Belg. J. Zool., 135 (2) : 205-207
July 2005
Analysis of the post-vitellogenic oocytes of three species
of Danubian Acipenseridae
Mirjana Lenhardt1, Roderick Nigel Finn2, Predrag Cakic1, Jelena Kolarevic3, Jasmina Krpo-
Cetkovic3, Ivica Radovic3 and Hans Jørgen Fyhn2
1 Institute for Biological Research "Sinisa Stankovic", 29 novembra 142, 11000 Belgrade, Serbia and Montenegro
2 Department of BiOlogy, University of Bergen, Allegt 41, N-5020 Bergen, Norway
3 Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia and Montenegro
Corresponding author : Mirjana Lenhardt, e-mail : lenhardt@ibiss.bg.ac.yu
ABSTRACT. Post-vitellogenic oocytes of beluga (Huso huso Linnaeus, 1758), Russian sturgeon (Acipenser
gueldenstaedtii Brandt, 1883) and sterlet (Acipenser ruthenus Linnaeus, 1758), sampled downstream of the "Iron
Gate II" dam on the Danube River, were characterised according to diameter, dry mass, water and protein contents.
All oocytes examined were ovoid in shape with the major diameter being measured in the animal-vegetal axis. The
beluga oocytes were the largest, with major and minor diameters of 4.18 ± 0.13 and 3.61 ± 0.14 mm, respectively.
The oocytes of the Russian sturgeon were the next largest, with major and minor diameters of 3.69 ± 0.16 and 3.36
± 0.15 mm, respectively, while those of the sterlet were the smallest, with major and minor diameters of 2.40 ± 0.10
and 2.14 ± 0.07 mm, respectively. Values for oocyte wet and dry mass (mg/ind) ranged from 25.9-32.1 for wet mass
and 12.2-15.5 for dry mass of the beluga oocytes, 18.9 ± 1.4, and 9.01 ± 0.12 for wet and dry mass of the Russian
sturgeon oocytes, to 6.5 ± 0.3 and 3.07 ± 0.14 of the sterlet oocytes. The water content of the oocytes of all three
sturgeons was very similar (51-53% of wet mass). The protein content (% of dry mass) was highly conservative
among the species at 53.0 ± 2.0, 55.9 ± 3.8 and 50.0 ± 1.2 for the oocytes of beluga, Russian sturgeon and sterlet,
respectively.
KEY WORDS : Russian sturgeon, beluga, sterlet, oocytes, Acipenseridae, yolk proteins
INTRODUCTION
The most significant research on the biology and culti-
vation of sturgeons was conducted in the former Soviet
The Acipenseriformes live almost exclusively in the
Union (DETTLAFF et al., 1993). Since the collapse of the
Northern Hemisphere with half of the extant number of
Soviet Union, however, virtually all research effort has
species occurring in Europe, mostly in the Ponto-Caspian
ceased through lack of funding.
region (Billard & Lecointre, 2001). Recently several asso-
The situation is very much the same in the countries
ciations have recommended that the status of beluga (Huso
bordering the lower region of the Danube (Serbia and
huso) be upgraded to Appendix I under current CITES list-
Montenegro, Romania, Bulgaria and Ukraine). Despite
ings since it has almost been extirpated from the Black Sea
the research effort of DETTLAFF et al. (1993), some
(Vecsei et al., 2002). Indeed many of the stocks of stur-
problems regarding normal growth and success of fer-
geons have dramatically decreased, primarily as a result of
tilization of the oocytes and eggs still exist. According
over fishing and habitat deterioration. Habitat loss is pre-
to AMIRKHANOV (1974) earlier studies suggested that
dominantly caused by pollution and damming of rivers,
protein concentration in oocytes can be a valid indica-
which blocks migration and access to proper spawning
tor of the staging, quality and successful fertilization
grounds (STEVENSON & SECOR, 1999 ; Vecsei et al., 2002).
