11

Stress Physiology
P.K. Reddy and J.F. Leatherland
Department of Biomedical Sciences, Ontario Veterinary College, University of
Guelph, Guelph, Ontario N1G 2W1, Canada.

INTRODUCTION
For vertebrate animals, stress is commonly defined as a state or condition in
which the homeostasis of an individual is disturbed as a result of the actions of
external stimuli, termed stressors. Stressors elicit changes in the animals
physiological state, which is interpreted as the stress response. Although this
concept appears to be relatively simple, it is not well understood as a
physiological process, and is commonly poorly defined in the scientific and
popular literature. One major problem is usually the definition of a stressor.
Depending on the physiological state of an animal at any given time, exposure to
the same stressor could elicit negligible, marked or extreme responses, with the
latter possibly leading to death.
All vertebrate animals exhibit adaptive stress as they respond constantly to
changes in environmental conditions, and thus they attempt to maintain a
relative constancy of the characteristics of their blood, as defined by the
paradigm of homeostasis (Hoar, 1983). For each blood parameter there is a
tolerance range of environmental challenge within which the animal is able to
regulate its blood composition. As the environmental challenges exceed the
ability of the animal to preserve constancy, there is resistance to further change,
but the physiological processes are pushed beyond normal limits. If the
environmental challenge persists, the upper or lower limits of resistance are
reached and the animal dies. The literature dealing with the concept of stress and
the response to stressors in animals usually does not address the degree of impact
of a given stressor in a given situation, and the boundary between stress and
distress has not been adequately differentiated.
The study of stress physiology is hampered by the imprecise quantification
of the biological impact of a given stressor, and by the inability to assess the
relative impact of different stressors. The intensity of the impact of a stressor
on an animal is usually based on intuitive beliefs, rather than on scientific
measurement. Stressors that may elicit profound responses in mammals, may
CAB INTERNATIONAL 1998. Fish Diseases and Disorders, Volume 2:
Non-infectious Disorders (eds J.F. Leatherland and P.T.K. Woo)

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not result in comparable physiological changes in fish, and vice versa. Even
among fish species, there is considerable variation in the level of response to a
specific stressor. Moreover, the level of response, even within the same species,
may vary with season and stage of development. Consequently, although the
concept of stress has some value as a loose definition, it should not be viewed as
a predetermined physiological state that is common to all species.
To determine (i) whether a particular treatment is stressful or not, and (ii) the
severity of the stressor, it is usual to monitor an animals physiological response
to that treatment (see below). The logic of this reasoning is questionable, since
the animal is subjected to a treatment that is presumed to be stressful, and
therefore the responses that we measure are assumed to be responses to stress;
ergo, we confirm that the treatment is, indeed, stressful! A more meaningful way
of viewing the concept of stress is to identify the biological value of the
physiological responses that are elicited by exposure of the animal to a particular
type of stressor. Some responses appear to have obvious adaptive value. For
example, the increased plasma glucose concentration evident in many fish
species following chase and capture is assumed to provide the fuel that is
necessary to increase the chance of the fish escaping capture. However, the
biological value of other physiological responses to stressors challenge is not
always self-evident. Nevertheless, it is usually assumed that the response to a
stressor challenge, in some manner, allows an animal to adjust its biological
activity. This is often achieved by energy reallocation countering the pressure
imposed by the stressor. Despite the controversies surrounding the concept and
definition of stress (see above), there is no doubt that in the aquacultural setting,
some standard animal husbandry practices are recognized as stressors that have
suppressive or damaging effects on growth, reproduction and health of fish, and
thus affect the cost of fish production.
Most experimental work in stress physiology has focused on responses of
the neuroendocrine system to stressors. This is largely because it is possible to
measure changes in the secretion of hormones, and the subsequent metabolic
responses to these hormones. Using these quantifiable approaches to identify
changes in neuroendocrine function, it is theoretically possible to develop
management practices that minimize stressful experiences.
The responses to stressors have been catalogued as an acute primary
response, a chronic secondary response and a prolonged chronic tertiary
response (Pickering, 1981; Wedemeyer et al., 1990). The primary response
involves the activation of the neuroendocrine system resulting in the release of
catecholamine and corticosteroid hormones. The secondary responses include
physiological responses to these hormones (e.g. increased cardiac output, O2
uptake and mobilization of energy reserves), and the tertiary response is
identified at the level of the whole organism, and includes the inhibition of
growth, impaired reproductive success and impaired immune responses.

