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Anaerobic metabolism during sub-maximal swimming in salmonids Burgetz, Ingrid Joanna 1996

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ANAEROBIC METABOLISM DURING SUB-MAXIMAL SWIMMING IN SALMONIDS by IN GRID JOANNA BURGETZ B.Sc, The University of Waterloo, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER; OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1996 © Ingrid Joanna Burgetz, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The extent of anaerobic metabolism required to support sub-maximal swimming below the critical swimming speed ( U ^ in rainbow trout (Oncorhynchus mykiss) was investigated by monitoring the concentration of lactate and phosphocreatine within the white muscle. Trout were swum to 70%, 80% and 100% of U c r i t. Lactate and phosphocreatine were measured using conventional methods, from the white muscle of exercised trout and sockeye salmon (O. nerka), and the chemical shift of inorganic phosphate (Pi) and quantity of phosphocreatine from the muscle were determined using 3 1P nuclear magnetic resonance (NMR) spectroscopy. The predictive relationship between lactate and Pi is described by the second-order regression equation: lactate (umols g"1 tissue)=856.38-306.67*P/+27.80*P/2 (r2=0.834). The relationship between the phosphocreatine (PCr) estimate obtained using 3 1P-NMR and the concentration of intramuscular PCr is described by the equation: PCr (umols g"1 tissue)=0.562+0.021 *PCr (ppm) (r=0.800). Estimates of lactate and PCr were obtained from trout exercised to 0% (rest), 70%, 80%) and 100% U c r i t, using these relationships. Anaerobic metabolism, as determined by lactate concentration, is required to support activity at and above 70% of U c r i t. With increasing exercise, the concentration of lactate also increased, indicating increased anaerobic" metabolism. Additionally, the concentration of lactate and phosphocreatine varies along the length of the body. The energy associated with anaerobic metabolism during sub-maximal swimming was estimated and found to be significant. i i T A B L E O F C O N T E N T S ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENTS vii C H A P T E R 1: G E N E R A L INTRODUCTION 1 Upstream spawning migration 4 C H A P T E R 2: A N O V E L M E T H O D O F UTILIZING 3 1 P - N M R T O E S T I M A T E M U S C L E L A C T A T E AND P H O S P H O C R E A T I N E IN RAINBOW T R O U T (ONCORHYNCHUS MYKISS) A N D S O C K E Y E S A L M O N (O. NERKA) F O L L O W I N G E X E R C I S E 6 INTRODUCTION 6 MATERIALS AND METHODS 10 Animals 10 Swimming Protocol 10 NMR-Spectroscopy 11 NMR Spectrum Analysis 12 Tissue Analysis 12 Statistics 13 RESULTS 16 DISCUSSION 26 Lactate-Pi relationship 26 Phosphocreatine-NMR-phosphocreatine relationship 31 Advantages and disadvantages of NMR spectroscopy to monitor muscular metabolites in salmonids 32 C H A P T E R 3 : A C C U M U L A T I O N O F L A C T A T E AND PHOSPHOCREATINE WITHIN T H E W H I T E M U S C L E O F RAINBOW T R O U T (ONCORHYNCHUS MYKISS) F O L L O W I N G E X E R C I S E 35 INTRODUCTION 35 MATERIALS AND METHODS 41 Animals 41 Swimming Protocol 41 NMR Spectroscopy and Analysis 42 Analysis and Statistics 43 iii RESULTS 45 Phosphocreatine accumulation following exercise 46 PCr/Lactate following exercise 47 DISCUSSION 56 Differential lactate and PCr concentrations along the length of the body 57 Variability in lactate and PCr concentrations between fish 65 Implications to energy budgets 66 General applications 68 CHAPTER 4: GENERAL DISCUSSION 69 Support of exercise by glycolytic muscle fibres 69 Effects of fish size 70 Temperature 71 Estimate of anaerobic metabolic energy costs 74 LITERATURE CITED 84 iv LIST OF TABLES Table 1. Swimming speed attained at each level of activity, and the size of rainbow trout in each group 44 v LIST O F FIGURES Figure 1. Schematic setup of fish placement within the bore of the magnet 14 Figure 2. Representative (full) spectrum obtained using 3 1P-NMR from a) resting rainbow trout, and b) exhausted rainbow trout. 18 Figure 3 . Representative spectrum obtained using experimental protocol (partial spectrum), using 31P-NMR. 20 Figure 4. Intramuscular lactate concentration (measured by biochemical methods) as a function of 3 1P-NMR derived inorganic phosphate chemical shift 22 Figure 5. 3 1P-NMR derived phosphocreatine as a predictor of biochemically measured intramuscular phosphocreatine concentration 24 Figure 6. Intramuscular pH from trout following exercise, as a function of intramuscular lactate concentration. 28 Figure 7. Mean white muscle lactate concentration in rainbow trout white muscle following exercise, at different positions along the body. 48 Figure 8. Variability in intramuscular lactate concentration at different positions along the body, following exercise. 50 Figure 9. Mean intramuscular phosphocreatine estimated from 3 1P-NMR in rainbow trout following exercise, at different positions along the body. 51 Figure 10. Variability in intramuscular phosphocreatine concentation at different positions along the body, following exercise. 53 Figure 11. The mean intramuscular phosphocreatine-lactate ratio in rainbow trout, following exercise, at different positions along the body. 54 Figure 12. The rate of oxygen consumption (M02) during swimming in rainbow trout at different proportions of U c r i t 77 vi ACKNOWLEDGEMENTS First, I must thank David Randall for his encouragement, interest, insight, and for providing a positive environment to learn and explore (and for many interesting stories over coffee that allowed me to stop worrying about the outcome of experiments). Thanks to Scott Hinch for the opportunity to pursue an interesting physiological question with ecological implications, and for his encouragement, excitement and support during my project. For being willing to devote a few hours on weekends and in the evening to set up the NMR so I could complete the experiments, and for being so understanding and humorous about "failed fish," I thank Dr. Anibal Rojas-Vargus. Also, I thank Troy Hallman for delivering the blows to many fish, carrying the fish, and teaching me how to analyse the NMR spectra data. Joelle Harris deserves thanks for her many hours of encouragement, during work hours and after hours. Without the collaborative spirit of the physiologists in both Zoology and Animal Science, I would not have been able to complete this study. Specific thanks for allowing me to use your equipment is due to the Iwama, Hochachka and Jones labs. For fruitful discussions and encouragement, and allowing me to sometimes worry, I thank the Randallites. For giving parts of this manuscript a helping and encouraging hand, I thank Tara Law. Also, for social support I thank the graduate students in the Jones lab, and those peripherally associated with them. I thank my parents and sisters for their encouragement, support and love through this experience. Thanks also to the Adult Salmon Passage Project, which is funded through the Fraser River Action Plan by Canada's Green Plan for providing funding through the course of my studies. vii CHAPTER 1: GENERAL INTRODUCTION Fish swim at a variety of speeds, from "sustained swimming" (>200 minutes) at relatively low velocities, to "burst swimming," which only lasts a few seconds (Beamish, 1978). Between sustained and burst swimming, at intermediate swimming speeds, fish can often swim for extended periods of time, but eventually they will fatigue. This type of swimming is termed "prolonged", and in the laboratory it can last between 2 seconds and 200 minutes. In the wild, fish utilize a number of different styles of swimming to cope with changing stimuli. Burst swuriming is associated with predator avoidance and prey capture (see Beamish, 1978), whereas sustained swimming is associated with extended migrations. Prolonged swimming is probably associated with a number of activities that change on a more rapid basis, and is difficult to differentiate from sustained swimming in the wild (Beamish, 1978). Sustained swimming at relatively low speeds relies exclusively on the generation of power by aerobic 'red' muscle fibres (Hudson, 1973; Johnston et al, 1977; Bone et al., 1978; Rome etal, 1984). These fibres have small cross-sectional areas (or diameters) (e.g., mature rainbow trout, Oncorhynchus mykiss, 1,283 ±180 um2, Kiessling et al, 1990; carp, Cyprineous carpio L., 564 ± 38 um2, Johnston and Bernard, 1984) and are found along the length of the fish's body in a superficial layer that is thickest near the lateral line. Red muscle fibres are aligned longitudinally in most fish (Alexander, 1969). The mitochondrial content in red muscle fibres of teleosts is around 30-35% of fibre volume (Johnston, 1981; Moyes et al, 1993), and they are highly vascularized with 1.5- 4.4 capillaries/fibre in carp (15°C and 28°C, respectively, Johnston and Bernard, 1984; Johnston, 1982). The proportion of red muscle fibres to the remaining skeletal muscle varies with species, and has been proposed to be related to the swimming activity 1 of the species (Mosse and Hudson, 1977; Gill et al, 1989). The majority of skeletal muscle fibres in fish are 'white' glycolytic fibres that are primarily responsible for powering burst swimming. The white muscle 'mass' in fish is typically composed of fibres of large diameter (e.g., in mature rainbow trout, 4 898±265 urn2, Kiessling et al., 1990, 1,267± 145 urn2 in carp, Johnston and Bernard, 1984), with few mitochondria, high levels of glycolytic enzymes, and are poorly vascularized, with 0.35-2.9 capillaries/fibre, in carp (15°C, 28°C, summarized in Bone, 1975; Johnston, 1982; Johnston and Bernard, 1984)). In rainbow trout, the proportion of small to large white fibres is dependent on the location of the sample. For example, a greater number of large fibres are found in the white muscle closest to the lateral line relative to fibres examined more dorsally at the same longitudinal section (Kiessling et al., 1990). The smallest white muscle fibres are similar in diameter to red muscle fibres, but retain properties similar to other white muscle fibres (Hudson, 1973; Johnston et al, 1975). Exercise metabolism in fish has been extensively studied in attempts to understand the biochemical consequences and interactions that affect both activity and fatigue. Many studies have investigated the metabolic state of the blood and muscle of fish following exhaustive or severe exercise (e.g. Black et al., 1959, 1960; reviewed in Wood and Perry, 1985; Milligan and Wood, 1986a,Z>; Moyes etal., 1992; reviewed in Moyes et al., 1993). Less is known about the consequences of non-exhaustive exercise on the metabolic state of fish. Recovery from exhaustive exercise involves a suite of metabolites that must be restored to their resting state. During slow or low speed sustained exercise, aerobic metabolism is responsible for powering swimming. However, as the swimming speed increases and becomes exhausting, white muscle fibres are recruited. Because white muscle fibres contain few . 2 mitochondria, the adenosine triphosphate (ATP) demand exceeds the aerobic capacity of these fibres. Thus, ATP production proceeds via anaerobic metabolism. Initially, phosphocreatine (PCr) is hydrolysed throught the creatine kinase reaction to produce ATP (Dobson et al., 1987), but resting levels of PCr in trout muscle are relatively low (13-27 umol/g (Milligan and Wood, 19866; Dobson et al., 1987; Boutilier etal, 1988; Mommson and Hochachka, 1988; Parkhouse et al, 1988; Wang et al, 1994 )), and are quickly depleted. When PCr is depleted, anaerobic glycolysis is initiated and the production of ATP from glycogen is accompanied by the production of lactate and protons resulting in an acidosis within the muscle. The paired reactions of these processes are: PCr + ADP + FT * Cr + ATP ATP ADP + FT + Pz Glycogen + 3 ADP + 3 Pi - 3 ATP + 2Pyruvate + 4H+ 2NAD+ + 4FT 2NADH(H+) 2Pyruvate + 2NADH(H+) ^ 2Lactate + 2H+ + 2NAD+ PCr Cr + Pz Glycogen + 3 ADP + 3Pi - 2Lactate + 3 ATP + 2H+ Following an episode where anaerobic glycolysis has supported activity, the levels of lactate within the white muscle are elevated, and the intramuscular pH is reduced. The time required for the metabolites to return to resting levels varies with species, although this may be more a reflection of the activity level of the fish. The benthic starry flounder, Platichthys stellatus, requires 8 hours to recover from strenuous exercise (Milligan and Wood, 1987). In salmonids, metabolites return to resting levels in 12-24 hours, following exhaustive exercise (Milligan and Wood, 1986a,6; Milligan and McDonald, 1988; Mommsen and Hochachka, 1988). The faster recovery of the white muscle following exercise in the inactive flounder than in the active trout 3 may be more due to a lower production of lactate during exercise, rather than a difference in rate of recovery (Milligan and Wood, 1986*, 1987). Intramuscular lactate is gradually metabolized, in situ by gluconeogenesis (Turner et al., 1983a,Z>; Dunn and Hochachka, 1986; Milligan and Wood, 1986; Milligan and McDonald, 1988; Pagnotta and Milligan, 1991; Milligan and Girard, 1993), over 8 to 24 hours (Black et al, 1962; Milligan and McDonald, 1988; Pagnotta and Milligan, 1995). The recovery of lactate to resting levels has been monitored with the concurrent increase in muscle glycogen ( Milligan and Wood, 1987; Schulte etal, 1992). Quantitatively, the disappearance of accumulated lactate from white muscle can account for the glycogen that is resynthesised during recovery (Parkhouse et al, 1988; Milligan and Wood, 1987; Arthur et al, 1992; Schulte et al, 1992; Wang et al, 1994). Thus, lactate can be used as an estimate of the quantity of glycogen consumed to produce ATP via anaerobic metabolism during a swimming bout. Upstream spawning migration Anadromous Pacific salmon return to freshwater to spawn, and often must undertake a long migration through freshwater to reach the spawning grounds. For instance, early Stuart sockeye salmon (Oncorhynchus nerka), a Fraser River stock in British Columbia, Canada, must migrate 1200 km to reach the spawning grounds. This is one of the first Fraser sockeye stocks to migrate up-river in the summer, and because of the timing of the migration, may face difficult passage due to high discharge and variable temperatures. The course of this migration goes through the Fraser canyon, and the Hell's Gate fishway's; a region known to create energetically demanding passage for sockeye salmon (Hindi et al, 1996). Hell's Gate represents perhaps the most arduous portion of the migration, as it is the narrowest portion of the Fraser River and has 4 surface velocities exceeding 6 m/s. An energy budget for upstream migrating sockeye salmon has been constructed with the aid of electromyogramy (EMG) radio telemetry to estimate swimming speeds (Hinch and Rand, 1996). One of the major drawbacks of this particular energy budget and most energy budgets for fish in general, is that they are based only on aerobic metabolism. Hinch and Rand (1996) use a relationship between the EMG signal and tailbeat frequency to estimate swimming speeds, and laboratory swim tunnel results to convert swimming speed to estimates of oxygen consumption. There is some indication that anaerobic metabolism occurs during the course of migration; fish caught and sampled just beyond Hell's Gate fishways had elevated levels of lactate and protons (A. Kiessling, personal communication. Dept. of Agriculture and Dept. of Food Science, Swedish University of Agricultural Sciences). In addition, the anaerobic component of sub-maximal swimming may be significant during migration. Because salmon cease feeding prior to entering the river, the energy stores for migration decrease over the course of migration, due to the maturation of gonads and swimming. Estimation of the additional costs associated with anaerobic metabolism during upstream migration is lacking (Brett, 1995), and are needed to refine energy budgets for fish during river migration. 