Aquaculture Nutrition

2014 20; 595–608

..........................................................................................
doi: 10.1111/anu.12113

1,2 1 1 3 1 4

5
1
Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran; 2 Department of Fisheries,
Faculty of Natural Resources, Urmia University, Iran; 3 Department of Comparative Histology & Embryology, Faculty of
Veterinary Medicine, Urmia University, Urmia, Iran; 4 Artemia & Aquatic Animals Research Institute, Urmia University,
Urmia, Iran; 5 Institut de Recerca i Tecnologia Agroalimentaries (IRTA),Centre de Sant Carles de la R
apita, Sant
Carles de la R

apita, Spain

Sturgeons are very important commercial fish because of

The study on histological characteristics of the digestive sys-
their roe, which is made into caviar. The Caspian Sea con-
tem of Beluga (Huso huso) was conducted from hatching until
tains large stocks of these species, but some of them are
50 days posthatching at 16.5 °C. Development of the diges-
becoming extinct, resulting in the development of the inten-
tive system in this species followed the general pattern
sive culture of different species of sturgeons in countries
described for other Acipenserids, although there were differ-
neighbouring the Caspian Sea basin. Sturgeons are a group
ences in the timing of organ development among species. At
of slow-growing fishes that mature very late in life, and
hatching, the mouth was opened and digestive system was
consequently, they are particularly vulnerable to overfishing
represented by a gastric cavity filled with yolk and lined by
and to other threats, like the loss of their natural habitat,
endodermal cells, and a partially differentiated hindgut. Gas-
river damming and deterioration of water quality (Kynard
tric glands started to differentiate at 46.5 degree- days post-
1997; May et al. 1997). As a consequence, wild stocks of
hatching (ddph), the earliest appearance time among sturgeon
sturgeons are decreasing dramatically during these last dec-
fishes studied to date. At the onset of exogenous feeding
ades (Billard & Lecointre 2001), and sturgeons are consid-
(144.9 ddph), yolk sac reserves were not completely depleted
ered as one of the most threatened group of animals on the
in the stomach, suggesting a period of mixed nutrition. The
IUCN Red List of Threatened SpeciesTM (IUCN 2013). The
complete development of the digestive system was not accom-
great sturgeon or Beluga, Huso huso Linnaeus 1758, is cate-
plished until 235.2 ddph when it showed all histomorpho-
gorized as critically endangered fish species according to
logical features typical of juvenile specimens. According to
the IUCN. The Beluga, H. huso, is an important commer-
histological results, it seems advisable to start co-feeding
cial species in the Caspian Sea and a good candidate for
H.huso larvae with inert diets at the onset of exogenous feed-
aquaculture because of their good growth performance,
ing, because exocrine pancreas and glandular stomach are
market price, propagation in captivity and high marketable
fully differentiated, although the complete substitution of live
value of their caviar, so it has been cultured for aquacul-
prey by inert feed is not recommended until 235 ddph.
ture and restocking purposes in Iran since 1991 (IFO
2002). The species was historically known from the Cas-
KEY WORDS: Beluga, co-feeding, digestive system, histology,
pian, Black, Azov and Adriatic Sea basins. Based on fish-
Huso huso, ontogeny
ery data and number of recorded spawning individuals, it
has been estimated that the wild native populations of this
Received 28 March 2013; accepted 19 July 2013
species have declined over 90% in the past three genera-
Correspondence: R. Asgari, Department of Fisheries, Faculty of Natural
Resources, University of Tehran, 31585-4314 Karaj, Iran. E-mail:
tions (ca. 60 years), and overfishing for meat and caviar
pikeperch@yahoo.com will soon cause global extinction of the remaining natural

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ª 2014 John Wiley & Sons Ltd

