Till S. Harter

Validation of the i-STAT system for the analysis of blood parameters in fish

The i-STAT system, a portable clinical analyzer, is increasingly being used to assess blood parameters in fish. This study validated the i-STAT system on rainbow trout blood under a broad range of conditions. Results indicate that the i-STAT is not an appropriate tool for assessing most blood parameters in rainbow trout.

Validation of the i-STAT system for the analysis of blood gases and acid–base status in juvenile sandbar shark (Carcharhinus plumbeus)

We validated the i-STAT system for the analysis of blood parameters in sandbar shark. Results indicate that it is a useful tool for measuring blood pH and could be suitable for operation in most field settings. However, it is not recommended for the assessment of gas tensions in shark blood.

Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium

ABSTRACT Oxygen supply to the heart of most teleosts, including salmonids, relies in part or in whole on oxygen-depleted venous blood. Given that plasma-accessible carbonic anhydrase (CA) in red muscle of rainbow trout has recently been shown to facilitate oxygen unloading from arterial blood under certain physiological conditions, we tested the hypothesis that plasma-accessible CA is present in the lumen of coho salmon (Oncorhynchus kisutch) hearts, and may therefore assist in the luminal oxygen supply to the spongy myocardium, which has no coronary circulation. We demonstrate a widespread distribution of CA throughout the heart chambers, including lumen-facing cells in the atrium, and confirm that the membrane-bound isoform ca4 is expressed in the atrium and ventricle of the heart. Further, we confirm that CA catalytic activity is available to blood in the atrial lumen using a modified electrometric ΔpH assay in intact atria in combination with either a membrane-impermeable CA inhibitor or specific cleavage of the Ca4 membrane anchor. Combined, these results support our hypothesis of the presence of an enhanced oxygen delivery system in the lumen of a salmonid heart, which could help support oxygen delivery when the oxygen content of venous blood becomes greatly reduced, such as after burst exercise and during environmental hypoxia. Highlighted Article: Results from research in coho salmon support the presence of an enhanced oxygen delivery system in the salmonid heart, which could help support cardiac function when oxygen supply to this vital organ becomes limiting.

Characterizing the metabolic capacity of the anoxic hagfish heart

ABSTRACT Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia at 10°C. Such anoxia tolerance demands the mobilization of anaerobic fuels and the removal of metabolic wastes – processes that require a functional heart. The purpose of this study was to measure the metabolic response of the excised, cannulated hagfish heart to anoxia using direct calorimetry. These experiments were coupled with measurements of cardiac pH and metabolite concentrations, at multiple time points, to monitor acid–base balance and anaerobic ATP production. We also exposed hagfish to anoxia to compare the in vitro responses of the excised hearts with the in vivo responses. The calorimetry results revealed a significant reduction in the rate of metabolic heat production over the first hour of anoxia exposure, and a recovery over the subsequent 6 h. This response is likely attributable to a rapid anoxia-induced depression of aerobic ATP-production pathways followed by an upregulation of anaerobic ATP-production pathways such that the ATP production rate was restored to that measured in normoxia. Glycogen-depletion measurements suggest that metabolic processes were initially supported by glycolysis but that an alternative fuel source was used to support the sustained rates of ATP production. The maintenance of intracellular pH during anoxia indicates a remarkable ability of the myocytes to buffer/regulate protons and thus protect cardiac function. Altogether, these results illustrate that the low metabolic demand of the hagfish heart allows for near-routine levels of cardiac metabolism to be supported anaerobically. This is probably a significant contributor to the hagfishs exceptional anoxia tolerance. Summary: Anoxia exposure of the excised Pacific hagfish heart causes an immediate decrease in metabolic activity but this fully recovers during the subsequent 6 h of anoxia.

Validation of the i-STAT and HemoCue systems for the analysis of blood parameters in the bar-headed goose,Anser indicus

Our results indicate that the i-STAT and HemoCue systems can be useful tools to measure many (but not all) blood-gas and acid-base variables in the bar-headed goose. The accuracy of generated results can be improved by using the correction equations provided here, although extrapolation beyond the tested conditions should be avoided.

Isoenergetic Replacement of Fat by Starch in Diets for African Catfish (Clarias gariepinus): Effect on Water Fluxes in the Gastro Intestinal Tract

The effect of an isoenergetic replacement of dietary fat by starch, on chyme characteristics and water fluxes in the gastro intestinal tract (GIT) was assessed. Adult African catfish (Clarias gariepinus) were fed a starch (SD) or fat (FD) diet and groups of fish were dissected at 2, 5 and 8 h after the consumption of a single meal. Chyme was collected quantitatively and was analysed for osmolality and dry matter (DM) content. Postprandial water fluxes were calculated, while using yttrium oxide (Y2O3) as an inert marker to account for the absorption of DM along the GIT. The largest differences in chyme characteristics between diets were observed in the stomach and decreased towards subsequent compartments. A high initial osmotic pressure was measured in the stomach for both diets (up to 498±2 mOsm kg−1) and was likely the driver for the endogeneous water influx to this compartment. Large additions of water were recorded to the stomach and proximal intestine for both diets and absorption of water took place in the mid- and distal intestine. Interestingly, the dietary treatment had an impact on water balance in the stomach and proximal intestine of the fish, but not in the mid- and distal intestine. A strong complementary relationship suggested that 59% of the water fluxes in the proximal intestine could be explained by previous additions to the stomach. Therefore, a higher dietary inclusion of starch led to a shift in water additions from the proximal intestine to the stomach. However, the sum of water additions to the GIT was not different between diets and was on average 6.52±0.85 ml water g−1 DM. The interactions between osmoregulation and digestion, in the GIT of fed freshwater fish, deserve further attention in future research.

