Aquaculture 467 (2017) 28–40

Contents lists available at ScienceDirect

Aquaculture

journal homepage: www.elsevier.com/locate/aquaculture

Understanding fish muscle growth regulation to optimize

aquaculture production
E.J. Vélez, E. Lutfi, Sh. Azizi, M. Perelló, C. Salmerón, M. Riera-Codina, A. Ibarz, J. Fernández-Borràs, J. Blasco,
E. Capilla, I. Navarro, J. Gutiérrez ⁎
Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Aquaculture has become an agronomic activity with noticeable development around the world to respond to the
Received 1 February 2016 simultaneous decrease of fish captures and the increasing demand of aquatic products for human consumption.
Received in revised form 3 June 2016 However, different problems limit the development of this industry and one of those is the time required for most
Accepted 3 July 2016
of the cultured fish species to achieve economically viable the commercial size. The knowledge up to date of the
Available online 4 July 2016
regulatory systems involved in controlling growth has improved very much but, it is still necessary to devote ef-
Keywords:
forts to transform the basic information in application to fish culture production. The aim of the present review is
Muscle growth to summarize the knowledge acquired with the studies about the GH/IGF axis and other hormones regarding
Endocrine regulation their function on the regulation of fish muscle development and growth. To this end, GH and IGFs effects in mus-
GH cle cells on metabolism and development are examined, as well as the contribution of IGF-I binding proteins, IGF-
IGFs I receptors and their downstream regulated molecules like TOR and its relation with cell proliferation and differ-
Myogenesis entiation and the myogenic regulatory factors. The effect of regulatory molecules on cultured myocytes are
MRFs reviewed as well as in vivo responses, including the model of sustained and maintained swimming. Key aspects
Exercise
we consider should be further investigated to complete the scenario of the regulation of fish muscle are also
proposed.
© 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2. Endocrine regulation of fish muscle growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.1. Growth hormone effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2. IGFs actions on growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.3. Metabolic effects of IGFs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.4. Other endocrine regulators of muscle growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.4.1. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.4.2. Thyroid hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.4.3. Steroids and adrenergic agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3. Regulation of fish myogenesis by IGFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4. New markers of muscle growth and quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5. Exercise and muscle growth in fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6. Perspectives on fish growth and aquaculture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

⁎ Corresponding author at: Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain.
E-mail address: jgutierrez@ub.edu (J. Gutiérrez).

http://dx.doi.org/10.1016/j.aquaculture.2016.07.004
0044-8486/© 2016 Elsevier B.V. All rights reserved.
 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
1. Introduction
The double role of muscle, mechanic and metabolic, requires specific
regulatory systems that furthermore will change as a function of age, re-
productive stage and during annual cycles. It should be also considered
that under the name of muscle there is a big variety of tissues
performing different functions including, for instance, the slow and
fast skeletal muscles; although most of the information available refers
to fast skeletal muscle that is also the most interesting part of body fish
in terms of aquaculture product (Mommsen, 2001).
Among the regulatory systems, hormones play an essential role
through a systemic action that will affect all target tissues. But there
are also the local actions that in the case of the growth hormone (GH)
and insulin-like growth factors (IGFs) modulate effects in specific tis-
sues such as the muscle. In fact, the GH/IGF axis is considered the
most important endocrine system regulating skeletal growth, although
modulation of other hormones like insulin, thyroid hormones, steroids,
etc. allows the fine control of muscle growth and development as well
as the adaptation to endogenous and external changes (Moomsen and
Moon, 2001).
The myogenic regulatory factors (MRFs) represent another impor-
tant group of molecules that exert a significant role specially for muscle
development but also during the period of compensatory growth after
fasting or reproductive stage, as well as for tissue regeneration after
an injury. MRFs, myocyte enhancer factors (MEFs) and myostatin are
the best known molecules involved but, there are other factors less
specific for muscle that also have important roles during myogenesis
(e.g. FGF, HGF, PAX, Sox, etc.) (Fuentes et al., 2013). Moreover, in recent
years, the target of rapamycin (TOR) complex has appeared as an im-
portant level of integration between nutritional and endocrine inputs
to improve growth (Vélez et al., 2014, 2016), which is of significant in-
terest for fish culture.
Due to the dynamic role of muscle from a metabolic point of view,
the endogenous proteolytic systems, which include the calpains, the ca-
thepsins and the ubiquitin-proteasome system, are considered also key
regulatory factors controlling growth potential. They are very important
systems in a tissue that often changes its metabolic role from an anabol-
ic and synthetic side to a proteolytic mode to provide the organism with
a supplementary load of amino acids and energy.
Muscle growth in fish has also a differential trait from other verte-
brates seen in many species, that of continuous growth. This fact deter-
mines that after sexual maturation fish continue to grow still with
important rates of muscle hyperplasia in comparison with most mam-
mals, which can change muscle mass only by hypertrophy (Stickland,
1983). Furthermore, this also occurs in a reduced group of fish species
(e.g. zebrafish, Danio rerio) that has been dubbed as a determined
growth species (Biga and Goetz, 2006). Continuous growth in fish offers
an interesting model of research on the role of stem cells, and also gives
valuable possibilities for application to aquaculture.
In the last years, different publications have claimed the interest of
exercise as a mechanism to improve fish aquaculture production, pro-
viding good results in growth and flesh quality. In mammals, it is well
known that exercise stimulates a GH response that consequently pro-
vokes skeletal growth. Thus, it is interesting to consider the possibility
of applying exercise during fish growth, which furthermore, due to
the high level of muscle plasticity, can respond with hyperplasia,
which has been demonstrated to be very important for flesh quality
(Blasco et al., 2015).
Our group has a long history in the research of fish metabolism and
endocrinology and in the last years has focused its attention in the reg-
ulation and improvement of fish growth by means of in vitro and in vivo
approaches. The aim of this review is to summarize the current informa-
tion in fish muscle growth regulation, focusing primarily in GH/IGF axis
and its systemic and local actions on myocytes, and to propose new
molecules and approaches that can be interesting for basic research
and its application for fish aquaculture.
29
2. Endocrine regulation of fish muscle growth
2.1. Growth hormone effects
The GH/IGF axis is the main regulator of growth in vertebrates; its
role in fish has been demonstrated in many species and previously
reviewed (Company et al., 2001; Reinecke et al., 2005; Reinecke,
2010; Reindl and Sheridan, 2012; Fuentes et al., 2013). GH exerts signif-
icant metabolic effects, as the regulation of muscle protein synthesis,
determining an increase on growth by both hyperplasia and hypertro-
phy. There are many references describing the effects of GH treatment
on muscle protein synthesis, muscle growth acceleration, hyperplasia,
muscle accretion, etc. (reviewed in: Moomsen and Moon, 2001). Fur-
thermore, it should be taken into account that the indirect effects of
GH in fish growth can increase appetite or the intestinal nutrient up-
take. An interesting aspect to consider is what part of the GH effect cor-
responds to its direct action on muscle and what is mediated through
IGFs secreted by the liver under GH stimulation.