wild populations if conservation measures are not taken
(Gessner et al. 2010). Independently of the final purpose of
this activity (conservation and restocking programmes or
commercial purposes), a reliable larval rearing technique
must be developed to ensure consistent production of good
quality fry. However, the high mortality rate during
H. huso larval rearing, up to 80% in some cases, has been
identified as one of the most serious bottlenecks at present
for the culture of this species. Therefore, the success and
development of aquaculture activity devoted to H. huso
culture still demand improvements in rearing techniques,
especially those affecting larval rearing practices such as
the partial or complete replacement of live prey with a
compound microdiet. Thus, to enhance the success of lar-
val rearing of this sturgeon species and to facilitate over-
coming one of the major bottlenecks of fish hatcheries, the
description of the ontogeny of the larval digestive enzymes
is a necessary tool. Artificial diets for larvae have been used
for intensive commercial culture of several Acipenserid spe-
cies from the onset of exogenous feeding onwards (Charlon
& Bergot 1991; Giovannini et al. 1991; Hung 1991; Gisbert
& Williot 1997).
Sturgeon embryos show intraembryonic yolk, and their
alimentary tract develops from the yolk sac endoderm
(Dettlaff et al. 1993). This is a result of holoblastic cleav-
age in sturgeon, contrary to the teleost fish in which the
egg is subjected to meroblastic cleavage, and in which the
alimentary tract develops independently from the extraem-
bryonic yolk sac. The postembryonic development of the
alimentary tract of the sturgeon takes place at the time of
endogenous feeding. Differentiation proceeds from the pos-
terior towards the anterior part. The spiral intestine is the
first to develop and the stomach (gastric stomach) the last
(Buddington 1985). From a nutritional point of view, post-
hatch developing larvae remain absolutely dependent on
yolk sac reserves. As yolk is depleted, larval sturgeon, as
other fish species, must begin to ingest external food to sat-
isfy increasing energy demands for growth. When sturgeon
larvae begin to feed they possess an anatomically complete
and functional digestive system with a high degree of mor-
phological organization (Buddington & Doroshov 1986;
Dettlaff et al. 1993; Gawlicka et al. 1995). Development of
the alimentary tract is similar among sturgeons (see reviews
in Buddington 1991; Dettlaff et al. 1993). However, there
are some differences concerning the time of differentiation
of various structures, and thus also the moment of func-
tional ability of food utilization by the digestive system.
Although the basic mechanisms of organ and system devel-
opment are similar among Acipenserid species, there are
considerable interspecific differences regarding the relative
timing of differentiation, development and functionality
during early ontogeny. Hence, there is a need to conduct
specific studies on the ontogenesis of digestive system of
fish for each species to better understand their nutritional
physiology. Among the 26 species belonging to the family
Acipenseridae, the development of the digestive system has
been mainly studied in the representatives of the genus Aci-
penser, such as Russian sturgeon A. gueldenstaedti (Dettlaff
et al. 1993), white sturgeon A. transmontanus (Gawlicka
et al. 1995), Siberian sturgeon A. baerii (Gisbert et al.
1998, 1999), green sturgeon A. medirostris (Deng et al.
2002; Gisbert & Doroshov 2003), Adriatic sturgeon A. na-
carii (Boglione et al. 1999; Camacho et al. 2011; Sanz et al.
2011) and Atlantic sturgeon A. oxyrinchus (Ostaszewska
et al. 2011). Although the knowledge of the digestive sys-
tem developmental changes associated with food assimila-
tion is essential for understanding the nutritional
physiology of larval fish (Segner et al. 1993) and there exist
several general descriptions of the development of the
digestive system of some several species of the genus Aci-
penser (see review in Buddington 1985; Dettlaff et al.
1993), there has been no comprehensive study on the diges-
tive tract and accessory digestive organs in the larval stages
of the genus Huso and in particular on the H. huso. Thus,
the aim of the present study was to describe in detail the
early organization, differentiation and development of the
different regions of the digestive system of H. huso to com-
pare it with other sturgeon species, achieve a better under-
standing and knowledge of its organization and
functionality and consequently, improve H. huso larval
rearing procedures and efficiency.
Beluga, H. huso, specimens used in the present study were
obtained by induced spawning of a pool of two females
and three males with LHRHa2 hormone. Fish were reared
in the Shahid Marjani Sturgeon Center (Gorgan), Iran
(37°, 4′, 30.62″ N: 54°, 40′, 10.45″ E). Eggs were incubated
at a temperature ranging from 11 to 12 °C in a closed
freshwater recirculation system. The fertilized eggs were
incubated in Yushchenko incubators for hatching and after
8 days of incubation at 12 °C, 68% of larvae hatched, and
1800 individuals [average body weight (BW): 15.6 
0.7 mg; average total length, TL: 11.2  0.4 mm] were
placed in per 500 L circular fibreglass tank connected to a
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Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
 flow-through freshwater system. During the rearing period,
the average values for water temperature, dissolved oxygen,
pH and flow rate were 16.5  0.2 °C, 10.7  0.3 mg L 1,
7.8  0.1 and 5.7  0.4 L min 1, respectively.
The feeding protocol used for H. huso larval rearing
from hatching to the juvenile stage is summarized in Fig. 1.
In brief, larvae were fed by mixed of Artemia nauplii (EG,
INVE, Dendermonde, Belgium) and Daphnia sp. from 12
to 25 dph (199.2–432.5 degree days posthatch, ddph) (500–
800 nauplii/larvae/day). A short co-feeding phase based
on Cladocerans and a compound diet (D1- particle
size = 0.5 mm; Biomar, Brande, Denmark) was conducted
from 25 to 30 dph (432.5–534 ddph), whereas from this
age onward, fish were fed only with the inert feed (D2- par-
ticle size = 0.8 mm; Biomar) at a feeding rate of 30% and
20% of stocked fish biomass, respectively.
Proximate composition of live prey (Artemia nauplii and
Daphnia sp.) was conducted in three samples per type of
