PHYSIOLOGY: FISH
Turner Jr J. W., Nemeth R., Rogers C. (2003): Measurement of fecal glucocorticoids in parrotfishes to assess stress. General and Comparative Endocrinology 133: 341-352.
FULL TEXT
Abstract
Coral reefs are in decline worldwide from a combination of natural and human forces. The environmental compromises faced by coral reef habitats and their associated fishes are potentially stressful, and in this study we examined the potential for assessing stress levels in coral reef fish. We determined the feasibility of using fecal casts from parrotfishes for remote assessment of stress-related hormones (cortisol and corticosterone), and the response of these hormones to the stress of restraint and hypoxia. Measurement of these hormones in fecal extracts by high performance liquid chromatography (HPLC) was validated using mass spectrometry, chemical derivitization, and radioactive tracer methods. In aquarium-adapted parrotfish, baseline levels of cortisol and corticosterone averaged 3.4 ± 1.1 and 14.8 ± 2.8 ng/g feces, respectively, across 32 days. During 13 days of periodic stress these hormones, respectively, average 10.8-fold and 3.2-fold greater than baseline, with a return to near baseline during a 23-day follow-up. Testosterone was also measured as a reference hormone which is not part of the stress–response axis. Levels of this hormone were similar across the study. These fecal hormones were also measured in a field study of parrotfish in 10 fringing coral reef areas around the Caribbean Island of St. John, US Virgin Islands. Extracts of remotely collected fecal casts of three parrotfish species revealed no difference in respective average hormone levels among these species. Also, there was no difference in respective hormone levels between aquarium and field environments. However, levels of both cortisol and corticosterone, but not testosterone, were elevated in two of the 10 reef sites surveyed. This study demonstrates that parrotfish fecals can be collected in aquarium and field conditions and that steroid hormones in these fecals can be extracted and reliably measured. The study also demonstrates that cortisol and corticosterone in parrotfish fecals can be used as an indicator of the stress–response which is unlikely to be masked by intrinsic variability in the sample source, environment or methodology.
Ellis T., James J. D., Stewart C., Scott A. P. (2004): A non‐invasive stress assay based upon measurement of free cortisol released into the water by rainbow trout. Journal of Fish Biology 65: 1233-1252.
FULL TEXT
Abstract
A procedure previously used for sex steroids was adapted to extract free cortisol and cortisone from water samples taken from rainbow trout Oncorhynchus mykiss tanks. Both corticosteroids could be readily detected by radioimmunoassay (RIA), with cortisol being predominant. All stages of the sampling, extraction and RIA procedure were validated for cortisol. An intermittent problem with poor replication was traced to the use of diethyl ether during the extraction procedure, and was overcome by the use of ethyl acetate. Other modifications were also introduced to speed up the procedure. The concentration and time course of release of both corticosteroids were shown to be related to the degree of stress that the fish had been subjected to. It was confirmed that cortisol concentrations in water and estimated cortisol release rates increased in response to handling stress, and that both were correlated with plasma cortisol concentrations. The potential for using water cortisol concentration and release rates to assess the primary stress response of fishes as a non‐invasive alternative to blood sampling is discussed.
Sebire M., Katsiadaki I., Scott A. P. (2007): Non-invasive measurement of 11-ketotestosterone, cortisol and androstenedione in male three-spined stickleback (Gasterosteus aculeatus). General and Comparative Endocrinology 152: 30-38.
FULL TEXT
Abstract
The androgen 11-ketotestosterone (11-KT) plays an important role in reproductive physiology and behaviour in male teleosts. In the three-spined stickleback, Gasterosteus aculeatus, the plasma concentrations of 11-KT are related to the breeding status of the fish. Sticklebacks are relatively small (generally less than 1 g) and in order to obtain sufficient plasma for assay of 11-KT, it has been necessary in the past to sacrifice the fish. In this paper, we report on the development of a non-invasive procedure for measuring 11-KT, cortisol and androstenedione (Ad) in the three-spined stickleback. Validation of the procedure included the demonstration that the rate of release of steroids into the water was correlated to their plasma concentrations. Ten males that were kept at a low temperature and short photoperiod were moved to high temperature and long photoperiod to initiate spermatogenesis and breeding. Every two to four days, for a total of 53 days, males were removed and placed in a beaker containing 50-ml water for 30 min. The water was then processed by solid phase extraction for radioimmunoassay. Males were presented with females on days 13/14, 18/19 and 44/45. 11-KT was originally undetectable but built up gradually to reach an average release rate of between 1 and 2.5 ng/g/h between days 16 and 45 and then started to decline (but non-significantly). Ad release reached a plateau of 1 ng/g/h about day 20. However, from days 44/45 to 51, there was a highly significant rise in the rate of release of Ad to 5 ng/g/h. On days 44/45, five of the males mated successfully and five did not. However, there were no significant differences in 11-KT or Ad release rates between the two groups. Cortisol release rates fluctuated with no pattern throughout the study. The results show that it is possible to make measurements on sex and stress steroid production in sticklebacks without recourse to anaesthesia, bleeding or sacrificing the fish. The procedure is potentially a powerful tool for the study of the link between steroids and behaviour in this useful sentinel species.
