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Development of industrial ﬁsh culture in Belarus
Technical Report · January 2013

DOI: 10.13140/RG.2.1.4626.8885
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Belarusian State Agricultural Academy
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Recirculation technologies in indoor and outdoor systems
HANDBOOK
Based on the presentations of the AQUAREDPOT workshop held

in Vilnius, Lithuania, on 13-14 May 2013
Research Institute for Fisheries, Aquaculture and Irrigation

Szarvas
2013
Edited by:
Peter Lengyel
Denes Gal
Gergo Gyalog
Vilmos Jozsa
Published by HAKI, Szarvas, 2013
Printed by:
Fazekas Nyomda, Szarvas
Contents
Introduction 2
Ideas behind the AQUAREDPOT project

Gál, D., Gyalog, G., Váradi, L. 3
Research projects and demonstration activity planned in the RAS to be

established in the frame of AQUAREDPOT Project
Péteri, A., Gyalog, G., Diviki. S., Kucska, B., Gál, D. 6
Production of stocking material in RAS as unity of selective breeding and

technology
Golod, V. М., Terentyeva, Е. G., Krupkin, V. Z 11
Nitrite-induced methemoglobinemia of freshwater fishes reared in

recurculating aquaculture systems
Khuda, L.V., Khudyy, A. I 22
Application of recirculating aquaculture systems (RAS) in Polish sturgeon

culture
Kolman, R., Zdanowski, B 30
Development of industrial fish culture in Belarus

Kostousov, V.G., Barulin, N.V. 44
The first experiment to obtain sex reversed rainbow trout in RAS conditions
in the Republic of Belarus
Slukvin, А. М., Metalnikova, K. V., Kostousov, V. G., Koneva, О. Yu.,
Rovba, Е. А. 49
Recirculation aquaculture systems in the Center of Aquaculture and

Environment Engineering, Warmia and Mazury University in Olsztyn,
Poland
Kucharczyk, D., Żarski, D., Krejszeff, S. 78
Actual issues in recirculating aquaculture –

Summary of the roundtable discussions at the AQUAREDPOT
workshop 92
1
INTRODUCTION
HAKI is a regional centre of excellence in aquaculture research

implementing a multidisciplinary programme. While HAKI is an important
representative of the CEE region in various European initiatives aiming at
aquaculture development (e.g. EATIP and various FP6 and FP7 projects), the
institute also has a leading role in the exchange of knowledge and networking
of aquaculture centres in the CEE region. HAKI was the initiator of the
establishment of the Network of Aquaculture Centres in Central and Eastern
Europe (NACEE) in 2004. One of the major objectives of NACEE is the
integration of the aquaculture centres in the CEE region into the European
Research Area (ERA).
In 2013, connected to the 4th General Assembly Meeting of NACEE held in
Lithuania, a 2-day-long workshop on ”Recirculation technologies in indoor
and outdoor systems” was organized. The first day was devoted to lectures on
the basic theory of water recirculation, while on the second day, more
practice-oriented research resultswere presented about viable species and
application of water recirculation elements in pond culture. Two separate
roundtable sessions were dedicated to the discussion of the use of
recirculation technologies in the research and aquaculture of Central and
Eastern Europe.
The lecturers included one scientist from the Aquaculture and Fisheries
Group of Wageningen University (AFI) (Developments in recirculating
aquaculture systems), one scientist from AquaBioTech (Application of RAS
to Eastern European aquaculture production), one scientist from NOFIMA
(Research objectives and design of RAS in NOFIMA), one scientist from
Denmark (RAS case stories), as well as 11 researchers from CEE (in the
fields of fish production in RAS and case studies for outdoor recirculation
systems in CEE) and two scientists from HAKI.
2
Ideas behind the AQUAREDPOT project
Dénes Gál, Gergő Gyalog, László Váradi
Research Institute for Fisheries, Aquaculture and Irrigation, Szarvas,

Hungary
Aquaculture is a recognised future option to provide animal protein to the

increasing population since it is an efficient user of feed and water resources,
and fish is a unique source of unsaturated fatty acids that have significant
health benefits.
In spite of the rapid growth of aquaculture on a global scale, the EU
aquaculture production is stagnating. More than sixty percent of the
consumed seafood in the EU derives from import. The new „Strategy for the
Sustainable Development of European Aquaculture” tries to give an impetus
to the growth of aquaculture in the EU and the European Commission will
pursue efforts in aquaculture R&D, and allocate a sufficient EU budget to
aquaculture projects to further develop the knowledge base for sustainable
and competitive aquaculture practices. The EU aquaculture industry of the
future should be at the forefront of sustainable development. Appropriate
measures must be put into place to ensure that the aquaculture industry can
take a lead role in the "blue revolution", whether this concerns the production
of aquatic food itself, technology and innovation, or the setting of standards
and certification processes at EU and international levels.
Freshwater aquaculture development has been recognised as an unexplored
opportunity in the Central and Eastern European (CEE) region to contribute
to the improvement of rural livelihoods and also to increase of fish
consumption, which is unacceptably low in the CEE region. The low
technological level of regional (CEE) aquaculture provides an exceptional
scope for innovation. In these countries, the freshwater extensive and semi-
intensive carp-based pond fish production is the dominant type of
aquaculture. In spite of significant efforts in the CEE countries aiming at the
modernization of the sector using the European Fisheries Fund (EFF) and the
R&D Framework Programs (FP6 and FP7), there is still a significant gap
between the technical level, intensity and quality of aquaculture production in
Western and Eastern Europe. Since the increasing competition for space and
3
water resources represents a major challenge for further developing or even
maintaining freshwater fish farming, the increase of the intensity level and
the recirculation/reuse of water and nutrients have been identified in the
Strategic Research Agenda (SRA) of EATIP as a strategic direction in the
sustainable development (in terms of ecological, economical and social
aspects) of freshwater aquaculture technologies.
The required modernisation of aquaculture production in the CEE region
can be enhanced only by research, innovation and development of the
extension capacity. The reinforcement of research capacity is a major
precondition of aquaculture development in the region by the facilitated
cooperation between regional research entities.
HAKI is a regional centre of excellence in aquaculture research
implementing a multidisciplinary programme. While HAKI is an important
representative of the CEE region in various European initiatives aiming at
aquaculture development (e.g. EATIP and various FP6 and FP7 projects), the
institute also has a leading role in the exchange of knowledge and networking
of aquaculture centres in the CEE region. HAKI was the initiator of the
establishment of the Network of Aquaculture Centres in Central and Eastern
Europe (NACEE) in 2004. One of the main objectives of NACEE is the
integration of the aquaculture centres in the CEE region into the European
Research Area (ERA).
The objective of the AQUAREDPOT project is to strengthen the research
potential of HAKI in order for it to become the leading research and
innovation knowledge centre in the field of freshwater aquaculture
development in the CEE region, which could subsequently act as a driving
force of technological development and improvement of the fish product
supply in this region.
The challenge of the AQUAREDPOT project is to strengthen HAKI’s S&T
research and innovation potential. In the frame of the project, HAKI will
acquire new knowledge, competences in the field of research, innovation and
intellectual property development, build strategic partnerships with
outstanding research partner organisations, mobilise human resources, recruit
new researchers, enhance research capacity and develop its research
infrastructure to improve its innovation impact to the regional aquaculture. In
order to achieve the enhancement of its innovation potential, HAKI will
collaborate actively with three leading European research entities
4
(Aquaculture and Fisheries Group of Wageningen University, Institut
Français de Recherche pour l’exploitation de la Mer, Norwegian Institute of
Fisheries and Aquaculture Research) and an SME (Aqua Biotech Ltd) during
the implementation of the project activities.
The promotion of regional aquaculture development by improving
scientific activity and transferring the latest research results to farmers and
investors is an accentuated aim of the AQUAREDPOT project. Improved
knowledge transfer should be based on more practice-oriented R&D work
taking into account the socio-economic characteristics of the region. Thus
this workshop organised together with NACEE and the Fisheries Service of
the Ministry of Agriculture of the Republic of Lithuania is dedicated to the
novel recirculating aquaculture technologies aimed to bridging the gap
between regional aquaculture research priorities and the needs of farmers and
investors (the fish farming sector of the future) by gathering researchers and
farmers of the CEE region.
5
Research projects and demonstration activity planned in the RAS to be
established in the frame of AQUAREDPOT Project
András Péteri, Gergő Gyalog, Sándor Diviki, Balázs Kucska, Dénes Gál
Research Instutute for Fisheries, Aquaculture and Irrigation, Szarvas,

Hungary
Fish production and development options

Production of cyprinids and some carnivorous/predatory fish species gives
the majority of freshwater-originated table fish consumed in Central and
Eastern Europe (CEE). A continuous increase in the production of these
species in the region and the growth of fish imports in last decade allow
forecasting that the demand for fish will increase also in next years.
Moreover, it can be predicted that the consumer preferences will shift
towards carnivorous/predatory fish species.
Supporting a better utilization of water bodies used for fish production
(450 000 ha fish ponds and reservoirs in Central and Eastern Europe) and
encouraging introduction of RAS suitable for mass production of fish more
valuable than the table size carp might provide a firm background for
aquaculture development in the region.
Establishment of technical facilities for fulfilling the above tasks

HAKI must fulfill three targets. The Institute will carry out research and
demonstration/training work related to RAS. Consequently, systems planned
to be constructed during the AQUAREDPOT project have to be suitable for
examining factors affecting fish rearing with sufficient repetitions necessary
for research work. Additionally, these systems must be available for
demonstration of the production methods in semi-industrial size in order to
convince target groups that the presented techniques are also suitable for a
large-scale production of fish. Production of fingerlings and adult fish of
some valuable species (catfish, sturgeons, etc.) will be an important side
activity of the systems.
6
For fulfilling the above tasks, three systems are planned to be constructed in
the building of the old RAS of the Institute (Figure 1):
 broodfish system with a total of 6-10 m3 tank volume;
 nursing unit for rearing fish up until fingerling size with about 18 m3
total tank volume (Figure 2);
 rearing unit for large size fingerling/table fish with about 30 m3 tank
volume, comprising of two tanks (Figure 3).
Both the broodfish unit and the nursing system will have two-three sets of
replaceable tanks which will provide an opportunity for running trials with
different sizes of fish, in different repetitions, at different water temperatures.
Additionally, a RAS comprising of 27 small tanks will be constructed for the

examination of the effects of immuno-stimulants on stress and disease
resistance of fish (Figure 4). In this unit, the total water volume of tanks will
be 3.5 m3. It will be possible to run all the tanks in one water circle and also
to separate the tanks and operate them as two independent water recirculation
systems.
Program for the utilization of these facilities

