Initially, this study examined the mechanisms by which Na+ and Cl-are regulated in freshwater rainbow trout during exercise and stress. Aerobic exercise (~2 body lengths sec⁻¹) caused a brief increase in diffusive Na⁺ efflux (Jₒᵤₜᴺᵃ⁺) and a brief decline in plasma Na+ and Cl⁻. This disturbance was rapidly compensated by a 3 fold increase in Jᵢₙᴺᵃ⁺ and Jᵢₙᶜˡ⁻(over the first 10-12 h exercise), and by a reduction in Jₒᵤₜᴺᵃ⁺ to 40% of routine by 7 h of exercise. The compensation produced a significant increase in whole body Na⁺ while whole body Cl⁻ remained unchanged. In contrast, confinement stress (for 4 or 8 h) caused an 8 fold increase in Jₒᵤₜᴺᵃ⁺and Jₒᵤₜᶜˡ⁻ which was sustained for at least the first 5 h of stress and resulted in large decreases in whole body Na+ and Cl-. Compensation of the losses was not complete until 24 h post-stress and was achieved by increases in Jᵢₙᴺᵃ⁺ and Jᵢₙᶜˡ⁻ (of similar magnitude and timing to that of exercise) as well as reductions in Jₒᵤₜᴺᵃ⁺ and Jₒᵤₜᶜˡ⁻ to nearly zero. We conclude that Jᵢₙ increased because of an activation of inactive transport sites in the gills while Jₒᵤₜ was reduced by a reduction in branchial ionic permeability, both responses mediated hormonally. Although the hormonal control mechanisms are as yet poorly defined, we argue that growth hormone and prolactin are responsible for the Jᵢₙ and Jₒᵤₜ regulation, respectively, and rule out either cortisol or epinephrine as having any role, at least with respect to the rapid NaCI regulation evident during exercise. The ability of rainbow trout to rapidly regulate ion balance was then investigated to determine whether it is unique to rainbow trout, exists in stream-dwelling animals or whether it is wide spread in fish regardless of preferred habitat. Common shiners, considered to be an active species and smallmouth bass, considered to be less active, were the two species of comparison. Common shiners demonstrated rapid increases in Jᵢₙᴺᵃ⁺ during exercise and confinement, a lack of change in whole body Na⁺ and Cl⁻ during exercise and a large Na⁺ and Cl⁻ loss during stress. In contrast, smallmouth bass experienced minimal increases in Jᵢₙᴺᵃ⁺ during exercise and no change during stress with ion loss occurring during both exercise and stress. It was concluded that the relative ability to regulate ion balance in response to stress and exercise may reflect the frequency with which the animal experiences that challenge in its natural habitat. Consequently, common shiners probably possess a similar uptake mechanism to that of rainbow trout while the mechanisms in smallmouth bass may exist, but in attenuated form. The final analysis investigated whether or not exercise training affected the magnitude of the disturbance to Na⁺ balance produced by both acute and chronic stress. This was important in that it could be applied to the improvement of fish stocking techniques. Trained fish demonstrated the ability to reduce ion loss produced by stress despite significantly high levels of cortisol, glucose and oxygen consumption. Similar results were produced by both acute and chronic stress and it was established that the rainbow trout's ability to regulate ions during stress, without altering the release of cortisol and catecholamines was improved by training. / Thesis / Master of Science (MSc)

The alligator gar Atractosteus spatula is a euryhaline fish found in the Gulf of Mexico and surrounding drainages. However, the extent of its hypo-osmotic abilities are not well understood. To determine effect of salinity on growth, metabolic rate, and osmoregulation abilities the following question was developed: when does the alligator gar have the osmoregulatory capabilities to survive in hyperosmotic environments? To answer this question, two different age groups (60 and 330 days after hatch [DAH]) of juvenile alligator gar were exposed to 4 different salinities (0, 8, 16, and 24 ppt) for a 30-day period. Specific growth rate, oxygen consumption rate, plasma osmolality, plasma ion concentrations, tissue Na+, K+-ATPase activities, and drinking rate were measured. I determined that the 60 DAH alligator gar had a greater ability to grow and regulate ions than did the 330 DAH alligator gar in increased salinity.

