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Cortisol and Glucose: Reliable indicators of fish stress?

MARCEL MARTÍNEZ-PORCHAS1, LUIS RAFAEL MARTÍNEZ-CÓRDOVA2 & ROGELIO
RAMOS-ENRIQUEZ3
1
Centro de Investigaciones Científicas y de Educación Superior de Ensenada. CICESE. Departamento de Acuicultura.
Km. 107 Carretera Tijuana–Ensenada. 22860. Ensenada Baja California, México. Email: marcelmp_6@hotmail.com
2
Universidad de Sonora. Departamento de Investigaciones Científicas y Tecnológicas de la Universidad de Sonora.
DICTUS. Bldv. Luis Donaldo Colosio. 83000. Hermosillo, Sonora
3
Universidad de Sonora. Laboratorio de Análisis Clínicos e Investigación de la Universidad de Sonora. LACIUS. Bldv.
Luis Donaldo Colosio. 83000. Hermosillo, Sonora.

Abstract. Stress in fish has been widely studied. Cortisol and glucose are two of the most
common stress indicators. In spite of the extended use of these indicators and their acceptance,
some inconsistencies have been reported in the results of several experimental studies, much of
them associated to undefined and uncontrolled variables which may alter the response in secretion
of cortisol and glucose into the bloodstream. Most of those factors are not considered as direct
stressors but have an effect on the intensity of the response which makes them a source of error.
Some of those factors are related to metabolic changes in the organisms as an adaptation or
acclimation mechanism; other are extrinsic to the fishes; other sources of error are caused
unconsciously by the researcher during manipulation or due to inadequate control of variables, and
may lead to intrinsic changes. The present paper is a contribution on the review of the most
evident factors that may affect results when using cortisol and/or glucose as fish stress indicators.
Some suggestions to avoid or minimize erroneous results in such investigations are also presented.

Keywords: Blood chemistry, blood parameters, blood sugar, biochemical responses, corticoids,
stress indicators.

Resumen. ¿Cortisol y glucosa: fiables indicadores de estrés de los peces? El estrés en los peces
ha sido ampliamente estudiado. El cortisol y la glucosa son dos de los indicadores de estrés más
comunes. A pesar del extenso uso de estos indicadores y su aceptación, se han reportado algunas
inconsistencias en los resultados de muchos experimentos, algunos de ellos asociados a variables
que no son controladas, las cuales pueden alterar la respuesta de secreción de cortisol y glucosa.
Muchos de esos factores no son considerados estresores directos, pero tienen un efecto en la
intensidad de respuesta, lo cual los vuelve una fuente de error. Algunos de esos factores están
relacionados con cambios metabólicos en los organismos, como un mecanismo de aclimatación o
adaptación; otros son extrínsecos a los peces y otros más son causados inconscientemente por el
investigador mediante la manipulación o inadecuado control de variables, lo cual puede provocar
cambios intrínsecos. El presente manuscrito es una contribución en la revisión de los factores más
evidentes que pueden afectar los resultados cuando se usa cortisol y/o glucosa como indicadores
de estrés en peces y a su vez se mencionan algunas sugerencias para evitar o minimizar resultados
erróneos.

Palabras clave: Azúcar sanguínea, corticoides, indicadores de estrés, parámetros sanguíneos,
química sanguínea, respuestas bioquímicas.

Introduction scientists dedicated to the research of environmental
influences on health (Barreto et al. 2006).
In recent years the concept of stress as There are discrepancies from the different
applied to fish has awaked the interest among authors about the stress definition. One of the most