HAKI will carry out research work in the planned systems for
adaptation/development of fish seed production technologies of species
which might be considered for commercial use. All aspects of fish rearing,
including effects of environmental factors on fish, selection of most suitable
feeding methods, health management during the rearing period, economy of
production, etc. will be examined. In spite of that the primary goal will
always be the supporting of seed production, trials for the development of
table fish production technologies will also be carried out. Methods for mass
production of endangered fish species will be elaborated in these systems,
too.
As an additional activity of the above-described work, performance of
different configurations of RAS will be examined using installed water
analyzing instruments and the existing laboratory background of the Institute.
Examination of environmental aspects of RAS operation will have a high
priority. Since, in the CEE region, RAS-based intensive fish rearing will be
implemented in most of cases for supporting fish production in pond fish
7
farms by supplying them with stocking material, the majority of RAS will be
most probably established in vicinity of fish ponds. Consequently, effluents
of RAS could be discharged into fish ponds directly or through wetlands.
Intensive research will be carried out for the determination of all aspects of
such type of effluent treatment.
Hungarian and foreign MSc and PhD students will be involved in the above-
detailed research works. Moreover, continuous efforts will be done to carry
out research work in frame of international collaborations.
These activities will mainly be financed from different domestic and
international grants.
HAKI plans to organize regular trainings for interested aquaculturists from
CEE countries. The curriculum of these trainings will include technical
aspects of RAS construction and operation and details of fish production
technics. Economic aspects of RAS operation will also be emphasized in
these trainings. In addition to local experts, internationally acknowledged
experts of different fields will be invited as lecturers. Language barriers will
be overcome by inviting professional interpreters or English-speaking
aquaculturists from the countries of participants.
Training costs will be covered by different local and international sources.
Moreover, participants arriving from profit-oriented fields of fishery industry
will be requested to pay their training costs.
Expected benefits
After the completion of the AQUAREDPOT program, HAKI will be able to:
 Give valuable technical support for fish producers both in Hungary
and other countries of the CEE region;
 Increase theinternational reputation of the Institute through RAS-
related publications;
 Develop a closer contact with aquaculturists of the CEE region
through RAS trainings;
 Increase the intensity of scientific relationships both with CEE and
Western European countries;
 Generate income for HAKI through grants, advisory work, training
fees and marketing valuable fingerlings.
8
Figure 1. Location of the planned systems in the building of the old RAS of HAKI
Figure 2. Upper view and cross-section of the RAS planned for fingerling production
9
Figure 3. Upper view and cross-section of the RAS planned for large-size fingerling/table
fish production
10
Production of stocking material in RAS as unity of selective breeding
and technology
Golod, V. М.,Terentyeva, Е. G., Krupkin,V. Z.
Federal Centre of Fish Genetics and Selection, Ropsha, Russia
Abstract
The Federal Centre of Fish Genetics and Selection (FCFGS), situated in a suburb of Saint
Petersburg, is a major rainbow trout seed supplier in the north-west of Russia. The main
focus of the enterprise is the development of fish breeds for industrial fish culture.
Five recirculating aquaculture systems (RAS) encompassing the entire production cycle of
trout from broodstock rearing to seed production and marketing have been constructed and
set into operation here in the recent years.
The high quality of the seed is reached through a combination of selective breeding and the
use of the possibilities offered by RAS. The selective breeding work combines methods of
mass selection and family selection with inbreeding and progeny selection. As a result,a
continuous production cycle of the three main groups of stocking material (fry with an
average weight of 5-10 g, fingerlings with an average weight of 50-70 g and yearlings with
an average weight of 100-300 g) has been established.
Keywords: Rainbow trout, fish seed, RAS, selection
During the many centuries of the history of humankind, the demand for fish
as food was mainly satisfied from natural-water fishing. The share of
artificial fish farming only became significant by the end of the 20th century,
while in the coming years, aquaculture is expected to become dominant.
Many different forms of fish rearing have been developed, with the new
forms not replacing the old ones but often existing alongside with them. Due
to this peculiarity, modern fish culture exists in various forms, which
conditionally can be classified into two basic groups: ranching and industrial
fish culture.
A special feature of industrial fish culture is the maximum concentration of
fish production in small areas, high mechanization of all fish farming
processes and maximum mobilization of the whole potential of the fish
organism for the achievement of a maximum productivity. The most
advanced form of industrial fish farming is the use of recirculating
aquaculture systems (RAS). These systems provide a complete biological
11
treatment of the used water and maintain optimum temperature, oxygen,
hydrological and hydrochemical conditions for the cultured species.
Studies and experimental work on the creation of recirculating fish farms
were conducted in the USSR already in the 1970s. Unfortunately, the
developed theoretical bases of this technology were not implemented in
practice and later, in the period of the collapse of the Soviet Union, all the
experimental facilities were lost. The interest in water recirculation systems
revived only in the 21st century. Currently, several such facilities operate in
Russia, addressing local challenges. At the same time, in countries such as
the United States, Germany, Finland and Denmark, RAS are widely used for
the large-scale fry production and grow-out of many cultured species (trout,
sturgeon, eel, etc.).
Water use and wastewater production are hundreds of times lower in RAS
than in flow-through tank farms. This greatly increases the number of water
sources suitable for the establishment of fish farms, allowing to bring the
places of fish production closer to those of consumption. The reduction of the
specific costs of water heating allows to organize year-round rearing of fish,
repeatedly using the same technological equipment and providing a
scheduled delivery of the products to customers, while the optimization of the
cultivation mode allows to reduce the feed conversion ratio. The low water
consumption, coupled with a complete mechanical and biological wastewater
treatment, makes RAS safe for the environment.
To advance the introduction of such plants in Russia, it was decided to
implement a pilot project to study the functioning of modern RAS in the
natural, climatic, institutional and legal conditions of Russia, to develop
model projects and initiate the training of personnel.
The Federal Centre for Fish Genetics and Selection (FCFGS) located in the
village of Ropsha, the nearest suburb to St. Petersburg, was selected as the
base for this project. This institution, which has a federal status, has
functioned as an independent organization since the early 1990s. Its main
task is to conduct breeding and genetic research, develop new fish breeds and
produce fish seed for growout farms. However, due to the fact that its
premises used to be an experimental station of the State Research Institute of
Lake and River Fisheries, the staff of the Centre has a long experience in
other fields of the fish farming science and practice as well.
12
Rainbow trout was introduced to Ropsha in the late 19th century. It was
reared on a small scale and was very expensive, rather a luxury food than an
everyday food item. Trout was reared in a practically artisanal way and the
production of portion fish (125-150 g) took as much as 3-4 years.
The establishment of industrial trout culture in Ropsha dates back to the
early 1930s, when the All-Russian Trout Hatchery was opened here. That
decade saw the establishment of the broodstock and the laying of the
foundations of the trout culture in the country. Unfortunately, the ponds were
destroyed and the broodstock was lost during the Second World War, and
therefore, after it ended, everything had to be started almost from scratch.
In 1948, 80,000 rainbow trout eggs were brought to Ropsha from a farm in
Germany, which were then incubated and reared in ponds on a natural diet to
obtain 9,500 fingerlings. A broodstock was created from these fish, which
served as the basis for the development of the country's trout culture: eggs
from Ropsha were supplied to all over the Soviet Union. As a result of a long
selection work with this broodstock, Rofor (short for "Ropshinskaya forel",
i.e. Ropsha trout), one of the two officially registered trout breeds owned by
FCFGS, was developed.
Another breed was developed from the middle of the 1970s on the basis of
steelhead trout, the migratory form of rainbow trout. In the course of the
selection work, family selection methods were widely used along with
progeny selection and inbreeding over several generations. The structure of
the breed includes an outbred and an inbred component. It is called Rostal
(short for "Ropshinskiy stal'nogolovyy", i.e. Ropsha Steelhead).
The breeds were developed against a backdrop of gradually changing
rearing biotechnology. Initially, the entire cycle (except for spawning) took
place in ponds on a natural food base, then earthen raceways and cages
installed in ponds appeared. Application of artificial feeds, first spleen-based
paste feeds, then pellets, already started at that time. Water temperature, the
most important factor for fish culture, showed little variation: since the area
is rich in underground springs, summer temperatures do not rise above 15° C,
while the mean annual temperature is about 7° C.
With the start of the new century, a rapid growth of market trout production
began in Russia, and one of the obstacles to this process was the lack of seed.
In this period, the technical capacities of FCFGS did not allow to produce
more than 1.5 million 1-g fry by the middle of May (the time of peak demand
13
for trout seed). At the same time, the production cost of fry was very high
due to the costs of water preparation (heating, degassing and oxygenation). In
this situation, growout farms preferred to purchase large fingerlings from
Finland. FCFGS had to face the obvious need to fundamentally change its
fish rearing technology.
It was clear from the very beginning that this reconstruction should be
based on water recirculation. First, the temperature of the springwater
fluctuates around 6° C throughout the year and thus, it must be heated for
intensive fry rearing. Second, the discharge of the springs is steadily
decreasing and water has to be used rationally. After studying different RAS
models, a horizontal water circulation type widely used in Denmark was
selected. One of the main arguments in favour of such a system was its
reliability and simplicity of design, which is important in Russia.
In 2008, the Ministry of Agriculture of the Russian Federation provided
financing for this purpose and work started on the reconstruction of the
hatchery unit.
First of all, a small area within the hatchery unit was assigned for the
construction of a module for fry nursing from the start of active feeding to an
average weight of 1 g.
This unit was made of sheet polypropylene and includes 12 tanks, each 9 m
long and 1 m wide. The depth of the water is 80 cm. The water flow,
provided by an airlift pump, is 150 l/s, while the amount of makeup water is
just 1-2 l/s. Water discharged from the tanks passes over a feces collector
where the heavy fractions of the pollutants are deposited. They are removed
by a feces pump daily. Then the water flows into the biological treatment
unit, which includes a fluidized bed filter, an expanded clay filter and low-
pressure aerators, after which it is returned into the tanks by an airlift pump.
The fry is fed by Biomar feeds using belt feeders with a 24-hour clock.
Each tank of the module is stocked with 100,000 fry with an average
weight of 300 mg. Nursing to 1 g is usually done at 16-17° C and takes about
two weeks. Each week, 300-500 thousand fish are stocked into the module
and the same quantity (minus the loss) is transferred for further rearing into
the fingerling rearing module. Thus, under regular operating conditions, the
standing stock of the module is more than 1 million fry, while the fish
biomass is 1 t. It should be emphasized that the quantity of fish grown in a
14
single cycle in a module of slightly more than 200 m2 is as much as was
produced in the entire hatchery unit before reconstruction.
In the next phase of the reconstruction, a fingerling rearing module and a
broodstock module were built in the hatchery unit.
The fingerling rearing module is made of concrete and has 14 tanks with an
area of 2 x 10 m and a depth of 1 m. Its design is identical to the fry nursing
module, the only difference is that the fecal traps are not cleaned by pumps
but the sludge flows into the collector by gravity. An airlift pump circulates
700 litres of water per second, the quantity of makeup water does not exceed
4 l/s. Feeding is done using T-Dram 2000 feeders with pneumatic feed
dispensers.
As already mentioned, 90-100 thousand 1-g fry are stocked into each tank.
Depending on the situation, fish are reared to 5-10 g in this module. The
maximum fish load of a tank can reach 1 t, that of the module as a whole can
be up to 10 t. In winter, 5-g fingerlings are stocked first to adaptation tanks,
then to the wintering module located outdoor in front of the hatchery unit. In
spring, fry from the last propagation sessions is sold to customers directly
from the hatchery. All fish transfers are carried out using fish pumps. After
the seed is sold to growout farms, the module is used for rearing pure-bred
fry and stocking material for selling as fingerlings or yearlings.
Machine sorting of the fish to four size groups is done when the average
weight of the pure-bred fingerlings is between 30-50 g. The larger fish
amounting to 20-30% are left for further rearing, with the exception of
record-size fish, which are transferred to the broodstock module. This latter
one is practically identical to the fingerling rearing unit by its design and
parameters, but it contains two tanks less, and the depth of the tanks is 20 cm
more. The airlift pumps of both modules are powered by one blower with an
engine power of 30 kW and a gauge pressure of 400 mbar.
The open-air module is used for the storage of the fry before selling, as well
as rearing fingerlings and yearlings.
Thus, the technology of fish production on the premises is as follows: 10-12
l/s of spring water with a temperature of 6° C is heated to 16-18° C by an
autonomous gas boiler. The heated water enters two tanks with a volume of
6 m3 each, mounted above the incubation module. For the removal of excess
gases, water in the tanks is aerated. If necessary, the warm water in the tanks
can be mixed with cold water. Up to 700,000 larvae are reared in the
15
incubation module in one cycle. The water used in the incubator is collected
and pumped into the nursing, fingerling rearing and broodstock modules. In
turn, about 8-10 l/s of the water used in these facilities is pumped into the
outdoor module. Due to the supply of rather large volumes of warm water,
the winter temperature in the outdoor module varies around 7oC, never
decreasing below 5oC, even in a strong frost. The wastewater discharged
from the fecal traps of all modules and during the cleaning of the expanded
clay load of the filter is collected in a sedimentation pond. During periods of
maximum load (in April-May, before selling of the fish seed) a water
exchange of 12 l/s allows to rear simultaneously up to one million larvae, 30
tonnes of juvenile fish with average weights ranging from 300 mg to 50 g and
40 tonnes of yearlings with an average weight of 200-300 g.
As a result of introducing the new technology, a substantial change in the
temperature of fish rearing has taken place for the first time in the history of
Ropsha trout breeds: it has become nearly optimal during the entire life cycle.
The temperature is 10-12°C in the period of spawning and egg incubation,
and generally 16-17°С during fry nursing. As a result of such a radical
change in the environmental conditions, specialists faced a new selective
breeding task: to reach better survival and growth rates of the juveniles
during their intensive rearing.
The breeding stock for the new breed was selected from 5-year-old females
of the two best families of the Rostal breed in February-March 2010. The
body weight of the females ranged between 1.6 kg and 3.5 kg (average: 2.3
kg), their working fecundity was between 3,700 and 9,000 eggs (average:
5,500), the weight of one egg was between 40 mg and 80 mg (average: 56
mg). Females whose offspring was selected for further breeding differed little
in terms of their body weight from the average values for the family, while
strongly differed from each other in terms of their working fecundity, as seen
from the data below:
No.of Body weight, kg Fecundity, pcs. Egg weight, mg
female
1 2.0 4450 57.5
2 2.3 6360 58.8
3 2.5 9080 64.1
16
These specific females were selected, in the first place, due to the stably
high viability indices of their offspring.
Males for the paired matings were selected from 4-year-old fish of the
outbred component of the breed. Fish with body weight slightly above the
average, maximum sperm volume (from 11 to 30 ml) and high sperm quality
were used for mating.
A total of 14 pair matings were set up. Families were evaluated according
to their survival and growth rates at different development stages.
Eggs were incubated at 10°C, fish were kept at 11°C. After the larvae
started external feeding, the temperature was raised to 18°C. After reaching
an average weight of 1 g (01.06.2010), five families selected in advance were
stocked into the fry rearing module.
Summer rearing was done at temperatures of 13-15°C (the boiler room was
closed off in summer) and, by October 1, the average weight of fingerlings
differed considerably in the different families, as seen from the below data:
No. of family Quantity, ind. Mean weight, g
8 1180 131
9 1100 166
12 1270 174
13 1240 113
14 2460 113
The total load of the fry rearing module was maintained at a level of 2 t
over the entire summer (pure-bred fry from mass matings was reared in the
free areas). Three full-sib families were reared to maturity.
Males reached maturity at one-year-old age; part of them was discarded and
sold, while the three best families, whose characteristics are shown below (as
of 22.02.2011), were stocked into the broodstock module:
No. of family Quantity, ind. Mean weight, g
8 1050 670
12 720 960
14 1520 700
The females of Family 14 (founder female 3) matured in autumn of the
same year, at the age of 18 months. Their average weight exceeded 1 kg, the
working fecundity was 4200 eggs, the relative fecundity was 2100 eggs/kg,
and the average egg weight was 47.4 mg. It should be noted that such body
17
weight and fecundity values corresponded to those of 3-year-old and 4-year-
old females reared by the old technology, respectively.
The females of the two other families did not mature at this age. Is is
interesting that the females that produced the offspring that matured at the
age of two years and those that produced the offspring that did not mature at
that age (Families 8 and 14) were full sibs, and thus, the difference between
the families in this respect was apparently attributable to the unrelated males.
Taking into account that growout farms may need stocking material with
females reaching maturity at different age (depending on whether they
produce only meat or also eggs), all the three above-described families were
kept for further testing.
The following twelve months of rearing took place in the broodstock
module. After a preliminary evaluation, Family 8 was excluded from further
breeding at the beginning of spawning season. The characteristics of 3-year
old females of Family 12 (matured for the first time) and Family 14 (matured
repeatedly) are shown in Tables I and II.
Table I. Characteristics of 3-year-old females of Family 12