5 C H A P T E R 2: A N O V E L M E T H O D O F UTILIZING 3 1 P - N M R T O E S T I M A T E M U S C L E L A C T A T E AND P H O S P H O C R E A T I N E IN RAINBOW T R O U T (ONCORHYNCHUS MYKISS) AND S O C K E Y E S A L M O N (O. NERKA) F O L L O W I N G E X E R C I S E INTRODUCTION 31P-nuclear magnetic resonance (NMR) spectroscopy has been used extensively over the past 15 years to monitor biological and physiological phenomenon. Limited use has been made of this technology with aquatic organisms (van Waarde and van den Thillart, 1994), but it has been used exhaustively in medical and sport research and in cell culture (e.g., Moon and Richards, 1973; Radda, 1992; Sahlin, 1992). Muscle bioenergetics during exercise has been studied extensively in humans using 3 1P-NMR (reviewed in Sapega et al, 1987, and McCully et al, 1994). The advantage of using human subjects are many. Spectra can be obtained during the course of exercise, and the change in metabolite levels and pH can be monitored without taking muscle biopsies. This technology has also been used in physio-pathological applications (reviewed in Cozzone and Bendahan, 1994). Obviously, monitoring muscle bioenergetics during exercise by this method is difficult to apply with fish, because of the limitations in using aquatic organisms with this technology. 31P (and other nuclei)-NMR spectroscopy has the advantage of being a non-invasive and non-terminal technique that is used to monitor intracellular components. This allows for the potential of using the same animal in a number of different treatment groups, and as a control, which may remove some of the inherent variability that occurs within a population or sample of 6 subjects. This approach allows for a decrease in the number of animals required for a set of experiments, without a decrease in the ability to determine differences between treatments. There are many reviews available that cover the mechanisms behind nuclear magnetic resonance spectroscopy (e.g., Rabenstein, 1978; Gadian, 1982; Sapega et al, 1987; van Waarde and van den Thillart, 1994), so this review will be brief, and is based on the aforementioned reviews. Nuclear magnetic resonance is based on the phenomenon that some nonradioactive, naturally occurring atomic isotopes display magnetic properties. Under a stationary magnetic field, these nuclei, which possess a property known as spin, align with the magnetic field. Each atomic nuclei has a resonant (or specific) frequency that induces the nuclei to absorb and emit electromagnetic energy as the nuclei move between magnetic field orientations (or quantum energy states). For example, both 'H and31P have a spin quantum number of Vi, thus these nuclei can have one of two orientations with respect the applied field. In NMR spectroscopy, the sample and the probe are placed within a strong, uniform magnetic field. A short, strong pulse of radiation at resonant frequency is transferred to the sample through a transceiver coil. This results in the excitation of the target nuclei (e.g., 31P); but because the energy is delivered in a pulse, the excitation is transient. The emission of energy from the nuclei after excitation follows a specific pattern, the so-called free induction decay (FID). The FID of target nuclei is sampled at a specific time after the pulse and following Fourier transformation, results in a spectrum that can be interpreted. Mobile molecules containing the target nuclei result in narrow signals (i.e. intramuscular Pz', PCr, and ATP); immobilized compounds containing phosphorus, like DNA and phospholipids, result in very broad signals that cannot be identified from background noise. Usually it is necessary to obtain more than one FED to produce a spectrum that can be 7 interpreted. Multiple pulses are often required, and each pulse is followed by the same delay period prior to sampling. The FLDs are then added together and result in a suitable and analysable spectrum. Target nuclei experience a local magnetic field, due to differences in the chemical environment and the adjacent atoms and molecules. Each type of molecule containing the target nuclei within a homogenous sample is subject to the same local magnetic field, resulting in a peak that can be compared to a reference compound. The separation of resonance frequencies from the reference frequency (i.e., PCr in 31P-NMR) is termed the chemical shift. The area under each peak is proportional to the number of nuclei that contributed to that signal from the sample. In 3 1P-NMR, PCr is normally used as the reference frequency because it can usually be unambiguously identified and it has a resonance frequency that is insensitive to changes in pH within a physiological range (Gadian, 1982). The chemical shift (5) of the inorganic phosphate peak is used to indicate the intracellular pH because it is easily observable in most 3 1P-NMR spectra, and it is especially sensitive to pH within physiological range. The pH estimates from the chemical shift of Pz agree closely with micro-electrode pH measurements from within cells, and has been shown to represent cytosolic pH and not intra-mitochondrial pH (reviewed in Sapega et al, 1987). The relationship between pH and the chemical shift of Pz (6-Pz) can be described by the Henderson-Hasselbalch equation and is dependent on temperature (Moon and Richards, 1973): pH = pKa + log([6-Pz - 5a]/[5b - 6-P/]) where 5 a and 6b are the chemical shifts of the fully protonated and the fully dissociated Pz', respectively. Confirmation of this relationship is usually accomplished by analysing solutions that 8 imitate the intracellular environment at various known pH values (Malhotra and Shapiro, 1993). The depletion of PCr during the initial recruitment of white muscle fibres and subsequent increase in protons (and lactate) due to anaerobic glycolysis can easily be monitored using 3 1P-NMR spectroscopy. Because the primary biochemical reactions that occur during exercise contain phoshorus, exercise physiology is an excellent candidate for investigations using 31P-NMR, and has been used to monitor changes within muscle in human subjects and in animal muscle preparations (Sapega et al, 1987; Baker et al, 1989; McCully et al, 1994). The purpose of this aspect of my study was to investigate the possibility of using the chemical shift of inorganic phosphate obtained through 3 1P-NMR spectroscopy, to determine the quantity of intramuscular lactate, after exercise, in salmonids. Specifically, the relationship between biochemically measured intramuscular lactate from the white musculature of rainbow trout and sockeye salmon, and the chemical shift of P/, from 31P-NMR spectroscopy was studied. In addition, the relationship between intramuscular phosphocreatine concentration and the area of the PCr peak obtained using 3 1P-NMR was examined. 9 MATERIALS AND METHODS Animals Rainbow trout and sockeye salmon of both sexes were obtained from West Creek Trout Farm, Abbotsford, British Columbia. Rainbow trout had a mean body weight (±standard deviation) of 431.8 ±81.63 g, and the sockeye salmon had a mean body weight of 575 ± 51.2 g. Rainbow trout and sockeye salmon were held seperately in outdoor 2 000 1 round plexiglass tanks supplied with aerated, flow-through dechlorinized Vancouver tapwater. Fish were acclimitized to these conditions for a minimum of two weeks prior to the experiments. Experiments using rainbow trout were performed in January through April, 1995. Experiments using sockeye salmon were performed in May 1995. Rainbow trout were fed with a commercial trout food and kept on a maintenance ration. Sockeye salmon were fed fish food that was provided by West Creek Trout Farm, and were kept on a maintenance ration. Holding temperature ranged between 7°C and 10.5°C. Swimming Protocol Fish were transferred from the outdoor tank into a Brett-style respirometer (Gehrke et al. 1989) a minimum of 12 hours prior to the initiation of the swimming protocol. Some fish were transferred from the tank into black plexiglass flow-through boxes inside, and were held for 12 to 24 hours prior to being transferred to the respirometer. Upon transfer into the respirometer, fresh water was flushed through the respirometer and fish were left to acclimatize to the respirometer for 12 hours, with the water velocity at approximately 1 body length per second (Ls"1). Fish were exercised to exhaustion by increasing the water velocity to the maximum 10 swimming speed the fish could maintain. When the fish could no longer sustain this speed (was unable to move off of the downstream electrified (5 V) grid), the water velocity was decreased by approximately 30%. If the fish could maintain this swimming speed for a period of time (approximately 5 minutes), then the water velocity was again increased. This pattern of increasing and decreasing the water velocity was maintained until the fish either lost control of balance or could not move off the back grid or swim at 1 Ls"1. Alternately, fish were swum to critical swimming speed (Ucrit), (n=2) by incrementally increasing the water velocity by steps. The initial step was approximately 0.5 L, and all subsequent increases were approximately 0.25 L. Each velocity was maintained for 30 minutes. Swimming was terminated when the fish could not move off the downstream electrified grid. NMR-Spectroscopy Fish were immediately transferred to a clear plexiglass box equipped with flow-through water; fish were lightly restrained, and the box sealed (Figure 1). The fish was quickly transferred into the NMR, placed on top of a 4 cm, copper surface 3 1P-NMR coil. The NMR consists of a 1.89 Tesla horizontal superconducting magnet (Oxford Instruments, Oxford, U.K.) which is connected to a Nicolet 1280 spectrometer. The 3 1P-NMR coil was used with a phosphoric acid standard solution for shimming (fine calibration), at a frequency of 32.5 MHZ (tuned for 31P). Each spectrum was obtained in 60 seconds, under the following conditions: 32 individual scans, nominal 90° pulse (42 \is), spectral window of 500 Hz, 1024 data points and a I second delay between pulses. The NMR was initially shimmed by Dr. Anibal Rojas-Vargas (Department of Zoology, U.B.C.), and during the experiments, the NMR was maintained/operated by either Dr. Rojas-Vargas or Troy M. Hallman (Dept. of Zool., U B C ) . 11 Following NMR analysis, the fish were then sacrificed by cephalic blow and muscle samples were excised and freeze-clamped in liquid nitrogen. NMR Spectrum Analysis The raw data obtained from the NMR was baseline corrected with a Gaussian Multiplication factor of 20, zero-fielded to 4000 points, Fourier transformed and phase shifted prior to deconvolution of the PCr and Pz' peaks. The PCr peak was designated to be 0 ppm to allow measurement of the chemical shift of the inorganic phosphate peak (Pz). Raw data were analysed three times to increase the precision of the determinations. If the coefficient of variation (C.V.) for each sample was greater than 10%, the sample was reanalysed. This analysis was performed by Troy Hallman in the winter of 1995 and spring 1996 (Dept. ofZoology, U.B.C.). Tissue Analysis Approximately 700 to 1 000 mg of white muscle were chipped from the muscle sample under liquid nitrogen. The sample was ground to a coarse powder under liquid nitrogen in a prechilled mortar and pestle. The tissue samples were subsequently transferred into preweighed tubes containing 4.0 ml of ice cold 7% perchloric acid, which were then reweighed to determine the precise mass of tissue. Samples were then homogenized with a Brinkman homogenizer, while held in a beaker containing an ice and salt water slurry which maintained the temperature between -5 and 0°C. Tissue samples were homogenized in three 15 seconds pulses with 30 seconds between homogenisations to allow the tissue to cool. The homogenized samples were then centrifuged at 10 000 x g for 10 minutes at 4°C, returned to the ice-salt slurry, and the supernatant was transferred to a clean, pre-chilled tube and neutralized with 3 M K 2 C0 3 , 0.1 M triethanolamine to a pH of 7.5. The neutralized samples were centrifuged at 10 OOOxg for 10 12 minutes (at 4°C) and the supernate was aliquanted (0.25 ml) into eppendorf tubes and frozen in liquid nitrogen. The extracted tissue samples were stored at -80°C until analysis. Extracts were assayed for lactate and PCr on a Molecular Devices THEPvMOmax microplate reader spectrophotometer equipped with SOFTmax or SOFTmax Plus software. All assays were run in triplicate and concentrations of lactate and PCr were validated by the use of appropriate standards. The standards were validated by assaying standard concentrations using a Shimadzu UV-Visible recording spectrophotometer UV-160. Enzyme assays were modified from Bermeyer (1985) for use with microplates. The mean and C.V. were calculated for each sample, and if the C.V. was greater than 14%, the sample was reanalysed. Most sample analyses had a C.V. less than 10%. Statistics Systat (v. 5, Systat Inc.) and Sigma Stat (v. 1.01 and 2.0, Jandel Scientific) were used for analysis purposes. The mean value from the triplicates analysed for lactate, PCr, 5-Pz and PCr area were used in the analysis. Because of the preselection for precise estimates, there was not a significant difference between the relationships when the mean, the minimum or the maximum estimate of lactate or PCr was used. Simple linear regression was used to determine the relationship between PCr and PCr area, and polynomial regression was used to determine the relationship between lactate and S-Pz. Multiple linear regression was used to test the effect of weight, species and temperature on the ratio of lactate-S-Pz, and PCr-PCr. Unless otherwise stated, a probability of making a type I error (a) was set to 0.05. 