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The effect of temperature and ammonia exposure on swimming performance of brook charr (Salvelinus fontinalis)
Comparative Biochemistry and Physiology, Part A 156 (2010) 523–528
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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
The effect of temperature and ammonia exposure on swimming performance of brook charr (Salvelinus fontinalis) C. Tudorache ⁎, R.A. O'Keefe, T.J. Benfey Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada, E3B 5A3
a r t i c l e
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Article history: Received 17 February 2010 Received in revised form 14 April 2010 Accepted 14 April 2010 Available online 29 April 2010 Keywords: Raceway Critical swimming speed Migration Toxicology Physiology Behaviour
a b s t r a c t The effects of water temperature and ammonia concentration on swimming capacity of brook charr (Salvelinus fontinalis, Mitchill, 1814) were determined by measuring gait transition speed (Ugt, cm s− 1), maximum burst speed (Umax, cm s− 1), tail-beat amplitude (a, cm), tail-beat frequency (f, Hz), maximum acceleration of bursts (Amax, cm s− 2), number of bursts, distance of bursts (cm) and total swimming distance (cm) in a 4.5 m long experimental raceway with increasing upstream water velocity. Temperatures other than the acclimation temperature of 15 °C significantly reduced swimming characteristics of gait transition, i.e. Ugt and Amax, while increased ammonia concentration reduced the measures of swimming after Ugt: Umax, the relationship between f and swimming speed above Ugt, a, Amax and the distance travelled with each swimming burst above Ugt. This study, using a novel raceway set-up shows various effects of temperature and ammonia exposure on the swimming performance of brook charr and can be used to establish threshold values for environmental management. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Critical swimming speed (Ucrit; Brett, 1964) is often used to assess the impact of environmental factors such as temperature, hypoxia, diseases or contaminants on fish performance (Brett and Glass, 1973; Beamish, 1978; Waiwood and Beamish, 1978; Thomas and Rice, 1987; Nikl and Farrell, 1993; Hammer, 1995). This is because it is generally assumed that maximum sustainable oxygen uptake occurs at Ucrit (Webb, 1975; Farrell and Steffensen, 1987; Keen and Farrell, 1994; Gregory and Wood, 1999). In the laboratory, Ucrit is commonly determined using increasing water velocity tests to measure the ability of fish to respond in confining swim tunnels. However, Ucrit measured in confining swimming tunnels can be influenced behaviourally (McFarlane and McDonald, 2002; Peake and Farrell, 2006, Tudorache et al, 2007). Fish may refuse to continue swimming when forced to maintain position against a water speed too high for steady and too low for unsteady locomotory gait (Peake and Farrell, 2006, Tudorache et al, 2007). Also, burst-and-glide swimming, which involves the generation of a positive ground speed (Muller et al., 2000; Peake and Farrell, 2004), cannot be maintained efficiently within a confining swim tunnel (Tudorache et al, 2007). An alternative measurement of swimming energetics is the gait transition speed (Ugt), at which the transition from steady cruising to burst-and-glide swimming mode occurs (Videler, 1993). When
⁎ Corresponding author. Tel.: + 1 31 6 20362185. E-mail address: [email protected] (C. Tudorache). 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.04.010
swimming in cruising mode, the predominant muscle groups involved are red aerobically driven muscles, while at switching into burst-andglide swimming mode white anaerobically powered muscles are engaged (Videler, 1993). Therefore, gait transition as an indicator for swimming performance bears both ecological and physiological importance (Peake, 2008, Tudorache et al., 2007). Gait transition is characterised by the first burst, typically as (1) a large and discrete increase in upstream motion, (2) increased tail-beat amplitude and (3) increased tail-beat frequency (Tudorache et al., 2007). Using a novel raceway that allows the fish to swim freely against increasing water speeds (see: Haro et al., 2004; Castro-Santos, 2004, 2005; Peake and Farrell, 2006; Peake, 2008), Ugt can be a more reliable measurement of maximum aerobic swimming speed than Ucrit (Peake, 2008). White muscles used to power burst-and-glide swimming are very susceptible to ammonia (NH3) toxicity, and ammonia exposure decreases swimming performance in both steady swimming (Beaumont et al., 1995; Shingles et al., 2001) and unsteady swimming (Tudorache et al, 2008; McKenzie et al, 2009). Especially salmonids are known to be susceptible to even low ammonia concentrations in freshwater (Shingles et al., 2001). The potential for toxicity is determined by dissolved ammonia concentration since it diffuses into the fish across their gills (Shingles et al., 2001). As water ammonia level rises, plasma ammonia levels increase in the fish due to a decreased excretion by means of Rhesus (Rh) proteins (Weihrauch et al., 2009; Wright and Wood, 2009). Increased NH3 levels within the fish alter the metabolic status, which may lead to premature muscle fatigue due to partial depolarisation of the
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membrane potential in the white muscles (Beaumont et al. 1995, 2000b) and, hence, a reduction in swimming performance (Beaumont et al., 2000a). High ammonia concentrations might, therefore, alter gait transition speed by impairing anaerobic capacity. Another factor strongly influencing gait transition is water temperature. Maximum cardiorespiratory performance is temperature dependent (Brett 1964, 1971; Randall and Brauner 1991; Taylor et al. 1996). The temperature optimum of MO2 in salmonids is close to their upper temperature limit (Farrell, 2007), as salmonids are unable to compensate for naturally decreasing water oxygen content with increasing temperatures through increased gill irrigation (Brett, 1971). And even though gill oxygen diffusing capacity increases with exercise, gas exchange is perfusion limited on the blood side of the gills during exercise (Daxboeck et al. 1982; Randall and Daxboeck 1982). With increasing water temperature in resting rainbow trout (Oncorhynchus mykiss), arterial blood saturation decreases (Heath and Hughes 1973), a finding that could reflect a perfusion limitation (Farrell, 2007). Also, with increasing temperatures, acidosis, hypoxia and hyperkalemia of the venous blood worsen with exercise (Kiceniuk and Jones 1977; Holk and Lykkeboe 1998), as shown in rainbow trout (Jain and Farrell 2003), leading to a decrease of maximum cardiac pumping because of the inhibition of contractility (Driedzic and Gesser, 1994). It was hypothesized that ammonia exposure and temperature will have an effect on swimming physiology and behaviour in brook charr (Salvelinus fontinalis), by altering gait transition speed, maximum burst speed, tail-beat amplitude, tail-beat frequency, maximum acceleration of bursts, number of bursts, distance of bursts (cm) and total swimming distance in a 4.5 m long experimental raceway with increasing upstream water velocity. An integrative approach was chosen in order to find threshold values which in combination with other data can be used for management of freshwater bodies.
2. Material and methods Brook charr (12.22±0.12 cm, fork length, LF, and 23.49±0.67 g body mass±S.D.) were obtained from the Miramichi Salmon Conservation Centre and held in dechlorinated municipal water (pH=8.50±0.09, alkalinity = 80.95 ± 0.49 mg L− 1, [Ca] = 35.6 ± 0.35 mg L− 1, [Mg] = 3.92 ± 0.08 mg L− 1, [Na]= 13.13 ± 1.17 mg L− 1, hardness = 103.00 ± 1.00 mg L− 1), in the aquaculture facilities at the University of New Brunswick (Fredericton campus) for at least 3 weeks prior to exposure. Water temperature was kept at 15.5 °C (range ±0.8 °C) and water speed in the tanks was low (0–0.5 cm s− 1). Fish were held in 140 L tanks in groups of 12 individuals. A flow-through configuration was used with a turnover rate of 100 L d− 1. Fish were fed a salmonid grower diet (Corey Feed Mills Ltd., Fredericton, NB, Canada) ad libitum.
2.1. Ammonia and temperature pre-exposure For the ammonia experiment, fish were exposed to ammonia (total ammonia, i.e. NH3 and NH+ 4 ) at 15 °C and a pH of 9.10 ± 0.12 for a total of 96 h. This coincides with the maximum exposure period for brown trout in Tudorache et al. (2008) and has proven to affect fast starts significantly. The concentration of NH3 in the water was 25.62 µmol L− 1. The total volume of 140 L of water was spiked with the required amount of NH4Cl (Sigma-Aldrich Chemical Co., Toronto, Canada) to reach concentrations of 14.38 (15.08 ± 0.06), 28.76 (29.36 ± 4.69), 43.14 (42.86 ± 5.87) and 57.53 (58.13 ± 4.11) µmol L− 1 total ammonia (NH3 and NH+ 4 ), which were maintained via in-flow addition from a stock solution. For the temperature experiment, fish were kept at 15 °C as described above and transferred to the experimental temperatures in the raceway without acclimation.
