Physiological Stress Response in Bonytail and

Razorback Sucker to Handling and Transport

Final Report

December 2013

Lower Colorado River Multi-Species Conservation Program

Steering Committee Members

Federal Participant Group

California Participant Group

Bureau of Reclamation
U.S. Fish and Wildlife Service
National Park Service
Bureau of Land Management
Bureau of Indian Affairs
Western Area Power Administration

California Department of Fish and Game

City of Needles
Coachella Valley Water District
Colorado River Board of California
Bard Water District
Imperial Irrigation District
Los Angeles Department of Water and Power
Palo Verde Irrigation District
San Diego County Water Authority
Southern California Edison Company
Southern California Public Power Authority
The Metropolitan Water District of Southern
California

Arizona Participant Group

Arizona Department of Water Resources
Arizona Electric Power Cooperative, Inc.
Arizona Game and Fish Department
Arizona Power Authority
Central Arizona Water Conservation District
Cibola Valley Irrigation and Drainage District
City of Bullhead City
City of Lake Havasu City
City of Mesa
City of Somerton
City of Yuma
Electrical District No. 3, Pinal County, Arizona
Golden Shores Water Conservation District
Mohave County Water Authority
Mohave Valley Irrigation and Drainage District
Mohave Water Conservation District
North Gila Valley Irrigation and Drainage District
Town of Fredonia
Town of Thatcher
Town of Wickenburg
Salt River Project Agricultural Improvement and Power District
Unit B Irrigation and Drainage District
Wellton-Mohawk Irrigation and Drainage District
Yuma County Water Users Association
Yuma Irrigation District
Yuma Mesa Irrigation and Drainage District

Other Interested Parties Participant Group

QuadState County Government Coalition
Desert Wildlife Unlimited

Nevada Participant Group

Colorado River Commission of Nevada
Nevada Department of Wildlife
Southern Nevada Water Authority
Colorado River Commission Power Users
Basic Water Company

Native American Participant Group

Hualapai Tribe
Colorado River Indian Tribes
Chemehuevi Indian Tribe
Conservation Participant Group
Ducks Unlimited
Lower Colorado River RC&D Area, Inc.
The Nature Conservancy

Lower Colorado River

Multi-Species Conservation Program

Physiological Stress Response in Bonytail and

Razorback Sucker to Handling and Transport
Final Report
Prepared by: Catherine L. Sykes, USFWS, Southwestern Native Aquatic
Resources and Recovery Center
In partial fulfillment of Agreement Number R11PG30012 between Bureau of
Reclamation and US Fish and Wildlife Service

Lower Colorado River

Multi-Species Conservation Program
Bureau of Reclamation
Lower Colorado Region
Boulder City, Nevada
http://www.lcrmscp.gov

December 2013

TABLE OF CONTENTS
Section

Page

Executive Summary .........................................................................................................................1

Introduction ......................................................................................................................................3
Methods............................................................................................................................................4
Experimental fish .................................................................................................................4
Experimental design.............................................................................................................5
Sample collections ...............................................................................................................8
Analysis of plasma constituents ...........................................................................................9
Data analysis ........................................................................................................................9
Results and Discussion ....................................................................................................................9
Recommendations ..........................................................................................................................16
Acknowledgments..........................................................................................................................17
Literature Cited ..............................................................................................................................18
Figures............................................................................................................................................22

EXECUTIVE SUMMARY
Bonytail (Gila elegans) and razorback sucker (Xyrauchen texanus) are propagated at the
Southwestern Native Aquatic Resources and Recovery Center (SNARRC) in support of recovery
efforts for these fish species in the lower Colorado River. In preparation for stocking, the fish are
harvested from ponds, moved to outdoor raceways, sorted, tagged, loaded in transport tanks and
hauled to a stocking location. SNARRC staff have developed and implemented handling
protocols for native species with the goal of minimizing the amount of stress experienced by the
fish during this process. A main consideration of the protocol is providing an adequate recovery
period between each of the handling events to allow the fish to recover physiologically before
they are transported. The purpose of this study is to evaluate the severity and duration of the
physiological stress response experienced by bonytail and razorback sucker exposed to current
handling and hauling protocols used at SNARRC and to monitor the fish for 21 d after transport
to discern the level of recovery.
Increased plasma cortisol, glucose, and lactate levels observed at each handling event
compared to resting levels clearly demonstrated a physiological stress response in both species to
handling protocols in place at SNARRC. However, the levels in those plasma parameters were
generally comparable to or approaching resting levels by 48 h post-handling. Plasma chloride
levels and osmolality fluctuated among all the sampling times suggesting that ionic and osmotic
imbalance was present through much of the study period. In the bonytail, three stress-related
mortalities were observed between 12 and 21 d post-hauling, but there were no mortalities in
razorback sucker during the study
The results from this study support the current handling protocols in place at SNARRC.
Fish culture activities include sufficient time between each handling event to allow recovery of
1

the fish before exposure to the next stressor. For all facilities rearing these species, we
recommend a recovery period of no less than 48 h between sequential handling events, but
suggest 5-7 d if stocking schedules allow. In addition, a minimum of 48 h recovery at the
stocking site is strongly recommended before a full release into their native waters. Future
research into the effects of stress on disease susceptibility, especially with bonytail, would be
beneficial in furthering our understanding of the long-term effects of handling on these fish.

