Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

Previous studies at the Key Lake uranium mill (Saskatchewan, Canada) suggested the complex effluent discharged alters energetic stores of resident fish species. A second study at the same site demonstrated certain fish from lakes downstream of the mill produce larvae with elevated incidence of developmental deformities. The mechanisms by which energy homeostasis is affected in fish downstream of the Key Lake uranium mill are unknown, and the effects of deformities and altered metabolism on swimming ability have not been explored. Therefore, the overall objective of this thesis was to investigate whether effluent exposed fish exhibited differences in swimming performance and energy homeostasis. To achieve this objective two experiments were conducted. In the first experiment juvenile spottail shiner (Notropis hudsonius) were collected from a lake downstream of the Key Lake uranium mill, and compared to fish collected from a nearby reference lake. In the second experiment larvae were collected from laboratory raised fathead minnow (Pimephales promelas) exposed to 5% diluted uranium mill effluent or control (dechlorinated municipal) water, and reared in the same treatments to 60 days post hatch (dph). No gross deformities were observed in any fish, and only shiner collected from the exposure lake in the field experiment had enlarged heart ventricles relative to body size compared to fish from the reference lake. Swimming performance was similar between shiner from the exposure and reference lakes in the field study, but effluent exposure impaired swimming ability in 60 dph fathead minnow in the laboratory experiment compared to fish from the control water treatment. After swimming performance tests fish were considered fatigued and metabolic endpoints were compared to non-fatigued fish. In both non-fatigued and fatigued shiner, liver glycogen was significantly greater in fish collected from the exposure lake compared to the reference lake. There was no difference in liver triglycerides in non-fatigued shiner between lakes, but liver triglycerides decreased after swimming in the field study reference fish. Muscle energy stores were unaffected by site or swimming in the field experiment. Conversely, whole body triglycerides and glycogen were similar between treatments in non-fatigued fathead minnow in the laboratory experiment. Swimming significantly decreased whole body triglycerides in fathead minnow from both treatments, but whole body glycogen was unaffected. In the field experiment blood endpoints (hematocrit, plasma glucose, lactate) in fatigued and non-fatigued shiner from both lakes further supported the possibility of altered intermediary metabolism or blunted stress response in fish downstream of the Key Lake uranium mill. In the field study, shiner muscle citrate synthase activity (an indicator of tissue aerobic capacity) was similar between lakes, but muscle âhydroxyacyl coenzyme A dehydrogenase activity (an indicator of tissue lipolytic capacity) was elevated. In contrast, laboratory fathead minnow whole body âhydroxyacyl coenzyme A dehydrogenase activity was similar between treatments, but citrate synthase activity was significantly lower in fathead minnow from the 5% effluent treatment. In summary, shiner from the exposure lake in the field experiment had similar swimming endurance and greater energy stores compared to fish from the reference lake, despite metabolic alterations. Fathead minnow from the 5% effluent treatment in the laboratory experiment had reduced swimming endurance that was matched by reduced whole body citrate synthase activity, but no other metabolic alterations were observed. Therefore, effluent exposure caused metabolic alterations in both fathead minnow and shiner, but specific effects between experiments were inconsistent. Overall, the physiological significance of the metabolic and swimming effects of effluent exposure is unclear, but suggests discharged effluent has the potential to negatively affect wild fish survivability.

Previous studies at the Key Lake uranium mill (Saskatchewan, Canada) suggested the complex effluent discharged alters energetic stores of resident fish species. A second study at the same site demonstrated certain fish from lakes downstream of the mill produce larvae with elevated incidence of developmental deformities. The mechanisms by which energy homeostasis is affected in fish downstream of the Key Lake uranium mill are unknown, and the effects of deformities and altered metabolism on swimming ability have not been explored. Therefore, the overall objective of this thesis was to investigate whether effluent exposed fish exhibited differences in swimming performance and energy homeostasis. To achieve this objective two experiments were conducted. In the first experiment juvenile spottail shiner (Notropis hudsonius) were collected from a lake downstream of the Key Lake uranium mill, and compared to fish collected from a nearby reference lake. In the second experiment larvae were collected from laboratory raised fathead minnow (Pimephales promelas) exposed to 5% diluted uranium mill effluent or control (dechlorinated municipal) water, and reared in the same treatments to 60 days post hatch (dph). No gross deformities were observed in any fish, and only shiner collected from the exposure lake in the field experiment had enlarged heart ventricles relative to body size compared to fish from the reference lake. Swimming performance was similar between shiner from the exposure and reference lakes in the field study, but effluent exposure impaired swimming ability in 60 dph fathead minnow in the laboratory experiment compared to fish from the control water treatment. After swimming performance tests fish were considered fatigued and metabolic endpoints were compared to non-fatigued fish. In both non-fatigued and fatigued shiner, liver glycogen was significantly greater in fish collected from the exposure lake compared to the reference lake. There was no difference in liver triglycerides in non-fatigued shiner between lakes, but liver triglycerides decreased after swimming in the field study reference fish. Muscle energy stores were unaffected by site or swimming in the field experiment. Conversely, whole body triglycerides and glycogen were similar between treatments in non-fatigued fathead minnow in the laboratory experiment. Swimming significantly decreased whole body triglycerides in fathead minnow from both treatments, but whole body glycogen was unaffected. In the field experiment blood endpoints (hematocrit, plasma glucose, lactate) in fatigued and non-fatigued shiner from both lakes further supported the possibility of altered intermediary metabolism or blunted stress response in fish downstream of the Key Lake uranium mill. In the field study, shiner muscle citrate synthase activity (an indicator of tissue aerobic capacity) was similar between lakes, but muscle âhydroxyacyl coenzyme A dehydrogenase activity (an indicator of tissue lipolytic capacity) was elevated. In contrast, laboratory fathead minnow whole body âhydroxyacyl coenzyme A dehydrogenase activity was similar between treatments, but citrate synthase activity was significantly lower in fathead minnow from the 5% effluent treatment. In summary, shiner from the exposure lake in the field experiment had similar swimming endurance and greater energy stores compared to fish from the reference lake, despite metabolic alterations. Fathead minnow from the 5% effluent treatment in the laboratory experiment had reduced swimming endurance that was matched by reduced whole body citrate synthase activity, but no other metabolic alterations were observed. Therefore, effluent exposure caused metabolic alterations in both fathead minnow and shiner, but specific effects between experiments were inconsistent. Overall, the physiological significance of the metabolic and swimming effects of effluent exposure is unclear, but suggests discharged effluent has the potential to negatively affect wild fish survivability.