live prey. Crude protein was determined by the Kjeldahl
method (N 9 6.25) using an automatic Kjeldahl system
(Behrotest WD 40, D€ usseldorf, Germany), and crude fat
content was determined by Soxhlet method (Folch et al.
1957). Total protein and lipid content in Artemia were
656  14 g kg 1 and 151  2 g kg 1, respectively, whereas
Daphnia sp. contained 526  19 g kg 1 of proteins and
111  1 g kg 1 of lipids. The proximate composition of
compound diets (D1 and D2) was 630 g kg 1 and
580 g kg 1 of total protein, 110 g kg 1 and 150 g kg 1 of
total lipids, and 116 g kg 1 and 115 g kg 1 total carbohy-
drate, respectively (data provided by feed manufacturer).
The compound diet was given to larvae between 4 and 6
times per day, and feed ration and particle size were pro-
gressively adjusted for fish size from 25 to 50 dph (432.5–
906.5 ddph). Since the mixed feeding phase, the inner
Figure 1 Feeding protocol and larval growth in total length and
wet weight in Beluga (Huso huso) from hatching until 906.5 ddph
(50 dph). ○: Weight- Δ: Length.
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Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
surfaces of the tanks were scrubbed and simultaneously
siphoned out to remove uneaten feed and faeces three times
a day.
Larvae were daily sampled (n = 40) from hatching to
306 ddph (18 dph) and then with 2-d intervals to
906.5 ddph (50 dph). Larvae were anesthetized with an
overdose of tricaine methanosulphonate (MS-222; Sigma-
Aldrich, Munich, Germany), rinsed in distilled water,
measured in length and weight and fixed in 10% phos-
phate-buffered formalin. Larval TL and BW were mea-
sured to the nearest 0.01 mm and 0.1 g by means of a
stereomicroscope equipped with a drawing tube and micro-
metre (Zeizz, Oberkochen, Germany) and analytical micro-
balance (Mettler toledo, XS 205; Zurikh, Switzerland),
respectively. For histological procedures, fixed samples
were dehydrated in graded series of ethanol and embedded
in paraffin. Then, a total of 20 serial sagittal sections of
whole larvae were cut at 7 lm thick using microtome (Le-
ica RM 2265; Leica Microsystems, Nussloch, Germany),
mounted on glass slides (6–9 serial per slide), air-dried and
stained with hematoxilin–eosin (H&E) for general histo-
morphological observations (Drury & Wallington 1980),
while periodic acid-Schiff (PAS) was used to detect neutral
and acid mucosubstances in mucous cells and glycogen
deposits in the liver (Pearse 1985). Microscopic analyses
were performed with a Nikon Eclipse 90i microscope con-
nected to a Nikon Digital Sight DS-U1 camera (Nikon
Corporation, Tokyo, Japan).
Total length and wet weight of Beluga larvae averaged
12.1  1.01 mm and 11.02  0.47 mg, respectively, at
hatching, and increased to 47.5  6.5 mm and 2.5  0.8 g
at 906.5 ddph (50 dph) (Fig. 1).
Buccopharynx At hatching time, the buccopharynx was
opened, and posterior section of its lumen was filled with
small yolk platelets (Fig. 2a). First goblet cells appeared
at 94.2 ddph (6 dph; 16.14 mm TL). The first signs of
differentiation of taste buds in the oral cavity at the level
(a)
(b) (c)
(d) (e)
of the entrance of the gills were observed at 94.2 ddph
(6 dph). Taste buds were also observed at the beginning of
the oral cavity (Fig. 2b). At 111.3 ddph (7 dph; 19.01 mm
TL), first PAS-positive goblet cells were detected in the
buccopharynx, which increase in number and reactivity as
fish grew, and at the onset of exogenous feeding were pre-
sented through the epithelium of the buccopharynx
(Fig. 2c). At the end of study, taste buds lining through
the epithelium of the buccopharynx close to the opercular
cavity (Fig. 2d). Furthermore, differentiation of canine-like
teeth proceeded from the base of the buccopharyngeal epi-
thelium at 46.5 ddph (3 dph; 14.75 mm TL) and eosin-
positive reaction was clearly observed at 490 ddph (28 dph)
(Fig. 2e).
Oesophagus One-day-old larvae (12.07 mm TL) did not
present a differentiated oesophagus. At 46.5 ddph (3 dph),
the oesophagus appeared as a simple columnar epithelium
Figure 2 Longitudinal histological sec-
tions of the buccopharynx of Beluga
(Huso huso) at different stages of devel-
opment. (a) General view of a larvae at
hatching showing the buccopharynx.
Note the opening of mouth (arrow)
and differentiation of gill arches (BG)
in the posteroventral region of the buc-
copharyngeal mucosa. (b) Taste buds
(arrow heads) at the beginning of the
oral cavity. Asterisk indicate oral cav-
ity. (c) Detail of buccopharynx mucous
cells containing neutral mucosubstances
(arrow heads) positively stained with
periodic acid-Schiff (PAS) and taste
buds (arrows) in a 14-dph larva. Aster-
isks indicate opercular cavity. (d) Taste
buds (arrows) lining through the epi-
thelium of the buccopharynx close to
the opercular cavity in 42-dph larvae.
(e) Teeth at 490 ddph (28 dph) with
eosin-positive reaction. Abbreviations:
B, buccopharynx cavity; BG, branchial
grooves; BL, buccopharyngeal lumen;
BP, buccal papillae; C, cartilage; E,
eye; FB, fore brain; HB, hind brain;
M, muscular layer; OV, otic vesicle;
PN, pronephros; T, tooth; YS, yolk
sac. Staining: all slides were stained
with haematoxylin–eosin with the
exception of 2c that was stained with
PAS.
with a narrow and short lumen. A large number of oesoph-
ageal cells contained supranuclear vacuoles with acidophilic
yolk inclusions and pigment granules. At this age, serial
histological sections revealed that the oesophagus was in
differentiation, and its connection with future stomach was
not yet established (Fig. 3a). At 111.3 ddph (7 dph), the
connection between the oesophagus and the cardiac stom-
ach took place due to the resorption of a yolk mass that
separated both regions of the digestive tract (Fig. 3c). At
this moment, the oesophagus had elongated, and two dif-
ferentiated regions associated with secretion (anteriorly)
and food transport functions (posteriorly) could be distin-
guished with abundant goblet and ciliated cells, respectively
(Fig. 3b). Goblet cells and the brush border of oesophagus
were strongly stained with PAS staining, showing that muc-
ins secreted by oesophageal goblet cells were rich in neu-
tral mucopolysaccharides (PAS-positive). The anterior
oesophageal region at 28 dph larvae was lined by a pseud-
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Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
 (a) (b)