Parameter lim Хave.±mx V,%
Body weight, g 2370 - 5200 4080 ± 114.5 18.2
Body length, cm 50.5 – 69.0 62.6 ± 0.64 6.7
Working fecundity, 2891 - 10712 7824 ± 277.6 23.0
no. of eggs
Relative fecundity, 794 - 2616 1929 ± 54.4 18.3
eggs/kg
Egg weight, mg 46.3 – 80.6 62.1 ± 1.07 11.2
Table II. Characteristics of 3-year-old females of Family 14

Body weight, g 1920 - 4300 3098 ± 85.9 18.6
Body length, cm 51.5 – 64.5 58.5 ± 0.47 5.3
no. of eggs
eggs/kg
Egg weight, mg 51.5 – 84.7 64.2 ± 0.94 9.8
As seen from the data in the tables, the body weight and working fecundity
of the females belonging to the family matured for the first time was 32% and
38% higher, respectively, than in the repeatedly matured family, as expected.
18
The families did not differ in terms of their relative fecundity and the average
egg weight.
Nineteen pair matings were set up at the peak of the spawning season,
including 9 matings within Family 12 and 10 matings within Family 14.
Broodfish were selected according to a standard scheme: first by their body
weight (to be somewhat higher than the average), then females were selected
by their working fecundity (those with maximum values) and egg weight (not
to be below the average), while males were chosen by the volume of their
sperm portion and the concentration of spermatozoa therein (those with
maximum values).
The evaluation of the families of the first inbred generation was also done
according to a scheme proven to be effective, taking into account the survival
of the embryos, larvae and fry, as well as their growth rate. Practically all
families showed survivals higher than the normative values (82 to 97% per
rearing stage); families with lower survival and growth rates were excluded
from further rearing. At an average weight of 2-3 g, mass selection was
carried out in the selected families with a severity coefficient of about 50%.
Fingerlings were reared in the fry rearing module, their characteristics as of
01.09.2013 are shown in Table III.
Table III. Characteristics of the families of the first inbred generation

No. of selected No. of founder N, ind. Mean body
family family weight, g
3 12 3020 170
18 12 4170 120
22 12 1500 160
8 14 1730 140
9 14 2170 180
10 14 2510 190
The studies carried out during the creation of the Rostal breed showed that
careful progeny selection of the breeding stock and choosing the families
with the best adaptability indices (fitness indices), including mainly the
survival rate, growth rate and fecundity, for further rearing make it possible
to reach significant results within some generations, to fix the new traits and
to avoid the negative consequences of inbreeding. At the same time, it is
desirable to have a certain reserve gene pool for later use. For this reason, in
addition to the described work conducted by methods of family selection
19
both breeds (Rofor and Rostal) are also bred using mass selection methods
for increasing their adaptability to the new rearing conditions while
maintaining their initial genetic variability.
The methodology was basically the following: Before spawning, breeders
were selected on the basis of their body weight: fish with a body weight
slightly above average (within 1.5 standard deviations of the mean) were
chosen for mating. The mating itself took place at the peak of spawning. No
less than 30 females and 30 males with maximum working fecundity (sperm
volume) were used for breeding. Fingerlings were selected for body weight
with a moderate severity coefficient (20-30%). After changing to the new
rearing technology, two- and three-year fish started to constitute the core of
the heterogeneous pure-bred broodstock instead of four- and five-year old
individuals, as before.
The average body weight of the RAS-reared 2-year-old females of both
breeds maturing for the first time (Tables IV and V) exceeded 2 kg. The
working fecundity of the Rostal and Rofor breeds was 4300 eggs and 3500
eggs, respectively. These values correspond to the breed standard developed
on the basis of 4-year-old repeatedly maturing females. The relative fecundity
of the Rostal and Rofor breeds was over 2000 eggs and 1700 eggs per kg of
body weight, respectively, which is also very high and corresponds to the
differences between the breeds. The only parameter by which these females
were inferior to the breed standard was the egg weight; however, the embryo
survival during the incubation exceeded the normative 80%.
Table IV.Characteristics of 2-year-old Rostal trout females

Body weight, g 1480 - 3030 2092 ± 79.4 17.8
Body length, cm 47.0 – 54.5 50.5 ± 0.44 4.1
eggs
eggs/kg
Egg weight, mg 37.3 – 52.1 43.0 ± 0.81 9.0
20
Table V.Characteristics of 2-year-old Rofor trout females
Parameter lim Хср.±mx V,%
Body weight, g 1450 - 2630 2139 ± 78.5 14.2
Body length, cm 46.0 – 55.0 51.3 ± 0.69 5.2
eggs
eggs/kg
Egg weight, mg 38.1 – 54.6 46.7 ± 1.10 9.1
Thus, the combining of the technical possibilities of RAS and targeted

selective breeding work, as well as the production of monosex (female)
stocking material and an interbreed cross showing heterosis for viability,
make it possible to set up large-scale production of rainbow trout fry with
good production indices, which is indispensable for winning the competition
for consumers. Only the use of recirculation allows to minimize the
production cost of the stocking material categories that are in the highest
demand in market fish production: fry with an average weight of 5-10 g,
yearlings of 100-300 g in May and fingerlings of 50-70 g in October.
21
Nitrite-induced methemoglobinemia of freshwater fishes reared in
recirculating aquaculture systems
Khuda,L.V., Khudyy,A. I.
Yuriy Fedkovych Chernivtsi National University, Chernivtsi, Ukraine
Abstract
The methemoglobin content and the functioning of the methemoglobin reduction system
were studied in the erythrocytes of freshwater fishes under conditions of nitrite intoxication.
It was demonstrated that, under NaNO2concentrations ranging between 7.25 mmol/l and
217.5 mmol/l, non-fermentative mechanisms with the participation of low-molecular-weight
compounds such as reduced glutathione and ascorbate become of primary importance in
methemoglobin reduction.
Keywords: nitrites, methemoglobin, glutathione, ascorbic acid.
Introduction
Among the principal factors of water quality in recirculating aquaculture
systems (RAS), nitrogen indices require special attention and constant
monitoring. It is well-known that nitrogen is present in aquaculture systems
in the form of ammonia (NH3), ammonium ions (NH4+), nitrites (NO2-) and
nitrates (NO3-). Nitrogen compounds in the aquatic environment are partly
supplied by the vital functions of the cultured species, as ammonium is the
main product of protein metabolism in fish. In addition, feeds with high
protein content, which are used in intensive fish rearing in RAS, act as
additional nitrogen sources in the aquatic environment.
A well-tuned operation of biofilters is an efficient way of controlling the
levels of ammonium and nitrites, which are highly toxic to fish. The two-
stage nitrification process whose first stage involves the conversion, by
bacteria of the genus Nitrosomonas, of ammonium to nitrites, which are
further oxidized to nitrates through the activity of Nitrobacter, another group
of nitrifying bacteria, takes place in the biofilters. As the first stage of
ammonium oxidation to nitrite yields significantly more energy than the
chemical reaction of nitrite oxidation to nitrate, the microflora responsible for
the first nitrification stage grows much faster. Thus, nitrite accumulation in
the environment becomes possible, which occurs especially frequently in the
22
first 4–8 weeks of the startup of a biofilter. In addition, even a minor oxygen
shortage can result in an imbalance of nitrification processes with the
subsequent possibility of ammonium-nitrate poisoning of fish, especially
under the conditions ofincreased stocking densities in RAS (Masser, 1999).
An increased production of methemoglobin (MtHb) in erythrocytes is the
main manifestation of nitrite intoxication. Hemoglobin transformed into
methemoglobin by the turning of the heme iron into an Fe3+ form loses its
main function of transporting oxygen, leading to the development of hemic
hypoxia (Raja and Sapkal, 2011). The resistance of the hemoglobin of fishes
to oxidation is low, and thus, their MtHb level can change in a wide range
even under physiological conditions (Soldatov, 2002). The lack of visible
symptoms of poisoning is explained by the efficient functioning of a multi-
component methemoglobin reductase system. Glutathione and ascorbic acid
are involved in the non-fermentative reduction of methemoglobin. Yet, the
main factor controlling the level of hemoglobin oxigenation and
deoxigenation is the activity of the NADH-dependent methemoglobin
reductase enzyme (NADH-Н cytochrome-b5 reductase, EC number: 1.6.2.2.),
which specifically transfers electrons from NADH through cytochrome-b5 to
methemoglobin. However, the activity of the system of methemoglobin
reduction to hemoglobime can be blocked if nitrite concentrations are high.
The objective of this research was to determine the effect of nitrite
intoxication on the methemoglobin content and the functioning of the
methemoglobin reduction system in the erythrocytes of freshwater fishes.
Material and methods

Isolated erythrocytes of Carassius gibelio (Bloch), Cyprinus carpio L.,
Hypophthalmichthys molitrix (Valenciennes) and Acipenser ruthenus L. were
used to model the effect of the nitrites. Blood was taken from the dorsal aorta
using heparine as an anticoagulant. The erythrocytes were separated from the
plasma by centrifugation at 500g and washed three times in Ringer’s
solution. The separated erythrocytes were divided into 6 groups: a control
and 5 treatment groups incubated in Ringer’s solution with the following
NaNO2 concentrations: 7.25 mmol/l (Group І), 14.5 mmol/l (Group ІІ), 72.5
mmol/l (Group ІІІ), 145.0 mmol/l (Group IV) and 217.5 mmol/l (Group V). It
is known that the median lethal dose of nitrite ions in water is 1,45 mmol/l
for several freshwater fishes (Alexander et al., 2009; Svobodova et al., 2000).
23
Considering that a ten-fold accumulation of nitrite ions in the blood plasma is
typical for fish (Kroupova et al., 2006), the NaNO2 concentrations in the
incubation medium of the erythrocytes were increased accordingly.
The methemoglobin content was measured spectrophotometrically using
the acetone cyanohidrin method (Ministry of Agriculture and Food of the
Russian Federation, 1999). The activity of methemoglobin reductase was
determined on the basis of the rate of methemoglobin reduction in the
presence of NADH. The reduced ascorbic acid content was determined from
the difference between the quantities of all ascorbate forms and the sum of
the dehydroascorbic and diketogulonic acids (Goryachkovskiy, 2005). The
reduced glutathione content was determined from its reaction with 5,5′-
dithio-bis(2-nitrobenzoic acid) (Rakhmanov et al., 2009). The total protein
content and the hemoglobin content were determined by the Lowry method
and the hemoglobin cyanide method, respectively (Ministry of Agriculture
and Food of the Russian Federation, 1999).
Results and discussion