INTRODUCTION
Southwestern Native Aquatic Resources and Recovery Center (SNARRC) in Dexter,
New Mexico maintains bonytail (Gila elegans) and razorback sucker (Xyrauchen texanus)
broodstock and produces larval and sub-adult fish for distribution to partners participating in fish
augmentation activities of the Lower Colorado River Multi-species Conservation Program (LCR
MSCP). Captive propagation and annual stockings are imperative for the survival of these two
species. Habitat loss and introduction of nonnative fish are two of the major factors impacting
their recovery. Although some success has been achieved in the recovery of razorback sucker,
the recovery efforts for bonytail have little to show after almost two decades of captive
propagation and augmentation to the wild (Bestgen et al. 2008; Karam and Marsh 2010).
Annually, SNARRC cultures 4,000-6,000 sub-adult bonytail and 500-1,000 sub-adult
razorback sucker for stocking into the LCR MSCP boundaries of the lower Colorado River. All
fish reared for stocking receive a wire tag or passive integrated transponder (PIT) tag and are
transported by truck for 12 to 14 h for release in the lower Colorado River. These activities
subject the fish to various stressors such as handling, confinement, and water quality changes
(Barton et al. 1980; Barton and Peter 1982; Nikinmaa et al. 1983; Davis and Parker 1986; Barton
et al. 2003). The effects of stress can be reduced fitness, increased susceptibility to disease and
vulnerability to predation (Wedemeyer and Wood 1974; Wedemeyer et al. 1990). Although fish
are designed to deal with acute stress through adaptive responses in their biological activities
such as the release of cortisol and increased blood sugar, the ability of the fish to cope may be
overwhelmed if the stress response is provoked through a series of events or becomes long-term.
Key components to understanding how fish cope with stress events include defining the intensity

of the stress response at the point of handling and determining the ability of the fish to regain a
pre-stress homeostatic balance.
The purpose of this study is to evaluate the severity and duration of the physiological
stress response experienced by bonytail and razorback sucker exposed to current handling and
hauling protocols originally developed for Colorado pikeminnow at SNARRC (Dexter 2004).
The first research objective is to characterize the stress response during harvest, tagging, and
before, during and after a long-distance transport by measuring plasma cortisol, lactate, glucose,
and chloride levels, and osmolality. Plasma cortisol is used to measure the primary stress
response, which involves a perception of a stress event at the hypothalamus subsequently
initiating the release of cortisol from interrenal tissue. Secondary responses include changes in
plasma glucose and lactate levels, which are measured to determine the degree of increased
energy demands and anaerobic metabolism, respectively. Measurements of chloride ions and
osmolality reveal changes in the ionic and osmotic responses to stress.
The second objective of this study is to monitor bonytail and razorback sucker held for 21
d after transport to discern the level of recovery using the same stress indicators (plasma cortisol,
glucose, lactate, chloride and osmolality) previously described and documenting any incidence
of delayed mortality. The results from this study will be used to evaluate SNARRCs current
handling protocols and, if necessary, develop and test revised protocols and submit handling
recommendations for bonytail and razorback sucker.

METHODS
Experimental fish. - The first segment of the study was conducted in 2011 with 3-yearold bonytail (mean total length and weight of 335 mm and 319 g, respectively) and the second

segment in 2012 with 4-year-old razorback suckers (mean total length and weight of 372 mm
and 621 g, respectively). Both species were spawned at SNARRC and reared in outdoor ponds.
The fish were maintained on a pelleted feed formulated for razorback suckers with the pellet size
and amount fed based on age of fish and total biomass of fish being cultured per pond (% body
weight/day and water temperatures). Fish rearing densities at SNARRC range from 2,000 to
2,700 fish per surface acre. Feed was stopped 48 h before dewatering of the pond began.
Experimental design. The study was conducted May 9 to June 15, 2011 with bonytail
and March 7 to April 25, 2012 with razorback sucker. The difference in the months in which
each segment was conducted reflects normal year-to-year operations required to meet yearly
production and stocking goals for multiple species recovery programs. Bonytail and razorback
sucker were handled from harvest to stocking according to standard protocols at SNARRC
(Dexter 2004). An overview of the experimental design is presented in Figure 1. To characterize
the resting state of the fish before the multiple handling events, a subsample of fish were
collected by seine from the pond and transferred to four indoor circular tanks (n = 5 each) and
held for 26 (bonytail) and 20 d (razorback sucker) to allow recovery from handling before blood
was collected. The 20 d minimum acclimation period was established based on Sykes et al.
(2011).
For each of the handling events, the fish collected for bleeding were sampled from among
the last fish to be handled in order to capture the point of maximum disturbance. The immediate
stress response following each handling was assessed by sampling 10 fish from each of the two
raceways (n = 20). The sampled fish were held in four 110 L tubs (5 fish each) with oxygen for
0.25 h to allow the cortisol to spike before blood samples were collected (Sykes et al. 2011). Fish
for the 12-, 24-, and 48-h recovery samples were held indoors in 500 L circular tanks (4
5

