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D.Sc. / Please refer to full text to view abstract

The pattern of alkaline phosphatase activity in the gastro-intestinal tract of steelhead trout as revealed by Gormori-Takamatsu’s cobalt sulphide method indicates that this enzyme is intimately concerned with differentiation. During the early stages of development, the enzyme is widely distributed and localized mainly in the cytoplasm and nuclei of the gut primordium, later on, however, it tends to become electively localized in those areas where active differentiation is taking place, and disappears from other regions. Regional accumulation and loss of alkaline phosphatase throughout the period of gut differentiation points to the characteristic discontinuity in enzyme development. In the fully developed gut, phosphatase activity is localized in the gastric and intestinal regions in the lamina propria, brush border, and nuclei of the tunica mucosa. The probable role of phosphatase in these locations is discussed. Topographically variable patterns of enzyme activity obtained under different pH conditions suggest that phosphatases present in the brush border, lamina propria, and nuclei have different pH optima and are active over different pH ranges. The use of inhibitors in various concentrations in the incubating medium shows that the phosphatase present in the brush border is more resistant to high concentrations of inhibitors than that in the lamina propria. Nuclear phosphatase, on the other hand, is remarkably sensitive to lower inhibitor concentrations. Under moderate starvation, an increase in the enzyme activity is indicated in the brush border of the intestinal mucosa; acute starvation brings about structural deterioration of the mucosa and desquamation of the columnar epithelial cells, leading to diffuse phosphatase reaction. Probable factors inducing phosphatase accumulation following starvation are discussed. Differential patterns of phosphatase activity in tissue sections obtained under different substrates, pH values, inhibitor concentrations, and levels of starvation strengthen the belief of 'enzyme plurality'. The nuclear and extranuclear phosphatases behave differently and probably are not identical. / Science, Faculty of / Zoology, Department of / Graduate

By superimposing an artificial water flow over the gills out of phase with the natural breathing movements of the fish, it has been possible to demonstrate that bradycardia and cardiorespiratory synchrony develop in response to decreased peripheral oxygen levels. Further evidence that peripheral and not central receptors are involved was furnished by injecting deoxygenated blood into the dorsal aorta; no effect on heart rate or breathing was observed. Bradycardia still develops in response to hypoxia at the respiratory surface even during the absence of branchial blood flow demonstrating that the circulatory system is not involved in this reflex. Numerous tastebud-like receptors have been found lining the anterior faces of the gill bars. These are innervated by the branchial branches of the vagus nerve. Stimulation of the cut central ends of these nerves results in responses similar to those obtained when environmental oxygen levels are decreased. It is suggested that these tastebud-like organs are the receptors and that the branchial branches of the vagus nerve form one afferent pathway for reflex bradycardia and cardiorespiratory synchrony. Other possible afferent pathways are suggested and the functional significance of the reflex is discussed. / Science, Faculty of / Zoology, Department of / Graduate

Mitochondria were isolated from heart and mosaic muscle of rainbow trout (Salmo qairdneri R.). State 3 respiratory rates were determined at 5 and 15°C using pyruvate, malate, lactate, glutamate or acetyl—carnitine as substrate. The final three substrates were used to generate pH profiles. Pyruvate was oxidized at high rates in all cases, indicating good potential for aerobic carbohydrate metabolism. At 15°C, malate was an equally good substrate for heart mitochondria, while all substrates were oxidized at similar rates to pyruvate in muscle mitochondria. Maximal oxidation rates of heart mitochondria were greater than or equal to those of muscle. State 3 Q₁₀ for oxidation of most substrates in heart was approximately 2, except for malate which had a Q₁₀ of 3. Mitochondrial oxidation tended to be more sensitive to decreased temperature in muscle than in heart, particularly with respect to acetyl—carnitine and glutamate oxidation which in muscle had Q₁₀ values of 4 and 7, respectively. Based on RCR values at 5 and 15°C, there was no indication that membrane permeability to H⁺ ions was altered by a 10°C change in temperature in mitochondria from either tissue. At pH above 7.6 respiratory rates decreased with increasing pH. State 3 respiratory rate increased in heart mitochondria as pH decreased, below 7.6 while in muscle mitochondria, no such pH dependence was observed. RCR values were above 4 in all experiments except at high pH. Muscle mitochondria were the more sensitive to extreme pH with respect to RCR. Heart mitochondria had higher oxidative rates than those of muscle and were less sensitive to decreased temperature, in keeping with the greater oxidative demands of that tissue relative to mosaic muscle. Muscle mitochondria which typically face larger fluctuations in extramitochondrial pH in vivo than do those of heart, were less sensitive to pH in vitro. It was concluded that substrate utilization patterns and response to changes in temperature and extramitochondrial pH in the two mitochondrial populations was different and reflected both the intracellular environment of the mitochondria and the different needs of each tissue for aerobic energy supply. / Science, Faculty of / Zoology, Department of / Graduate

