Critical Dissolved Oxygen Tolerances of Whirling ...

North American Journal of Aquaculture 78:366–373, 2016 © American Fisheries Society 2016 ISSN: 1522-2055 print / 1548-8454 online DOI: 10.1080/15222055.2016.1201556

ARTICLE

Critical Dissolved Oxygen Tolerances of Whirling Disease-Resistant Rainbow Trout Eric R. Fetherman* Colorado Parks and Wildlife, 317 West Prospect Road, Fort Collins, Colorado 80526, USA

Jonathan A. Wardell U.S. Fish and Wildlife Service, 427 Lakeview Drive, Orangeburg, South Carolina 29115, USA

Chris J. Praamsma Colorado Parks and Wildlife, Bellvue Fish Research Hatchery, 5500 West County Road 50C, Bellvue, Colorado 80512, USA

Marta K. Hura Colorado Parks and Wildlife, 317 West Prospect Road, Fort Collins, Colorado 80526, USA

Abstract

A low concentration of dissolved oxygen (DO) is commonly the limiting factor in fish culture systems. Hypoxia tolerance in Rainbow Trout Oncorhynchus mykiss can be affected by both history of domestication and growth rate. As such, selecting strains for specific characteristics such as growth rate or disease resistance could potentially affect DO tolerance, making culture difficult. Here we used two experiments to examine the differences in tolerance to lower DO concentrations among four Rainbow Trout strains and crosses selected for resistance to whirling disease Myxobolus cerebralis. The first experiment examined differences in critical DO concentrations of fry (≥73 mm total length [TL]) when exposed to rapid decreases in DO at 30, 60, 90, and 120 d postswim-up. In addition, since formalin is a common chemical used in aquaculture to treat for external parasites, the effect of exposure to formalin on tolerance to low DO was evaluated. The second experiment evaluated critical DO concentrations among four strains and crosses exposed to a prolonged decrease in DO at age 7 months (averaging 178 mm TL). Formalin exposure had an effect on low-DO tolerance, with DO concentrations that resulted in a loss of equilibrium decreasing with an increase in formalin concentration. In addition, low-DO tolerances were diminished with an increase in fish size, with larger fish losing equilibrium at higher DO concentrations. Differences in DO concentrations resulting in loss of equilibrium and mortality were evident among the strains and crosses in the second experiment. This experiment demonstrated that DO concentrations must be below 2.0 mg/L before loss of equilibrium is observed. However, if fish are soon returned to well-oxygenated water, losses can be minimized. Additionally, other hatchery practices that compromise hypoxia tolerance may increase mortality more quickly following low-DO exposure, and care should be taken to correct low-DO issues shortly after loss of equilibrium is observed.

A low concentration of dissolved oxygen (DO) is commonly the limiting factor in fish culture systems (Kindschi et al. 1991). Consumption of DO by cultured fish is affected both by factors associated with the hatchery environment (Alabaster et al. 1957; Elliot 1969; Cameron 1971; Hughes 1981; Rombough 1988; Watten et al. 1991) and by the

*Corresponding author: [email protected] Received February 17, 2016; accepted May 31, 2016

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biology and behavior of the culture species (Beamish 1964; Parker 1973; Klar et al. 1979; Thomas and Gilmour 2012). As such, determining tolerances can help optimize culture and increase poststocking survival when rearing multiple strains for recreational or conservation stocking purposes. Dissolved oxygen consumption and tolerances