replicates of 5 fish for each of the 3 sample times, n = 20/sample) with aerated single-pass flowthrough well water. The pre-tagging and pre-loading events were assessed by sampling six fish
from each of the two raceways (n = 12). All fish used for blood collections at SNARRC were
removed from the study and maintained separately for future stocking.
The first handling event was harvesting the fish from the pond. The pond was dewatered
over 48 h with fresh water added to the catch basin to aide in acclimation of the fish to SNARRC
well water and offset any conditions related to deteriorated water quality during harvest.
Handling of the fish included concentrating them in a concrete catch basin at the lowest point in
the pond and harvesting them by seine, dip net and use of a crane operated bucket. The fish were
loaded into the bucket, hoisted to a hauling tank containing fresh oxygenated well water with
0.5% sodium chloride (NaCl), and transported approximately 0.40 km to two outdoor covered
raceways with aerated single-pass flow-through well water at 18C (pH 6.9; hardness 3,325
mg/L CaCO3; alkalinity 162 mg/L CaCO3). The use of 0.5% NaCl as a prophylactic treatment
after handling and during transport is prescribed in the handling protocol (Dexter 2004) to aid the
fish in osmotic regulation and recovery from handling stress (Barton and Zitzow 1995; BurgdorfMoisuk et al. 2011). The size and number of fish harvested required two separate transport
events with the first group stocked into raceway A and the second group into raceway B. The
stress response was assessed after transport to raceways by sampling the fish as previously
described. Fish in both raceways were given a prophylactic treatment of 0.5% NaCl for
approximately 1 h. The fish were allowed a recovery period (bonytail 7 d, razorback sucker 12 d)
before they were handled for tagging. The handling protocol (Dexter 2004) mandates a minimum
7 d recovery period between each handling event, but the actual time may be extended based on

work activities and propagation priorities each year. During the recovery period and immediately
before tagging, blood samples were collected as previously described.
The second handling event involved tagging the fish (bonytail were wire tagged and
razorback sucker were PIT tagged). The fish were anesthetized in 100 mg/L tricaine
methanesulfonate (MS-222) and 0.5% NaCl for tagging and lengths and weights were recorded.
Following tagging, the fish received daily static bath treatments of 0.5% NaCl and 15 ppm
Furanace for 6 to 8 h for 2 consecutive days to relieve handling stress and prevent potential
infection of the injection site. The fish were allowed a second recovery period (bonytail 9 d,
razorback sucker 14 d) with all handling and recovery blood samples collected as previously
described. The third handling event was loading the fish into transport tanks. The fish were
collected from the raceways with dip nets and loaded at a density of 179 kg/m3 into two separate
trucks equipped with two tanks each. Pre-load and 0.25 h post-load blood samples were collected
for this event. Achii Hanyo Native Fish Rearing Facility (Achii Hanyo) in Parker, AZ was
chosen as the location for the simulated stocking event and the 21 d monitoring period. The trip
from SNAARC to Achii Hanyo is 790 miles with a drive time of approximately 13 h in a fish
transport truck. Both trucks left SNARRC at the same time to transport the fish to Achii Hanyo.
At the 6 h time point in transport (approximately half-way to Achii Hanyo) blood was collected
from six fish in each of two tanks (n = 12) on one truck. The fish were returned to the same truck
and hauled back to SNARRC for stocking at a later date. This protocol prevented disturbing the
fish in the second truck that were being transported to Achii Hanyo for the simulated stocking
and 21 d monitoring period.
Upon arrival at Achii Hanyo, the fourth handling event was a simulated stocking. Six fish
each were sampled directly from the two hauling tanks (n = 12) to characterize the condition of
7

the fish at the end of the transport. A second sample of six fish each (n = 12) was bled 0.25 h
after the fish were netted from the truck and released into holding tanks, representing the
stocking event. The fish were held at Achii Hanyo in 900 L circular tanks equipped with aerated,
single-pass flow-through Colorado River water (pH 6.5-6.8; hardness 296 mg/L CaCO3;
alkalinity 130 mg/L CaCO3) delivered from the irrigation canal. The recovery tanks were set up
in duplicate for sampling at 24- and 48-h, and 7-, 14-, and 21-d post-handling. Two additional
tanks were setup with flow-through well water (pH n/a; hardness 330 mg/L CaCO3; alkalinity
207 mg/L CaCO3) as a negative control for pathogens. The last two tanks contained bonytail
reared at Achii Hanyo to serve as a control for fish acclimated to river water. To monitor the
response of handled fish exposed to aquatic pathogens resident in canal water, the fish did not
receive any form of treatment to alleviate the effects of stress, disease or parasites while at Achii
Hanyo. Feeding was resumed 5 d after arrival at Achii Hanyo.
The fish transported from SNARRC to Achii Hanyo were maintained under quarantine
status while at Achii Hanyo. All equipment used in handling the fish remained in isolation from
the rest of the facility to eliminate potential cross-contamination between the study fish and fish
reared at Achii Hanyo. As fish were sampled they were euthanized, frozen and disposed of
according to USFWS policy. At the end of the study all tanks and associated equipment were
scrubbed and disinfected with Virkon Aquatic (Western Chemical, Ferndale, WA) or bleach.
Sample collections. At the time of each sampling, fish were quickly captured by dip net
and anesthetized in 200 mg/L tricaine methanesulfonate (MS-222, Argent Chemical
Laboratories, Redmond, WA). Blood was collected from the caudal vein using heparinized 25G
needles and 1 mL syringes and transferred to 1.5 mL microcentrifuge tubes. Whole blood was
centrifuged after each sample collection and plasma separated from the packed red cells. Each
8