(c) (d)

Figure 3 Longitudinal histological sections of the oesophagus of Beluga (Huso huso) at different stages of development. (a) Detail of the
developing oesophagus in 3-dph larvae. (b) Detail of the pseudostratified oesophageal epithelium containing neutral mucosubstances posi-
tively stained with periodic acid-Schiff (PAS) in 7-dph larvae showing two differentiated zones, a secretory anterior region with goblet cells
(AE) and a posterior segment with large microvilli specialized in food transport (PE). (c) Connection of the buccopharynx with developing
stomach in 7-dph larvae. (d) Detail of the oesophageal epithelium completely lined by goblet cells (arrowheads) in 28 dph larvae. Asterisk
indicates oesophageal lumen. Abbreviations: A, atrium; AE, anterior oesophagus; B, barble; BC, buccopharynx cavity; BG, branchial
grooves; E, oesophagus; L, liver; NS, non-glandular stomach; PE, posterior oesophagus; SV, sinus venosus; V, ventricle; YL, yolk. Staining:
all slides were stained with haematoxylin–eosin with the exception of 4b and d that were stained with PAS.

ostratified cylindrical epithelium, while the posterior region,
which connected the oesophagus with the cardiac stomach,
consisted of a simple stratified epithelium (Fig. 3d).
Stomach and pyloric caeca At hatching, the large endoder-
mal yolk sac of H. huso was surrounded by a thin squa-
mous basophilic epithelium. One furrow (invaginating
epithelium) appeared at hatching in the dorsal posterior
region of the yolk sac, merging and dividing the yolk sac
into two compartments between 14.5 and 46.5 ddph
(1–3 dph) (Fig. 4a). The anterior wall of the furrow lined
with a squamous epithelium became the ventral lining of
the stomach, while its posterior wall lined with a columnar
epithelium became the dorsal lining of the intestine.
The pyloric (non-glandular) stomach started differentia-
tion at 30.4 ddph (2 dph; 14.25 mm TL) in the anterior
ventral region of the yolk sac from a fold of multilayered
squamous epithelium (Fig. 4a). This region of the stomach
was well differentiated at 199.2 ddph (12 dph; 21.66 mm
TL), with mucosal folds surrounded by a prominent tunica
muscularis (Fig. 4c). The epithelium of the non-glandular
stomach consisted of columnar cells with basal nuclei,
some apical vacuoles and PAS-positive acidophilic microv-
illi (Fig. 4f). The cardiac (glandular) stomach started to
..............................................................................................
differentiate at 46.5 ddph (3 dph) (Fig. 4b). The most rele-
vant changes in the histological organization of this part
of the stomach were the change from a squamous to a
columnar epithelium surrounding the gastric lumen filled
with yolk remnants and the appearance of the first rudi-
ments of gastric glands. Between 111.3 and 235.2 ddph (7–
14 dph; 19.01–22.99 mm TL), the glandular stomach
greatly developed occupying most part of the abdominal
cavity, and abundant gastric tubular glands were arranged
along numerous longitudinal folds (Fig. 4c). These tubular
glands were composed of a single type of secretory cells
devoid of microvilli on their apical border and lining their
base with a simple cubic epithelium. The number of gastric
glands and thickness of mucosa layers increased during lar-
val feeding phase, while their histochemical properties
remained the same. The platelet yolk was present in the
glandular stomach until 235.2 ddph (14 dph). From this
time onwards, glandular stomach with a lumenally exposed
cubical simple epithelium with basal nuclei and prominent
microvilli with PAS-positive mucosal cells was well devel-
oped, and numerous simple and tubular gastric glands
were noted (Fig. 4e). At the junction of the pyloric and
cardiac stomach, suddenly reducing muscle thickness of
non-glandular stomach and gastric glands in glandular

Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
(a) (b)
Figure 4 Longitudinal histological sec-
tions of the stomach and pyloric caeca
of Beluga (Huso huso) at different
stages of development. (a) division of
the yolk sac by a furrow separating
intestine and stomach at 2 dph. Note
the melanin granules inclusions
(arrows) in the yolk and the developing
non-glandular stomach and liver in
(d)
ventral region of the yolk sac. (b)
Developing gastric glands in yolk sac
epithelium (arrows) in 3-dph larvae. (c)
General view of the digestive system in
12-dph larvae. (d) Reduction in the
diameter of the muscle layer (double
arrows) and increment in gastric glands
(asterisks) in the transition from joint
pyloric to the glandular stomach in 14-
dph larvae. (e) Detail of glandular
stomach with mucous cells containing
neutral mucosubstances positively
stained with periodic acid-Schiff (PAS)
(arrowheads) in 18-dph larvae. (f)
Detail of non-glandular stomach with
mucous cells containing neutral muco-
(c) substances (arrowheads) positively
stained with PAS in 18-dph larvae. (g)
(e) (f)
Non-glandular stomach separated from
the anterior intestine by an epithelial
fold in 23-dph larvae. (h) Detail of
pyloric caeca with mucous cells con-
taining neutral mucosubstances posi-
tively stained with PAS (arrowheads) in
14-dph larvae. Abbreviations: AI, ante-
rior intestine; BS, bile sac; EP, body
epithelium; GS, glandular stomach; H,
(g) (h)
heart; II, Intermediate intestine; L,
liver; ML, muscular layer; NS, non-
glandular stomach; P, pancreas; PC,
pyloric caeca; PN, pronephros; S,
sphincter; SI, spiral intestine; V, vein;
YL, yolk; YS, yolk sac. Staining: all
slides were stained with haematoxylin–
eosin with the exception of 5e, f and h
that were stained with PAS.

stomach epithelium was observed (Fig. 4d). The pyloric
sphincter appeared as an epithelial fold that separated the
stomach from the anterior intestine (Fig. 4g). From
235.2 ddph (14 dph) to the end of the study, the stomach
increased in size and complexity by means of an increase
in the size and number of mucosal folds in both cardiac
and fundic regions, as well as by an increase in gastric
glands in the cardiac and the thickness of the tunica musc-
ularis in the pyloric region, but no relevant histological
modifications were observed. On 12 dph larvae, PAS-posi-
tive reaction observed in the gastric epithelial cell surface
that was not observed from 14 dph onwards.
At 77 ddph (5 dph), pyloric caeca start to form as an
epithelial ridge that separated the non-glandular stomach
from the anterior intestine. The ridge was visible as a series
of small pocket-like invaginations of the intestinal wall,
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Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
 (a) (b)