13 Figure 1. Schematic setup of fish placement within the bore of the magnet (not to scale). Fish is placed within the plexiglass box and lightly restrained using a plate with perfusion holes and a balloon (not shown). Continuous exchange of dechlorinized water occurs, with the inflow tube placed either in the fish's mouth or above the fish. The box is placed on the sled containing the probe coil, and the box and sled are placed within the bore of the magnet. Readings are taken using NMR spectroscopy, and data is acquired and saved to the computer system. 14 15 RESULTS A typical 3 1P-NMR spectrum obtained from rainbow trout muscle at rest and following U c r i t are shown in Figure 2 a, b. The first peak is the Pz peak, the second large peak is the PCr peak and the subsequent peaks correspond to ATP. The very small peak that is located on the left side of the Pz peak, and is partially overlapped by the Pz peak, corresponds to sugar phosphates; this peak can only be seen clearly in the spectrum from exercised muscle. The sugar phosphate and ATP peaks were not analysed for this study. These are examples of the complete set of data that can be obtained using 3 1P-NMR with rainbow trout and the phosphate compounds are relatively easily observable. These spectra were obtained by combining 512 individual scans using a spectral window (S.W.) of 1500 Hz. The remaining parameters for obtaining the spectra are the same as previously described. For the data obtained in this study, fewer scans were combined (36) and the SW was 500 Hz. This resulted in a clear spectra for obtaining the chemical shift of Pi and the area under the PCr curve, but the signal-to-noise ratio is too low to discern the ATP and monophosphate and phosphodiester proportions, and the spectral window does not include the third peak associated with ATP (Figure 3). A larger window would require additional scanning time. By combining fewer scans, a spectra was obtained rapidly (1 minute, total time). Because the rate of change of metabolite levels within white muscle is rapid following exercise, to use this method to estimate post-exercise concentrations of lactate and PCr the elapsed time to obtain each spectra was reduced as much as possible. Spectra were clear enough to analyse the chemical shift of Pz and the area under the phosphocreatine peak, which were the two parameters of interest. 16 The relationship between white muscle lactate concentration and the 5-Pz', as measured by 31P-NMR spectroscopy, in both rainbow trout and sockeye salmon is illustrated in Figure 4. As the inorganic phosphate chemical shift peak decreases, the amount of lactate increases. This relationship can be described by the equation: lactate (umols/g tissue) = 856.381-306.669*S-Pz +27.798*S-Pz'2 (i2=0.834, adjusted r^O.827). The ratio of lactate (umols/g tissue) to the chemical shift of Pz was tested to see if it was related to the species of fish, weight of the fish, temperature of the experiment, section of the fish monitored and the swimming treatment used. No species effect was found (P=0.1408). Although there was a difference in the weight of the animals, this was found not to affect the ratio of lactate to Pz chemical shift (P=0.9369). Temperature was also not found to affect the ratio either (P=0.3694). Both treatment (swimming protocol) and section monitored were found to have a signifcant effect on the lactate-S-Pz ratio (P<0.001, P=0.03, respectively). The relationship between biochemically measured PCr and the area under the PCr peak derived from a 3 1P-NMR generated spectrum can be described by the linear equation: PCr (umols/g tissue) = 0.56195 + 0.02.0882*PCr (ppm) (r2=0.800) (Figure 5). Multiple linear regression was used to test if the ratio between biochemically obtained PCr and 3 1P-NMR derived PCr was influenced by the species of fish, weight of the fish, temperature of the experiment, and treatment or the section where PCr was monitored. In all cases except the section monitored, there was no significant affect of these factors on the PCr-PCr ratio. 17 Figure 2. Representative (full) spectrum obtained using 31P-NMR from a) resting rainbow trout, and b) exhausted rainbow trout. Note the difference in the chemical shift of inorganic phosphate (Pi) from PCr in resting trout compared to the exhausted trout. Also note the height of the PCr peak between the two conditions. Sugar phosphates can only be clearly seen in the spectrum from the exhausted fish. The three peaks corresponding to ATP (y, a, P) are greatly reduced in the exhausted fish. 18 19 Figure 3. Representative spectrum obtained using experimental protocol (partial spectrum), using 31P-NMR. This spectrum is from scanning the white muscle of a fish exercised to 70% of U c r i t. Note that the baseline is noiser than the baseline in Figure 2a) and b), and also note that the Pz and PCr peaks can be clearly determined. 20 -1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 1 1 1 i 1 i 1 1 r 10 5 0 -5 -10 PPM 21 Figure 4. Intramuscular lactate concentration (measured by biochemical methods) as a function of 3 1P-NMR derived inorganic phosphate chemical shift in rainbow trout (circles, n=6) and sockeye salmon (squares, n=4). Each fish was sampled a number of times, and therefore not all values are independent. Mean value of triplicate measurements ± the standard error for both intramuscular lactate and the P/' chemical shift. The solid line represents the polynomial regression line of best fit, and the hatched lines represent the 95% confidence intervals. 22 100 - i 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 Chemical shift of inorganic phosphate peak 23 Figure 5. 3 1P-NMR derived phosphocreatine as a predictor of biochemically measured intramuscular phosphocreatine concentration in rainbow trout (circles, n=5), and sockeye salmon (squares, n=5). The mean and the standard error are presented for both intramuscular PCr and ° NMR derived PCr. The solid line represents the linear regression line of best fit, and the hatched lines represent the 9 5 % confidence intervals. 2 4 35 PCr (umols/g)=0.56195+0.020882*(NMR-PCr) r 2=0.800 30 -\ 25 20 a 15 OH 10 H 0 J I I I I I i 1 1 n 1 0 150 300 450 600 750 900 1050 1200 1350 N M R PCr 25 DISCUSSION Lactate-Pi relationship White muscle Pz chemical shift and lactate concentration were monitored at rest and following exercise to assess if there was a relationship between the two metabolites. Biochemically measured intramuscular lactate was related to the chemical shift of intramuscular Vi, measured using 31P-NMR spectroscopy, and this relationship was best described by a second-order regression whereby lactate was predicted by 5-Pz (r2=0.834, adjusted r2=0.827, Figure 4). This predictive relationship represents a novel method of estimating lactate accumulation in vivo following exercise in salmonids. Previously, the only method available to monitor intramuscular lactate following exercise was to terminally sample salmonids. This invasive method provides in vitro information of lactate accumulation following experimental protocols, and may be contaminated by artifacts that occur during sampling (van den Thillart et al. 1990). Non-invasive measurements of 8-Pz via 3 1P-NMR provides an estimate of the in vivo lactate accumulation following exercise that can be directly compared to literature values. 3 1P-NMR has been used extensively to monitor the intracellular pH (pHz) in many systems (see Gadian, 1982, and Malhotra and Shapiro, 1993) through the relationship between pHz and the chemical shift of Pz'. The relationship between pHz and S-Pz has not been previously extended to anaerobic metabolites, such as lactate. Lactate production during glycolysis is associated with a large production of protons, and therefore is an appropriate metabolite for inference from 6-Pz following exercise. A measured linear relationship between pHz and lactate exists in human skeletal muscle (Sahin, 1978). A somewhat similar relationship can be derived for fish by combining pHz and lactate measurements from a number of studies (Figure 6). This 26 combined relationship can be fitted with a second order regression (r^O.848). The 6-P/' obtained in this study were converted to pH/ using the Henderson-Hasselbach equation fitted for 7°C and 10°C (Kost, 1990). This data set can also be fitted with a second order regression (r2=0.848), however, the curve is shifted to the right, indicating greater concentrations of lactate detected within white muscle of fish used in this study. No statistical difference exists in the slopes or intercepts of these two curves, as detected by ANCOVA. Therefore, the data obtained from the literature and the present study can be combined, and fitted with a second order regression (r2=0.675). The slight differences observed between the data collected in this study and the combined data from the literature, are due to a number of factors. The pH/ and lactate values obtained from the literature are calculated means from a number of fish in each study. Some of the data present are estimated lactate or pH; values obtained from figures presented in the literature (identified with an asterix (*)). These values have additional variation associated with them, as the estimates have limited precision. Lactate concentrations obtained from Milligan and Wood (1986Z>) and Tang and Boutilier (1988), were converted from mmol/intracellular fluid volume (ICFV) to umol/g tissue, using ICFV for pre- and post-exercise (in Milligan and Wood, 1986). Additionally, different techniques were used in obtaining the pH/' measurements in the studies (i.e., DMO (5,5-dimethyl-2-4-oxazolidinedione), homegenate assays). Some of the data used in the relationship were obtained from studies that monitored the change in pH/' and lactate following exercise for 24 hours post-exercise. 27 Figure 6. Intramuscular pH from trout following exercise, as a function of intramuscular lactate concentration. Intramuscular pH from the present study was derived using the chemical shift of Pz (see text for details). The intramuscular lactate concentration was determined using standard biochemical methods. The combined data sets result in the second-order regression equation pH=7.334-0.016*[lactate]+(7.30*10"5)*[lactate]2 (r^O.675). Data identified with an asterix (*) were obtained from figures presented in the literature. 28 O Milligan and Wood, 1986/3 H Schulte et al, 1992 ^ Parkhouse et al, 1988 20 40 60 80 100 Lactate (^irnol/g tissue) 29 This may be a problem in that pHz and lactate may not remain associated following exercise. During recovery from exercise there are a number of different metabolic processes occurring, some of them may produce H + , like the rebuilding of PCr stores to resting levels. The creatine phosphokinase reaction consumes protons during the production of ATP, but produces them during production of PCr. The production and consumption of ATP also involves protons; therefore, using pH to estimate the intramuscular lactate concentration during recovery will probably result in inaccurate estimates because lactate and protons are no longer directly associated. The calibration solution that was used to construct the pH-temperature conversion was desgined to be of similar ionic strength as the myocardial intracellular milieu (Kost 1990). For the estimation of carp muscle pH/, calibration curves have been constructed between measured pH/ of a similar solution that contained 30% carp muscle homogenate. The resulting curve agrees exactly with the pH calibration from Kost at 20°C (van den Thillart, 1989). Some error may be associated with this conversion factor when compared to determination of intramuscular pH by more traditional biochemical methods (e.g., Portner et al, 1990). Despite all of these possible errors in measurements, calculations and differences due to technique and protocol, the relationship between pH/' and lactate in white muscle of rainbow trout obtained from the literature and from the present study was not found to statistically differ. Other NMR and MRI methods are available but, in general, are not applicable to the determination of pH/, PCr or lactate in fish muscle. 'H-NMR has been used in humans and other non-aquatic animals to monitor lactate dynamics. This technology may not be a very applicable method to examine lactate dynamics within fish muscle. Because of the abundance of naturally occurring ! H in biological compounds, the spectrum that is obtained is difficult to interpret. 30 There are many overlapping peaks and the spectrum is quite narrow (a few ppm) (reviewed in van Waarde and van den Thillart, 1994). Additionally, if ^-NMR is used to obtain estimates of lactate, often the levels of lactate are too low to be detected because of the compounding effects of strong 'H lipid and H 2 0 signals (van den Thillart and van Waarde, 1996). Recently, there have been technological advances resulting in multi-dimensional analysis. Combined 31P, 'H NMR and MRI can obtain spectra corresponding to lactate, PCr, Pz and pHz images within one hour, using rat gastrocnemius muscle (Morikawa et al. 1994). This is not a practical method of estimating lactate, pHz or PCr from fish white muscle following exercise, as recovery is initiated immediately, and although recovery is lengthy, the initial changes following exercise are important parameters that should be monitored within minutes, not hours. Thus, 31P-NMR is, at present, the method that best suits the determination of post-exercise metabolites in fish muscle. Carp, goldfish (Carassius auratus) and tilapia (Orechromis mossambicus) have been used extensively in hypoxia studies using NMR spectroscopy ( van den Thillart et al, 1989, 1990; van Waarde et al. 1990a, b; van den Thillart and van Waarde, 1993; van Ginneken et al, 1995), and to a more limited extent, the freshwater loach, Cobitis biswae, (Chiba et al, \990a,b). There has been limited, or non-existent use of these techniques on salmonids because of their violent response to being restrained. Phosphocreatine-NMR-phosphocreatine relationship The linear relationship between NMR-PCr area and the PCr levels measured biochemically is useful in a number of ways. Firstly, it is important as a confirmatory measure to ensure that the integrated area under the PCr resonance peak is related to biochemically 31 deterrnined intramuscular PCr concentration ([PCr]). Not only does this ensure that the NMR-PCr values estimate intramuscular PCr concentration, but additionally this provides a relationship that can be used to convert the area of the PCr peak to a concentration. This allows comparison of NMR derived PCr values with biochemical measurements reported in the literature. 