Introduction
Oxygen is a limiting factor both in the environment and production systems, so reduction may become a stressor. Diel cyclic hypoxia may occur with varying frequency and duration in freshwater habitats and in the context of climate change, the frequency of this situations will increase . Under a stressful situation fish activate the hypothalamic-pituitary-interrenal axis (HPI) which triggers the release of cortisol that induces secondary and tertiary responses. The level of activation of this response will depend on the intensity and the duration of the stressor Also, the recovery of individuals subjected to such stressors depends on their ability to modulate physiological, biochemical, and behavioural responses to maintain metabolic functions and homeostasis. Since episodes of repeated hypoxic stress have been less studied, the aim of this study is to determine the haematological, physiological and molecular response of rainbow trout under repeated hypoxia observe whether there is a habituation response.
Materials and methods
Rainbow trout juveniles were acclimated to AQUAB fish facilities (Universitat Autònoma de Barcelona, UAB), After the acclimation period, f ish were randomly divided in 5 different treatment groups, 2 control groups (absolute control (AC) and manipulated control (MC)) and 3 hypoxia groups: H1 that only received 1 hypoxic exposure, group H2 which received 2 hypoxic exposures and group H3 which received 3 hypoxic exposures.
Every exposure to hypoxia consisted in reducing the water oxygen level in the tanks from 8-9 mg/L to 2 mg/L by removing the aeration pumps and bubbling N2 into the system. Then fish were left in the tanks for 1 hour and sampled subsequently at 1,6 and 24 post exposure. Water oxygen levels were continuously monitored during the experiment. After the challenge, 9 fish per treatment, and time-point were euthanized with an overdose of MS-222. Samples of blood, skin, gills, and intestine were taken.
Results
Haematological parameters as red blood cells number or haemoglobin show that manipulation increase these levels, while hypoxia decreases them . Interestingly, fish are able to recover the control level of haematological parameters after 3 shocks (Table 1).
Respect to the physiological response we observe that cortisol level, show and increase at 1 hour post exposure. Also, looking at lactate levels we observe that there is an increase in H1 and H2 at 1 hour , but not in H3 suggest ing a metabolic habituation to hypoxic exposures. Finally, regarding glucose levels there is an increase at 1 hour , and a later recovering of control levels at 24 hours.
Finally changes in gene expression were also observed in crh and gr1 stress response genes and in l1b, il10 immune response genes.
Discussion
The results of our experiment on induced hypoxia suggest that trout subjected to repeated acute hypoxia could cope with oxygen levels down to 2mg/L of oxygen in water up to 24 hours after the stressor, and that subjecting the fish to the same stressor, trout shows a trend for habituation. Thus, among the hematological variables, red blood cells (RBC) and hemoglobin (HGB) show an increase in the CM group which may be due to the handling of the animals as observed in Acerete et al (2004). On the contrary, as observed in H1, hypoxia produces a decrease in RBCs, probably because the oxygen delivery system is excessively altered and then the overall metabolism and activity decreases (Pichanvart et al 2002). The increase of hematocrit levels after 1 hour would be associated to balancing out the extra need for oxygen (Muszee et al 1998).
Glucose is used as a substrate for glycolytic activity leading to the release of cell energy so that as time progresses these reserves are used. Nevertheless, in most cases an acute stress induces an increase in plasma glucose as a result of the energetic needs derived from the stress situation (Abdel-Twaab et al 2019). L actate levels increase facing a situation of hypoxia, as to maintain the cellular energy balance. This may be explained by the fact that oxygen-independent mechanisms are needed since oxygen availability is 15 times lower under hypoxic conditions (Richard J.G, 2011).
References
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