The conducted research showed that the incubation of erythrocytes in
media with increasing NaNO2 concentrations resulted in an increase of the
hemoglobin content in comparison with the control (Figure 1).
% C. gibelio
90 C. carpio
H. molitrix
80
A. ruthenus
70
60
50
40
30
20
10
0
K I II III IV V
Figure 1. Methemoglobin content in the erythrocytes of freshwater fishes at different NaNO2

concentrations
Notes (hereinafter): К – control group, І – 7.25 mmol/l NaNO2, ІІ – 14.5 mmol/l, III – 72.5
mmol/l, IV – 145.0 mmol/l, V – 217.5 mmol/l.
24
It should be noted that the erythrocytes of all studied fish species react to
NaNO2 concentrations close to median lethal doses (Groups II and III)
virtually identically. Specific differences can be observed when applying
lower (Group I) or much higher nitrite concentrations (Groups IV and V). As
seen in the figure, the most pronounced reaction to the increasing NaNO2
concentration is shown by silver carp erythrocytes.
Taxonomical differences in the character of methemoglobin accumulation
have been demonstrated by several authors (Alexander et al., 2009;
Svobodova et al.; 2000; Saleh and McConkey, 2012). It may be assumed that
the observed species-specific differences in methemoglobin accumulation are
based on functional characteristics of the methemoglobin reductase systems
of the erythrocytes, which ensure the reduction of methemoglobin.
Under physiological conditions, the main component of the methemoglobin
reductase system in the erythrocytes of all vertebrates, including fish, is the
NADH-Н cytochrome-b5 reductase (NADH-methemoglobin reductase),
which ensures the transformation of 70-90% of MtHb back to hemoglobin
(Saleh and McConkey, 2012; Prodanchuk and Balan, 2007). Yet, as was
demonstrated by the authors before, the activity of methemoglobin reductase
remained unchanged (sterlet erythrocytes) or decreased (Prussian carp
erythrocytes) with the growing methemoglobin concentration in all the used
variations of toxic nitrite concentrations (Khuda et al., 2012а; Khuda et al.,
2012b). The interaction of nitrites with the iron ion of the heme in the
hemoglobin allows to hypothesize the possibility of similar interactions with
other heme-containing proteins, in particular, catalase and cytochrome-b5 as a
component of methemoglobin reductase (Moraes et al., 2002).
It is highly probable that, at high nitrite concentrations, the MtHb-reducing
function is mainly assumed by low-molecular-weight components of the
system: reduced glutathione and ascorbic acid.
The process of hemoglobin oxidation is known to generate active
compounds and free radicals: superoxide anion radical, NO, ONOO-, Н2O2,
etc. Glutathione, whose redox system (GSH-GSSG) acts as a buffer
protecting against the destructive effects of active oxygen forms, significantly
contributes to the antioxidant potential of erythrocytes.
A mild oxidation of the sulfhydryl groups of reduced glutathione protects
the SH groups in the hemoglobin and several proteins and enzymes of the
erythrocytes from free-radical oxidation. Thus, the possibility of direct
25
reduction of methemoglobin and the antioxidant properties of GSH determine
its important role in the system of maintaining the hemoglobin structure and
function. High GSH concentration was demonstrated to be characteristic of
fish erythrocytes. They have a much higher GSH/Hb ratio than mammals
(Soldatov, 2002). The reduced glutathione content in fish can change within a
wide range (reaching levels of up to 10 mmol/mg protein in some fish),
which is explained by the significant adaptive capacity of the erythrocytes of
aquatic animals due to their existence in the changing conditions of the
aquatic environment.
The results of the conducted studies showed that the level of reduced
glutathion stayed within the above limits in all studied species. E.g. the GSH
content in the erythrocytes of Prussian carp and silver carp was similar,
around 0.3 mmol/mg protein, while for sterlet and common carp, the
respective values were 0.1 mmol/mg protein and 0.85 mmol/mg protein,
respectively (Figure 2).
C. gibelio
1 C. carpio
0.9 H. molitrix
A. ruthenus
0.8
0.7
mmol/mg protein
0.6
0.5
0.4
0.3
0.2
0.1
0
K I II III IV V
Figure 2. Reduced glutathione content in the erythrocytes of freshwater fishes at different
NaNO2 concentrations
A clear tendency toward the decrease of the reduced glutathione level in the
erythrocytes of common carp and Prussian carp was observed in the first four
experimental groups as compared with the control. The obtained results
indicate an active oxidation of glutathione and, accordingly, its role in the
defense processes against antioxidants as well as in methemoglobin reduction
in the erythrocytes in the presence of nitrites. It should be noted that the GSH
26
content in the erythrocytes of all studied fish species did not differ from the
control when incubated with the highest used NaNO2 concentration (217.5
mmol/l). As regards silver carp and sterlet, decreased GSH levels were
observed only in the experimental Groups ІІІ and ІV. It is probable that the
high glutathione concentrations in the erythrocytes of Groups I and II ensure
an active operation of the ascorbate redox system, as the reduction of
dehydroascorbic to ascorbic acid is a sufficiently fast process in the presence
of sulfhydryl compounds such as glutathione or cysteine.
In order to test this hypothesis and to evaluate the role of ascorbic acid in
the erythrocítes during nitrite intoxication, the content of reduced ascorbate
was studied under the specified conditions.
The obtained results indicated a significantly lower concentration of
ascorbic acid in sterlet erythrocytes than in other studied fishes (Figure 3).
In contrast with humans, primates and some mammals (guinea pig), which
lack two enzymes (D-glucuronate reductaseand L-gulono-γ-lactone oxidase)
ensuring the synthesis of ascorbic acid from glucose, cyprinids can
synthesize ascorbic acid. However, literature sources contain some doubtful
data on the activity of gulono-lactone-oxidase, the last enzyme of the
ascorbate biosynthesis pathway, in sturgeons (Gy. Papp et al., 1995;Verlhac
and Gabaudan, 2010). Although some authors demonstrated the activity of
that enzyme and, accordingly, the possibility of ascorbate synthesis in some
sturgeons, there are facts suggesting the necessity of additional
supplementation of C-vitamin from external sources.
C. gibelio
80 C. carpio
H. molitrix
70 A. ruthenus
60
50
µmol/ml
40
30
20
10
0
K I II III IV V
Figure 3. Reduced ascorbate content in the erythrocytes of freshwater fishes at different
NaNO2 concentrations
27
The studies showed a decreased content of reduced ascorbate in sterlet
erythrocytes at all used concentrations, with a cca. 1.5-fold decrease observed
already at the lowest concentration. It is obviously related to the
transformation of reduced ascorbic acid into the form of dehydroascorbic
acid after having been used as a reduced agent and an antioxidant.
It should be noted that a significant decrease of the ascorbic acid content
was characteristic of all the studied fish species when their erythrocytes were
incubated an nitrite concentrations close to LC50. Thus, in addition to the
well-known role of ascorbic acid as an important component of the
antioxidant defense of erythrocytes, its participation in methemoglobin
reduction and defense from the toxic effects of nitrites should also be noted.
Accordingly, a sufficient nutritional ascorbate uptake is absolutely essential
during fish rearing in RAS.
References
Alexander, J., Benford, D., Cookburn, A., 2009. Nitrites as undesirable

substances in animal feed. The EFSA Journal, 1017:1–47.
Goryachkovskiy, А.М., 2005. Clinical biochemistry in laboratory
diagnostics. Odessa, Ekologiya, pp. 616. (In Russian)
Gy.Papp, Zs., Jeney, Zs., Jeney, G., 1995. Comparative studies on the
effect of vitamin C feeding of European catfish (Silurus glanis L.) and
sturgeon hybrid (Acipenser ruthenus L.×Acipenser baeri L.). J. Appl.
Ichthyol., 11: 372–374.
Khuda, L.V., Khudyy, О.І., Khachman, Ya.Yu., 2012а. Nitrite-induced
methemoglobin accumulation in sterlet erythrocytes. Actual problems of
theoretical and practical ichthyology. Proceedings of the VInternational
scientific and practical conference of ichthyology dedicated to the memory
of I.D. Shnarevych (Chernivtsi, 13-16 September 2012). Chernivtsi,
Knigi-ХХІ, pp. 241–243. (In Ukrainian)
Khuda, L.V., Marchenko, М.М.,Khachman,Ya.Yu., Khudyy, О.І.,
2012b. Effect of nitrite intoxication on the methemoglobin reduction
system in erythrocytes of Prussian carp. Biologichni systemy, 4 (4): 393–
396. (In Ukrainian)
Kroupova, H., Machava, J., Piackova, V., 2006. Nitrite intoxication of
cоmmоn carp (Cyprinus carpio L.) at different water temperatures. Acta
Vet. Brno. 75: 561–569.
28
Ministry of Agriculture and Food of the Russian Federation, 1999.
Methodological indications on conducting hematological studies on fish.
Minselkhozprod RF, no. 13-4-2-/1487, pp. 20. (In Russian)
Moraes, G., Avilez, I.M., Altran, A.E., 2002. Biochemical effects of
environmental nitrite in matrinxa (Brycon cephalus). Aquatic Toxicology:
Mechanisms and Consequences. International Congress on the Biology of
Fish. Symposium Proceedings. Vancouver, p. 15-26.
Prodanchuk, G.N., Balan, G.M., 2007. Toxic methemoglobinemias:
mechanisms of development and ways of optimizing the treatment.
Sovremennye problemy toxikologii, 1: 37–45. (In Russian)
Raja, I.A. Sapkal, H.P., 2011. Blood and electrolyte responses in Clarias
batrachus exposed to nitrogen pollution. Biosci. Biotech. Res. Comm, 4
(2): 219–222.
Rakhmanova, T.I., Matasova, L.V., Semenikhina, A.V., Safonova, O.A.,
Makeeva, A.V., Popova, T.N. (eds.), 2009. Methods of evaluating the
oxidative status. Voronezh State University, pp. 62. (In Russian)
Saleh, M.C., McConkey, S., 2012. NADH-dependent cytochrome b5
reductase and NADPH methemoglobin reductase activity in the
erythrocytes of Oncorhynchus mykiss. Fish Physiol. Biochem., 38 (6):
1807–1813.
Soldatov, А.А., 2002. Specific features of the structure, polymorphism ad
resistance to oxidation of fish hemoglobins. Zhurnal evolyutsionnoy
biokhimii i fiziologii, 38 (4): 305–308. (In Russian)
Svobodova, Z., Machova, J., Poleszczuk, G., 2000. Nitrite poisoning of fish
in aquaculture facilities with water-recirculating system. Acta Vet. Brno.,
74: 129–137.
Verlhac, V., Gabaudan, J., 2010. The effect of vitamin C on fish health.
Centre for Research in Animal Nutrition, Saint-Louis Cedex, pp. 35.
29
Application of recirculating aquaculture systems (RAS) in Polish
sturgeon culture
Kolman, R., Zdanowski, B.
The Stanisław Sakowicz Inland Fisheries Institute, Olsztyn-Kortowo, Poland

Abstract
The development of sturgeon farming in Poland is closely related to recirculating

aquaculture systems (RAS). The most critical stages of sturgeon rearing, i.e. artificial
propagation and seed production, take place in RAS. The application of RAS during the
development of female stocks for caviar production is also quite justified economically in
Poland. Finally, recirculating aquaculture systems are used in the ichthyological work related
to the project currently conducted in Poland on the rehabilitation of the Baltic population of
Atlantic sturgeon.
Keywords: RAS, sturgeons, artificial propagation, rearing, egg collection
Introduction
The history of recirculating aquaculture systems in Poland goes back to the
first half of the 1970s, when the Inland Fisheries Institute started
experimental work on the development of efficient systems for seed
production of autumn-spawning trout (Kolman 1978; Kolman 1989а). The
technological launching of the first recirculating aquaculture unit for
industrial rearing of trout fry took place in 1976 (Kolman 1989b). By the time
work on sturgeons started in Poland, i.e. late 1980s, several RAS for rearing
of larvae and fry of different fish species had been in operation in our
fisheries. This accelerated and facilitated the work on development of
intensive rearing technologies of juvenile stages of sturgeons (Kolman 1993;
Kolman i Szczepkowski 1995).
The first batch of fertilized eggs of sturgeons: Russian sturgeon Acipenser
gueldenstadti and bester Huso huso x Acipenser ruthenus, was imported to
Poland from the Soviet Union in 1989. It was placed into Weiss jars in the
recirculating hatchery of the Dgał Experimental Centre of the Inland
Fisheries Institute, situated in Pieczarki, near Giżycko. RAS units for egg
incubation, rearing of larvae and fry, as well as rearing of the broodstock and
keeping of the breeders before artificial propagation had been constructed in
this centre inaugurated in 1986. The Dgał Centre developed first the
30
biotechnology of intensive rearing of juvenile stages of sturgeons, then, as
they grew and matured, that of broodstock development, and finally, the
technology of their artificial propagation in RAS conditions (Kolman et
al.1998; Kolman et al. 1999).
Fields of RAS application in Polish sturgeon culture