3 1P-NMR studies investigating metabolites within fish muscle typically report the integrated area under the PCr peak as a percentage of resting PCr peak area (van den Thillart et al, 1989; van Waarde et al. 1990a; van den Thillart and van Waarde, 1993; van Ginneken et al., 1995). This makes comparisons with other studies that report the concentration of intramuscular PCr difficult. In biopsy samples from human muscle tissue, a discrepancy from NMR-PCr has been noted, and is assumed to be due to hydrolysis during the time between obtaining the sample and freezing (Dawson, 1982). Although one of the advantages of NMR spectroscopy is fewer artifacts due to sampling procedure that are usually present in metabolite estimates using analytical methods (van den Thillart et al. 1990), by utilizing the relationship between NMR-PCr and biochemically measured [PCr]i; the two methods of monitoring PCr become comparable. Advantages and disadvantages of NMR spectroscopy to monitor muscular metabolites in salmonids Some of the advantages of using 31P-NMR spectroscopy to estimate white muscle lactate are inherent to this technology. It is a non-invasive technique that monitors metabolites in vivo, and many phosphorylated compounds above a concentration of 0.5 mM can be detected simultaneously (reviewd in Sapega et al, 1987). Results can be obtained very quickly, in this study each spectrum was obtained within one minute. This rapid measurement resulted in a lower signal-noise ratio, because the number of scans (N) is relatively small and the signal 32 increases with the number of scans, and the noise decreases by (Gadian, 1982). In rainbow trout and sockeye salmon, the small N does not inhibit the ability to obtain a clear spectrum for analysing the chemical shift of Pz and the area of the PCr peak. However, more scans are usually required to obtained a satisfactory spectrum for ATP analysis (100-1000) (van Waarde and van den Thillart, 1994). The use of this method for the measurement of white muscle pHi is somewhat problematic in measurements of resting fish. Resting levels of muscle metabolites (such as lactate, PCr and Pz) from fish are difficult to obtain, and this technology presents no exception. Analytical techniques are problematic due to handling stress and muscle stimulation during termination and excision of muscle samples; PCr declines rapidly and an increase in the concentration of Pz occurs (reviewed in van Waarde and van den Thillart, 1994). Obtaining spectra from the various sections of the musculature of resting fish is straightforward, however the spectrum may not always produce a clear result. The peak that corresponds to Pz is very small in resting muscle, and as such, more scans may have to be accumulated to get a clear signal so that the chemical shift between Pz and PCr can be measured. An additional method of obtaining resting Pz chemical shift values is to increase the delay time before acquisition of data, following the magnetic pulse. This allows for proportionally more of the phosphorus nuclei to recover from the magnetic pulse prior to detection and typically results in an increased signal-to-noise ratio. With a 'cleaner' spectra, the Pz peak is more easily identified, and the chemical shift can be analysed. At present the focus of the magnetic pulse results in a relatively large area of the muscle mass being examined. With stronger magnets and smaller coils, the section monitored can be 33 reduced, without compromising the quality of the obtained spectra. In recent years, additional improvements have occurred that allow for NMR spectroscopy to differentiate between muscle fibre types, and in the case of multiple-volume localizations, metabolite content in several muscles can be measured simultaneously ( Bailes et al, 1987; Vandenborne et al, 1991). The primary drawback of this technology is the cost in obtaining a suitably powerful magnet and spectrometer. 34 C H A P T E R 3: A C C U M U L A T I O N O F L A C T A T E AND PHOSPHOCREATINE WITHIN T H E W H I T E M U S C L E O F RAINBOW T R O U T (ONCORHYNCHUS MYKISS) F O L L O W I N G E X E R C I S E INTRODUCTION Swimming at low speeds is accomplished through the sequential contraction of muscle fibres along the length of the body and on alternating sides. Prior to the contraction of muscle fibres, there is a wave of excitation that can be monitored using EMG. The wave of exitation follows the same pattern as the recruitment of muscle fibres. The muscle fibres that are recruited to produce the power necessary for swimming are slow, oxidative ('red') fibres. These fibres are superficial and form a thicker triangular mass near the lateral line. Activity from these red muscle fibres has been monitored in a number of species of fish during slow swimming (Hudson, 1973; Johnston et al., 1977; Bone et al., 1978; Rome et al, 1984). When faced with elevated water velocities or when required to rapidly escape from predators or chase prey, fish may require additional power to propel themselves. If the required swim speeds are very high, then the fish may be to employ a different behaviour that is characterized by a period of initial acceleration followed by steady swimming. This style of "burst" swiniming is often used in predator avoidance, or prey capture situations, and is used in any situation when a rapid burst of swimming is required. Glycolytic fibres support burst swimming, which can only be maintained for very short periods (<20 s) (reviewed in Beamish, 1978). Prolonged swimming has been defined as the type of swimming that results in fatigue but can occur for a duration between 20 seconds and 200 minutes (Beamish, 1978). This type of 35 swimming is mimicked in laboratory swimming studies that examine the critical swimming speed, Ucri,. U^t is the maximum velocity that a fish can swim at for a set period of time (Brett, 1964). In testing U c r i t, incremental increases in water velocity are imposed on the fish; this process results in the fish recruiting red muscle fibres to swim at the lower speeds, and sequentially recruiting more fibres as the water velocity increases, until white muscle fibres are sequentially recruited, and eventually fatigue ensues. Previous studies have demonstrated the recruitment of white muscle fibres below U c r i t (e.g.., Hudson, 1973; Johnston et al., 1977; Bone et al., 1978; Brill and Dizon, 1979; Johnston and Moon, 1980). Teleost white muscle fibres are arranged in a complex manner, with deep fibres spiralling within myotomes at angles up to 30° to the longitudinal axis. In some fish, the white muscle fibres may form helices across myomeres. The myotomes are cones of fibres that are folded into a convoluted 'W shape, which overlaps other myomeres (Alexander, 1969). With such a complex design, white muscle fibres are not expected to be recruited in only one pattern. With different sizes of muscle fibres, in various areas within the musculature, the function of these fibres may differ. However, no experimental evidence obtained in vivo has at this point clearly identified any specific difference in the function these divergent fibres. The innervation of white muscle fibres has been examined in a number of fish species, and some generalizations have been made (Hudson, 1969; Bone, 1978). In elasmobranches, holosteans, chondrosteans and some primitive teleost fish, the white muscle fibres have been shown to be focally innervated by a single, 'basket-like' endplate (for review, see Johnston, 1981). In these fishes, white muscle fibres are recruited only for burst-type activity, and quickly result in fatigue. This has been shown in Pacific herring, Clupea harengus pallasi Valenciennes, 36 and dogfish, Squalus acanthias, (Bone 1978; Bone et al, 1978). Other teleost fish have white muscle fibres that are multiply innervated, and two types of electrical activity have been recorded from these fibres, spike potentials and junction potentials (Hudson, 1973). Multiply innervated fibres have been shown to be active during sustained swimming (Hudson, 1973; Johnston et al, 1977; Bone et al, 1978; Jayne and Lauder, 1995). Muscle fibres innervated with multiple motonerurons have been studied to determine relationships between the motoneurons and the corresponding fibres that are innervated by the motoneurons. In mammalian muscle fibres a 'size principle' exists. This principle relates the size of the neuronal cell to the diameter of the motoneuron to the muscle fibres it innervates. The basic components of this principle are as follows. A motoneuron only innervates one type of muscle fibre (e.g., slow oxidative (red) or fast glycolytic (white); Wuerker et al, 1965). The diameter of the motoneuron is related to the neuronal cell size and to the number of muscle fibres it innervates ( Henneman et al, \965a,b; McPhedran et al, 1965; Wuerker et al, 1965). In addition, an inverse relationship between the excitability of a motoneuron and the neuron's cell size has been observed (Henneman et al, 1965a). Thus, small motoneurons have low thresholds, and innervate few muscle fibres. When large and small motoneurons are exposed to stimuli of equal magnitude, small motoneurons are recruited first, followed by larger motoneurons (Henneman et al, 1965a). Additionally, because of the lower threshold for activation of small motoneurons, these motoneurons are recruited more often than larger motoneurons. The small, highly used motoneurons have been found to innervate muscle fibres with high ATPase activity (i.e. slow, red muscle fibres), whereas larger motoneurons innervate larger fibres that have lower ATPase activity (i.e., large 'pale' motor units) (Henneman and 37 Olson, 1965). Inhibition is also cell size-dependent, with larger motoneurons being more susceptible to inhibititory stimuli than small motoneurons (Henneman et al, 1965&). The 'size principle' of recruitment of motoneurons and subsequently muscle fibres has been indirectly tested in fish with multiply innervated muscle fibres (Rome et al, 1984; Jayne and Lauder, 1994). EMG studies have demonstrated that slow sustained swimming requires the recruitment of some red muscle fibres. As swimming speed increases, more red fibres are recruited until all of the fibres are being used to power swimming. At this point, if swimming speed is increased some white muscle fibres are also recruited. The order of white muscle recruitment should follow the size principle, whereby motoneurons that innervate few fibres will be recruited first. Increased recruitment of white muscle fibres will continue until burst swimming occurs, at which point all the white muscle fibres are activated. Experiments using carp and scup, Stenotomus chrysops, have illustrated this theory up to the point where white muscle fibres are initially recruited (Rome et al, 1984; Rome et al, 1985; Rome, 1990). There is additional evidence that this order of recruitment is maintained at varying temperatures; at cold temperatures the order is the same, but is compressed (Rome et al, 1985; Sisson and Sidell, 1987; Rome et al, 1990). However, the pattern of white muscle fibre recruitment has not been as well documented, and the correlation of motoneuron size to number of fibres has not been investigated. A slight modification in this theory on the order of muscle fibre recruitment in fish has been proposed recently (Jayne and Lauder, 1995). In these studies, muscle recruitment in bluegill sunfish (Lepomis macrochirus) was examined using EMG electrodes in fish swimming in a wider variety of patterns, including during the recruitment of white muscle fibres (Jayne and 38 Lauder, 1994). This study demonstrated that during slow, sustained swimming, only red muscle fibres are recruited; with increased swimming speed, an increase in the length of the EMG signal ("burst duration") was recorded, up to approximately 70% of the longest duration (from those red muscle fibres, for each fish). When the threshold for white muscle fibre recruitment is reached, red muscle fibres are recruited simultaneously with some white muscle fibres. Once all red muscle fibres are recruited (maximal burst duration), an increase in swimming speed results in additional white fibre recruitment, up to 30-40% of the maximal burst duration for those white muscle fibres. Futher increases in swimming speed result in a decrease in red muscle fibre activity, from 100% to about 25% of maximal burst duration. During burst swimming, white muscle activity increases independently of red muscle fibres (Jayne and Lauder, 1994). These two models of fish muscle recruitment may not be mutually exclusive, as the previous studies using carp and scup investigated only swimming that recruited red muscle fibres up to initial white fibre recruitment. Additionally, muscle fibre arrangement within myomeres and the relative proportions of white muscle fibres of differing diameter can vary among species (Alexander, 1969; Johnston et al, 1977). The motoneurons that activate muscle fibres in fish have not been thoroughly examined, therefore, no conclusions can be made regarding the. overall appropriateness of the mammalian size principle to fish muscle recruitment. The goal of this part of my study was to estimate the extent of white muscle fibre recruitment in rainbow trout during sub-maximal swimming speeds. This was achieved through monitoring the concentration of a metabolically important end-product, lactate, within the white musculature. This information was used to infer the relative contribution of anaerobic metabolism to sub-maximal swimming. The use of EMGs to indicate muscle activity have the 39 advantage of indicating when white muscle fibres are activated, but cannot monitor the physiological ramifications of white muscle recruitment. In this part of my study, I investigated the effect of swimming at 70, 80, and 100% of U c r i t on the concentration of lactate and phosphocreatine within the white muscle, in three areas along the body of rainbow trout. This was examined in order to determine at what proportion of critical swimming speed white muscle fibre recruitment becomes significant, and whether this recruitment was consistent along the length of the body. 40 MATERIALS AND METHODS Animals Rainbow trout were obtained from Spring Hill Trout Farm (Abbotsford, B.C.) in December 1995. Both Spring Hill and West Creek obtain rainbow trout from the same source, and therefore, the fish were from the same stock as the fish used in the previous experiments (Chapter 2; Al Ludwig, West Creek Trout Farm, Abbotsford, B.C., personal communication). Rainbow trout were kept in an outdoor 2 000 L round plexiglass tank with a flow-through dechlorinated water supply system at the Department of Zoology, U.B.C. Feeding regime was similar to the previously described protocol (Chapter 2). Rainbow trout were 35.6± 1.38 cm total length and 482.7 ± 54.5 g. Fish were not fed for at least 2 days prior to removal for use in experiments. The holding temperature changed seasonally from 7°C to 10°C. Experiments were performed in May and June, 1996, at 10°C. Swimming Protocol Rainbow trout were removed by net, lightly anaesthetized with buffered tricane methanesulfonate (Syndel), and were measured for mass and total length. The anaesthetized fish was then transferred to either a black plexiglass box with a flow-through water supply or directly into the respirometer. The fish quickly regained balance, and when a low water velocity was imposed, began to swim. After 10 to 20 minutes at a low speed, the water velocity was increased to approximately 1 Ls"1, and