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have the potential to differ among strains or subspecies as a result of origin (Kindschi et al. 1991), phenotypic variations obtained through selective breeding (Klar et al. 1979), or adaptations to particular environments (Wagner et al. 2001). Specifically, hypoxia tolerance in Rainbow Trout Oncorhynchus mykiss can be affected by both history of domestication (Kindschi et al. 1991) and growth rate (Roze et al. 2013). Therefore, selecting strains for specific characteristics, such as growth rate or disease resistance, could potentially affect tolerance to low DO concentrations, making culture difficult. Whirling disease Myxobolus cerebralis-resistant Rainbow Trout are a relatively new addition to the state aquaculture system in Colorado. The German Rainbow (GR; also known as Hofer) is a hatchery-derived strain that had been exposed to M. cerebralis over multiple generations in Germany (Hedrick et al. 2003), developing a “resistance” to M. cerebralis. Although GR can be infected with M. cerebralis, parasite burdens are usually low (Hedrick et al. 2003; Schisler et al. 2006; Fetherman et al. 2012) and the strain can survive and reproduce in the presence of M. cerebralis. Although domestication facilitated pathogen resistance, the strain’s viability in the wild was uncertain (Schisler et al. 2006), which led to its experimental crossing with the Harrison Lake (HL) strain (Schisler 2006). The HL (origin: Harrison Lake, Montana) exhibits enhanced resistance to M. cerebralis relative to other Rainbow Trout strains, which could be related to ancestry (Vincent 2002; Wagner et al. 2006). Resistance was increased significantly when HL fish were crossed with GR fish (Schisler 2006). Both the GR and HL strains, as well as various crosses of these strains, are maintained as broodstock in Colorado’s state hatchery system and are produced and stocked for recreational purposes statewide. Two experiments were conducted to determine whether lowDO tolerance differed among the GR and HL strains and their crosses. The first experiment examined differences in critical DO concentrations, that is, those at which a fish lost equilibrium when exposed to rapid decreases in DO. In addition, anecdotal evidence suggests that GR fish exhibit sensitivity to formalin during treatment for external parasites. The chemical oxygen demand of formalin is thought to affect DO tolerance, leading to respiratory compromise of fish during treatment (Speare et al. 1996). Formalin exposure can also cause dramatic intracellular edema and increase diffusion distances across gill lamellae (Smith and Piper 1972; Wedemeyer and Yasutake 1974). Therefore, the effect of formalin exposure on low-DO tolerance was also evaluated in the first experiment. The second experiment evaluated critical DO concentrations among the strains and crosses exposed to a prolonged decrease in DO. The objectives of these experiments were to determine whether strain or cross differences in low-DO tolerance existed, to determine whether formalin exposure affected low-DO tolerance, and, ultimately, to inform and optimize culture of M. cerebralis-resistant Rainbow Trout stocks.

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METHODS Rapid decrease in dissolved oxygen concentration and interactions with formalin.—Four M. cerebralis-resistant Rainbow Trout strains and crosses were used to determine differences in critical DO concentrations and effects of formalin exposure: GR, HL, GR×HL 50:50, and GR×HL 75:25. Fish were spawned and reared at the Colorado Parks and Wildlife (CPW) Bellvue Fish Research Hatchery (BFRH; Bellvue, Colorado). Fish were created using pooled family groups, with each family originating from a unique male– female pair. Eighteen families were used to create the GR fish, 21 families were used to create the HL fish, 20 families were used to create the GR×HL 50:50 fish, and 37 families were used to create the GR×HL 75:25 fish. All fish were held at the BFRH until they reached 30, 60, 90, or 120 d postswimup. One week before beginning the DO trials for a given age group, fish were transported from the BFRH to the CPW Aquatic Toxicology Laboratory (Fort Collins, Colorado) using 19-L water coolers filled with hatchery water and supplied with air from a pump connected to the transport vehicle’s auxiliary power system. Total loading and transport time was approximately 30 min. On arrival at the lab, water from within the lab was mixed with water from the hatchery to allow fish to slowly acclimate to lab water temperature and chemistry (pH 7.4; alkalinity and hardness, 34 and 38 mg/L CaCO2 equivalent, respectively). Fish were held in four 110-L aquaria, one for each strain or cross. Well water was supplied at a rate of 18.93 L/min and maintained at a temperature of 12°C. Thirty- and 60-d-old fish were fed a diet of Rangen size 0 soft moist feed, and 90- and 120-d-old fish were fed Rangen size 1 or 2 feed. Fish were fed at 2.5% of their body weight, and feeding proportions were recalculated on a daily basis according to tank density. When fish were not being used in the DO trials, they were fed four times a day using Fish Mate automatic feeders (Pet Mate, Ltd., Surrey, UK). On the day in which a fish was used in a trial, it was transferred out of the holding tanks before first feeding and food was withheld for the duration of the trial (24 h). Rainbow Trout averaged 33 ± 4 mm (mean ± SD) TL and 0.29 ± 0.10 g at 30 d postswim-up, 45 ± 5 mm TL and 0.91 ± 0.34 g at 60 d postswim-up, 55 ± 7 mm TL and 1.68 ± 0.71 g at 90 d postswim-up, and 73 ± 9 mm TL and 4.12 ± 1.71 g at 120 d postswim-up. Experiments were conducted at each of four ages (30, 60, 90, and 120 d postswim-up) to determine whether age or fish size played a role in DO tolerance, and critical DO concentrations were measured in the absence (0) and presence of formalin (167 and 250 mg/L). Ten replicates from each strain or cross were tested at each formalin concentration for a total of 30 trials per strain or cross at each of the four ages. Formalin treatment and strain or cross were randomly assigned to a tank, and the order in which the trials were conducted was randomized before initiation of the trials within an age group. Dissolved oxygen trials were simultaneously conducted in two 9.5-L glass aquariums insulated on four of the six sides to

