The water parameters measured in this study, except for temperature, were in accordance with levels required for pirarucu rearing (Sousa et al. 2017).
Blood is a dynamic tissue that exhibits the most rapid response in regard to the health and nutritional status of fish exposed to the different environments in which they live (Fazio, 2019). Cultivation environments offer thermal variations throughout the day and seasons that are capable of promoting significant blood changes due to the control exercised over erythropoiesis processes and respiratory demand (Ahmed et al. 2020). In the present study, exposure of pirarucu to experimental temperatures for 96 h resulted in a decrease in the levels of RBC, Hb and Ht at 37 ºC, compared to the other temperatures evaluated, suggesting anemia in the animals under these conditions. Shahjahan et al. (2018) observed a reduction in RBC and Hb levels and an increase in the number of circulating erythroblasts in striped catfish (Pangasianodon hypophthalmus) after three days of exposure to 36 ºC, suggesting failure of the hematopoietic system in cell maturation as a result of thermal stress. Reductions in hemoglobin levels were also observed for specimens of the species Labeo rohita exposed to 37.5 ºC for two weeks (Roychowdhury et al., 2020). These authors concluded that temperature-induced anemia was associated with the observed liver damage of the species and the escape of iron, important for the synthesis of Hb. We believe that these effects may not have occurred in the pirarucu in our study, even in the face of thermal stress, since MCH levels were elevated in the species, which suggests that anemia was not being induced by the absence of iron, but occurring as a result of the period of circulation and elimination of cells in the bloodstream. The findings of Cheng et al. (2015) show that exposure of pufferfish (Takifugu obscurus) to 34 ºC for 72 hours increases erythrocyte apoptosis rates by up to 15%. In addition, the temperature range tested in the present study (± 9 ºC) and the exposure period contributed to the observed response because, even in climate change scenarios with temperatures varying by up to ± 3 ºC and exposure to ammonia for 28 days, the species showed no changes in its hematological responses, which suggests that the thermal effect is time dependent for the species (Ramírez et al. 2023).
The stress produced by the elevation in temperature requires increased energy expenditure for the regulation of cellular and body functions and maintenance of homeostasis at the expense of higher metabolic costs (Kumar et al., 2015). In the present study, our data show that the increase in temperature demands a greater amount of energy from the pirarucu, as shown by the plasma glucose levels. Our data are in line with studies conducted for the species Horabagrus brachysoma (Dalvi et al., 2017) and pufferfish (Takifugu obscurus) (Cheng et al., 2018), which indicate increased energy requirements in hot environments. The elevation of plasma lactate levels in the pirarucu at 37 ºC suggests that the species uses anaerobic metabolism as a strategy to cope with warmer water temperatures. We believe that raising the temperature to these levels produces muscle hypoxia, recycling lactate into the plasma to ensure ATP production via pyruvate oxidation (Nelson and Cox, 2013). Similar results were reported for European seabass (Dicentrarchus labrax) exposed to 32 ºC for 10 days (Islam et al., 2020) and specifically reinforce that the pirarucu is capable of using different strategies for energy supply during a shorter exposure time.
In this study, plasma triglyceride levels decreased with the increase in temperature, while plasma cholesterol remained unchanged. Studies show that elevated temperatures can reduce plasma concentrations of cholesterol and triglycerides, thus indicating the occurrence of impaired lipid metabolism in acute exposure, or even during heat shock, as a consequence of stress (Li et al. 2019; Liu et al. 2022). We thought that the increase in temperature would produce an elevation of these metabolites as a result of the mobilization of substrates for energy generation and the maintenance of the homeoviscosity of cell membranes. However, the reduction of triglycerides to critical levels, as observed at 37 ºC, suggests that stress is high and reserves are being applied as a substrate for energy generation. Therefore, the energy preference of pirarucu is being changed in order to economize in the use of proteins as an energy source, since the species requires high levels of dietary protein and has high protein turnover (Ituassú et al., 2005; Pelster et al., 2020). On the other hand, our data contrast with the observations of Liu et al. (2019), who found an increase of both metabolites in lenok (Brachymystax lenok) exposed to heat stress from 16 ºC to 24 ºC for between one and seven days. For these authors, the impact of temperature on lipid metabolism is temporal and dependent on energy preferences when in stressful situations.