the fish was left to recover and become accustomed to the swim tube for 10 to 12 hours. Following this initial period, the swimming protocol was initiated, and the fish was forced to swim either to (as described in Chapter 2), 80% U c r i t or 70% U c r i t. The mean body weight, 41 total length, swimming speed and % U c r i t of the rainbow trout in each group is summarized in Table 1. Each fish was used for only one swimming trial, with a minimum of 6 fish per group. Following the prescribed swimming regime, the fish were transferred from the respirometer to a plexiglass box (previously described in Chapter 2). Struggle was minimized by the use of a plastic bag filled with water at ambient temperature, into which the fish swam, and was gently lifted out of the respirometer and into the plexiglass box. Resting fish were placed into the plexiglass box 10 to 12 hours prior to obtaining readings to allow for recovery from handling stress. The box was placed in the vertical position, to ensure that the fish was in an upright postition. Fresh, dechlorinized water flowed into the box, and paper towels were placed over the front top and sides, and black plastic was placed over the whole box to block out most of the sunlight and room lights. Following recovery, the box was gently rotated 90° and placed into the bore of the magnet. NMR Spectroscopy and Analysis Measurements were obtained at rostral (0.281,-0. ML), mid (0.44L-0.54Z) and caudal (0.64L-0.74Z,) sections of the musculature. The length of time to obtain the 1024 spectra per sample was one minute, as previously described. The remaining parameters for the NMR spectroscopy were identical to the parameters used for determining the relationship between lactate and P/ (Chapter 2). The NMR was maintained and operated by Dr. Anibal Rojas-Vargas (Dept. of Zoology, U.B.C.). Following the measurements, the fish was removed from the plexiglass box and placed in a black box to recover. Resting fish were also measured in three areas, similar to the sections measured for exercised trout. In some cases, additional spectra at the three sections were obtained. This was 42 to ensure resolution of the Pz peak. Additional spectra were obtained over 3.36 minutes under the following conditions: 128 individual scans, nominal pulse 90° (42 us), spectral window of 500 Hz, 1024 data points and a 1 second delay between pulses. The additional scans allowed for easier differentiation of the Pz peak from background noise. Analysis of spectra was performed by Troy M. Hallman (Dept. of Zoology, U B C . ) and myself, as previously described (Chapter 2). Analysis and Statistics The chemical shift of Pz and the area under the PCr curve were analysed three times, and each estimate was converted to either lactate (umol/g) (from 5-Pz) or PCr (umol/g) (from PCr area) using the equations developed in Chapter 2. The mean, standard deviation and coefficient of variation (C.V.) were calculated for the converted values. If the C. V. was greater than 10%, the sample was reanalysed (3 times) to increase the precision of the estimate. One-way repeated measures ANOVA was used to test for differences between sections at each swimming speed (0, 70, 80, 100% Ucrit). Although each measurement on the fish is not a repeated measure in the strict definition, measurements within the same fish will be more similar than measurements between fish. It was desired to detect a different in the pattern of metabolites between the sections, as well as the absolute differences between the sections. One-way ANOVA was used to test for differences between swimming speed within each section. Any differences were detected using the Student-Newman-Keuls post-hoc multiple comparisons test. Sigma Stat (v. 1.01 and v. 2.0, Jandel Scientific) were used. Significant differences are P<0.05 unless otherwise stated. 43 - H pq 4=1 - H - H a T 3 • S <t> £ CL CO c/3 I—] PQ - H Tt Tt T t in r-in c n in o © © © ON o -Tt © ON 0 0 v o c n <n c n c n in c n in c n Tt T t 0 0 o\ c n o r-~ c n T—1 o o r—1 Tt CN 0 0 in m c n t--Tt ON Tt CN Os Tt v o Tt OO Tt in Tt OO c--VO Tt OO O © v o v o c n ' 1 VO o < <n c n in in o o © T—1 o © © o c n i—i ON v q 0 0 Tt CN r—H r-- OO o o VO CN c n o © © © © © 100.00 Tt in Os t> VO Tt Os VO o o © 44 RESULTS Lactate Concentrations within white muscle Each fish had three areas measured to estimate the lactate concentration within that portion of the white musculature. Regardless of the section of musculature monitored, the largest concentration of lactate was detected following swimming to critical swimming speed. Generally, there was a greater concentration of lactate within the rostral musculature than in the mid or caudal sections, when differences due to swimming treatment were considered (Figure V) Following swimming for 30 minutes at approximately 70% U c r i t, the estimated lactate concentration within the muscle mass was not significantly different from levels estimated from resting fish (Figure 7). Within the rostral section, a significantly greater concentration of lactate was estimated following 30 minutes of swimming at 80% LL ,^ than the resting levels. Swimming to U c r i t resulted in significantly greater lactate concentrations in all sections when compared to resting levels. Swimming at 70% U c r i t resulted in significantly greater concentrations of lactate within the mid and rostral sections when compared to the caudal region (Figure 7). This pattern was not found following the other swimming protocols, however, swimming at 80%o U c r i t did result in a significantly greater concentration of lactate rostrally than caudally. No difference was observed between sections following U c r i t or in resting fish. The range of lactate concentration that was associated with each swimming protocol is presented in Figure 8. Swimming at 70% U c r i t resulted in a narrow range of [lactate] within the white muscle. Swimming at approximately 80% U c r i ( results in a larger range of [lactate], 45 overlapping with the range of both 70% U c r i t and Ucr i t. The range of [lactate] estimated following swimming to U c r i t covers a smaller range than 80% LJ r i t. Resting [lactate] did not vary much between individuals or sections. Phosphocreatine accumulation following exercise The mean intramuscular phosphocreatine, as estimated from the relationship between 3 1P-NMR PCr area and biochemically determined PCr is presented for the three monitored sections within the musculature following exercise (Figure 9). Following exercise, the general trend across all muscle sections was a significantly greater [PCr] in resting muscle than in muscle from fish forced to exercise. Also, there was significantly less PCr remaining within the white muscle following exercise to U c r i t compared to 70% and 80% U c r i t. No difference in the [PCr] following swimming at 70% and 80% U c r i t was detected when all muscle sections are taken into account. The mean intramuscular [PCr] estimated in resting muscle had a significancy higher [PCr] in the mid region than caudally (Figure 9). Following swimming to 70% U c r i t, this pattern was repeated, however there was also a significantly lower [PCr] within the rostral region when compared to the mid region. Exercise to U c r i t and 80% Uc r i t resulted in lower [PCr] with no detectable difference between the muscle sections. Within a section, all swimming treatments resulted in significantly lower [PCr] than in resting muscle. No difference was detected between the [PCr] following 70% and 80% U c r i t in any section. Within the rostral section there was a significant difference between the [PCr] following swimming at 70% or 80% U c r i t and swimming to U c r i t. This pattern was repeated in all sections. The range of PCr concentrations that were estimated following each swimming treatment 46 are illustrated in Figure 10. The range of [PCr] estimated from resting fish is separated from the range of concentrations associated with swimming at 70% U c r i t. The range from resting fish is quite narrow compared to the range of concentrations detected following swimming at either 70% or 80%) U c r i t. Swimming to U c r i t also resulted in a narrow and separated range of [PCr] detected within the white muscle. The range associated with swimming at 70% U c r i t is very-broad and overlaps with almost all of the range of concentrations detected following swimming at 80%) U c r i t. The range of concentrations associated with swimming at 80% of U c r i t is narrower than 70%) U c r i t, and although nearly completely contained within the range of [PCr] associated with 70%) U c r i t, shows a more definite pattern of greater [PCr] within the mid section. PCr/Lactate following exercise The mean ratio of PCr to lactate from each swimming treatment for each section is presented in Figure 11. The ratio of PCr to lactate associated with resting fish muscle was significantly greater than following swimming (all sections). Within the caudal section, swimming at 70% U c r i t resulted in a higher ratio of PCr to lactate than following swimming at U c r i t . Within the mid-section, both 70% and 80% Lt had significantly higher ratios than following swimming at U c r i t. However, within the rostral region, there were no significant differences between 70%, 80% and 100%) U c r i t, although the trend was similar to the other regions (resting ratio>70% Ucri> 80% Ucri> Ucrit). 47 Figure 7. Mean white muscle lactate concentration in rainbow trout white muscle following exercise, at different positions along the body. Lactate estimates were obtained through application of the relationship developed between 3 1P-NMR derived Vi and intramuscular lactate concentation (see Figure 4, Chapter 2). The 'a' indicates a significant difference from the resting lactate concentration, within a given position. The asterix (*) indicates a significant difference from the caudal section, within the swimming group. 48 Rest Rostral Mid Caudal 49 80-1 70-3 60-Q £ f 50-I B 40-30-20-10-0-E3 Rest E3 70% Ucrit 3 80% Ucrit 100% Ucrit 1 1 1 1 Posterior Mid Anterior Figure 8. Variability in intramuscular lactate concentration at different portions along the body, following exercise. Individual fish are represented by unique symbols, within each treatment. 50 Figure 9. Mean intramuscular phosphocreatine estimated from 3 1P-NMR in rainbow trout following exercise, at different positions along the body. The 'a' indicates a significant difference from the resting phosphocreatine concentration, within a given position. The letter 'b' indicates a significant difference from the phosphocreatine concentration following U c r i t, within a given position. The asterix (*) indicates a significant difference from the mid section, within a swimming treatment. 51 52 40 35-30-oo 5 | 25-J J 20-1 § 15H 10-4 5 J Y/S^^/SSSS/SS/SSS/S/S. A w / / / / / / / / / / / / / / / / / / / / / . — I — Caudal @ Rest [ H 70% Ucrit 80% Ucrit 100% Ucrit Mid —I 1 Rostral Figure 10. Variability in intramuscular phosphocreatine at different positions along the body, folloing exercise. Individual fish are idnetified by unique symbols within treatment. 53 Figure 1 1 . The mean intramuscular phosphocreatine-lactate ratio in rainbow trout, following exercise, at different positions along the body. Both PCr and lactate concentrations were estimated using 31P-NMR. The asterix (*) indicates a significant difference from the caudal section, within the swimming treatment. 54 55 DISCUSSION White muscle lactate was monitored in fish subjected to swimming speeds corresponding to 70, 80 and 100% of U c r i t to determine at what proportion of maximum swimming speed anaerobic metabolism was occurring in the white muscle. In rainbow trout, exercised in 10°C water, significant increases in intramuscular lactate concentration occurred following exercise at 80% U c r i t when compared to resting levels (Figure 7). A number of studies that have demonstrated that white muscle fibres are recruited at sustainable speeds (e.g., Hudson, 1973; Bone et al, 1978; Jayne and Lauder, 1995). It has been suggested that at 80% U c r i t there is some contribution of energy from anaerobic glycolysis, in trout swimming at 15°C, however it was concluded that this contribution was negligible (Webb, 1971). Although 80%) U c r i t has been previously suggested as the threshold for white muscle activation, this is not the case in all fish species. There is evidence that fish with multiply innervated white muscle fibres recruit these fibres during sustained swimming, whereas focally innervated fibres are only recruited during near-maximal swimming (e.g., Bone, 1975; Bone et al, 1978). Through the use of EMG direct evidence has been found for activation of white muscle fibres during sustained swimming (Hudson, 1973; Bone et al, 1978; Brill and Dizon, 1979; Rome et al, 1985). The results from EMG studies have shown that in rainbow trout, during sustained swimming, some slight but regular activity is detectable from the electrodes placed in the white muscle (Hudson, 1973). EMG activity has been detected from the white muscle fibres of carp during swimming even at very slow sustained speeds (0.5 Ls"1, 25-30 cm FL, Bone etal, 1978). Indirect evidence also suggests that white muscle fibres are increasingly recruited as sustained swimming speeds increase in the coalfish (Gadus virens L.; Greer Walker 56 and Pull, 1973). Additionally, increased concentrations of white muscle lactate have been found in small coalfish during sustained swimming above 2 Ls"1, well below the estimated mean maximum sustained swimming speed for these fish (Johnston and Goldspink, 1973/3). Differential lactate and PCr concentrations along the length of the body The accumulation of lactate within the white muscle mass of exercised rainbow trout was not the equal along the length of the body (Figure 7). Three different regions were monitored for intramuscular lactate and PCr concentrations. The pattern of lactate concentration along the length of the body differed from that of PCr concentration. Significantly greater concentrations of lactate were detected in the rostral region when compared to the caudal region following exercise to 70 and 80% U c r i t. No statistical difference was detected between sections in resting fish or in fish exercised to U c r i t. The general trend observed in sub-maximally exercised fish muscle was an increase in concentration of lactate from caudal to rostral. This result suggests that recruitment of white muscle fibres during sub-maximal swimming is complex, and not the same throughout the body. Some possible mechanisms whereby greater concentrations of lactate would result rostrally than caudally during exercise are: (1) more anaerobic fibres are recruited rostrally, (2) the same number of fibres are recruited along the length of the body, but in the rostral musculature they are recruited for a longer period of time, (3) these fibres are recruited to a greater extent within the rostral portion of the white