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prevent temperature fluctuations. Before commencement of a trial, aquaria were filled with water supplied from the holding tanks of the strain or cross being used in that trial. A trial was begun by randomly selecting a fish from the holding tank and placing it into an experimental tank, where it was allowed to acclimate for 1 h. Water temperature and DO concentrations within the experimental tank were maintained at 12°C and 100% saturation, respectively, during the 1-h acclimation period. Tank temperatures were maintained throughout the trial by using temperature regulators, custom-shaped titanium chilling rods, and peristaltic pumps to move ice water from a 2-gal (7.6-L) water cooler containing an ice bath through the chilling rods when needed. Following the acclimation period, helium, regulated to 125 mL/min, was injected into the tanks with the objective of reducing the DO concentration to less than 10% saturation over the course of the 1-h trial. Helium was used instead of nitrogen to prevent inducing gas bubble disease (per Colorado State University IACUC protocol 13-4000A), which could have increased delayed mortality (see below). If the trial included exposure to formalin, this also was added at the end of the acclimation period. Corning magnetic stirrers and Teflon-coated magnetic stir bars were used to circulate the water in the tanks and maintain temperature. The DO concentration (mg/L) and saturation (%) were measured using YSI Pro Optical DO (ODO) sensors, which logged both quantities, as well as temperature and barometric pressure, every minute throughout the experimental trial. Critical DO concentrations were defined as the point at which a fish lost its equilibrium for a period of 10 s. Upon reaching critical DO concentration, DO was recorded from the YSI Pro ODO meter, and the fish was immediately transferred to water held at 100% saturation to recover. Fish were held for 24 h after experimentation to determine whether delayed mortality occurred after exposure to low DO concentrations, formalin, or both. After 24 h, fish were removed from their holding tanks, euthanized with an overdose of tricaine methanesulfonate (MS-222), measured, and weighed. Before the start of a new trial, tanks were flushed and rinsed twice to ensure that no formalin residue remained. Prolonged decrease in dissolved oxygen concentration.— The same four M. cerebralis-resistant Rainbow Trout strains and crosses (GR, HL, GR×HL 50:50, and GR×HL 75:25) were used to determine differences in critical DO concentrations when decreases in DO were prolonged. Rainbow Trout for this experiment were spawned at the BFRH in December 2013 and reared for approximately 7 months in separate hatchery troughs by strain or cross. Rainbow Trout averaged 178 ± 35 mm TL and 74 ± 48 g at the time of experimentation. A hatchery trough (366 cm long × 48 cm wide × 30 cm deep), enclosed in plastic tarps to prevent inducing a physiological stress response to movement or light, was used to conduct the prolonged decrease in DO concentration experiments at the BFRH. The trough was divided into two equal sections using a perforated screen. The day before being used