2.2. Experimental set-up The experimental set-up consisted of a plexiglas raceway with gradually increasing water velocity due to a slight inclination (see Peake, 2008 for example). The raceway was 450 cm long, 12.5 cm wide and 50 cm deep. Each end was fitted with a trapezoid holding tank, 75 cm long, 50 cm wide and 50 cm deep. Water was pumped from the downstream tank to the upstream tank by means of a submersible pump (Alita Model PV-800; Aracadia, CA, U.S.A.). The total volume of water was 250 L. For the ammonia experiment, the water was spiked with the necessary amount of NH4Cl to reach concentrations of 14.38 (13.89 ± 0.16), 28.76 (28.16 ± 2.37), 43.14 (45.16 ± 6.17) and 57.53 (57.78 ± 6.36) µmol L− 1 total ammonia (NH3 and NH+ 4 ). The water had a pH of 9.11 ± 0.13. The raceway was inclined by 3.5°, resulting in an upstream water depth of 5 cm and a water velocity of 110 cm s− 1 and a downstream water depth of 40 cm and a velocity of 10 cm s− 1. The water velocity was measured every 10 cm and showed a linear decline towards the downstream end of the raceway. The control water temperature of 15 °C and the experimental water temperatures of 10 and 20 °C were maintained with an in-line chiller (Aqua Logic Model DS-7; San Diego, CA, U.S.A.) and dissolved oxygen concentration did not go below 7.5 mg L− 1. Eight cameras (SNG SED-CAM-YC26S, Sharpe Electronics Corp., Osaka, Japan), connected to a digital video recorder (EDVR 16 D3, Everfocus, Taipei, Taiwan), were mounted below the raceway to record images of a 70 cm section with overlapping fields. The original footage was recorded in mp4 format and transformed into avi format using ImageViewer (Everfocus, Taipei, Taiwan). A grid on the bottom over the entire length of the raceway facilitated orientation and scaling. Because the camera lenses were wide-angled, the resulting hemispherically convex distorted images were adjusted with a digital concave distortion using Adobe AfterEffects CS3 Professional version 8.0.2.27 (Adobe Systems Inc., San Jose, CA, USA) without any loss of image quality. The position of the centre of mass determined the position of the fish along the raceway, as described by Tudorache et al. (2008). Fish were introduced in the downstream holding tank of the raceway 1 h before the experiment started. Access to the raceway was blocked with a grid that was removed when the experiment started. The cameras were set on and footage recorded over the following 2 h. At the end of the experiment, fish were gently removed from the raceway and prepared for final measurement of LF, weight and blood and muscle ammonia concentrations. Fish from the experimental group exposed to ammonia were killed with a sharp blow on the head. A sample of white muscle tissue was removed from the left dorsocaudal section of the trunk, snap frozen in liquid nitrogen, packed in aluminium foil and stored at −80 °C for the determination of the ammonia concentration in the muscle. The total time of killing and sampling took only b1 min per fish in order to avoid additional ammonia build up in the muscles due to excessive thrashing (Tudorache et al. 2010). The use of animals was approved by the UNB Animal Care Committee, following guidelines established by the Canadian Council on Animal Care. The number of fish per experiment was ten. 2.3. Analysis Total ammonia concentrations in water were determined using the salicylate–hypochlorite method according to Verdouw et al. (1978). Total muscle ammonia was extracted according to the method described by Wright et al. (1995). Total muscle and plasma ammonia was measured using an enzymatic kit (Sigma 171 C, Sigma-Aldrich Chemical Co.). The XY coordinates of the centre of mass were digitized for three randomly chosen forward swimming movement, one from the beginning, one from the middle and one from the end of video sequences (avi files), and exported into Vernier logger pro 3.4.6 (Vernier
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Fig. 1. Representative plot of total swimming speed against water speed for a control fish at 15 °C. The fish starts with increasing swimming velocity in cruise mode and then changes at the gait transition speed (Ugt) into burst-and-glide mode. Burst-and-glide is characterised by rapid accelerations (burst) followed by decelerations until near zero ground speed (i.e., swimming speed equals water speed). The solid line indicates Ugt and the dotted line indicates zero ground speed, i.e. where the swimming speed is equal to the water speed.
Software & Technology, TX, USA) to perform a manual tracking of each fish's movements. Locomotor variables were calculated using the digitized coordinates, and the following parameters were assessed: 1) gait transition speed (Ugt, cm s− 1): maximum swimming speed observed that was supported entirely using a steady undulatory locomotory gait. The first three burst sequences thereafter initiated burst-and-glide swimming mode. The start of a burst was defined in terms of: (a) a large and discrete increase in upstream motion, (b) increased tail span and (c) increased tailbeat frequency (Tudorache et al., 2007). 2) maximum swimming speed (Umax, cm s− 1): the highest swimming speed in cm s− 1 possible at burst swimming mode before the fish fell back. 3) tail-beat frequency (f, Hz): the number of full tail beats per second. 4) tail-beat amplitude (a, cm): the distance between the maximum deflections of the caudal tail edge to both sides. 5) maximum acceleration after gait transition (Amax, cm s− 2): the maximum acceleration at burst swimming mode. 6) number of bursts: the number of fast forward accelerations after Ugt before Umax was reached. 7) distance of bursts (cm): the distance covered while bursting forward. 8) total swimming distance (cm): the distance covered by a continuous forward movement including aerobic as well as anaerobic swimming modes.
Fig. 2. Gait transition speed (Ugt, black) and maximum swimming speed (Umax, white) at 10, 15 and 20 °C. Letters indicate significant differences (N = 10, p b 0.05).
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Fig. 3. Gait transition speed (Ugt, black) and maximum swimming speed (Umax, white) at different total ammonia (NH+ 4 and NH3) concentrations. Letters indicate significant differences (N = 10, p b 0.05).
2.4. Statistics All assumptions for parametric tests were met. All parameters were compared using a multiple ANOVA with a Bonferroni post-hoc test. The significance level for all tests was set at p b 0.05. Linear regression analysis was used to evaluate the relationship between swimming speed and tail-beat frequency or amplitude.
3. Results Upon entering the raceway, fish swam in steady cruise mode, supported by slow and rhythmic undulations of the body and caudal fin, increasing their swimming speed steadily until gait transition. After gait transition, they accelerated rapidly and swam in burst-andglide mode. Bursts were characterised by a large and discrete increase in upstream motion and increased tail-beat amplitude and frequency, followed by a passive glide. The next burst was initiated when ground speed reached nearly zero. The swimming speed in burst-and-glide mode increased rapidly, reaching Umax at ca. 80% of the entire forward swimming period. At the end of the forward movement, the fish reached a standstill before falling back in the raceway (Fig. 1). Ugt for the control group at 15 °C in water with no added ammonia was 37.43 ± 1.47 cm s− 1, and was reduced at both 10 °C and 20 °C (p b 0.05, Fig. 2). However, Umax at 10 and 20 °C did not differ significantly from control (p N 0.05, Fig. 2).
Fig. 4. Representative plot of tail-beat frequency (f) against swimming speed (U) for a control fish at 15 °C. The vertical line indicates gait transition speed and the resulting linear relationship can be described by the linear function f = 1.5557+ 0.1033U (r2 = 0.92) before and f = 1.5793+ 0.102U (r2 = 0.85) after gait transition (N = 3, p b 0.05).