muscle mass, or (4) there is significantly less PCr rostrally, so anaerobic glycolysis is required sooner rostrally than caudally. Additionally, these four mechanisms may be combined in a number of ways to result in the pattern of lactate observed in this study. Investigation into the concentration of PCr from the same regions of the fish muscle 57 following exercise indicates that at rest, a greater PCr concentration occurs in both the mid and rostral regions than caudally (Figure 9). During exercise (70 and 80% Ucrit), the pattern of PCr concentration is retained, with the mid section having a significantly greater concentration of PCr than both the rostral and caudal sections. The different PCr concentrations along the length of the fishes body that were observed in this study may indicate that whole-body estimates of PCr concentration from white muscle following sub-maximal swimming and at rest, are usually overestimated. This overestimation would occur in studies where the muscle sample is removed from the mid-lateral musculature. The concurrent changes of PCr and lactate concentrations within the white muscle are illustrated in Figure 11. No significant difference was detected in the metabolite ratio following exercise between sections. Resting levels of PCr within the caudal muscle fibres is less than from either the mid or rostral sections. Thus, it does not appear that any of the sections have a significantly different store of PCr that would alter the metabolic requirements of that section during sub-maximal or maximal swimming. PCr is quickly depleted, prior to the metabolism of glycogen to lactate. Therefore, the high PCr concentration within the rostral muscle fibres does not indicate an earlier recruitment of anaerobic glycolysis within the rostral section. The higher concentrations of lactate within the rostral white muscle may be due to differences in the relative proportion of red and white muscle along the length of the body. The relative proportions of red and white muscle fibres within various sections have been examined in a number of fish species. The proportion of total skeletal mass that the red fibres occupy in small fish (7.5-15 cm fork length), has been calculated as increasing in the caudal musculature (4-16%), with little change in proportion between rostral (1-5%) and mid regions (2-6%; Gill 58 et al, 1989). These trends are species dependent. The only salmonid examined by Gill et al. (1989) (lake whitefish, Coregonus clupeaformis), had intermediate red muscle proportions for all sections. The fractional mass of white muscle in sculpin, Myoxocephalus scorpius L., has been measured at various sections along the body (22-25 cm TL, 15 ° C). The distribution of the white muscle mass follows a bell curve, with the greatest proportion of white muscle fibres (77%) found between 0.37-0.66 L (Johnston et al., 1995). Although these studies have concentrated on species other than rainbow trout, the general shape and anatomy of fish indicate that muscle will be thickest at the widest point along the body, generally in the mid section. If the fractional mass of white muscle fibres within rainbow trout is similar to that of the sculpin, a proportionately greater quantity of lactate should be found within the mid section; however, in this study the concentration of lactate within the middle section was found not to be greater than the rostral or caudal sections, and consistent quantities of muscle were analyzed from all three sections. The greater accumulation of lactate within the rostral muscle section than in either the mid or caudal section is probably an indication of greater recruitment of white muscle fibres from this region. This could occur through either the recruitment of more fibres rostrally or the same number of fibres being recruited for a longer period of time, during sub-maximal swimming. One method of testing this without estimating actual force and power production in vitro, would be to investigate the pattern of innervation of muscle fibres along the length of the fish's body. The size principle of recruitment of motoneurons (Henneman et al, 1965a,/3) may help explain the neuronal mechanism of muscle recruitment that could result in the pattern of lactate concentration that was observed in this study. Mammalian motoneurons are recruited from 59 smallest to largest, the diameter of a motoneuron is correlated with the number of fibres it innervates, and the neuronal cell size. Therefore, if this size principle is applicable in fish, and there is some indication it is (e.g., van Raamsdonk et al, 1983; Rome et al, 1984; Fetcho, 1986), the smallest motoneurons, which innervate few muscle fibres, are recruited first, and at a lower threshold. Sequentially larger motoneurons are recruited, activating more muscle fibres. If the rostral white muscle mass consists of fibres innervated with relatively smaller motoneurons than in the caudal, then for a given stimulus, more rostral fibres would be recruited than caudal fibres, as the signal moves from rostral to caudal. Alternatively, rostral white muscle fibres may be innervated with similar sized motoneurons as caudal fibres, however, quantitatively more of these fibres may be located within the rostral musculature. White muscle fibres in zebra fish, Brachydanio rerio, and goldfish, Carassius auratus, are innervated by motoneurons of relatively large diameters, but are quite variable in size. Some small motoneurons that innervate white muscle fibres are of similar size as the motoneurons that innervate red muscle fibres. The largest motoneurons primarily supply the deep white fibres (van Raamsdonk et al, 1983; Fetcho, 1986). Few studies have investigated the composition of muscle fibres and motoneurons in fish, and no study has investigated the motoneuron organization of trout red and white muscle. There is little information regarding the detailed pattern of innervation of white muscle fibres within fish, nor has the size of motoneurons in relation to the size of muscle fibres within the white muscle along the length of the fish been studied. There is some indication that differences in the composition of white muscle fibres occur within the white musculature. A dorso-lateral difference in the composition of white muscle 60 fibres in mature rainbow trout has been detected. White muscle fibres examined closest to the lateral line had the largest diameter, and those examined from a more dorsal section had smaller diameter fibres (Kiessling et al, 1990). However, the fibre diameters from the rostral and caudal muscle have not yet been examined. Within the sequential contraction of muscle fibres from rostral to caudal a pattern of muscle fibre recruitment and subsequent power production (force*sarcomere distance/time) might exist that may help with the interpretation of the pattern of lactate concentration observed in this study. Recently, a number of studies have investigated the production of power by red and white muscles (typically using excised blocks of muscle) from different portions along the length of the body (Altringham et al, 1990; van Leeuwen et al, 1990; Rome et al, 1993; Davies et al, 1995; van Leeuwen, 1995, Wardle et al, 1995). Research that tests the theories on power production within fish muscles have shown species dependent results, and rainbow trout muscle power production has not yet been modelled. Measurements of the actual force development during muscle fibre recruitment in vivo and in vitro are at present lacking, as well as precise measurements of the distance of sarcomere movement in vivo. Power production in the muscle is dependent on when in the shortening-lengthening cycle activation of the muscle fibre occurs. In cyclical swimming, differences in the activation of muscle fibres along the length of the body have been demonstrated, resulting in different amounts of work and power being produced (Altringham, 1990; van Leeuwen et al, 1990; Rome et al, 1993; Davies et al, 1995). The estimated power production in superficial red muscle fibres from various sections along the body of carp showed a decreasing profile of power from rostral to caudal (van Leeuwen et al, 1990). The generation of power of scup occurs in the caudal and 61 mid musculature (i.e., an increasing power profile, from rostral to caudal) (Rome et al, 1993). Scup have a different body shape and a less undulatory style of swimming than carp (Rome et al, 1992). These differences in power production may be primarily due to differences in the swimming profiles between the two species. In white muscle, a pattern of decreasing power production from rostral to caudal has been observed in cod, Gadus morhua L., and saithe, Pollachius virens (Davies et al, 1995; Altringham et al, 1990, 1993). In contrast, no change in the power production of white myotomal fibres of the bottom-dwelling sculpin has been detected (Johnston et al, 1993). If trout has a similar swimming pattern as saithe, as suggested by Wardle et al. (1995), then the expected propagation of power from the rostral and transmission by the caudal muscle to the tail blade may be an appropriate model. If this is the case, the greater power production would be observed rostrally, with a decreasing profile to either a negative or near-zero power production caudally. Negative or near-zero power production occurs if the activation of the muscle fibres occurs during the lengthening cycle (stiffening). If the caudal muscle acts primarily to transmit power to the tail blade, then the expected activation and contraction of white muscle fibres may be negligible. This could result in the profile of lactate concentrations observed in this study. If increased power production occurs rostrally, this could be the result of a number of factors. The contraction may be occurring only during the contraction of the sarcomeres, thus only producing positive power. Alternatively, the total time of activation could be different between sections along the length of the body. There is some indication that differences in the relaxation time of muscle fibres occurs; rostral muscle in scup have a faster intrinsic relaxation time than do caudal fibres, however, no mechanism for this difference has been investigated 62 (Rome et al, 1993). No correlation between power generation and biochemical measurements of anaerobic end products have been studied thus far, therefore, it is difficult to interpret power production profiles in terms of biochemical end-products and reactions. One study that does not rely on assumptions of sarcomere length changes and force development by muscle fibres investigated the role of skin and axial skeleton on swimming performance (Long et al, 1994). In the pumpkinseed sunfish (Lepomis gibbosus), the rostral muscle can generate most of the required locomotory power, which is then transmitted to the caudal fin via the skin and axial muscle. If this pattern of power generation and transmission applies to rainbow trout (and other salmonids), then the results of this study may be explained by this pattern of power generation. That is, if only the rostral muscle is required to power swirnming, then more muscle fibres will be recruited rostrally than caudally. Additional studies are necessary to determine the pattern of power production and generation that occur during undulatory swimming in rainbow trout. EMGs are often used to obtain information regarding the recruitment of muscle. Marked increases in the amplitude of EMG bursts have been recorded during increases in speed, and have been assumed to be due to recruitment of additional muscle fibres (Grillner, 1974; Grillner and Kashin, 1976). By studying the differences in EMG signals during different swimming speeds and from various portions along the body, a correlation between the intensity and duration of the EMG signal and the number and size of muscle fibres that contribute to the signal may be constructed. No relationships of this kind have yet been reported. One recent study investigated the onset and offset time of white and red muscle activity within rostral and caudal muscle sections of bluegill sunfish. The differences in the EMG signals between the red and white muscle 63 at the same position were compared, but unfortunately, the white muscle activity in the rostral and caudal muscle mass were not compared (Jayne and Lauder, 1994). The representative EMGs that were reported however, appear to have differences in the intensity of white muscle fibre activity between the rostral and caudal during burst and glide swimming. The total duration of EMG signals during swimming at intermediate speed appears to be longer rostrally than caudally (Figure 2A-C, Jayne and Lauder, 1994). An extended duration of muscle recruitment rostrally during swimming may result in either white muscle fibres being recruited for a longer duration, or more white muscle fibres being recruited during this time. Either recruitment pattern would result in a greater concentration of lactate rostrally because of the increased recruitment of glycolytic white muscle fibres and subsequent anaerobic production of ATP. The quantification of EMG signals is required to test this theory. Muscle fibre recruitment has been monitored within various positions of myomeres to determine if fibres within a myomere are synchronously recruited. Muscle fibres within a myomere are recruited simultaneously during burst swimming, in largemouth bass (Micropterus salmoides; Jayne and Lauder, 1995). Semi-steady swimming resulted in the detection of activity from one (epaxial) site of a myomere, despite the lack of activity at the corresponding hypaxial site (Jayne and Lauder, 1995). Muscle fibres within a myomere appear to be recruited independently, or as subgroups within the myomere. This independent recruitment of muscle fibres indicates that there is some plasticity in the recruitment of white muscle fibres during sub-maximal swimming. This aspect of swimming mechanics has not been investigated thoroughly, and an increase in the understanding of the design constraints on muscle fibres within and between myomeres would help to expand our understanding of the finite details of the mechanics 64 of fish swimming. These models of muscle fibre recruitment suggest mechanisms by which the pattern of lactate concentration following sub-maximal swimming might have occurred. Evidence exists that muscle fibres can be recruited differentially within a myomere (Jayne and Lauder, 1995), and it seems plausible that muscle fibres within different myomeres should also be differentially recruited. There is a possibility that more muscle fibres may be recruited rostrally than caudally (innervated with smaller motoneurons, which have a lower activation threshold, larger EMG pulses), or fibres being recruited for a longer period of time (longer EMG pulses). If the muscle mechanics and power production and transmission in trout are similar to pumpkinseed, then the rostral musculature is more important for generating power to swim