The role of carbonic anhydrase in carbon dioxide excretion and acid-base regulation in the rainbow trout, Salmo gairdneri has been investigated. While a significant amount of carbonic anhydrase was found in the blood of the trout, calculations based on red cell hemolysates suggest that the probable circulating levels of carbonic anhydrase activity in blood may not be sufficient to account for the observed carbon dioxide excretion. An analysis of carbonic anhydrase activity in whole blood from the trout revealed that intact fish erythrocytes, unlike mammalian erythrocytes totally fail to facilitate the dehydration of extracellular bicarbonate. The possible mechanism of this phenomenon has been examined; however the salient point was that fish red blood cells do not appear capable and therefore by implication apparently not necessary for the excretion of carbon dioxide at the gills of trout. The observed excretion of carbon dioxide in the trout was found to be accounted for by the gills and their compliment of carbonic anhydrase. This finding was based on the following observations. (1) Depletion of circulating blood carbonic anhydrase levels during severe anemia was without effect on carbon dioxide excretion rates or blood acid-base status. (2) Introduction of the carbonic anhydrase inhibitor, diamox into anemic fish produced a severe acid-base disturbance associated with a fall in observed carbon dioxide excretion. (3) Isolated perfused gill preparations excrete carbon dioxide at rates comparable to those observed in vivo from free swimming fish. (4) Carbon dioxide excretion in isolated gill preparations is abolished by diamox. The excretion of carbon dioxide in fish occurs via the movement of plasma bicarbonate into the branchial epithelium, where it is subsequently dehydrated into molecular carbon dioxide and excreted. A model is proposed and supportive evidence presented to account for the coupling of ionic exchange occurring across the gill with carbon dioxide excretion. The proposed model distinguishes between control of plasma hydrogen ion activity and regulation of plasma total carbon dioxide concentration per se. The functional significance of this pattern of carbon dioxide excretion for aquatic animals is discussed along with the implications for air breathing fish. / Science, Faculty of / Zoology, Department of / Graduate