Lupica S. J., Turner Jr J.W. (2009): Validation of enzyme‐linked immunosorbent assay for measurement of faecal cortisol in fish. Aquaculture Research 40: 437-441.
FULL TEXT
Abstract
Quantification of glucocorticoid (GC) levels in faeces has become an established method for the non‐invasive assessment of adrenocortical activity. These hormones are frequently determined in plasma samples as parameters of adrenal activity and response to stress. Because GCs are metabolized and excreted with both intact hormone and their metabolites present in faeces, the concentration of GCs can be measured in excreta. Faecal samples present the advantages of easy collection, no stress to the animal and elimination of the issue of potentially misleading acute GC spikes. The aim of this study was to determine if an enzyme‐linked immunosorbent assay (ELISA) for cortisol was appropriate for monitoring adrenocortical activity in faecal casts of fishes. Performance of the cortisol ELISA was validated by comparison to high‐performance liquid chromatography, which is an established method for measuring free GCs and GC metabolites in faeces. Parallelism and sample extraction efficiency were compared for the two methods. Pearson’s correlation across samples for these methods was 0.996. Results demonstrated that the ELISA was an efficient, sensitive and reliable method for cortisol measurement in faecal extracts, which should permit integration of non‐invasive stress monitoring into studies of fish behaviour and physiology.
Easy R. H., Ross N. W. (2010): Changes in Atlantic salmon Salmo salar mucus components following short‐and long‐term handling stress. Journal of Fish Biology 77: 1616-1631.
FULL TEXT
Abstract
This study examined changes in Atlantic salmon Salmo salar epidermal mucus proteins following short- and long-term handling stress. Short-term stress consisted of a single removal of fish from water for 15 s with long-term stress consisting of daily removal of fish from water for 15 s over 21 days. In the long-term handling stress study, there was a high level of individual variability with respect to mucus alkaline phosphatase, cathepsin B and lysozyme activities, with no correlation to treatment group. There was limited or no positive correlation between lysozyme, cathepsin B or alkaline phosphatase activities and plasma cortisol. There was a significant difference in lysozyme activity for both control and stressed fish at day 21 compared to other sampling days. In the short-term study, there was again high variability in mucus enzyme activities with no difference observed between groups. Immunoblotting also showed variability in mucus actin breakdown products in both short- and long-term handling stress studies. There appeared, however, to be a shift towards a more thorough breakdown of actin at day 14 in the stressed group. This shift suggested changes in mucus proteases in response to long-term handling stress. In summary, there were correlations of some mucus enzyme/protein profiles with stress or cortisol; however, the variability in S. salar mucus enzyme levels and actin fragmentation patterns suggested other triggers for inducing changes in mucus protein composition that need to be investigated further in order to better understand the role of mucus in the response of S. salar to external stressors.
Zuberi A., Ali S., Brown C. (2011): A non-invasive assay for monitoring stress responses: A comparison between wild and captive-reared rainbowfish (Melanoteania duboulayi). Aquaculture 321: 267-272.
FULL TEXT
Abstract
The stress response of wild and captive reared rainbowfish (Melanoteania duboulayi) following chasing by a simulated predator was examined. Cortisol release rate was monitored using a flow through system by measuring water borne hormone levels. Tests using known cortisol concentrations revealed that the technique yielded 95% of the cortisol present in the water. Cortisol release rates increased several fold in both populations after being chased but peaked at different time periods. Wild fish showed a typical stress response with release rate rising to (2.29 ± 0.22 ng g−1 h−1) 2 h after exposure followed by rapid recovery. The captive-reared population by contrast showed an atypical response with cortisol release rate peaking 4 h post exposure but reaching only half the level of the wild fish (1.19 ± 0.11 ng g−1 h−1). The implications for the release of hatchery-reared fish for stock enhancement are discussed.
