Integrating Water Flow, Locomotor Performance and Respiration of Chinese Sturgeon during Multiple Fatigue-Recovery Cycles

The objective of this study is to provide information on metabolic changes occurring in Chinese sturgeon (an ecologically important endangered fish) subjected to repeated cycles of fatigue and recovery and the effect on swimming capability. Fatigue-recovery cycles likely occur when fish are moving through the fishways of large dams and the results of this investigation are important for fishway design and conservation of wild Chinese sturgeon populations. A series of four stepped velocity tests were carried out successively in a Steffensen-type swimming respirometer and the effects of repeated fatigue-recovery on swimming capability and metabolism were measured. Significant results include: (1) critical swimming speed decreased from 4.34 bl/s to 2.98 bl/s; (2) active oxygen consumption (i.e. the difference between total oxygen consumption and routine oxygen consumption) decreased from 1175 mgO2/kg to 341 mgO2/kg and was the primary reason for the decrease in U crit; (3) excess post-exercise oxygen consumption decreased from 36 mgO2/kg to 22 mgO2/kg; (4) with repeated step tests, white muscle (anaerobic metabolism) began contributing to propulsion at lower swimming speeds. Therefore, Chinese sturgeon conserve energy by swimming efficiently and have high fatigue recovery capability. These results contribute to our understanding of the physiology of the Chinese sturgeon and support the conservation efforts of wild populations of this important species.


Introduction
Water flow is commonly believed to be of an important environmental factor to fish. Natural selection favors locomotor and respiration strategies appropriate for an existing flow [1]. Chinese sturgeon (Acipenser sinensis, Gray 1834) has been listed as Class I Endangered Species by the Chinese Government [2] and is also listed as Critically Endangered (CR) in the IUCN Red List of Threatened Species (version 2013.1). The species is anadromous and currently occurs only in the middle and lower reaches of the Yangtze River and near-shore in the Yellow and East China Seas [3]. Although captive breeding of Chinese sturgeon is now feasible [4,5], wild populations have declined significantly because of dam construction, overfishing and pollution [6][7][8][9][10]. Chinese sturgeon once migrated further than any other sturgeon, over 3,200 km in the Yangtze River. Restoration of the wild sturgeon populations is difficult due to long maturation time, infrequent spawning and low productivity [11][12]. Although the species has a life span of 50-60 years, the species breeds only three or four times and the hatchling survival rate is estimated at ,1%.
Dam construction has blocked spawning migration [13] and the sturgeon populations have declined dramatically [10].
Augmenting wild populations by release of captive breed sturgeon is a great achievement for species conservation but not sufficient. Building fishways that allow migration is another important part of the effort to maintain wild populations [14,15]. Data on swimming capability and the physiological response to exercise and fatigue provide important design criteria for fishways [16][17][18][19].
Publications on Chinese sturgeon have been more frequent in recent years [20] but have focused on biochemistry and habitat assessment. Research on swimming capability and excess postexercise oxygen consumption, for one repetition, has been reported (e.g., [21][22][23][24]. However, fish typically undergo multiple fatigue-recovery cycles while moving through fishways of large dams [25] and data on the effects of repeated fatigue-recovery is necessary for the design of effective fishways. In this study, the effect of repeated fatigue-recovery cycles on swimming capability and metabolism were investigated using a stepped velocity test in a Steffensen-type swimming respirometer. The study documents the change in respiration and swimming capability of fish undergoing repeated fatigue-recovery cycles. These results contribute to our understanding of the physiology of the Chinese sturgeon and support the conservation efforts of wild populations of this important species.

Ethics Statement
This study was conducted in strict accordance with the laws governing animal experimentation in China. The protocol was approved by the China Three Gorges University. All efforts were made to minimize suffering.

Test fish
Juvenile sturgeon were obtained from the institute of Chinese sturgeon research, China Three Gorges Project Corporation, in Yichang, China (30u569 N; 111u159 E) and were of the second filial generation of cultured Chinese sturgeon. The sturgeon (body length: 10.8960.11 cm and body weight 8.3860.27 g, mean 6 S.E.) were maintained in cylindrical tanks (80 cm in diameter and 90 cm deep). They were fed to satiation each morning at 8:00 am with Tubificidae. Water temperature varied from 19.3uC to 20.8uC and dissolved oxygen (DO) maintained above 7.0 mg/L. Ammonia-N and nitrite-N were lower than 0.050 mg/L and 0.007 mg/L, respectively. Forty eight hours prior to the experiment, feeding was interrupted to prevent the elevated oxygen consumption rate associated with digestion [26].