In this sense, several studies in salmonids have demonstrated that a
GH chronic treatment causes an increase in plasma IGF-I levels (Biga et
al., 2005; Raven et al., 2012; Kling et al., 2012). Moreover, such a treat-
ment provoked an increase in IGF-I gene expression in the liver in
coho salmon (Oncorhynchus kisutch) (Raven et al., 2012) as well as
also in muscle and other tissues in rainbow trout (Oncorhynchus mykiss)
(Biga et al., 2004b), thus supporting both systemic and local paracrine/
autocrine actions for GH and IGFs.
The studies on GH receptors and their signal transduction are rather
recent, and have demonstrated the existence of two main receptors
(GHR-I and GHR-II) with complementary functions (Fuentes et al.,
2013). Truncated variants of GHR-I (tGHR-I) have been characterized
in turbot (Scophthalmus maximus) (Calduch-Giner et al., 2001), and in
other related species as Japanese flounder (Paralichthys olivaceus)
(Nakao et al., 2004) or fine flounder (Paralichthys adspersus) (Fuentes
et al., 2012). In the last case, tGHR-I has been postulated as responsible
for the slow growth of this species. Two different GH receptors were
also described in several other fish species (Tse et al., 2003; Benedet et
al., 2005; Saera-Vila et al., 2005; Jiao et al., 2006; Walock et al., 2014)
and in gilthead sea bream muscle (Sparus aurata), different responses
to fasting and re-feeding were described (Saera-Vila et al., 2005). We
have recently observed that both muscle GH receptors play different
roles in gilthead sea bream adapted to exercise (Vélez et al., 2016).
These results agree with the fact that, in gilthead sea bream, GHR-I
seems to be more involved in anabolic signals, while GHR-II is directed
towards energy depot mobilization, although more studies are required
in different species and conditions to generalize these results. Fish GH
receptor signaling is even a younger discipline and Fuentes et al.
(2013) have reviewed the state of the art. Although the main pathways
are well conserved among vertebrates, it seems that both isoforms use
slightly different transduction molecules (Jiao et al., 2006; Kittilson et
al., 2011; Fuentes et al., 2012) with GHR-I working more through
STAT5 and GHR-II through ERK (Kittilson et al., 2011). Fuentes et al.
(2012) pointed out that in fine flounder, the JAK2/STAT5 pathway is
inactivated during fasting but reactivated with nutritionally favorable
conditions, which is in agreement with the GH receptor division of func-
tion observed in gilthead sea bream.
There are not many studies on the direct effect of GH on muscle but
Rius-Francino et al. (2011) demonstrated the stimulatory effect of
gilthead sea bream GH on proliferation of cultured gilthead sea bream
myocytes (Fig. 1 insert). Interestingly stronger stimulatory effects on
myocytes proliferation were obtained when GH was administrated to-
gether with IGF-I or IGF-II (Fig. 1). The GH transgenic coho salmon
(Devlin et al., 1994) has represented a valuable model to study the biol-
ogy of growth, metabolism and behavior (Abernathy et al., 2015; de la
García et al., 2015; Chen et al., 2015; Kim et al., 2015a, 2015b) showing
that GH overexpression produces both muscle hyperplasia and hyper-
trophy. Levesque et al. (2008) summarized the main facts that explain
30 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
Fig. 1. (A) Immunocytochemical detection of proliferating cell nuclear antigen (PCNA) in gilthead sea bream myocytes at day 4. Cell proliferation was determined after 6 h in the absence
(control, CT) or presence of mammalian GH 10 nM, IGF-I 100 nM, IGF-II 100 nM or combined treatments of GH and IGFs (IGF-I or IGF-II). Results are expressed as percentage of PCNA
positive cells (mean ± SEM). The insert figure shows the proliferative effects of sea bream GH (sbGH) on PCNA in the same conditions. Different letters indicate significant differences
(P b 0.05) and asterisks indicate differences with control (P b 0.001). (B) Representative microscope image indicating negative (arrow) and positive (arrow heads) cells. Bar =
200 μm. Adapted from Rius-Francino et al. (2011).
the higher growth in GH transgenic Atlantic salmon (Salmo salar), com-
prising the higher numbers of myogenic precursor cells, their prolifera-
tion rates and their direct proliferative response to GH treatment. GH
transgenic fish have been obtained for other species like rainbow
trout, carp (Cyprinus carpio), tilapia (Oreochromis niloticus), channel cat-
fish (Ictalurus punctatus) or zebrafish (Devlin et al., 2001; Rasmussen
and Morrissey, 2007; Figueiredo et al., 2007).
The different experiments of GH treatments (Raven et al., 2012;
Kling et al., 2012; Biga et al., 2004a, 2004b, 2005; Gahr et al., 2008) dem-
onstrated the effects of this hormone on metabolism and muscle
growth. Biga et al. (2004a) found that GH treatment in rainbow trout in-
creases myosin protein levels, thus regulating the expression of the
most abundant muscle protein. Gahr et al. (2008) reported that even
after short-term treatment, GH augmentation alters the expression of
genes involved in metabolism and growth regulation in rainbow trout.
Nevertheless, Biga and Mayer (2009), showed a differential regula-
tion of the growth-related genes as effect of GH treatment, in a compar-
ative experiment with an indetermined and a determined growth
fish species (i.e. giant danio, Danio aequipinnatus and zebrafish,
respectively). In this work, the IGF-I and GHR-I expression was higher
in giant danio muscle that in zebrafish. However, the same treatment
increased the myostatin expression in zebrafish, whereas it was
down-regulated in the case of giant danio. These results suggest that
myostatin could be responsible for limiting the growth stimulation
caused by GH treatment in this determinate-growth species (zebrafish).
Interestingly, GH transgenic fish were unable to respond to GH treat-
ment, indicating that transgenic fish show certain level of saturation on
stimulatory growth pathways (Raven et al., 2012). Recently, we have
treated gilthead sea bream fingerlings and juveniles with GH (Vélez et
al., unpublished data) and results demonstrated also in this species a
growth increase and diminution of fat depots.
On the other hand, GH immunoneutralization in rainbow trout
(Fauconneau et al., 1996) decreased mainly muscle protein synthesis,
together with a decrease in body weight. In zebrafish, Silva et al.
(2015) achieved a double mutant for GH and GHR that resulted in
lower weight and a strong decrease of the somatotrophic axis intracel-
lular signaling by diminishing its signal transducer (STAT5.1).
McMenamin et al. (2013) identified a zebrafish GH1 mutant, vizzini,
which exhibited abnormal small body size and increased accumulation
of adipose tissue. All these results support the direct action of GH on
muscle growth stimulating protein synthesis, hyperplasia and hypertro-
phy, which in turn increase body weight.