Stress Physiology

DIAGNOSTIC MEASUREMENT OF THE STRESS

RESPONSE
Activation of the neuroendocrine system in fishes triggers the release of
catecholamine and corticosteroid hormones from the interrenal tissue
(Donaldson, 1981; Mazeaud and Mazeaud, 1981). These, in turn, cause the
mobilization of carbohydrate and lipid reserves, and stimulate the production of
energy reserves via gluconeogenesis (Larsson, 1973; Sheridan, 1986, 1987;
Suarez and Mommsen, 1988), all of which may have an adaptive value to the
stressed fish. Changes in plasma catecholamine or corticosteroid hormones
or in blood nutrient levels have been used to monitor the stress response.
Catecholamine levels are less frequently used because the hormone is relatively
difficult to measure, since it relies on chromatographic separation techniques,
and is therefore expensive to perform. In contrast, cortisol levels are relatively
easy to measure using commercial diagnostic kits, and it is generally easy to
differentiate between the usually low basal (unstressed) plasma cortisol levels
and post-stressor levels which can be 10100 fold higher than basal values
(Barton and Iwama, 1991). Because plasma cortisol titres are the most
frequently used diagnostic tool (Pickering, 1981), glucocorticoids, such as
cortisol (and to some extent, catecholamine hormones also) are sometimes
erroneously described as stress hormones. Elevated plasma cortisol levels
should not be considered as a general indicator of a stressful state in fish. Whilst
it is true that certain stressful situations elicit an increased cortisol release,
presumably effecting metabolic changes that have adaptive value, some chronic
and debilitating stressful situations resulting in the death of the afflicted animal
do not elicit a change in cortisol secretion (Laidley et al., 1988).
Plasma cortisol levels rise rapidly (usually within a few minutes) following
acute exposure to some stressors. They return to basal levels in about an hour,
whereas chronic exposure to the stressor results in the plasma cortisol levels
being elevated for longer periods. Peak hormone levels following acute
exposure to a stressor are not normally maintained for the duration of the chronic
application of the same stressor. A major drawback with using cortisol as the
indicator of stress levels is that chasing, capture and blood sampling procedures
are also stressors, and thus may cause increases in blood levels of the hormone.
Moreover, in several species of fishes, basal blood cortisol levels vary markedly
depending on the stage of development (particularly during the reproductive
phase), level of feeding and time of day. Thus, elevated plasma cortisol levels
may not necessarily be indicative of exposure to a stressor. An understanding of
the normal cortisol physiology of a given species is usually needed to assist in
the interpretation of blood cortisol data.
Post-stressor increases in blood glucose levels have also been used as
monitors; glucose levels are easy to measure, and relatively inexpensive, and the
most commonly measured indicator of the secondary phase stress response in
fish (Wedemeyer et al., 1990). Since these increases result, in part, from cortisolinduced gluconeogenesis, blood glucose changes have sometimes been used as
indirect measures of altered cortisol secretion.
Post-stressor increases in muscle and plasma lactate levels, and decreases in

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blood pH values are also found in some fish species (see below); these responses
are associated with the release of catecholamine hormones (Milligan and Girard,
1993), and may thus be used as indirect measures of catecholamine secretion.
Other simple methods, suitable for field studies, may also be useful indicators of
responses to stress, including haematocrit, plasma chloride and sodium
concentrations and physical condition indices, such as condition factor, growth
and various organosomatic indices (HSI, GSI etc.). However, these need to be
validated for a given fish species under experimental situations before they can
be applied generally as a diagnostic measure.

CONSPECIFIC COMPETITION
Conspecific social interactions of fish are found in both wild and captive
situations. In the wild, these interactions are the expression of the evolution of
characteristics that have adaptive value in terms of competition for food,
protection against predators and selection of breeding partners. However, in
captive stocks held in confined spaces, the opportunities for flight or
concealment are limited and the individuals may be exposed to constant social
stress (Noakes and Leatherland, 1977) and subordinate fish commonly die.
Clearly, aggressive behaviour or other forms of interspecific competition, social
dominance and territoriality have no adaptive value for fish held in captivity and
they may become injurious, cause reductions in growth rate and impair the
functions of the immune system. This is of particular concern in the husbandry
of socially aggressive and territorial species, such as salmon and trout.
Confinement in small groups results in the development of marked
hierarchy (Yamagishi, 1962), and subdominant or submissive fish are
characterized by slower growth rate (Li and Brocksen, 1977), darker coloration
and enhanced interrenal activity (Noakes and Leatherland, 1977), loss of
lymphocytes from haemopoietic tissues (Peters and Schwarzer, 1985), elevated
plasma cortisol levels and increased mortality rates (Laidley and Leatherland,
1988) when compared with dominant fish.
In the aquacultural setting the challenge is to identify potential problems and
provide rearing conditions under which conspecific behaviour is no longer a
factor in the acquisition of food, space, and other resources. Periodic size
grading has been used successfully to remove larger dominant fish and thus to
enhance the growth of remaining smaller fish. Similarly the aggressive
behaviour of stocks can be reduced by regularly adjusting stocking densities.
Optimal stocking densities differ depending on the species, and thus specific
recommendations cannot be provided. Fish that exhibit schooling behaviour are
clearly less prone to stocking density stress than solitary species. However, at
least for cultured salmonid species, very low and very high stocking densities are
to be avoided. Intermediate levels of stocking density tend to result in decreased
agonistic behaviour, and thus growth rate and health-related problems are
decreased (Brown et al., 1992).
Under certain situations, feeding itself can be considered to be stressful
because of conspecific competition for the food. Post-prandial increases in

Stress Physiology

plasma cortisol levels have been reported in rainbow trout (Oncorhynchus

mykiss) adapted to a single time of day of feeding (Reddy and Leatherland,
1994). Even when food is provided on demand to well-acclimated animals, the
early morning feeding activity of rainbow trout is accompanied by increases in
plasma cortisol levels, that are most likely caused by conspecific interactions
(Boujard and Leatherland, 1992a,b). However, these periods of increased
plasma cortisol levels tend to be short-lived, and the peak levels are generally
not as high as those found following exposure to handling and chase stressors.
Thus, conspecific interactions associated with feeding periods may not, ipso
facto, represent a major stressor.