plasma sample was aliquoted into four 0.5 mL cryotubes to reduce the number of freeze-thaw
cycles when conducting the multiple blood chemistry assays. The plasma samples were stored in
a -80 C freezer until analysis was performed. At Achii Hanyo, plasma samples were maintained
in liquid nitrogen until transported back to SNARRC and stored in a -80 C freezer.
Analysis of plasma constituents. - Plasma cortisol concentrations of individual fish were
measured at New Mexico State University by means of a solid-phase radioimmunoassay
(Diagnostic Products Corporation, Los Angeles, CA). Plasma glucose and lactate concentrations
were measured using colorimetric assay kits from Eton Bioscience Inc. (San Diego, CA)
designed for a 96-well microplate reader. Plasma osmolality was measured with a 5520 VAPRO
vapor pressure osmometer calibrated with manufacturers standard solutions and chloride ion
concentrations with a digital chloridometer (Labconco Corporation, Kansas City, MO).
Data analysis A one-way analysis of variance (ANOVA) was conducted in SPSS
version 21 (IBM, Pittsburgh, PA) to compare response variables (cortisol, glucose, lactate,
osmolality, and chloride) at each sampling time. Dunnett T3 posthoc test was used to identify
where differences occurred. Statistical significance was determined at P 0.05 for all tests.
Values reported are means standard errors of the mean.

RESULTS AND DISCUSSION

Harvest, tagging, and hauling procedures employed at captive propagation facilities are
all unavoidable stress events for fish species. Optimizing handling protocols to reduce the
intensity of the stress, the duration of the stress, and the synergistic effect of multiple stressors is
a major factor to releasing healthy fish for successful repatriation. A rapid and intense
physiological stress response to handling using standard hatchery protocols at SNARRC was
9

clearly exhibited by both bonytail and razorback suckers (Figures 2 and 3). Although the timing
of the handling events varied between the two species, a similar response and recovery pattern
was observed in both. However, there were differences between the two species in the condition
of the resting fish at the time they were sampled. Bonytail are a highly reactive fish species. The
immediate and dramatic flight response when a disturbance occurs is readily observed in
bonytail within a captive setting. Because of that strong reaction to disturbances, working with
this species as research animals is challenging. Despite efforts to minimize disturbances while
holding the resting fish indoors, the mean plasma cortisol levels of 125.05 14.52 mg/mL and
glucose levels of 85.06 8.46 mg/dL appeared to be elevated. In a previous study conducted
with 1-year-old bonytail at SNARRC, resting levels of 56.3 12.22 ng/mL cortisol and 43.7
1.03 mg/dL glucose were reported (Sykes et al. 2011). The range of resting plasma lactate levels
(3.0 to 19.6 mM) also suggests some fish were exhibiting a stress response, but it is difficult to
fully evaluate this data for bonytail because no reports have been published for comparison. In
comparing other cyprinids, plasma lactate resting levels ranged from 2 to 6 mM (Pottinger 2010).
Regardless of the elevated resting levels in the plasma constituents, our ability to distinguish
differences between the handling events and recovery periods was not diminished.
The mean resting levels for the plasma constituents in the razorback suckers were 59.89
22.74 ng/mL cortisol, 66.35 mg/dL glucose, and 4.25 0.42 mM lactate. Published data is not
available for comparison on resting levels for these parameters in razorback sucker and little is
available for catostomids in general, but these values are within resting levels reported for a
variety of species. Across Order Cypriniformes a wide range of values in resting plasma cortisol
(3 to 92 ng/mL) and plasma glucose (54 to 114 mg/dL) have been reported (Bracewell et al.

10

2004; Cho et al. 2009; Pottinger 2010) and resting lactate levels are generally reported as ranging
from below 1 mM to 6 mM (Pottinger 1998; Pottinger 2010; Kortan et al. 2011).
A significant stress response in both bonytail and razorback sucker was clearly exhibited
at each handling event (Figures 2 and 3). A threefold increase in plasma cortisol in bonytail was
observed at harvest (393.10 31.97 ng/mL), tagging (441.20 26.02 ng/mL) and loading
(403.03 30.58 ng/mL), and a twofold increase at stocking (265.53 26.59 ng/mL). In bonytail
submitted to a single net stressor, Sykes et al. (2011) reported similar mean plasma cortisol
levels of 405.98 25.66 ng/mL. Razorback sucker exhibited a four- to fivefold increase in
plasma cortisol at harvest (271.04 30.32 ng/mL) and stocking (297.85 37.34 ng/mL) and a
sevenfold increase at tagging and loading (417.42 25.51 and 408.36 26.78 ng/mL). Hunt et
al. (2012) reported plasma cortisol levels of 333.5 31.4 ng/mL for bonytail and 224.6 58.5
ng/mL for razorback sucker (at 20 C) entangled in trammel nets for 2 h, but it is likely that the
initial peak response was not captured. The plasma cortisol response in bonytail (Sykes et al.
2011), as well as in other species (Tanck et al. 2000; Jentoft et al. 2005; Fagundes and Urbinati
2008), has been observed to occur rapidly with peak levels captured before 1 h post-stress.
A significant stress response was also observed in plasma glucose levels in both species
at harvest (143.94 4.89 and 185.81 21.58 mg/dL in bonytail and razorback, respectively) and
tagging (152.04 8.15 and 139.26 14.89 mg/dL in bonytail and razorback, respectively). Even
though a detectable response was measured at the 0.25 h sample, the maximum levels may not
have been captured as plasma glucose generally requires more time than plasma cortisol to reach
a peak response (Figures 2 and 3). After a single net stressor, Sykes et al. (2011) reported a high
of 286.62 19.77 mg/dL plasma glucose in bonytail at 3 h post-stress; following a multiple net
stressor, a high of 398.57 60.70 was not observed until 6 h post-stress. Mazik et al. (1991) and
11

