The Effect of AQUI‐S® Sedation on Primary,Secondary, and Tertiary Stress Responses during Salmon Smolt,Salmo salar L., Transport and Transfer to Sea

The purpose of this study was to determine the effect of AQUI‐S^® (5.0 mg/L) sedation compared with nonsedation on the primary (plasma cortisol), secondary (osmoregulation), and tertiary (mortality) stress responses in Atlantic salmon smolts during transport and transfer to sea. AQUI‐S sedation during on‐ and off‐loading sufficiently reduced the primary stress response to lower mortality (2.5%) during transfer to sea compared with unsedated fish, which experienced a mortality rate above 11.5%. The unsedated fish experienced an acute mortality, which only stabilized 16 d after the transport. None of the secondary stress responses measured in this experiment could contribute in explaining this phenomenon, with the possible exception of plasma magnesium. While osmolality and chloride increased in both groups during the experiment, it did not seem to be related to the difference in the primary stress response documented in the sedated and unsedated groups. It instead appeared to be an effect of the salinity change from freshwater to seawater after transport. Plasma magnesium differed between the groups, while plasma magnesium in the AQUI‐S‐sedated group returned to prestress levels 12 h after transport. The unsedated group showed no such recovery even 1 wk after transport, which may indicate a disturbance in hydromineral balance and give plausible explanation for delayed mortality this group experienced. AQUI‐S shows promise as a stress‐reducing sedative for Atlantic salmon smolts and if used properly could improve animal welfare and survivability during and after common aquaculture‐related incidents.     

Potential benefit of clove oil sedation on animal welfare during salmon smolt, Salmo salar L. transport and transfer to sea

The purpose of this study was to determine the effect of clove oil (4.0 mg L⁻¹) sedation, compared with non-sedation, on the primary (plasma cortisol), secondary (osmoregulation) and tertiary (mortality) stress responses in Atlantic salmon smolts during transport and transfer to sea. Clove oil sedation during on- and off-loading sufficiently reduced the primary stress response to lower mortality (2.1%) during transfer to sea compared with unsedated fish, which experienced a mortality rate above 12.2%. The unsedated fish experienced an acute mortality that stabilized only 6 days after the transport. None of the secondary stress responses measured in this experiment could contribute towards explaining this phenomenon, with the possible exception of plasma magnesium (Mg²⁺). Plasma Mg²⁺ differed between the groups; while plasma Mg²⁺ in the clove oil sedated group returned to pre-stress levels 72 h after transport, the unsedated group showed no such recovery even 1 week after transport, which may indicate a disturbance in the hydromineral balance, and provides a plausible explanation for the delayed mortality in this group. Eugenol-based anaesthetics appear to be promising as a stress-reducing sedative for Atlantic salmon smolts, and, if used properly, this chemical could improve animal welfare and survivability during and after common aquaculture-related incidents.     

Anesthesia induces stress in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>), Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) and Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3–4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2–2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.     

Whole-body corticosteroid and plasma cortisol concentrations in larval and juvenile Atlantic cod Gadus morhua L. following acute stress

Methods were developed to assess whole‐body immunoreactive corticosteroid concentrations (IRC) and plasma levels of cortisol in Atlantic cod subjected to several common, acute stressors. A measurable corticosteroid stress response was observed at the first sampling in whole bodies of 8‐day post‐hatch (dph) larvae. Two groups of juveniles (～5 and 30 g) were subjected to a 30 s net stressor and whole‐body IRC and plasma cortisol levels were determined. Post‐stressor IRC in smaller fish rose approximately 14‐fold, peaked at 1 h, were sustained for 3–6 h and returned to pre‐stressor levels within 24 h. Post‐stressor plasma cortisol levels in larger fish rose approximately 18‐fold, peaked at 0.5–1 h, were sustained for 1–3 h and then returned to near pre‐stressor levels after 24 h. Immunoreactive corticosteroid concentrations appeared to remain elevated longer than plasma cortisol levels suggesting that steroids other than cortisol were contributing to total immunoreactivity in homogenates. Juveniles exposed to either a grading procedure or high density transport had maximal IRC and plasma cortisol levels within 90 min which returned to pre‐stressor levels within 24–72 h. A reduction in water salinity (20 g L^?1) did not moderate the corticosteroid response in juveniles. The results show that Atlantic cod respond to common, acute stressors in a manner similar to other teleosts. Whole‐body homogenates can be used to identify changes in IRC in response to acute stress in cod with the caveat that recovery IRC may differ from plasma cortisol concentrations.     