GROWTH
In fish, as in other vertebrates, growth is an extremely complex phenomenon that
is influenced by a plethora of biotic and abiotic factors. Optimal growth depends
on the availability of adequate levels of appropriate nutrients, but a common
response to stressors is for fish to cease feeding, and this behavioural change is a
commonly used indicator of a problem in farmed species. Several other factors
are also known to affect growth rates, including the level of activity of the fish,
the extent of conspecific interactions, the quality of the habitat, and season of the
year (usually linked to photoperiod and water temperature). In addition,
semi-lunar rhythms of growth rate changes, superimposed on the circannual
rhythms, have also been found (Leatherland et al., 1993).
The energy partitioning events that affect somatic and skeletal growth,
including the events giving rise to the periodicity of growth in fishes, is closely
regulated by the neuroendocrine system. Several hormone systems are involved
including the adrenal (interrenal) and gonadal steroid hormones, thyroid
hormones, growth hormone (GH), prolactin (PRL), insulin-like growth factor
(IGF), insulin, glucagon, epinephrine, parathyroid hormone and calcitonin. It
must therefore be emphasized that stressor-induced changes in the animals
physiology that result in the release of nutrient reserves will likely involve
altered activity of several components of the endocrine system; these are
secondary compensatory events, and need not necessarily be under the direct
influence of the stressor. For example, a possible route for the growth inhibiting
actions of cortisol (see below) is its proposed action of inhibiting GH and
triiodothyronine (T3) secretion (Leatherland, 1988; Bjrnnson, 1997). However,
the evidence for a direct cause-effect link between cortisol and altered blood GH
and T3 levels is not particularly strong, and any shifts in GH and thyroid
hormone levels are more likely to be compensatory responses to induced
gluconeogenic events.
Some situations in which growth of fish was inhibited may be incorrectly
identified as stressful. High stocking density is intuitively identified as a stressor
for some salmonid fish species, even when feed is provided ad libitum and water
quality is maintained. Reduced growth performance and changes in blood
hormone levels have been associated with high stocking situations for several
species (Pickering and Stewart, 1984; Leatherland and Cho, 1985; Vijayan and

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Leatherland, 1988; Vijayan et al., 1990). However, the pattern of change of the
hormones closely resemble those seen in fish fed a restricted daily ration of food
(Farbridge et al., 1992). There is some evidence to suggest that sight feeding
species do not grow well at high stocking density because their feeding
efficiency is reduced (Wedemeyer, 1976; Refstie, 1977). Consequently, even
when feed is available in excess, they may be, ipso facto, receiving a reduced
ration (Leatherland, 1993). An alternative, but a related explanation for the
effect of high stocking density on growth is the impact of increased social
interactions (Refstie and Kittelsen, 1976) and the resulting increase in activity
levels which results in diversion of energy from growth. In some species, such as
Arctic char (Salvelinus alpinus), growth rate is positively correlated with
stocking density (Jrgensen et al., 1993). This apparent paradox is explained in
part by the observation that agonistic behaviour in this species is reduced as
stocking density is increased, and thus the observation lends support to the
indirect relationship between growth performance and rearing density. In
addition to stocking density, growth rates of fishes have been shown to be
negatively impacted by deterioration of water quality and confinement, and by
repeated netting, handling and transportation procedures. As for stocking
density, the depression of growth rate following such treatments can be
explained on the basis of their impact on some aspect of feeding behaviour.
Some stressors may also affect growth performance of fish, both in the wild and
in aquaculture situations, by affecting digestive efficiency, metabolic processing
of nutrients, or the secretion of anabolic or catabolic hormones. Consequently,
although a change in growth rate, per se, may be a useful indicator of a general
problem (see Chapter 13), it may not be a particularly good indicator of the
action of specific types of stressors.

