang et al. 2015) and Oncorhynchus mykiss (Foss et al. 2012). Glucose concentrations did not change. Glucose is considered an important indicator of stress along with cortisol (Sopinka et al. 2016). Considering that there was an increase in cortisol, the lack of glucose response can be due to the time between the beginning of exposure to the cold and blood collection (24 h). It is possible that higher levels of glucose occurred more acutely and returned to initial levels when fish were sampled.

Regarding immune system biomarkers, in the 1st exposure the RAL increased in both groups of fish and was reestablished after 5 days of recovery. This suggests that it was the change in the water flow regime, which was common to both groups, that caused the cell activation and not the temperature. In the 2nd exposure, activation only occurred in fish exposed to cold, a response that disappeared after re-establishment of the control temperature. Fish exposed to cold for the 2nd time maintained their ability to activate this immune response. RAL is a response of phagocytic cells, and under stress conditions macrophages can be activated as an important defense mechanism. When activated, these cells produce large amounts of ROS with bactericidal activity (Secombes et al. 1996).

In the control fish, the complement system was activated after the 2nd exposure to cold; this was probably associated with the change in the water flow regime, though we did not find the same response in fish exposed to 16 ºC. In relation to lysozyme, in the control group, concentrations increased after the 2nd change in the water flow, while in fish exposed to 16 ºC the increase occurred after the recovery of the 1st and 2nd cold exposures (5 days and 48 h, respectively), suggesting a delayed compensatory response to the temperature lowering. Other studies that diverge from our data showed reduced activities of the complement system and lysozyme in Oncorhynchus mykiss (Nikoskelainen et al. 2004) and Epinephelus coioides (Cheng et al. 2009) exposed to cold.

Low temperature can alter the body homeostasis and promote changes in the liver's antioxidant capacity, resulting in oxidative stress (Kaushik and Kaur 2003; Ye et al. 2015). Components of the antioxidant defense system, such as the enzymes SOD, CAT and GPx, play a role in scavenging superoxide anions, free radicals, hydroxyl, and other radicals, protecting cells from oxidative damage (Bartoskova et al. 2013; Lortz et al. 2000). The exposure of pacu to 16 ºC activated CAT and SOD in the 1st and 2nd cold exposures, respectively, and GPx was activated in fish after the 2nd exposure compared to control fish. In the control fish, at 28 ºC, the 1st change in the water flow regime reduced the GPx activity. The SOD-CAT system is considered the first line of defense against excess ROS (Pandey et al. 2003). SOD acts by catalyzing the dismutation of superoxide into peroxide, which is decomposed by CAT into water and oxygen (Aebi 1984; Van Der Oost et al. 2003). The increase in SOD and CAT was observed in Etroplus suratensis (Joy et al. 2017) and Danio rerio (Malek et al. 2004) exposed to low temperatures. GST activity was not affected by temperature and was reduced in both groups of fish 5 days after the reestablishment of the control temperature when the water flow was opened.

The antioxidant GSH increased at both temperatures after the 1st cold exposure and recovered thereafter, suggesting it was caused by the change in the water flow regime, which was common to both groups of fish, and not by the temperature. In fish, GSH concentrations can increase through biosynthesis or by increasing its regeneration by glutathione reductase (GR) as a protective response (Peña-Llopis et al. 2002). In relation to LPO levels, the high variations observed in the values of fish exposed to cold do not allow confirmation of the occurrence of oxidative stress despite an increase of 60–70% after the 1st exposure coinciding with the increase in GSH. In control fish, this increase was significant. From there, the LPO returned to the initial values. Temperature variation affects antioxidant defenses, as observed in zebrafish exposed to a lower temperature (a decrease of 28°C to 18°C), resulting in increased activities of antioxidant enzyme genes such as GPx, Cu, Zn-SOD, Mn-SOD, GST (Malek et al. 2004).

Triglyceride levels increased after 5 days of recovery from the 1st cold exposure in both groups of fish, probably from food. The higher levels remained stable in fish exposed to cold for the 2nd time, suggesting that this energy source was preserved during the temperature lowering. At the same time, we observed moderate mobilization in visceral fat stores in the 2nd exposure in those fish. From the 2nd recovery period, the energy mobilization normalized. Regarding the increased liver glycogen in the control group in the 2nd cold exposure, this probably represents the reserve from the food consumed during the 5 days of recovery, while in fish exposed to cold this energy source remained unchanged throughout the experiment. Glycogen reserves were preserved.