in a trial, fish were moved to the upper half of the trough to prevent accidental feeding or handling stress. Dissolved oxygen trials were conducted in the lower half of the trough in two 41.6-L plastic containers. Water (12°C) flowed through the trough at a rate of 18.93 L/min to maintain temperature within the plastic containers during the trials. Fluorescent lights (two 1.2-m-long 32-W bulbs) held 0.3 m above the water surface provided constant and consistent lighting within the enclosed trough. In addition, a Logitech Web camera was mounted above each tank so that end points could be determined after a trial had been conducted, preventing observer movement from increasing fish stress levels and affecting critical DO concentrations. Because the duration of these trials exceeded typical exposure periods for formalin treatment (Piper et al. 1982), the effects of formalin exposure were not evaluated in this experiment. At the start of a trial, both plastic containers were filled with water directly from the trough. Fish from the upper half of the trough were transferred to the containers, one fish per container, allowing two trials to be conducted simultaneously. A perforated lid allowing oxygen exchange at the water surface was placed on top of each container and secured for the 1-h preexperiment acclimation period. In addition, oxygen was introduced to both tanks through a Sweetwater fine-pore diffuser at a rate of 3–4 mL/min to allow the fish to acclimate to the tanks, to reduce physiological effects of handling, and to increase DO concentrations to 100% saturation. Saturation was confirmed using two YSI Pro ODO meters. After the acclimation period, the perforated lid was removed from the tanks and replaced with a clear Plexiglas lid. The Plexiglas lid had only two holes, one for the air hose and one for the YSI Pro ODO meter. Both holes were cut to fit equipment exactly, allowing the plastic containers to become sealed chambers and prevent oxygen exchange at the water surface. To reduce DO concentrations, nitrogen gas, used because gas bubble disease was not a concern in an experiment with an endpoint of mortality (see below), was introduced to the tank at a rate of 50 mL/min. The rate of nitrogen introduction had been determined before experimentation as the rate that would reduce DO levels in the tanks to less than 1.0 mg/L within 4 to 6 h. Oxygen depletion was confirmed by using the logging function of the YSI Pro ODO meters, which produced DO curves that could be analyzed after each trial. In addition, the logging function of the meters was synchronized with the time function in the Logitech cameras so that endpoints could be determined postexperimentation. Trials were completed once mortality occurred. Three endpoints were observed: initial loss of equilibrium (ILOE), final loss of equilibrium (FLOE), and mortality. Initial loss of equilibrium occurred at the first time a fish lost its equilibrium, turning upside down for 2 s but recovering shortly thereafter. Final loss of equilibrium occurred when a fish could not recover its equilibrium for longer than 10 s. Mortality occurred when fish movement, including its operculum, fins, or tail, was no longer visible. At each of these

TOLERANCE TO DISSOLVED OXYGEN CONCENTRATIONS IN RAINBOW TROUT

endpoints, when the endpoint occurred, the DO concentration (mg/L and percent saturation) and temperature were recorded. After a trial, fish were removed from the tank, measured, and weighed. Only one trial was conducted per tank per day. Ten replicate trials were conducted for each strain or cross, for a total of 40 trials. Recovery after exposure to low dissolved oxygen.—Using the same groups of fish as those used in the prolonged decrease in DO experiment, an additional set of trials were conducted to determine whether fish could recover once they had reached the average concentration at which FLOE had occurred in the previous trials (1.2 mg/L; 13% saturation). All experimental procedures were similar to those described above. However, once tanks reached 1.2 mg/L DO, fish were immediately removed from the tank and placed in a separate trough with fresh-flowing well-oxygenated water. Fish were held for 24 h to determine whether they would recover. At the end of the 24-h monitoring period, fish were measured and weighed and their status (dead or alive) was recorded. Four replicate trials were conducted for each strain or cross, for a total of 12 trials. Statistical analyses.—Statistical analyses were conducted using the GLM procedure in SAS (SAS Institute 2015). In the experiment examining the effects of rapid decreases in DO concentration and formalin exposure, differences in critical DO concentrations among the strains and crosses, formalin concentrations, and ages (including interactions) were analyzed using a three-factor analysis of covariance (ANCOVA), with body weight as a covariate (N = 477). Differences in delayed (24 h) mortality among the same three factors (with interactions) were also analyzed using a three-factor ANCOVA but with critical DO concentration as a covariate, accounting for the fact that lower concentrations may have caused physiological complications or gill damage that prevented recovery (N = 477). In the experiment examining the effects of prolonged decreases in DO concentration, differences in endpoints (ILOE, FLOE, and mortality) and among strains or crosses (including interactions) were analyzed using a two-factor repeated measures analysis of covariance (RM ANCOVA), with body weight as a covariate. Recovery status following low DO exposure was compared among strains and crosses using an ANCOVA, with body weight as a covariate. Values for all analyses were reported from the Type III sum of squares. If significant (P < 0.05) effects were identified, the least-squares means method was used to determine which treatments caused significant differences in critical DO concentration, mortality, among the strains or crosses, or endpoints. RESULTS Rapid Decrease in Dissolved Oxygen Concentration and Interactions with Formalin Critical DO concentrations did not differ among strains or ages, and body weight did not have a significant effect on critical DO concentration. In addition, critical DO concentrations were