Pottinger (2010) also observed increased plasma glucose levels in three cyprinid species at 6 h
post-stress. In the current study, however, a sufficient number of tanks were not available to hold
fish for additional samplings between the 0.25 h and 12 h sample times. Despite the sampling
time limitations, the increases observed in plasma glucose at each handling event are evidence of
a metabolic stress response. Plasma lactate levels also increased significantly at all handling
events for both species except in the bonytail at loading, indicating the occurrence of anaerobic
metabolism in response to handling.
Documenting the intensity of the stress response to handling is important, but monitoring
the ability of fish to recover is of greater value for evaluating the effect of handling practices on
endangered fish species. Although both species exhibited a strong stress response to handling,
they also demonstrated resilience in recovery over a 48 h period. Bonytail plasma cortisol,
glucose and lactate levels decreased through the 12 and 24 h post-handling samples and by 48 h
post-handling were comparable to resting levels (Figure 2). One anomaly noted was an
unexplained increase in plasma lactate at 48 h post-harvest. At pre-tagging another irregularity
was measured when bonytail perceived a stressor that initiated a rise in cortisol before the fish
could be anesthetized. The low glucose and lactate levels measured at that sample time suggest
that this was most likely an acute response to our sampling technique. The measured plasma
constituent levels in razorback suckers were comparable to resting levels by all 24 h posthandling samples with the exception of post-tagging plasma glucose (Figure 3). Although not
statistically significant, plasma glucose remained elevated at the 48 h post-tagging, but a
complete recovery was documented by the pre-load sample. Regardless of noted irregularities,
the results from plasma cortisol, glucose and lactate measurements suggest that bonytail and
razorback sucker recovered between handling events and that the recovery period was sufficient
12

to prevent a cumulative effect. In addition, no mortalities in bonytail or razorback sucker were

recorded at SNARRC during the harvest, tagging and loading events. However, results from the
plasma chloride and osmolality measurements suggest both species continued to experience ionic
and osmotic disturbances throughout the study while confined to tanks and raceways (Figures 2
and 3). Plasma chloride and osmolality fluctuated in bonytail throughout the study with no
distinguishable pattern observed. Plasma chloride in razorback sucker remained significantly
elevated above resting levels in 84% of the sample times and osmolality in 74%. Part of the
explanation may be the use of NaCl after each handling event and during the transport.
A point of interest involving the use of NaCl during transport was observed in the rapid
decrease in plasma cortisol levels from loading to 6 h post-loading (mid-trip of transport),
especially in the bonytail (Figure 2). Barton and Zitzow (1995) reported lower cortisol levels in
walleye (Stizostedion vitreum) that were allowed to recover in 0.5% NaCl water after a handling
stress compared to the fish placed in freshwater. Although the authors did not observe a
difference in the initial stress response at 1 h post-handling, by 3 h post-handling significantly
lower plasma cortisol levels were noted in fish held in 0.5% NaCl. The 0.5% NaCl used in the
transport water of this study appears to have had a similar effect on bonytail and possibly
razorback sucker, although razorback sucker cortisol recovery was generally faster than bonytail
recovery at all sampling times in this study. Bonytail plasma cortisol levels decreased 54.3% in
the 6 h between the loading event and the mid-trip sample compared to a decrease of 6.6% and
33.1% in the 12 h post-harvest and post-tagging samples, respectively. Both species were
exposed to 0.5% NaCl in the raceways during the prophylactic treatment given after the harvest
and tagging events, but the resident time of the NaCl is only 1 to 2 h in flow-through raceways
(M. Ulibarri, SNARRC, personal communication). That short exposure time may have limited
13