The aquaculture of sturgeons has three main directions (objectives):
 obtaining fertilized eggs and rearing seed for stocking into natural
waters or on-growing in aquaculture;
 intensive rearing of table fish;
 developing and using all-female stocks for caviar production.
A full-cycle rearing of sturgeons requires an adequate site providing
optimum conditions for the growth of individual development stages of these
fish. As shown by the experience of intensive sturgeon culture in Poland, the
main production of market-size sturgeon can be done in raceways with
natural thermal regime. The production cycle in raceways can be reduced to 2
years if stocking is done in mid-May and the average weight of the stocking
material is more than 10 g (Figure 1) (Kolman 1999).
2500
2000
Average weight [g]
1500
1000
500
0
0 100 200 300 400 500 600 700 800
Days of rearing
Figure 1. Rearing cycle of market-size Siberian sturgeon in flow-through raceways for trout
The changes of average weight shown in the figure were determined on the
basis of the results of rearing Siberian sturgeon Acipenser baerii in a trout
farm of the northern part of Poland. The average weight of the fish had
exceeded 400 g by the end of the first rearing season. During the wintering,
which can last up to 6 months in Poland, the body weight of the fish
decreased by 12%. By the end of the next season, the average weight of the
31
sturgeon had reached about 1500 g, i.е. some of the faster-growing fish
weighing about 2.0 kg could be sold already, while the rest reached a
marketable size after wintering and 2 to 3 months of intensive on-growing. In
connection with this, an early spawning with subsequent incubation and
larval rearing is advisable. As the period suitable for early spawning is
characterized by natural water temperatures significantly lower than required
for either the final maturation of sturgeon broodfish or the development and
growth of embryos and larvae, the artificial propagation of sturgeons and the
rearing of the fry should be done in fully controlled environmental
conditions, which are provided by recirculating aquaculture systems (RAS).
The optimal values of environmental parameters along with adequate feeding
guarantee high survival and growth rates of the sturgeon larvae and fry
(Kolman et al. 1996). The biotechnology of intensive seed rearing in RAS,
which has been developed in Poland, allows to produce Siberian sturgeon and
hybrid fry with an average weight of about 10 g within 55 to 60 days from
the date of hatching (Figure 2).
16
14
Average weight [g]
12
10
8
6
4
2
0
35 40 45 50 55 60
Days of rearing
Figure 2. Growth rate of Siberian sturgeon fry in RAS tanks
The composition of individual technological lines for seed production

depends on their function, which does not only apply to fish rearing devices,
but also to water treatment equipment. This is related to the character of
changes in water quality caused by certain technological production
processes. For this reason, a sturgeon seed rearing centre should consist of
fully independent technological lines situated in separate units.
32
Egg collection and fry rearing of sturgeons
As mentioned above, these biotechnological stages of sturgeon rearing
should take place entirely in RAS.
Broodfish can already be selected for propagation in autumn, after the
natural water temperature decreases below 10-12oС. In vivo methods of
evaluating gonad maturity are generally used for this purpose, such as
ultrasound diagnostics (Figure 3) or biopsy (Figure 4).
Figure 3. Studying the maturity stage of a sturgeon female by ultrasound diagnostics.
Figure 4. Studying the maturity stage of a sturgeon female using a probe.
33
The selected fish, i.e. mature males and females with PI (polarity index) of
the oocytes below 0.15, were moved to the tanks of the broodfish keeping
unit at least two weeks before the planned date of artificial propagation. The
unit is designed for short-term keeping of the broodfish, during which, the
thermal and hormonal stimulation of their final maturation and, finally, the
collection of their gametes takes place. The unit generally has at least two
tanks for separate keeping of the males and females. Rotationally molded
tanks with a diameter of about 3.0 m and an effective depth of about 1.0 m
have performed well (Figure 5).
Figure 5. Rotationally moulded tanks in the broodfish keeping unit.
As adult sturgeon are characterized by a relatively low intensity of

metabolism, moreover, they are not fed during conditioning, the water
treatment system does not need to be highly efficient. Oxygen loss in the
circulating water can be fully compensated by aeration, while the small
quantities of ammonia excreted by the fish can be removed by a simple
diatomaceous earth filter. However, the unit must be equipped with an
efficient system for full control of the temperature, allowing to provide the
required water temperature in the preset regime. This is especially important
in the first period after stocking the broodfish into the tanks, as the rate of
increase of the water temperature may not exceed 1оСh-1.
After conditioning of the broodfish at spawning temperatures, i.e. 12–18оС
depending on the species, hormonal stimulation of the last oocyte maturation
stage and ovulation can start in 10 to 12 days. Injections of a suspension of
34
common carp hypophysis or synthetic GnRH analogues are generally used in
Poland for this purpose (Kolman 2006).
Eggs are collected with the Podushka (1999) method, i.е. by cutting the
oviducts (Figure 6). The stickiness of the fertilized eggs is eliminated by a
tannin solution, after which, they are moved into incubation jars of the
hatchery. After colection of the gametes, the water temperature in the RAS
should be gradually decreased to a value close to that outside. Fish are then
moved outside for wintering and, for the next interspawning interval, are
stocked to ponds with a natural water temperature regime.
Figure 6. Egg collection with the Podushka (1999) method.
Egg incubation unit

Sturgeon eggs are incubated in Weiss or McDonald jars in Poland (Figure
7). The latter are especially suited for the purpose, as their construction
prevents the aggregation of the eggs and their attachment to the walls of the
jar, which otherwise often happens in the first incubation period.
The developing embryos are in contact with the environment through the
semi-permeable egg membranes. The toxic products excreted during
metabolic processes are removed from the oocytes by osmosis. Oxygen
uptake by the eggs is made possible by osmosis, too. In relation to this, the
water flowing through the incubation jars must be free of toxic metabolites:
ammoniaandcarbondioxide, while its oxygen saturation must be over 70%.
Therefore, special attention should be paid to good aeration of the water in
recirculating aquaculture systems designed for egg incubation, while a
35
submerged diatomaceous earth filter adsorbing ammonia should be sufficient
for water treatment. The hatched larvae are caught in hapas placed in the
receiving tanks, from where they are gradually transferred to larval and fry
rearing tanks.
Figure 7. Russian sturgeon eggs in McDonald jars
Larval and fry rearing unit

The biotechnology applied in Poland involves a two-stage rearing process
of sturgeon seed:
 from the start of active feeding to an average weight of about 0.5 g;
 from an average weight of 0.5 gto an average weight of 10-20 g.
This separation of the larval rearing and fry rearing stages is mainly
justified by the behaviour and feeding of fish, in particular, by the digestion
physiology (Żółtowska et al. 2003). In the initial rearing period, the larvae are
very sensitive to the feed quality and the feeding method. The tank bottoms
should be frequently cleaned in this period to remove the sediment consisting
of uneaten feeds and excrements. In the next stage, after shifting to industrial
high-protein feeds, feeding issues become less complicated and the fry clean
the tank bottom themselves. Therefore, the seed rearing unit should be
divided into two independent recirculating aquaculture systems (Figure 8).
The RAS where the first rearing stage takes place should be equipped with
36
troughs or rotationally moulded tanks with diameters of up to 2.0 mand water
depths adjustable between 0.2 and 0.5 m. For the second stage, rotationally
moulded tanks with diameters of 2.0–3.0 m and water depths adjustable
between 0.5 and 0.8 m are the most convenient.
Figure 8. RAS in the sturgeon larval and fry rearing unit
The juvenile development stages of fishes and, in particular, sturgeons, are

very sensitive to the quality of the aquatic environment. In addition, they are
characterized by high metabolic rates, and, as a consequence, high oxygen
consumption and high specific amounts of excreted ammonia. The dynamics
of the changes in metabolic rate as a function of fish size is demonstrated by
the data of Table 1: sturgeon fry with a mean weight of 0.8 g and sturgeons
with a mean weight of 450 g consume an average of 709.1 mg and 177.7 mg
О2 kg-1 h-1 and excrete 67.7 mg and 1.9 mg NH4 kg-1 h-1, respectively.
Therefore, the water treatment system should be very efficient in the sturgeon
larval and fry rearing unit. This mainly applies to ammonia removal and
oxygenation facilities.
37
Table I. Oxygen consumption and ammonia excretion by Siberian sturgeon (after
Szczepkowski et al. 2000)
Weight Average oxygen Average ammonia

of fish consumption excretion
(g) [mg kg-1 h-1] [mg kg-1 h-1]
0.8 709.1 ± 195.3 67.7 ± 17.1
1.2 756.2 ± 183.9 61.7 ± 18.3
3.4 508.6 ± 93.1 34.3 ± 12.6
5.9 598.6 ± 109.4 58.2 ± 8.8
11.9 660.9 ± 64.0 36.3 ± 4.4
21.8 465.3 ± 46.1 35.3 ± 3.8
30.1 403.7 ± 75.8 34.7 ± 7.5
73.4 279.7 ± 50.1 23.0 ± 4.5
104.4 227.9 ± 15.0 3.5 ± 2.4
167.2 186.7 ± 6.4 3.5 ± 1.5
222.4 154.2 ± 12.0 3.1 ± 0.7
341.8 173.2 ± 7.6 1.7 ± 0.7
449.0 177.7 ± 7.8 1.9 ± 0.7
The main RAS functions of incubation and larval and fry rearingare
ensured by the systems of water circulation, thermoregulation and water
preparation, including water treatment. The first consists of appropriately
selected pumps, tubes and retention tanks (Figure 9). Moreover, it must
combine lifting of the water with pumps with its gravitational flow. This
allows to equalize the partial pressure of water-dissolved gases, thus
preventing the development of gas bubble disease in the earliest development
stages of sturgeons. In addition, such a circulation system allows to precisely
regulate the velocity of water flow in the technological facilities.
The water preparation in recirculation systems for incubation and fry
rearing should mainly include the removal of toxic water-dissolved products
of sturgeon metabolism (mostly ammonia and carbon dioxide) and suspended
matter (digested and undigested feed residues), as well as water oxygenation
and disinfection.
38
Figure 9. Scheme of a RAS for rearing of sturgeon fry.
1– fish rearing tanks; 2 – microscreen drum filter; 2’- sedimentation tank; 3 – lower retention
tank; 4– fluidized-bed biofilter; 5 - oxygenator;
Removal of ammonia
Ammonia is the end product of both protein metabolism in the fish
organism and the mineralization of protein compounds in the feed residues.
The studies of many authors indicate the high toxicity of ammonia gas to
fish. Ammonia is mostly present in water in a dissociated form and only a
small part of it remains in the gas phase. As both our own experience and
literature data show, sturgeons and salmonids are the most sensitive to
ammonia. A negative effect on larvae was already observed at a
concentration of 0.005 mg NH3l-1 (Kolman 1992).
The most efficient and most frequently applied method of ammonia
removal is through biological nitrification. The nitrification process goes on
very well in moderately loaded biofilters.
Fluidized-bed biofilters are used both in intensive seed production and in
table fish production. The external frame of the biofilter has a cylindrical
shape ending in a cone. Inside, there is a centrally located cylinder filled with
polyethylene chips with a specific weight slightly lower than that of water,
whish serve as a substrate for biofilm development. A continuous water flow
mixes the media, preventing an excessive growth of the biofilm. The excess
biofilm and the suspended organic matter (the remains of the digested and
39
undigested feeds) settle in the lower part of the conical section of the
biofilter, from where they are periodically removed.
Water oxygenation
Both the metabolic oxygen consumption by the fast-growing fish and the
oxygen consumption by the processes of mineralization and nitrification are
very high in a intensive fry-rearing RAS. In addition, juvenile stages of
sturgeons are very sensitive to low oxygen contents in the water. In
connection with this, the methods of oxygen enrichment of the water used in
such systems should be efficient enough. Therefore, devices using pure
oxygen need to be applied. Different types of contact chambers are used for
adding oxygen to water (Kolman 2010).
Development and use of all-female stocks for caviar production

The above-mentioned aspects of RAS application for propagation and seed
production of sturgeons do not include all the possible uses of recirculating
aquaculture systems in sturgeon culture. The experimental work performed in
Poland in the end of the 20th century and the computer simulations based on
them showed that the development of all-female aquaculture stocks for caviar
production makes RAS rearing of both table fish and broodfish economically
feasible. This direction of sturgeon culture has developed very dynamically
in the last decade, and currently, the volumes of caviar production in
aquaculture have reached the maximum level of production of this luxury
food item obtainable from natural sturgeon populations (Bronzi 2012).
Poland has also joined the countries producing caviar from aquaculture-
reared females. The using of RAS technology allows to significantly reduce
the time required for obtaining mature females. In Siberian sturgeon, А.
baeri, or in bester, eggs can be first obtained from the broodfish at the age of
6–7 years. The shortening of the production cycle needed for the
development of the female broodstock results in a significant cost reduction.
Egg collection for caviar production is done in Poland without sacrificing
the fish, with the Podushka (1999) method. After egg collection the fish are
placed into flow-through earthen ponds with natural water temperature.
Similarly to egg collection for propagation purposes, females are selected in
autumn and the selected mature females are kept at temperatures lower than
40
those required for spawning. From this stock, groups are chosen whose size is
limited by the size of the broodfish keeping unitand the caviar processing
capacity, which are then stocked into RAS tanks. The further procedure is the
same as in the case of the stimulation of artificial propagation, described
before. As a result of the application of recirculation technologies with full
temperature control, i.е. water heating and cooling, the period of egg
collection can be extended to several months, allowing to optimize the
organization of this process while limiting technological costs.
According to the technology developed in Poland, recirculating aquaculture
systems for table fish and broodfish rearing are equipped with round tanks
with diameters of 6–8 mand a depth of 1.5 m, which, based on the
observations, provide good conditions for the sturgeons, contributing to
better growth rates and faster maturation (Figure 9).
Conclusion
In conclusion, it should be noted that such a rapid and efficient growth of
market sturgeon production would have been impossible without using RAS.
In addition, the developed technologies of full-cycle intensive sturgeon
culture have paved the way for a new direction of sturgeon culture in Poland,
i.е. restitution of Baltic sturgeon populations (Kolman et al. 2008, Kolman et
al. 2011).The work in this direction started in 2004. The starting material for
the ichthyological research consisted in fertilized eggs of Atlantic sturgeon
Acipenser oxyrinchus from the Saint John River. According to the results of
genetic studies, the same species recently inhabited the Baltic Sea as well
(Ludwig et al. 2002; Stankovič et al. 2007). New, purpose-made RAS are
used for producing seed which is then stocked into tributaries of the Oder and
Vistula, аs well as for rearing broodstocks from which broodfish will be
selected for propagationin the coming years (Kolman et al. 2013). Thus, it
can be stated that the use of recirculating aquaculture systems in Polish
sturgeon culture has also become a means for active protection of the extinct
population of the most valuable Baltic species – Atlantic sturgeon.
41
References
Kolman R., 1978. Podchów wylęgu pstrąga jesiennego tarła w układzie