Pan-American Journal of Aquatic Sciences (2009), 4(2): 158-178

Cortisol and Glucose: Reliable indicators of fish stress? 159

accepted is described as chemical and physical et al. 1999). In spite of the extensive use of cortisol
factors causing body reactions that may contribute to and glucose levels as stress indicators, there are
disease and-or death (Rottmann et al. 1992). Stress some inconsistencies in the results of various
is also known as “the nonspecific response of the experiments that in some cases would be attributed
body to any demand made upon it” (Selye 1973). to unknown situations.
Although there are several definitions, most of them This is a review on the effectiveness of
refer to an “altered state” which increases the energy glucose and cortisol as stress indicators in fish and
demand. According to Selye (1985) stress should be we attempt to identify possible errors within
divided into two phases: “eustress” or the healthy different scenarios and make some
stress and “distress” or bad stress. Eustress occur as recommendations.
a response of the organism undergoing situations Cortisol.
that provoke physiological changes that optimize its Cortisol is the principal glucocorticoid
biological performance, for example exercise. secreted by the interrenal tissue (steroidogenic cells)
Distress occurs when certain factor promotes located in the head-kidney of teleost fish (Iwama et
physiological changes into an organism that may al. 1999). This hormone is released by the activation
compromise organism’s integrity. Major part of of the hypothalamus-pituitary-interrenal axis (HPI
stress research is focused on distress phase. axis) (Mommsen et al. 1999). When an organism
The response to stress in fish is undergoes stress conditions, the hypothalamus
characterized by the stimulation of the releases corticotropin-releasing factor (CRF) toward
hypothalamus, which results in the activation of the blood circulation. This polypeptide further
neuroendocrine system and a subsequent cascade of stimulates secretion of adrenocorticotrophic
metabolic and physiological changes (Wedemeyer hormone (ACTH) from the anterior pituitary gland
1990, Lowe & Davison 2005). These changes (Fryer & Lederis 1986) which finally activates the
enhance the tolerance of an organism to face an release of cortisol by the interrenal tissue
environmental variation or an adverse situation (Mommsen et al. 1999).
while maintaining a homeostasis status (Mazeaud et Cholesterol is the precursor of cortisol; this
al. 1977, Pickering, 1981). sterol is transformed to pregnenolone by the action
Under conditions of stress, the body of the of the enzyme P450 side chain cleavage (P450SCC)
fish emits immediate responses recognized as in the inner mitochondrial membrane. Then
primary and secondary responses. The primary pregnenolone is further converted into 11-
response is the perception of an altered state by the deoxycortisol by steroidogenic enzymes and this
central nervous system (CNS) and the release of the product is finally converted to cortisol by enzyme
stress hormones, cortisol and catecholamines 11b-hydroxylase (Miller 1998, Castillo et al. 2008).
(adrenaline and epinephrine) into the bloodstream by The secretion of cortisol is slower than
the endocrine system (Randall & Perry 1992). catecholamines, but its effects are more prolonged
Secondary responses occur as a consequence of the (Gamperl et al. 1994a, b; Waring et al. 1996),
released stress hormones (Barton & Iwama 1991), combining mineral and glucocorticoid actions to
causing changes in the blood and tissue chemistry, restore homeostasis (Wendelaar-Bonga 1997, Maule
e.g. an increase of plasma glucose (Barton 1997, et al. 1993, Colombe et al. 2000). Cortisol activates
Begg & Pankhurst 2004). This entire metabolic glycogenolysis and gluconeogenesis processes in
pathway produces a burst of energy to prepare the fish; but also causes that chromaffin cells increase
fish for an emergency situation (Rottmann et al. the release of catecholamines which further increase
1992). glycogenolysis and modulate cardiovascular and
Some plasma chemicals may be useful tools respiratory function (Reid et al. 1992, Reid et al.
to evaluate the health and/or stress condition of the 1998). This whole process increases the substrate
fishes (Sadler et al. 2000a, Campbell 2004, Wagner levels (glucose) to produce enough energy according
& Congleton 2004). Because stress has been with the demand.
reported to elevate plasma cortisol (Pottiner & Factors that can affect the intensity of
Mosuwe 1994, Wendelaar-Bonga 1997, Pottinger et response. The intensity of response is not always
al. 2003, Haukenes et al. 2008) and glucose levels caused by a specific stressor in any experiment;
(Silbergeld 1974, Wedemeyer & Yasutake 1977, instead it may be modulated or affected by alien
David et al. 2005), many researchers consider as a factors that are not considered as direct stressors
“rule of thumb” that fishes undergoing stressful (Frisch & Anderson 2005) but that may further
situations exhibit plasmatic increases of cortisol and impact cortisol secretion (Fig. 1). Those factors that
glucose (Hattingh 1977, Balm et al. 1989, Barcellos affect/modulate the response may be from intrinsic

Pan-American Journal of Aquatic Sciences (2009), 4(2): 158-178

(1998) made a review about the secretion as their ancestors (Pottinger & Carrick adrenergic response in fish and mentioned that the 1999). Extrinsic factors may affect a over or sub estimating the cortisol response (Table variety of biochemical functions within the fish I). Pottinger catecholamine in the production of glucose. Environmental color is reported to have an previously to the beginning of any experiment. 1998. 1994b. influenced by adverse internal or external conditions Sakakura et al. for example. (1995) and necessitate an “alteration in the capacity to identified sexual maturity as a factor related with the express the adrenergic stress response”. nutritive immunoreactive cortisol concentrations during the stress. salinity. 1987. 4(2): 158-178 . into cortisone (Kime 1978) which is significantly experiments may provide erroneous results. 2003). intensity of response in fishes. Heritability is considered as a tryptophan (serotonin precursor) (Lepage et al. Figure 1. (+) means et al. (1998) hypothesized that the rearing temperature for eggs and larvae of fish can influence the induction of cortisol response. response. external factors. Lankford et al. Vijayan & Leatherland 1990). Also Stouthart et al. nutritional status. 2006) to avoid Extrinsic. Iwama et al. Liver is the key organ for subordinate organisms showed a higher response cortisol disposal with the hepato-biliary system as than dominant ones. (2002) reported an increase of in the history of the fish (anoxia. Stouthart et al. This last argument can be transition from larval to juvenile stage of yellowtail explained because those organisms require energy (Seriola quinqueradiata). nutritional Another factor that may affect results is the state. modulator with progeny groups of high response and 2002. In some species the magnitude of the stress response varies with respect of a previous thermal acclimation or acclimatization (Strange et al. pollution. serotonin which is a HPI axis regulator increases when administered dietary Intrinsic. Nutritional status (Pottinger 1998. Pan-American Journal of Aquatic Sciences (2009). fact that in some cases cortisol is rapidly converted If a modulator of response is not identified. thereby less immunoreactive than cortisol. 1998. 1999). 1977. physical stress). A higher intensity of cortisol response is to laboratory conditions (temperature. Differences in the intensity of response might occur in domesticated organisms as compared with non-domesticated ones (Jentoft et al. Some authors it is indispensable to know cortisol basal levels of have reported increases in the concentrations of any experimental species. socially subordinate salmonids exhibit enhanced However the efficiency of that process is reported to CRF mRNA in the preoptic area. 2003). Gilmour et al. this is in agreement with Doyon the main biochemical pathway for cortisol clearance et al. Pottinger et al. as general guidelines (Barton & Imawa 1991. etc (Mommsen et al. water quality. As an example Koldkjær et al. 2003) is another factor that may affect the positive modulation and (–) means negative modulation. maturity. nature when some factors depend basically on the tanks as compared with those in gray and white genotype or phenotype of the organism and from tanks when fish were exposed to crowding stress extrinsic nature when response is affected by (Rotllant et al. (2003) report. We suggest standardizing physiological and organism such as cortisol biosynthesis and release biochemical status off all experimental organisms rates. Gamperl et al. Also age has been identified as one of those regulation in the production of stress hormones is factors (Pottinger & Mosuwe 1994). be altered by stress. Patiño et al. For effect on cortisol secretion (Van der Salm et al. 2003).160 MARTINEZ-PORCHAS ET AL. instance: a prior acclimation of experimental fishes 2004). 1992). dissolved documented in Pargus pargus acclimated in black oxygen. for instance. where they documented that (Wilson et al. (2004) reported differences in plasma cortisol of rainbow trout (Oncorhynchus mykiss) when comparing results in warm months versus cold months. low response showing a similar intensity of cortisol Reid et al. Briefly view of the dynamics of cortisol and 2005). Pottinger et al. (2005) The rate of cortisol clearance is another step mentioned that cortisol response is variable even in in the cortisol cycle that may be influenced by salmonid fishes of the same stock and that environmental factors. There are species-specific plasma cortisone of stressed fishes (Weisbart & and stressor-specific cortisol values that may serve McGowan 1984.