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not affected by interactions between strain and formalin concentration; formalin concentration and age; or strain, formalin concentration, and age. Formalin concentration, however, did have a significant effect on low-DO tolerance (ANCOVA; F2, 428 = 3.31, P = 0.031). Dissolved oxygen concentrations resulting in loss of equilibrium were significantly higher for fish that were not exposed to formalin (1.37 mg/L) than for fish exposed to 250 ppm of formalin (1.29 mg/L; P = 0.012). Exposure to increasing formalin concentrations appeared to lower critical DO concentration, with exposure to 167 ppm formalin resulting in a mean critical DO concentration of 1.31 mg/L. However, this concentration did not differ significantly from that for either fish not exposed to formalin (P = 0.068) or fish exposed to 250 ppm of formalin (P = 0.454). Despite nonsignificance, the interaction between strain or cross and fish age revealed some interesting patterns. In general, critical DO concentration increased with an increase in age within a strain or cross (Figure 1). However, this increase in critical DO concentration was significant only in the HL strain (P = 0.034). The HL strain was also the only strain wherein critical DO concentrations were significantly lower at 30 d postswim-up than at 60, 90, and 120 d (P ≤ 0.034); however, critical DO concentrations did not differ among HL fish at 60, 90, or 120 d postswim-up (P ≥ 0.359). No significant differences were observed among ages within the other three strains or crosses (P ≥ 0.057). At 30 d postswim-up, critical DO concentrations for the HL strain were significantly lower than for both the GR (P = 0.050) and the GR×HL 75:25 (P = 0.042) but not for the GR×HL 50:50 (P = 0.614). At 90 d postswim-up, critical DO concentrations for the HL strain were significantly higher than for the GR (P = 0.006) and the GR×HL 50:50 (P = 0.032) but not for the GR×HL 75:25 (P = 0.199). At 60 and 120 d postexposure, no differences were observed between any of the strains or crosses (P ≥ 0.133; Figure 1). Delayed (24 h) mortality occurred occasionally in each of the age groups. Fish within each of the strains and crosses exhibited delayed mortality during the experiments, and delayed mortality was observed at least once in each of the formalin treatments (Table 1). Incidence of delayed mortality did not differ among any of the factors or interactions included in the ANCOVA. Although significant differences in critical DO concentrations were apparent (F1, 429 = 4.60, P = 0.033), the lack of significance in the main effects suggests that DO concentration did not affect delayed mortality. Prolonged Decrease in Dissolved Oxygen Concentration Overall, body weight had a significant effect on critical DO concentration (F1, 107 = 7.52, P = 0.007) for prolonged exposure, and results suggest that the larger the fish, the less tolerant it was of low DO concentrations. Dissolved oxygen concentrations differed among the strains and crosses (F3, 107 = 11.41, P < 0.001; Table 2). The GR×HL 50:50 strain was more tolerant of lower DO concentrations, reaching a lower average DO concentration than either the HL or GR×HL 75:25 but not the GR strain.