zamkniętego obiegu wody. Gosp. ryb. 7: 12-15.
Kolman R., 1989a. Badania modelowe przydatności zamkniętego obiegu
wody do podchowu wylęgu ryb. Roczn. Nauk Roln. seria H, 102, 1: 55-
70
Kolman R., 1989b. Podchów wylęgu pstrąga jesiennego tarła w
podchowalni ryb Ośrodka Zarybieniowego w Młynowie. Roczn. Nauk
Roln. seria H, 102, 1: 88-107.
Kolman R., 1992. Efektywność biologicznego filtru półkowego
zastosowanego do uzdatniania wody w systemie recyrkulacyjnym przy
wychowie pstrąga. Archiwum Rybactwa Polskiego. Vol.1, supl.1, s.37.
Kolman R. 1993. Wyniki intensywnego chowu wylęgu i narybku bestera w
warunkach zamkniętego obiegu wody. Kom. Ryb. nr. 5, s. 10-13.
Kolman R., 2006. Rozród ryb jesiotrowatych. P.A.U.. Prace Komisji Nauk
Rolniczych, Leśnych i Weterynaryjnych. 7: 23-30.
Kolman R., 2010. Jesiotry chów i hodowla – Poradnik hodowcy. Wydanie II
uzupełnione. Wydawnictwo IRS.: 134s.
Kolman R., M. Szczepkowski. 1995. Badania eksploatacyjne obiegu
zamkniętego z biologicznym złożem fluidalnym. Kom.Ryb.s: 23-25.
Kolman R., Stanny A., Szczepkowski M., 1996. Comparison of the effects
of rearing sturgeon fry using various starters. Arch. Ryb. Pol. V.4, F.1.,
45-56.
Kolman R., B. Szczepkowska, M. Szczepkowski, 1998. Dojrzewanie ryb
jesiotrowatych w DOZ “Dgał”. Kom. Ryb. 5, s: 9-11.
Kolman R. M. Szczepkowski, B. Szczepkowska, 1999. Podchów wylęgu
jesiotra na paszy sztucznej i mieszanej. Kom. Ryb. 1, s: 10-12.
Kolman R., Kapusta A., Szczepkowski M., Duda A., Bogacka-Kapusta
E., 2008. Jesiotr bałtycki Acipenser oxyrhynchus oxyrhynchus Mitchill.
Wyd. IRS.: 73.
Kolman R., Kapusta A., Duda A., Wiszniewski G., 2011. Review of the
current status of the Atlantic sturgeon Acipenser oxyrinchus oxyrinchus
Mitchill 1815, in Poland: principles, previous experience, and results. J.
Appl. Ichtyol. 27:186-191.
Ludwig A., Debus L., Lieckfeld D., Wirigin I., Benecke N., Jenneckens I.,
Willot P., Waldmann J.R., Pitra C. 2002. When the American sea
sturgeon swam east – Nature. 493: 447-448.
Podushka S.B., 1999. Poluchenie ikry u osetrovykh s sokhraneniem zhizni
proizvoditeley. Naychno-Tekhnicheskij Byuleten’ Laboratorii Ikhtiologii
INENKO. St. Peterburg, vyp.2: 4-19.
42
Stankovič A., Panagiotopoulou H., Węgleński P., Popovič D. 2007.
Badania genetyczne nad jesiotrem w związku z programem jego restytucji
w wodach Polski. W: Restytucja jesiotra bałtyckiego. Ryszard Kolman
(Red). Wyd. IRS. Olsztyn. P. 21-26.
Szczepkowski M., B. Szczepkowska, R. Kolman, 2000. Comparison of
oxygen consumption and ammonia excretion by Siberian sturgeon
(Acipenser baeri Brandt) and its hybrid whith green sturgeon (Acipenser
medirostris Ayres). Arch. Ryb. Pol. 8,2: 205-212.
43
Development of industrial fish culture in Belarus
Kostousov, V.G.1, Barulin, N.V.2

1
Institute of Fisheries, Minsk, Republic of Belarus
2
Belarussian State Agricultural Academy, Gorki, Republic of Belarus
Abstract
The current status and development prospects of RAS-based industrial fish culture are
analyzed in the Republic of Belarus. The implementation of projects on the rearing of trout,
sturgeon and African catfish is considered an alternative to traditional pond fish farming
methods in supplying internal markets with new fish products and minimizing the volume
of anthropogenic effluents entering river catchments.
Keywords: fish, industrial fish culture, recirculating aquaculture systems
The fish and fish product supply of the human population used to be
increased mainly through increasing the exports of seafood. The prospect of
depleting the biological resources of the coastal sea regions and the objective
increase of the raw material prices due to the growing costs of the
development of high sea fishing make it necessary to actively increase the
inland water production and, in particular, aquaculture production.
Aquaculture is recognized as the fastest-growing sector of dietary animal
protein production, which, in many respects, is a consequence of the
impossibility to satisfy the growing demand for fish products from natural
resources. The aquaculture of Belarus traditionally developed in the direction
of carp-based pond farming. The potential of this development direction is
now practically fully exploited, so further growth is only possible through the
expansion of production capacities by building new pond areas. In addition,
the internal marketdemand requires an increase of the rangeof products made
of the reared fish species, including luxury food. Due to several economic
and nature conservation reasons, the growth of the development of pond
aquaculture has stopped in most countries of the Baltic region. At the same
time, the technological development of new fish culture methods, fish
feeding and feed production along with the requirement to minimize biogenic
effluents from fish culture enterprises have contributed to the development of
the industrial fish culture direction as an independent market segment in the
production of trout, sturgeons and some other fishes.
44
The European Union has chosen industrial aquaculture as a flagship project
of the EU Strategy for the Baltic Sea Region. The Baltic Sea Region
Programme (AQUABEST) 2007–2013 finances projects that contribute to
the implementation of the EU Strategy for the Baltic Sea Regionand is the
first regional consortium of the states of the region, which includes state
structures, production organizations, educational institutions and consumers
in order to develop a common strategy of sustainable aquaculture production
in the region. Participation in the project has a great practical importance for
the Republic of Belarus, which is juststarting to intensively develop such
aquaculture technologies, as the implementation of the programme is based
upon the development and adaptation of the development conception of
Danish intensive technologies of rearing high-value fish species in
recirculating aquaculture systems (Barulin, 2013). Denmark is known to be a
world leader in the technological level of aquaculture, where a rather unique
concept of recirculating aquaculture systems, the so-called „model fish
farms”, has been developed.
Industrial fish culture based on water reuse and water recirculation began to
develop in Belarus in the late 1980s when the construction of tank modules
for market fish rearing started at several industrial enterprises having cheap
heat and electric energy, as well as technical oxygen. A total of nine such
water-reuse or recirculating fish production plants had been established by
01.01.1996 (Konchits, 2002). The production capacities of the constructed
farms allowed to produce up to 1590 tonnes of fish, but their utilization rate
was only 1.3–17%, and the annual market fish production ranged between 20
and 270 tonnes (Figure 1). The main aquaculture species was common carp,
whose stocking material was supplied from pond farms. Most of these units
had gone out of business by the early 2000s because of their low economic
efficiency.
Since 1998, private individuals and organisations have started to establish
new fish culture enterprises based on recirculating aquaculture systems
(RAS) using more sophisticated technologies. As the costs of fish rearing
have remained high, the rearing has focused on the more expensive
sturgeons. Four such facilities are currently in operation in Minsk (ТМ Ltd.),
Minsk Region (Private Enterprise „Akvatoriya” and Rosich JSC) and
Mogilev (Remona Ltd.). The production volume of these enterprises was
45.6 tonnes in 2012. A small trout-producing RAS (with a capacity of
45
10 tonnes) have been built by a private entrepreneur in Grodno Region. The
first African-catfish-producing plant with a capacity of 25 tonnes (Prosoma
Ltd.) was set in operation in the same province.
300
250
200
тонн
150
100
50
0
1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000
Figure 1. Dynamics of fish production in RAS, 1987–2000.
A new stage in the development of this direction started with the adoption
and implementation of the 2011-2015 State Fisheries Programme. In the
frame of the Programme’s implementation, the establishment of 16
specialized industrial water-reuse and recirculating aquaculture complexes is
planned, including 8 plants for trout production, 2 plants for sturgeon
production, 3 plants for African catfish production and 3 specialized
hatcheries supplying seed to the market fish production complexes. As a
result of the implementation of the Programme, a RAS-based trout
production complex with an annual capacity of up to 100 tonnes was set in
operation in 2011 in Vitebsk Region. Its actual production volume amounted
to 56 tonnes of table fish in 2012. In 2012, the Fish Farming Investment
Group Ltd. (Israel) installed the first stage of a RAS-based industrial unit for
the cultivation of African catfish in Brest Region, using the MegaFlow low-
head recirculation system. The planned final production capacity of the
enterprise will be 700 mt/year after the work is finished. The first 20 tonnes
of table fish were already produced in the end of 2012. In September 2012,
the first RAS-based salmonid hatchery with the capacity of 3 million fish was
set in operation at the Belarussian State Agricultural Academy (Mogilev
Region) in order to supply market trout production farms with stocking
material. The first batch of fish seed amounting to about 150 000 fish with an
average weight of 50 g is expected in April 2013. A total of 129 tonnes of
46
fish were produced in RAS in 2012, including 68.1 tonnes of trout,
37.5 tonnes of sturgeons and 32.9 tonnes of African catfish.
The development prospects of industrial fish culture in the coming years are
linked to the implementation of the following investment projects.
Catfish rearing. The project „Construction of an industrial complex for
producing 500 tonnes of African catfish in a low-head recirculating
aquaculture system in Svetlogorsk, Gomel Region” is identical to the Israeli
project in terms of technology. An investment business plan of this project
has already been elaborated and negotiations are under way to receive state
guarantees from the Government of the Republic of Belarus for obtaining a
foreign line of credit.
The reconstruction of an existing tank complex for catfish rearing with an
annual production capacity of 37 tonnes started in Brest Region in 2012
(Dneprobugskiy Fish Farm JSC).
Trout rearing. The construction of 2 industrial market fish producing
complexes for rearing salmonids with a total planned annual capacity of
568 tonnes of fish has started (Alba JSC in Minsk Region and „Holding
Mogilevvodstroy” JSC in Mogilev Region). The project documents and
budget for the construction of a 200-tonne trout farm of the „Khazar Fish”
Joint Ltd. (Minsk Region) have been developed and all preparatory work has
been finished.
In addition, the planning of 2 further trout complexes with a total annual
production capacity of 400 tonnes of table fish in Minsk Region (Volma Fish
Farm JSC) and Mogilev Region („Holding Mogilevvodstroy” JSC) has been
practically finished.
Sturgeon rearing. The investment business plan of the construction project
of a sturgeon-rearing industrial complex with an annual production capacity
of 241 tonnes has been developed for Brest Region, which is currently
undergoing the required coordination process for attracting foreign lines of
credit. Planning work is under way for the reconstruction of the existing tank
space for sturgeon rearing with a capacity of up to 100 tonnes of table fish in
Brest Region (Selets Experimental Fish Farm JSC).
Seven industrial complexes with a total annual production capacity of
952 tonnes of table fish are planned to be set in operation in 2013, including
4 complexes for trout production, 2 complexes for sturgeon production and
one complex for catfish production. By 2016, the production volume of RAS-
47
reared fish is planned to be increased to 3800 tonnes or, taking into account
the regional development programmes, to 4740 tonnes (Figure 2).
5000
4500
4000
3500
3000 по Госпрограмме
тонн
2500
с учетом региональных
2000 программ
1500
1000
500
0
2012 г. 2016г.
Figure 2. Growth prospects of RAS production
References
Barulin, N.V., 2013. The significance of the AQUABEST programme in the
development of aquaculture and restoration of valuable fishes of
Belarus.Proceedings of the 2nd International Scientific Conference
„Restoration of natural populations of valuable fish species”, Saint
Petersburg, p. 43. (In Russian)
Konchits, V.V., 2002. Status and development perspectives of industrial and
collective fish farming in Belarus.Voprosy rybnogo khozyaystva, 18: 5-
15. (In Russian)
48
The first experiment to obtain sex-reversed rainbow trout in RAS
conditions in the Republic of Belarus
Slukvin, А. М.1, Metalnikova, K. V.2, Kostousov, V. G.3, Koneva, О.