Guardiola F. A., Cuesta A., Esteban M. Á. (2016): Using skin mucus to evaluate stress in gilthead seabream (Sparus aurata L.). Fish and Shellfish Immunology 59: 323-330.
FULL TEXT
Abstract
The welfare of farmed fish is influenced by environmental and management factors, which may substantially increase stress levels of the animals and even endanger their survival. Three experiments to simulate different stress conditions (acute crowding, anaesthetic agents and air exposure) were developed. Cortisol levels were measured in fish skin mucus and the values obtained were correlated with those obtained in serum from the same fish specimens and also in the water surrounding the fish. Concomitantly, the effect of these stressors on immunoglobulin M (IgM) and several enzymes related to immunity were also determined in the serum and skin mucus of gilthead seabream (Sparus aurata L.) in order to identify potential reliable, non-invasive stress bioindicators. Our results demonstrated that skin mucus and water are good non-invasive matrices for detecting and measuring cortisol in stressed fish. Interestingly, a time lag in cortisol levels between serum, skin mucus and the surrounding water was detected. While IgM levels and protease activity were affected by all the stressors in serum and skin mucus, peroxidase activity increased in both matrices but only in fish exposed to acute crowding. The present findings could be relevant for fish aquaculture and underline the importance of skin mucus not only for assessing fish immune status but also for acting as a potential fish stress biomarker. Some of the studied enzymes could be used as biomarkers not only of fish stress in general, but also for understanding the type of stress suffered by the fish.
Fernández-Alacid L., Sanahuja I., Ordóñez-Grande B., Sánchez-Nuño S., Viscor G., Gisbert E., Herrera M., Ibarz A. (2018): Skin mucus metabolites in response to physiological challenges: A valuable non-invasive method to study teleost marine species. Science of the Total Environment 644: 1323-1335.
FULL TEXT
Abstract
Knowledge concerning the health and welfare of fish is important to conserve species diversity. Fish mucosal surfaces, and particularly the skin, are of utmost importance to protect the integrity and homeostasis of the body and to prevent skin infections by pathogens. We performed three trials simulating different environmental and anthropogenic challenges: fish capture (air exposure), bacterial infection and fasting, with the aim of evaluating epidermal mucus as a non-invasive target of studies in fish. In this initial approach, we selected three well-known marine species: meagre (Argyrosomus regius), European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata) for our study. Mucus viscosity was measured in order to determine its rheological properties, and mucus metabolite (glucose, lactate, protein and cortisol) levels were analysed to establish their suitability as potential biomarkers. Skin mucus appeared as a viscous fluid exhibiting clearly non-Newtonian behaviour, with its viscosity being dependent on shear rate. The highest viscosity (p < 0.05) was observed in sea bream. Mucus metabolites composition responded to the different challenges. In particular, glucose increased significantly due to the air exposure challenge in meagre; and it decreased during food deprivation in sea bream by a half (p < 0.05). In contrast, mucus protein only decreased significantly after pathogenic bacterial infection in sea bass. In addition, mucus lactate immediately reflected changes closely related to an anaerobic condition; whereas cortisol was only modified by air exposure, doubling its mucus concentration (p < 0.05). The data provided herein demonstrate that mucus metabolites can be considered as good non-invasive biomarkers for evaluating fish physiological responses; with the glucose/protein ratio being the most valuable and reliable parameter. Determining these skin mucus metabolites and ratios will be very useful when studying the condition of critically threatened species whose conservation status prohibits the killing of specimens.
Carbajal A., Reyes-López F. E., Tallo-Parra O., Lopez-Bejar M., Tort L. (2019): Comparative assessment of cortisol in plasma, skin mucus and scales as a measure of the hypothalamic-pituitary-interrenal axis activity in fish. Aquaculture 506: 410-416.