and normal growth of the fertilized eggs (BRASHE,
In Yugoslavia there was a significant drop in catch of
1964 ; FEDOROVA & GRUDANOV, 1968). More recently,
all sturgeon species after 1970 and 1984, when the lower
CHEBANOV (2001) conducted similar research on Rus-
stretch of the Danube was dammed by, respectively, Djer-
sian sturgeon (Acipenser gueldenstaedtii) in which he
dap I and II hydropower stations (JANKOVIC, 1993 ; VEC-
followed protein and water contents in the oocytes over
SEI et al., 2002). Today, species of Acipenseridae, except
a ten year period. Together with other physiological
sterlet, occur in Serbia and Montenegro only in a 17.8 km
parameters, these findings helped him to evaluate
stretch of the Danube River, from the Djerdap II dam to
reproductive quality of the natural and artificially
the border with Bulgaria, close to the Timok River mouth.
reared populations.
Presently sturgeon farming, outside of Serbia and Mon-
Consequently, the goal of this investigation was to
tenegro, yields more than 2,000 tons of fish per year and
make a preliminary comparative study of the biometry,
about 15 tons of caviar. Such efforts could contribute to a
gravimetry and protein contents of the oocytes of three
reduction of fishing pressure on wild stocks (BILLARD &
species of Danube sturgeons : beluga, Russian sturgeon
LECOINTRE, 2001).
and sterlet (Acipenser ruthenus).

---

206
M. Lenhardt, R.N. Finn, P. Cakic, J. Kolarevic, J. Krpo-Cetkovic, I. Radovic and H.J. Fyhn
MATERIAL AND METHODS
All females were sampled during the spring and fall of
2001 in the Danube, downstream of "Iron Gate II" dam
(863 km from the Danube delta). Oocytes were sampled
from three beluga females (BeB, BeC, BeD) caught in
April and May, total length (TL) = 282, 287, 304 cm and
wet mass (W) = 161, 194.5, 159.6 kg respectively, one
Russian sturgeon (GuA) caught in October, TL = 180 cm,
W = 25 kg and two sterlets (RuA, RuB) caught in April,
total length (TL) = 64, 68 cm and wet mass (W) = 1.3, 1.5
kg. The second sterlet (RuB) had undergone final oocyte
maturation and provided ovulated eggs.
Major (D ) and minor (D ) diameters were measured in
1
2
0.9% NaCl. Wet masses were determined after removal
of excess ovarian fluid, and the samples were frozen and
stored at ­20°C until lyophilisation, dry mass and protein
content analyses.
Oocyte volume (V) was calculated using the formula :
2
4
v = - ·
D
·
1
D
·
2
3
---
---
2
2
where D and D represent the major and minor diame-
1
2
ters respectively.
Oocyte wet and dry masses were measured to the near-
est 0.1 mg. Lyophilisation was performed for 48 hours.
Oocyte proteins were precipitated with 1 mL 6%
trichloro-acetic acid TCA to remove free amino acids,
then centrifuged (10,000 x g, 5 min, 4° C). The precipitate
was washed once in 6% (TCA), then solubilised in 1M
NaOH. Solubilisation was accomplished with sonification
(3 x 15 sec at 400 Hz). Prior to analyses, 1 mL double-
distilled water was added to give a final concentration of
0.5M NaOH. Following the addition of the Lowry rea-
gents (LOWRY et al., 1951), triplicate samples were read at
650 nm with a Pye Unicam spectrophotometer. Bovine
Fig. 1. ­ Major (D ) and minor (D ) diameter, volume, wet
1
2
serum albumin was used as standard.
(WM) and dry (DM) mass, water and protein content of beluga
(Be) oocytes, sterlet (RuA) oocytes, sterlet (RuB) eggs and
Russian sturgeon (GuA) oocytes.