Performance tests are used to gauge swimmer fitness and guide training prescription. While some traditional protocols, such as, best average swimming (Bavg) lack scientific support, the three-minute all-out test (3MT) is validated to measure critical speed (CS) and distance capacity above CS (D’) from a single maximal swimming bout. In collegiate swimming, a 3MT could be convenient and time efficient. Yet, issues arise with calculating 3MT parameters. Moreover, anthropometry, resistance training, and stroke technique may influence swimming performance and physiological thresholds including 3MT parameters. Hence, this dissertation focused on evaluating the 3MT parameter calculation methods using the interval and lap split methods. Then, we assessed the influence of body anthropometrics, resistance training, and stroke techniques on 3MT parameters throughout a season. Bland-Altman plots showed that CS did not present remarkable behavior while D’ had systematic bias. Furthermore, there were no statistical differences between CS calculation methods (p = 0.83). However, D’ had moderate effect differences (p = 0.01, d = -0.70). Bavg showed very large correlations with maximum sprint speed (MSS) (r = 0.78) and CS (r = 0.81), but improvements after 6-weeks of concurrent training (p < 0.001, d = -0.85) seemed mainly driven by CS (p < 0.001, d = -1.68). Significant moderate to near perfect correlations were found between anthropometrics, 3MT parameters, and medicine ball pulldown throw (MBT) performance during pre- and post-season. Body composition changes may have influenced MBT performance changes (r = 0.46 – 0.55). CS increased at the expense of D’ while both reached stabilization point with no changes in MSS. There were moderate to large differences in 3MT parameters for the backstroke, but only MSS and CS differed in breaststroke, with no D’ differences in either group. Large to near perfect correlations were found between freestyle and secondary stroke 3MT parameter counterparts (r = 0.62 – 0.93), except for D’ in backstroke. Actual and predicted CS in both backstroke (p = 0.27) and breaststroke (p = 0.97) did not significantly differ. The lap split method during a 3MT seems like a practical and useful protocol to monitor CS and D’ in collegiate swimmers.

3 ABSTRACT Title: Comparison of lactate level at the same load in a swimming treadmill and on a bicycle ergometer Subject: 6 students (3 girls and 3 boys) from the first year of Bachelor's study at the Faculty of Physical Education and Sport of the Charles University at the age of 20,5 years ± 3 years took part in this research. Goal of the research: To compare demands of the load in the swimming flume with the same load on the bicycle ergometer, mainly at the level of the anaerobic threshold. Methods: We tested students by the method Critical Swimming Speed (CSS) for finding out their anaerobic threshold. Then students swam physical tests in the swimming treadmill - flume. The test consists of three 6 min parts. The first part of the swimming test was under the level of the CSS, the second part was at the level of the CSS and the third part was above the level of the CSS. We used the device for measuring the beat frequency (sport-testers) for obtaining information about changes of the heart rate (HR) during tests. Afterwards we accomplished tests on the bicycle ergometer, where we maintained HR at the same levels as in the load in the flume. We measured level of blood lactate (LA) after every 6 min part in the flume and on the ergometer. We statistically processed resulted values of LA by the Wilcoxon...

