Gaining meaningful blood samples from water-breathing fish is a significant challenge. Two main methods typically used are grab 'n' stab and surgical cannulation. Both methods have benefits, but also significant limitations under various scenarios. Here we present a method of blood sampling laboratory fish involving gradual induction of anaesthesia within their home tank, avoiding physical struggling associated with capture, followed by rapid transfer to a gill irrigation system to maintain artificial ventilation via adequate gill water flow and then followed by sampling the caudal vasculature. This method negates many blood chemistry disturbances associated with grab 'n' stab (i.e. low pH and oxygen, elevated lactate, CO2 and stress hormones) and generates results that are directly comparable to cannulated fish under a wide range of experimentally-induced acid-base scenarios (acidosis and alkalosis). Crucially this method was successful in achieving accurate acid-base blood measurements from fish ten times smaller than are typically suitable for cannulation. This opens opportunities not previously possible for studies that relate to basic physiology, sustainable aquaculture, ecotoxicology, conservation, and climate change.

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2-1 kPa CO2 (2,000 - 10,000 µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate the internal acid-base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid-base regulatory responses in marine fish are scarce. We observed that upon sudden exposure to ~1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ~40 minutes, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ~2 hours, which is one of the fastest acid-base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3- in blood, which increased from ~4 to ~22 mM. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid-base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3- and pH, likely because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid-base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments.

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2 and 1â€
kPa CO2 (2000-10,000â€
µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate for the internal acid-base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid-base regulatory responses in marine fish are scarce. We observed that upon sudden exposure to ∼1â€
kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ∼40â€
min, thus restoring haemoglobin-O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ∼2â€
h, which is one of the fastest acid-base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3- in blood, which increased from ∼4 to ∼22â€
mmolâ€
l-1. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid-base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3- and pH, probably because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid-base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments.

Globally, aquaculture development is leading the way in the ‘Blue Revolution’ with it rapidly outstripping wild-capture fisheries. However, in the face of climate change and greenhouse gas emissions, it is vital that going forward, aquaculture develops in a sustainable and ecologically aware manner. One aspect of this is ensuring farms are operating at maximum efficiency, particularly from a biological perspective. Here we present an assessment of the impact that elevated environmental CO2 often found in fish farms can have on the digestive physiology of salmonids and the development of new tools for the field of fish physiology.
Chapter 2 presents an experimental investigation into how exposure to elevated CO2 interacts with the metabolic costs of digestion. Utilising unsealed intermittent flow respirometry it was observed that rainbow trout (Oncorhynchus mykiss) are metabolically robust to elevations in environmental CO2 across the scope of one large meal and 6 small meals. This indicates that the reduction in growth observed in salmonids exposed to elevated CO2 is not due to an increased metabolic cost of digestion and therefore may be due to other aspects of a fish’s energy budget.
The contents of Chapter 3 validate a novel method of fish phlebotomy that opens up a range of possibilities for assessing physiological responses that have thus far been unattainable with current methods. By comparing the novel method to grab ‘n’ stab and cannulation it was found that the novel method was able to avoid the acute stress responses associated with grab ‘n’ stab and generate data comparable with cannulation. This method particularly allows assessment of accurate acid-base responses to stimuli in a more ‘natural’ state and in fish smaller than cannulation can typically achieve. Notably, this allows for fish to be voluntarily feeding before blood samples are taken, opening up routes for novel methods of assessing physiological responses to feeding.
Utilising the method developed in Chapter 3, Chapter 4 assesses the internal physiological processes associated with feeding and how these processes are impacted by exposure to elevated CO2. Utilising a combination of post-prandial acid-base whole animal flux measurements, and characterisation of post-prandial blood acid-base state, haematology, and osmotic components, a full characterisation of the physiological processes of digestion were assessed. This chapter found significant alterations to typical transport processes associated with feeding and indicates that elevated CO2 may cause switching of the routes of gastric bicarbonate excretion. There was also evidence of elevated CO2 impacting protein utilisation and assimilation supporting the evidence presented in Chapter 2.

The Mekong Delta is host to a large number of freshwater species, including a unique group of facultative air-breathing Anabantiforms. of these, the striped snakehead (Channa striata), the climbing perch (Anabas testudineus), the giant gourami (Osphronemus goramy) and the snakeskin gourami (Trichogaster pectoralis) are major contributors to aquaculture production in Vietnam. The gastrointestinal responses to feeding in these four species are detailed here. Relative intestinal length was lowest in the snakehead, indicating carnivory, and 5.5-fold greater in the snakeskin, indicating herbivory; climbing perch and giant gourami were intermediate, indicating omnivory. N-waste excretion (ammonia-N + urea-N) was greatest in the carnivorous snakehead and least in the herbivorous snakeskin, whereas the opposite trend was observed for net K+ excretion. Similarly, the more carnivorous species had a greater stomach acidity than the more herbivorous species. Measurements of acid–base flux to water indicated that the greatest postprandial alkaline tide occurred in the snakehead and a potential acidic tide in the snakeskin. Additional findings of interest were high levels of both PCO2 (up to 40 mmHg) and HCO3− (up to 33 mM) in the intestinal chyme of all four of these air-breathing species. Using in vitro gut sac preparations of the climbing perch, it was shown that the intestinal net absorption of fluid, Na+ and HCO3− was upregulated by feeding but not net Cl− uptake, glucose uptake or K+ secretion. Upregulated net absorption of HCO3− suggests that the high chyme (HCO3−) does not result from secretion by the intestinal epithelium. The possibility of ventilatory control of PCO2 to regulate postprandial acid–base balance in these air-breathing fish is discussed.