the effect of NaCl on plasma cortisol compared to the longer constant exposure experienced
within the transport tank. An additional impact of NaCl on bonytail may be observed in the
attenuated plasma cortisol response at stocking in comparison to the other handling events
(harvest, tagging, and loading). Walleye maintained in 0.5% NaCl water that were subsequently
exposed to a second stressor also exhibited a lower stress response than fish maintained in
freshwater (Barton and Zitzow 1995).
In contrast to the presumed beneficial effect of NaCl on plasma cortisol levels, plasma
glucose levels were observed to increase in bonytail and razorback sucker during transport
(Figures 2 and 3). Similar effects of NaCl treatments on plasma glucose have been reported by
Burgdorf-Moisuk et al. (2011) in goldfish (Carassius auratus) and Chagas et al. (2012) in pacu
(Colossoma macropomum). Conversely, substantial decreases in plasma glucose levels were
observed in striped bass (Morone saxatilis) transported for 5 h in 1% NaCl water (Mazik et al.
1991). Sodium chloride has been used regularly in aquaculture to reduce stress and treat fish for
external maladies, but the full physiological consequences of its use on specific fish species is
rarely understood. It is doubtful the increased plasma glucose levels had long-term impacts on
the bonytail or razorback sucker due to the transient nature of the spike, but further research is
needed to determine the extent of any positive or negative effects of NaCl based on
concentration and other environmental factors such as water chemistry (e.g. temperature,
hardness, and alkalinity).
In addition to documenting the stress response and the short-term recovery of the fish,
another important aspect of this study is the long-term impact of handling and stress on these
endangered fish species. Gaining an understanding of the condition of the fish post-stocking is
critical for resource managers to evaluate the ability of the fish to adjust to a new environment.
14

Although we were unable to maintain the fish under a setting that mimics natural post-stocking
conditions, holding the bonytail and razorback suckers for 21 d at Achii Hanyo provided the
opportunity to monitor the metabolic recovery of the fish and document any incidence of delayed
mortality. Between 24 and 48 h post-hauling, plasma cortisol, glucose and lactate levels in
bonytail and razorback sucker returned to or below resting levels and generally remained
comparable to resting levels through the 21 d sample time (Figures 2 and 3). The spike in plasma
cortisol in both the bonytail and razorback suckers at 21 d post-hauling was associated with
difficulty in netting the fish from the recovery tanks.
During the 21 d monitoring period at Achii Hanyo, no mortalities were observed in the
razorback sucker. In the bonytail from SNARRC maintained in Colorado River water, one
mortality was observed at 12 d post-hauling. In another tank, two additional mortalities were
recorded, one each at 18 and 19 d post-hauling. Three bonytail also were lost at 24 h posthauling when they jumped out of the well water control tanks during the night. The dead fish had
lesions on their abdomens, but external parasites and pathogens were not observed. However, at
the 21 d post-hauling sample, visual observations of ich (Ichthyophthirius multifiliis) and anchor
worms (Lernaea cyprinacea) were recorded on all remaining fish from SNARRC maintained in
Colorado River water (Figures 4A and B). Anchor worms were also present on the bonytail
reared at Achii Hanyo and maintained in Colorado River water, but ich was not observed on
those fish. The bonytail from SNARRC maintained in well water did not exhibit ich or anchor
worm infestations. Ich can be a pathogen of concern at Achii Hanyo when handling fish (M.
Olson, Willow Beach National Fish Hatchery, personal communication) and the water
temperatures at the time of the study with the bonytail (21 to 25 C) were optimum for the
outbreak. We recognize that the artificial conditions at Achii Hanyo under which the fish were
15

held presented a greater exposure risk to ich compared to a natural environment, but the lack of
an infestation on the Achii Hanyo bonytail held under the same parameters suggests that the
nave fish from SNARRC were more susceptible. Although published reports have documented
the effect of stress on fish susceptibility to disease (Snieszko 1974; Wedemeyer and Wood 1974;
Ellsaesser and Clem 1986; Dror et al. 2006), one unanswered question from this study is whether
the prior handling stress contributed to the outbreak of ich on the bonytail transported from
SNARRC. In contrast, there were no pathogens observed on the razorback sucker at any time at
Achii Hanyo. That may be partially attributed to an earlier transport date and cooler water
temperatures (16 to 19 C), but differences have been previously reported between the
susceptibility of bonytail and razorback sucker to ich (Tyus et al. 1999). Determining the impact
of handling stress to the sensitivity of bonytail subsequently exposed to pathogens ubiquitous in
their native waters may shed additional light on the lack of success in repatriation attempts for
this species.
RECOMMENDATIONS
The current handling protocols in place at SNARRC did elicit stress responses in both
bonytail and razorback sucker, but the time between each handling event was sufficient to allow
recovery of the fish before exposure to the next stressor. Plasma cortisol, glucose, and lactate
levels in bonytail and razorback suckers were comparable to resting levels by 48 h post-handling,
but plasma chloride and osmolality levels remained variable throughout the study. We
recommend a recovery period of no less than 48 h between sequential handling events at
hatcheries rearing these species, but suggest 5-7 d if stocking schedules allow. Also, a minimum
of 48 h recovery at the stocking site is strongly recommended before a full release into their
native waters.
16

Further investigation into the effects of stress on disease susceptibility, especially with
bonytail, is warranted for a better understanding of the long-term implications of handling stress.
The lack of success in the bonytail recovery program is no doubt multi-faceted, but the link
between stress and disease may have a significant role in the inability of artificially-propagated
fish to survive after stocking. Also, exploring the physiological benefit versus cost aspect of
using specific concentrations of NaCl after handling might provide further insight into optimal
handling procedures for these species.

ACKNOWLEDGEMENTS
Funding for this project was provided by the U.S. Bureau of Reclamation in partial fulfillment of
a cooperative agreement with the Lower Colorado River Multi-Species Conservation Plan under
Work Task C11. Sincere thanks goes to a first-class fish bleeding crew (Teresa Lewis, Izhar
Khan, Renee Martin, Jason Nachtmann, Justin Merrifield, Rene Gallindo, and Andrea Montony)
and to Manuel Ulibarri, William Knight, Ian Paige, and Kim Yazzie for their tireless assistance
with this study. A special thank you is given to Mark Olson for providing tanks at Achii Hanyo
and the Willow Beach/Achii Hanyo crew for their assistance with the 21 d monitoring period.