METABOLISM
The elevated plasma cortisol levels following acute or chronic exposure of fish
to some stressors may elicit a gluconeogenic action, stimulating the mobilization
of amino acids and nonprotein molecules, such as lipids, to be used as substrates
for energy production (Leach and Taylor, 1982; Davis et al., 1985; Sheridan,
1986; Vijayan and Leatherland, 1989; Vijayan et al., 1994a,b; Reddy et al.,
1995). This redistribution of energy reserves indirectly influences growth
performance by directing resources away from anabolic events. The conclusion
is supported experimentally; significant suppression of growth rate and condition factor were found in cortisol-treated fish (Pickering and Duston, 1983;
Davis et al., 1985; Barton et al., 1987; Foo and Lam, 1993a).
Cortisol lowers the levels of carbohydrate reserves in fish, particularly
hepatic glycogen stores (Davis et al., 1985; Barton et al., 1987; Vijayan and
Leatherland, 1989; Vijayan et al., 1991). These reserves are an extremely
important source of readily available metabolite that is used to supply immediate
energy needs. Consequently, any situation or condition that results in the
long-term depletion of hepatic glycogen reserves is potentially detrimental to the
animals, since their ability to tolerate additional stressors is compromised

Stress Physiology

(Vijayan and Moon, 1992). Hepatic glycogen levels are decreased in stressed
fish (Paxton et al., 1984; Vijayan et al., 1990) in a manner similar to that seen in
cortisol-treated fish. When the exposure to a stressor is of short duration, these
responses may not be damaging since hepatic glycogen reserves are rapidly
replenished when external energy sources become available (Farbridge and
Leatherland, 1992b), however, with chronic exposure to a stressor the loss of
carbohydrate reserves may make the animal vulnerable to further stressor
challenges.
Many of the responses to cortisol probably represent direct actions of
cortisol at the tissue level; this is supported by the findings of altered activities of
key metabolic enzymes in the liver and other organs or tissues following cortisol
administration or after stressor challenge (Foster and Moon, 1986; Vijayan et al.,
1991, 1993, 1994a,b). However, in addition, cortisol may potentiate the effects
of other catabolic hormones. For example, it plays a permissive role in the
actions of catecholamine hormones and insulin on the liver of rainbow trout and
the sea raven (Hemitripterus americanus) (Reid et al., 1992; Vijayan et al.,
1993).
In addition to the action of catecholamine hormones on intermediary metabolism, these hormones also influence the oxygen carrying capacity of
haemoglobin. Stressor-induced elevated epinephrine and nor-epinephrine levels
enhance blood oxygen transport directly by elevating or maintaining
haemoglobin-oxygen affinity (Claireax et al., 1988) via increasing the number
and affinity of -adrenoreceptors in the erythrocytes.

DISEASE SUSCEPTIBILITY
Commonly encountered stressors in an aquacultural setting, such as chasing,
handling, netting, crowding, transportation and confinement, are known to
suppress the immune system of teleost fishes (Maule et al., 1987), making them
less resistant to infection (Pickering and Duston, 1983; Schreck et al., 1985;
Maule et al., 1989). There is a well-documented association between environmental stressors and outbreak of disease in fish, probably linked to the
immunosuppressive actions of stressors. The immunosuppression is generally
linked to the post-stressor elevations in plasma cortisol levels since
corticosteroids are known to play a significant immunosuppressive role via their
membrane stabilization actions.
Examples of the increased disease-susceptibility of teleost fish with
experimentally induced hypercortisolism include a marked increase in
susceptibility of brown trout (Salmo trutta) to fungal and bacterial infections and
increased mortality (Pickering and Duston, 1983; Pickering and Pottinger,
1989), and the study by Woo et al. (1987) who demonstrated that rainbow trout
implanted with cortisol were more susceptible to the parasite, Cryptobia
salmositica and had lower antibody titres than infected control fish. It is
interesting to note, however, that although heavy infestations of C. salmositica
can (and does) result in the death of the host fish, plasma cortisol levels were not
elevated in experimentally-infected rainbow trout that were debilitated by the

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infections (Laidley et al., 1988). Rand and Cone (1990) came to the same
conclusion in rainbow trout experimentally infected with the fungus
Ichthyophonus hoferi. The fungus causes a chronic systemic disease with the
formation of granulomas in the kidneys, spleen, liver and heart. Thus, although
cortisol does appear to suppress the immune response, not all pathogens, even
those that are clearly causing severe stress responses, stimulate increased
corticosteroid secretion.
Although the immunosuppressive effects of cortisol have been clearly
demonstrated, albeit indirectly, in several species of fish, the molecular
mechanisms of glucocorticoid-mediated immunosuppression have received
only limited attention in the field of fish immunology. Lymphocytopenia occurs
following the transient elevation of cortisol levels; the reduction in the number
of circulating lymphocytes may be an important link between acute stress
response and the onset of disease (Pickering and Pottinger, 1985). Similarly,
Ellsaesser and Clem (1987) showed that both the number and immunological
competence of circulating lymphocytes in the blood of channel catfish (Ictalurus
punctatus) was reduced following the chronic elevation of plasma cortisol
levels. Further, the cortisol-induced increased susceptibility of Atlantic salmon
(Salmo salar) to Vibrio salmonicida was correlated with a marked reduction in
the number of circulating lymphocytes and thrombocytes (Wiik et al., 1989).
Pickering and Pottinger (1987) also observed a marked and prolonged reduction
in circulating thrombocytes in both brown trout and rainbow trout subjected to
chronic crowding stress, but these authors argued that mechanisms other than
cortisol-induced immunosuppression were responsible.
In addition to suppressing the proliferation of lymphocytes, cortisol has
been shown to suppress immunoglobulin production by lymphocytes and reduce
the production and action of various intercellular mediators such as
prostaglandins and lymphokines (Kaattari and Tripp, 1987; Tripp et al., 1987).
Immunoglobulins (IgMs) play several key roles in the immune response,
including their effects on the neutralization of bacteria, rendering them more
susceptible to phagocytosis. Plasma IgM concentrations are decreased markedly
in masu salmon (Oncorhynchus masou) following administration of cortisol.
Nagae et al. (1994) has shown that plasma immunoglobulin (IgM) production
was significantly lower in cortisol-treated fish. They proposed that increases in
the susceptibility to infectious diseases in stressed fish may be due to effects of
cortisol on IgM production. In coho salmon (Oncorhynchus kisutch), the
cortisol-associated reduced IgM production is due to a suppression of the
mitogenic response of lymphocytes and a reduction in the number of precursor
antibody producing cells (APC) as a result of suppressed lymphokine
production or activity in lymphocytes (Kaattari and Tripp, 1987; Tripp et al.,
1987). These studies also showed that cortisol suppression of APC production
in salmon is caused by inhibition of an interleukin-like (interleukin-1)
molecule secreted by macrophage-like cells which activates antigen-specific
B-cell precursors.
Macrophages in fish have a wide range of functions including phagocytosis,
processing and presentation of antigens to lymphocytes and secretion of
cytokines (MacArthur and Fletcher, 1985). Consequently, factors that modulate