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FIGURE 1. Average critical DO concentrations (mg/L; SE bars) among the strains and crosses at the four ages tested in the rapid decrease in DO concentration and formalin interaction experiment. GR = German Rainbow strain; HL = Harrison Lake strain.

Critical DO concentrations did not differ among the GR, HL, or GR×HL 75:25 fish. The DO concentrations also differed among the endpoints (F2, 107 = 89.99, P < 0.001; Table 2). Initial loss of equilibrium and FLOE did not occur at significantly different DO concentrations. However, mortality occurred at a significantly lower DO concentration than either ILOE or FLOE. The interaction between strain or cross and endpoint was significant (F6, 107 = 5.11, P < 0.001). Within a strain or cross, DO concentrations resulting in ILOE did not differ from those TABLE 1. Number of delayed mortalities (out of 10) occurring in the four strains and crosses of Rainbow Trout exposed to formalin concentrations of 0, 167, and 250 mg/L at 30, 60, 90, or 120 d postswim-up (age) in the rapid decrease in DO concentration and formalin interaction experiment. GR = German Rainbow strain; HL = Harrison Lake strain.

Formalin, (mg/L) 0

167

250

Age (d)

GR

HL

GR×HL 50:50

GR×HL 75:25

30 60 90 120 30 60 90 120 30 60 90 120

0 0 1 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 1 0 1 0 0 0

1 1 0 0 0 0 0 0 0 1 1 1

0 0 0 0 0 0 0 0 0 1 0 0

resulting in FLOE (P ≥ 0.145; Figure 2). DO concentrations resulting in ILOE and FLOE were significantly lower in the GR×HL 50:50 strain than in any of the other strains or crosses (P ≤ 0.012). Initial loss of equilibrium and FLOE did not differ among the GR, HL, and GR×HL 75:25 (P ≥ 0.092). The DO concentrations resulting in mortality differed among the strains and crosses (Figure 2). Mortality in the HL strain occurred at a significantly higher DO concentration than in the GR, GR×HL 50:50, or GR×HL 75:25 fish (P ≤ 0.032). However, DO concentrations resulting in mortality did not differ among the GR, GR×HL 50:50, and GR×HL 75:25 fish (P ≥ 0.067). Within all of the strains and crosses, DO concentrations resulting in mortality were significantly lower than the concentrations resulting in ILOE or FLOE (P ≤ 0.006). However, differences between concentrations that resulted in ILOE, FLOE, and mortality were larger in the GR and GR×HL 75:25 than in the HL and GR×HL 50:50 (Figure 2).

TABLE 2. Average DO concentrations (mg/L; SE in parentheses) tolerated by each of the strains or crosses, and where the endpoints of initial loss of equilibrium (ILOE), final loss of equilibrium (FLOE), or mortality occurred in the prolonged decrease in DO concentration experiment. Within each column, DO concentrations with the same letters do not differ significantly from each other. GR = German Rainbow strain; HL = Harrison Lake strain.

Strain or cross GR GR×HL 50:50 GR×HL 75:25 HL

DO 1.08 0.98 1.17 1.16

(0.04) (0.03) (0.03) (0.04)

zy z z y

Endpoint

DO

ILOE FLOE Mortality

1.25 (0.02) y 1.19 (0.02) y 0.85 (0.02) z

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FIGURE 2. Average DO concentrations (mg/L; SE bars) at which initial loss of equilibrium (ILOE), final loss of equilibrium (FLOE), and mortality occurred among the strains and crosses in the prolonged decrease in DO concentration experiment. GR = German Rainbow strain; HL = Harrison Lake strain.

Recovery after Exposure to Low Dissolved Oxygen In general, fish could recover after being exposed to DO concentrations of 1.2 mg/L. Of the 12 fish tested, only 2, a GR and a GR×HL 75:25, died after being exposed to a DO concentration of 1.2 mg/L. The ability to recover did not differ among the strains and crosses (F3, 7 = 0.39, P = 0.767), and body weight did not contribute to the ability of fish to recover from low DO concentrations (F1, 7 = 0.17, P = 0.694).