Respirometer
The sturgeon were tested in a sealed 14 L Steffensen-type swimming respirometer submerged in a 55.7 L tank (84.0 cm long 639.9 cm wide 617.0 cm deep) to maintain temperature. A schematic diagram of the respirometer was included in an earlier publication [27]. Respirometer components include a motor with speed controller and propeller and a rectangular swimming chamber (4.5 L, 35.5 cm long 611.0 cm wide 611.5 cm deep) with a Lucite multi-aperture rectifier at the entrance to maintain laminar flow and a wire grid at the exit. Normal respirometer operating assumptions were made: (1) swimming speed (U) is equal to water flow speed as measured with an acoustic Doppler velocity meter (Nortek AS, Oslo, Norway), and (2) oxygen consumption can be calculated from the change in dissolved oxygen, as measured by an oxygen electrode (Hach HQ30d, Loveland, USA).

Protocol for stepped velocity test
Ten sturgeons were tested and the body length (bl), fork length, total length and body mass of each sturgeon were measured. Before testing, fish were acclimated overnight in the respirometer at a very low flow speed (0.3,0.5 bl/s) and then allowed to swim at 0.5 bl/s from 7:00-8:00 am the next morning [12,28]. When testing began, flow speed was increased by 0.5 bl/s every 20 min and dissolved oxygen was measured at 5 min intervals. When the sturgeon was exhausted (swimming ceased and the fish rested against the wire grid for 20 s), flow speed was adjusted to 0.5 bl/s during the 30 min recovery period [27]. This comprises a single stepped velocity test and each sturgeon tested was subjected to four tests in succession to simulate a repeated cycle of fatigue and recovery.

Data collection and analyses
All experimental data was expressed as mean 6 S.E. and was analyzed and fitted using Origin (Version 8.1). The level of significance was determined using ANOVA Fisher LSD. Fit to mathematical models was evaluated following the Akaike information criterion AIC [29,30] using the model correcting for finite sample size and frequently applied in behavioral ecology [31]; where RSS is the residual sum of squares from the fitted model, n is sample size, and k is the total number of parameters. Its use helps prevent overfitting and offers a relative estimate of the information lost by a given model. The value of AICc is inversely related to the goodness of fit of a given model. Based on data from the stepped velocity test, the critical swimming speed (U crit ) of the test fish is calculated as described by Brett [32]. Oxygen consumption rate (MO 2 ) is calculated using Equation 2; where V (L) is the volume of the respirometer, m (kg) is the mass of the sturgeon, d(DO)/dt (mgO 2 /(L6h)), which is the slope of the linear regression of DO decreasing over time (R 2 .0.99), is the rate of change in dissolved oxygen in the respirometer during the step test and d(DO)'/dt (mgO 2 /(L6h)) is the rate of change in dissolved oxygen in the control (respirometer with no fish). Routine oxygen consumption rate (MO 2, routine ) of each sturgeon is the MO 2 at 0.5 bl/s (Cai et al., 2013b). Maximum oxygen consumption rate (MO 2, max ) of each sturgeon is the maximum value of MO 2 during the stepped velocity test. The aerobic scope (AS) is the difference between MO 2, max and MO 2, routine and is related to the energy potentially available for swimming [33,34]. During the swimming period, variation of MO 2 with U was obtained by fitting the data to Equation 3 [35,36]: where a, b and c are constants. Active oxygen consumption (AOC) is the difference between aerobic oxygen consumption and routine oxygen consumption during the period and was obtained by computing the area under the MO 2 curve during the period. During the recovery period, variation of MO 2 over time (t) was obtained by fitting the data to Equations 4 and 5 [37,38]: where a, b and c are constants. Excess post-exercise oxygen consumption (EPOC) is the difference between aerobic oxygen consumption and routine oxygen consumption during recovery from exhaustion and was obtained by computing the area under the MO 2 curve during the period. Assuming that EPOC is the oxygen consumed to recover from anaerobic metabolism, total oxygen consumption was obtained by adding EPOC to aerobic oxygen consumption [32]. Total oxygen consumption was calculated by the iterative method of Lee et al. [37] until EPOC and the area between the MO 2 curve and the broken line (representing anaerobic metabolism) differed ,0.5%. The broken line was then used to estimate U at the onset of anaerobic respiration (Ua).
Metabolic parameters were displayed in Table 1. Differences in MO 2, routine among the four tests were not significant. And differences in MO 2, max were also not significant; values for MO 2, routine were similar but MO 2, max and AS decreased with test number.
As shown in Fig. 2, U crit varied exponentially with AS: U crit~2 :78448z0:00114|e 0:0224AS (F~1882,P~0:00163, Data for the variation of MO 2 with U was fitted to Equation 3 and the results are shown in Table 2. The speed coefficient (c), a measure of swimming efficiency, increased during tests 2 and 3 and then decreased in test 4. Figure 3 illustrates the results of further analysis of the physiological changes that occurred during the four stepped velocity tests. The decreases in EPOC indicates that anaerobic capacity decreases with successive cycles of fatigue and recovery; the decrease in the last test was significant larger than in the second and third tests. The onset of anaerobic metabolism occurred at lower swimming speeds (absolute U a , 1.82, 1.79, 1.64, 1.57 bl/s, respectively, for tests 1,4). However, because the decrease in U crit was more pronounced, relative U a increased with successive testing (41.94%, 47.48%, 50.15% and 52.68%).
During the recovery period, MO 2 decreased rapidly over time; the relationship fitted to Equations 4 and 5 is shown in Table 3.  Juvenile sturgeon, such as shortnose sturgeon Acipenser brevirostrum [39] and Amur sturgeon (Acipenser schrenckii) [27], generally display U crit levels of 2 bl/s,3 bl/s. However, the U crit of Chinese sturgeon was found to be above that range, indicating that Chinese sturgeon have better swimming capability. In addition, there was a linear decrease (R 2 = 0.975) in U crit of the Chinese sturgeon with repeated fatigue-recovery cycles (Fig. 1). However, the difference in U crit declined with repeated testing (D Ucrit 0.57, 0.50, 0.29 bl/s) and the recovery ratio increased from 86.9% to 91.1%. A better fit could have been obtained using a higher order equation but would have resulted in overfitting according to AIC analysis. The recovery ratio for Chinese sturgeon is comparable to sockeye salmon (Oncorhynchus nerka), with a recovery ratio of approximately 85% after a resting time of 1 hour [40]. Because exertion decreases U crit , the slot velocities of upstream fishways should be lower than those in downstream fishways.  Table 3. Variation of oxygen consumption rate over time during the recovery period*. Differences in MO 2, routine over the four test cycles were small and erratic (Table 1), indicating little, if any, influence of fatigue on the routine metabolic rate. While differences in MO 2, max were not significant, the changes were more pronounced and decreased with each test cycle. This leads to a decrease in AS and is consistent with the observed decrease in the U crit . The relationship between AS and U crit is shown in Fig. 2, indicating an exponential increase in U crit with AS.