17

LITERATURE CITED
Barton, B. A., A. H. Haukenes, B. G. Parsons, and J. R. Reed. 2003. Plasma cortisol and chloride
stress responses in juvenile walleyes during capture, transport, and stocking procedures.
North American Journal of Aquaculture 65:210-219.
Barton, B. A., and R. E. Peter. 1982. Plasma cortisol response in fingerling rainbow trout, Salmo
gairdneri Richardson, to various transport conditions, anesthesia, and cold shock. Journal
of Fish Biology 20:39-51.
Barton, B. A., R. E. Peter, and C. R. Paulencu. 1980. Plasma cortisol levels of fingerling rainbow
trout (Salmo gairdneri) at rest, and subjected to handling, confinement, transport, and
stocking. Canadian Journal of Fisheries and Aquatic Sciences 37:805-811.
Barton, B. A., and R. E. Zitzow. 1995. Physiological responses of juvenile walleyes to handling
stress with recovery in saline water. Progressive Fish-Culturist 57:267-276.
Bestgen, K. R., K. A. Zelasko, R. I. Compton, and T. E. Chart. 2008. Survival, condition, habitat
use, and predation on stocked bonytail (Gila elegans) in the Green River, Colorado and
Utah. The Southwestern Naturalist 53:488-494.
Bracewell, P., I. G. Cowx, and R. F. Uglow. 2004. Effects of handling and electrofishing on
plasma glucose and whole blood lactate of Leuciscus cephalus. Journal of Fish Biology
64:65-71.
Burgdorf-Moisuk, A., M. A. Mitchell, and M. Watson. 2011. Clinical and physiological effects
of sodium chloride baths in goldfish (Carassius auratus). Journal of Zoo and Wildlife
Medicine 42:586-592.

18

Chagas, E. C., L. D. de Araujo, L. C. Gomes, J. C. O. Malta, and A. M. B. Varella. 2012. Effect

of sodium chloride on physiological responses and monogenean control in tambaqui
(Colossoma macromum). Acta Amazonica 42:439-444.
Cho, S. J., C. A. Caldwell, and W. R. Gould. 2009. Physiological stress responses of Rio Grande
silvery minnow: effects of individual and multiple physical stressors of handling,
confinement, and transport. North American Journal of Fisheries Management 29:16981706.
Davis, K. B., and N. C. Parker. 1986. Plasma corticosteroid stress response of fourteen species of
warmwater fish to transportation. Transactions of the American Fisheries Society
115:495-499.
Dexter National Fish Hatchery and Technology Center. 2004. Fish handling and transport
protocol developed for Colorado pikeminnow.
Dror, M., M. S. Sinyakov, E. Okun, M. Dym, B. Sredni, and R. R. Avtalion. 2006. Experimental
handling stress as infection-facilitating factor for the goldfish ulcerative disease.
Veterinary Immunology and Immunopathology 109:279-287.
Ellsaesser, C. F., and L. W. Clem. 1986. Haematological and immunological changes in channel
catfish stress by handling and transport. Journal of Fish Biology 28:511-521.
Fagundes, M., and E. C. Urbinati. 2008. Stress in pintado (Pseudoplatystoma corruscans) during
farming procedures. Aquaculture 276:112-119.
Hunt, T. A., D. L. Ward, C. R. Propper, and A. C. Gibb. 2012. Effects of capture by trammel net
on Colorado River native fishes. Journal of Fish and Wildlife Management 3:133-141.
Jentoft, S., A. H. Aastveit, P. A. Torjesen, and O. Andersen. 2005. Effects of stress on growth,
cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and
19

domesticated rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and

Physiology 141A:353-358.
Karam, A. P., and P. C. Marsh. 2010. Predation on adult razorback sucker and bonytail by
striped bass in Lake Mohave, Arizona-Nevada. Western North American Naturalist
70:117-120.
Kortan, J., J. Blahova, K. Kruzikova, and Z. Adamek. 2011. Stress responses of carp pond fish
stock upon hunting activities of the great cormorant (Phalacrocorax carbo sinensis L.).
Aquaculture Research 42:322-330.
Mazik, P. M., B. A. Simco, and N. C. Parker. 1991. Influence of water hardness and salts on
survival and physiological characteristics of striped bass during and after transport.
Transactions of the American Fisheries Society 120:121-126.
Nikinmaa, M., A. Soivio, T. Nakari, and S. Lindgren. 1983. Hauling stress in brown trout (Salmo
trutta): physiological responses to transport in fresh water or salt water, and recovery in
natural brackish water. Aquaculture 34:93-99.
Olson, M. 2011. Willow Beach National Fish Hatchery, Willow Beach, Arizona, personal
communication.
Pottinger, T. G. 1998. Changes in blood cortisol, glucose and lactate in carp retained in anglers
keepnets. Journal of Fish Biology 53:728-742.
Pottinger, T. G. 2010. A multivariate comparison of the stress response in three salmonid and
three cyprinid species: evidence for inter-family differences. Journal of Fish Biology
76:601-621.
Snieszko, S. F. 1974. The effects of environmental stress on outbreaks of infectious disease of
fishes. Journal of Fish Biology 6:197-208.
20