Stress Physiology

the activity of these cells are likely to affect immunocompetence and thus the
potential susceptibility of the animal to disease. Narnaware et al. (1994) showed
that acute exposure to several types of stressors can significantly depress the
macrophage activity in rainbow trout by depressing the phagocytically active
macrophages in the spleen and kidney. These changes are thought to be effected
by elevated catecholamine hormone levels, rather than by changes in cortisol
levels. Narnaware and Baker (1996) also demonstrated that immunological
stress-related responses were annulled by the injection of physiological
concentrations of cortisol during acute stress. They believe that the injection of
cortisol exerts an acute protective effect on the immune cells, possibly by
reducing the release of catecholamines from the sympathetic nervous system.
However, in long term stress, cortisol has deleterious actions upon the activity of
the immune system.
Resistance to disease changes with stage of development in salmonid
species. Smoltification is one example of a developmental event that is linked to
marked variations in disease susceptibility. As coho salmon undergo smoltification they become less resistant to vibriosis. The concomitant reduction in
relative number of splenic lymphocytes and circulating white blood cells
suggest that the general immune system may be impaired (Maule et al., 1987).

REPRODUCTION
The factors involved in the regulation of reproduction in vertebrates are
extremely complex, and there is still much to be learned about the interactions of
the biotic and abiotic factors that influence reproductive success in fish. It is
beyond the scope of this chapter to deal with all known parameters that regulate
reproduction in teleost fishes, but for species in the wild, successful reproduction
requires adequate nutrients for gamete production, the integration of environmental cues with the hypothalamuspituitary glandgonad neuroendocrine
cascade that bring about the growth and maturation of the gametes, and the
integration of environmental cues and the expression of conspecific behaviour
of courting, mating, gamete release and subsequent parental activities in some
species (Fig. 11.1). For salmonid and anguillid species the preparation for
reproduction is complicated by migratory behaviour which, in itself, is
extremely complex and is affected by a myriad of environmental factors. Many
other teleostean species exhibit reproductive strategies that require special
behavioural and physiological adaptations. These include hermaphroditism,
ovoviviparity, strategies to protect the embryos, such as egg deposition at
specific stages of tidal cycles and migration to riffle beds, and many forms of
parental care of the embryos (e.g. mouth brooding, brood pouch development,
nest building, aeration of the embryos by fanning, etc.). Stressors that impact on
any aspect of the physiological or behavioural events can therefore impair
reproductive success. It must also be emphasized that reproduction and
spawning may be, themselves, extremely stressful events, and are associated
with depressed plasma immunoglobin titres (Suzuki et al., 1997), increased
disease susceptibility (Pickering and Christie, 1980), marked lymphocytopenia

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288

Environmental
factors
Photoperiod
Nutritional state
Temperature

Hypothalamuspituitary gland
gonad axis

Puberty
Gametogenesis
Gamete maturation
Courting behaviour
Male

Environmental
factors
Temperature
Spawning substrate
O2 levels
Water turbidity
Pheromones
[Contaminants]

Female

Mating

Courting behaviour
Ovulation/spermiation
Release of gametes
Fertilization

Parental care

Fig. 11.1. Schematic diagram indicating the various factors involved in the reproduction of
fishes. Stressors that act on any of the physiological or behavioural processes have the potential
to impact on reproductive success of a given species.