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Quantitication of stress requires the use of a stress indicator that is easy to measure, and which can be readily interpreted in terms of the potential long-term effects to an organism. This study evaluates the suitability of metabolic rate as an indicator of the stress response in fish. By comparing the metabolic with the cortisol stress response, the most commonly used indicator of stress in fish, it was possible to assess the suitability of metabolic rate as a stress indicator. Changes in metabolic rate were used to predict the long-term effects of transport-related stressors. This study also detennined the baseline metabolic rates of the tish. The standard and the active metabolic rates of juvenile P. cummersonnii were 0.16 ± 0.02 (mean ± S.D, n = 6) mg O₂g⁻¹h⁻¹, and 0.56 ± 0.04 mg O₂g⁻¹h⁻¹, respectively, whereas the routine metabolic rate for the fish was 0.25 ± 0.03 mg O₂g⁻¹h¹. The relationship between metabolic rate and body weight was described by the equation ϺO₂ = 0.64 W⁻°·³⁸. 24-h oxygen consumption measurements showed that juvenile P. commersonnii exhibited diel rhythmicity in oxygen consumption rate, the higher rates occurring at night and the lower rates during the daytime. The higher nocturnal metabolic activity may have been due to increased activity induced by an endogenous rhythm related to feeding. Diel rhythmicity has direct implications for the measurement of baseline metabolic rates since it could result in overestimation or underestimation of these rates. 24-h continuous oxygen consumption measurements enabled the detection of the rhythmicity in oxygen consumption rate, and thereby ensured a greater degree of accuracy in the estimation of these parameters. The metabolic stress response in juvenile P. commersonnii was best described by the equation, y = -0.0013 x² + 0.0364 x ÷ 0.3052, where x = time after application of stressor, and y = oxygen consumption rate. Using the derivative of this equation, the metabolic stress response was estimated to peak approximately 14 min after application of a simulated capture and handling stressor. Oxygen consumption increased by about 300 % as a result of the stress. Approximately 15 min after application of a similar stressor, plasma cortisol levels in stressed fish was 200 % higher than baseline levels. However, cortisol levels in fish sampled 30 min after the disturbance was similar to the baseline cortisol levels, indicating that full recovery had occurred. Although the patterns in the metabolic and cortisol stress responses were similar, metabolic rate could be measured continuously, thereby ensuring accurate interpretation of the data. Furthermore, increases in metabolic rate during the stress response are a culmination of physiological events from the primary to the tertiary levels of biological organization and are, therefore, easier to interpret in terms of long-term effects on the fish. Different transportation procedures elicited variable degrees of stress in juvenile P. commersonnii. The cost of metabolism attributed to the effects of capture and handling was twice as much as that attributed to acute temperature elevation. Acute temperature decrease resulted in a signiticant reduction in the oxygen consumption rate (ANOVA, P < 0.05). Oxygen consumption by the fish was not affected by fish density (ANOVA: F = 2.002, P = 0.5), or by oxygen depletion at dissolved oxygen concentrations above the critical level. Below this level, however, oxygen consumption decreased linearly with further decrease in dissolved oxygen concentration. These results showed that the highest energetic cost to juvenile P. commersonnii was incurred as a result of capture and handling. The results also showed that by subjecting fish to different stressors, it was possible to categorize them according to their relative metabolic costs to the fish. At 25º C, the effective concentration of 2-phenoxyethanol to fully anaesthetize (Stage IV, McFarland 1960) juvenile P. commersonnii was 0.4 ml l⁻¹ and the most appropriate concentration for deep sedation (Stage II, McFarland 1960) of the fish for at least 24 h was 0.2 ml l⁻¹. A maximum of 3 minutes was required by the fish to recover from the effects of the anaesthetic. There was no correlation between fish weight and the rate of induction of anaesthesia (r² = 0.001, p = 0.3). At the peak of the metabolic stress response, oxygen consumption was twice as high in the un-anaesthetized fish compared to the fish anaesthetized after the application of the simulated capture and handling stressor, suggesting that anaesthetization with 2-phenoxyethanol may have reduced the effect of the disturbance on the fish. Similar oxygen consumption rates for the fish anaesthetized prior to capture and the non-stressed fish suggested that the increases in metabolic rate could be linked to the struggling associated with attempts by fish to escape from the perceived stressor. Anaesthetization of juvenile P. commersonnii with 0.3 ml l⁻¹ 2-phenoxyethanol resulted in a more than 200 % increase in plasma cortisol concentration. The elevated levels of plasma cortisol in the anaesthetized fish suggested a manifestation of 2-phenoxyethanol as a stressor. At the time of capture, cortisol levels in fish that were anaesthetized prior to capture were the same as those measured in the disturbed fish at the peak of the stress response (ANOVA, p = 0.95), suggesting that the anaesthetized fish were already experiencing considerable stress at the time they were captured. Undisturbed juvenile P. commersonnii that were anaesthetized for 1 h also had cortisol levels that were five times higher than those measured in undisturbed-unanaesthetized fish, indicating that the duration of exposure to the anaesthetic had a significant effect on plasma cortisol levels. The results presented in this study demonstrate the usefulness of metabolic rate as an indicator of acute stress in fish. This was achieved by comparing the metabolic and the cortisol stress responses. The ease and accuracy with which oxygen consumption of fish could be measured made it possible to measure the stress response more accurately than by plasma cortisol concentration. It was also possible to monitor metabolic rate continuously over a long duration using polarographic oxygen sensors, thus enabling a better evaluation of the stress response. These results, thus, suggest that metabolic rate measurements could be a more practical way to quantify the effects of acute stressors on juvenile fishes. By detailing the profile of the metabolic stress response in P. commersonnii, this study makes a contribution towards understanding the physiological effects of stress in fishes. The study also contributes towards the quantification of baseline metabolic rates of this species under captivity. This study also contributes towards understanding the effects of 2-phenoxyethanol on the stress physiology of fish. By anaesthetizing fish under different conditions of stress, it was possible to evaluate the effect of 2-phenoxyethanol on the metabolic stress response. The ability of 2-phenoxyethanol to reduce physical activity of the fish, and thereby reduce the impact of acute stress on the metabolic stress response, makes it a good agent for the mitigation of stress during the capture and handling of fish. However, the increase in plasma cortisol concentration during prolonged anaesthetization using this drug suggests that the anaesthetic might be a stressor to fish and may, therefore, not be suitable for long-term sedation.

