Automated determination of critical oxygen concentration for routinely active fish

Synopsis We describe an automated respirometer and control system that determines critical (metabolism-limiting) oxygen concentration for routinely active fish. A microcomputer monitors fish metabolic rate as oxygen concentration in the closed respirometer declines; the critical oxygen concentration is signaled by metabolic-rate change, which is resolved via statistically based rules that consider both magnitude and consistency of rate deviations. After the critical oxygen concentration is found, data are written to disk, the respirometer is reoxygenated and another trial is initiated. This sequence can be repeated indefinitely without human intervention, allowing replicate estimates from a single fish. Ideally, metabolic rate should be calculated frequently during a trial, to minimize exposure of the fish to sub-critical oxygen concentrations. However, precision of measurement is limited by ‘noise’ related to length of time interval over which changes in oxygen concentration are determined, respirometer chamber volume, and fish respiration rate. Short time intervals lead to excessive noise, whereas long time intervals result in insufficient numbers of rate measurements. In a respirometer chamber of calculable optimum volume, measurements made by averaging oxygen readings taken very rapidly over two to three minute intervals provide a good compromise. We present data from experiments with bluegill,Lepomis macrochirus, to illustrate the method and show that critical oxygen concentrations identified by the system are consistent with estimates made by humans viewing graphs of the same experiments     

A lab-built respirometer for plant and animal cell culture

A very simple off-line respirometer was developed to measure oxygen consumption rates of low respiring and shear-sensitive cell suspensions. The respirometer is composed of a 10 mL glass syringe in which the plunger was substituted with a polarographic dissolved oxygen probe. Mechanical agitation is provided by means of a magnetic stirring bar inside the measuring chamber and a stir plate placed below the respirometer. Abiotic oxygen fluxes occurring in the measurement chamber such as oxygen diffusion and probe oxygen consumption were investigated. The apparent oxygen uptake rate was then corrected for abiotic oxygen fluxes, leading to accurate measurements of respiration rates ranging from 0.5 to 25.0 mM x h(-1). Additionally, the effect of the stirring bar shape and of the test length on the integrity of plant (Eschschzoltzia californica) and animal (NS0) cells was evaluated. Animal cells showed a higher resistance to mechanical stirring inside the respirometer compared to plant cells (0% of broken cells and 78.1% respectively for a polygonal stirring bar and a 15 min test). For plant cells, cell damage inside the measurement chamber was reduced by optimizing the stirring bar shape and reducing the test length to 5 min or less. This very simple design was shown to provide reliable and low-cost quantification of the oxygen uptake rate of plant and animal cells and can be use even for more demanding measurements such as oxygen affinity studies.     

An automatic respirometer for determining oxygen uptake in crayfish (Austropotamobius pallipes (Lereboullet)) over periods of 3-4 days.

1. An automatic respirometer has been developed for continuous measurements over 3-4 days on 1-15 g crayfish. The sensor is a modified Mackereth oxygen electrode. Respiration is recorded on a millivolt potentiometric pen recorder during closed periods when the O2 concentration in the medium falls to a predetermined level. A solenoid-operated valve is then opened via a relay circuit energized by a reed switch mounted on the recorder. Medium flows through the respirometer until the O2 concentration is raised back to another predetermined level. Artificial media containing little or no nutrient salts are used to restrict the growth of microbes. 2. Respiration was determined chiefly on crayfish (Austropotamobius pallipes) with body wet weights of 7-0-12-5 g at 10-0 degrees C. In unrestrained but relatively quiescent animals, standard metabolism is described by the regression equation, ln O2 uptake = 3-3037 + 1-002 ln body wt. In restless crayfish active metabolism is described by the equation, ln O2 uptake = 4-4412 + 0-861 ln body wt.     

An underwater respirometer and programmable data logger for in situ energy budget studies

An in situ microprocessor-controlled underwater respirometer which was used for monitoring oxygen exchanges during the determination of the energy budget for a Caribbean coral, is described. Sensors were used to monitor levels of oxygen, temperature and underwater light. The 6502-based micro-computer was programmed to record these values on a tape cassette and to flush sea water through the respirometer at selected intervals. Final analyses of the data recorded during a 24- or 36-h in situ run were made with a shore-based microcomputer. The apparatus is inexpensive to construct and the programming flexibility opens the possibility for use in a range of other in situ monitoring situations.     