(Pickering, 1986) and increased perispawning mortality (Mills, 1971). In

migratory salmonid species, plasma cortisol levels are markedly elevated during
the spawning migration (Morrisson et al., 1985; McBride et al., 1986) and may
account for some of the perispawning pathology. However, it is still not clear
whether these markedly elevated plasma cortisol levels are, ipso facto, stressor
related. Moreover, in spite of the hypercortisolism and post-spawning mortality,
the final stages of gamete maturation, ovulation/spermiation and spawning are
usually successfully completed if appropriate spawning areas are available.
Further, for captive species which can be stripped of gametes, the eggs fertilized
and the embryos reared under hatchery conditions, stressors that impact on

Stress Physiology

behavioural events are less significant, although stressors that affect gamete
development and growth are still a concern.
Although reproductive success is influenced by the disruptive effects of
stressors, most studies have been made using captive species, and have generally
focused on the effect of stressors on the hypothalamuspituitary glandgonad
axis. Very little information is available on the effect of stressors on key aspects
of reproductive behaviour. Therefore, we will focus on the reported impact of
stressors on reproductive endocrinology; however, the possibilities of environmental factors influencing reproductive success by impacting behaviour must
also be considered.
As with most other areas of stressor actions, much attention has been paid to
the effect of elevated cortisol levels on the reproductive physiology. It is
generally argued that gonadal steroid hormone secretion in vertebrates is
suppressed by elevated plasma glucocorticoid levels (review by Moore and
Devinche, 1988), but in fish, the endocrine mechanisms involved are still not
clearly understood.
In traditional aquacultural practice, stressors associated with the impairment
of reproductive success include chasing, capture, handling, anaesthetization (for
purposes of sexing and checking for ovulation/spermiation), and confinement.
In controlled studies using repeated application of these stressors, a range of
responses related to impaired reproductive success have been recorded. These
include gonadal atresia (Northern pike, Esox lucius: Scott, 1979), failure to
mature (milkfish, Chanos chanos: Marte and Lacanilo, 1986), delayed ovulation
(several salmonid species: Redding and Patio, 1993; rainbow trout, brown
trout: Campbell et al., 1992, 1994), decreased gonadal weight and reduced levels
of pituitary GtH, plasma gonadal steroid hormones and plasma vitellogenin
concentrations (rainbow trout, brown trout: Pickering et al., 1987; Sumpter et
al., 1987; Carragher et al., 1989), decreased pituitary and plasma GtH levels
(goldfish [Carassius auratus]: Gillet et al., 1981), reduced egg size and reduced
quality of gametes (rainbow trout, brown trout: Campbell et al., 1992, 1994) and
irregular survival of embryos (rabbitfish, Siganus guttatus: Ayson, 1989).
However, not all studies have demonstrated deleterious effects of such
procedures on parameters of reproductive function; for example Wilson et al.
(1995) found no effects on chasing, capture and confinement on spawning
performance, egg fertilization rates or egg hatching rates of cod, Gadus morhua.
Cortisol administration can bring about similar changes in reproductive
physiology, including reduced gonad size and oocyte growth (tilapia,
Oreochromis mossambicus: Foo and Lam, 1993a), and lowered plasma gonadal
steroid levels (tilapia: Foo and Lam, 1993b; rainbow trout, brown trout:
Carragher et al., 1989). These responses have been interpreted as indicative of
an effect of cortisol on GtH secretion. Supporting evidence for this was the
finding of lowered in vitro release of GtH from the pituitary gland (rainbow
trout: Carragher and Sumpter, 1990) and lowered pituitary GtH content (rainbow
trout, brown trout: Carragher et al., 1989). In addition, there are reports of an
impact of cortisol on ovarian follicle as evidenced by reduced basal and
GtH-stimulated in vitro secretion rates of gonadal steroids by the ovarian
follicles (rainbow trout: Carragher and Sumpter, 1990; P.K. Reddy, R. Renaud

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290

and J.F. Leatherland, unpublished data), suggesting a direct effect of cortisol on

gonadal steroidogenesis (Fig. 11.2). However, other studies have not supported
these findings. For example, Barry et al. (1995b) found no effect of cortisol on
oocyte maturation or ovulation in walleye, Stizostedion vitreum, and Pankhurst
et al. (1995), working with goldfish, carp and a snapper, Pagrus duratus, found
no evidence of a cortisol effect on ovarian steroidogenesis or the ability of the
theca/granulosa cells to respond to GtH. These two studies argue against cortisol
actions at the level of GtH secretion and gonadal steroidogenesis, respectively.
In female fish, an additional possible site of action for cortisol on
impairment of gonadal function is the liver (Fig. 11.2). The number of hepatic

Hypothalamus

Pituitary
gland

**
GtH

Gonadal growth

Ovary

**

Vitellogenin

**
**

**

Gametogenesis

Sex
steroids

Liver

Testis

Sex
steroids

E2

Fig. 11.2. Schematic diagram of the hypothalamuspituitary glandgonadal axis, including the
ovaryliver association showing the possible sites of action of cortisol on endocrine function.