<p> The development of Na+ and Ca++ transport, ammonia excretion, and respiration in larval rainbow trout were studied over the period of yolk sac absorption and shortly after the onset of exogenous feeding. The gills and the yolk sac epithelium of larvae were examined for any indication of the presence of mitochondria-rich cells and/ or Na-K-ATPase activity. </p> <p> Whole body Na+ content of the larvae increased throughout yolk sac absorption and after the onset of exogenous feeding. During this period there was an increase in Na+ influx, efflux, transporter capacity Umax> and transporter affinity (Km). Jmax increased 4.6 times during yolk sac absorption, and increased another 2.8 times 248 degree·days after the onset of exogenous feeding. Km decreased over the first 168 degree·days and stayed constant for the remainder of the experimental period. The gills were the primary site of Na+ efflux. Na+ accumulation from the water began the first day post hatch. </p> <p> Ca++ transport capacity increased throughout yolk sac absorption, but then decreased after the onset of exogenous feeding. Jmaxca++ exhibited a 15.5 fold increase over 257 degree·days, which then decreased to 30% of that value after exogenous feeding commenced. There was a 162 fold increase in Ca++ Km over yolk sac absorption, which then decreased 15.6 times after the onset of exogenous feeding. Whole body Ca++ began to increase approximately 200 degree·days post-hatch and exhibited a large increase after the onset of exogenous feeding. It is possible that during yolk sac absorption the larvae does not begin to accumulate Ca++ from the external environment until its internal reserves are depleted, and with the onset of feeding the larvae then obtains its Ca++ from its diet. </p> <p> Ammonia excretion in larval trout increased 3.6 times over yolk sac absorption, and increased another 2.8 times after exogenous feeding commenced. There was no evidence of a link between Na+ uptake and NJ-:4+ excretion in larval fish as NJ-:4+ excretion was not stimulated in response to increasing external Na+ concentrations. Measurements of the ammonium concentration at different surfaces of the larvae revealed a larger ammonium concentration next to the gills than next to the yolk sac and skin. This may indicate that the gills are the primary site for ammonia excretion in the larval fish. </p> <P> Initially, branchial and cutaneous surfaces contributes to respiration, but as the gills grow and develop and the yolk sac decreases in size and the skin thickens, the contribution of the gills to this function increases. </p> <p> The gills of first day hatchlings are comprised of gill arches and filaments. Lamellae do not begin to develop until a few days after hatching. The gill filaments contain mitochondria-rich cells and have Na-K-ATPase activity as determined through binding of the fluorescent dye anthroylouabain. The yolk sac epithelium contained cells with concentrated regions of mitochondria, but significant Na-K-ATPase activity was not detected when compared to branchial staining. </p> / Thesis / Master of Science (MSc)

This study investigated the regulation of Na⁺ Ca²⁺ Cl⁻ during development in embryonic rainbow trout (Oncorhynchus mykiss). Because there is a close relationship between pH regulation and ion uptake mechanisms in adult teleosts, pH, pCO₂, and NH₄⁺ levels in unstirred layers (USLs) adjacent to whole eggs and dechorionated embryos were determined using double-barrel ion-selective microelectrodes (ISMEs). Whole eggs accumulated Na⁺, Ca²⁺ and Cl⁻ during the last 20 days of embryonic development, suggesting an ionoregulatory ability prior to hatching. Na⁺ uptake by whole eggs was linearly related to external [Na⁺], suggesting that Na⁺ crosses the chorion by diffusion. The uptake by dechorionated embryos was saturable, indicating the presence of active transport or facilitated diffusion mechanisms on the surface of embryos prior to hatching. Ca²⁺ uptake by whole eggs and dechorionated embryos was saturable, suggesting that specific pathways or binding sites are present in the chorion, and that active transport or facilitated diffusion mechanisms are present at the surface of embryos. The much higher Jₘₐₓ for whole eggs than dechorionated embryos suggests a role of the perivitelline fluid (pvf) or chorion in ion uptake. Jₘₐₓ was lower in dechorionated embryos than in hatchlings suggesting that Ca²⁺ uptake mechanisms may not be fully developed in dechorionated embryos. Low pH and high [NH₄⁺] were measured in the USLs adjacent to whole eggs and dechorionated embryos, relative to the bulk water. A high Pcₒ₂ measured adjacent to the gills suggests that CO₂ excretion was the primary source of USL acidification. pH was lower in the USL adjacent to the gills and trunk than in the USL adjacent to the yolk sac. [NH₄⁺] was highest adjacent to the gills and trunk than adjacent to the yolk sac. There was no change in the extent of USL acidification or in [NH₄⁺] over the last half of embryonic development. After hatching, however, there was a significant increase in [NH₄⁺] adjacent to the gills, which was not accompanied by a change in USL pH. It is possible that a change in permeability of the transporting surfaces (i.e. gills, trunk, yolk sac) occurs after hatching. / Thesis / Master of Science (MS)