Short-term handling stress affects the humoral immune responses of juvenile Atlantic cod,Gadus morhua

Juveniles of Atlantic cod, Gadus morhua L., weighing 30–50 g were subjected to short-term handling stress by exposing them to air for 3 min and immediately placing in the rearing tank for recovery. Indices for the primary stress response as well as selected immune- and metabolism-related parameters were measured in the plasma before the application of the stressor and at 1-h, 24-h and 7-day post-stress. Plasma cortisol significantly increased at 1-h post-stress then returned to the prestress levels at 24-h post-exposure. Plasma glucose and total antioxidant capacity had two peaks at 1 h and 7 days after handling stress. Alkaline phosphatase significantly increased at 24-h post-exposure, while no significant changes in myeloperoxidase activity was observed. Lysozyme and antiprotease activities were significantly downregulated at 1- and 24-h post-exposure, respectively. These findings showed that short-term handling stress in Atlantic cod juveniles resulted in temporal elevation of the primary stress response and metabolism with a corresponding decrease in its defense against bacterial pathogens.     

Stress responses of Atlantic salmon (Salmo salar L.) elicited by water level reduction in rearing tanks

The Atlantic salmon (Salmo salar) stress response was examined by measuring plasma cortisol, glucose and chloride in fish after water level reduction within rearing tanks. Maximum plasma cortisol levels (366±43 (SD) nM and 534±280 nM for Groups 1 and 2, respectively) were observed 20 min after application of the stressor. Cortisol levels were down to control levels 24 h later. The pattern for cortisol changes observed within these two groups were comparable. Plasma glucose and chloride concentrations did not change significantly in either of the experimental groups. The fish in group 2 were then repeatedly subjected to the same stressor every third day. After the fifth exposure to the stressor, blood was obtained from fish at times corresponding to sampling after the first exposure. Their maximum plasma cortisol level only reached 223 (±96) nM, and was down to prestress levels within 2 h. Plasma glucose and chloride concentrations did not change significantly in this second experiment. This challenge test revealed an acute primary response in Atlantic salmon without any apparent harmful secondary responses, that may thus serve as a standardized reference stressor using other fish groups under comparable conditions.     

Stress response to anthropogenic noise in Atlantic cod Gadus morhua L.

The potential effects of anthropogenic noise on the physiology of Atlantic cod have not been well described. The aim of the present study was to investigate the impact of anthropogenic noise on Atlantic cod stress response using cortisol as a biomarker as well as on broodstock spawning performance. Results showed that artificial noise consisting of a linear sweep from 100 to 1000 Hz can induce a transient and mild cortisol elevation with a clear noise intensity dose response. In all cases plasma levels returned to baseline levels <1 h post sound exposure. Daily exposure to a similar intensity and frequency noise range applied habitually to a broodstock population during the spawning window resulted in a significant reduction in total egg production and fertilisation rates thus reducing the total production of viable embryos by over 50%. In addition, a significant negative correlation between egg cortisol content and fertilisation rate was observed. These results confirm that cod can perceive noise generated within a frequency range of 100–1000 Hz and display a heightened cortisol plasma level. In addition, anthropogenic noise can have negative impacts on cod spawning performances.     

Changes in selected stress and immune‐related genes in Atlantic cod,Gadus morhua,following overcrowding

Using Atlantic cod, Gadus morhua, as a model for the stress response in gadoid fish, the changes in the expression of some stress and immune genes as well as the profiles of plasma cortisol were examined. Adult fish were kept at a density of ca. 100 kg m^?3 by lowering the water level in the rearing tank for 1 h and this short‐term crowding stress was repeated thrice over a 12‐h interval period. Blood samples were collected before exposure and at 2, 24 and 72 h post crowding. Plasma cortisol level significantly increased at 2 h post crowding but returned to pre‐crowding levels 24 h after exposure. The relative expression of the stress response genes, glucose transporter‐3 and a putative heat shock protein 70 significantly increased at 2 and 24 h post crowding respectively. Significant up‐regulation of the pro‐inflammatory cytokines, interleukin (IL)‐1β and IL‐8, as well as anti‐bacterial genes, g‐type lysozyme and bactericidal permeability‐increasing protein/lipopolysaccharide‐binding protein (BPI/LBP) was also observed at 2 h and the levels were maintained until 72 h post exposure, except for BPI/LBP which had maximum up‐regulation at 24 h. The present observations have implications with respect to fish welfare and assessment of the health status of the farmed fish.     