2006. This trout (O. Metz et al. This culminates in less cortisol secretion than Although these are common sense expected. & McEntire been implicated in cortisol release during the chronic 2006). 2005). glucose and cortisol levels immediately after which is another source of error that appears when handling and then returned to the basal level after 8 the fish has been acclimated to conditions of stress hours (Morales et al. Carp (Cyprinus carpio) or was acclimatizated to a certain stress factor in its increased plasma cortisol when retained in anglers´ environment that the collector did not notice. but To illustrate this. functioning as an emergency system. but decreased to the basal stress status after chronic stress experiments. 1998). to test genetically modified organisms. acclimated to chronic confinement (70 ng mL-1) than It has been suggested that after stress. 2005) and some regularly becomes weak or disappears some hours sympathetic nerve fibers (Arends et al. In consonance. 1999). (1987) suggestions. color and shape of 2005. Fast et al. that during the first 6-48 hours after an organism In chronic-stress experiments some fish undergoes adverse conditions it suffers changes in showed a weak increase of cortisol (Barton et al. which is called Pan-American Journal of Aquatic Sciences (2009). (1987b) hypothesized an acclimation of the HPI axis Stein-Behrens & Sapolsky 1992). cortisol response would not be a Cortisol levels of red drum during some handling sufficient but rather a less reliable tool to examine procedures grew rapidly. 4(2): 158-178 . the cortisol response is rapid but al. observed that cortisol levels of juvenile rainbow leading to abnormal results. Thus. Ramsay 2006). For variability in response from one organism to another example. 1987). different hormones such as on the stress response. In experiments endorphin from the pars intermedia (PI) (Lamers et of acute stress. to basal levels after 6 hours (Iwama et al. 1992) or an collection.Cortisol and Glucose: Reliable indicators of fish stress? 161 photoperiod. Therefore. fishes from polluted sites returned to basal after a chronic exposure. Organisms accustomed to et al. weight. state within 48 hours (Robertson et al. alpha-melanocyte-stimulating hormone (MSH). Barton et al. Previous experience to stress conditions Common dentex (Dentex dentex) increased its should also be considered as a chronic exposure. In most fishes. Acute and chronic stress. Pickering & Pottinger damage of homeostatic mechanisms (Dickhoff 1989. 1994. may have a different response than those acclimated Although there are exceptions (Gil-Barcellos to laboratory conditions. Rainbow avoid tissue damage (Wendelaar-Bonga 1997). but it is Perhaps this means that the fish used to stress indispensable to measure cortisol immediately after require a lesser amount of cortisol to reach the same stress and over time. phase in fishes. 1999) have after the exposure to stress (Davis Jr. Mommsen et al. others that thrive in environments without predators. cortisol test assessed by changes in plasma cortisol levels. the using clonal groups of fishes (Plaut & Gordon release of cortisol decreases because the interrenal 1994). because in the absence of the harassing of predators will show a weak stress the ACTH some other pituitary hormones can response during the experiments as compared with increase the secretion of cortisol (Wilson et al. Barton keep nets but returned to basal levels within 4 hours et al. organisms should be from exhaustion of the endocrine system as a result of the same progenies or at least from the same place of prolonged hyperactivity (Hontela et al. cortisol reaches highest However if the sub-optimal condition persists this concentration 1 hour after being stressed. trout increased in acute exposure to stress. because a single and/or a late quantity of energy (glucose). mykiss) submitted to a 6-week exercise damage has been observed in salmons. where high program showed less cortisol concentration than levels of cortisol cause death in Pacific salmon unexercised trout when both were in rest condition (Oncorhynchus spp) by tissue degeneration and (Woodward & Smith 1985). (2005) observed a less intense response of fish (Pottinger 1998). blood chemicals (cortisol increase). There are other several situations that have an effect For instance. when an organism undergoes suboptimal (even from the same species) may be avoided by conditions for a considerable period of time. and returns system may be also depleted. 1992. test will have a high probability to be far from the On the other hand. 2006). It also has been suggested that high habituation of the organism to that condition. size. the fish acclimated to low density (139 ng mL-1) when cortisol levels of fishes return to basal levels to submitted both to acute handling stress. is a good option in acute stress experiments. for example (Vijayan and Leatherland 1990. sometimes these are not followed. However this is a difficult task and would not tissue of stressed fishes becomes less sensitive to the be possible in many cases and also clonal groups action of ACTH or other pituitary hormones may be only useful in specific studies. 2008) probably caused by experimental containers). Selye (1936) reported real response.