The H+-ATPase rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. In the present study, a ratiometric imaging technique using the pH indicator dye BCECF was developed to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo. Initial validation experiments demonstrated that HR cells subjected to respiratory acidosis (1% CO2) or metabolic alkalosis (20 mM NH4Cl) exhibited changes in BCECF 513/438 emission ratios which were consistent with the expected effects of these treatments on pHi. Subsequent experiments focussed on the involvement of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (isoform NHE3b; zslc9a3.2) and the H+-ATPase (atpv1aa) in pHi regulation. Additionally, the role of HR cell carbonic anhydrase (“CA2-like a”) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. To do so, relative HR cell pHi changes were monitored during acid-base challenges in shams and in fish experiencing morpholino gene knockdown of either NHE3b, H+-ATPase or “CA2-like a”. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalization were altered markedly in fish experiencing knockdown of “CA2-like a”, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. Knockdown of “CA2-like a”, although presumed to limit H+ availability to NHE3b and H+-ATPase, yielded qualitatively similar results to knockdown of either single H+ excretory pathway. The metabolic alkalosis and subsequent acidification associated with NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.

[ Truncated abstract] The purpose of this series of studies was to further our understanding of the relationship between muscle buffer capacity and training. Study one was performed to determine if there were differences in muscle buffer capacity (βmin-vitro) between females of differing training status (i.e., team-sport, endurance-trained and untrained subjects). Studies two and three were then performed to determine if short-term training could improve muscle buffer capacity and what type of training best improves this muscle characteristic (i.e., high-intensity, moderate-intensity or resistance training). Studies four and five were performed to determine if the accumulation of H+ during exercise was a mechanism by which training improved muscle buffer capacity. Below is a summary of these five studies. Study 1. The team-sport group had a significantly higher βmin-vitro than either the endurance-trained or the untrained groups (181 ± 27 v 148 ± 11 v 122 ± 32 'mol H+?g dm-1?pH-1 respectively; P<0.05). The team-sport group also completed significantly more relative total work (299 ± 27 v 263 ± 31 v 223 ± 21 J?kg-1, respectively; P<0.05) and absolute total work (18.2 ± 1.6 v 14.6 ± 2.4 v 13.0 ± 1.9 kJ, respectively; P<0.05) than the endurance-trained or untrained groups during a repeated sprint ability (RSA) test. There was a significant correlation between βmin-vitro and RSA (r=0.67; P<0.05). These findings show that young females competing in team sports have a larger βmin-vitro than either endurance-trained or untrained females. This may be the result of the intermittent, high-intensity activity during training and the match play of team-sport athletes... Conclusions. Our results show that training intensity is an important determinant of muscle buffer capacity. High-intensity interval training (120-150% LT) improved muscle buffer capacity (9 27%), however, neither moderate-intensity or resistance training improved muscle buffer capacity. In contrast higher training intensity (150-180% LT) may negatively affect muscle buffer capacity. Differences in the accumulation of H+ during high-intensity interval training does not appear to be the underlying mechanism for changes to muscle buffer capacity, however changes to this muscle characteristic were associated with pre-training muscle buffer capacity.

Goldfish undergo an adaptive morphological change in their gills involving the reversible growth and loss of a mass of cells (interlamellar cell mass, ILCM) in between the lamellae depending on oxygen demand, which can be altered by the environment or metabolic demands of the individual. The ILCM contributes to decreased passive Na+ efflux across the gill. Active uptake is maintained by the re-distribution of the ionocytes expressing Na+-uptake relevant genes (NHEs and H+-ATPase) to the outer edge of the ILCM where they can establish contact with the external environment and/or lamellar epithelium. This adaptation is thought to be partly responsible for the extreme anoxia tolerance demonstrated by goldfish, which they experience on a seasonal basis living in a pond environment. Hypoxia and hypercapnia are frequently encountered in such freshwater environments and as such, the effect of the ILCM on the capacity for acid-base regulation was evaluated. Differences in the time course of acid excretion to the environment without effect on systemic pH regulation were likely the result of the ILCM.