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Table 1a Regression values for tail-beat frequency (f, Hz) plotted against swimming speed (U, cm s− 1) with the formula f=a+bU at different water temperatures. Temperature (°C)
10
15 (control)
20
a (before Ugt) a (after Ugt) b (before Ugt) b (after Ugt)
1.56 ± 0.13 1.58 ± 0.18 0.104 ± 0.042 0.105 ± 0.039
1.57 ± 0.14 1.56 ± 0.11 0.104 ± 0.031 0.106 ± 0.024
1.58 ± 0.15 1.58 ± 0.13 0.102 ± 0.020 0.107 ± 0.024
Ammonia concentration did not affect Ugt. Umax showed a gradual decline with increasing ammonia concentration, reaching about 75% of control values at 57.53 µmol L− 1 (Fig. 3). Tail-beat frequency (f) for the control group at 15 °C in water with no added ammonia was positively correlated with swimming speed (U) before (r2 = 0.90, p b 0.05) and after (r2 = 0.88, p b 0.05) gait transition (Fig. 4) and could be described by the regression equation
Fig. 5. Tail-beat amplitude (a) at different total ammonia (NH+ 4 and NH3) concentrations before (black) and after (white) gait transition. Letters indicate significant differences (N = 10, p b 0.05).
f = a + bU where a is the intercept of the regression and b is the slope. Slope and intercept were not affected by temperature (Table 1a) or ammonia concentrations below 43.14 µmol L− 1 (p N 0.05, Table 1b), but the intercept was significantly decreased and slope significantly increased at ammonia concentrations of 43.14 and 57.53 µmol L− 1 at speeds higher than Ugt (Table 1b). For the control group at 15 °C in water with no added ammonia, tailbeat amplitude (a) did not vary with swimming speed, but it did vary with swimming mode, increasing from 2.04 ± 0.34 cm when cruising to 3.20 ± 0.41 with burst-and-glide swimming (p b 0.05). It was not affected by temperature but declined with increasing ammonia concentration at speeds higher than Ugt, with values above 0.5 mg L− 1 significantly decreased. Values of a below Ugt were not significantly different from control (Fig. 5). Maximum acceleration of bursts after gait transition (Amax) was significantly reduced by 20% at 10 °C compared to the control at 15 °C, but at 20 °C Amax did not differ from control (Fig. 6a). Increased ammonia concentrations also affected Amax, which was significantly reduced from control values at 43.14 and 57.53 µmol L− 1 by about 15 and 20%, respectively (Fig. 6b). With temperature as a factor, the number of forward bursts until the end of the raceway was reached did not differ from the control (3.00 ± 0.38). However, the number of bursts was significantly increased (p b 0.05) with ammonia concentrations of 43.14 and 57.53 µmol L− 1 (3.37 ± 0.33 and 3.93 ± 0.49, respectively). Distance of burst was significantly reduced (p b 0.05) from control (89.34 ± 13.24 cm) with ammonia concentrations of 43.14 and 57.53 µmol L− 1 (76.13 ± 7.19 and 65.40 ± 7.66 cm, respectively). The total swimming distance was not significantly different between the treatments or the control (371.50 ± 11.91 cm). Also, the total distance covered in burst-and-glide mode was not significantly different from the control (274.62± 32.93 cm). 3.1. Plasma and tissue ammonia Ammonia levels in exposed fish were significantly elevated in the plasma at exposure levels of 0.5 mg L− 1 and higher and in white muscle at levels of 44.04 µmol L− 1 and higher (Table 2).
4. Discussion The results of this study show that linear swimming is affected by both temperature and ammonia concentration. Gait transition speed (Ugt), the speed at which the transition from cruise to burst-and-glide swimming occurs, was lower at 10 and at 20 °C than at 15 °C. Brook charr show a maximum aerobic capacity at around 15 °C (Beamish, 1978). However, Ugt was not affected by increased ammonia concentrations in the water, a finding in accordance with the prediction that ammonia has little effect on red aerobic muscles. The maximum burst speed (Umax), on the other hand, was not affected by temperature, a result that is in accordance with Blaxter and Dickson (1959), Brett (1964), Beamish (1978) and Videler (1993). Webb (1978) reported an increase in Umax of escape fast starts; however, a positive correlation of Umax with temperature was not detected in the current study, possibly due to the low frame rate of the filming footages used. However, Umax, the relationship between tailbeat frequency and swimming speed above Ugt, tail-beat amplitude, maximum acceleration after gait transition and the distance travelled with each swimming burst above Ugt were all significantly reduced at the higher ammonia concentrations, suggesting that ammonia reduces white muscle performance (Tudorache et al., 2008, McKenzie et al., 2009). The observed significant decrease of the intercept and the increase of the slope of the tail-beat frequency at speeds higher than Ugt with elevated ammonia concentrations also suggests an increased cost of transport after Ugt. Shingles et al. (2001) showed that the critical swimming speed of rainbow trout is reduced at elevated ammonia concentrations. Critical swimming speed swimming is the speed at which maximum sustainable oxygen uptake occurs (Webb, 1975; Farrell and Steffensen, 1987; Keen and Farrell, 1994; Gregory and Wood, 1999) and measures aerobically as well as anaerobically powered swimming performance (Brett, 1964; Beamish, 1978; Videler, 1993). The present study shows that swimming performance before Ugt is less affected by ammonia than anaerobically powered swimming performance, up to a concentration of 58.72 µmol L− 1. This suggests that elevated ammonia concentrations may lead to a decreased Ucrit by reducing swimming performance after
Table 1b Regression values for tail-beat frequency (f, Hz) plotted against swimming speed (U, cm s− 1) with the formula f = a + bU under the influence of elevated ammonia concentrations in the water. *Indicates significant difference from control (one-way ANOVA, p b 0.05, N = 10). Concentration (µmol L− 1)
0 (control)
14.38
28.77
43.15
57.53
a (before Ugt) a (after Ugt) b (before Ugt) b (after Ugt)
1.57 ± 0.14 1.56 ± 0.11 0.104 ± 0.031 0.106 ± 0.024
1.61 ± 0.15 1.57 ± 0.09 0.111 ± 0.025 0.106 ± 0.032
1.58 ± 0.13 1.58 ± 0.12 0.112 ± 0.020 0.113 ± 0.024
1.54 ± 0.17 0.48 ± 0.13* 0.113 ± 0.012 0.125 ± 0.014*
1.53 ± 0.12 0.24 ± 0.16* 0.118 ± 0.013 0.130 ± 0.016*
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Fig. 6. Maximum acceleration (Amax) at different temperatures (a) and total ammonia (NH+ 4 and NH3) concentrations (b). Letters indicate significant differences (N = 10, p b 0.05).