Figure 5 Longitudinal histological sec-

tions of the intestine of Beluga (Huso
huso) at different stages of develop-
ment. (a) Division of the yolk sac by a
furrow (arrow) separating intestine and (c) (d)
stomach at 3 dph. (b) Detail of ante-
rior and intermediate intestine with
lipid deposits in enterocytes in 7-dph
larvae. Note the yolk particles in non-
glandular stomach (arrow). (c, e) Detail
of anterior and intermediate intestine
in 18 and 14-dph larvae, respectively.
(d, f) Decrease in lipid deposition in
intestinal villi (asterisks) along ontog-
eny (d, 9 dph; f, 32 dph). Abbrevia-
tions: AI, anterior intestine; B, brush
border; GG, gastric glands; GS, glan- (e) (f)
dular stomach; H, heart; I, intestine; II,
intermediate intestine; L, liver; M, mus-
cular layer; MP, melanin plug; NS,
non-glandular stomach; P, pancreas;
PC, pyloric caeca; PN, pronephros; S,
developing stomach; Y, yolk; YS, yolk
sac. Staining: all slides were stained
with haematoxylin–eosin with the
exception of 7e that was stained with
periodic acid-Schiff.

which later developed into the finger-like projections typi-
cal of these intestinal appendages. The mucosa of the pylo-
ric caeca was similar from its intestinal counterpart and
was lined by a columnar epithelium covered with a dense
layer of microvilli. Over the next few days, the number of
caeca increased as fish grew, as well as the density of the
PAS-positive mucous cells in the pyloric caeca epithelium.
This resulted in a thick layer of mucus covering the colum-
nar epithelium of this digestive appendage. On the
235.2 ddph (14 dph), pyloric appendages fully extended
and were completely formed (Fig. 4h).
Anterior and intermediate intestine The differentiation of
the intestinal wall started at hatching, progressing in a pos-
teroanterior direction between 14.5 and 46.5 ddph
(1–3 dph) (Fig. 4a). However, the anterior region of the
intestine was filled with yolk and did not differentiate until
111.3 ddph (7 dph) (Fig. 5a). During yolk resorption,
supranuclear lipidic vacuoles in the cells of the intestinal
..............................................................................................
epithelium increased in size and number and were present
until the beginning of exogenous feeding at 199.2–
235.2 ddph (12–14 dph) (Fig. 5c). They were completely
absent from 14 dph onwards (Fig. 5f). The first goblet cells
(PAS-positive) in the posterior and anterior intestinal
regions appeared between 94.2 and 167 ddph (6–9 dph;
17.14–19.76 mm TL), respectively. The number of goblet
cells increased with differentiation of the mucosa, and they
were more abundant in the posterior region (Fig. 5e). At
46.5 ddph (3 dph), the middle intestine started to differenti-
ate showing numerous epithelial folds. Enterocytes of this
intestine section had basally located nuclei and supranu-
clear vacuoles filled with yolk platelets. During develop-
ment, the height of folds decreased, and yolk platelets
disappeared from the epithelial cells. The epithelium of
the middle intestine is similar to the anterior with exception
of the number and size of mucosal folds, which were
lower and less numerous (Fig. 5c,e). Also, in the anterior
intestine, enterocytes were less vacuolated. No relevant

Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
histological changes were observed after 235.2 ddph
(14 dph).
Spiral valve and rectum At hatching, the posterior intes-
tine, the so-called spiral valve in sturgeons, was primarily
differentiated (Fig. 6a). The lumen of spiral valve was lined
with a simple ciliated columnar epithelium containing
supranuclear eosinophilic yolk inclusions. At this stage,
goblet cells were already visible, and they had similar histo-
chemical properties to those in the anterior and intermedi-
ate intestine. The lumen of the spiral valve was initially
filled with yolk and melanin (Fig. 6b); but, it became
devoid of yolk as larvae developed in concomitance with a
major deposits of melanin that formed the melanin plug.
The anus opened at 235.2 ddph (14 dph) coinciding with
the complete resorption of the residual yolk in the spiral
valve and the ejection of the melanin plug. The rectum
(hind gut) was also distinguishable at this age as a short
intestinal flattened region devoid of mucosal folds (Fig. 6c).
At this level, the urinary bladder emerged outside the diges-
tive tract posterior to the anus (Fig. 6d).
Liver and pancreas Accessory digestive glands were absent
at hatching time. The liver rudiment appeared at 14.5 ddph
(1 dph) in the ventral portion of the endodermal yolk sac
(Fig. 7a). At the same time, the rudiment of the exocrine
pancreas developed dorsally to the furrow dividing the yolk
sac into the stomach and intestine. In this study, direct
connection between the coronary sinus of the heart and the
liver was clearly observed in the first few days (Fig. 7b).
During the endogenous feeding phase, hepatocytes were
(a) (b)
(c) (d)
spherical with centrally located basophilic nuclei and
slightly eosinophilic homogeneous cytoplasm, achieving a
polyhedral shape as the larvae developed (Fig. 7c). After
the onset of exogenous feeding, hepatocytes increased in
size and number, and were tightly packed between sinu-
soids, often around a central vein. The level of hepatic lipid
deposits increased from 44.2 to 400.2 ddph (9–23 dph;
19.76–23.55 mm TL), whereas after this age, the level of
fat vacuoles greatly decreased (Fig. 7d), and PAS-positive
glycogen deposits gradually increase up to end the experi-
ment (Fig. 7e). As the larvae grew, the liver increased in
size and occupied most of the anterior part of the abdomi-
nal cavity.
The first signs of pancreatic differentiation were observed
on the first day posthatching (14.5 ddph). Exocrine pancre-
atic cells were arranged in acini around the small intercellu-
lar lumina. Acinar cells had eccentric (basal) nuclei and
strongly basophilic cytoplasm. Eosinophilic and PAS-posi-
tive zymogen granules were observed in acinar cells at
77 ddph (5 dph; 16.95 mm TL). At this time, pancreatic
acinar cells were grouped in rosette patterns around central
canals that anatomized with large pancreatic ducts. The
most relevant changes in the histological organization of
the pancreas were the increase in size and number of pan-
creatic acini as larvae grew (Fig. 7f).
As there are interspecific variations in the timing of organ
formation, development and functionality, it is necessary to
conduct studies of organogenesis for individual species to
Figure 6 Longitudinal histological sec-
tions of the posterior intestine and rec-
tum of Beluga (Huso huso) at different
stages of development. (a, b) Detail of
posterior intestine (a: at hatching; b: 5-
dph). Note the accumulating melanin
granules and yolk particles. (c) Detail
of posterior intestine and rectum in 14-
dph larvae. Note the anus opening
(asterisk). (d) Detail of rectum of 32-
dph larvae. Abbreviations: AP, anal
pore; HG, hind gut; I, intestine; II,
intermediate intestine; MN, metaneph-
ros; MP, melanin plug; PU, presumable
urinary duct; SI, spiral intestine; U, uri-
nary bladder; UD, urinary duct; YS,
yolk sac. Staining: haematoxylin–eosin.
..............................................................................................
Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
 (a) (b)
Figure 7 Longitudinal histological sec-
tions of the accessory digestive organs
of Beluga (Huso huso) at different
stages of development. (a) Splitting the
liver from the yolk sac epithelium in 1-
dph larvae (the circular cross-section).
(b) Direct relation between coronary
sinus and liver in 3-dph larvae (the cir-
cular cross-section). (c) Liver in 9-dph
larvae showing large lipid deposits (c) (d)
(asterisk) within hepatocytes (unstained
deposits within hepatocytes corre-
sponded to lipids dissolved during the
embedding process of the larvae in par-
affin); note the displacement of the
nuclei to the periphery of the cell
(arrows). (d) Reduction in fat deposits
in 28-dph larvae with regard to 9-dph.
(e) Periodic acid-Schiff (PAS)-positive
glycogen deposits in 28-dph larvae. (f)
(e) (f)
Detail of the exocrine pancreas at the
time of exogenous feeding (14-dph lar-
vae). Abbreviations: A, pancreatic aci-
nar cells; C, connective tissue; EP,
epithelium; H, heart; L, liver; S, liver
sinusoids; YS, yolk sac; Z, zymogen
granules. Staining: haematoxylin–eosin
with the exception of 7e that was
stained with PAS.