Regarding hematological biomarkers, we did not observe significant changes in the number of total leukocytes except for an increase in the 2nd cold exposure compared to the control values, which inverted when recovery started. In contrast, in Epinephelus coioides exposed to 19 ºC, 27 ºC, and 35 ºC, for 3–96 h, total WBC count, respiratory burst, and phagocytic activity decreased 3, 48, and 96 h after fish were transferred to 19 ºC and 35°C (Nikoskelainen et al. 2004). In our study, cold was associated with the profile of monocytes, which increased during the recovery from the 1st cold exposure, reduced in the 2nd exposure and increased again after that. Monocytes are cells in transit in the peripheral bloodstream. During the inflammatory process they migrate to the connective tissue where they transform into macrophages (Cuesta et al. 1999). The macrophage is probably the most important cell in the immune response because in addition to being a phagocytic cell it produces and releases cytokines involved in the signaling between cells during the triggering of immune responses and is involved in presenting antigens to lymphocytes (Secombes and Fletcher 1992; Cuesta et al. 1999).

Temperature shifts, among other stressful procedures in aquaculture, regularly promote infections of farmed fish, activating functional mechanisms of fish antimicrobial host defenses. In fish, macrophage-lineage cells are integral to immune responses (Grayfer 2018). Phagocytic cells’ ROS response is an attribute of these cells’ antimicrobial defense, and the efficiency of this response often reflects the ability of macrophages to eliminate microorganisms. The cold also affected the granulocyte response in pacu, which increased throughout the experiment compared to the control temperature condition from the 1st exposure. Granulocytes act in the recognition and elimination of pathogens (Ellis 1999). In the case of eosinophils, we did not observe changes in fish exposed to cold, but their values were always lower than those observed in fish kept at 28 ºC. Although the function of eosinophils is not fully understood, these cells act in the processes of inflammation and cellular defense through degranulation (Urbinati et al. 2020).

In conclusion, the intermittent exposure of pacu acclimated to 28 ºC to cold (16°C) activated the stress pathway, with an increase in cortisol; activated the immune system, increasing the respiratory activity of leukocytes and the lysozyme response; increased the number of circulating monocytes and granulocytes; and affected the antioxidant system by increasing the activity of the enzymes SOD, CAT and GPx. Pacu was sensitive to cold but was able to display protective responses.

Funding declaration

This project was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [Grants Code 001].

Data availability statement

The data will be made available on request.

Statement of Ethics

This experiment was carried out following the Ethical Principles in Animal Experimentation adopted by the National Council for the Control of Animal Experimentation (CONCEA) and approved by the Committee on Ethics in the Use of Animals (CEUA) (Protocol 016291/19), Faculty of Agrarian and Veterinary Sciences, UNESP, Jaboticabal.

Author Contribution

C.F.P.F.: Conceptualization, Investigation, Methodology, Validation, Formal Analysis, Software, Data Curation, Writing - Original Draft. B.U.A.: Methodology, data collection and analysis. E.C.U.: Supervision, Conceptualization, Resources, Data Curation, Writing - Review & Editing, Visualization.All authors reviewed the manuscript.

Acknowledgement

The authors thank the Aquaculture Center of Sao Paulo State University (Caunesp) for supplying the fish.

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Physiological responses of pacu (Piaractus mesopotamicus) to intermittent cold exposure: A comprehensive analysis of stress, immunity, antioxidant, and metabolic adaptations

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Abstract

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Introduction

Material and methods

Animals and experimental design

Samplings and analyses

Biomarkers of stress

Biomarkers of innate immunity

Biomarkers of the antioxidant system and oxidative stress

Metabolic biomarkers

Biomarkers of blood cells

Statistical analysis

Results

Biomarkers of stress

Biomarkers of innate immunity

Biomarkers of the antioxidant system and oxidative stress

Metabolic biomarkers

Blood cell markers

Discussion

Conclusion

Declarations

Author Contribution

Acknowledgement

References

Additional Declarations

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