DISCUSSION Tolerance to low DO concentrations differed among the GR and HL strains, likely a result of differences in their history of domestication, selection for certain traits such as growth and disease resistance, and the proportion of these traits retained in the GR×HL crosses. In addition to being M. cerebralis resistant, the GR is a fast-growing strain relative to other wild strains of Rainbow Trout (Fetherman et al. 2011). Roze et al. (2013) showed that fast-growing fish are more hypoxia tolerant than slow-growing fish. In addition, phenotypic varieties can differ in oxygen consumption rate and ambient oxygen concentration at fatigue (Klar et al. 1979). Although the GR reached similar DO concentrations for the ILOE and FLOE endpoints, mortality occurred at significantly lower DO concentrations than in the wild, slower growing HL strain. In fact, DO concentrations at which mortality occurred in the GR×HL 50:50 and 75:25 strains were lower than in the HL, suggesting that the tolerance of the GR strain is maintained in these crosses. This tolerance may have also led to the larger differences in DO concentrations between FLOE and mortality in the GR and GR×HL 75:25 (higher proportion of GR) relative to the GR×HL 50:50 and HL (lower proportion of GR).

These results suggest that hatchery managers may have more time to correct issues with low DO concentrations after observing loss of equilibrium in the GR and GR×HL 75:25 than they would after observing loss of equilibrium in the HL or GR×HL 50:50. Although ILOE and FLOE occurred at lower DO concentrations in the GR×HL 50:50, mortality occurred shortly thereafter. Therefore, higher losses would be expected in the HL and GR×HL 50:50 if actions were not taken to quickly resolve issues with low DO relative to the GR and GR×HL 75:25. Because exposure to low DO elicits a stress response (Caldwell and Hinshaw 1994) and can increase the probability of mortality from exposure to disease (Sniesko 1974; Caldwell and Hinshaw 1995), it is likely that strain differences would be even more pronounced if low DO becomes an issue in M. cerebralis-positive hatcheries. Overall, the critical DO tolerances exhibited by the strains and crosses are similar to those reported previously for Rainbow Trout (Burdick et al. 1954; Davison et al. 1959; Rombough 1988) but are lower than those reported for Cutthroat Trout O. clarkii (Wagner et al. 2001). Fish body size played a role in low-DO tolerance in both experiments. Generally, more oxygen per unit size is required by smaller fish than by larger fish (Elliot 1969; Piper et al. 1982). As fish become larger, their DO requirement per unit size decreases (Kindschi et al. 1991). Nilsson and ӦstlundNilson (2008) observed that bigger individuals have a greater ability to survive in hypoxic conditions than do smaller ones because the ability to produce ATP anaerobically increases with size; thus, small fish run out of glycogen or reach lethal levels of metabolic end-products faster than big fish do, due to higher mass-specific metabolic rate. The results of our experiment, however, differ from those cited above in that larger individuals