Physiological short-term response to sudden salinity change in the Senegalese sole (Solea senegalensis)

The physiological responses of Senegalese sole to a sudden salinity change were investigated. The fish were first acclimated to an initial salinity of 37.5?ppt for 4?h. Then, one group was subjected to increased salinity (55?ppt) while another group was subjected to decreased salinity (5?ppt). The third group (control group) remained at 37.5?ppt. We measured the oxygen consumption rate, osmoregulatory (plasma osmolality, gill and kidney Na⁺,K⁺-ATPase activities) and stress (plasma cortisol and metabolites) parameters 0.5 and 3?h after transfer. Oxygen consumption at both salinities was higher than for the control at both sampling times. Gill Na⁺,K⁺-ATPase activity was significantly higher for the 55?ppt salinity at 0.5?h. Plasma osmolality decreased in the fish exposed to 5?ppt at the two sampling times but no changes were detected for high salinities. Plasma cortisol levels significantly increased at both salinities, although these values declined in the low-salinity group 3?h after transfer. Plasma glucose at 5?ppt salinity did not vary significantly at 0.5?h but decreased at 3?h, while lactate increased for both treatments at the first sampling time and returned to the control levels at 3?h. Overall, the physiological response of S. senegalensis was immediate and involved a rise in oxygen consumption and plasma cortisol values as well as greater metabolite mobilization at both salinities.     

Cortisol response of golden shiners (Notemigonus crysoleucas) fed diets differing in lipid content

The purpose of this study was to characterize the plasma cortisol response in golden shiners (Notemigonus crysoleucas) under crowding stress, and to determine whether dietary lipid affects the cortisol response. The main differences between the diets were the relative amounts and types of fatty acids of the n-3 and n-6 families. In experiment 1, triplicate groups of golden shiners were fed diets with 4 or 13% menhaden fish oil, or 4 or 13% poultry fat at 3% body weight for 4 weeks before the crowding stress. In experiment 2, fish were fed diets with 8% lipid from soybean oil (SBO), cod liver oil (CLO), (SBO+CLO), or cottonseed oil (CSO) for 6 weeks before the stress test. Plasma cortisol was measured initially, two hours after the stressor was applied, and two hours after the stressor was removed. The overall pattern of the cortisol response was similar to that seen in other warmwater fishes, but the rate and magnitude of the response were not affected by diet in either experiment. Golden shiners have an intermediate cortisol response compared to other fish that have been exposed to similarly stressful conditions.     

Effects of dorsal aorta cannulation on the stress response of channel catfish (Ictalurus punctatus)

The stress response to dorsal aorta cannulation and serial blood sampling was examined in the channel catfish Ictalurus punctatus. Channel catfish cannulated and repetitively sampled once a day for 7 days did not exhibit a change in response to surgery or to the 24h sample regime as measured by plasma cortisol, glucose, and chloride. In fish that were either serially bled (0, 1, 3, 6, 9, 12, 24, and 48h) immediately after surgery or allowed to recover 6 days before being serially bled, overall plasma cortisol levels were higher than those of fish bled every 24h. Catfish serially bled immediately after surgery had significantly higher plasma glucose levels compared with catfish allowed to recover from surgery 6 days before serial sampling. Although channel catfish recover from cannulation surgery in 24h, a longer recovery period may be needed prior to serial sampling if the samples are taken more frequently than every 24h.     

Osmoregulatory balance in Murray cod, Maccullochella peelii peelii (Mitchell), affected with chronic ulcerative dermatopathy

This study examined the osmoregulatory capability of Murray cod, Maccullochella peelii peelii, affected by chronic ulcerative dermatopathy (CUD) in intensive aquaculture. This condition appears to arise only in facilities utilizing groundwater, with the causative agent suggested to be a water-borne factor. Healthy Murray cod (~ 700 g) were transferred to a CUD-affected farm to monitor the progression of the syndrome and began to show signs of CUD after approximately five months. In order to evaluate possible effects of CUD on osmoregulation; plasma electrolyte concentrations, osmolality, and Na⁺,K⁺-ATPase activities were measured, and gill histology and immunohistochemistry were analyzed. Plasma electrolyte concentrations and osmolality of CUD-affected Murray cod were consistent with reference values determined for non CUD-affected fish. A greater number of gill mucous cells were observed in Murray cod cultured at the CUD-affected farm compared to non CUD-affected fish. We also found an un-identified cell type that was present solely in the gills of CUD-affected Murray cod. Gill Na⁺,K⁺-ATPase activity was significantly higher in severely CUD-affected Murray cod compared to individuals transferred to the CUD-affected farm. While there appeared to be some minor changes in the gills of CUD-affected fish, this study demonstrated that Murray cod were able to effectively osmoregulate, although, perhaps at an energetic cost.     