Speed exponent, c
The exponent c (Equation 3) is inversely related to swimming efficiency [34,38]. In the first test, c was 1.20, similar to those of the white sturgeon, lake sturgeon and Amur sturgeon [27,41]; not surprising as the four Acipenser species are closely related [42]. In the three successive tests, the values of c did not change consistently (c = 1.70, 1.71, 1.42, respectively, for Tests 2, 3, and 4). There are numerous reports on the effect of fish morphology on swimming efficiency [43,44]. In this investigation morphology was consistent among the tests and results indicate that repeated fatigue-recovery cycles affect physiological function. It has been reported that different fish use different swimming strategies [45,46], but it remains unclear as to whether the sturgeon are adjusting swimming strategy in response to repeated fatiguerecovery cycles. Swimming efficiency was highest in Test 1, lower in Tests 2 and 3, with some recovery displayed in Test 4.

Excess post-exercise oxygen consumption, EPOC
During fatigue recovery the variation of oxygen consumption rate with time is usually described by Equation 4 [27,37]. The data fit for Test 1 and Test 2 was good (Table 3) but data for Test 3 and  Test 4 could not be fit to Equation 4 because the fit did not converge. After fitting the data to several other functions Equation 5 was selected, based on fit and AIC analysis, to estimate the onset of anaerobic respiration and its contribution to U crit .
Information on EPOC is helpful for establishing design criteria for the resting pools of fishways [27,47]. The EPOC is inversely related to the recovery capability from exhaustion [37]. The EPOC in the first test was 35.7 mgO 2 /kg, much lower than for salmon but similar to Amur sturgeon [27,37], indicating high recovery capability for Chinese sturgeon. Although EPOC decreased with repeated recovery from fatigue, it does not necessarily follow that the recovery capability increased because U crit and AS also decreased and AOC decreased by 70% from Test 1 to Test 4 ( Table 1).
The EPOC is directly related to anaerobic respiration [37] and the decrease in EPOC indicates that repeated recovery from fatigue lowers capacity for anaerobic metabolism. With each fatiguerecovery cycle anaerobic respiration begins at a lower swimming speed but contributes less to achieving U crit . However, it can be seen in Fig. 3 and Table 1 that oxygen consumption is primarily from aerobic respiration and the decrease in aerobic respiration is the primary cause for the decrease in U crit . It was reported that salmon pass fishways using primarily aerobic respiration [25], consistent with our results.
Swimming speed at onset of anaerobic respiration, U a Aerobic metabolism with red muscle fiber provides propulsion in general, but white muscle fiber contributes to achieving high speeds [36,48,49]. It was reported that anaerobic swimming in cyprinids begins to augment propulsion when swimming speed reaches 30,50% U crit [50]. However, aerobic power is different in different fish species [37,50,51]. In this study, the relative U a was between 41.94% and 52.68% U crit (absolute U a , 1.82, 1.79, 1.64, 1.57 bl/s, respectively, for tests 1,4) similar to the higher range of cyprinids. So, with repeated step tests, white muscle (anaerobic metabolism) began contributing to propulsion at lower absolute swimming speeds.

Conclusions
Repeated cycles of fatigue and recovery affect the swimming capability of Chinese sturgeon; U crit , AOC and absolute U a decrease with decreasing swimming capacity. These results contribute to our understanding of the physiology of the Chinese sturgeon and support the conservation efforts of wild populations of this important species.