The liver is an important organ for detoxification, storage and metabolization in fish. The enzymes ALT (alanine aminotransferase) and AST (aspartate aminotransferase) are markers of cell damage in the liver and heart tissue. When injured, the cells release these enzymes into the bloodstream, indicating the occurrence of some form of dysfunction in the activity of these organs (Nelson and Cox, 2013). The increase in temperature is described as regulating the increase in the activities of these enzymes (Li et al. 2022). Studies have shown that exposure to increased temperatures produces elevated AST and ALT levels in rainbow trout (Oncorhynchus mykiss), rahu (Labeo rohita), lenok (Brachymystax lenok) and red cusk-eel (Geypterus chilensis) at different temperature ranges and for different exposure times (Dettleff et al., 2022; Li et al., 2022; Liu et al., 2019b; Roychowdhury et al., 2020). In the present study, ALT and AST levels increased at temperatures of 34 ºC and 37 ºC compared to the others. This indicates that the hepatic metabolism of pirarucu has a greater demand placed on it and may become impaired with increases in temperature, which suggests the impairment of the organ and its basic physiological functions, as found for chocolate mahseer (Neolissochilus hexagonolepis) by (Dash et al. 2023). Additionally, the withdrawal of the thermal stimulus and the return to environmental temperature levels could normalize the activity levels of the enzymes, since at temperatures of 34 ºC and 37 ºC, the fish could not choose ranges that produced thermal comfort (Li et al. 2022).
Elevated temperatures demand a greater amount of oxygen for metabolic processes and act as disruptors of lipids, proteins and nucleic acids, due to the imbalance in the production of mitochondrial reactive oxygen species, which can lead to the occurrence of oxidative stress (Birnie-Gauvin et al., 2017; Lu et al., 2016; Lushchak, 2011). In these situations, there is an increase in the body’s antioxidant response (e.g., SOD, CAT, GPx, ascorbic acid, tocopherol), which acts to neutralize these oxidizing agents, although a pattern between species is not observed (Lushchak and Bagnyukova 2006; Cui et al. 2014; Baldissera et al. 2020).
In this study, the increase in temperature caused the elevation of oxidative stress levels in pirarucu, although in a nonlinear way. This is because, between 31 ºC and 37 ºC, an increase in lipid peroxidation levels was observed, similar to what has been reported in other species (Madeira et al., 2016, 2013; Nitz et al., 2020; Vinagre et al., 2014, 2012), while no differences were observed between Tenv, 34 ºC and 37 ºC in the present study. The same pattern was found for the Antarctic fish species Notothenia coriiceps exposed to temperatures between 0 ºC and 4 ºC for 24 hours (Klein et al., 2017). However, the data of these authors demonstrate that, after 6 days of exposure under these same conditions, the elevation of oxidative stress levels can be observed as the temperature increases. In the present study, the increase in lipid peroxidation levels for pirarucu at the environmental temperature may be linked to the fact that a temperature fluctuation of 27.1 ºC to 29.9 ºC was observed between the beginning and the end of the thermal exposure of the species. This occurrence denotes that the short thermal buoyancy is able to induce the rapid occurrence of oxidative stress in this species, which explains the similarity between temperatures. Similar results were found for specimens of goldfish (Carassius auratus) exposed to a thermal shock of 35 ºC for 6 hours (Lushchak and Bagnyukova 2006). The enzymes SOD, CAT and GPx responded differentially to temperature. The elevation of SOD and GPx enzymes to the 34 ºC and 37 ºC groups in relation to the Tenv group suggests that under these conditions the antioxidant system is acting to neutralize oxidative damage in the pirarucu according to the increase in temperature, which corroborates previous findings (Hu et al., 2015; Li et al., 2019, 2022; Wood et al., 2013). However, at 37 ºC the CAT enzyme was reduced in relation to temperatures of 31 ºC and 34 ºC, which we believe is linked to increased GPx activity, since both act on peroxides. In addition, in competition with other peroxidases, CAT activity tends to be reduced, since GPx has a greater effect on fatty acid hydroperoxides (Lin and Shiau 2005; Li et al. 2019).