2.2. IGFs actions on growth
IGF-I and IGF-II have clear and important effects on fish muscle
growth. In the first studies on IGF-I receptors in fish muscle it was
surprising the abundancy of them (Párrizas et al., 1994a, 1995), espe-
cially when comparing with the number of insulin receptors. This was
true for white and red skeletal muscle, cultured myocytes and heart
(Moon et al., 1996; Baños et al., 1997; Castillo et al., 2002, 2004), and
confirmed in muscle of different poikilotherms. However, in
homeotherms, the situation reverted with higher levels of insulin recep-
tors that through evolution have gained importance in the regulation of
muscle function. Thus, the role of IGFs in fish muscle is facilitated by
their higher number of IGF-I receptors. Furthermore, to understand
IGFs action, together with IGFs receptors, the function of IGF-I binding
proteins should be considered. IGFBPs represent a key step in the regu-
lation of IGFs action, but in fish there is limited information. Cunming
Duan has done extensive work in fish, mainly zebrafish and with cell-
line models (Duan and Xu, 2005; Wang et al., 2009; Ocampo Daza et
al., 2011). As in mammals, at least six different IGFBPs have been de-
scribed that exert a double function of growth promotion and inhibition
in fish. A long variety between species and tissues can be found in terms
of IGFBPs expression (see below).
The effects of IGFs in in vivo approaches have been demonstrated in
different studies. By administration of IGF-I through implanted mini-os-
motic pumps, it was shown that IGF-I treatment increases the body size
of juvenile coho salmon (McCormick et al., 1992). A significant correla-
tion between plasma IGF-I levels and growth rates in salmon was dem-
onstrated (Beckman et al., 1998; Dyer et al., 2004; Kawaguchi et al.,
2013) and Beckman proposed that these IGF-I plasma levels can provide
a useful index of growth, although it is necessary to precisely define the
fish groups to compare (Beckman, 2011). Little information is available
regarding circulating IGF-II blood levels in fish (Gentil et al., 1996;
Wilkinson et al., 2004) but a positive correlation between IGF-I, IGF-II
and body weight was also observed in Atlantic salmon under nutritional
restriction (Wilkinson et al., 2006).
In vivo approaches to study IGFs face the difficulties to differenti-
ate between the effects of circulating levels of IGFs from hepatic ori-
gin and those from IGFs locally secreted by the muscle. In this sense,
in vitro studies enable to analyze the effects of a specific treatment
without systemic influences. IGF muscle gene expression is regulat-
ed by GH, and Vong et al. (2003) found in carp that GH increased
IGF-I and mainly IGF-II expression in muscle. Jiménez-Amilburu et
al. (2013) found in gilthead sea bream myocytes, that GH increases
total IGF-I expression and that GH plus IGF-I also increases IGF-II ex-
pression. However, there was a clear dependency of the time and
treatment conditions. Bower and Johnston (2010b) found in Atlantic
salmon myocytes that the addition of IGF-I and amino acids to
starved cells results in an 18-fold increase in IGF-I mRNA, indicating
a positive feedback mechanism. Recently, Azizi et al. (2016) have
demonstrated in sea bream myocytes that both IGF-I and IGF-II stim-
ulate the gene expression of IGF-I but not that of IGF-II. The complex
 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40 31

Fig. 2. Effects of insulin (INS) 1 μM, IGF-I 100 nM or IGF-II 100 nM on 2-deoxyglucose (A) and L-alanine (B) uptake in gilthead sea bream myocytes at day 4. The treatments were for 2 h in
the case of L-alanine and 1 h for the 2-deoxyglucose. Data are expressed in percentage over the basal (mean ± SEM). Different letters indicate significant differences (P b 0.05). Adapted
from Montserrat et al. (2012).

network of peptides, binding proteins and receptors provides an ef-
ficient regulatory system of muscle function.
Fauconneau and Paboeuf (2000) and Gabillard et al. (2010) demon-
strated, by means of BrdU technique, the proliferative properties of IGF-I
and IGF-II in a dose dependent manner in rainbow trout muscle satellite
cells. Castillo et al. (2004) using the thymidine technique found the
same effects for IGF-I in rainbow trout myocytes and Codina et al.
(2008) demonstrated that IGF-II stimulated also thymidine incorpora-
tion in the same model. Rius-Francino et al. (2011) found in gilthead
sea bream myocytes (by means of PCNA immunocytochemistry) that
IGF-I and IGF-II stimulate proliferation, producing IGF-II stronger effects
than IGF-I (Fig. 1).
Cleveland and Weber (2010) in rainbow trout primary cultured
muscle cells observed that IGF-I stimulates protein synthesis and in-
hibits proteolysis via PI3K/protein kinase B signaling. Upton et al.
(1998) also demonstrated the IGF-I inhibition of proteolysis in salmon
cell lines.
Overall, the abundance of IGF-I receptors in muscle tissues an-
nounced an important role for these growth factors in fish muscle de-
velopment that has been demonstrated later using in vitro and in vivo
studies. Thus, the IGFs stimulatory effects on proliferation and protein
synthesis shown in different species has been reinforced with their pos-
itive correlation with fish size and weight growth.
2.3. Metabolic effects of IGFs
The higher number of muscle IGF-I receptors compared to insulin re-
ceptors in fish suggested that IGFs could play some metabolic functions
in this tissue besides their proliferative and differentiation effects. The in
vitro culture of myocytes offered the possibility to investigate such ef-
fects. Castillo et al. (2004) found that IGF-I stimulates glucose uptake
in trout myocytes at a higher level than insulin. The same effect was ob-
served for IGF-II in rainbow trout myocytes (Codina et al., 2008) and in
gilthead sea bream myocytes (Montserrat et al., 2007b, 2012) (Fig. 2a).
Later, the stimulatory effect of IGF-I in protein expression of glucose
transporter 4 (Glut4) was also found both in rainbow trout myocytes
(Díaz et al., 2009) and gilthead sea bream myocytes (Montserrat et al.,
2012).
Similarly, IGF-I and IGF-II stimulated the uptake of alanine in
myocytes of rainbow trout (Castillo et al., 2004; Codina et al., 2008)
and gilthead sea bream (Montserrat et al., 2007a) at a stronger level
than insulin, indicating their strong anabolic role (Fig. 2b). Also, free
fatty acids were regulated by IGF-I: uptake in rainbow trout myocytes
was increased by this growth factor in a stronger way than insulin
(Sánchez-Gurmaches et al., 2010). Thus, it is clear that together with
the role of IGFs stimulating proliferation and differentiation of muscle
cells, IGFs also play a remarkable function for the regulation of

Fig. 3. Schematic representation of IGFs signaling pathways to control substrates uptake, protein synthesis and muscle cell proliferation. IGFs through the activation of the PI3K/AKT
pathway stimulate the entrance of amino acids (AA) and glucose (GLU) into the cell, which will provide substrates and energy for protein synthesis and cell proliferation via TOR and
MAPK signaling, respectively.
32 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
metabolism in fish, facilitating the uptake of substrates that will contrib-
ute to the growth of muscle (Fig. 3).
2.4. Other endocrine regulators of muscle growth
Moomsen and Moon (2001) reviewed the effects of several hor-
mones in the regulation of fish growth. It is not the objective of this re-
view to cover in depth this topic but rather to focus in muscle
regulation. Together with GH/IGF axis other endocrine molecules
exert their influences on muscle growth and will be briefly commented
in the lines below.