optimize larval rearing techniques and feeding conditions.
Studies on larval organogenesis, especially those devoted to
the digestive system, are considered as an useful tool for
establishing the functionality of the main organ systems
and physiological requirements of larvae to ensure optimal
welfare and growth under aquaculture conditions, which
might be useful for improving current larval rearing prac-
tices (Padros et al. 2011). In this sense, this information is
of value for designing larval feeding protocols that are able
to synchronize the state of development of the larvae with
the rearing protocol, as well as provide insight into the
proper weaning time.
Acipenseriformes (sturgeons and paddlefishes) differ
from the modern teleosts by holoblastic cleavage, by intra-
cellular platelet yolk in the embryo and by differentiation
of the digestive system from the yolk sac anlagen (Dettlaff
et al. 1993). Immediately after hatching, the alimentary
tract of sturgeons is divided into two regions (Ostaszewska
& Dabrowski 2009). The anterior section consists of the
buccal cavity, the occluded oesophagus and the future
anterior part of the gut filled with yolk mass, and sur-
rounded by the endodermal cells, which will develop into
..............................................................................................
the stomach walls and intestine. The posterior section of
the alimentary tract consists of the posterior intestine,
which will develop into the spiral intestine and the anus.
The postembryonic development of the alimentary tract of
sturgeon species takes place at the time of endogenous
feeding. Differentiation proceeds from the posterior
towards the anterior part. The spiral intestine is the first to
develop and the glandular stomach and pyloric caeca the
last (Buddington 1985). The basic mechanisms of organ
and system development are similar among all Acipenseri-
formes, although this study showed the existence of consid-
erable interspecific differences regarding the relative timing
of differentiation, development and functionality of the
digestive tract and accessory digestive organs during early
ontogeny among H. huso and the representatives of the
genus Acipenser.
At hatching, the mouth of H. huso larvae was already
opened contrary to the species of the genus Acipenser
described so far, which opened their mouths during the
endogenous feeding stage and before the onset of exoge-
nous feeding (Table 1). In all described species, epithelial
cells lining the buccopharynx showed vacuoles filled with

Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
 References: 1Present study; 2Boglione et al. (1999); 3Camacho et al. (2011); 4Sanz et al. (2011); 5Cataldi et al. (2002); 6Gisbert et al. (1998); 7Dettlaff et al. (1993); 8Gisbert & Doro-
Atlantic sturgeon
remnants of acidophilic yolk and pigment granules, as a

(A. oxyrinchus)10
consequence of the holoblastic cleavage of the embryo and
contribution of yolk-rich endodermal cells in the digestive
tract development. However, these yolk inclusions disap-
114
114

114

114

190
190
190
19
NA

NA

NA

NA
NA
pear during the first day after hatching as they are used by
developing cells (Dettlaff et al. 1993). This process is cou-
(A. ruthenus)9

pled with the development of goblet cells and taste buds

lining the buccopharyngeal epithelium that is similar
Sterlet

among Acipenserid species, although its timing varies

32
80
96
48

162
48

64

16
112

144
144
144
144
among species (Table 1). The development of buccopharyn-
(A. medirostris)8
Green sturgeon

geal goblet cells containing a mixture of neutral and acidic

mucins, as well as those from the oesophageal epithelium,
128–144

144–160
144–160 is of special relevance, because their secreted mucins may
96–112

32–48

serve as a lubricant, as fish do not have salivary glands

112
192

224

240
NA
32

32

16
found in higher vertebrates. Mucosubstances produced by
(A. gueldenstaedti)7

goblet cells may have the same functions as mammalian

saliva in protecting the mucosa of the entire alimentary
Russia sturgeon

canal from mechanical, physical and chemical abrasive fac-

148.8–167.4
148.8–167.4

148.8–167.4

tors (Sarasquete et al. 2001), whereas the presence of sialic

Table 1 Comparison of major developmental events of the digestive system ontogeny in different sturgeon species