than the caudal, because the power is transmitted via the axial skeleton and the skin. These possibilities require further investigation to determine the mechanisms that result in greater concentrations of lactate in the rostral white musculature following sub-maximal swimming. Variability in lactate and PCr concentrations between fish The variability in lactate and PCr concentrations following exercise at 70% and 80% U c r i t are illustrated in Figures 9 and 11. This variability can be explained primarily by differences in behaviour between individual fish. If a fish, at a given water velocity accelerates to the front of the tube and then glides toward the back, this behaviour requires more energy than swimming steadily in the same place. Any differences in behavior over the trial time period will result in variation in the recruitment of white muscle fibres and thus the concentration of lactate and PCr in the white muscle. The variability in the concentration of PCr is greater, and this probably reflects the nature of PCr hydrolysis and anaerobic glycolysis. PCr hydrolysis occurs prior to 65 anaerobic glycolysis, and therefore, if any differences in the activity of the fish occurred, PCr would be hydrolyzed to varying degrees. If PCr is completely hydrolysed, anaerobic glycolysis would occur, and some production of lactate would ensue. Therefore, PCr concentration can range from near resting levels to near exhausted levels during sub-maximal swimming because PCr hydrolysis is the initial reaction that occurs when white muscle fibres are activated. Lactate concentrations do not vary as extensively, because only after the hydrolysis of PCr, will lactate be produced. Therefore, the range of lactate concentrations will be more limited during sub-maximal swimming. Swimming to 80% and 100% U c r i t resulted in a greater variability in the concentration of lactate than PCr; this is because once anaerobic glycolysis has been initiated, the hydrolysis of PCr will be nearly complete (Hochachka, 1985) and lactate production will vary with any variations in activity. Additionally, U c r i t was determined for a sub-sample of the fish used in this study, and the mean U c r i t was calculated from this subset and used to estimate the U c r i t for each fish. Therefore, each fish may be forced to swim at slightly different proportions of its individual U c r i t resulting in variability in the extent of anaerobic metabolism that was required to support activity. Implications to energy budgets Webb (1971) concluded that the contribution of anaerobic metabolism to the overall energy efficiency and budget of the fish in increasing-velocity (Ucrit) tests was negligible despite stating that anaerobic metabolism was required to support activity above 80% U c r i t. The present study shows that a statistically greater concentration of lactate occurs within the white muscle of rainbow trout following swimming at 80% of U c r i t for 30 minutes (Figure 7). Although statistically significant, this increase in lactate concentration may not be of biological importance. 66 The biological importance of anaerobic glycolysis occurring during sub-maximal swimming may be minor for a fed fish in a water treadmill. For a starving, migrating, salmon that is using its energy reserves for gonad production while migrating for extended periods of time through diverse and sometimes arduous sections of river to reach the spawning grounds, the anaerobic component of sub-maximal swimming is probably important. As yet, the anaerobic component of swimming during the spawning migration has not been adequately investigated. Energy budgets are generally based on aerobic considerations, and assume that the anaerobic portion of swimming as negligible. An energy budget has been constructed for the early Stuart sockeye salmon spawning migration based on data obtained from EMG telemetry (Hinch and Rand, 1996). These radio transmitters provide data on temperature and tailbeat frequency, information which is put into a standard bioenergetic equation to predict energy expenditure (e.g., Beauchamp et al, 1991). The present study demonstrates that above 70% of the critical swimming speed, lactate accumulates within the white muscle mass, at 80% of U c r i t, this becomes significantly greater than resting values. Although these trials only lasted 30 minutes at each increment, the accumulation of lactate within the muscle indicates that for a long-term migration, the anaerobic component may be significant, and probably should be incorporated into energy budgets. The conversion of the concentration of lactate within the white muscle fibres to an energy equivalent that can be used in energy budgets is required. During anaerobic exercise, muscle glycogen is metabolised to produce three ATPs and two lactates. Thus, for every two moles of lactate detected within the fish muscle, three moles of ATP were produced. From this conversion to ATP, a caloric equivalent may be applied in order that the anaerobic component 67 can be added to the overall energy budget. General applications The statistically significant increase in muscle lactate concentrations following swimming at 80% U c r i t in adult rainbow trout at 10°C may be applied to energetic models of other fish species with caution. This relationship between swimming speed and white muscle activity may be consistent in other species, however, this has not been studied. If swimming form and lifestyle is similar to hatchery raised rainbow trout, then application may be acceptable. The mean U c r i, for the rainbow trout in this study, 2.13 Ls"1, is significantly less (P<0.001) than the estimated U c r i t for a similarly sized sockeye (at the same temperature, from a 60 minute U c r i t test; 2.71 Ls"1; Brett and Glass, 1973). This is expected, because sockeye salmon are generally strong swimmers. Many stocks of sockeye undergo long spawning migrations, often longer than the spawning migrations of many pink salmon stocks and some steelhead (anadromous rainbow trout) stocks. The U c r i t estimate for pink salmon, 2.19 Ls"1 (Brett, 1982), which can swim approximately 80% of the speed of sockeye, is not significantly different (P=0.160) from the mean U c r i t obtained in this study. Thus, it may be appropriate to assume that pink salmon also recruit white muscle fibres during sub-maximal swimming and statistically different concentration of lactate from that at rest occur following swimming at 80% U c r i t. Further studies may be required to confirm that these results also apply to sockeye swimming at sub-maximal speeds. 68 CHAPTER 4: GENERAL DISCUSSION Support of exercise by glycolytic muscle fibres Recruitment of white muscle fibres during exercise has been demonstrated in a number of fish species. In fish that have white muscle fibres that are multiply innervated, the recruitment of these white muscle fibres has been demonstrated during swimming at speeds below burst swimming (i.e., carp, trout, saithe, cod, scup; Hudson 1973; Bone et al., 1978; Rome et al., 1984, 1990). The present study has demonstrated that anaerobic glycolysis is required to support activity above 70% U c r i t in rainbow trout at 10°C. Other studies have suggested that swimming at 80% U c r i t results in the recruitment of white muscle fibres; typically these studies have employed EMG electrodes. If differential recruitment of white muscle fibres within myomeres (Jayne and Lauder, 1995) and between myomeres occurs during sub-maximal swimming, it is possible that these studies may not have detected some activity because of the placement of the electrodes. The extent of recruitment of white muscle fibres is not known, because no relationship between EMG signals and the number and intensity of white muscle fibres recruited has been established. Quantification of EMG signals, specifically the duration and amplitude of the signal, in terms of the extent of recruitment of white muscle fibres in the region of the electrode and the total timing of activation may be useful information to aid in the interpretation of EMG signals during sub-maximal swimming. If the relative proportions of white muscle fibres recruited during sub-maximal swimming could be measured or estimated, any differences in the extent of recruitment of muscle fibres along the length of the body could then be monitored. Regardless, measurement of intramuscular lactate provides a direct estimate of anaerobic metabolism, which 69 EMG signals cannot provide. This study has demonstrated that there appears to be greater recruitment of white muscle fibres within the rostral muscle mass during sub-maximal swimming than in the caudal or mid regions, as indicated by the accumulation of the anaerobic end-product, lactate. Swimming to U c r i t results in essentially the same amount of recruitment of white muscle fibres throughout the length of the body; this is demonstrated by the similar lactate concentrations in the white muscle. Thus, a similar production of ATP occurred on a per gram basis, throughout the white muscle when the fish was forced to swim to U c r i t. Effects of fish size The fish that were used in these studies ranged from approximately 300 g to 650 g. This range of fish sizes was found not to affect the relationship between 3 1P-NMR derived 5-Pz' and lactate. Although this range is relatively limited in terms of the maximum and minimum mass that rainbow trout and sockeye salmon can achieve, it does serve to provide some indication that expanding the relationship to larger fish should not be problematic. There are a number of studies that have investigated the effect of size of fish on a number of mechanical and biochemical aspects of swimming (Brett, 1964, 1965; Brett and Glass, 1973; Goolish, 1991). As a fish grows, the maximum swimming speed that can be attained increases, however when swimming speed is expressed in terms of body lengths per second, the swimming speed decreases as the fish length increases. A relationship has been established between size of fish and the expected U c r i t at a given temperature; speed = a(body length)b, where b is the slope and approximates 0.5, which indicates the relative ability to maintain a sustained speed decreases as the size increases (Brett 1965). The ratio of active to standard metabolic rate is the metabolic scope for activity of a fish. 70 With increasing size, this ratio increases, although not linearly. This increase is due to a gradual decrease in the standard metabolic rate with increasing size (Brett, 1965). Thus, a large difference exists between the metabolic scope of very small fish and very large fish. The anaerobic scope of an animal is defined as the difference between the aerobic energy production (by the red muscle in fish) and the energy demands during burst swimming (see Goolish, 1991). The anaerobic scope increases with increasing size . Very small fish have a small anaerobic scope, and therefore, recruitment of anaerobic fibres is not expected during sub-* maximal swimming,. As a result, the accumulation of lactate following exhaustive exercise in small fish is less (2-40g, 7-24 umol/g; reviewed in Goolish, 1991) than in intermediate and large fish (>150 g; 27-50 umol/g; Conner et al, 1964; Milligan and Wood, 1986/3; Parkhouse et al, 1987; Milligan and Girard, 1993). Therefore, because of the anaerobic scope of small fish is reduced compared to larger fish, the threshold of activation of glycolytic white muscle fibres is probably much higher than 80% U c r i t. Neither the relationship between intramuscular lactate and NMR derived P/ nor the initiation of anaerobic glycolysis within the white musculature determined in this study should be applied to fish below 150 g. In addition to differences in anaerobic scope and swimming performance, small fish pose a logistical problem for use with NMR technology. In the present NMR setup, the smallest fish that produces a clear signal is approximately 300 g. Below this size, the signal becomes very noisy. The cross-sectional area of very small fish would be smaller than the available coils, and therefore, any signal that could be obtained would not be from white muscle only, but would be whole body estimates. Temperature 71 Swimming performance is temperature dependent, thus the critical swimming speed changes with temperature. The optimum temperature to maximize swimming performance is the temperature at which the greatest aerobic scope exists—the largest difference between the standard and active metabolic rates. Optimum temperatures have been assessed in some species. For sockeye salmon, the optimum temperature is 15°C (Brett, 1964). The critical swimming speed is fastest at the optimum temperature, and decreases with increasing or decreasing temperature (Brett, 1964). A similar trend is seen with coho salmon, O. kisutch, with an optimum temperature close to 20°C (Griffiths and Alderdice, 1972; Glova and Mclnerney, 1977). This alteration in U c r i t with temperature also results in a change in the initial recruitment of white muscle fibres. The speed at which white muscle fibres are initially recruited changes with temperature; in rainbow trout, the fastest speed of recruitment, monitored over a limited temperature range (3, 11, and 18°C), was at 11 °C (Taylor et al., 1996). Striped bass, Morone saxatilis, also experience the effect of acclimation temperature on white muscle recruitment, with faster swimming speeds prior to the initial recruitment of white muscle fibres being obtained at warmer acclimation temperatures (Sisson and Sidell, 1987). Thus, both U c r i t and initial recruitment of white muscle fibres are affected by temperature, however, the relative proportion of U c r i t at which white muscle fibres are recruited at different temperatures is at present, unknown. Some general trends have emerged from studies investigating the effect of acute temperature changes on swimming speed and the initial recruitment of white muscle fibres. The order in which muscle fibres are recruited apparently remains the same regardless of temperature, however, with a reduction in temperature, the speed over which muscles fibres are recruited 72 becomes compressed (Rome et al, 1984). Recruitment of white muscle fibres occurs at a lower swimming speed when fish are forced to swim at a reduced temperature than the acclimation temperature (Rome et al., 1985). When acutely exposed to warmer temperatures, the maximum swimming speed that is attained before white muscle fibres are recruited is greater than at the lower temperature (Rome et al, 1984, 1985, 1990; Sisson and Sidell, 1987). Direct comparison between the speed attained at a given temperature reveals that fish acclimated to cold temperatures swim better at the cold temperature than fish acclimated to warm temperatures (Rome et al, 1985). If fish are forced to swim at an intermediate temperature, between the two acclimation temperatures, the cold-acclimated fish swims faster prior to recruiting white muscle fibres than the warm-acclimated fish (Sisson and Sidell, 1987). The increased ability to swim at cold temperatures following cold-acclimation, reflects modifications within the musculature. An increase in the number of red muscle fibres has been documented in goldfish (Johnston and Lucking, 1978), and an increase in the mitochondrial volume occupied within red muscle fibres increases with acclimation to cold temperatures (reviewed in Johnston and Dunn, 1987). Rainbow trout also experience modifications in red muscle fibre proportion and blood flow to the musculature due to acclimation to cold temperature. Acclimation to cold temperature (4°C) results in an increase in the proportion of red to white muscle from that observed at 18°C. Additionally, the blood flow to the white muscle is reduced at 4°C (Taylor et al, 1996). Other studies have not documented any changes due to temperature acclimation in rainbow trout within the myofibrillar proteins (Dean, 1969; Penney and Goldspink, 1981). Therefore, additional studies are needed to establish the changes due to temperature acclimation within rainbow trout musculature. 