Yu.1, Rovba, Е. А.1
1
Institute of Genetics and Cytology of the National Academy of Sciences of
Belarus, Minsk, Republic of Belarus
2
Russian Federal Research Institute of Fisheries and Oceanography,
Moscow, Russian Federation
3
Institute of Fisheries, Scientific and Practical Centre for Animal Husbandry,
National Academy of Sciences of Belarus, Minsk, Republic of Belarus
Abstract
The objective of the research was to provide a scientific foundation for the methods of
increasing the biological productivity of rainbow trout through studying and modifying its
genome expression.
The effect of androgen on the growth, development and sex reversal of rainbow trout fry
and fingerlings was studied in the recirculating aquaculture system (RAS) of the
Bogushevskiy Fish Hatchery, Vitebsk Region, Republic of Belarus. The application of a
7 mg/kg feed dosage of the preparation during an exposition period of 3 months was found
to result in sex reversal-related changes in the gonads of trout fry, with frequencies of
occurrence of 25.0% to 42.9% of the studied specimens. Trout reared in RAS and treated
with androgensdid not differ from the control in terms of their morphometric indices.
Ichthyophthiriosis was diagnosed in the fry and mortality occurred both in the treated and
the control stock. In such conditions, the survival of androgen-treated fry was 11.55 higher
than that of the control. It was confirmed that the results of molecular genetic research on
the Y-specific locus OmyY1 allowed to efficiently remove live males at the fingerling stage.
The results of histological and genetic sexing studies in trout fry showed a 100% match.
New knowledge was obtained on the growth, survival and possibilities of modifying the
genome expression using androgens in the fry of salmonids kept under RAS conditions.
Keywords: RAS, rainbow trout fry, methyltestosterone, genome expression, sex-reversed

trout fingerlings.
Introduction
An explosive growth of commercial salmon production and culture-based
salmon fisheries has taken place in the last decades (Hisar et al., 2012). The
market production of salmonids, mostly females for roe production, as well
as the stock enhancement of rare and endangered species by releasing farmed
49
females can nowadays be put on a commercial basis using cryopreservation
of the sperm of sex-reversed fish with female genome.
The aim of the presented research was to support the implementation of the
provisions of the State Programme for the Development of Fisheries
Activities in 2011-2015, approved by the Resolution 1453 of 07.10.2010 of
the Council of Ministers of the Republic of Belarus, concerning import
substitution measures including the increasing of the domestic production
and consumption of top-quality fish and fish products (Council of Ministers
of the Republic of Belarus, 2010).
While only about 40 tonnes of trout were produced annually in the
Republic of Belarus before 2010, the State Programme for the Development
of Fisheries Activities in 2011-2015 envisages the construction of 11
specialized trout farms with an overall capacity of 1,500 tonnes of table trout
per year, including fish for trout caviar production. Altogether, the
implementation of the State Programme for the Development of Fisheries
Activities in 2011-2015 will allow to increase the production volume of high-
value fish species, such as salmonids and sturgeons, to an annual
2,500 tonnes in the country.
Material and methods

The study material was collected at the recirculating aquaculture system
(RAS) of the Bogushevskiy Fish Hatchery, Vitebsk Region, Republic of
Belarus.
The design capacity of the RAS trout farm is 100 tonnes of fish. The
facility contains outdoor fish tanks made ofprecast reinforced concrete blocks
and site-cast monolithic reinforced concrete (Figures 1а, 1b). The water is
supplied by an artesian well with a water temperature of +4oС and a feeder
canal with water intake from the bottom layers of the supply reservoir. The
discharge of the artesian well is 29 l/s. The concrete tank system is divided
into two units: a nursery unit (tanks for rearing trout fry to a fingerling stage)
consisting of 11 fish tanks of 11.5 х 2.0 х 1.2 m and 2 technological reserve
tanks (Figure 2), and a grow-out unit for table fish rearing. The effective
volume and surface area of the nursery tanks is 304 m3 and 253 m2,
respectively. The nursery unit includes two 1.5 х 1.5 х 14.8 m tanks for
fluidized bed filters. Seven tanks of 2.0 х 8.4 m contain filters loaded with
9 mm long plastic capsules with a diameter of 7 mm. The nursery unit has
50
sludge cones for the removal of uneaten feed and metabolic wastes of trout
fry. A unit of three low-pressure aerators with an overall surface of 24 m2 is
installed for gas stripping and oxygenation of the water. The process water is
circulated by an airlift with a capacity of 700 l/s. If the water temperature
exceeds +18oС, an OXYPLUS E 017100 device oxygenates the water. Liquid
oxygen is supplied to the facility by road. The oxygen concentration in the
water is controlled and regulated by an automatic OXYGuard-Commander
system. TheрНof the water is controlled by an OXYGuardрНManta system.
The grow-out tank unit (Figures 3а, 3b,3c) consists of 4 fish tanks with
dimensions of 31.4 m х 3.5 m and an effective volume of 571 m3.
Figure 1а. RAS of the Bogushevskiy Fish Hatchery
Figure 1b. Airlift at the Bogushevskiy Fish Hatchery
51
Figure 2. Nursery unit of the RAS
Figure 3а. Grow-out unit of the RAS
52
Figure 3b. Filters at the grow-out unit of the RAS
Figure 3c. Airlift at the grow-out unit of the RAS
The surface area of the tanks is 440 m2. The grow-out unit contains also 2
tanks with fluidized bed filters, whose dimensions are 1.5 х 1.5 х 11.5 m, and
seven 6 m х 2 m tanks containing biofilters with plastic capsule loading. The
total area of the filters is 84 m2. Sludge cones for the removal and utilization
of uneaten feed and metabolic wastes of market-size trout have also been
53
installed in the table-fish-producing unit. There are also three low-pressure
aerators with an overall surface of 24 m2. The water is circulated by an airlift.
There are 4 oxygenation devices. Hydrochemical parameters are controlled
using a Polaris portable temperature and oxygen meter.
The fish rearing cycle in RAS is as follows: 280,000 trout fry with an
average weight of 5 g are stocked into the nursery unit in spring and reared to
fingerlings with a mean weight of over 100 g by November. In November,
trout fingerlings are harvested and moved to wintering cages in a warmwater
canal of the Novolukoml Power Station. After wintering, 75,000 yearlings
with an average weight of 400 g are returned to the RAS for rearing to the
market size of 1 kg.
Our studies were done on rainbow trout (Oncorhynchus mykiss W.) fry.
After reaching an average weight of 5 g, all the experimental and control
stock was moved to the Bogushevskiy Fish Hatchery for further ongrowing.
Only two concrete tanks (one treatment tank and one control tank) were
used for ongrowing at the Bogushevskiy Fish Hatchery.
10,100 and 10,000 fry with an average weight of 5.0 g were stocked into
the experimental Tank 6 and the control Tank 7, respectively.
The fry was fed trout feeds by Aller Aqua (manufactured in Poland) and
Raisio (manufactured in Finland), as well as an experimental production feed
formulated by the Institute of Fisheries, with the addition of a solution of
methyltestosterone in ethanol in a concentration of 7 mg/kg feed. Androgen-
supplemented feeds were fed to the fry for a period of 3 months (from
20 April to 19 July).
The feeding rates were recalculated for both raceways once in three days,
taking into account the planned weight gain during the calculations. During
and at the end of the experiment, the following parameters were determined:
feed efficiency, survival rate, growth indices.
During the work, the environmental quality, the correspondence of growth
conditions to normative requirements, the indices of linear growth and weight
gain, and the biochemical composition of tissues in the treated and control
groups, including “red” and “white” components of the blood, were
observed. The extent of morphometric differences between fry that was
treated with the hormone preparation and fry that was not was determined on
the basis of measurements of live fish and studies of the fixed samples.
54
The hydrochemical parameters in the raceways and tanks of the nursery
unit of the trout farm were analyzed according to standard hydrochemical
study methodologies (State Committee on Science and Technology of the
Council of Ministers of the USSR, 1978).
The dissolved oxygen content, pH value and temperature were measured
with «Horiba-U-7» and «Hanna» water quality checkers.
The growth rate and morphometric indices were calculated using the
methodologies of ichthyological studies described by Pravdin (1966).
Biochemical and haematological studies were done following standard
methodologies (Limanskiy et al., 1984).
A total of 20 water samples were collected for complete analysis, as well as
525 trout fry for the analysis of fisheries biological and biochemical
parameters.
For histological studies, the trout fry was fixed alive in Chamberlain or
Bouin solutions after resection of the peritoneum of each fish. The fixation
was done according to the protocol described by Roskin and Levinson (1957)
and Pausheva (1988). The samples were then immersed in 70% rectified
ethanol in biopsy cassettes, each sample in a separate cassette. Each cassette
was placed into a glass vessel filled with 70% ethanol and closed with a
ground glass stopper for a period of 48 hours. The tissue processor is shown
in Figure 4.
Figure 4. STP-120 carousel-type tissue processor for histological processing of the tissues
55
The tissues in the biopsy cassettes were processed in a carousel-type tissue
processor, model STP-120 (Figure 4); paraffin embedding was done using an
ЕС-350 embedding centre (Figure 5).
Figure 5. ЕС-350 embedding centre
Longitudinal sections with a thickness of 6 µm were made using a

MICROM HM 440 E sledge microtome (Figure 6). The obtained sections
were stained with Gill’s Hematoxylin No. 2 [BIOVITRUM, 2012] followed
by eosin staining.
Figure 6. MICROM HM 440 E sledge microtome and a slide warmer
The photographs of the ready histological slides were taken by a Leica

microscope connected to a computer system and an automatic Leica DC
camera, with a 10x eyepiece magnification and 10x, 20x, 40x and 100x
objective magnifications (Figure 7). Digital microphotographs of the
56
histological samples were taken using the DC Viewer programme combined
with the PhotoShop 4.0 image processor. A total of 59 samples of rainbow
trout fry from Bogushevskiy Fish Hatchery were processed.
Figure 7. Microscope with a Leica DC computer system
Molecular genetic studies were done on caudal fin fragments of trout fry
preserved in 96% ethanol. The research objective was live identification of
rainbow trout males in different fry and fingerling development stages using
AFLP primers.
Y-specific DNA sequences in rainbow trout were first found by Brunelli et
al. (2008, 2010). A male-specific DNA fragment was first identified in
Chinook salmon using AFLP technology. This fragment was extracted from
the gel and repeatedly amplified using an appropriate selective combination
of AFLP primers. The amplified fragments were directly sequenced and
cloned by ligating them into a pGEM-T vector. The cloned inserts were then
sequenced and matched with primers.
In order to identify a Y-specific DNA sequence in rainbow trout, a
combination of primers for the OtY2 locus of the Y chromosome of Chinook
salmon was used in the first stage. As a result, a PCR product of 995 base
pairs was obtained, which was subsequently sequenced. The homology of
this DNA region of rainbow trout with the corresponding DNA region of
Chinook salmon was 96%. The genome libraries of rainbow trout were
checked for clones continuing the product in question, which were then
isolated and sequenced. This allowed the identification and characterization
of a 21 kb DNA sequence of the Y chromosome of rainbow trout, which was
57
highly similar to the sequence of the Y chromosome in Chinook salmon that
was situated next to the OtY2 locus. The homologous sequence starts at the
OtY2 semi-specific junction formed by the insertion of the “ReO_6-like”
non-LTR retrotransposon / non-LTR retrovirus in Chinook, coho, chum and
sockeye salmons. This retrotransposon element is absent in the locus of
rainbow trout homologous with OtY2, which was marked by the authors as
OmyY1 (Brunelli et al., 2010).
The amplification of the OmyY1 locus of rainbow trout using the primers
proposed by Brunelli et al. (2008) yields a PCR product of 792 base pairs.
The accuracy of sex determination using this molecular genetic marker is
about 96.5% according to data in Brunelli et al. (2008, 2010).
Thus, an attempt was made to evaluate the accuracy of male identification
by a molecular genetic approach in the trout fry used in our experiment. For
this purpose, the OmyY1 locus was genotyped in the collected rainbow trout
samples. The obtained data were compared to the results of parallel
histological research done by colleagues at VNIRO (Moscow, Russia).
DNA was extracted from the fish fins by phenol-chloroform extraction. The
concentration and purity of the extracted DNA were determined by
spectrophotometry using an Ultrospec 3300 pro UV/Visible
spectrophotometer (Biochrom Ltd.) (Figure 8). A spectrophotometric
analysis of the levels of protein pollution in the obtained DNA preparations
performed on the basis of the А260/А280 correlation of absorption
coefficients (normal range between 1.8 and 2.0) confirmed an adequate
purification level of the obtained DNA preparations. The average А260/А280
ratios of absorption coefficients were 1.88±0.03 and 1.54±0.05 in the June
and August samples, respectively. The average DNA concentrations in the
fish fin preparations were 1483.47±151.18 µg/ml and 1004.72±174.69 µg/ml
in the June and August samples, respectively.
58
Figure 8. Ultrospec 3300 pro UV/Visible spectrophotometer (Biochrom Ltd.)
The quality of the extracted DNA was checked with electrophoresis on a

2% agarose gel (Agarose D1 Low EEO, Conda) (Figure 9).
Figure 9. Gel with extracted rainbow trout DNA.