Sykes, C. L., C. A. Caldwell, and W. R. Gould. 2011. Physiological effects of potassium

chloride, formalin, and handling stress on bonytail. North American Journal of Fisheries
Management. 31:291-298.
Tanck, M. W. T., G. H. R. Booms, E. H. Eding, S. E. Wendelaar Bonga, and J. Komen. 2000.
Cold shocks: a stressor for common carp. Journal of Fish Biology 57:881-894.
Tyus, H. M., W. C. Starnes, C. A. Karp, and J. F. Saunders III. 1999. Effects of invasive tissue
collection on rainbow trout, razorback sucker, and bonytail chub. North American
Fisheries Management 19:848-855.
Ulibarri, M. 2013. Southwestern Native Aquatic Resources and Recovery Center, Dexter, New
Mexico, personal communication.
Wedemeyer, G. A., and J. W. Wood. 1974. Stress as a predisposing factor in fish diseases. Fish
Disease Leaflet 38. U.S. Fish and Wildlife Service. 8 p.
Wedemeyer, G. A., B. A. Barton, and D. J. McLeay. 1990. Stress and acclimation. Pages 451489 in C. B. Schreck and P. B. Moyle, editors. Methods for fish biology. American
Fisheries Society, Bethesda, Maryland.

21

Stocking

21 Days
Post-Hauling

21-d

14-d

7-d

48-h

Monitoring Period
24-h

End-transport

Haul

0.25-h

14.5
Hours

Mid-transport

Loading
0.25-h

Pre-Loading

48-h

Recovery Period
24-h

Tagging

9 Days (BT)
14 Days (RB)

12-h

48-h

24-h

12-h

Recovery Period

0.25-h

7 Days (BT)
12 Days (RB)

Pre-Tagging

Sampling
Times

Resting

Handling
Events

0.25-h

Pond Harvest

FIGURES

Figure 1. Schedule of handling events and blood collection times for bonytail (BT) and
razorback sucker (RB) harvested from ponds and held in outdoor raceways in preparation for
stocking. After a 7 d (BT) or 12 d (RB) recovery period fish were tagged. Following a second
recovery period (9 d for BT and 14 d for RB), fish were loaded into a transport truck, hauled to
Achii Hanyo as a simulated stocking trip, and held for a 21 d monitoring period.

22

7d
Recovery

9d
Recovery

*
*

14.5 h
Haul

21 d
Monitoring

7d
Recovery

9d
Recovery

14.5 h
Haul

21 d
Monitoring

*
*

*
*

* *

*
*

* *
*
*

*
*

*
*
*

Figure 2. Average plasma cortisol, glucose, lactate, chloride, and

osmolality levels in resting bonytail and bonytail subjected to harvest,
tagging, and hauling protocols through 21 d post-hauling. Open boxes
designate handling events and the vertical dotted lines delineate the
recovery periods. Values are means SEs based on two replicates and
either 6 or 10 fish per replicate (n = 12 or 20). Asterisks denote
detectable difference from resting levels.

23

7d
Recovery

9d
Recovery

14.5 h
Haul

7d
Recovery

21 d
Monitoring

*
*

9d
Recovery

14.5 h
Haul

21 d
Monitoring

*
* *

* * *

* *
* *

* *

*
*

* *

*
*
*

*
*
*

* *

* * * * *

* * *

Figure 3. Average plasma cortisol, glucose, lactate, chloride and

osmolality levels in resting razorback sucker and razorback sucker
subjected to harvest, tagging, and hauling protocols through 21 d posthauling. Open boxes designate handling events and the vertical dotted
lines delineate the recovery periods. Values are means SEs based on
two replicates and either 6 or 10 fish per replicate (n = 12 or 20).
Asterisks denote detectable difference from resting levels.

24

Figure 4. Ichthyophthirius multifiliis (A) and Lernaea cyprinacea (B) on bonytail transported from SNARRC and maintained for 21 d in Colorado
River water at Achii Hanyo.

25

BUDGET
Project Charges for 2011
Materials and Supplies
Utilities & Equipment Maintenance
17% Admin Overhead

$ 45,032.38
3,940.82
8,325.44

Net Funds available for 2011

Subtotal Expended as of Sept. 30, 2011

(100,000.00)
57,298.64

Carryover to 2012

(42,701.36)

Project Charges for 2012

O&M Labor Costs
Materials and Supplies
17% Admin Overhead
Net Funds available for 2012
Subtotal Expended as of Sept. 30, 2012
Carryover to 2013

$ 60,957.10
14,795.72
12,877.98
(142,701.36)
88,630.80
(54,070.56)

Project Charges for 2013

O&M Labor Costs
Materials and Supplies
Utilities & Equipment Maintenance
17% Admin Overhead

$ 60,497.36
26,434.76
1,607.31
15,051.70

Net Funds available for 2013

Subtotal Expended as of Sept. 30, 2013

(104,070.56)
103,591.13

Total Project Funds

Total Project Expenditures

(250,000.00)
249,520.57

Unused balance

(479.43)

26