Ugt. In order to test this, future studies of the critical swimming speed should include the measurement of gait transition speed. The results at different water temperatures also show that Ugt is reduced, possibly due to a reduction in aerobic swimming capacity, while swimming capacity after gait transition is not affected. Therefore, a measured reduction in Ucrit (e.g.: Beamish, 1978; Videler, 1993) may be due to a reduction of aerobic swimming capacity, as the protocol for Ucrit tests includes swimming aerobic as well as anaerobic swimming capacities. Both long-distance and short-distance migratory fish species use waterways for migration (Lukas and Baras, 2001). Physical barriers can obstruct free migration and can cause fragmentation and finally extinction of fish populations (Warren and Pardew 1998; Toepfer et al. 1999; Warren et al. 2000). Fish ladders and culverts designed to facilitate migration are often introduced to migratory paths. In order to be able to pass such devices, the water speed must be adjusted to the swimming potential of the migrating fish species. In the past, the standard water velocity was equal to, or lower than, the critical swimming speed obtained in forced swimming tests, as the critical swimming speed is assumed to be the swimming speed at which the maximum sustainable oxygen uptake occurs (Webb, 1971; Farrell and Steffensen, 1987; Keen and Farrell, 1994; Gregory and Wood, 1999). However, some authors have criticized this method (e.g. Plaut, 2001, Castro-Santos, 2004, 2005; Peake and Farrell,
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2006), proposing the use of Ugt instead (Peake and Farrell, 2006; Peake, 2008), as this represents the highest speed that is obtained by using only red aerobically powered muscles (Videler, 1993; Peake, 2008). The tilted raceway used in this study enables not only the measurement of Ugt, but also other parameters, such as maximum swimming speed, acceleration at bursts and kinematic parameters such as tail-beat frequency and amplitude under different environmental conditions. The factors assessed in this study, temperature and ammonia concentration, are not only important environmental factors but also represent factors influencing aerobically and anaerobically powered swimming performance. The results show that to give a good estimate of swimming capacity and the ability to negotiate obstacles on the migration path, Ugt is not sufficient, as it concerns only aerobically powered swimming performance. The present study shows that in estimating total swimming capacity (both aerobic and anaerobic), critical swimming speed gives a better approximation of the effect of factors which influence anaerobic swimming speed as well (Beaumont et al., 1995; Shingles et al., 2001). Therefore, the use of critical swimming speed as an indicator for swimming capacity is still crucial, especially when measuring effects on both aerobic as well as anaerobic swimming performance. The results of swimming capacity and performance is not only determined physiologically but also behaviourally. Gait transition is employed when the swimming speeds become too fast to be maintained by red aerobic muscles alone (Peake and Farrell, 2006; Tudorache et al. 2007). However, as the switch from cruising to burstand-glide swimming occurs, Ugt has a strong volitional component and the bioenergetics of swimming cannot be fully understood if disconnected from its behavioural component (Farrell, 2007). It has therefore been proposed that Ugt measured in confined swimming tunnels is not comparable with Ugt measured in volitional raceways set-ups (Farrell, 2007; Peake, 2008). Also, absolute values of Ugt and Ucrit measured in confined swimming tunnels cannot be extrapolated to free swimming fish and therefore cannot be used as measures of swimming capacity in the wild. The present methodology gives more ground for extrapolation in freely swimming fish in the wild as they are not confined by the size of regular Brett- or Blazka-type swimming tunnels (Peake, 2008). However, if comparing physiological, ecological or genetic factors in fish, traditional swimming tunnels are sufficient. Also, confined swimming tunnels are superior to large raceways as they allow the measurement of dissolved oxygen and other metabolic gasses and more invasive methods, such as cannulation. Such methodologies are impossible or very difficult to be realised in a 4.5 m long raceway. The ammonia concentration used in this study, i.e. maximum 57.53 µmol L− 1 of total ammonia is the equivalent of 1 mg L− 1 and is thus the same as used by Tudorache et al. (2008), resulting in a reduction of fast start performance in brown trout. However, 57.53 µmol L− 1 of total ammonia results in a concentration of NH3 of 15.57 µmol L− 1 at a pH of 9.1. A slightly higher concentration used by Shingles et al. (2001; 20 µmol L− 1) proved to be sufficient to reduce critical swimming speed in rainbow trout significantly. This value coincides with 50% LC50 of rainbow trout of 0.5 g body weight (Shingles et al. 2001). According to Ye and Randall (1991) a pH of 9 does not affect swimming capacity in salmonids and brook charr is especially tolerant for high pH values (Daye and Garside, 1975). The results of this study suggest that linear swimming characteristics in brook charr swimming in a raceway with increasing upstream
Table 2 Plasma and white muscle ammonia levels in brook charr after exposure to 0, 14.68, 29.36, 44.04 and 58.72 (µmol L− 1 total ammonia for 96 h. Different letters indicate significant difference (p b 0.05, N = 10). Ammonia concentration (µmol L− 1)
0 (control)
14.38
28.77
43.15
57.53
Plasma ammonia (µmol L− 1) White muscle ammonia (µmol g− 1)
175.53 ± 58.64a 1.42 ± 0.28a
233.92 ± 47.46a 1.49 ± 0.31a
329.48 ± 67.33b 1.88 ± 0.42ab
443.84 ± 52.00c 2.05 ± 0.52b
503.63 ± 59.32c 2.52 ± 0.42b
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water velocity are altered by temperature and ammonia exposure. The present methodology in combination with studies on population and ecological level can be used for the establishment of threshold values for environmental management. Acknowledgements Thanks are extended to S.J. Peake for his contributions to this research and to J. Peake for technical support. This research was partially supported by an NSERC Discovery Grant awarded to T.J. Benfey. C. Tudorache was supported by a grant of Manitoba Hydro awarded to S.J. Peake (Grant number: G215). References Beamish, F.W.H., 1978. Swimming capacity. Fish Physiol. 7, 101–187. Beaumont, M.W., Butler, P.J., Taylor, E.W., 1995. Plasma ammonia concentration in brown trout in soft acidic water and its relationship to decreased swimming performance. J. Exp. Biol. 198 (10), 2213–2220. Beaumont, M.W., Butler, P.J., Taylor, E.W., 2000a. Exposure of brown trout, Salmo trutta, to a sub-lethal concentration of copper in soft acidic water: effects upon muscle metabolism and membrane potential. Aquat. Toxicol. 51, 259–272. Beaumont, M.W., Taylor, E.W., Butler, P.J., 2000b. The resting membrane potential of white muscle from brown trout (Salmo trutta) exposed to copper in soft, acidic water. J. Exp. Biol. 203, 2229–2236. Blaxter, J.H.S., Dickson, 1959. Observations on the swimming speeds of fish. J. Mar. Sci. 24, 472–479. Brett, J.R., 1964. The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Board Can. 21, 1183–1226. Brett, J.R., 1971. Energetic responses of salmon to temperature. A study of some thermal relations in the physiology and freshwater ecology of sockeye salmon. Am. Zool. 11, 93–113. Brett, J.R., Glass, N.R., 1973. Metabolic rates and critical swimming speeds of sockeye salmon (Onchorhynchus nerka) in relation to size and temperature. J. Fish. Res. Board Can. 30, 379–387. Castro-Santos, T., 2004. Quantifying the combined effects of attempt rate and swimming performance on passage through velocity barriers. Can. J. Fish. Aquat. Sci. 61, 1602–1615. Castro-Santos, T., 2005. Optimal swim speeds for traversing velocity barriers: an analysis of volitional high-speed swimming behavior of migratory fishes. J. Exp. Biol. 208, 421–432. Daxboeck, C., Davie, P.S., Perry, S.F., Randall, D.J., 1982. Oxygen uptake in spontaneously ventilating blood-perfused trout preparation. J. Exp. Biol. 101, 33–45. Daye, P.G., Garside, E.T., 1975. Lethal levels of pH for brook trout, Salvelinus fontinalis. Can. J. Zool. 53 (5), 639–641. Driedzic, W.R., Gesser, H., 1994. Energy-metabolism and contractility in ectothermic vertebrate hearts — hypoxia, acidosis, and low-temperature. Physiol. Revs. 74 (1), 221–258. Farrell, A.P., 2007. Cardiorespiratory performance during prolonged swimming tests with salmonids: a perspective on temperature effects and potential analytical pitfalls. Phil. Trans. Royal Soc. B. 362, 2017–2030. Farrell, A.P., Steffensen, J.F., 1987. An analysis of the energetic cost of the branchial and cardiac pumps during sustained swimming. Fish Physiol. Biochem. 4, 73–79. Gregory, T.R., Wood, C.M., 1999. Interactions between individual feeding, growth, and swimming performance in juvenile rainbow trout (Oncorhynchus mykiss) fed different rations. Can. J. Fish. Aquat. Sci. 56, 479–486. Hammer, C., 1995. Fatigue and exercise tests with fish. Comp. Biochem. Physiol. 112, 1–20. Haro, A., Castro-Santos, T., Noreika, J., Odeh, M., 2004. Swimming performance of upstream migrant fishes in open-channel flow: a new approach to predicting passage through velocity barriers. Can. J. Fish. Aquat. Sci. 61, 1590–1601. Heath, A.G., Hughes, G.M., 1973. Cardiovascular and respiratory changes during heat stress in rainbow trout (Salmo gairdneri). J. Exp. Biol. 59 (2), 323–338. Holk, K., Lykkeboe, G., 1998. The impact of endurance training on arterial plasma K+ levels and swimming performance of rainbow trout. J. Exp. Biol. 201 (9), 1373–1380. Jain, K.E., Farrell, A.P., 2003. Influence of seasonal temperature on the repeat swimming performance of rainbow trout Oncorhynchus mykiss. J. Exp. Biol. 206, 3569–3579. Keen, J.E., Farrell, A.P., 1994. Maximum prolonged swimming speed and maximum cardiac performance of rainbow trout, Oncorhynchus mykiss, acclimated to two different water temperatures. Comp. Biochem. Physiol. 108, 287–295.