73 Although the effect of temperature on U c r i t and oxygen consumption in salmonids is well documented (Brett, 1964; Brett and Glass, 1973), the effect of temperature on initial white muscle recruitment is not. There appears to be a concurrent depression in the initial recruitment of white muscle fibres due to acclimation temperatures different from optimum (Taylor et al, 1996). It is not known if the relative proportion of U c r i t that corresponds to the initial recruitment of white muscle fibres changes in the same manner with temperature. Therefore, it is imperative that further studies investigating the effects of temperature on initial white muscle recruitment during sub-maximal swimming be performed. Because of this deficit in our knowledge, application of the results of this study on the initiation of white muscle fibre recruitment during sub-maximal swimming to different temperature regimes should be executed with caution. Additionally, the relationship between the intramuscular lactate concentration and the chemical shift of P/ was constructed with fish acclimated to a narrow range of temperatures (7.5-10.5°C), and although no effect of temperature was detected, further investigation as to the effect of a wider range of temperatures is required to confirm this. Estimate of anaerobic metabolic energy costs The aims of my studies were to estimate the extent of anaerobic metabolism during sub-maximal swimming, in relation to the critical swimming speed. This was achieved through monitoring the lactate concentration in rainbow trout white muscle following various swimming regimes. Intramuscular lactate was chosen as the metabolite of interest because the quantity of lactate produced during exercise can be related to the quantity of glycogen depleted (2 moles lactate = 1 mole glycogen; Arthur et al., 1990; Moyes et al, 1993). Additionally, the quantity of ATP produced during this reaction is known, as is the ATP required to metabolise the lactate 74 to glycogen during recovery (3 ATP per glycogen are produced during anaerobic glycolysis, 5 ATP are required to produce glycogen during recovery; Moyes et al, 1990; Moyes et al, 1993). Therefore, the quantity of ATP produced at each level of activity can be estimated from the monitored lactate concentration. From the calculated production of ATP, whole body energy estimates of anaerobic metabolism can be calculated. An oxygen equivalent for ATP can be used to compare energy production from anaerobic glycolysis to the oxygen consumption associated with the corresponding activity level. Oxygen consumption varies with swimming intensity, and relationships have been constructed between oxygen consumption and swimming speed or tailbeat frequency (Brett, 1964; reviewed in Beamish, 1978). Thus, the anaerobic metabolism that occurs at various levels of activity can be expressed in terms of the estimated oxygen consumption. From this oxygen consumption equivalent, the energy associated with anaerobic glycolysis can be presented as the percentage equivalent of the total oxygen consumption estimated at that level of activity, representing an energy 'tax'. Oxygen consumption estimates for different levels of activity are required, for rainbow trout of similar size and at a similar temperature (10°C), as used in the present experiments. By combining available data from the literature, a relationship between the oxygen consumption during swimming and the activity level (proportion of Ucrit) can be constructed (Figure 12). The data used in this relationship used fish ranging in size (250g-500g), and at various temperatures (10°C-15°C); in spite of the variations in temperature and weight, the regression is significant (PO.001, r^O.861). The data from Webb (1971) that was used in constructing this relationship correspond to the oxygen consumption of fish swimming up to and including approximately 80% 75 U c r i t . Data above 80% LJrit from Webb (1971) were not included in the present relationship because the oxygen consumptions of these fish deviated substantially from the other data. Therefore, application of the relationship presented in Figure 12, beyond 80% of U c r i t, should be done so judiciously. The mean lactate concentration from each section monitored, at each swimming speed, can be used to estimate the whole body lactate production during 30 minutes of exercise. White muscle makes up approximately 60% of the total mass (Randall and Daxboeck, 1982); this white muscle mass estimate can be subsequently used in the estimation of whole body lactate production. Mean whole body lactate can be calculated either by averaging the lactate concentration for each section, or by weighting each section by approximately how much muscle mass is be associated with that section. The distribution of white muscle mass should be reflected by the shape of the fish, and crude determinations (n=3) indicate that the relative distribution of muscle between the rostral, mid and caudal sections in rainbow trout is approximately 34, 45, and 21%>, respectively. These approximate proportions of muscle were used in calculating the weighted estimates of whole body lactate from the three sections monitored. The mean lactate concentration for each section was calculated and the whole body lactate was estimating using either the unweighted estimate of muscle mass or the weighted estimates. The conversion of whole body lactate to whole body ATP was calculated using two different sets of assumptions. The quantity of ATP produced (and subsequently consumed) during anaerobic glycolysis, 1.5 ATP per lactate, was used to convert whole body lactate to whole body ATP production. 76 Figure 12. The rate of oxygen consumption (M02) during swimming in rainbow trout (250-500g) at different proportions of U c r i t, and at a range of temperatures (10-15°C), from data obtained from the literature. The solid line represents the linear regression line of best fit, and the dashed lines represet the 95% confidence intervals for the regression line. 77 1 0 0 0 -1 V Weatherley etal., 1982; Rogers and Weatherley, 1983 Webb, 1971 Bushnell et al., 1984 Brauner et a l , 1994 log(M0 2) = 1.935+0.719*(Proportion Ucrit) r2=0.861 v 0 . 0 0 . 2 0 . 4 0 . 6 Proportion of Ucrit 0 . 8 1 . 0 78 This calculation assumes that gluconeogenesis does not occur during the swimming period, and does not account for the aerobically supplied ATP that is required for the resynthesis of intramuscular glycogen following exercise (2.5 ATP per lactate; Moyes, et al., 1992). The cost of gluconeogenesis as well as the anaerobic production of ATP following exercise is also presented. Although the cost of gluconeogensis is temporally seperated from the ATP production due to anaerobic glycolysis, this cost must be accounted for, especially for the application of energy estimates associated with anaerobic glycolysis to energy budgets. To compare the quantity of ATP that is produced during anaerobic glycolysis to the energy demand of aerobic swimming, an oxygen equivalent for ATP was used (6 ATP per 02. Moyes et al., 1993). This calculated oxygen equivalent can then be directly compared to oxygen consumption data at the same swimming speed. During 30 minutes of swimming at 70% U c r i t, a medium sized rainbow trout (see Table 1) expends, due to anaerobic glycolysis, on average the equivalent of 12.95 mg 02kg"1. If the cost of recovery is included in the estimate, the average equivalent is 34.54 mg 02kg"1. Using the relationship between oxygen consumption and U c r i t (Figure 12), the energy associated with the production of lactate within the white muscle during 70% U c r i t is equal to approximately 19.7%) of the estimated oxygen consumption due to aerobic metabolism during the 30 minute trial. The total cost, although not incurred during the 30 minutes of swimming, is equivalent to approximately 52.6% of the estimated oxygen consumption. If the weighted average is used, the equivalent is 14.3 mg 02kg"1, or 21.8%, due to anaerobic glycolysis, and the equivalent including the cost of recovery is 38.2 mg 02kg"' or 58.3%. The calculation of the percentage equivalent of the oxygen consumption due to anaerobic 79 glycolysis and gluconeogenesis during 30 minutes is appropriate because the oxygen consumption data used were obtained from studies that imposed steps of short duration (30-60 minutes), and it is unlikely that gluconeogenesis occurred during the swimming trial. Therefore, no cost associated with glyconeogenesis is included in the oxygen consumption measurements used to construct the relationship between oxygen uptake and swimming speed (Figure 12). Because gluconeogensis requires aerobically supplied ATP, the oxygen consumption associated with gluconeogenesis is elevated from the oxygen consumption associated only with aerobic swimming ("oxygen debt"). This cost must be accounted for in energetics models, therefore it is presented as a percentage equivalent of the oxygen consumption due to aerobic swimming. If gluconeogenesis is occurring during swimming, then these estimates of the energetic costs are gross underestimations. Swimming at 80% of U c r i t for a period of 30 minutes was preceded by swimming at approximately 10% of U c r i t for 30 minutes, in these experiments. If no gluconeogenesis is assumed to have occurred during the course of the swimming trials, the oxygen equivalents for anaerobic glycolysis and anaerobic production of ATP and the recovery metabolism can be calculated. If the lactate produced during swimming at 70% U c r i t is not metabolised during the swimming trial, and only the cost of anaerobic glycolysis is calculated, the effect of swimming at 80%o U c r i t results in an equivalent of 18.9 mg 02kg"' (18.53 mg 02kg"', weighted average), or 25.0%) (24.5%), weighted). If the cost of gluconeogenesis is included in these calculations, the resulting oxygen equivalent is 50.39 mg 02kg_1 (49.4 mg 02kg_1) or 66.6%> (65.3%, weighted). The expression of the oxygen equivalents associated with anaerobic metabolism and recovery as a percentage of the oxygen consumption at 80% U c r i t accounts for the ensuing oxygen debt. 80 Another possibility exists regarding the production and consumption of lactate during sub-maximal swimming. If the lactate produced during swimming at 70% of U c r i t was either partially or entirely resynthesised to glycogen during the trial, then only the estimate of the energy associated with anaerobic glycolysis and not gluconeogenesis is required. This is because if gluconeogenesis occurs during the swim trials, oxygen consumption measurements at 80% U c r i t are overestimations of the aerobic demand for swimming, because of the increase in oxygen consumption due to gluconeogenesis. Therefore, if the lactate produced during 70% U c r i t is metabolised during the exercise trial, the lactate produced due to swimming at 80% U c r i t results in the oxygen equivalent of 31.55 mg 02kg"1, or 41.7%) (30.9 mg Qkg 1 or 40.8%, weighted average). If the quantity of lactate produced at U c r i t is equivalent to that produced during an episode of burst swimming, the oxygen equivalent for this activity can be calculated. If no correction for ATP produced during swimming at 80% is applied, then the oxygen equivalent for the ATP produced during the burst is 76.9 mg 02kg"' (77.5 mg C^kg"1, weighted average). If only one burst activity occurs during a time period of 30 minutes, then the equivalent percentage of oxygen consumption due to the anaerobic activity is approximately 69.3% (69.8%, weighted average). When the cost associated with the resynthesis of glycogen are included in the oxygen equivalent, the cost of burst activity is 206.2 mg 02kg_1 (206.6 mg 02kg"1), or 184.8% (186.1%, weighted average) of the aerobic cost of swimming for 30 minutes. Burst activity occurs over a much smaller time scale, therefore the actual equivalent oxygen consumption percentage for the total duration of an episode of burst swimming would be much greater. This calculation was made using the relationship illustrated in Figure 12. Some anomalous oxygen consumption data 81 exists between 80% and 100% and was therefore excluded from the relationship developed in Figure 12. If the percentage of the total aerobic energy that is equivalent to the production of energy anaerobically is calculated using the anomalous oxygen consumption (from Webb, 1971), the equivalent is 65.5% (65.9%, weighted), or 43.0% (43.3%, weighted average) with the inclusion of recovery costs. This difference is quite substantial, although the fish used in for obtaining oxygen consumption were exercised at 15°C, and were not as large as the fish used in the present study. These two differences may account for part of the variation. However, additional information is clearly required to determine which estimate of anaerobic cost is more accurate. Recovery metabolism is thought to be inhibited following exhaustive exercise by the release of Cort isol (Pagnotta et al, 1994). When plasma Cortisol is prevented from being released (e.g., injection of a Cortisol blocker), the intramuscular pH/, lactate and glycogen recover within two hours (Pagnotta et al., 1994). Although the release of Cortisol during sub-maximal swimming is unknown, if Cortisol is released only during burst (or exhaustive) swimming, but not during sub-maximal swimming, the duration of recovery from anaerobic metabolism may be shorter. Therefore, if Cortisol is absent during swimming at sub-maximal speeds, the recovery dynamics during this activity may mimic the dynamics of cortisol-blocked fish; recovery of pH/, lactate and glycogen may occur within two hours. This would indicate that some recovery within the white muscle is occurring during swimming, and therefore, the estimates of the energy associated with anaerobic metabolism at 70% U c r i t are underestimates. 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