Evaluation of the Total Carbonaceous Biochemical Oxygen Demand Method, Using a Compensated Recording Respirometer

The total carbonaceous biochemical oxygen demand test is described, and experimental data demonstrating its stoichiometry, precision, and accuracy are presented. The test is more reproducible and faster than the current 5-day biochemical oxygen demand test procedure, and if a respirometer is used, the effects of toxic chemicals, pH changes, and nutrient imbalances can be routinely monitored. The design principles of a multichannel compensated recording respirometer suitable for this test are described.     

The influence of gradual water acidification on the oxygen consumption pattern of fish

1. Carp (Cyprinus carpio) and tilapia (Oreochromis mossambicus) were kept in a flow-through respirometer for a week, while continuous oxygen consumption measurements were carried out, at a constant water O₂ concentration.2. Our measurements showed that tilapia decreased its standard metabolic rate (SMR), its average oxygen consumption, and its maximum metabolic rate in acid water (pH 4.0), whereas carp did not.3. It is proposed, based on our results, that carp and tilapia probably have different strategies to survive pH depression episodes: whereas tilapia avoids the additional stress of exercise, carp tries to escape the acid conditions.     

Cardiorespiratory performance during prolonged swimming tests with salmonids: a perspective on temperature effects and potential analytical pitfalls

A prolonged swimming trial is the most common approach in studying steady-state changes in oxygen uptake, cardiac output and tissue oxygen extraction as a function of swimming speed in salmonids. The data generated by these sorts of studies are used here to support the idea that a maximum oxygen uptake is reached during a critical swimming speed test. Maximum oxygen uptake has a temperature optimum. Potential explanations are advanced to explain why maximum aerobic performance falls off at high temperature. The valuable information provided by critical swimming tests can be confounded by non-steady-state swimming behaviours, which typically occur with increasing frequency as salmonids approach fatigue. Two major concerns are noted. Foremost, measurements of oxygen uptake during swimming can considerably underestimate the true cost of transport near critical swimming speed, apparently in a temperature-dependent manner. Second, based on a comparison with voluntary swimming ascents in a raceway, forced swimming trials in a swim tunnel respirometer may underestimate critical swimming speed, possibly because fish in a swim tunnel respirometer are unable to sustain a ground speed.     

Energetics of osmoregulation: I. Oxygen consumption by Fundulus heteroclitus

We have developed a flow-through method for measuring oxygen consumption in fish which allows continuous monitoring over periods of days with good accuracy. Our goal was to determine the changes in basal metabolic rate in estuarine fish as a function of salinity. We show that in Fundulus heteroclitus, the oxygen consumption drops by 50% during the first 12 hr in the respirometer, as the fish cease exploratory movements. We have determined the influence of temperature and body size on resting respiratory rate, but failed to find any circadian or tidal rhythm in aerobic respiration. With these variables controlled, we determined that changing from 10 to 30 ppt water had no demonstrable effect on oxygen uptake. Since there must be a large change in osmotic flux due to this change in salinity, it appears that the fish might be diverting energy from other uses rather than increasing aerobic energy production to meet the increased osmoregulatory work load.     

Steady-state measurements of respiration rate with an oxygen electrode.

An oxygen electrode was developed which measures steady-state respiration rates in a volume of 0.25 ml and at oxygen concentrations as low as 0.1 μm. The steady state was achieved by pumping air-equilibrated buffer into the respirometer at various rates. The method is most suitable for tissue slices.     

Development of respirometry methods to assess the microbial activity of thermophilic bioleaching archaea

Respirometry methods have been used for many years to assess the microbial activity of mainly heterotrophic bacteria. Using this technique, the consumption of oxygen and evolution of carbon dioxide for heterotrophic carbon catabolism can be used to assess microbial activity. In the case of autotrophic bioleaching bacteria, carbon dioxide is used as a carbon source resulting in the consumption of both oxygen and carbon dioxide. The use of such respirometry techniques at high temperatures (up to 80 degrees C) for the investigation of bioleaching Archaea, however, poses particular difficulties. At these elevated temperatures, the solubility of oxygen into the liquid phase is particularly poor. This work details specific methods by which high temperature constraints are overcome while monitoring the activity of thermophilic Archaea using a Micro-Oxymax respirometer (Columbus Instruments). The use of elevated headspace oxygen concentrations, in order to overcome low oxygen solubility, is demonstrated as well as the effect of such elevated oxygen concentrations on microbial oxygen consumption rates. The relative rates of oxygen and carbon dioxide consumption are also illustrated during the oxidation of a chalcopyrite concentrate. In addition, this paper details generic methods by which respirometry data can be used to quantify inhibitory effects of a compound such as Na(2)SO(4). The further use of such data in predicting minimum hydraulic reactor retention times for continuous culture bioleaching reactors, as a function of concentration of potentially inhibitory compounds, is also demonstrated.     