Kiceniuk, J.W., Jones, D.R., 1977. Oxygen-transport system in trout (Salmo gairdneri) during sustained exercise. J. Exp. Biol. 69, 247–260. Lukas, M.C., Baras, E., 2001. Migration of Freshwater Fishes, 1st ed. Blackwell Science, Oxford, UK. McFarlane, W.J., McDonald, D., 2002. Relating intramuscular fuel use to endurance in juvenile rainbow trout. Physiol. Biochem. Zool. 75, 250–259. McKenzie, D.J., Shingles, A., Claireaux, G., Domenici, P., 2009. Sublethal concentrations of ammonia impair performance of the teleost fast-start escape response. Physiol. Biochem. Zool. 82 (4), 353–362. Muller, U.K., Stamhuis, E.J., Videler, J.J., 2000. Hydrodynamics of unsteady fish swimming and the effects of body size: comparing the flow fields of fish larvae and adults. J. Exp. Biol. 203, 193–206. Nikl, D.L., Farrell, A.P., 1993. Reduced swimming performance and gill structural changes in juvenile salmonids exposed to 2-(thiocyanomethylthio)benzothiazole. Aquat. Toxicol. 27, 245–263. Peake, S.J., 2008. Gait transition speed as an alternate measure of maximum aerobic capacity in fishes. J. Fish Biol. 72, 645–655. Peake, S.J., Farrell, A.P., 2004. Locomotory behaviour and post-exercise physiology in relation to swimming speed, gait transition and metabolism in free-swimming smallmouth bass (Micropterus dolomieu). J. Exp. Biol. 207, 1563–1575. Peake, S.J., Farrell, A.P., 2006. Fatigue is a behavioural response in respirometerconfined smallmouth bass. J. Fish Biol. 68, 1742–1755. Plaut, I., 2001. Critical swimming speed: its ecological relevance. Comp. Biochem. Physiol. 131 (1), 41–50. Randall, D.J., Brauner, C., 1991. Effects of environmental factors on exercise in fish. J. Exp. Biol. 160, 113–126. Randall, D.J., Daxboeck, C., 1982. Cardiovascular changes in the rainbow trout (Salmo gairdneri Richardson) during exercise. Can. J. Zool. 60 (5), 1135–1140. Shingles, A., McKenzie, D.J., Taylor, E.W., Moretti, A., Butler, P.J., Ceradini, S., 2001. Effects of sublethal ammonia exposure on swimming performance in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 204 (15), 2691–2698. Taylor, S.E., Egginton, S., Taylor, E.W., 1996. Seasonal temperature acclimatisation of rainbow trout: cardiovascular and morphometric influences on maximum sustainable exercise level. J. Exp. Biol. 199, 835–845. Toepfer, C.S., Fisher, W.L., Haubelt, J.A., 1999. Swimming performance of the threatened leopard darter in relation to road culverts. Trans. Am. Fish. Soc. 128 (1), 155–161. Thomas, R.E., Rice, S.D., 1987. Effect of water-soluble fraction of Cook Inlet crude- oil on swimming performance and plasma-cortisol in juvenile coho salmon (Oncorhynchus kisutch). Comp. Biochem. Physiol. C 87, 177–180. Tudorache, C., Viaenen, P., Blust, R., De Boeck, G., 2007. Longer flumes increase critical swimming speeds by increasing burst–glide swimming duration in carp Cyprinus carpio, L. J. Fish Biol. 71, 1630–1638. Tudorache, C., Blust, R., De Boeck, G., 2008. Social interactions, predation behaviour and fast start performance are affected by ammonia exposure in brown trout (Salmo trutta L.). Aquat. Toxicol. 90, 145–153. Tudorache, C., O'Keefe, R.A., Benfey, T.J., 2010. Flume length and post-exercise impingement affect anaerobic metabolism in brook charr Salvelinus fontinalis. J. Fish Biol. 76, 729–733. Verdouw, H., Vanechteld, C.J.A., Dekkers, E.M.J., 1978. Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12, 399–402. Videler, J., 1993. Fish Swimming, 1st ed. Chapman & Hall, London, UK. Waiwood, K.G., Beamish, F.W.H., 1978. Effects of copper, pH and hardness on the critical swimming speed of rainbow trout (Salmo gairdneri, Richardson). Water Res. 12, 611–619. Warren, M.L., Pardew, M.G., 1998. Road crossings as barriers to small-stream fish movement. Trans. Am. Fish. Soc. 127 (4), 637–644. Warren, M.L., Burr, B.M., Walsh, S.J., Bart, H.L., Cashner, R.C., Etnier, D.A., Freeman, B.J., Kuhajda, B.R., Mayden, R.L., Robison, H.W., Ross, S.T., Starnes, W.C., 2000. Diversity, distribution, and conservation status of the native freshwater fishes of the southern United States. Fisheries 25 (10), 7–31. Webb, P.W., 1971. The swimming energetics of trout I. Thrust and power output at cruising speeds. J. Exp. Biol. 55, 489–520. Webb, P.W., 1975. Acceleration performance of rainbow trout Salmo gairdneri and green sunfish Lepomis cyanellus. J. Exp. Biol. 63, 451–465. Webb, P.W., 1978. Fast-start performance and body form in 7 species of teleost fish. J. Exp. Biol. 74, 211–226. Weihrauch, D., Wilkie, M.P., Walsh, P.J., 2009. Ammonia and urea transporters in gills of fish and aquatic crustaceans. J. Exp. Biol. 212, 1716–1730. Wright, P.A., Wood, C.M., 2009. A new paradigm for ammonia excretion in aquatic animals: role of Rhesus (Rh) glycoproteins. J. Exp. Biol. 212, 2303–2312. Wright, P.A., Pärt, P., Wood, C.M., 1995. Ammonia and urea excretion in the tidepool sculpin (Oligocottus maculosus) — sites of excretion, effects of reduced salinity and mechanisms of urea transport. Fish Physiol. Biochem. 14 (2), 111–123. YE, X., Randall, D.J., 1991. The effect of water pH on swimming performance of rainbow trout (Salmo gairdneri). Fish Physiol. Biochem. 9, 15–21.
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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a
The effect of temperature and ammonia exposure on swimming performance of brook charr (Salvelinus fontinalis) C. Tudorache ⁎, R.A. O'Keefe, T.J. Benfey Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada, E3B 5A3
a r t i c l e
i n f o
Article history: Received 17 February 2010 Received in revised form 14 April 2010 Accepted 14 April 2010 Available online 29 April 2010 Keywords: Raceway Critical swimming speed Migration Toxicology Physiology Behaviour
a b s t r a c t The effects of water temperature and ammonia concentration on swimming capacity of brook charr (Salvelinus fontinalis, Mitchill, 1814) were determined by measuring gait transition speed (Ugt, cm s− 1), maximum burst speed (Umax, cm s− 1), tail-beat amplitude (a, cm), tail-beat frequency (f, Hz), maximum acceleration of bursts (Amax, cm s− 2), number of bursts, distance of bursts (cm) and total swimming distance (cm) in a 4.5 m long experimental raceway with increasing upstream water velocity. Temperatures other than the acclimation temperature of 15 °C significantly reduced swimming characteristics of gait transition, i.e. Ugt and Amax, while increased ammonia concentration reduced the measures of swimming after Ugt: Umax, the relationship between f and swimming speed above Ugt, a, Amax and the distance travelled with each swimming burst above Ugt. This study, using a novel raceway set-up shows various effects of temperature and ammonia exposure on the swimming performance of brook charr and can be used to establish threshold values for environmental management. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Critical swimming speed (Ucrit; Brett, 1964) is often used to assess the impact of environmental factors such as temperature, hypoxia, diseases or contaminants on fish performance (Brett and Glass, 1973; Beamish, 1978; Waiwood and Beamish, 1978; Thomas and Rice, 1987; Nikl and Farrell, 1993; Hammer, 1995). This is because it is generally assumed that maximum sustainable oxygen uptake occurs at Ucrit (Webb, 1975; Farrell and Steffensen, 1987; Keen and Farrell, 1994; Gregory and Wood, 1999). In the laboratory, Ucrit is commonly determined using increasing water velocity tests to measure the ability of fish to respond in confining swim tunnels. However, Ucrit measured in confining swimming tunnels can be influenced behaviourally (McFarlane and McDonald, 2002; Peake and Farrell, 2006, Tudorache et al, 2007). Fish may refuse to continue swimming when forced to maintain position against a water speed too high for steady and too low for unsteady locomotory gait (Peake and Farrell, 2006, Tudorache et al, 2007). Also, burst-and-glide swimming, which involves the generation of a positive ground speed (Muller et al., 2000; Peake and Farrell, 2004), cannot be maintained efficiently within a confining swim tunnel (Tudorache et al, 2007). An alternative measurement of swimming energetics is the gait transition speed (Ugt), at which the transition from steady cruising to burst-and-glide swimming mode occurs (Videler, 1993). When
⁎ Corresponding author. Tel.: + 1 31 6 20362185. E-mail address: [email protected] (C. Tudorache). 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.04.010