FULL TEXT
Abstract
Cortisol, the end product of the hypothalamus-pituitary-interrenal (HPI) axis, has been traditionally measured in blood as indicator of stress in fish, however, the degree of invasiveness inherent to blood collection is not always possible or desirable. Instead, cortisol measurement in skin mucus is far less invasive, but as blood, this method potentially provides only a brief window of information of the HPI axis activity. The newly described method of cortisol measurement from scales may serve as a long-term, integrated measure of the HPI axis activity in fish. While skin mucus and scales cortisol measurement present practical and conceptual advantages, there are still several unclear issues related to their biological relevance that need deeper study. Accordingly, we aimed to evaluate whether skin mucus and scales cortisol levels can be reliably used as stress indicators by subjecting fish to prolonged, continuous stressful conditions. The present study demonstrates that the measurement of cortisol in skin mucus reflect circulating cortisol concentrations when fish are responding to stress with an intense activation of the HPI axis. Results also revealed that cortisol content in scales strongly correlates to circulating cortisol levels in chronically stressed fish. Besides, we provide further support that scales cortisol assessment offer a retrospective measure of the past stress experience in fish. While this study provides a good basis for future research applying the methods presented, our results open the question of whether these matrices have additional sources of cortisol other than blood, and the route of incorporation or diffusion. Further knowledge about the general robustness and stability of scales cortisol in fish subjected to prolonged stress would largely help strengthen the interpretation of hormone fluctuations in this matrix.
Carbajal A., Soler P., Tallo-Parra O., Isasa M., Echevarria C., Lopez-Bejar M., Vinyoles D. (2019): Towards non-invasive methods in measuring fish welfare: the measurement of cortisol concentrations in fish skin mucus as a biomarker of habitat quality. Animals 9: 939.
FULL TEXT
Abstract
Cortisol levels in fish skin mucus have shown to be good stress indicators in farm fish exposed to different stressors. Its applicability in free-ranging animals subject to long-term environmental stressors though remains to be explored. The present study was therefore designed to examine whether skin mucus cortisol levels from a wild freshwater fish (Catalan chub, Squalius laietanus) are affected by the habitat quality. Several well-established hematological parameters and cortisol concentrations were measured in blood and compared to variations in skin mucus cortisol values across three habitats with different pollution gradient. Fluctuations of cortisol in skin mucus varied across the streams of differing habitat quality, following a similar pattern of response to that detected by the assessment of cortisol levels in blood and the hematological parameters. Furthermore, there was a close relationship between cortisol concentrations in skin mucus and several of the erythrocytic alterations and the relative proportion of neutrophils to lymphocytes. Taken together, results of this study provide the first evidence that skin mucus cortisol levels could be influenced by habitat quality. Although results should be interpreted with caution, because a small sample size was collected in one studied habitat, the measurement of cortisol in skin mucus could be potentially used as a biomarker in freshwater fish.
Bulloch P., Schur S., Muthumuni D., Xia Z., Johnson W., Chu M., Palace V., Su G., Letcher R., Tomy G. T. (2020): F2-isoprostanes in fish mucus: a new, non-invasive method for analyzing a biomarker of oxidative stress. Chemosphere 239: 124797.
FULL TEXT
Abstract
F2-isoprostanes (F2-isoPs) are a reliable biomarker class for oxidative stress in vivo in animals. These compounds are traditionally measured in matrices like liver and plasma, however social and environmental pressures warrant the development of non-lethal and non-invasive methods to assess animal health. Therefore, this study aimed to develop a high-performance liquid chromatography tandem mass spectrometry (HPLC-ESI-MS/MS) method to separate and detect F2-isoPs in fish mucus. The method was developed and validated for four native F2-isoP isomers using Northern pike mucus (Esox lucius). Linearity was observed between 5 and 1000 pg/μL. The limits of detection of the four F2-IsoP isomers ranged from 0.63 to 2.0 ng/g. Recoveries ranged from 78 to 95%, and matrix effects were small (<10%). The between-day and within-day repeatability for all target analytes was lower than 20% RSD. Endogenous F2-isoPs were measured in the pike mucus (5.3–28.8 ng/g). A preliminary study of baseline F2-isoP concentrations in lake trout (Salvelinus namaycush) captured from five lakes at the IISD-Experimental Lakes Area in Northwestern Ontario, Canada, was also conducted to test the interspecies applicability of the method. Endogenous F2-isoPs were quantified in lake trout (6.3–132 ng/g). Lake trout samples displayed large variability within and between the different lakes, which suggests sampling methods may require adjustment for this species. This work developed a sensitive analytical method for measuring F2-isoPs in fish mucus, however several further studies are required to determine its ability to accurately measure oxidative stress in fish species.