Enhanced hemoglobin–oxygen unloading in migratory salmonids

Recent findings indicate that some teleost fishes may be able to greatly enhance hemoglobin–oxygen (Hb–O2) unloading at the tissues under conditions that result in catecholamine release. The putative mechanism relies on the high pH sensitivity of teleost hemoglobin (Hb), intracellular red blood cell (RBC) pH regulation via β-adrenergic Na+/H+ exchanger (β-NHE) activity, and plasma-accessible carbonic anhydrase at the tissues that short-circuits RBC pH regulation. Previous studies have shown that in rainbow trout, this system may double Hb–O2 unloading to red muscle compared to a situation without short-circuiting. The present study determined that: (1) in rainbow trout this system may be functional even at low concentrations of circulating catecholamines, as shown by conducting a dose–response analysis; (2) Atlantic and coho salmon also possess β-NHE activity, as shown by changes in hematocrit in adrenergically stimulated cells; and (3) with β-NHE short-circuiting, Atlantic and coho salmon may be able to increase Hb–O2 unloading by up to 74 and 159%, respectively, as determined by modeling based on O2 equilibrium curves. Together, these results indicate that a system to enhance Hb–O2 unloading may be common among salmonids and may be operational even under routine conditions. In view of the life histories of Atlantic and coho salmon, a system to enhance Hb–O2 unloading during exercise may help determine a successful spawning migration and thus reproductive success.

Beyond just hemoglobin: Red blood cell potentiation of hemoglobin-oxygen unloading in fish.

Teleosts comprise 95% of fish species, almost one-half of all vertebrate species, and represent one of the most successful adaptive radiation events among vertebrates. This is thought to be in part because of their unique oxygen (O2) transport system. In salmonids, recent in vitro and in vivo studies indicate that hemoglobin-oxygen (Hb-O2) unloading to tissues may be doubled or even tripled under some conditions without changes in perfusion. This is accomplished through the short circuiting of red blood cell (RBC) pH regulation, resulting in a large arterial-venous pH difference within the RBC and induced reduction in Hb-O2 affinity. This system has three prerequisites: 1) highly pH-sensitive hemoglobin, 2) rapid RBC pH regulation, and 3) a heterogeneous distribution of plasma-accessible CA in the cardiovascular system (presence in the tissues and absence at the gills). Although data are limited, these attributes may be general characteristics of teleosts. Although this system is not likely operational to the same degree in other vertebrates, some of these prerequisites do exist, and the generation and elimination of pH disequilibrium states at the RBC will likely enhance Hb-O2 unloading to some degree. In human disease states, there are conditions that may partly satisfy those for enhanced Hb-O2 unloading, tentatively an avenue for future work that may improve treatment efficacy.

The time-course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout, Oncorhynchus mykiss

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O2) unloading for cardiovascular O2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (β-NHE) create H+ gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pHi) must recover during venous transit (31-90 s) to enable O2 loading at the gills. The halftimes ( t1/2) and magnitudes of RBC β-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pHi, were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O2 as a sensitive indicator for changes in RBC pHi. In addition, the recovery rate of RBC pHi was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t1/2 of β-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t1/2 of RBC pHi recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H+ gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O2 unloading from pH-sensitive Hb at the tissues, while protecting O2 loading at the gills; the resulting increase in O2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

A novel technique for the precise measurement of CO2 production rate in small aquatic organisms as validated on Aeshnid dragonfly nymphs

ABSTRACT The present study describes and validates a novel yet simple system for simultaneous in vivo measurements of rates of aquatic CO2 production (ṀCO2) and oxygen consumption (ṀO2), thus allowing the calculation of respiratory exchange ratios (RER). Diffusion of CO2 from the aquatic phase into a gas phase, across a hollow fibre membrane, enabled aquatic ṀCO2 measurements with a high-precision infrared gas CO2 analyser. ṀO2 was measured with a PO2 optode using a stop-flow approach. Injections of known amounts of CO2 into the apparatus yielded accurate and highly reproducible measurements of CO2 content (R2=0.997, P<0.001). The viability of in vivo measurements was demonstrated on aquatic dragonfly nymphs (Aeshnidae; wet mass 2.17 mg–1.46 g, n=15) and the apparatus produced precise ṀCO2 (R2=0.967, P<0.001) and ṀO2 (R2=0.957, P<0.001) measurements; average RER was 0.73±0.06. The described system is scalable, offering great potential for the study of a wide range of aquatic species, including fish. Summary: In vitro and in vivo validation of a simple respirometry system for simultaneous measurements of oxygen consumption and carbon dioxide production rate, in small aquatic organisms (2 mg wet mass).