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were less tolerant of low DO concentrations than were smaller individuals. In the first experiment, we observed an increase in critical DO concentrations with an increase in fish age. However, differences among the ages within a strain were less pronounced than expected, likely because within about 30 d posthatch, critical DO concentrations reach an asymptotic low (Rombough 1988). In the second experiment, body weight also had a significant negative effect on tolerance of low DO concentrations. One potential explanation may be the design of the experiments. All of the experiments we conducted were enclosed, preventing fish from being able to supplement DO intake at the water surface. Rainbow Trout show increases in both rate and amplitude of breathing in response to low DO (Holeton and Randall 1967), and standard oxygen uptake increases linearly with body weight (Beamish 1964). Therefore, larger individuals may have more rapidly consumed the small amounts of DO remaining in the tank or may have felt the physiological effects and succumbed to them more quickly than smaller fish due to an increase in breathing rate. Formalin was included in the first experiment because Colorado hatchery managers had reported higher mortality rates after formalin treatment, especially among GR and GRcross fish, a common observation among hatchery operators (Piper and Smith 1973; Speare and Ferguson 1989). Formalin is thought to affect DO tolerance due to its chemical oxygen demand, leading to respiratory compromise of fish during treatment (Speare et al. 1996). In addition, dramatic intracellular edema and increased diffusion distance across lamellae have been observed during single treatments of Rainbow Trout with formalin at 167 or 250 mg/L (Smith and Piper 1972; Wedemeyer and Yasutake 1974). Both effects would be expected to diminish the tolerance to low DO concentrations in the presence of formalin; however, that was not the case here. In this study, formalin had a marginally sparing effect similar to that observed by Speare et al. (1996). Earlier experiments conducted by Wedemeyer (1971) showed that metabolic oxygen consumption was depressed after formalin treatment and also noted a decline in ventilation rate in formalin-treated salmonids. Speare et al. (1996) suggested that an observed decline in ventilation frequency or volume during treatment, possibly a nociceptive reflex, may lead to a reduction in oxygen uptake. In either case, the results of this study suggest that exposure to formalin does not diminish the tolerance of Rainbow Trout to low DO concentrations and that additional mortality can be avoided if fish are soon returned to well-oxygenated water. Formalin exposure is also known to cause gill damage (Smith and Piper 1972), and gill pathological changes have been associated with changes in ionoregulation in exposed fish (Wedemeyer and Yasutake 1974). Although there were no differences in postexposure (24 h) mortality in this experiment, formalin treatment can result in posttreatment mortality spikes (Piper and Smith 1973; Wood 1979; Spear and Ferguson 1989), potentially a result of gill damage. Gill damage could affect DO uptake and therefore could result in

mortality at higher DO concentrations following formalin exposure. It is possible that the 24-h monitoring period was not long enough to observe delayed mortality as a result of pathological changes to the gills. Problems with low DO in aquaculture can arise at the worst possible times, for example, during formalin treatment. This experiment demonstrates that DO concentrations must get below 2.0 mg/L before loss of equilibrium is observed and DO issues start to need to be corrected. Exposure to formalin during a low-DO event may slightly buffer the fish against effects, but it also lowers the concentration at which loss of equilibrium will be noticeable, potentially causing mortality to occur more quickly. However, if fish are soon returned to welloxygenated water, losses can be minimized. Depending on the stress levels of the fish, e.g., from crowding or disease, low DO concentrations may increase ventilation rate and decrease the amount of time available to correct an issue. This effect is especially concerning in the GR×HL 50:50 and HL strain, where mortality is likely to occur shortly after loss of equilibrium. Additionally, hatchery practices that compromise hypoxia tolerance may increase mortality more quickly after exposure to low DO concentrations. Therefore, care should be taken to correct low-DO issues shortly after loss of equilibrium is observed. ACKNOWLEDGMENTS This work was sponsored in part by Colorado Parks and Wildlife and the Colorado Cooperative Fish and Wildlife Research Unit at Colorado State University, and funding was provided in part by the Federal Aid in Sport Fish Restoration program, Project F-394R. We thank J. Anderson, T. Davis, B. Neuschwanger, and G. Schisler for technical assistance and rearing of the Rainbow Trout strains and crosses used in this experiment. We also thank S. Brinkman for his help in rearing fish, providing technical assistance and advice, and providing the lab space and equipment for conducting the DO and formalin interaction experiment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Colorado State University IACUC protocol 13-4000A. REFERENCES Alabaster, J. S., D. W. M. Herbert, and J. Hemens. 1957. The survival of Rainbow Trout (Salmo gairdnerii Richardson) and perch (Perca flaviatilis L.) at various concentrations of dissolved oxygen and carbon dioxide. Annals of Applied Biology 45:177–188. Beamish, F. W. H. 1964. Respiration of fishes with special emphasis on standard oxygen consumption. II. Influence of weight and temperature on respiration of several species. Canadian Journal of Zoology 42:177–187. Burdick, G. E., M. Lipschuetz, H. J. Dean, and E. J. Harris. 1954. Lethal oxygen concentrations for trout and Smallmouth Bass. New York Fish and Game Journal 1:84–97. Caldwell, C. A., and J. Hinshaw. 1994. Physiological and haematological responses in Rainbow Trout subjected to supplemental dissolved oxygen in fish culture. Aquaculture 126:183–193.

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