18.6
111.6

130.2
130.2
130.2
130.2

acid residues in mucous prevents viruses from recognizing

NA

NA
NA

NA

their receptor determinants and also preserves the mucosa

Siberian sturgeon

from the attack of sialidase produced by bacteria (see

review in Zambonino-Infante et al. 2008).
(A. baeri)6

The ontogenic development of the oesophagus in H. huso

For comparative purpose, larval development is shown in degree days posthatching (ddph).
18
54
54
54

18

18
108–126
NA
NA

216
162
180–198
162

was faster than in other sturgeon species described

(Table 1), although the process of differentiation is similar
among them. In this sense, the complete differentiation of
1353,112.54
Adriatic sturgeon
(A. naccarii)2,3,4,5

this region, characterized by the complete resorption of

342–453,4

342–453,4
682
682
90–1053
90–1053
172

1353–1504–1685

1354
3853
153,4,172, 215
NA

yolk remnants and the establishment of the communication

between the oesophagus and the stomach occurred depend-
ing on the species considered, between 1.5 and 65.5 ddph
earlier than in species from the genus Acipenser. The early
shov (2003); 9Wegner et al. (2009); 10Ostaszewska et al. (2011).
Great sturgeon

differentiation of the oesophagus and its differentiation

(Huso huso)1

At hatching

At hatching

into two functional regions, the anterior part for the secre-
tory function and the posterior part participating in food
199.2
46.5

46.5
46.5
14.5

16.5

storage and transport (Gisbert et al. 1998), is of impor-

235

235
235
62

77

tance because this region of the alimentary canal of young

Morphologically complete digestive system
Appearance of incipient liver and pancreas

fish is able to respond to changes in environmental condi-

Average rearing temperature (centigrade)
Expel of melanin plug and anus opening
Appearance of zymogen granules in the

tions and maintain osmotic balance when gill arches are

not completely functional (Sarasquete et al. 2001). In Aci-
penser medirostris, the oesophagus has been reported to
Appearance of gastric glands

Onset of exogenous feeding

Oesophagus differentiation

reduce water osmolality and facilitating its absorption in

Appearance of Taste buds

Appearance of intestine

the intestine and the acclimation of juvenile fish to hyper-

NA, data not available.
Developmental events

Appearance of teeth

osmotic environments (Allen et al. 2009), which is of

Yolk sac exhaustion

importance in this group of anadromous species. Further-

Mouth opening

more, neutral glycoconjugates secreted by oesophageal gob-

pancreas

let cells are considered to cooperate in the digestion of

food and its transformation into chyme, as well as in the

..............................................................................................

Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
 absorption of easily digested substances, such as disaccha-
rides and short-chain fatty acids (Sarasquete et al. 2001).
Intestinal differentiation in H. huso was similar to other
sturgeon species, proceeding from its posterior region
(spiral valve) to the anterior intestine (Buddington 1985;
Heming & Buddington 1988). However, differences in the
timing of organ differentiation were found between Aci-
penserid species (Table 1). Histological observations
revealed that the intestine achieved its definitive organiza-
tion just before the onset of exogenous feeding, as indi-
cated by the level of folding of the mucosa and the
appearance of goblet cells in the anterior and intermediate
intestinal regions. In this study, no relevant differences
were observed in the histological organization of the ante-
rior and posterior regions of the intestine, with the excep-
tion of the number and size of mucosal folds, which were
longer and deeper in the posterior intestine, as well as the
degree of fat accumulation that was more important in the
posterior intestine. Intestinal fat deposits gradually accu-
mulated in the ciliated anterior intestine epithelium and in
the liver, as a result of yolk digestion (Dettlaff et al. 1993),
whereas they tended to decrease along larval development
and completely disappeared at 198–215 ddph, indicating an
increase in lipolytic capacities with age and/or a change in
lipid requirements (Rønnestad et al. 2013). Intestinal lipid
inclusions in fish larvae are considered a temporary storage
form of absorbed fatty acids (Watanabe & Sawada 1985)
in cases when the lipid absorption rate exceeds the rate of
lipoprotein synthesis (Sheridan 1988) or because of an
inability to metabolize them (Kjørsvik et al. 1991). The
development of enterocytes during larval growth is com-
bined with increasingly effective lipoprotein synthesis,
which is accompanied by a considerable decrease in the
number of large lipid vacuoles in the enterocytes, as well as
an important increase in the number of small lipid particles
in the intercellular spaces (Deplano et al. 1991; Gisbert
et al. 2008). Present results indicated that although H. huso
was fed with feeds formulated for trout, these diets seem to
cover basic nutritional requirements of the species and did
not unbalance the digestive physiology of the animal,
because no signs of intestinal steatosis linked to an unbal-
ance between lipid absorption and transport enterocytes’
capabilities were observed, contrary to other species
(Gisbert et al. 2005; Trevi~no et al. 2011).
In H. huso, the gastric region started to differentiate
between 15.5 and 46.5 ddph, a process characterized by the
development of an epithelial furrow in the posterior region
of the yolk sac that separates the intestine from the future
stomach. Similarly, to other sturgeon species, the non-
..............................................................................................
Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd
glandular stomach started to developed earlier (33 ddph)
than the glandular stomach (46.5 ddph) in H. huso. In this
sense, the appearance of gastric glands normally indicates
the formation of a functional stomach (Stroband & Kroon
1981), which is also a histological criterion to differentiate
fish larvae from juveniles (Tanaka 1971). In addition, the
presence of mucous cells in the epithelium lining the stom-
ach lumen, producing and secreting neutral glycoconju-
gates, confirmed that between 111.3 and 235.2 ddph, the
stomach was probably functional in this species, because
neutral mucosubstances may protect the stomach from
autodigestion processes caused by HCl and enzymes pro-
duced by gastric glands (see review in Zambonino-Infante
et al. 2008). These histological findings are in agreement
with biochemical analyses on digestive enzyme activities
(Asgari et al. 2013).The differentiation of the glandular
stomach took place earlier than in other sturgeon species
(Table 1) and even in other larval fish studied so far
(Rønnestad et al. 2013). Similarly, to other fishes, the
development of the pyloric caeca, together with that of the
stomach, are the last steps in the differentiation of the ali-
mentary canal in H. huso, as well as being the anatomical
digestive features that mark the end of the larval phase and
the beginning of the juvenile period in this species. In this
context, the larval period in terms of nutrition ends with
the completion of a functional stomach (Segner et al.
1993), and consequently, the stomach and pyloric caeca dif-
ferentiation could be considered as a decisive event in the
nutritional physiology of larvae and could be used as an
external marker for the start of weaning (Verreth & van
Tongeren 1989; Segner et al. 1993). Hence, it might be rec-
ommended to start introducing inert diets under a co-feed-
ing regime from the onset of exogenous feeding in H. huso
larval rearing, whereas the complete replacement of live
prey by microdiets is not advisable until 235.2 ddph, when
the larval period ends from a nutritional physiology point
of view, and there is a putative transition from alkaline to
acid digestion. However, further research must be con-
ducted on assessing the functionality of the digestive sys-
tem, considering the production of pancreatic, stomach and
intestinal enzymes to confirm present histological observa-
tions and to enlarge the knowledge on the digestive physi-
ology of this species under different nutritional conditions.
Similar to other sturgeon species (Buddington & Doro-
shov 1986; Dettlaff et al. 1993; Gisbert & Doroshov 2003
among others), the accessory digestive glands in H. huso
were absent at hatching, The liver primordium in H. huso
started to form during the first day after hatching as in
A. nacarii and A. ruthenus (Boglione et al. 1999; Wegner
et al. 2009), whereas in A. medirostris, this process takes
place later (Gisbert & Doroshov 2003). In all cases, the
liver achieved its definitive histological organization by the
onset of first feeding. The level of hepatic lipid deposition
in H. huso progressively increased during the endotrophic
and exotrophic phases, whereas they progressively
decreased after 380 ddph. In this sense, changes in the his-
tological organization of digestive organs (intestine and
liver) along larval development or under different feeding
conditions are considered as valuable biomarkers to evalu-
ate the nutritional condition and assess the effects of die-
tary imbalances in larvae and juveniles (Gisbert et al.
2008). In this regard, the liver is considered a good target
tissue, because its histological organization is particularly
sensitive to diet quality and quantity. Thus, changes in the
level of lipid accumulation in the liver of H. huso after 380
ddph may be attributed to a dietary change, because at this
time larvae were progressively transferred from Artemia
nauplii to Daphnia sp. Changes in lipid deposition may be
associated to the lower lipid content of cladocerans with
regard to Artemia nauplii (Macedo & Pinto-Coelho 2001;
Barata et al. 2005; Øie et al. 2011), as well as higher pro-
portion of sterols and phospholipids (Bychek et al. 2005).
Thus, changes in the lipid deposition in the liver of H. huso
seemed to be attributable to the nature of the dietary lip-
ids, because phospholipids are generally more easily metab-
olized than neutral lipids (Gisbert et al. 2005). Similarly to
the liver, 1 day after hatching the pancreas primordium
appeared dorsally from the cleft dividing the yolk sac.
Zymogen granules into a part giving rise to the stomach
and into another part from which the intestine will
develop. The zymogen granules in the acinar cells in
H. huso appeared earlier than in other sturgeon species
with the exception for A. ruthenus (Table 1). In some spe-
cies like A. gueldenstaedti (Ostaszewska & Dabrowski 2009)
and A. transmontanus (Gawlicka et al. 1995), the zymogen
granules were present in the acinar cells at older ages and
just before the onset of the exogenous feeding and even
after exogenous feeding in A. medirostris (Gisbert &
Doroshov 2003), which indicated time-developmental dif-
ferences among sturgeon species.
The results of present study indicate that organization and
sequence of differentiation of the digestive tract and acces-
sory digestive organs in H. huso larvae were similar to
other sturgeon species. In brief, at the onset of exogenous
feeding the digestive system consists of a well-developed
buccal cavity, oesophagus, glandular and non-glandular
stomach, anterior intestine, spiral intestine and anus.
However, the complete development of the digestive sys-
tem in H. huso was not accomplished until 235.2 ddph
when it showed all histomorphological features typical of
juvenile specimens. Similar to other sturgeon species, the
melanin plug is expelled from the spiral intestine lumen as
a consequence of first feeding, which indicated that this
process cannot be used as a criterion for start feeding
H. huso larvae (Gisbert & Williot 2002). According to his-
tological results and recent results from Agh et al. (2011)
and Asgari et al. (2013), it seems advisable to start co-
feeding H. huso larvae with inert diets at the onset of
exogenous feeding, because exocrine pancreas and glandu-
lar stomach are fully differentiated, although the complete
replacement of live prey by inert feed is not recommended
until 235 ddph. However, further research is needed to
define the best co-feeding and weaning strategy for this
species. In addition, the organogenesis of the digestive sys-
tem in H. huso described in this study and future studies
of digestive enzymes activities may not only increase
knowledge on the developmental features and digestive
capabilities of this species, but also is an important tool
to develop a histological model that would serve as a ref-
erence when normal developmental patterns might be
altered due to rearing and/or nutritional conditions.
We would like to thank the Shahid Marjani Sturgeon rear-
ing centre for the assistance in obtaining Beluga brood-
stocks for spawning and Hamid Eshaghzadeh for help in
larval rearing and sampling during this study.
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Aquaculture Nutrition, 20; 595–608 ª 2014 John Wiley & Sons Ltd