2.4.1. Insulin
Insulin is in mammals an important regulator of muscle function, but
it seems that in fish such a role is not so well developed, with IGFs serv-
ing part of its metabolic control. As mentioned above, the number of in-
sulin receptors in the muscle of all fish species examined is clearly low
compared to mammals (Párrizas et al., 1995). Different studies showed
that carnivorous fish species presented lower insulin receptors than
omnivorous species (Párrizas et al., 1994b), correlating the type of diet
with the role of the muscle to allocate postprandial nutrients. While it
was demonstrated that the number of insulin receptors was up-regulat-
ed by diet or in response to insulin (Párrizas et al., 1994a) the receptor
number never reached the level of IGF-I receptors and the action of in-
sulin in fish is considered less important than in mammals. Neverthe-
less, there are different studies that demonstrate the anabolic function
of insulin stimulating in vivo the uptake of amino acids in muscle
(Tashima and Cahill, 1968; Inui and Ishioka, 1983). Using in vitro cul-
tured myocytes from rainbow trout and gilthead sea bream, it has
been demonstrated that insulin increases the uptake of amino acids
(Castillo et al., 2004; Montserrat et al., 2012). In the same model, insulin
increased glucose entrance in myocytes by activating Glut4 (Díaz et al.,
2009). Although at a lower level than IGF-I, insulin stimulated the up-
take of free fatty acids from the culture medium in trout myocytes
(Sánchez-Gurmaches et al., 2010). However, insulin did not provoke
thymidine uptake by rainbow trout myocytes suggesting a rather
minor role in cell proliferation, and focused its emerging function in me-
tabolism regulation.
2.4.2. Thyroid hormones
There is abundant information on the synergic effects of thyroid hor-
mones (TH) with other factors (e.g. GH, steroids) on the stimulation of
fish growth (Donaldson et al., 1979). Duarte-Guterman et al. (2014)
reviewed such cross-talk between hormones, mainly for TH and sex ste-
roids, regulating development and adult endocrine systems. In fact, a
prominent function of TH, common to all vertebrates, is their role in dif-
ferentiation of specific tissues and in general in the development to the
adult phenotype. During the process of metamorphosis from larvae to
juvenile, fish undergoes numerous changes in the morphology and
physiology that are accompanied by surges in TH. This is especially rel-
evant in the metamorphosis of the flatfish (Campinho et al., 2015;
Gomes et al., 2015).
TH receptors (THR) are characterized in different fish species and in
flounder (Paralichthys olivaceus) THRα and THRβ show complementary
profiles in muscle metamorphosis (Moomsen and Moon, 2001) contrib-
uting to changes towards the adult phenotype (Laudet, 2011). Treat-
ment with TH accelerates metamorphosis (Power et al., 2001) while
goitrogenic thiourea inhibits it.
Direct effects in fish muscle are described, with increases in protein
and RNA synthesis in a dose dependent manner in tilapia (Tilapia
mossambica) (Matty et al., 1982). In general, TH administration in-
creases the rate of muscle growth and the length and weight in fish
(Matty et al., 1982) although it is difficult to separate the direct effects
of TH and those via GH or IGFs and this will require further research.
Little and Seebacher (2013) showed in zebrafish that hypothyroidism
treatment decreased mRNA concentrations of myosin heavy chain
(MHC) fast isoforms and affected general swimming performance. GH
and IGF-I exert important effects on vertebrate thyroid function that
in general are stimulatory. Data in fish are scarce, but synergic effects
of GH and TH on growth stimulation have been demonstrated in differ-
ent fish species, with several studies suggesting also that T4 can stimu-
late GH release from pituitary somatotrophs (Donaldson et al., 1979).
Furthermore, both hormones exert important metabolic effects; there-
fore, it is difficult to understand fish growth without GH and thyroid
hormones contribution.
2.4.3. Steroids and adrenergic agonists
Glucocorticoids and gonadal steroids exert significant effects in the
muscle of vertebrates. Cortisol is the main glucocorticoid in fish and it
is involved importantly in the stress response. During stress, cortisol
maintains the balance in energy homeostasis through stimulation of
processes such as gluconeogenesis and inhibition of others such as di-
gestion and immune response (Schjolden et al., 2009). It is also involved
in muscle proteolysis in different conditions, like spawning migration,
smoltification, exercise, etc. (Ellis et al., 2012).
Corticosteroid receptors are expressed either in the cell membrane,
for rapid actions or into the nucleus to regulate gene transcription. Tel-
eost fish express two kinds of corticosteroid receptors, glucocorticoids
(GR) and mineralocorticoids (MR), which bind to cortisol with different
affinities. GR show two isoforms, GR1 and GR2 that play different roles
(Ellis et al., 2012).
Cortisol is a key controller of fish intermediary metabolism, increas-
ing oxygen uptake and gluconeogenesis and inhibiting glycogen synthe-
sis. Such an increase in metabolic rate would contribute to reduce
growth and in support of this Ellis et al. (2012) described a negative cor-
relation between growth and cortisol levels. Cortisol also regulates mus-
cle glycogen synthesis, and rainbow trout subjected to exhaustive
exercise presented a decrease of white muscle glycogen stores in paral-
lel to the rise of plasma cortisol levels (Milligan, 1997). Increased plas-
ma cortisol levels also suppress appetite, reducing food intake, food
conversion efficiency, and growth in juvenile rainbow trout (de Boeck
et al., 2001).
Cortisol possesses multiple functions in fish (osmoregulation, repro-
duction, behavior, etc.) but also interacts with GH/IGF to affect fish
growth (Leung et al., 2008; Kajimura et al., 2003; Pierce et al., 2011;
Won and Borski, 2013). Recently, Madison et al. (2015) suggested that
the cortisol growth-suppressing effects in rainbow trout result from re-
duced food intake mediated partially by increases in liver leptin-a1,
brain corticotropin-releasing hormone, and a sustained increase in he-
patic IGFBP-1 expression that reduces GH/IGF axis actions from mobiliz-
ing energy reserves. Nevertheless, it would be helpful to know more
about the indirect effect of cortisol on GH/IGF axis mediated thru the
levels of blood metabolites.
The sex steroids have been related with changes in muscle protein
synthesis in fish linked to reproductive cycles (Moomsen and Moon,
2001). Anabolic steroids are growth promoters mainly in juveniles,
stimulating muscle protein synthesis, amino acid uptake in intestine, in-
crease in nitrogen retention and ammonia excretion diminution
(Moomsen and Moon, 2001). Suppression of interrenal cells activity
by androgens has been observed in fish (Pottinger et al., 1996) thus
preventing cortisol proteolytic activity. Much of the information of sex
steroids in growth arrives from the interaction with GH/IGF axis
(McCormick et al., 2005). 17 β-estradiol (E2) is catabolic reducing
liver response to GH by decreasing IGF-I expression and production, as
well as activating proteolytic mechanisms in muscle (Cleveland and
Weber, 2011). Contrary, testosterone (T) is anabolic, increasing hepatic
GH sensitivity and IGF-I production. Both steroids exert their effects in
muscle differentially regulating the sensitivity to IGF-I (Hanson et al.,
2012). Recently, Cleveland and Weber (2015) identified in salmonids
the mechanisms to respond to sex steroids and the disparate effects of
estrogens and androgens in fish growth. In fact, sex steroids affected dif-
ferently IGF-I, IGF-II and IGFBPs expression in liver and muscle of
 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
Fig. 4. Relative gene expression profile throughout gilthead sea bream myocyte culture
development of total IGF-I, IGF-II and the splice variants IGF-Ib and IGF-Ic. The splice
variant IGF-Ia was not detectable. Mean ± SEM. Adapted from Jiménez-Amilburu et al.