The effect of temperature and mass on routine metabolism in Sarotherodon (Tilapia)mossambicus (Peters)

A reappraisal of oxygen uptake by Sarotherodon mossambicus was undertaken using a continuous flow respirometer. Measurements were obtained over the temperature range 16°C–37°C for fish weighing between 10 g and 150 g. Oxygen uptake was converted to energy equivalents ( Q _ox) using the value 13.68 J mg O₂^–1and the routine metabolic energy expenditure can be described by the equation E =0.0086 t ^{2 0783} M ^{0 652} where E is the energy requirement for routine metabolism expressed in J h^-1, t the temperature in °C and M the mass in g.     

Oxygen-stabilized enzyme electrode for d-glucose analysis in fermentation broths

A new principle for the construction of oxygen-dependent enzyme electrodes is presented. The enzyme electrode is based on a galvanic oxygen electrode which is furnished with an electrolysis anode covered by immobilized enzyme and placed close to the oxygen-sensing surface. An ordinary oxygen electrode is used as a reference electrode. The enzymatic consumption of oxygen in the enzyme electrode generates a potential difference between the electrodes which is utilized to control electrolytic generation of oxygen from water in such a way that zero differential potential is maintained. Thus, the enzyme electrode operates under ambient oxygen tension and does not suffer from oxygen limitation. The electrolytic current in this system gives a measure of the concentration of substrate surrounding the enzyme electrode. The electrode has been applied for continuous d-glucose analysis in situ during batch cultures of Candida utilis.     

Physiological responses to swimming fatigue in juvenile largemouth bronze gudgeon Coreius guichenoti

Stepped velocity tests were conducted on juvenile largemouth bronze gudgeon Coreius guichenoti in a swim tunnel respirometer, and oxygen consumption increased with swimming speed to fatigue and then decreased during recovery. Serum levels of total protein, glucose and triglycerides initially decreased, increased at fatigue and then decreased during recovery. Levels stabilized after 120 min, corresponding to the time necessary to recover from fatigue.     

The effect of temperature on routine metabolism in Tilapia rendalli boulenger

Using a continuous flow respirometer it was shown that young Tilapia rendalli had three distinct phases of oxygen consumption over the temperature range of 17–40° C. In the first phase (17–28° C) the metabolic energy demand followed the normal logarithmic increase with increasing temperature. Between 28 and 37° C the increased oxygen uptake was suppressed and showed a relatively small increase with increasing temperature. This feature was believed to be a significant energy saving function important to the growth of these fish which feed in the warm eulitoral margins of lakes during the day. The final phase shows a return to the original logarithmic increase in oxygen consumption. These results were related to, and compared with, actual changes in biomass at various temperatures and the theoretical and actual biomass changes were found to compare favourably.     

Routine metabolic rate of southern bluefin tuna (Thunnus maccoyii).

Routine metabolic rate (RMR) was measured in fasting southern bluefin tuna, Thunnus maccoyii, the largest tuna species studied so far (body mass=19.6 kg (+/-1.9 SE)). Mean mass-specific RMR was 460 mg kg(-1) h(-1) (+/-34.9) at a mean water temperature of 19 degrees C. When evaluated southern bluefin tuna standard metabolic rate (SMR) is added to published values of other tuna species, there is a strong allometeric relationship with body mass (423 M(0.86), R(2)=0.97). This demonstrates that tuna interspecific SMR scale with respect to body mass similar to that of other active teleosts, but is approximately 4-fold higher. However, RMR (not SMR) is most appropriate in ram-ventilating species that are physiologically unable to achieve complete rest. Respiration was measured in a large (250,000 l) flexible polypropylene respirometer (mesocosm respirometer) that was deployed within a marine-farm sea cage for 29 days. Fasted fish were maintained within the respirometer up to 42 h while dissolved oxygen dropped by 0.056 (+/-0.004) mg l(-1) h(-1). Fish showed no obvious signs of stress. They swam at 1.1 (+/-0.1) fork lengths per second and several fed within the respirometer immediately after measurements.     