(2002) Rainbow trout female Oncorhynchus mykiss Trapping 57 764 Acute Clements et al. (2003) . (2000b) Atlantic salmon (triploid) Salmo salar Confinement 27 124 Acute Sadler et al. (2001) Walleyes Stizostedion vitreum Capture and transport 33-315 380-480 Acute Barton et al. 4(2): 158-178 162 Table I. Plasma cortisol values of different species of fishes before and after being stressed.Pan-American Journal of Aquatic Sciences (2009). (2002) Rainbow trout (diploid) Oncorhynchus mykiss Handling and confinement 77 698 Acute Benfey & Biron (2000) MARTINEZ-PORCHAS ET AL. Rainbow trout male Oncorhynchus mykiss Trapping 16 380 Acute Clements et al. (2002) Atlantic salmon Salmo salar Sea lice challenge 99 339 Chronic Bowers et al. (2000) Rainbow trout Oncorhynchus mykiss Chemical exposure 49 110 Chronic Benguira et al. (2002) Sea bream Sparus aurata Crowding 13 358 Chronic Ortuño et al. Cortisol (nmol/l) Species Stressor Prestress Poststress Exposure References Atlantic char Salvelinus alpinus Handling 5 449 Acute Lyytikäinen et al. (2000) Scaphirhynchus albus Pallid sturgeon Scaphirhynchus albus Handling 5 8 Acute Barton et al. (2000b) Brook trout (diploid) Salvelinus fontinalis Handling and confinement 19 242 Acute Benfey & Biron (2000) Brook trout (triploid) Salvelinus fontinalis Handling and confinement 2 146 Acute Benfey & Biron (2000) Common carp Cyprinus carpio Density 19 206 Acute Ruane et al. (2002) Pallid sturgeon Confinement 5 16 Acute Barton et al. (2000) Atlantic salmon (diploid) Salmo salar Confinement 27 151 Acute Sadler et al.

mechanisms”. activating alarm reactions producing a primary (2006) studied the impacts of three pharmaceuticals and a secondary response (Brown 1993). Roche & interrenal tissue. a cytosolic ligand- Pan-American Journal of Aquatic Sciences (2009). ibuprofen. blood chemicals return to normal (general prevails. but the duration of this mechanism depends on the availability of energy reserves. and salicylic acid) in salmon (Salmo salar). Gravel & Vijayan fish. the GAR symptoms appear again caused by adaptation syndrome) due to some modifications in energy depletion (Fig. 2004. which plasma cortisol levels. If those conditions the metabolism. pathways which eventually will influence neural and the expression of StAR and P450SCC decreased in interrenal tissue functions. Several pollutants can stress the due the action of chemicals. However cortisol may be reported as Several studies have corroborated the complementary data of chronic experiments. Some chemicals affect metabolic ranging from pesticides to pharmaceuticals. which is involved in explained that “the exposure to chemicals may steroidogenesis of cortisol (Hontela 2006). 2). the fish modifies and regulate the biochemical processes to restore the homeostasis (GAR). experiments. These findings were also tested in Bogué (1996) argued that one of the most frequent vitro and observed that salicylic acid produced a responses in fish blood to specific chemical depression of ACTH stimulation in cortisol secretion intoxication is cortisolemia. if the stressful environment continue. When Pickering & Pottinger (1987b) can be affected by environmental contaminants experimented with salmonids under crowding because xenobiotic chemicals such as DDT are conditions. In Atlantic (acetaminophen. Hontela primary stress indicator in acute rather than chronic 1997). composition were better stress indicators than resulting in secretion of less bioactive ACTH. impairment in the cortisol synthesis and secretion Chemicals. it has been observed that cortisol secretion hydrocarbon–receptor (AhR). the same directly compromise the stress response by author also stated that StAR protein may be a interfering with specific neuroendocrine control sensitive target of many environmental pollutants. The responses change with respect to time. But. Likewise we agree with those in turn will promote a minor cortisol release from authors and recommend that cortisol may be a the interrenal tissue (Aluru et al. Biochemical responses of fishes undergoing chronic stress (Selye 1936). 4(2): 158-178 . and a lower gene expression of steroidogenic acute Nevertheless Wendelaar-Bonga (1997) regulatory (StAR) protein. they concluded that changes in cellular toxicants targeting multiple sites along the HPI axis.Cortisol and Glucose: Reliable indicators of fish stress? 163 “general alarm reaction” (GAR). cortisol and glucose levels rainbow trout and supported the hypothesis that increased after being exposed to high aluminum these pharmaceuticals disrupt steroidogenesis in fish concentrations (Ytrestøyl et al. In agreement with this fish exposed to xenobiotics because they bind aryl finding. BIOCHEMICAL PARAMETERS BEGINNING OF STRESS EXPOSURE GENERAL ALARM  REACTION GENERAL ALARM  REACTION GENERAL  ADAPTATION  SYNDROME EXHAUSTION TIME Figure 2. Also. 2001).