(2013).
rainbow trout. Thus, estradiol down-regulated GH/IGF axis in liver,
while in muscle maintained growth signals. Contrary, androgens up-
regulated GH/IGF axis in liver but not in muscle.
β2-adrenergic agonists (BAAs) have similar actions to catechol-
amines, binding to the adrenergic receptors to produce specific actions
in target tissues. BAAs have been used in terrestrial farm animals to in-
crease muscle protein and to reduce muscle fat (Moomsen and Moon,
2001). In fish, BAAs administration has been investigated in several spe-
cies, which revealed lower effects than in mammals (Mustin and Lovell,
1993). Vélez et al. (unpublished results) found that formoterol activated
gilthead sea bream myocytes TOR phosphorylation. Salem et al. (2006)
treated rainbow trout with oral clenbuterol and ractopamine, analyzing
their effects on muscle proteases and proteins. The study concluded that
both BAAs show anabolic effects in muscle up-regulating protein syn-
thesis and down-regulating proteolytic systems, although the authors
claimed more research before the application of these components in
aquaculture. Abo et al. (2012) demonstrated that the rat muscle hyper-
trophy provoked by clenbuterol treatment was parallel to the up-regu-
lation of IGF signaling.
Thus, although the GH/IGF axis exerts the main role in the regulation
of growth, other hormones can modulate its action and need to be taken
into account to understand the final growth results. Natural reproduc-
tive cycles or stress episodes will determine steroid circulating levels
that directly or indirectly will influence GH/IGF axis consequently af-
fecting growth performance.
3. Regulation of fish myogenesis by IGFs
It is well known that IGF-I plays an important role in skeletal muscle
fiber regulation, including satellite cells activation, proliferation and dif-
ferentiation (Snijders et al., 2015). In fish, Johnston et al. (2011) and
Fuentes et al. (2013) reviewed the effects of IGFs on skeletal muscle
growth regulation. The effects of IGFs on proliferation and differentia-
tion in fish muscle cells have been also previously discussed (see
above; Fauconneau and Paboeuf, 2000; Castillo et al., 2004; Codina et
al., 2008; Gabillard et al., 2010) as well as the differences between
IGF-I and IGF-II (Rius-Francino et al., 2011; Azizi et al., 2016) (Fig. 1).
Through the myogenic stages there are clear variations in muscle cell
function. In our laboratory, we have studied the expression of IGFs dur-
ing the development of gilthead sea bream myocytes. IGF-II expression
was highest at the beginning of culture and decreased when the cells
started to differentiate, similar to effects observed for total IGF-I
(Fig. 4). In the case of IGF-I, among the splice variants described previ-
ously by Tiago et al. (2008), IGF-Ib showed a parallel expression pattern
as that of total IGF-I, whereas IGF-Ic showed its own profile during cul-
ture progression (Jiménez-Amilburu et al., 2013). Peterson et al. (2004)
found in channel catfish muscle that IGF-II gene expression was higher
than that of IGF-I; and in the same study, it was observed in two catfish
33
families that the fast-growing family expressed higher levels of IGF-II
than the slow-growing family, suggesting a specific role for IGF-II.
As indicated previously, IGFBPs and IGF-I receptors modulate the ac-
tion of growth factors. IGFBPs are a complex regulatory system includ-
ing six different proteins that furthermore can have paralogs with
specific functions (Johnston et al., 2011). IGFBP-1 was considered a neg-
ative regulator (Duan et al., 2000) and more recently other findings sup-
port this role in zebrafish muscle for both IGFBP-1a and IGFBP-1b being
these the only IGFBPs genes responding to a fasting–re-feeding (Amaral
and Johnston, 2011). A similar function was attributed to IGFBP-2 in
rainbow trout (Gabillard et al., 2006) and fine flounder (Safian et al.,
2012), but not in Atlantic salmon (Bower et al., 2008). Moreover,
IGFBP-3 was proposed as a growth stimulator, but the results in fine
flounder are variable (Safian et al., 2012). IGFBP-4 and IGFBP-5 in the
different species studied seem to play anabolic functions (Azizi et al.,
2016; Vélez et al., 2016) and can be considered pro-myogenic molecules
(Fuentes et al., 2013). The few studies which have analyzed the role of
IGFBP-6 (Gabillard et al., 2006; Bower et al., 2008; Safian et al., 2012)
point to it being a negative regulator of IGF-I. The regulation of IGFBPs
is not very well known in fish muscle and Bower and Johnston
(2010b) found that the addition of IGF-I, IGF-II or insulin and amino
acids to the medium of Atlantic salmon cells increased the expression
of IGFBP-5.2 and IGFBP-4, but not IGFBP-5.1. IGF-I and IGF-II, directly
stimulated IGFBP-6 gene expression but not when amino acids were
present. Azizi et al. (2016) observed in gilthead sea bream myocytes
that IGFBP-4 did not respond to IGFs treatment but IGFBP-5 was stimu-
lated by both IGFs. Thus, once specific roles have been defined for a spe-
cific species, IGFBPs can be good markers of the growth condition of the
fish.
Regarding IGF receptors, our group has investigated in different fish
species their binding characteristics (Párrizas et al., 1995; Méndez et al.,
2001) and signal transduction pathways (Castillo et al., 2006;
Montserrat et al., 2007b). With respect to IGF-II receptors, very little
studies are available in fish but our group (Méndez et al., 2001) reported
a very low number of them in membranes of brown trout (Salmo trutta
fario) embryos. In rainbow trout muscle, IGF-IRa expression did not re-
spond to fasting but increased in re-feeding and IGF-IRb expression per-
formed the inverse role (Montserrat et al., 2007a). Recently, Azizi et al.
(2016) have observed in gilthead sea bream that IGFs down-regulated
IGF-IRa gene expression at 6 h of treatment but the responses of IGF-
IRb were more variable as a function of the peptide and the duration
of the treatment. Overall, it seems that there is a certain level of func-
tional distribution and regulation between both IGF-I receptor isoforms
that deserves further investigation.