The effects of steady-state, aerobic swimming exercise upon blood volume and flow distribution in the rainbow trout {Salmo gairdneri) were examined. Isotopic Rubidium-86, and radiolabelled microspheres were injected into trout forced to swim against a current at 80% of their critical velocity (U[sub=crit]) in a Brett-type water tunnel respirometer. The results gathered from experiments using these radioactive tracers within the circulatory system of the trout indicated that blood flow during exercise was redistributed to favour working muscles, at the expense of diminished blood flow to those organs and tissues in the systemic circulation which could tolerate periods of transient hypoxia. Active hyperaemia in the skeletal muscle and vasoconstriction of the coeliacomesenteric artery, via adrenergic receptor mechanisms are proposed as the main sites of the control for blood volume and flow redistribution in the systemic circulation in trout during exercise. The gills of these fish must be able to maintain adequate gas transfer in order to keep pace with the increased metabolic demands of the working muscles during exercise. An isolated, saline-perfused trout head preparation and a spontaneously ventilating, blood-perfused whole trout preparation were developed in order to study how increases in the pulsatility of input and increases in the cardiac output through these gills; cardiovascular alterations known to occur during exercise in vivo in these fish, affect fluid flow distribution through, and within the branchial vasculature, and gas exchange across the gills. Data from these preparations indicated that pulsatility of flow increased venolymphatic fluid drainage from within gill tissues, as well increasing the fluid flux/reflux across the branchial microvasculature. However, these changes in fluid distribution associated with increased pulse pressure did not significantly change the rate of gas transfer across the gills. Although gill vascular resistances to flow were very sensitive to alterations in pulse pressure and flow rate, only perfusion flow rate through the gills could cause significant changes in the rate of mass transfer of gases across the gills. The gills of trout therefore were found to be perfusion and not diffusion limited for gas transfer, under conditions which simulated those found at rest and during exercise, in vivo. It also was shown that, given oxygen uptake and cardiac output data from the literature, combined with those for blood flow redistribution during exercise from the present study, the working muscles, which were operational during steady-state, aerobic swimming exercise in rainbow trout, could account for nearly all the measured increase in the total oxygen uptake at this level of exercise. The circulatory system of the rainbow trout, both branchial and systemic, was shown to be quite efficient in its ability to take up and distribute oxygen to the tissues during prolonged, aerobic swimming exercise. The numerous cardiorespiratory adjustments noted during exercise account for this animal's ability to maintain swimming activity in the face of increased oxygen demands put upon the circulation by enforced activity. / Science, Faculty of / Zoology, Department of / Graduate

M.Sc. (Zoology) / This study was undertaken to determine the osmotic regulation of Labeo capensis and to investigate the effectiveness of the addition of salt to water in which the fish are transported. The blood of L. capensis in tap water has a lower pH value than that of the same species acclimatized to salt water. It seems that the pH of fish transported in salt water is not altered immediately as in the case of fish transported in fresh water. In both groups the lowest pH values were reached two days after the fish had been transported ...