Stress Physiology

17-oestradiol (E2) receptors (rainbow trout) and level of hepatic E2 cytosolic

binding (brown trout) was reduced following cortisol administration (Pottinger
and Pickering, 1990; Pottinger et al., 1991), suggesting an impact on vitellogenin production, and a subsequent impaired growth of oocytes. Paradoxically, in
migratory trout and salmon, in which plasma cortisol levels are chronically
elevated (see above), this vitellogenin-suppressive role of cortisol may suppress
vitellogenesis and redirect energy resources toward migratory activity.
In conclusion, whereas there is experimental evidence for a stressor effect
on some measures of reproductive biology, and in some instances these same
measures can be replicated following the administration of cortisol, the
responses tend to be inconclusive, and may not be applicable to all fish species.
Further, although there appears to be some consistency in the cortisol suppression of gonadal steroidogenesis, it is not yet resolved whether such responses are
at a level to impact negatively on gametogenesis or gamete maturation.

EARLY ONTOGENY
Recent studies in a number of teleost species have shown that thyroid hormones,
sex steroids and corticosteroids are present in relatively high concentrations in
the yolk of eggs and these hormones are presumed to be of maternal origin. The
significance of these hormones in the regulation of early development still
remains the subject of considerable speculation and controversy (reviewed by
Brown and Bern, 1989: Lam, 1994; Leatherland, 1994). In general, the levels of
the thyroid hormones decline from the time of fertilization until the endogenous
production of these hormones begins in the embryos which often occurs around
the time of complete yolk absorption. Recent evidence suggests that the embryos
of salmonid species are able to metabolize cortisol and gonadal steroids (Khan et
al., 1997a,b,c). This ability of the embryo to efficiently convert biologically
active steroids such as cortisol into less biologically active forms, lessens the
possibility of these hormones exerting deleterious effects on early development,
such as the neurotoxic actions of glucocorticoids seen in mammalian fetal
development (Sapolsky and Meaney, 1986). This concept of inactivation of
highly biologically active forms of steroids by converting them into less active
metabolites has also been proposed for mammalian models (Akinola et al.,
1996). In addition, cortisol may interfere with the metabolism of other steroids;
in in vitro studies using rainbow trout and Arctic char embryos, when cortisol
was added to the incubation medium there was a significant inhibition of
the conversion of 17-hydroxyprogesterone (17OHP) to either 17,20dihydroxy-4-pregnen-3-one (17,20P) or androstenedione (A4) (Khan et al.,
1997c). This is possibly due to the competition of the two substrates for the
limited number of receptor sites of the enzyme (17,20-desmolase), the same
enzyme that is involved in the metabolism of cortisol and 17OHP. Thus, elevated
cortisol exposure may compromise the ability of the embryo to metabolize yolk
hormones, and thereby impact on early developmental processes.
In fact, significant amounts of cortisol have been reported to be present in
the fertilized eggs, embryos and early juvenile stages of several species of

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teleosts (de Jesus et al., 1991; de Jesus and Hirano, 1992; Hwang et al., 1992;
Barry et al., 1995a; Sampath-Kumar et al., 1995), and therefore there is the
possibility of impaired development. However, in early juvenile stages, cortisol
appears to have a beneficial effect, acting to improve salinity tolerance in salmon
species (Nichols et al., 1985; Richman et al., 1987) and seabass (Lates
calcarifer) (Sampath-Kumar et al., 1993), and interacting with other hormones
to regulate some aspects of early development in the Japanese flounder,
Paralichthys olivaceus (de Jesus et al., 1990, 1991) and Pacific threadfin or moi
(Polydactylus sexfilis) (Brown and Kim, 1995). Thus, the presence of cortisol
may not necessarily be disadvantageous, and there may be differences in the
implications of an increased exposure of tissues to cortisol at different stages
during the early development. Some indication of the onset of a biological role
for corticosteroid hormones might be derived from studies of the first
appearance of the ability to secrete interrenal steroids by the developing embryo
or early juvenile stages.
In the lampreys, Lampetra planeri and Petromyzon marinus, corticosteroid
biosynthesis begins during metamorphosis of the ammocoete (Weisbart and
Youson, 1975; Seiler et al., 1983). This is also the case for the Japanese flounder
which also undergoes a form of metamorphosis (de Jesus et al., 1991). In
salmonid species, which tend to have a relatively long embryonic developmental
phase, the interrenals are capable of cortisol secretion prior to hatching (Pillai et
al., 1974; de Jesus and Hirano, 1992; Pottinger and Mosuwe, 1994). However, a
functional hypothalamuspituitary glandinterrenal (HPI) axis capable of
responding to stressors may not be developed until 2 weeks after hatching in
rainbow trout (Barry et al., 1995a,c) and 5 weeks after hatching in brown trout
(Pottinger and Mosuwe, 1994). The lack of a response of the HPI axis until
post-hatching may be due to the immaturity of the higher brain and hypothalamic centres (HPI axis) regulating the stress response. If this is the case, then
high cortisol levels in the eggs might have negative effects on the normal
development of the HPI axis. Equally, it might be argued that the delay of a
functional HPI axis has adaptive advantage, by avoiding stress-induced
disruption of other physiological processes during key ontogenic stages.