MRFs play a very significant role in the regulation of muscle cells de-
velopment, and it is necessary to know their relationship with IGFs
(Fig. 5). IGF-I and IGF-II in combination with amino acids stimulated
the gene expression of MyoD1c in Atlantic salmon myocytes (Bower
and Johnston, 2010b). MyoD2 and Myf5 increased their expression
after GH and IGF-II treatment, while myogenin and MRF4 increased
in response to IGF-I stimulation in gilthead sea bream myocytes
(Jiménez-Amilburu et al., 2013). Vélez et al. (2014) also observed in
gilthead sea bream myocytes that IGF-I was able to increase the pres-
ence of myogenin positive cells but not MyoD. Recently, de Mello et al.
(2015) also found in satellite cells of rainbow trout that IGF-I up-
regulated myogenin expression. Azizi et al. (2016) observed that IGF-I
decreased MyoD gene expression in myocytes of this species but IGF-II
did not. All these results suggest that at least in some fish species,
both IGFs regulate myogenesis, but while IGF-II exerts its influence on
the early stages, IGF-I seems to participate more in differentiation.
In this sense, Azizi et al. (2016) observed that IGF-I stimulated MHC
expression but not IGF-II.
Myostatin is a negative regulator of myogenesis and Seiliez et al.
(2012b) and Gabillard et al. (2013) found that it was able to decrease
the proliferative effect of IGF-I in rainbow trout cultured satellite cells.
Garikipati and Rodgers (2012) also observed in rainbow trout myocytes
34 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
Fig. 5. Schematic model of fish myogenesis. The proliferation phase of myoblasts is identified by elevated gene expression of proliferating cell nuclear antigen (PCNA) accompanied by
several transcription factors (Sox8, Myf5, MyoD2 and Pax7) that control myocytes activation and development. Then, cells initiate the differentiation and fusion process to become
myofibers and mature myotubes showing a rise on Myogenin and MRF4 gene expression, and corroborated by an increase in myosin heavy chain (MHC) mRNA levels. Adapted from
Vélez et al. (2015).
that myostatin appeared to partly mediate IGF-I stimulated myogenic
precursor cells differentiation. However, the effects of IGF-I on
myostatin expression are not clear and could be opposite according to
the isoform considered (MST1a or MST1b) or the experimental design.
Fuentes et al. (2013) pointed out the importance of standardizing cell
culture conditions to compare the respective findings and demonstrate
the specific regulatory role of this and other molecules.
Follistatin is a protein that is expressed almost by all tissues and it
has been demonstrated as a potent antagonist of members of the
transforming growth factor (TGF)-family, including myostatin. The
study of Medeiros et al. (2009) on follistatin transgenic rainbow trout
demonstrated that myostatin inhibition and follistatin overexpression
caused a greater and visible muscle development, opening a new and
promising line of research for transfer to aquaculture. Snijders et al.
(2015) reviewed the role of the different IGF-I splice variants on
human myogenesis regulation. Thus IGF-IEc or mechano growth factor
(MGF) stimulates proliferation and Myf5 expression, while IGF-IEa ex-
pression strongly correlates with MRF4 expression, known for its role
in satellite cell differentiation. The MGF has not been described in fishes
yet, but this kind of temporal association of IGF system regulating differ-
entially myogenesis remembers the distribution of functions among
both IGFs observed in gilthead sea bream.
Summarizing, it is clear that IGFs have a significant role on the regu-
lation of myogenesis although there are differences between the species
as well as it also appears to be very important the developmental stage
or the physiological conditions chosen to perform the study. Although
more research in fish is needed, the IGF system is fundamental to under-
standing the regulation of muscle growth as indicated previously
(Mingarro et al., 2002; Beckman, 2011), and can provide valuable
markers for the improvement of fish growth culture conditions.
4. New markers of muscle growth and quality
Early studies on IGFs and insulin effects demonstrated their stimula-
tory actions on MAPK and AKT phosphorylation in rainbow trout and
gilthead sea bream myocytes (Castillo et al., 2004; Montserrat et al.,
2007b; Codina et al., 2008). Fuentes et al. (2011) showed how circulat-
ing levels of IGF-I and nutritional status regulated both pathways in fine
flounder muscle. However, the response of both pathways to the in vitro
stimulation was different and MAPK phosphorylation was more respon-
sive to treatment in myoblast than in myotubes. On the other hand,
AKT response to stimulation was more stable or even increased in dif-
ferentiated myocytes and myotubes (Montserrat et al., 2007b). These
findings encouraged to continue the research for fish muscle growth
markers and indicators of differentiation and proliferation.
The TOR in mammals is well known as a key point to coordinate the
IGF-I signal with nutrient inputs (mainly amino acids) to regulate pro-
tein synthesis. However, in fish little information was available until
Seiliez et al. (2008) published the first study in rainbow trout, showing
an increase of TOR phosphorylation at 2.5 h of postprandial stage. In the
same study it was demonstrated that the phosphorylation of down-
stream molecules of TOR (4EBP1 and 70S6K) was also increased when
in vitro myotubes were treated with amino acids or insulin. In gilthead
sea bream myocytes, amino acids treatment but not IGF-I, was able to
stimulate TOR phosphorylation and gene expression and also that of
its downstream molecules (Vélez et al., 2014). Later Azizi et al. (2016)
verified that IGF-I does not provoke an increase on TOR gene expression
but IGF-II was able to stimulate it, which fits well with its relevant role
during proliferation when protein synthesis is more abundant.
Seiliez et al. (2013) demonstrated that cultured rainbow trout
myotubes treated with human myostatin prevents the TORC1 signaling
pathway by decreasing the phosphorylation of 4EBP1 and resulting in a
reduction of cell diameter. Recently, Fuentes et al. (2015) found in fine
flounder that the blockage of TORC1 decreased muscle and body
growth, muscle cellularity, IGF-I, IGF-II, IGFBP-4 and IGFBP-5 expres-
sion, while the expression of muscle growth inhibitors (e.g., atrogin-1,
murf-1, IGFBP-2, IGFBP-3) was up-regulated. These authors claimed
for the critical role of TORC1 in growth from the cellular to organism
level. de la García et al. (2015) found that in GH transgenic coho salmon
muscle, TORC1 downstream molecules (eif4e1 and eif4e2) were also
up-regulated. There are other interesting molecules to consider in this
review, like PAX7 and SOX8, however the information in fish is limited.
Bower and Johnston (2010a) showed PAX7 expression in Atlantic salm-
on muscle cells development, suggesting its role in proliferation and dif-
ferentiation. Froehlich et al. (2013a, 2013b) pointed to PAX7 and PAX3
as markers of myogenesis in zebrafish myocytes, since in association
with some transcription factors such as MyoD1, Myf5 and myogenin,
these molecules can play a key role in regulating the determined and
the indeterminate growth patterns of both zebrafish and giant danio
species, respectively. These authors suggest that PAX3 sustained ex-
pression, paired with the reduced Myf5 showed in giant danio, can ex-
plain the hyperplasia capacity in adults of indeterminate growth
species. de la García et al. (2014) described its profile in sea bream
myoctes development, placing PAX7 expression peak in the first part
of the myogenic process close to Myf5 and MyoD.
Muscle growth is a balance between proteogenic and proteolytic
systems and this is specially observed in fish which naturally suffer
 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
fasting periods. In this sense, the main proteolytic pathways in skeletal
muscle include the ubiquitin-proteasome system (UPS), the autopha-
gic-lysosome system (ALS) containing cathepsins, the calpain pathway,
and the caspase pathway.