Cai X., Zhang J., Lin L., Li Y., Liu X., Wang Z. (2020): Study of a noninvasive detection method for the high-temperature stress response of the large yellow croaker (Larimichthys crocea). Aquaculture Reports 18: 100514.
FULL TEXT
Abstract
At present, the methods for monitoring the health status of fish mostly utilize blood biochemical indicators. As the blood collection process often causes damage to the fish body (sometimes causing death), the need to develop a noninvasive detection method is urgent. Previous studies have shown that stress indicators can be detected in fish skin mucus or the surrounding water. In this experiment, the level of cortisol in the blood, fish skin mucus, and surrounding water of large yellow croaker was determined at 0, 0.5, 1, 1.5, and 2 h after high-temperature stress induction. Moreover, the levels of malondialdehyde (MDA), immunoglobulin M (IgM), and alkaline phosphatase (AKP) in the serum and fish skin mucus were also measured. The results were as follows: with increasing stress duration, the cortisol levels in the serum, fish skin mucus, and surrounding water increased first and then decreased, all reaching a peak at 1.5 h. The Pearson correlation coefficients for the cortisol levels between the serum and skin mucus, serum and surrounding water, and skin mucus and surrounding water were 0.936, 0.955, and 0.915, respectively, all with a significance level of P < 0.01. The MDA and IgM levels in the serum and fish skin mucus both increased first and then decreased, while the AKP levels in the serum and skin mucus decreased first and then increased. For the MDA, IgM, and AKP levels, the Pearson correlation coefficients between the serum and skin mucus were 0.586, 0.762, and 0.792, respectively, all with a significance level of P < 0.01. According to the results mentioned above, we can use the surrounding water or skin mucus as media to monitor early stress responses in large yellow croaker under high-temperature stress.
Makaras T., Razumienė J., Gurevičienė V., Šakinytė I., Stankevičiūtė M., Kazlauskienė N. (2020): A new approach of stress evaluation in fish using β-d-Glucose measurement in fish holding-water. Ecological Indicators 109: 105829.
FULL TEXT
Abstract
In general, the well-being of aquatic ecosystems is a key parameter reflecting ecosystem wealth. To avoid negative influences on fish physiology, the stress state of species must be monitored. β-d-glucose level is one of the key indicators of acute stress in rainbow trout (Oncorhynchus mykiss) juveniles and has been associated with different environmental conditions, including different amounts of chemicals. In the present study, we explore the possibility of assessing the β-d-glucose level in water as a reliable indicator of acute stress in the fish exposed to chemical contaminants. Amperometric biosensor utilizing immobilized into membrane glucose oxidase Aspergillus niger (GOx) for the measurement of low concentration of β-d-glucose in fish holding water was developed and described. Specific layers of the enzymatic membrane allowed to eliminate interfering signals and the biosensor have been fitted for β-d-glucose measurements in fish-holding water. Besides that, the biosensor exhibited several advantageous functional properties, i.e. a wide linear range (0.005–30 mM), high sensitivity (1.07 ± 0.01 µA mM-1cm−2), good selectivity and long-term stability. It was the combination of these biosensor properties that made the simultaneous measurement of β-d-glucose both at low (in water) and high (in fish blood) concentrations and operating in non-pretreated turbid media feasible. Fish were exposed to the primary complex metal (Zn – 0.1, Cu – 0.01, Cd – 0.0015, Ni – 0.034, Pb – 0.014 and Cr – 0.01 mg·L−1, respectively) mixture (MIX) (prepared at maximum-permissible-concentrations for inland waters in European Union (EU)) and MIX5↓ (5-fold reduced (↓) concentration of each metal in the mixture). The obtained results revealed that the blood-glucose level in fish did not change over time significantly, while the level of β-d-glucose released into water by the fish after 3 h exposure to MIX was found to have significantly increased. However, potential stress was observed in the fish exposed to both MIX and MIX5↓ metal mixtures revealing fish sensitivity and proving suitability of the glucose measurement procedure. No significant relationship was found to exist between blood-glucose and water β-d-glucose levels. The results showed that in contrast to blood-glucose measurements, the proposed non-invasive measurement of β-d-glucose in water was beneficial. Thus, this study provides a new approach for the development of a method for stress indication in fish using the measurement of glucose in holding water. Moreover, this glucose measurement procedure can prove to be another valuable tool for stress evaluation method in aquaculture, fish welfare and toxicological research, especially for studying short-term stress responses induced by chemicals.