RESULTS
The largest oocytes were obtained from beluga, and
smallest from sterlet, while Russian sturgeon had inter-
DISCUSSION
mediate sized oocytes (Fig. 1). The size classification was
also evident for wet mass and dry mass, and reflected the
The data concerning oocyte biometry and gravimetry
size of the female caught. However, despite the differ-
for three Danube sturgeon species were correlated with
ences in size, all oocytes had similar water content with
parental size. Beluga oocyte diameter, volume, wet and
values of 51-53% of wet mass. Similarly, the oocyte rela-
dry mass were the largest followed by Russian sturgeon,
tive protein content (% of dry mass) was also conserved
then sterlet.
between the species and ranged from 50-56%.
Average diameters of three beluga oocytes were
Overall the data show that irrespective of size, the pro-
4.18 mm for major and 3.61 mm for minor diameter. Data
tein content and cell water are closely regulated compo-
given by HOLCIK (1989) for this parameter were around
nents of the post-vitellogenic oocytes of the Danube stur-
3.8 mm for major and 3.4 mm for minor diameter, which
geons.
corresponded better to the data obtained for Russian stur-
From one species only, the sterlet, we had the opportu-
geon (3.7 and 3.4 mm). Between these two results are
nity to sample oocytes (RuA) and ovulated eggs (RuB).
those (4.0 and 3.6 mm) obtained by Dettlaff & Ginsburg
From Fig. 1 it is possible to see that ovulated eggs of ster-
(1954). According to BERG (1949) 3.5 mm and 3.0 mm
let had bigger values for diameter, volume and protein
are average values for major and minor diameters respec-
content compared to the oocytes of the RuA female.
tively.

---

Danube sturgeons' oocytes
207
There are several data given for sterlet oocyte diame-
DETTLAFF, T.A. & A.S. GINSBURG (1954). The embryonic devel-
ters by different authors. According to HOLCIK (1989)
opment of sturgeons (stellate sturgeon, Russian sturgeon,
oocyte diameter is around 2.5 mm. JANKOVIC (1958)
beluga) in connection with questions of their reproduction.
noted that oocyte diameters ranged from 2.0-2.9 mm for
Acad. Sci. Press, Moscow.
major axis and 1.8-2.8 mm for minor axis. Values
DETTLAFF, T.A., A.S. GINSBURG & O.I. SCHMALHAUSEN (1993).
obtained in the present study were 2.7 and 2.4 mm for the
Sturgeon fishes development biology and aquaculture. Hei-
delberg, Berlin, Springer-Verlag, New-York.
major and minor diameters of the ovulated eggs found in
FEDOROVA, L.S. & S.D. GRUDANOV (1968). Some aspects of the
the abdomen of RuB, and 2.4 mm and 2.1 mm for the
protein metabolism in beluga females from the spring
major and minor diameters of the post-vitellogenic
migration during gonad maturation. In : Elaboration of the
oocytes of RuA.
biological bases and biotechnics of rearing in sturgeon
Average wet mass of beluga oocytes was 29.3 mg, and
fisheries in bodies of water of the USSR, Astrakhan.
14.0 for its dry mass. Wet and dry mass for oocytes of
FINN, R.N., H.J. FYHN, B. NORBERG, J. MUNHOLLAND, & M.
REITH (2000). Oocyte hydration as a key feature in the adap-
Russian sturgeon were 18.9 mg and 9.0 mg, and 6.5 mg
tive evolution of teleost fishes to seawater. In : NORBERG,
and 3.1 mg for sterlet oocytes. CHEBANOV (2001) found a
KJESBU, TARANGER, ANDERSON & STEFANSSON (eds), Proc 6th
difference in Russian sturgeon oocytes wet mass in the
Int. Symp. Reprod. Physiol. Fish, Inst Mar Res & Univ Ber-
years 1991 - 2000. In that period, values ranged from 17.9
gen, ISBN : 82-7461-048-2 : 289-291.
to 20.4 mg, which is very similar to results presented
FINN, R. N., M. LENHARDT, A. FERRARA, P. CAKIC, J.J. ISELY, J.
here.