(heart puncture). Flodmark et al. In contrast. 1997). (LD50) (Sprague 1969). such as the case of small size Anesthetics have been used to reduce pain and fishes. 1995). metomidate and enguenol) in altering the cortisol production and secretion (Aluru fathead minnows. cortisol release. concluding that MS 222 did not et al. that some anesthetics per se (i. estimation of cortisol as a toxic stress indicator may (1995) intraperinoteally injected ACTH into Atlantic be in doubt. 222). A possible explanation It is also worth to mention that some of the for these abnormal results may be that oxygen above studies only tested a single dose of anesthetic. 2005). 1995) or enzymatic enzyme in the conversion of 11-deoxycortisol to activity (oxidative stress enzymes) (Pedrajas et al. In a recent rather than blocked (Bolasina 2006). a key blood parameters (Iwama et al. phenoxiethanol and tricaine are the two most used Pickering & Pottinger (1987a) observed that anesthetics. experiment Welker et al. Results are very contrasting. and if no response is observed in fishes salmon to promote cortisol secretion. the biochemical pathway of cortisol generation may Anesthesia and Sampling. Olsen et al. it change in plasma cortisol of neotropical fish was documented that clove oil did not block cortisol Prochilodus lineatus when exposed to different secretion in stressed sea bream (Pagrus major). for the level of pituitary hormones and thus increases example when fishes are sacrificed while sampling the possibilities to obtain less precise results. it is necessary to handle the limiting adaptation capacity. while difference in glucose levels blood cortisol in channel catfish exposed to were assessed. cortisol response is due to anesthetic and how much phenoxiethanol and tricaine increased blood cortisol is due to the stressor. with a consequent (2006) evaluated the effectiveness of three depress of steroidogenic enzyme activity and finally anesthetics (MS 222. concentrations of a glyphosphate-based herbicide at Isoeugenol was shown to diminish 60% different periods. tricaine and 2. Although 2- probably not be reflected in cortisol response. we do not recommend their use for the exposure of brown trout (Salmo trutta) to poor cortisol and glucose evaluation because of the quality water resulted in a 50% suppression of the possibility of erroneous increased results. induced transcription factor. confinement. Palić et al. strong blocker of cortisol increase in channel catfish Langiano & Martínez (2008) did not observed any (Ictalurus punctatus) (Small 2003). whereas metomidate showed greater These results suggest that the interrenal effectiveness in blocking cortisol release under high response may be contaminant specific. then it is not recommended to anesthetics reduce or block the activation of HPI use metyrapone nor isoenguenol. sacrificing precision for the survival of the However. instead they had secretion and even if the fish is under stress this will better results using metomidate. if more than one sample (from caudal awareness. concentration in water significantly decreases when Thus it is unknown if the kind of anesthetic per se is an anesthetic is added and HPI axis is activated inefficient or the dose was not adequate. Similar conclusion was assessed by Barton tests would be required and report how much of the & Peter (1982) when observed that 2.e. the efficacy of phenoxyethanol) are stressful and may raise plasma anesthetics seems to be species-specific and prior cortisol. (2002) mentioned experimental organisms. and perhaps leading organisms. Therefore many pollutants halt cortisol block the activation of HPI axis. In this case. metyrapone and etomidate halt cortisol histopathological indicators (Schwaiger et al. perhaps sampling process. (1997) reported an Some anesthetics though have been shown impairment of the cortisol stress-response in the to halt secretion of cortisol. When sampling compromise physiological status of organisms cortisol in fishes. Gorbi & Regoli 2004). Metyrapone has also shown successful results To mention some examples: medium lethal dose as a cortisol synthesis blocker (Hopkins et al. so blood chemicals would not be altered at anesthetics constitute better options. in the trout Salmo gairdneri. they reported under obvious stressful conditions.164 MARTINEZ-PORCHAS ET AL. and thus avoid metabolism enhancement vein) of every fish is needed or the investigator does (increase of cortisol or other parameters) in fish. Brodeur et al. these anesthetics may be organism and provokes an alarm reaction altering used if a single sample of every fish is required. Small (2003) documented that management ethics). (2007) tested four Pan-American Journal of Aquatic Sciences (2009). cortisol (Dang & Trainer 2007). 4(2): 158-178 . Clove oil showed to be a yellow perch (Perca flavescens) from polluted sites. But. secretion by inhibiting 11β-hydroxylase. To not intend to sacrifice the animal (animal this respect. whilst high should be replaced by more useful tests to evaluate cortisol levels were found when using tricaine (MS the effect of any chemical compound on any species. This hindrance in 1995. However. and maybe other axis. behavior (Sprague 1971). This handling eventually aware the them to death. then cortisol that metomidate blocked cortisol release. Thus the ammonium concentrations (Small 2004).

measured free cortisol released into water by In suboptimum or stressful conditions rainbow trout. Those stress hormones in conjunction with 170 to 2000 pg/g/h respectively. stress. but also explore the adequate dose. because “the concentration content is a less precise indicator of stress than of free steroid in the water equates to the cortisol (Wedemeyer et al. anesthetizing. which in cortisol in water to evaluate stress status in fish was turn can be expressed as mechanical energy (Lucas proposed (Scott et al. (2004) 1996). because apparently the time after not be used to measure individual cortisol levels. 2001) emphasized that care and not conjugated steroid fractions (sulphated and has to be taken when using plasma glucose as the glucuronidated steroids) have to be measured to only indicator. Ellis et al. 1990. this method does concentrations and temperatures. 1999. this method emerges 222 effectively blocked cortisol secretion of channel as an interesting alternative to substitute cortisol catfish during the first 20 minutes after measurements in plasma. Park et al. water cortisol measurement for what its use is of Non invasive methods. Despite the responses when administered it at different advantage of being non intrusive. methods have been used as indirect indicators of Glucose. the common carp (Cyprinus catecholamine hormones.Cortisol and Glucose: Reliable indicators of fish stress? 165 concentrations of MS 222 (0. 2005). and branchial routes play a secondary role in steroid Regardless of the wide use of glucose as an elimination (Idler & Truscott 1972. the method can should be taken. Therefore. Huntingford et al. success (Oliveira et al. some authors (Mommsen et al. because cortisol secretion the cortisol concentrations. Scott & Ellis also suggested that only free cortisol 1999. indicator of stress. of cortisol. This glucose production is However Scott & Ellis (2007) pointed out that in mostly mediated by the action of cortisol which some cases cortisol in water is too low to be stimulates liver gluconeogenesis and also halts measured by conventional methods. being necessary peripheral sugar uptake (Wedemeyer et al. being steroids was cortisol. Thus. After that. very similar tests should consider not only anesthetics biomasses are required in every experimental unit. (2008) proved the efficacy of clove oil in Also the major part of free cortisol releasing occurs anesthetizing fishes and their physiological through the gills (Ellis et al. (2007) found that the MS Despite those related problems. applying the anesthetic has an effect on the secretion unless tests of single organisms are carried out. but the levels tended to increase after Another non invasive method to measure 25 minutes. 2003). the measurement of transformed to chemical energy (ATP). finding that the not have yet the precision of direct evaluations optimal dose (lower cortisol and glucose secretion) (plasma cortisol levels. 1999) to cope with the minimal intervention. (2005) tagged two (internal or external) the chromaffin cells release species of fishes. Butler 1973). The best results were is in direct proportion to fish biomass and flow rate obtained with the dose of 90mg·l-1. cortisol mobilize and elevate glucose production in This method has the advantage that fishes fish through glucogenesis and glycogenolysis are not stressed up when sampling due to null or pathways (Iwama et al. cortisol is to measure it in feces. “fight of flight” reaction. but the treatment with the This method also faces the problem of fish highest level of anesthetic (180mg·l-1) also increased mass and water flow rate. In 1994. which is very close to the moment in cortisol level was found in the treatment without time that the sample is taken. cortisol in water increased from 70 to 400 and from 1998). prior modifies cortisol concentration. 2006) or decreased at higher temperatures. Pan-American Journal of Aquatic Sciences (2009). Welker et al. 2001). anesthetic (0mg·l-1). 4(2): 158-178 . 120 and 180mg·l-1) concentration of ‘physiologically active’ steroid in in the channel catfish and reported that the highest the plasma. Flodmark et al. adrenaline and carpio) and the rutilus (Rutilus rutilus) reporting that noradrenaline toward blood circulation (Reid et al. Moreover there is no necessity energy demand produced by the stressor for the to bleed and hurt the animal to measure cortisol. while renal through the insulin action (Nelson & Cox 2005). Lower et al. This procedure has Water temperature is another point of been reported by some authors. 90. to extract and concentrate cortisol from water. Furthermore. Also together with calculations considering flow rate (see it is important to establish the time at which samples Scott & Ellis 2007). and that one of those role in the bioenergetics of animals. Glucose is then released (from liver and muscle) because the highest proportion of cortisol is toward blood circulation and enters into cells eliminated through hepatic processes. 1990). but with limited concern at the time of administering any anesthetic. Pottinger 1998). Turner et al. It has been reported that glucose evaluate stress response. themselves. Non invasive limited practical value. Sorensen & Scott found that goldfish Glucose is a carbohydrate that has a major released steroid to the water.