Freret-Meurer N. V., Fernández do Carmo T., Cabiró G. (2021): Opercular beat: A non-invasive and rapid method to detect stress in seahorses. Journal of Applied Aquaculture 33: 291-299.
FULL TEXT
Abstract
Seahorses are commonly used in the aquarium trade and in seahorse-watching ecotourism in Brazil. Their manipulation and transportation may lead to significant stress, compromising their health. Assessing stress response during manipulation is difficult. Thus, the aim of the present study was to identify respiration patterns for these animals, providing baseline data for a health and well-being protocol to be applied during seahorse management. We conducted experiments in captivity and in the wild to evaluate transportation and handling as potential stressors. Significant differences between opercular beat after manipulation and recovery was observed in captivity and in the wild. Seahorses displayed a base opercular beat around 30/min in the wild and 45/min in captivity, increasing to 66 beats/min after manipulation in the wild and 64/min in captivity. No difference between sex or size was noticed. Opercular beat in captivity remained higher than in the wild 24 h after manipulation, suggesting that captivity protocols should include acclimation longer than 24 h.
Santymire R. M., Young M., Lenihan E., Murray M. J. (2022): Preliminary investigation into developing the use of swabs for skin cortisol analysis for the ocean sunfish (Mola mola). Animals 12: 2868.
FULL TEXT
Abstract
The ocean sunfish (mola; Mola mola) is the heaviest bony fish in the world. This slow-moving fish often is injured by fishing boats that use drift gillnets attributing to its listing as Vulnerable by the IUCN. The Monterey Bay Aquarium (Monterey, CA, USA) has a program that brings in smaller molas from the ocean and acclimates them for an exhibit. When they grow too large for the million-gallon Open Seas exhibit, they are returned to Monterey Bay through a “reverse” acclimatization. Our overall goal was to use skin swabs to evaluate mola stress physiology to better understand the effects of this program. Our objectives were to validate this non-invasive method by taking opportunistic swabs throughout acclimatization and during stressful events. We swabbed each individual (n = 12) in three different body locations. Swabs were analyzed using a cortisol enzyme immunoassay. We averaged the three swabs and examined the absolute change of cortisol from the first taken upon handling to during treatments and the different acclimation stages. We considered elevated cortisol concentrations to be ≥1.5-fold higher than the first sample. Overall, mean (±SEM) cortisol varied among individuals (564.2 ± 191.5 pg/mL swab (range, 18.3–7012.0 pg/mL swab). The majority (four of six) of molas swabbed within the first week or month had elevated skin cortisol compared to their first sample. All seven molas that were being treated for an injury or illness had elevated skin cortisol (range, 1.7- to 127.6-fold higher) compared to their post-acclimation sample. This is the first step in validating the use of non-invasive skin swabs for glucocorticoid analysis in the mola. Further biochemical analysis is needed to determine the specific steroids that are being measured.
Kennedy E. K., Janz D. M. (2023): Can scale cortisol concentration be quantified non-lethally in wild fish species?. Conservation Physiology 11: coac081.
FULL TEXT
Abstract
Cortisol, the primary glucocorticoid in fishes, is secreted into the bloodstream in response to stress. Circulating cortisol accumulates in scales, a durable calcified structure that can be easily sampled from many fish species. As such, the use of scale cortisol concentration (SCC) is currently being explored as a means of chronic stress biomonitoring in wild fishes. Scales serve an important role in fish physiology and thus the number of scales required for reliable cortisol analysis is a limiting factor in the non-lethal collection of such samples. To date, scale cortisol quantification has also only been performed non-lethally in captive fishes and due to differences in stress responsiveness SCCs likely differ in wild species. As such, this study aimed to (1) apply our fish scale processing and analysis protocol to wild fish species and (2) apply it to five north temperate fish species to provide information useful to future non-lethal scale sampling regimes. Cortisol was successfully measured in scales collected from wild northern pike (Esox lucius), walleye (Sander vitreus), whitefish (Coregonus clupeaformis), white sucker (Catostomus commersonii) and captive rainbow trout (Oncorhynchus mykiss). SCCs were significantly different between species and thus the sample mass required for reliable cortisol analysis differed as well. In addition to the size of the fish and the mass of their scales this is an important consideration for future scale cortisol analyses as these factors could make SCC an attainable non-lethal sample matrix in some species of fish but impractical in others.