swimming in cruising mode, the predominant muscle groups involved are red aerobically driven muscles, while at switching into burst-andglide swimming mode white anaerobically powered muscles are engaged (Videler, 1993). Therefore, gait transition as an indicator for swimming performance bears both ecological and physiological importance (Peake, 2008, Tudorache et al., 2007). Gait transition is characterised by the first burst, typically as (1) a large and discrete increase in upstream motion, (2) increased tail-beat amplitude and (3) increased tail-beat frequency (Tudorache et al., 2007). Using a novel raceway that allows the fish to swim freely against increasing water speeds (see: Haro et al., 2004; Castro-Santos, 2004, 2005; Peake and Farrell, 2006; Peake, 2008), Ugt can be a more reliable measurement of maximum aerobic swimming speed than Ucrit (Peake, 2008). White muscles used to power burst-and-glide swimming are very susceptible to ammonia (NH3) toxicity, and ammonia exposure decreases swimming performance in both steady swimming (Beaumont et al., 1995; Shingles et al., 2001) and unsteady swimming (Tudorache et al, 2008; McKenzie et al, 2009). Especially salmonids are known to be susceptible to even low ammonia concentrations in freshwater (Shingles et al., 2001). The potential for toxicity is determined by dissolved ammonia concentration since it diffuses into the fish across their gills (Shingles et al., 2001). As water ammonia level rises, plasma ammonia levels increase in the fish due to a decreased excretion by means of Rhesus (Rh) proteins (Weihrauch et al., 2009; Wright and Wood, 2009). Increased NH3 levels within the fish alter the metabolic status, which may lead to premature muscle fatigue due to partial depolarisation of the
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membrane potential in the white muscles (Beaumont et al. 1995, 2000b) and, hence, a reduction in swimming performance (Beaumont et al., 2000a). High ammonia concentrations might, therefore, alter gait transition speed by impairing anaerobic capacity. Another factor strongly influencing gait transition is water temperature. Maximum cardiorespiratory performance is temperature dependent (Brett 1964, 1971; Randall and Brauner 1991; Taylor et al. 1996). The temperature optimum of MO2 in salmonids is close to their upper temperature limit (Farrell, 2007), as salmonids are unable to compensate for naturally decreasing water oxygen content with increasing temperatures through increased gill irrigation (Brett, 1971). And even though gill oxygen diffusing capacity increases with exercise, gas exchange is perfusion limited on the blood side of the gills during exercise (Daxboeck et al. 1982; Randall and Daxboeck 1982). With increasing water temperature in resting rainbow trout (Oncorhynchus mykiss), arterial blood saturation decreases (Heath and Hughes 1973), a finding that could reflect a perfusion limitation (Farrell, 2007). Also, with increasing temperatures, acidosis, hypoxia and hyperkalemia of the venous blood worsen with exercise (Kiceniuk and Jones 1977; Holk and Lykkeboe 1998), as shown in rainbow trout (Jain and Farrell 2003), leading to a decrease of maximum cardiac pumping because of the inhibition of contractility (Driedzic and Gesser, 1994). It was hypothesized that ammonia exposure and temperature will have an effect on swimming physiology and behaviour in brook charr (Salvelinus fontinalis), by altering gait transition speed, maximum burst speed, tail-beat amplitude, tail-beat frequency, maximum acceleration of bursts, number of bursts, distance of bursts (cm) and total swimming distance in a 4.5 m long experimental raceway with increasing upstream water velocity. An integrative approach was chosen in order to find threshold values which in combination with other data can be used for management of freshwater bodies.
2. Material and methods Brook charr (12.22±0.12 cm, fork length, LF, and 23.49±0.67 g body mass±S.D.) were obtained from the Miramichi Salmon Conservation Centre and held in dechlorinated municipal water (pH=8.50±0.09, alkalinity = 80.95 ± 0.49 mg L− 1, [Ca] = 35.6 ± 0.35 mg L− 1, [Mg] = 3.92 ± 0.08 mg L− 1, [Na]= 13.13 ± 1.17 mg L− 1, hardness = 103.00 ± 1.00 mg L− 1), in the aquaculture facilities at the University of New Brunswick (Fredericton campus) for at least 3 weeks prior to exposure. Water temperature was kept at 15.5 °C (range ±0.8 °C) and water speed in the tanks was low (0–0.5 cm s− 1). Fish were held in 140 L tanks in groups of 12 individuals. A flow-through configuration was used with a turnover rate of 100 L d− 1. Fish were fed a salmonid grower diet (Corey Feed Mills Ltd., Fredericton, NB, Canada) ad libitum.
2.1. Ammonia and temperature pre-exposure For the ammonia experiment, fish were exposed to ammonia (total ammonia, i.e. NH3 and NH+ 4 ) at 15 °C and a pH of 9.10 ± 0.12 for a total of 96 h. This coincides with the maximum exposure period for brown trout in Tudorache et al. (2008) and has proven to affect fast starts significantly. The concentration of NH3 in the water was 25.62 µmol L− 1. The total volume of 140 L of water was spiked with the required amount of NH4Cl (Sigma-Aldrich Chemical Co., Toronto, Canada) to reach concentrations of 14.38 (15.08 ± 0.06), 28.76 (29.36 ± 4.69), 43.14 (42.86 ± 5.87) and 57.53 (58.13 ± 4.11) µmol L− 1 total ammonia (NH3 and NH+ 4 ), which were maintained via in-flow addition from a stock solution. For the temperature experiment, fish were kept at 15 °C as described above and transferred to the experimental temperatures in the raceway without acclimation.
2.2. Experimental set-up The experimental set-up consisted of a plexiglas raceway with gradually increasing water velocity due to a slight inclination (see Peake, 2008 for example). The raceway was 450 cm long, 12.5 cm wide and 50 cm deep. Each end was fitted with a trapezoid holding tank, 75 cm long, 50 cm wide and 50 cm deep. Water was pumped from the downstream tank to the upstream tank by means of a submersible pump (Alita Model PV-800; Aracadia, CA, U.S.A.). The total volume of water was 250 L. For the ammonia experiment, the water was spiked with the necessary amount of NH4Cl to reach concentrations of 14.38 (13.89 ± 0.16), 28.76 (28.16 ± 2.37), 43.14 (45.16 ± 6.17) and 57.53 (57.78 ± 6.36) µmol L− 1 total ammonia (NH3 and NH+ 4 ). The water had a pH of 9.11 ± 0.13. The raceway was inclined by 3.5°, resulting in an upstream water depth of 5 cm and a water velocity of 110 cm s− 1 and a downstream water depth of 40 cm and a velocity of 10 cm s− 1. The water velocity was measured every 10 cm and showed a linear decline towards the downstream end of the raceway. The control water temperature of 15 °C and the experimental water temperatures of 10 and 20 °C were maintained with an in-line chiller (Aqua Logic Model DS-7; San Diego, CA, U.S.A.) and dissolved oxygen concentration did not go below 7.5 mg L− 1. Eight cameras (SNG SED-CAM-YC26S, Sharpe Electronics Corp., Osaka, Japan), connected to a digital video recorder (EDVR 16 D3, Everfocus, Taipei, Taiwan), were mounted below the raceway to record images of a 70 cm section with overlapping fields. The original footage was recorded in mp4 format and transformed into avi format using ImageViewer (Everfocus, Taipei, Taiwan). A grid on the bottom over the entire length of the raceway facilitated orientation and scaling. Because the camera lenses were wide-angled, the resulting hemispherically convex distorted images were adjusted with a digital concave distortion using Adobe AfterEffects CS3 Professional version 8.0.2.27 (Adobe Systems Inc., San Jose, CA, USA) without any loss of image quality. The position of the centre of mass determined the position of the fish along the raceway, as described by Tudorache et al. (2008). Fish were introduced in the downstream holding tank of the raceway 1 h before the experiment started. Access to the raceway was blocked with a grid that was removed when the experiment started. The cameras were set on and footage recorded over the following 2 h. At the end of the experiment, fish were gently removed from the raceway and prepared for final measurement of LF, weight and blood and muscle ammonia concentrations. Fish from the experimental group exposed to ammonia were killed with a sharp blow on the head. A sample of white muscle tissue was removed from the left dorsocaudal section of the trunk, snap frozen in liquid nitrogen, packed in aluminium foil and stored at −80 °C for the determination of the ammonia concentration in the muscle. The total time of killing and sampling took only b1 min per fish in order to avoid additional ammonia build up in the muscles due to excessive thrashing (Tudorache et al. 2010). The use of animals was approved by the UNB Animal Care Committee, following guidelines established by the Canadian Council on Animal Care. The number of fish per experiment was ten. 2.3. Analysis Total ammonia concentrations in water were determined using the salicylate–hypochlorite method according to Verdouw et al. (1978). Total muscle ammonia was extracted according to the method described by Wright et al. (1995). Total muscle and plasma ammonia was measured using an enzymatic kit (Sigma 171 C, Sigma-Aldrich Chemical Co.). The XY coordinates of the centre of mass were digitized for three randomly chosen forward swimming movement, one from the beginning, one from the middle and one from the end of video sequences (avi files), and exported into Vernier logger pro 3.4.6 (Vernier
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Fig. 1. Representative plot of total swimming speed against water speed for a control fish at 15 °C. The fish starts with increasing swimming velocity in cruise mode and then changes at the gait transition speed (Ugt) into burst-and-glide mode. Burst-and-glide is characterised by rapid accelerations (burst) followed by decelerations until near zero ground speed (i.e., swimming speed equals water speed). The solid line indicates Ugt and the dotted line indicates zero ground speed, i.e. where the swimming speed is equal to the water speed.