Routine metabolic rate of southern bluefin tuna (Thunnus maccoyii)

Routine metabolic rate (RMR) was measured in fasting southern bluefin tuna, Thunnus maccoyii, the largest tuna species studied so far (body mass=19.6 kg (+/-1.9 SE)). Mean mass-specific RMR was 460 mg kg(-1) h(-1) (+/-34.9) at a mean water temperature of 19 degrees C. When evaluated southern bluefin tuna standard metabolic rate (SMR) is added to published values of other tuna species, there is a strong allometeric relationship with body mass (423 M(0.86), R(2)=0.97). This demonstrates that tuna interspecific SMR scale with respect to body mass similar to that of other active teleosts, but is approximately 4-fold higher. However, RMR (not SMR) is most appropriate in ram-ventilating species that are physiologically unable to achieve complete rest. Respiration was measured in a large (250,000 l) flexible polypropylene respirometer (mesocosm respirometer) that was deployed within a marine-farm sea cage for 29 days. Fasted fish were maintained within the respirometer up to 42 h while dissolved oxygen dropped by 0.056 (+/-0.004) mg l(-1) h(-1). Fish showed no obvious signs of stress. They swam at 1.1 (+/-0.1) fork lengths per second and several fed within the respirometer immediately after measurements.     

Oxygen consumptions and potassium contents of slices of rat renal cortex.

An oxygen electrode respirometer for determining the oxygen consumption of slices of mammalian renal cortex is described and assessed. Though rat renal cortical slices incubated in potassium-free medium for one hour lost 102 +/- 14 mmol of potassium/kg dry weight, there was only a small, nonsignificant fall in oxygen consumption. In contrast the oxygen consumption of slices incubated in potassium-free medium with 10 mmol.1-1 ouabain was markedly reduced (by 32 +/- 6%), while such slices lost 180 +/- 15 mmol of potassium/kg dry weight. These disproportionate effects on potassium loss and inhibition of oxygen consumption suggest that in renal cortical slices the loss of potassium in low potassium medium is not primarily due to inhibition of the conventional sodium pump.     

中华鳖幼体呼吸代谢的初步研究

本文利用改装后的Ｋａｌａｂｕｋｈｏｖ－Ｓｋｖｏｒｓｔｏｖ呼吸器,同时测定了中华鳖在自由潜水状态下的空气呼吸和水呼吸摄氧量。结果表明：在正常生活状态下,中华鳖主要依靠空气呼吸摄氧;水呼吸仅占总呼吸摄氧量为２．６８％左右。呼吸摄氧率无显著昼夜变化。     

Effects of cell density and temperature on oxygen consumption rate for different mammalian cell lines.

Oxygen consumption rates were measured in a respirometer for different mammalian cell lines (BHK, murine hybridoma, and CHO), and the effects of cell density (1-20 million cells/mL) and temperature (6 to 37 degrees C) on specific oxygen consumption rate were investigated. The specific oxygen consumption was cell line dependent. For a given temperature, the murine hybridoma cells had the lowest and the CHO cells had the highest oxygen consumption rate. The specific oxygen consumption rate was not affected by the cell concentration for cell densities between 1 and 20 million cells/mL. However, artificial trends implicating the effects of cell density were obtained when traditional analysis was used and the probe response time was neglected. A detailed mathematical analysis was presented to investigate the magnitude of errors originating from neglecting the probe response time for the calculation of oxygen consumption rate. The error was significant, especially when the probe response was slow and/or the oxygen consumption was fast. Temperature influenced the specific oxygen consumption rate similarly for the cells studied, and about 10% decrease was observed in specific oxygen consumption by 1 degrees C decrease in the temperature. Between 6 and 37 degrees C, the effect of temperature on oxygen consumption rate could be described using an Arrhenius model, i.e., qO2 = qoO2. e-E/RT. The activation energy, E, in this equation was similar for different cells (between 80 and 90 kJ/mol), indicating the action of a similar mechanism for the effect of temperature on oxygen consumption.     

Cost of venom regeneration in Parabuthus transvaalicus (Arachnida: Buthidae)

Scorpion venom has many components, but is mainly made up of water, salts, small molecules, peptides, and proteins. One can reasonably assume that the production and storage of this complex secretion is an expensive metabolic investment. However, to date, no study has addressed the costs associated with the regeneration of venom by scorpions. Using a closed-system respirometer, we examined the difference in oxygen consumption between milked and unmilked scorpions to determine the metabolic costs associated with the first 72 h of subsequent venom synthesis. During this time period, milked scorpions had a significantly higher (39%) metabolic rate than unmilked scorpions. The regenerated venom from a second milking had significantly lower (74%) protein concentration, suggesting that venom regeneration was incomplete after 72 h. The protein content in the regenerated venom was not correlated with oxygen consumption. The significant increase in oxygen consumption after milking supports existing hypotheses about the metabolic cost associated with venom regeneration and provides further insight on why scorpions appear to be judicious in their stinger use.