In mammals UPS is the main responsible pathway for degradation
of short-lived, damaged, and misfolded proteins by two steps:
ubiquitylation and proteasomal degradation. Muscle-specific RING-fin-
ger 1 (MURF1) and atrogin 1 (also known as MAFBX) are two muscle-
specific ubiquitin ligases, enzymes participating in the last step of the
ubiquitylation, and are markedly induced in almost all types of muscle
atrophy (Cohen et al., 2015). MURF1 and atrogin 1 are frequently used
as genetic markers of muscle proteolysis in fish (Cleveland and Weber,
2010; Seiliez et al., 2010; Bower et al., 2010; Salmerón et al., 2015).
ALS participates in the degradation of long-lived proteins and for the
elimination of redundant or damaged cellular structures. During fasting,
the PI3K-AKT-TOR signaling decreases leading to activation of FOXO-
mediated expression of atrogenes which trigger muscle proteolysis via
the UPS and autophagic-lysosome system (Cohen et al., 2015). Some ev-
idences that these systems are regulated by IGF-I in fish exist. Bower et
al. (2010) found that IGF-I down-regulated atrogin1 in muscle cells of
Atlantic salmon; similarly, IGF-I decreased the expression of atrogin1
and MURF1 in rainbow trout myocytes (Cleveland and Weber, 2010).
IGF-I stimulation in rainbow trout myotubes, inhibited FoxO1 activity
(Seiliez et al., 2010, 2011). The same author (Seiliez et al., 2012a) re-
ported in rainbow trout myocytes that amino acid deprivation is follow-
ed by a rapid increase of autophagosome formation and a slower
induction of the expression of several autophagy-related genes (LC3B,
gabarapl1, atg4b) via both TOR-dependent and -independent ways. To
investigate the relative contribution of ALS and UPS towards protein
degradation of myotubes in rainbow trout, Seiliez et al. (2014) found
that IGF-I prevented the up-regulation of both systems under serum
deprivation. Interestingly, ALS contribution to total protein degradation
was much higher than UPS. However, combination of both systems only
represents 55% of total protein degradation, suggesting the action of
other proteolytic systems. Two potential candidates of these other pro-
teolytic systems are the calpains, proteases related with disruption of
myofibrillar proteins into smaller fragments and accessible for the
UPS, and the caspases, endopeptidases that regulate cell death and in-
flammation; however, they are not very well known in fish muscle
(Macqueen et al., 2010; Preziosa et al., 2013; Salmerón et al., 2013,
2015).
Recently we have characterized in gilthead sea bream several mem-
bers of the calpains (Capn), lysosomal cathepsins (CTS) and UPS fami-
lies, and shown that fasting induces an increase in capns1b, MURF,
atrogin 1 and several CTSs expression but re-feeding reduced CTSs,
capn1, capn2, capns1a, capns1b and members of the UPS expression
(Salmerón et al., 2013, 2015). Dietary positive effect on texture was
significantly correlated with decreased capn1 and capns1a expression
(Fig. 6). Overall, deeper knowledge of the role that the different
proteogenic and proteolytic systems play in fish will help to improve
Fig. 6. Correlation between (A) calpain 1 (Capn1) or (B) calpain small subunit 1a (Capns1a) relative gene expression with muscle texture (maximal strength, N) of gilthead sea bream.
(A) r = −0.409, P = 0.043, (B) r = −0.449, P = 0.028. Adapted from Salmerón et al. (2013).
35
the growth and flesh quality in aquaculture, and its investigation will
provide useful applied information.
5. Exercise and muscle growth in fish
There is growing evidence that moderate and sustained exercise can
improve fish growth and feed conversion in salmonids (Davison, 1997;
Davison and Herbert, 2013) and in non-salmonid species (Palstra et al.,
2010, 2014, 2015; Ibarz et al., 2011; Martín-Pérez et al., 2012; Felip et al.,
2013). Most of these studies were performed in adult fish, but also in ju-
venile Atlantic salmon (McDonald et al., 1998), in fingerlings of yellow-
tail (Seriola quinqueradiata) (Yogata and Oku, 2000) or in fingerlings of
gilthead sea bream (Blasco et al., 2015; Vélez et al., 2016). In these last
studies our group has demonstrated an increase in body weight after
5 weeks of swimming.
In order to increase growth rate by means of moderate exercise, the
swimming speed of choice has been suggested to be the optimal (i.e.
lowest energy cost swimming, Uopt) (Davison and Herbert, 2013).
However, under these conditions, while salmonids increase their food
intake to offset costs (Davison, 1997; Felip et al., 2012) other species
as yellowtail (Seriola lalandy) (Palstra et al., 2015) or gilthead sea
bream enhance utilization of nutrients without food intake increase
(Ibarz et al., 2011; Felip et al., 2013). Thus, fish seem to adapt their me-
tabolism to nutritional regime and environmental conditions and de-
pend on their ability to match fuel supply to energy use to grow
(Magnoni et al., 2013). Sustained swimming can enhance the utilization
of dietary carbohydrates on a low-protein, high-carbohydrate regime,
and spare the use of dietary proteins for muscle growth in both rainbow
trout and gilthead sea bream (Martín-Pérez et al., 2012; Felip et al.,
2012, 2013). This sparing effect, in the case of juvenile sea bream, is
probably due to a lower entry of amino acids into the tricarboxylic
acid cycle as a reduction in citrate synthase (CS) activity, and an increase
of cytochrome-c-oxidase (COX) activity (Martín-Pérez et al., 2012).
However, in fingerlings of this species fed a high-protein diet under
sustained swimming, amino acid oxidation increased, as reflected by
the high mitochondrial CS activity at the same time of the decrease in
COX activity. All of this demonstrates the great metabolic plasticity of
fish to adjust tissue-specific demands in response to exercise and ener-
gy availability. Interestingly, regardless of oxidative nutrient strategy,
the response of fish to chronic submaximal swimming is, in general,
qualitatively similar to mammalian aerobic training, and the trend is to-
wards a more aerobic phenotype accelerating muscle growth (Johnston
and Moon, 1980; McClelland et al., 2006; LeMoine et al., 2010). This
growth can occur by hypertrophy, hyperplasia or a combination of
both depending on the species. We observed increases of capillarization
and fiber size of white muscle in gilthead sea bream under moderate
swimming indicating vascularization and hypertrophy of this muscle
(Ibarz et al., 2011) as reported previously in other species (Davison,
1997; Johnston, 1999; Johnston and Moon, 1980). Moreover, Palstra et
al. (2010, 2014) have also described that exercise effects in zebrafish
36 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
are directed by hypertrophy and angiogenesis. However, as Mommsen
(2001) suggested in an interesting review, zebrafish is not a good
model of indeterminate growth; and as other authors have demonstrat-
ed later (Biga and Goetz, 2006), zebrafish have a little capacity of post-
larval mosaic hyperplasia, being this one of the most important reason
to postulate zebrafish as a species with a determinate growth pattern.