Software & Technology, TX, USA) to perform a manual tracking of each fish's movements. Locomotor variables were calculated using the digitized coordinates, and the following parameters were assessed: 1) gait transition speed (Ugt, cm s− 1): maximum swimming speed observed that was supported entirely using a steady undulatory locomotory gait. The first three burst sequences thereafter initiated burst-and-glide swimming mode. The start of a burst was defined in terms of: (a) a large and discrete increase in upstream motion, (b) increased tail span and (c) increased tailbeat frequency (Tudorache et al., 2007). 2) maximum swimming speed (Umax, cm s− 1): the highest swimming speed in cm s− 1 possible at burst swimming mode before the fish fell back. 3) tail-beat frequency (f, Hz): the number of full tail beats per second. 4) tail-beat amplitude (a, cm): the distance between the maximum deflections of the caudal tail edge to both sides. 5) maximum acceleration after gait transition (Amax, cm s− 2): the maximum acceleration at burst swimming mode. 6) number of bursts: the number of fast forward accelerations after Ugt before Umax was reached. 7) distance of bursts (cm): the distance covered while bursting forward. 8) total swimming distance (cm): the distance covered by a continuous forward movement including aerobic as well as anaerobic swimming modes.
Fig. 2. Gait transition speed (Ugt, black) and maximum swimming speed (Umax, white) at 10, 15 and 20 °C. Letters indicate significant differences (N = 10, p b 0.05).
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Fig. 3. Gait transition speed (Ugt, black) and maximum swimming speed (Umax, white) at different total ammonia (NH+ 4 and NH3) concentrations. Letters indicate significant differences (N = 10, p b 0.05).
2.4. Statistics All assumptions for parametric tests were met. All parameters were compared using a multiple ANOVA with a Bonferroni post-hoc test. The significance level for all tests was set at p b 0.05. Linear regression analysis was used to evaluate the relationship between swimming speed and tail-beat frequency or amplitude.
3. Results Upon entering the raceway, fish swam in steady cruise mode, supported by slow and rhythmic undulations of the body and caudal fin, increasing their swimming speed steadily until gait transition. After gait transition, they accelerated rapidly and swam in burst-andglide mode. Bursts were characterised by a large and discrete increase in upstream motion and increased tail-beat amplitude and frequency, followed by a passive glide. The next burst was initiated when ground speed reached nearly zero. The swimming speed in burst-and-glide mode increased rapidly, reaching Umax at ca. 80% of the entire forward swimming period. At the end of the forward movement, the fish reached a standstill before falling back in the raceway (Fig. 1). Ugt for the control group at 15 °C in water with no added ammonia was 37.43 ± 1.47 cm s− 1, and was reduced at both 10 °C and 20 °C (p b 0.05, Fig. 2). However, Umax at 10 and 20 °C did not differ significantly from control (p N 0.05, Fig. 2).
Fig. 4. Representative plot of tail-beat frequency (f) against swimming speed (U) for a control fish at 15 °C. The vertical line indicates gait transition speed and the resulting linear relationship can be described by the linear function f = 1.5557+ 0.1033U (r2 = 0.92) before and f = 1.5793+ 0.102U (r2 = 0.85) after gait transition (N = 3, p b 0.05).
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Table 1a Regression values for tail-beat frequency (f, Hz) plotted against swimming speed (U, cm s− 1) with the formula f=a+bU at different water temperatures. Temperature (°C)
10
15 (control)
20
a (before Ugt) a (after Ugt) b (before Ugt) b (after Ugt)
1.56 ± 0.13 1.58 ± 0.18 0.104 ± 0.042 0.105 ± 0.039
1.57 ± 0.14 1.56 ± 0.11 0.104 ± 0.031 0.106 ± 0.024
1.58 ± 0.15 1.58 ± 0.13 0.102 ± 0.020 0.107 ± 0.024
Ammonia concentration did not affect Ugt. Umax showed a gradual decline with increasing ammonia concentration, reaching about 75% of control values at 57.53 µmol L− 1 (Fig. 3). Tail-beat frequency (f) for the control group at 15 °C in water with no added ammonia was positively correlated with swimming speed (U) before (r2 = 0.90, p b 0.05) and after (r2 = 0.88, p b 0.05) gait transition (Fig. 4) and could be described by the regression equation
Fig. 5. Tail-beat amplitude (a) at different total ammonia (NH+ 4 and NH3) concentrations before (black) and after (white) gait transition. Letters indicate significant differences (N = 10, p b 0.05).
f = a + bU where a is the intercept of the regression and b is the slope. Slope and intercept were not affected by temperature (Table 1a) or ammonia concentrations below 43.14 µmol L− 1 (p N 0.05, Table 1b), but the intercept was significantly decreased and slope significantly increased at ammonia concentrations of 43.14 and 57.53 µmol L− 1 at speeds higher than Ugt (Table 1b). For the control group at 15 °C in water with no added ammonia, tailbeat amplitude (a) did not vary with swimming speed, but it did vary with swimming mode, increasing from 2.04 ± 0.34 cm when cruising to 3.20 ± 0.41 with burst-and-glide swimming (p b 0.05). It was not affected by temperature but declined with increasing ammonia concentration at speeds higher than Ugt, with values above 0.5 mg L− 1 significantly decreased. Values of a below Ugt were not significantly different from control (Fig. 5). Maximum acceleration of bursts after gait transition (Amax) was significantly reduced by 20% at 10 °C compared to the control at 15 °C, but at 20 °C Amax did not differ from control (Fig. 6a). Increased ammonia concentrations also affected Amax, which was significantly reduced from control values at 43.14 and 57.53 µmol L− 1 by about 15 and 20%, respectively (Fig. 6b). With temperature as a factor, the number of forward bursts until the end of the raceway was reached did not differ from the control (3.00 ± 0.38). However, the number of bursts was significantly increased (p b 0.05) with ammonia concentrations of 43.14 and 57.53 µmol L− 1 (3.37 ± 0.33 and 3.93 ± 0.49, respectively). Distance of burst was significantly reduced (p b 0.05) from control (89.34 ± 13.24 cm) with ammonia concentrations of 43.14 and 57.53 µmol L− 1 (76.13 ± 7.19 and 65.40 ± 7.66 cm, respectively). The total swimming distance was not significantly different between the treatments or the control (371.50 ± 11.91 cm). Also, the total distance covered in burst-and-glide mode was not significantly different from the control (274.62± 32.93 cm). 3.1. Plasma and tissue ammonia Ammonia levels in exposed fish were significantly elevated in the plasma at exposure levels of 0.5 mg L− 1 and higher and in white muscle at levels of 44.04 µmol L− 1 and higher (Table 2).