DEAN, M.S. EVJEN & H.J. FYHN (2001). Yolk proteins and
free amino acids in Acipenseriformes. Abstract : Tenth Euro-
Oocyte water and relative protein content of all three
pean Ichthyological Congress in Prague 3-7th September
sturgeons were very similar. The water content results are
2001.
at the lower range of 50-70 % reviewed by KAMLER
FINN, R.N., G.C., ØSTBY, B. NORBERG, & H.J FYHN. (2002a). In
(1992) for freshwater fishes, and the protein content was
vivo oocyte hydration in Atlantic halibut (Hippoglossus hip-
similar to the lower range reviewed by KAMLER (1992).
poglossus). Proteolytic liberation of free amino acids, and
Indeed the values found here for beluga are considerably
ion transport are driving forces for osmotic water influx. J.
lower than the recomputed 63% reported by KAMLER
Exp. Biol., 205 : 211-224.
(1992). Unlike the pelagic eggs of marine fishes (F
F
INN et
INN, R.N., M. WAMBOLDT, & H.J. FYHN (2002b). Differential
al., 2000 ; 2002a ; 2002b), sturgeon oocytes do not
processing of yolk proteins during oocyte hydration in fishes
(Labridae) that spawn benthic and pelagic eggs. Mar. Ecol.
undergo proteolysis and hydration during final oocyte
Prog. Ser. 237 : 217-226.
maturation (FINN et al., 2001 ; 2002c). Our findings sug-
FINN, R.N., M., LENHARDT, A., FERRARA, J.J., ISELY, J. DEAN, &
gest that, despite the difference in size of both female and
H.J. FYHN (2002c). Yolk proteins and free amino acids in
oocyte, water content and protein levels are conservative
Acipenseriformes. Proc. 26th Larval Fish Conference of the
aspects of the reproductive biology of sturgeons.
American Fisheries Society. Bergen, Norway, 22-26 July.
HOLCIK, J. (1989). Freshwater fishes of Europe. General intro-
duction to fishes and Acipenseriformes. Aula Verlag, Wies-
REFERENCES
baden.
JANKOVIC, D. (1958). Ecology of Danubian sterlet (Acipenser
AMIRKHANOV, G.A. (1974). The dynamics of free amino acid
ruthenus). Institute for Biological Research, Belgrade.
content during the embryonic development of the sturgeon,
JANKOVIC, D. (1993). Populations of Acipenseridae prior and
Acipenser gueldenstaedtii. Vopr Ihtiol., 19 : 127-131.
after the construction of the HEPS Djerdap I and II. Ichthyo-
B
logia, 25 : 29-34.
ERG, L.S. (1949). The freshwater fishes of the USSR and adja-
cent countries. Akademia nauk USSR, Moscow & Lenin-
KAMLER, E. (1992). Early life history of fish. An energetic
grad.
approach. Chapman and Hall, Fish and Fisheries Series 4.
London
BILLARD, R. & G. LECOINTRE (2001). Biology and conservation
L
of the sturgeon and paddlefish. Rev Fish Biol Fish, 10 : 355-
OWRY O.H., N.J. ROSEBROUGH, A.L. FARR & R.J. RANDALL
(1951). Protein measurement with the folin phenol reagent. J
392.
Biol Chem, 193 : 265-275.
BRASHE, Z. (1964). Biochemical embryology. Inostr. Lit-ra,
STEVENSON, J.T. & D.H. SECOR (1999). Age determination and
Moscow.
growth of Hudson River Atlantic sturgeon, Acipenser
CHEBANOV, M.S. (2001). Ecological-technological principles of
oxyrinchus. Fish. Bull., U.S., 98 : 153-166.
stock enchancement and living gene bank formation for con-
VECSEI, P., R. SUCUI & D. PETERSON (2002) Threatened fishes of
servation of Caspian and Azov Sea sturgeon diversity.
the world : Huso huso (Linnaeus, 1758) (Acipenseridae).
Report of Krasnodar Research Institute of Fisheries.
Environ Biol Fish 65 : 363-365.

---