1999. with different lipid and protein content resulted in Sometimes no significant changes in plasma different responses of blood glucose of the orange. Vijayan & Moon (1994) suggests that “the h). 2). but the level avoid erroneous results we also suggest prior began to decrease over time until depleted (David et standardization of organisms before any stress al. Normally the (Costas et al. authors who concluded that blood glucose results As in the cortisol case it is indispensable to have to be interpreted with care. 2003). That affirmation is supported by other reversible under normoxic conditions. others found no change (Rotllant & Nutritional status is a factor that can have an Tort 1997. cortisol (acute experiments) or the organism be habituated and others may be influenced by internal or (chronic experiments). then. Catecholamines Measuring glucose just after an acute experiment is Pan-American Journal of Aquatic Sciences (2009). nutritive stress. Jentoft et al. sugar they may affect liver glycogen stores (Nakano & levels increase during stress. A decrease of glucose is external conditions in the history of the fish linked to depletion of reserve energy. 2005). 2004. The intake of diets (Wood et al. However it is possible that fish exposed to Glucose also varies between species and chronic stress suffer substrate depletion that leads to stage of development (Iwama et al. (catecholamine of glucose minutes or days after the stress (Pratap & action for instance) (Vijayan et al. because under stress the spotted Grouper (Epinephelus coioides) when it was fish is rapidly consuming the energetic substrates exposed to cold stress (Cheng et al. 2006). Some authors suggest that this increase of glucose in plasma is not as rapid as for increase in glucose may be attributed to a different cortisol. Barton et al. fishes should not be exercised (Reid et al. 2002). As mentioned. 2008). affect the stress response and glucose clearance (1998) also concluded that those effects are rates”. fold. nonetheless glucose response is still more variable However there have been observed increases than cortisol response. glucose may be observed. some factors can When Nilsson (1989) exposed crucian carp indirectly alter the response of glucose levels in (Carassius carassius) to anoxic conditions (76-169 blood. 1988. life stage. demand so that glucose cannot be accumulated stress hormones such as catecholamines. 2005). taking into account know basal or pre-stress levels of the species to be extrinsic factors such as diet. however some authors Tomlinson 1967. etc. 2006). 1990).166 MARTINEZ-PORCHAS ET AL. (2008) stated that glucose and cortisol “cannot glycogenolysis increase (Vijayan & Moon 1992). Hemre et a decrease on plasma glucose (Fig. al. Trenzado et al.. response. However Reid et al. To this respect. it has been Barcellos et al. 1998). plasma glucose may be attributed to a high energy On the other hand as previously stated. West et al. if catecholamine production or secretion is Factors that affect the intensity of modified also glucose response may be affected. (1993) argued that during versus 4. The generated (glucose) since the main function of the channel catfish under fasting conditions evidenced central nervous system (CNS) is to maintain hyperglycemia after 30 days of experiment (22. Cortisol is known to increase during experimental or acclimation period to avoid blood glucose. presented the maximum glucose exposed to net confinement and electroshock. whereas Simontacchi glycogen phosphorylase which results in a et al. time since studied (Table II). 4(2): 158-178 . To level in the fourth day of exposure. 1994. McEntire 2006). reported a weak rise of glucose (Davis Jr.8 homeostasis. because Energy demand. herein a disruption in cortisol an increase in energy demand. physical stress) of the experiment. in blood glucose whilst cortisol secretion is impaired Rapid measurements. pollution. and even a decrease effect in the glucose response. sugar levels (Wagner et al. this caution will secretion may conclude in an altered glucose reduce the risk to obtain abnormal results. Many researchers documented an increase mechanism of the action of cortisol. Woodward & Strange (1987) observed The freshwater fish rohu (Labeo rohita) that wild rainbow trout experienced a cortisol exposed to high fenvalerate (an insecticide) increase 3 times greater than hatchery fish when concentrations.7 ng·ml-1 in the control group) (Peterson & peak activity glucose use can increase by almost 30- Small 2004). last feeding and season of the year. Falahatkar & Barton 2007) demonstrated that catecholamines itself can increase because cortisol triggers glucose production. a weak or no change in experiment. Hemre & Krogdahl 1996. If it is not part (anoxia. From those results. Similar to cortisol. (1999) were skeptical about the use promote the phosphorylation of the enzyme of glucose as a stress indicator. be considered itself as reliable stress indicators”. a significantly decrease in stored noradrenaline in rearing history including nutritional status may kidney head was observed. 1990). 1991. McLeay 1977. & Wedemeyer et al. response. Wendelaar-Bonga 1990. Mommsen et al.

