Fluctuations in DO levels are common in natural and captive conditions with high freshwater fish stocking densities (Copatti et al. 2019; Pontin et al. 2020). For survival under these conditions, animals often need to be able to tolerate hypoxia and reoxygenation (Nitz et al. 2020b). When oxygen flow returns during reoxygenation, several tissues become shockedas toxicity increases, causing oxidation of biomolecules and overproduction of ROS (Lushchak et al. 2001; Giraud-Billoud et al. 2019) and consequent loss of function or physiological homeostasis. In our study, there was no mortality, demonstrating that pacu is resistant to hypoxia and reoxygenation (rapid or gradual), regardless of the temperature evaluated. Furthermore, the oxidative stress parameters showed that pacu copes well with rapid reoxygenation under lower temperature conditions. However, these parameters may take longer (12 h) to stabilize, which confirms our study hypothesis.
The antioxidant defense system is composed of different enzymatic and non-enzymatic components that act as detoxifying agents for peroxyl radicals, representing the general antioxidant status of organisms (Amado et al. 2009). Our study observed changes in ACAP levels at both reoxygenation rates and under different thermal conditions. For example, gills and liver ACAP content were higher at the two highest temperatures (23°C and 28°C) at 1 h after recovery. As oxidative stress commonly occurs soon after the first moments of recovery (Welker et al. 2013; Johannsson et al. 2018; Giraud-Billoud et al. 2019), this behavior of the antioxidant defense system was expected. An increase in ACAP at elevated temperatures is related to a physiological adjustment generated to intercept increased ROS production (Lushchak 2011; Zebral et al. 2016).
Furthermore, the reduction in ACAP levels at 18°C at 1 h after recovery found in the different treatments (except for gradual reoxygenation) in our study may have been a result of the use of antioxidant components to contain the increase in free radical production in a recent hypoxia setting (Clanton 2007). This scenario seems to have been avoided in the current study in both organs (gills and liver) under conditions of intermediate temperature (23oC) and rapid recovery (0.5 h), as well as in the liver in the interaction between 18oC and gradual reoxygenation (5 h). Similarly, an elevation in ACAP levels in pacu gills exposed to hypoxia at 23oC was verified by Nitz et al. (2020a).
In addition, at 12 h after recovery, gills and liver ACAP content were lower at 28ºC, although ACAP had a different behavior (12 h) for the organs evaluated. While for the gills, there was an increase in ACAP content in the gradual recovery compared to the rapid recovery, the opposite occurred in the liver at this same temperature (28oC). Such alterations must have been organ-specific. Due to different metabolic demands, other tissues have oxygen tensions (Johannsson et al. 2018) that can trigger different oxidative responses. One organ can compensate for the increase in oxidative stress in another organ.
Overall, ACAP can provide an understanding of the resistance of each organism to the toxicity caused by ROS (Amado et al. 2009). ROS plays a fundamental role in activating the defense system. The success of each species depends on its ability to activate mechanisms that will serve as a shield to deal with the period of DO deprivation. This success also depends on the animals efficiently removing ROS in the subsequent period when oxygen is reintroduced (Onukwufor et al. 2016). Our study shows a reestablishment of ACAP levels in fish exposed to 18oC at 12 h after recovery, indicating that ACAP changes were temporary at this temperature.
When reperfusion occurs, organs are oxygenated, and, in parallel, there is an increase in ROS generation with LPO induction, protein oxidation, and DNA damage (Hermes-Lima and Zenteno-Savin 2002). On the other hand, according to Johannsson et al. (2018), reoxygenation generally does not cause new oxidative damage in hypoxia-tolerant species such as pacu. In our study, the best conditions for reducing gills LPO levels 1 and 12 h after recovery were observed at 18°C in fish subjected to rapid reoxygenation and at 23°C for those exposed to gradual reoxygenation. In general, the increase in temperature caused an increase in LPO levels in the gills, which could cause oxidative damage in fish that underwent rapid reoxygenation at an intermediate temperature (23oC). However, this would be the best temperature for fish that underwent gradual reoxygenation, and oxidative damage to the gills would only be linked to the highest temperature (28oC). On the other hand, liver LPO levels were higher at 23oC at 1 and 12 h after recovery. Similarly, Lushchak et al. (2005) also observed increased lipid damage (TBARS) in the liver of common carp (Cyprinus carpio) 14 h after rapid reoxygenation (up to 30 min) in fish maintained under hypoxia for 5.5 h.
A reduction in water temperature could also increase oxidative stress due to reduced enzyme activity and membrane fluidity (Tattersall et al. 2012), increasing LPO levels (Rossi et al. 2017). However, when exposed to low temperatures, ectothermic organisms commonly develop strategies such as home viscous adaptation to maintain the fluidity of biological membranes and other adjustments such as increasing the degree of lipid unsaturation and accumulation of PUFAs (Bagnyukova et al. 2007). However, this higher concentration of PUFAs increases therisks of oxidative stress since these substances are primary targets of ROS, which act by removing a proton from the conjugated double bond system. This event creates a peroxyl radical, which triggers the onset of LPO reactions (Abele and Puntarulo 2004). Therefore, an LPO may have occurred in the liver of the pacu maintained at the lowest temperatures tested in the current study (mainly 23oC). In contrast, in a previous study with Cyphocharax abramoides, a hypoxia-tolerant species, no change in LPO was observed after 3 h of hypoxia, regardless of the reoxygenation rate (Johannsson et al. 2018).
In addition to fatty acids, proteins are also affected by the overproduction of ROS, resulting in carbonylation, aggregation, fragmentation, amino acid modification, change in electrical charge and inactivation of membrane enzymes, receptors, and transport proteins, and oxidation of sulfhydryl groups (Lushchak 2011; Madeira et al. 2013). Although the modification of proteins by free radicals is not as critical as LPO, it could lead to the loss of their functions and (mainly) their ability to communicate intra- and intercellularly (Lushchak et al. 2005; 2011).
In our study, a reduction in the gills PSH content of juveniles was observed at 18oC at 1 h after recovery. However, this tissue had no changes in PSH content at 12 h after recovery. In the liver, there was an increase in PSH values at an intermediate temperature (23ºC), which could have occurred in a stressful situation as a protective reaction for the cells. Under stress, changes in thiol content can occur to remove harmful components that are readily replaced by a disulfide (or -SS disulfide bridges) through enzymatic reduction (Dickinson and Forman 2002). In addition, in the current study, fish must have triggered protective response mechanisms in the interaction between 23°C and gradual reoxygenation, as evidenced by the increase in liver PSH content at 1 and 12 h after recovery.