NON-CORTICOSTEROID HORMONES
Plasma concentrations of several non-corticosteroid hormones (growth hormone [GH], prolactin [PRL], somatolactin [SL] and the thyroid hormones,
L-thyroxine [T4] and triiodothyronine [T3]) are known to change following
exposure to stressors, such as handling and confinement, as well as in response
to cortisol administration. However, it is not clear whether these changes are
directly in response to hypercortisolism, or indirect compensatory responses to
alterations in intermediary metabolism and energy processing. Moreover, for
most hormones examined, the reports of altered hormone levels are inconsistent,
even in closely related fish species, and therefore the biological significance of
such changes remains to be determined.
Although several studies have described post-stressor changes in GH, the

Stress Physiology

results have been very contradictory, with increases reported for goldfish, tilapia
and rainbow trout (Cook and Peter, 1984; Kakizawa et al., 1995; Reddy et al.,
1995), decreases for rainbow trout and tilapia, Oreochromis niloticus (Pickering
et al., 1991; Farbridge and Leatherland, 1992a; Leatherland, 1993; Auperin et
al., 1997) and no effects for chum salmon (Oncorhynchus keta) (Wagner and
McKeown, 1986). It is unlikely that the changes are a direct response to
hypercortisolism since Auperin et al. (1997) showed that the stressor-related
decrease in GH in tilapia was transitory, and not correlated with plasma cortisol
levels, and we have found an increase in plasma GH levels following cortisol
administration to rainbow trout (P.K. Reddy, A.C. Holloway and J.F.
Leatherland, unpublished data).
As for GH, the reports of altered plasma PRL levels in response to stressors
are extremely variable. Pottinger et al. (1992) and Kakizawa et al. (1995)
reported a decrease in PRL levels and no changes in response to handling/
confinement stressors in rainbow trout, respectively, whereas stressor-increased
PRL levels were reported for coho salmon (Avella et al., 1991) and tilapia, O.
niloticus (Auperin et al., 1997). It is highly likely that the reported responses are
indirectly related to physiological responses to stressors.
Similarly, exposure to chronic environmental stress commonly causes a
suppression of thyroidal activity in fish, but it is not clear whether the reduction
in plasma T4 and T3 concentrations is a result of chronic stress or due to reduced
food intake normally associated with chronic stress. One of the first measurable
responses to fasting or reduced food ration is a decline in plasma T3
concentration, presumably as an energy sparing strategy (Leatherland, 1994).
Thus, a stressor-induced depression of feeding activity would likely be followed
by compensatory changes in thyroid hormone economy.

MINIMIZING STRESS IN AQUACULTURE

Stress is an unavoidable component in intensive aquaculture. Therefore,
intensive aquacultural practice requires the careful management of the rearing
environment and is assisted by a thorough understanding of the physiology of
fish to minimize the deleterious effects of stress. Under intensive aquaculture
operations, the stock is routinely chased and netted, handled, graded, transported
and treated with drugs. In addition, the fish may also be subjected to chronic
overcrowding and deterioration of water quality. The latter can be eliminated as
a problem if the holding tanks are supplied with high quality water at rates
needed to supply the animals oxygen needs and to dilute metabolic wastes.
Further, since it is well known that the duration of stress response is proportional
to the intensity and duration of imposition of the stressor, by reducing the
duration of netting, grading and hauling protocols, the rate of recovery from
such stressors will be enhanced. Also, during handling, it is important to
maintain ambient water temperature, or even to lower it slightly. Increased water
temperature results in lowered oxygen availability and stressor-induced
mortality of fish is known to increase with increasing water temperature (Barton
and Schreck, 1987). An additional consideration is the exposure of animals to

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repeated stress situations; stressor effects are additive (Barton et al., 1986) and
therefore repeated stressors should be avoided to allow sufficient recovery time
between stressors.
To minimize the adverse responses to transportation, loading density,
temperature stability and provision of sufficient oxygen are key factors. In
addition, anaesthetics (such as MS222 and 2-phenoxyethanol), hyptonic drugs
(such as quinalbarbitone, sodium amytol, quinaldin, etomidate, metomidate)
and salts have been applied successfully as water additives to minimize
transportation stress and to improve survival. Anaesthetics and drugs are
primarily used to slow metabolism, thus reducing O2 consumption and the
accumulation of toxic waste produces. This can be assisted by the withdrawal of
food for a short period of time prior to confinement and transportation. Salt
formulations have been used as water additives to mitigate stressors and improve
survival by protecting the fish against the loss of ions that can result from ionoor osmoregulatory dysfunction that occurs during chronic exposure to stressors
(Carmichael, 1984).
Stress response in fish is considered to be a genetic feature and genetic
differences in the stress response have been reported in chinook salmon,
Oncorhynchus tshawytscha (Barton et al., 1986; Maule et al., 1989) and in
Atlantic salmon (Refstie, 1986; Gjedrem and Gjen, 1995). Therefore, it may be
possible to select for characteristics of improved tolerance to stressor exposure
which may make them better suited for intensive aquaculture conditions.
However, there is insufficient information available to determine at this time
whether the traits of reduced response to stressors are accompanied by
characteristics that are not advantageous for intensive culture.

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