9 6.4 Emerald rockcod Trematomus bernacchii Temperature 1.9 7. Glucose (mmol/l) Species Stressor Prestress Poststress Exposure References Atlantic cod Gadus morhua Nitrite exposure 0. (2006) Sunshine bass Morone chrysops x saxatilis Temperature and confinement 6. When Chronic/Acute appears. (2007) Rainbow trout Copper and air exposure 5.2 9 Acute Miller et al.4 Acute Barreto & Volpato (2006) Nile tilapia Social stressor 1. (2007) Coral trout Capture and handling Chronic Frisch & Anderson (2005) Plectropomus maculatus 1.17 0. In every line is indicated if the kind of exposure to the stressor was acute or chronic. Cortisol and Glucose: Reliable indicators of fish stress? Table II.6 7.1 7. 4(2): 158-178 Plectropomus leopardus 1.8 Acute Welker et al.6 1.5 Chronic/Acute Davis & Peterson (2006) White sturgeon Acipenser transmontanus Air exposure 1.5 10 Chronic Lowe & Davison (2005) Channel catfish Ictalurus punctatus Handling 1. it indicates that the fish was first acclimated to any condition and thereafter exposed to an acute stressor. (2008) 167 .2 Chronic/Acute Gagnon et al.7 Acute Barreto & Volpato (2006) Rainbow trout Oncorhynchus mykiss Pollutant 4.2 6.5 7.8 10 Acute Urbinati & Carneiro (2006) Nile tilapia Oreochromis niloticus Electroshock 2.9 Pan-American Journal of Aquatic Sciences (2009).7 2.7 Acute Zuccarelli et al.23 Chronic Siikavuopio & Sæther (2006) Bald notothen Pagothenia borchgrevinki Temperature 4.1 10. Plasma glucose values of different species of fishes before and after being stressed.5 Chronic Lowe & Davison (2005) Matrinxã Brycon amazonicus Handling and transportation 2.

(2004) reported an increase when the fish were exposed to argued that “none of the current indicators of stress. Some pyruvate carboxykinase. some anesthetics do not fit with that Szwajkowska et al. (2008) did not observe a rise of glucose in the us to consider that cortisol and glucose are not Atlantic salmon during acute experiments.4 mmol·l-1 when exposed to MS 222. but not by MS instance. Glutamine synthase for example. fructose 1. Also clove (Langiano & Martínez 2008). hours or even days later glucose when anesthetized with clove oil. but enough as stress indicators. carboxykinase (PEPCK) mRNA. but if the experimental units to an alteration in amino acid metabolism (Hopkins are limited and all the samples have to be taken from et al. 2-phenoxytehanol and quinaldine recognized as a stress indicator. Fast several factors that can affect these responses give et al. 2003). Dziewulska- Nevertheless.6-biphosphate. Those cortisol case.168 MARTINEZ-PORCHAS ET AL. considered a source of error. 2-phenoxytehanol and quinaldine) of cortisol in the common carp. (2003) exposed rainbow response. controversial results suggest that perhaps anesthetic For chronic experiments. 1996). study stress. 11. while a limited number of samplings Otherwise. In the case of cortisol it is known that in Blood samples can be extracted during the some fishes a small increase in plasma cortisol leads chronic experiments. A 3-week period of crowding including the stress hormones. also immune response was depressed probability of not measuring any change. adverse conditions Pan-American Journal of Aquatic Sciences (2009). however this is not a indicators of stress in fish. Ortuño et al.9 and 16. it is possible to measure should be established. 1995). the acclimation efficacy in halting glucose increase is species- period should be long enough to ensure that in case dependant and also previous tests may be required of observing a null or weak glucose response. intermediate enzymes of glycolysis (phosphoenol Anesthesia and sampling. rather it is increased glucose in another experiment with recommended to measure glucose over time as in the rainbow trout (Wagner et al. it is recommended that sampling is not cortisol (Reid et al. however regarding the rule and may vary among species and stressors. Iwama et al. it is recommendable to complement cortisol Therefore. Thus single measures oil and MS 222 blocked cortisol secretion but of glucose are not a real indicator. are 100% suitable in stress elevated cortisol and glucose in gilthead reflecting stressed states in fish”. Velíšek et al. 1998). (2003) documented an increase role. Some authors suggest 3 weeks of Previously we documented that many acclimation for laboratory experiments (Houston studies utilized cortisol and glucose as sole stress 1982. clove oil. for this reason it is plausible to consider the same tanks. prevent any negative impact on trout to cortisol treatments and reported an increase performance and thus measure real values of glucose in the abundance of phosphoenolpyruvate or other blood components (Pickering 1998). In another experiment. because there is the respectively. very frequent. (2008) increased 222 or quinaldine. (2003) exposed Atlantic salmon to other four at a rate of 2°C·h-1 and measured glucose every 2°C. glucose can and glucose with other stress indicators to establish a be measured immediately after a chronic exposure to more complete profile of the experimental organism. 4(2): 158-178 . that (see Anesthesia and sampling in cortisol section). Vijayan et al. response is not caused by the general adaptation Other stress indicators. On the other hand. unlike in acute experiments. Aqui-S™ and not finding statistical differences from 10 to 24°C (a Benzoak®) not finding any increase in plasma critical temperature). (2002) tested four anesthetics (MS of glucose 6-phosphatase when injected a high dose 222. response is weak. The catecholamines positively influence that process. syndrome (Fig. benzocaine. Perez-Casanova et al.6. These results appeared glucose. For ideal role of anesthetics is to minimize fish stress instance. in gilthead seabream and reported that basal glucose However. prolonged stress. 2).1. since cortisol and when anesthetics are used before sampling. anesthetics (metomidate. Iversen et water temperature of Atlantic cod (Gadus morhua) al. For by benzocaine and 2-phenoxyethanol. if it is necessary to measure glucose has been observed to increase with small response of over time. (2005) probably because the change in blood glucose levels reported a significant increase in rainbow trout might occurrs minutes. stressful conditions. irregularities have been reported in glucose response glucose 6-phosphatase). Nevertheless. because the consequent handling acid metabolism would be a complement or also a and manipulation of organisms may lead to more accurate indicators of stress even if cortisol erroneous results in the future samples. 2003). in consonance with seabream (Sparus auratus) (Tort et al. that. For instance catecholamines are benzocaine. there are other important level was 3. Johnsson et al.6 mmol·l-1 and increased to 5. parameters that should be taken into account to 11. it is not recommendable to measure that the activity of those enzymes involved in amino cortisol over time.