The current understanding of lactate metabolism in fish is based almost entirely on interpretation of concentration measurements that cannot be used to infer changes in flux. Moreover, the transporters regulating these fluxes have never been characterized in rainbow trout. My goals were: (1) to quantify lactate fluxes in rainbow trout under normoxic resting conditions, during acute hypoxia, and exercise by continuous infusion of [U-14C] lactate; (2) to determine lactate uptake capacity of trout tissues by infusing exogenous lactate in fish rest and during graded exercise, and (3) to clone monocarboxylate transporters (MCTs) and determine the effects of exhausting exercise on their expression. Such information could prove important to understand the mechanisms underlying the classic “lactate retention” seen in trout white muscle after intense exercise. In normoxic resting fish, the rates of appearance (Ra) and disappearance (Rd) of lactate were always matched (~18 to 13 µmol kg-1 min-1), thereby maintaining a low baseline blood lactate concentration (~0.8 mM). In hypoxic fish, Ra lactate increased from baseline to 36.5 µmol kg-1 min-1, and was accompanied by an unexpected 52% increase in Rd reaching 30.3 µmol kg-1 min-1, accounting for a rise in blood lactate to 8.9 mM. In exercising fish, lactate flux was stimulated > 2.4 body lengths per second (BL s-1). As the fish reached critical swimming speed (Ucrit), Ra lactate was more stimulated (+67% to 40.4 μmol kg-1 min-1) than Rd (+41% to 34.7 μmol kg-1 min-1), causing an increase in blood lactate to 5.1mM. Fish infused with exogenous lactate stimulated Rd lactate by 300% (14 to 56 μmol kg-1 min-1) during graded exercise, whereas the Rd in resting fish increased by only 90% (21 to 40 µmol kg-1 min-1). Four MCT isoforms were partially cloned and characterized in rainbow trout: MCT1b was the most abundant in heart, and red muscle, but poorly expressed in gill and brain where MCT1a and MCT2 were prevalent. MCT4 was more expressed in the heart. Transcript levels of MCT2 (+260%; brain), MCT1a (+90%; heart) and MCT1b (+50%; heart) were stimulated by exhausting exercise. This study shows that: (i) the increase in Rd lactate plays a strategic role in reducing the lactate load imposed on the circulation. Without this response, blood lactate accumulation would double; (ii) a high capacity for lactate disposal in rainbow trout tissues is elicited by the increased blood-to-tissue lactate gradient when extra lactate is administered; and (iii) rainbow trout may be unable to release large lactate loads rapidly from white muscle after exhausting exercise (lactate retention) because they poorly express MCT4 in white muscle and fail to upregulate its expression during exercise.

The current understanding of lactate metabolism in fish is based almost entirely on interpretation of concentration measurements that cannot be used to infer changes in flux. Moreover, the transporters regulating these fluxes have never been characterized in rainbow trout. My goals were: (1) to quantify lactate fluxes in rainbow trout under normoxic resting conditions, during acute hypoxia, and exercise by continuous infusion of [U-14C] lactate; (2) to determine lactate uptake capacity of trout tissues by infusing exogenous lactate in fish rest and during graded exercise, and (3) to clone monocarboxylate transporters (MCTs) and determine the effects of exhausting exercise on their expression. Such information could prove important to understand the mechanisms underlying the classic “lactate retention” seen in trout white muscle after intense exercise. In normoxic resting fish, the rates of appearance (Ra) and disappearance (Rd) of lactate were always matched (~18 to 13 µmol kg-1 min-1), thereby maintaining a low baseline blood lactate concentration (~0.8 mM). In hypoxic fish, Ra lactate increased from baseline to 36.5 µmol kg-1 min-1, and was accompanied by an unexpected 52% increase in Rd reaching 30.3 µmol kg-1 min-1, accounting for a rise in blood lactate to 8.9 mM. In exercising fish, lactate flux was stimulated > 2.4 body lengths per second (BL s-1). As the fish reached critical swimming speed (Ucrit), Ra lactate was more stimulated (+67% to 40.4 μmol kg-1 min-1) than Rd (+41% to 34.7 μmol kg-1 min-1), causing an increase in blood lactate to 5.1mM. Fish infused with exogenous lactate stimulated Rd lactate by 300% (14 to 56 μmol kg-1 min-1) during graded exercise, whereas the Rd in resting fish increased by only 90% (21 to 40 µmol kg-1 min-1). Four MCT isoforms were partially cloned and characterized in rainbow trout: MCT1b was the most abundant in heart, and red muscle, but poorly expressed in gill and brain where MCT1a and MCT2 were prevalent. MCT4 was more expressed in the heart. Transcript levels of MCT2 (+260%; brain), MCT1a (+90%; heart) and MCT1b (+50%; heart) were stimulated by exhausting exercise. This study shows that: (i) the increase in Rd lactate plays a strategic role in reducing the lactate load imposed on the circulation. Without this response, blood lactate accumulation would double; (ii) a high capacity for lactate disposal in rainbow trout tissues is elicited by the increased blood-to-tissue lactate gradient when extra lactate is administered; and (iii) rainbow trout may be unable to release large lactate loads rapidly from white muscle after exhausting exercise (lactate retention) because they poorly express MCT4 in white muscle and fail to upregulate its expression during exercise.