Cortisol stimulates intestinal fluid uptake in Atlantic salmon (Salmo salar) in the post-smolt stage

The fluid uptake rate of the posterior intestine of salmonids increases during the parr-smolt transformation. Intestinal fluid uptake in post-smolt Atlantic salmon was investigated after treatment with cortisol and growth hormone (GH), alone or together. Two replicate experiments were conducted in August 1991 and August 1992. Cortisol was emulsified in vegetable shortening and vegetable oil (1:1) and implanted into the peritoneal cavity. GH was administered as intraperitoneal injections in a saline vehicle on days 0 and 2. On days 5 and 6, plasma cortisol levels, gill Na⁺,K⁺-ATPase activity, andin vitro measurements of fluid transport rate (J_v) across the posterior intestine were measured. Implants of cortisol elevated the plasma cortisol levels within a physiological range, and resulted in elevated gill Na⁺,K⁺-ATPase activity, as expected. The fluid uptake rate across the posterior intestine was roughly doubled by cortisol treatment. GH treatment did not affect intestinal fluid transport, gill Na⁺,K⁺-ATPase activity, or plasma cortisol concentrations. The seawater-adapting increase in the rate of fluid uptake by the posterior intestine of smolting salmon is probably stimulated by elevated plasma cortisol concentrations.     

The acute stress response in fed and food deprived Atlantic cod, Gadus morhua L.

The aim of the present study was to evaluate the effect of stress and nutritional state (fed vs food deprived) on the generalized stress response and intestinal integrity in Atlantic cod (Gadus morhua L.). Cod in feeding or food deprived states were subjected to 15 min of acute stress (exhaustive exercise). Blood was collected at 9 intervals from before stress (t = 0), to t = 48 h post stress and analysed for blood haematocrit and haemoglobin, and plasma cortisol, lactate, glucose, osmolality, chloride, as well as the tissue damage indicators glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and thiobarbituric acid reactive substances. Intestinal segments were prepared for histology with the same intervals, while assessment of intestinal integrity and microbiology was performed at t = 0, 4 and 48 h post stress.Subjecting cod to exhaustive stress initiated a standard stress response including increased blood Hct and plasma cortisol, glucose, chloride, osmolality and lactate. Food deprived fish did in general have reduced stress resistance compared to fed fish. For many parameters, cod returned slowly to basal levels. Cellular indicators of tissue damage and oxidative stress increased in a biphasic manner following stress. Stress did not affect gut histology but did transiently increase gut permeability. Furthermore, stress had no effect on the adherent bacterial population level in midgut, but did cause a small decrease in hindgut (non-significant) and hindgut chamber (p < 0.05). Isolates belonging to Carnobacterium were predominant but not affected by stress.In conclusion, food deprived cod are less resistant to stress than fed cod. The magnitude of the response is less than in salmonids, but the effects are persistent (including tissue damage indicators and oxidative stress) and may have negative long term consequences. The gut is relatively resistant to stress, there is however a transient increase in the intestinal permeability and alterations in microbiota that may indicate lower protection against invading pathogens.     

Transport stress in Atlantic salmon (Salmo salar L.): anaesthesia and recovery

The effects of metomidate anaesthesia on levels of plasma cortisol, glucose, haematocrit and chloride in Atlantic salmon (1⁺) (Salmo salar L.), after a 2‐h transport and during a 48‐h recovery period were investigated. The use of metomidate anaesthesia during transport led to a reduced release of cortisol and significantly lower levels of plasma cortisol after a 48‐h recovery period. Plasma glucose did not return to basal level after a 48‐h recovery period, indicating that even longer recovery may be needed for the fish to return to a pre‐stress state. The results show that metomidate anaesthesia combined with a recovery period lessens the stress burden imposed by hauling and transport.     

Effect of acclimation temperature on the acute stress response in juvenile Atlantic cod, Gadus morhua L., and haddock, Melanogrammus aeglefinus L.

Juvenile Atlantic cod (Gadus morhua L.) and haddock (Melanogrammus aeglefinus L.) were subjected to 30 s air exposure stressors following acclimation to 4, 10 and 14 and 4, 8 and 14°C respectively. Both species responded to the stressor with increases in plasma cortisol at all temperatures tested. At 14°C cortisol levels peaked within 1 h post‐stressor, and returned to pre‐stressor levels within 24 h. In contrast, at 4°C, peak cortisol levels were not attained until 6 h post‐stressor in haddock and remained elevated beyond 24 h in both species. The rate of plasma glucose accumulation was greater at higher temperatures in both species and no increase was seen at 4°C. Lysozyme activity in cod, in response to the stressor, was lower than the values reported for some other species and increased slightly at 14°C. The results show that cod and haddock acclimated to different temperatures respond to common, acute stressors in a manner similar to other teleosts.     