4. Discussion The results of this study show that linear swimming is affected by both temperature and ammonia concentration. Gait transition speed (Ugt), the speed at which the transition from cruise to burst-and-glide swimming occurs, was lower at 10 and at 20 °C than at 15 °C. Brook charr show a maximum aerobic capacity at around 15 °C (Beamish, 1978). However, Ugt was not affected by increased ammonia concentrations in the water, a finding in accordance with the prediction that ammonia has little effect on red aerobic muscles. The maximum burst speed (Umax), on the other hand, was not affected by temperature, a result that is in accordance with Blaxter and Dickson (1959), Brett (1964), Beamish (1978) and Videler (1993). Webb (1978) reported an increase in Umax of escape fast starts; however, a positive correlation of Umax with temperature was not detected in the current study, possibly due to the low frame rate of the filming footages used. However, Umax, the relationship between tailbeat frequency and swimming speed above Ugt, tail-beat amplitude, maximum acceleration after gait transition and the distance travelled with each swimming burst above Ugt were all significantly reduced at the higher ammonia concentrations, suggesting that ammonia reduces white muscle performance (Tudorache et al., 2008, McKenzie et al., 2009). The observed significant decrease of the intercept and the increase of the slope of the tail-beat frequency at speeds higher than Ugt with elevated ammonia concentrations also suggests an increased cost of transport after Ugt. Shingles et al. (2001) showed that the critical swimming speed of rainbow trout is reduced at elevated ammonia concentrations. Critical swimming speed swimming is the speed at which maximum sustainable oxygen uptake occurs (Webb, 1975; Farrell and Steffensen, 1987; Keen and Farrell, 1994; Gregory and Wood, 1999) and measures aerobically as well as anaerobically powered swimming performance (Brett, 1964; Beamish, 1978; Videler, 1993). The present study shows that swimming performance before Ugt is less affected by ammonia than anaerobically powered swimming performance, up to a concentration of 58.72 µmol L− 1. This suggests that elevated ammonia concentrations may lead to a decreased Ucrit by reducing swimming performance after
Table 1b Regression values for tail-beat frequency (f, Hz) plotted against swimming speed (U, cm s− 1) with the formula f = a + bU under the influence of elevated ammonia concentrations in the water. *Indicates significant difference from control (one-way ANOVA, p b 0.05, N = 10). Concentration (µmol L− 1)
0 (control)
14.38
28.77
43.15
57.53
a (before Ugt) a (after Ugt) b (before Ugt) b (after Ugt)
1.57 ± 0.14 1.56 ± 0.11 0.104 ± 0.031 0.106 ± 0.024
1.61 ± 0.15 1.57 ± 0.09 0.111 ± 0.025 0.106 ± 0.032
1.58 ± 0.13 1.58 ± 0.12 0.112 ± 0.020 0.113 ± 0.024
1.54 ± 0.17 0.48 ± 0.13* 0.113 ± 0.012 0.125 ± 0.014*
1.53 ± 0.12 0.24 ± 0.16* 0.118 ± 0.013 0.130 ± 0.016*
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Fig. 6. Maximum acceleration (Amax) at different temperatures (a) and total ammonia (NH+ 4 and NH3) concentrations (b). Letters indicate significant differences (N = 10, p b 0.05).
Ugt. In order to test this, future studies of the critical swimming speed should include the measurement of gait transition speed. The results at different water temperatures also show that Ugt is reduced, possibly due to a reduction in aerobic swimming capacity, while swimming capacity after gait transition is not affected. Therefore, a measured reduction in Ucrit (e.g.: Beamish, 1978; Videler, 1993) may be due to a reduction of aerobic swimming capacity, as the protocol for Ucrit tests includes swimming aerobic as well as anaerobic swimming capacities. Both long-distance and short-distance migratory fish species use waterways for migration (Lukas and Baras, 2001). Physical barriers can obstruct free migration and can cause fragmentation and finally extinction of fish populations (Warren and Pardew 1998; Toepfer et al. 1999; Warren et al. 2000). Fish ladders and culverts designed to facilitate migration are often introduced to migratory paths. In order to be able to pass such devices, the water speed must be adjusted to the swimming potential of the migrating fish species. In the past, the standard water velocity was equal to, or lower than, the critical swimming speed obtained in forced swimming tests, as the critical swimming speed is assumed to be the swimming speed at which the maximum sustainable oxygen uptake occurs (Webb, 1971; Farrell and Steffensen, 1987; Keen and Farrell, 1994; Gregory and Wood, 1999). However, some authors have criticized this method (e.g. Plaut, 2001, Castro-Santos, 2004, 2005; Peake and Farrell,
527
2006), proposing the use of Ugt instead (Peake and Farrell, 2006; Peake, 2008), as this represents the highest speed that is obtained by using only red aerobically powered muscles (Videler, 1993; Peake, 2008). The tilted raceway used in this study enables not only the measurement of Ugt, but also other parameters, such as maximum swimming speed, acceleration at bursts and kinematic parameters such as tail-beat frequency and amplitude under different environmental conditions. The factors assessed in this study, temperature and ammonia concentration, are not only important environmental factors but also represent factors influencing aerobically and anaerobically powered swimming performance. The results show that to give a good estimate of swimming capacity and the ability to negotiate obstacles on the migration path, Ugt is not sufficient, as it concerns only aerobically powered swimming performance. The present study shows that in estimating total swimming capacity (both aerobic and anaerobic), critical swimming speed gives a better approximation of the effect of factors which influence anaerobic swimming speed as well (Beaumont et al., 1995; Shingles et al., 2001). Therefore, the use of critical swimming speed as an indicator for swimming capacity is still crucial, especially when measuring effects on both aerobic as well as anaerobic swimming performance. The results of swimming capacity and performance is not only determined physiologically but also behaviourally. Gait transition is employed when the swimming speeds become too fast to be maintained by red aerobic muscles alone (Peake and Farrell, 2006; Tudorache et al. 2007). However, as the switch from cruising to burstand-glide swimming occurs, Ugt has a strong volitional component and the bioenergetics of swimming cannot be fully understood if disconnected from its behavioural component (Farrell, 2007). It has therefore been proposed that Ugt measured in confined swimming tunnels is not comparable with Ugt measured in volitional raceways set-ups (Farrell, 2007; Peake, 2008). Also, absolute values of Ugt and Ucrit measured in confined swimming tunnels cannot be extrapolated to free swimming fish and therefore cannot be used as measures of swimming capacity in the wild. The present methodology gives more ground for extrapolation in freely swimming fish in the wild as they are not confined by the size of regular Brett- or Blazka-type swimming tunnels (Peake, 2008). However, if comparing physiological, ecological or genetic factors in fish, traditional swimming tunnels are sufficient. Also, confined swimming tunnels are superior to large raceways as they allow the measurement of dissolved oxygen and other metabolic gasses and more invasive methods, such as cannulation. Such methodologies are impossible or very difficult to be realised in a 4.5 m long raceway. The ammonia concentration used in this study, i.e. maximum 57.53 µmol L− 1 of total ammonia is the equivalent of 1 mg L− 1 and is thus the same as used by Tudorache et al. (2008), resulting in a reduction of fast start performance in brown trout. However, 57.53 µmol L− 1 of total ammonia results in a concentration of NH3 of 15.57 µmol L− 1 at a pH of 9.1. A slightly higher concentration used by Shingles et al. (2001; 20 µmol L− 1) proved to be sufficient to reduce critical swimming speed in rainbow trout significantly. This value coincides with 50% LC50 of rainbow trout of 0.5 g body weight (Shingles et al. 2001). According to Ye and Randall (1991) a pH of 9 does not affect swimming capacity in salmonids and brook charr is especially tolerant for high pH values (Daye and Garside, 1975). The results of this study suggest that linear swimming characteristics in brook charr swimming in a raceway with increasing upstream
Table 2 Plasma and white muscle ammonia levels in brook charr after exposure to 0, 14.68, 29.36, 44.04 and 58.72 (µmol L− 1 total ammonia for 96 h. Different letters indicate significant difference (p b 0.05, N = 10). Ammonia concentration (µmol L− 1)
0 (control)
14.38
28.77
43.15
57.53
Plasma ammonia (µmol L− 1) White muscle ammonia (µmol g− 1)
175.53 ± 58.64a 1.42 ± 0.28a
233.92 ± 47.46a 1.49 ± 0.31a
329.48 ± 67.33b 1.88 ± 0.42ab
443.84 ± 52.00c 2.05 ± 0.52b
503.63 ± 59.32c 2.52 ± 0.42b
528
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