Despite this data, it is not yet well established if sustained exercise en-
hances also hyperplasia processes, but we have recently observed an in-
crease of small fibers number of white muscle in fingerlings of gilthead
sea bream under moderate activity (Moya et al., unpublished data), and
it could be that the hyperplasia would be more suitable in species with
indeterminate growth. Global underlying changes in protein expression
of white and red muscle of sea bream demonstrated that exercise affect-
ed many aspects of muscle physiology (Martín-Pérez et al., 2012). An-
other interesting observation was the reduction of mesenteric fat
found in gilthead sea bream fingerlings after 5 weeks of moderate and
sustained exercise (Blasco et al., 2015).
The GH/IGF axis is tightly coupled to the energy status of the fish and
linked to feed intake via the expression of GH, IGF-I and their receptors
(Raven et al., 2008). However, the literature about the axis response to
moderate exercise in fish is scarce or partial. Although a close associa-
tion between exercise induced growth and circulating levels of GH
and IGF-I was not found for masu salmon (Oncorhynchus masou
masou) (Azuma et al., 2002), increased plasma GH for coho salmon,
and for rainbow trout under sustained swimming was observed
(Barrett and McKeown, 1988a, 1988b). We have found a significant in-
crease of circulating IGF-I concentration in gilthead sea bream juveniles
under moderate swimming (Sánchez-Gurmaches et al., 2013). In fin-
gerlings of this species, we have also observed an increased IGF-I/GH
ratio as a consequence of decreased GH and increased IGF-I plasma
concentration (Blasco et al., 2015). Furthermore, in the same study,
moderate swimming determined an increase of hepatic IGF-I gene
expression (mainly the IGF-Ia splice variant) in parallel to the
enhanced IGF-I levels in plasma. In muscle of gilthead sea bream
Fig. 7. Proposed model for muscle growth regulation in fish. The GH/IGFs system regulates
the muscle expression of IGFs and IGFBPs, which can act in both autocrine and paracrine
manner to control IGFs actions. In collaboration with myogenic regulatory factors
(MRFs), cell proliferation and differentiation will be controlled. IGFs modulate cell
metabolism (e.g. nutrients uptake) and the TOR signaling pathway, which controls
protein turnover with also the involvement of proteolytic systems. All these regulatory
factors will participate in the control of hyperplasic and hypertrophic growth of muscle.
fingerlings, the isoform increased was IGF-Ic together with the elevated
expression of the anabolic GH receptor, GHR-I (Vélez et al., 2016). More-
over, swimming stimulated the expression of IGFBP-5 in the anterior re-
gion of the muscle, whereas in caudal muscle enhanced IGF-II mRNA
levels, suggesting different response to exercise throughout the muscle.
Additionally, TOR activation and gene expression augmented in
the whole muscle mass, suggesting an increase in protein synthesis
that would sustain the higher growth rate in fish under moderate
swimming.
All these results confirm the positive growth effects of moderate and
sustained exercise and support the use of certain molecular markers
(such as GH/IGFs, IGFBP-5 or TOR) for monitoring the response of the
fish to growth trials.
6. Perspectives on fish growth and aquaculture
The future of aquaculture requires an improvement of the growth
and quality of the product combined with the sustainability of the activ-
ity. The endocrine control of growth includes several molecules and
pathways that we have summarized in Fig. 7. The GH/IGFs axis is the
predominant system and a very valuable tool to monitor the perfor-
mance of a determined cultured fish. IGF-I levels appear to be good
markers of adequate growth. However, very little information is avail-
able on IGF-II plasma levels and their function. Also better knowledge
on circulating IGFBPs is necessary in many fish species to be utilized as
growth markers.
Regulation of fish myogenesis is still poorly understood and more
studies on the control of MRFs during muscle development and growth
are required. Myostatin studies in fish are scarce but this could be a very
useful molecule for muscle growth improvement. The action of IGFs on
muscle cells and the role of local IGFBPs and IGF-I receptors can provide
suitable information to be applied in fish culture. All these molecules ac-
quire special relevance in the continuous growth exhibited by aquacul-
ture fish species, and gives additional interest to try to explain this
characteristic of fish muscle development, not only for aquaculture
but also for vertebrate muscle regeneration.
The search for quality muscle markers is another important area that
seems to be still poorly developed in aquaculture in comparison with
farm animals. TOR complex has become an essential regulator of muscle
growth and needs more investigation in fish to serve as an indicator of
the good balance between nutrition and growth during fish culture.
The data on proteolytic systems has lately increased but more studies
on their role in vivo in fish growth are required. Some of these mole-
cules can be chosen for genetic investigation to be utilized in the pro-
grams of strain and broodstock selection.
In recent years, the discovery of noncoding RNAs, such as microRNAs
(miRNAs), has revealed a new perspective on the regulation of gene ex-
pression. miRNAs are small RNA molecules (19–22 nt) that regulate
gene expression by inhibiting protein translation and inducing mRNA
degradation (Silahtaroglu and Stenvang, 2010). Indeed, it has been ex-
tensively demonstrated that miRNAs are critically relevant in situations
that require adaptation to new conditions and processes that involve a
high degree of adaptability such as, the regulation of metabolism during
exercise (Párrizas et al., 2015). A set of miRNA, called myomiRs, have re-
cently been implicated in muscle growth and development (Güller and
Russell, 2010), indicating that it may be possible to use miRNAs as a new
and valuable tool for understanding such processes. Although this field
is extensively studied in mammals, there is still little information avail-
able about the role of muscle-related miRNAs in fish and their implica-
tion on muscle growth.
Finally, exercise is indicating a very useful way to increase fish
growth and flesh quality that with an adequate diet can provide a sus-
tainable strategy to improve aquaculture that deserves further research.
In this review fundamental parts of skeletal growth regulation have
not been covered, but it is also necessary to acquire a better knowledge
 E.J. Vélez et al. / Aquaculture 467 (2017) 28–40
on the regulation of bone and cartilage development to be able to im-
prove final body weight, size and flesh quality.
In conclusion, in the last years there have been very significant ad-
vances in the basic knowledge of the GH/IGF system and its role in the
regulation of metabolism, myogenesis and growth in fish. However, it
remains still necessary to complete some of the aspects highlighted in
this review, and to transfer this information to fish culture, thus bring-
ing aquaculture to the level of quality and sustainability that the society
needs.
Acknowledgements
E.J.V. and E.L. are supported by a predoctoral fellowship from the
“Ministerio de Ciencia e Innovación” (MICINN). This study was support-
ed by the projects from the MICINN AGL2012-39768 and AGL2015-
70679-R to J.G., AGL2014-57974-R to I.N., the SGR2009-00402 and
2014SGR-01371 from the “Generalitat de Catalunya” and the “Xarxa
de Refèrencia d'R + D + I en Aqüicultura”. The authors would like to
thank Carlos Mazorra from Tinamenor S.L., Ramon Fontanillas from
Skretting and the personnel from the facilities at the School of Biology
for the maintenance of the fish.
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