This response comprises some experiments). These suggestions are not a rule of thumb Melanocyte stimulating hormone (α-MSH) is a and may be replaced or complemented with others. 1999. Thompson et al. Alvaenga & Volpato (1995) stated that integrity. 1984. to select an adequate anesthetic according alternative because they are indicators of the to the species. 1991) manner. and organisms are not exercised despite ventilation rate is a very sensitive test. 2003). repair and degradation of altered or denatured Anesthetics efficiency shows many proteins to maintain homeostasis. Grutter & Pankhurst 2000). produced from pyruvate via the enzyme Conclusions lactate dehydrogenase during exercise and is Cortisol and glucose cannot be eliminated considered as a stress indicator in fishes because its from the stress indicators list. measurements such as other stress hormones. To be required to assist the folding of nascent polypeptide used as stress indicator. This method is very Glucose measurements show many simple and if the water is clear enough the inconsistencies and should be a complement of measurements can simply be determined visually. fish also respond at the cellular blood-cell counts (preferably in chronic level to stressors. frequent. In that fishes (Kawauchi et al. non-invasive methods and/or others. but not how much. 1998. 2000. hsps. it is only useful to indicate experiments the sampling should not be very if the fish is being stressed or not. masking Non invasive methods such as measuring important differences among treatments and that the cortisol in water are a suitable alternative to avoid oxygen consumption and ventilation rate can anesthetic problems. Lamers et al. stress. because the handling and manipulation of Other non-invasive methods are the organisms may affect the future measurements. On the other hand. 2005. etc. the limitation that the severity of any stimulus or Repeated glucose measures have to be done stressor is not reflected in this parameter (Barreto & during or after acute exposures. the physiological status of chains. false results obtained by using any and proved to increase in stressful conditions particular stress test may be validated or contrasted (Arends et al. Evans et al. It also can has been observed that the ventilation rate and be used as a tool to provide a point of reference for a oxygen consumption increase with different particular species. It is important. inconsistencies and there is controversy about the On the other hand. released into blood stream (Iwama 2007). Eventually. 2008). etc. light intensity. Lactate is a chemical with others. The dose of the anesthetic and the water of metabolic differences derived from social stress temperature are subjects of concern. These non-invasive influence the stress response of the fish as long as methods are good candidates to complement the possible and to be certain they are not part of the cortisol and glucose as stress indicators. 2000). different non-invasive convenience to use an effective metabolism blocker methods can be used to complement the biochemical anesthetic or a non harmful anesthetic due to animal and molecular parameters. but due to their high levels are enhanced in under adverse situations variability they must be complemented with other (Thomas et al. usually show data with high variance. presence of predators. while causing a minimum damage to the animal However. It stress tests rather than a main indicator. 2004). although experiment. it has prior sampling blood. protein changes. stress status of any fish. air Results may be more realistic if exposure.Cortisol and Glucose: Reliable indicators of fish stress? 169 activates the HPI axis and catecholamines are methods to measure cortisol (see above). identify possible situations or factors that may 1994. (Brown et al. but during chronic Volpato 2004) and thus. standardization of organisms and experimental Sager et al. In cells of stressed organisms there is Cortisol may be useful only in acute stress an increase in the production of hsps which are experiments and monitored throughout time. as long as oxygen consumption represent an adequate possible. peptide produced in the pituitary cells of several depending on the nature of the stressor. 4(2): 158-178 . act as a molecular chaperone and mediate the organisms should be standardized. measurement of the excretion of nitrogenous In using both cortisol and glucose as stress compounds such as ammonia and urea. compound that plays a role in anaerobic metabolism of animals. 1999. the use of these two they have the same limitation as the non-invasive indicators as pollution or toxic stress indicators is Pan-American Journal of Aquatic Sciences (2009). for example an enhancement in in order to have a more complete profile about the heat shock protein (hsps) synthesis (Iwama et al. which effectively blocks metabolism metabolic rate due to high activity. stressors such as. The ventilation rate and management ethics. gas indicators the researcher needs to be careful to exchange (carbon dioxide) and others (Walsh et al. However conditions are done. complement the stress studies.

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