The ability of clove oil and MS-222 to minimize handling stress in rainbow trout (Oncorhynchus mykiss Walbaum)

The effects of 60‐mg L^?1 clove oil and 60‐mg L^?1 tricaine methanesulphonate (MS‐222) on the blood chemistry of rainbow trout were compared after exposure to handling stress via caudal puncture blood sampling. Fish sampled by caudal puncture and subsequently exposed to anaesthetics showed a typical handling stress response over a 48‐h period. There were no significant differences between the responses of fish exposed to equal concentrations of clove oil and MS‐222, with the following exceptions: the blood glucose at full anaesthesia, and lactate at full recovery increased significantly in the clove oil‐exposed fish. In a subsequent experiment, the stress response observed in fish sampled by caudal puncture and exposed to clove oil and MS‐222 was compared with a non‐anaesthetized control group. The increases in plasma cortisol levels were significantly lower at recovery in fish treated with either anaesthetic compared with the control fish. Fish exposed to MS‐222 had significantly higher cortisol levels at 1 h. These findings show that few differences exist between the anaesthetic effects of clove oil and MS‐222 on the physiological response of fish to stress. However, clove oil is more effective at reducing the short‐term stress response induced by handling and blood sampling, and is recommended as an alternative fish anaesthetic.     

Changes in arterial PO₂, physiological blood parameters and intracellular antioxidants in free-swimming Atlantic cod (Gadus morhua) exposed to varying levels of hyperoxia

Free-swimming Atlantic cod (Gadus morhua) were exposed to water oxygen pressures (P _wO₂) ranging from 18.1 to 41.5 kPa and sampled for blood using an indwelling caudal artery cannula. Arterial blood oxygen pressure (P _aO₂) increased with increasing P _wO₂, from 12.0 kPa in normoxia (18.1 kPa) to 34.2 kPa in the highest hyperoxic level tested (41.5 kPa). Blood CO₂ pressure and plasma bicarbonate concentration increased with P _wO₂, indicating reduced ventilation with increased P _wO₂. Plasma glucose, sodium and potassium were not affected by water oxygen level. Blood oxidative stress biomarkers, reduced glutathione, oxidized glutathione and the oxidative stress index (ratio between oxidized and total glutathione) differed intermittently between normoxia and hyperoxia. The oxidative stress index was higher in the blood of exposed compared to unexposed control cod. Together with elevated P _aO₂, these findings suggest increased production of reactive oxygen species and increased oxidative stress in Atlantic cod exposed to hyperoxia.     

Cortisol and hematological response in Brazilian codling, Urophycis brasiliensis (Pisces, Phycidae) subjected to anesthetic treatment

The effects of benzocaine anesthesia on hematological parameters (erythrocyte and platelet number, hemoglobin concentration and hematocrit), plasma electrolytes (Na⁺, K⁺ and Cl⁻), cortisol, glucose and cholesterol levels in Brazilian codling (Urophycis brasilensis) exposed to a benzocaine bath solution were investigated. Cortisol and glucose levels 1 h after capture from the wild and on days 7 and 14 during the acclimatization period (20 days) were also determined. During the second week, cortisol levels reached to basal values (9 ng ml⁻¹). The use of benzocaine causes a significant increase in the erythrocyte number, in the concentration of hemoglobin and sodium and a decrease in chloride. Plasma cortisol and cholesterol levels were higher in fish exposed to the anesthetic, and glucose levels decreased significantly comparing with the control group, showing strong fluctuations between individuals in all the cases. These results show that the use of benzocaine causes an acute stress response that could be reverted quickly and it has to be considered during the evaluation of stress in fish.     

Effect of handling and fish size on secondary changes in carbohydrate metabolism in Atlantic salmon, Salmo salar L.

Two size groups (0.5 and 3 kg) of Atlantic salmon, Salmo salar L., were stressed by netting and transport from sea cages into land-based tanks. Handling lasted for 1 h. Before onset of stress, basal concentrations of haematocrit and haemoglobin, plasma cortisol and glucose, and liver and muscle glycogen were measured. All levels were within ranges reported for salmon. After handling and at chosen time intervals during recovery, samples were taken to judge the impact of handling and whether secondary changes in carbohydrate metabolism were related of fish size. A low cortisol peak indicated a mild stress reaction after handling independent to fish size. Plasma glucose peaked as cortisol declined, and returned to basal levels within 48 h. Liver glycogen seemed to be the main source of plasma glucose. No changes were measured in muscle glycogen concentrations. The results indicate a high tolerance to handling stress in Atlantic salmon independent of fish size.