М – GeneRullerTM 100bp Plus DNA Ladder
molecular weight marker (Fermentas)
The obtained results (Figure 9) indicate a satisfactory quality of the

extracted DNA (presence of high-molecular-weight DNA).
Amplification. PCR with the combination of primers proposed by Brunelli
et al. (2008) was used for the evaluation of the genotype heterogeneity of the
studied rainbow trout specimens at the locus OmyY1.
The PCR solution (20 µl) consisted of 2.0 µl 10х PCR buffer (10x
DreamTaq buffer, Fermentas) containing 20 mM MgCl2; 2.0 µl 10х d’NTP-
mix (Primetech, Belarus); ≈10 pmol primer (Primetech, Belarus); 0.05 U/µl
DreamTaq DNA polymerase (Fermentas); 1 µl DNA matrix, diluted with
Milli-Q water to a total volume of 20 µl.
Amplification was done using a C1000TM thermal cycler (Bio-Rad, USA)
(Figure 10) under the conditions below (Figure 11).
59
Figure 10. MyCyclerTM thermal cycler (Bio-Rad, USA)
Figure 11. PCR protocol
Electrophoresis. The amplification products were separated by

electrophoresis on a 2% agarose gel (Agarose D1 Low EEO, Conda) with the
addition of ethidium bromide(0.5 µg/ml)in a 1х ТBЕ buffer using a Compact
L horizontal electrophoresis apparatus (Biometra, Germany).
Gel visualization was done using a GelDoc XR gel documentation system
(Bio-Rad, USA) (Figure 12).
60
Figure 12. Gel Doc 2000 system (Bio-Rad, USA)
The obtained images were processed with the Quantity One 4.4 software
(Bio-Rad, USA).
Methods or recording fish production parameters. When analysing the

results of our experimental work with androgens, the following fish
production parameters normally included in the normative and technological
documentation of fish farms were taken into account: amount of stocked
larvae (fry) (1000 ind.); average individual weight of fry at stocking and
harvest (g); total fish biomass (kg); survival rate of the fry, percentage of
morphological anomalies in fry.
The statistical analysis of the materials was done using the STATISTICA
6.0 software.
Results
The scheme of obtaining sex-reversed offspring, mainly females, is widely
known: at the first stage, sex-reversed individuals with female genome are
obtained from a small quantity of normal fry using testosterone analogues. At
the second stage, these sex-reversed indivisuals are cross-bred with normal
females, obtaining mainly female offspring (Metalnikova et al., 1989). The
number of sex-reversed fish may be restriced due to their high resilience, as
well as the high resilience of their – mostly female – offspring
(XXxXX>XX) (Metalnikova and Privezentsev, 2010).
61
These research results were produced in 2012 in cooperation with the
Institute of Fisheries of the Scientific and Practical Centre for Animal
Husbandry of the National Academy of Sciences of Belarus (Minsk,
Belarus). The histological studies were performed by the Russian Federal
Research Institute of Fisheries and Oceanography (Moscow, Russia).
Hydrochemical parameters during fry rearing in RAS.

The water temperature, which reached 10-14ºС on average from April to
September, was found to have decreased to 10ºС in October. The рН of water
ranged between 8.2 and 8.3, the dissolved oxygen content stayed within the
acceptable range of 6.5-10.0 mg/l. Such parameters as water hardness,
calcium, magnesium and iron were relatively stable without much
fluctuation. As to parameters with set limits, increased mineral nitrogen
content and a higher permanganate index were detected in the water.
Considering that a recirculating aquaculture system was used, the most
probable is that the biofilters could not cope with the increasing biogenic load
and could not ensure an adequate quality of water treatment. In October,
some mineral nitrogen forms in water, such as nitrite and nitrate nitrogen,
exceeded the optimal limits by factors of 4.8 and 2.0, respectively (Table I).
By November, the nitrite and nitrate nitrogen content, the permanganate
index and the phosphorus content of water decreased by 65%, 68%, 15% and
75%, respectively, as compared to September, which is related to the
decreasing water temperature and the slowing-down of mineralization
processes along with the rationing of fish feeds.
Thus, the conditions of fish rearing in the tanks of the fish production
complex were satisfactory by all parameters limited by the standard, with the
exception of the content of mineral nitrogen forms.
62
Table I. Hydrochemical parameters of water in the tanks of the trout production complex of
the Bogushevskiy Fish Hatchery
Content
limits under the
Parameter Unit
STB 1943-2009 Tank 6 Tank 7
standard
Temperature ºС ≤ 20 10.7 10.7
pH рН 7.0-8.0 8.3 8.3
Dissolved oxygen mg/l 9.0 6.89 7.12
Ammonium ion mg N/l 0.5 0.58 0.56
Nitrite ion mg N/l 0.02 0.048 0.009
Nitrate ion mg N/l 1.0 2.18 2.11
Phosphate ion mg Р/l 0.3 0.004 0.024
Total iron mg/l 0.5 0.02 0.018
Permanganate index mg О/l 10.0 11.30 12.44
Results of morphometric studies of androgen-treated rainbow trout fry. The

research included both measurements of live fish and studies on fixed
material. The growth characteristics of fish depended on the hydrochemical
regime and feeding, and thus, the hydrochemical conditions of fish rearing
were monitored during the grow-out period.
Examples of weight gain and linear growth in the treated and control
groups of trout are presented in Tables II and III.
According to the data of Table III, the average weight of rainbow trout fry
in the control and treatment raceways as well as in the tanks on a whole did
not differ during the methyltestosterone feeding period. Statistically
significant differences (p<0.05) in the weight of the rainbow trout fry were
only found in the initial feeding stages (27.04.2012, 12.05.2012). After that,
approximately the same growth rates were observed, which is clearly shown
in Figure 13.
63
Table II. Growth parameters of trout fingerlings in the summer grow-out period
Date Treated group Control group
length, cm weight, g n length, cm weight, g n
range mean range mean range mean range mean
06.06 6.8-9.4 8.15 4.8-11.6 7.64 15 - - - - -
28.06 5.9-10.1 8.99 3.0-11.0 8.43 16 7.2-10.0 8.5 5.5-12.0 8.7 15
11.08 12.5-13.1 12.8 31.5-32.6 32.0 4 - - - - -
Динамика изменения массы молоди радужной форели (W)

50,0
Среднее
± Стандартная ошибка
45,0 ±0,95Доверительный интервал
40,0 39,0
35,0
31,8
30,0
Масса, г
25,0
21,0
20,6
20,0
15,0 14,2 14,1

13,7
12,8
10,4
9,6
10,0
8,5
7,3
5,2
5,0 Опыт
3,6
1,9
1,7
2,7 Контроль
0,0
27.04.2012 22.05.2012 14.06.2012 05.07.2012 02.08.2012
12.05.2012 06.06.2012 27.06.2012 17.07.2012
Дата
Figure 13. Body weight changes in the treated and control groups of rainbow trout fry
The data in the chart showing the total length changes of the fry (Figure 14)
confirm no difference in the average lengths of rainbow trout fry in the
control and treatment tanks on a whole during the methyltestosterone feeding
period. Statistically significant differences (p<0.05) in the total length of the
rainbow trout fry (Table III) were only found in the initial feeding stages
(12.05.2012, 06.06.2012). After that, approximately identical growth rates
were observed.
Similarly, the data in the chart showing the standard length changes of the
fry (Figure 15) confirm no difference in the average standard lengths of
rainbow trout fry in the control and treatment tanks on a whole during the
64
methyltestosterone feeding period. Statistically significant differences
(p<0.05) in the total length of the rainbow trout fry (Table III) were also
found only in the initial feeding stages (12.05.2012, 06.06.2012). After that,
approximately identical growth rates were observed.
Table III. Dynamic comparison of the morphometric parameters of rainbow trout fry in the
treated and control groups
t-test for independent samples
Standard deviation,
Dates of measurements in the treated and Average Significance Sample size
SD
control groups level, p
Treated Control Treated Control Treated Control
Weight, g
27.04.2012{Treated} vs. 27.04.2012{Control} 1.9 1.7 0.047 51 50 0.47 0.43
Total length (L), cm
Standard length (l), cm
65
Динамика изменения длины тела молоди радужной форели ( L)
Среднее
± Стандартная ошибка 14,4
±0,95Доверительный интервал
14,0
13,2
12,0
11,2
11,0
10,7
10,4 10,4
10,4
Длина, см
10,0 9,7
9,4
8,7
8,0
8,0 7,7
7,7
6,7
6,1
6,0
5,5
5,4 Опыт
Контроль
27.04.2012 22.05.2012 14.06.2012 05.07.2012 02.08.2012

12.05.2012 06.06.2012 27.06.2012 17.07.2012
Дата
Figure 14. Total length changes in the treated and control groups of rainbow trout fry
Динамика изменения длины тела до конца

основания хвостового стебля (l) м олоди радужной форели
14,0
Среднее
13,0 ± Стандартная ошибка 12,8
±0,95Доверительный интервал
Длина до конца хвостового стебля ( l), см
12,0 11,7
11,0
10,0 9,8
9,5 9,6
9,2 9,3
9,2
9,0
8,7
8,4
8,0 7,7
7,0
7,0 6,8
6,0 5,7
5,2
5,0 4,8
4,6
4,0
Опыт
Контроль
3,0
27.04.2012 22.05.2012 14.06.2012 05.07.2012 02.08.2012
12.05.2012 06.06.2012 27.06.2012 17.07.2012
Дата
Figure 15. Standard length changes in the treated and control groups of rainbow trout fry
Thus, no effect of methyltestosterone on morphometric parameters of

rainbow trout fry was detected. It should be noted that the slowdown in the
66
growth of rainbow trout fry both in the control and treatment tanks between
26.07.2012 and 17.07.2012 (see charts) was probably related to an
Ichthyophthirius infection of the rainbow trout fry, as well as a bacterial
infection occurred in the same period.
A lower occurrence of morphological abnormalities (in particular, opercular
reduction) was noted in the individuals of the treated group. While the
percentage of morphological abnormalities was approximately the same in
the treated sample and the control (15.4% and 20.0 %, respectively) at the
beginning of the experiment (27.05.2012), the difference increased
significantly during the experiment. There were 64% of individuals with
morphological abnormalities in the control group, while the share of
individuals with morphological abnormalities never exceeded 24 % in the
treated group.
The changing growth rate and the shift to a predominantly female-type
morphogenesis as a result of hormonal preparations is expected to be
manifested in the change of the body proportions (plastic features), in
particular, those that reflect the exterior features and can act as secondary
sexual characteristics. These had never been studied in fingerlings before,
and thus, the possibility of their detection at early developmentr stages is of
interest. The results of the measurements of two sets of samples and their
statistical analysis are presented in Table IV.
The analysis of the data in Table IV showed a wider range of the values
regarded as secondary sexual characteristics and higher values of the standard
error in the treated group than in the control group. However, these
differences were not yet statistically significant (t< 3) in fingerlings, which
was probably related to their insufficient manifestation at that ontogenetic
stage. The research will be continued on two-year fish, at stages closer to
sexual maturity.
67
Table IV. Some plastic features of trout fry shown as percentage of the fork length
Treated group Control group
Feature t
lim M +/-m lim M +/-m
Predorsal length 43.3-47.5 45.31 0.28 42.9-46.6 44.71 0.30 1.5
Preventral length 44.5-54.4 50.98 0.66 47.0-51.1 49.47 0.27 2.1
Preanal length 63.0-70.9 67.59 0.52 63.4-68.2 66.32 0.28 2.2
Postdorsal length 32.0-37.6 35.06 0.36 33.3-37.5 35.41 0.36 0.7
Dorsal fin length 9.9-13.9 12.38 0.27 11.0-14.6 12.19 0.25 0.5
Anal fin length 7.8-12.8 9.58 0.33 8.0-10.2 8.98 0.17 1.6
Head length 21.8-32.2 24.13 0.59 20.6-25.0 22.37 0.31 2.6
Body depth 20.2-25.3 23.55 0.36 20.0-25.0 22.49 0.38 2.0
Note: lim – range of values; M – arithmetic mean; +/- m – standard error of the mean; t-
statistic (normalized deviate)
Results of biochemical and haematological research. As shown by the

results of biochemical analyses, no significant differences were found in the
dry matter and moisture content of the body between the treated and control
groups of fish. The crude protein content was 5.8% higher in the treated fish
than in the control (73.52% vs. 67.68%, respectively). This is probably
related to the fact that the protein content of the experimental feed was 7%
higher than that of the control. The crude fat content of the body of the
treated and control fish was virtually identical, 30.96% and 31.57%,
respectively, although the lipid content of the experimental feed was 13%
lower than that of the control feed. The crude ash content of the treated group
was 0.7% higher than in the control, in line with the fact that the content of
the minerals calcium and magnesium in the experimental feed was higher
than in the control feed.
The digestibility of the nutrients contained in the experimental feed by the
organism of the trout can be inferred from the efficiency of the use of
nutrients for growth. These indices express the relationship between the
consumed feed and the reaction of the animal thereto, which has a big
significance in evaluating the nutrition value of feeds.
Haematological research was also conducted in order to evaluate the
physiological status of trout fingerlings.
The following blood parameters were studied: total serum protein,
hemoglobin content, erythrocyte and leucocyte content, leukocyte formula.
As shown by haematological studies, the total serum protein level of trout
fingerlings had been very low before they were fed with the experimental
feed. The values of this parameter ranged between 2.29 and 4.20, with an
68
average of 3.15. Both the average and the maximum values were
significantly below the 6.2 g% norm.
The hemoglobin concentration was very unstable, changing within a wide
range – from 62 g/l to 91 g/l. The average of 76.7 g/l slightly exceeded the
reference range (71.0–73.0 g/l).
The reference range of the erythrocyte sedimentation rate (ESR) is between
1 mm/h and 4 mm/h, and the average of 1.85 mm/l stayed within these limits.
However, this parameter was also characterized by a wide range of values:
the minimum and maximum values were 1.0 and 5.0 mm/h, respectively. The
red and white blood cell counts were also within the reference range: while
the normal values for red and white blood cells are 1.15±0.08 million cells/µl
and 58.6±6.4 thousand cells/µl, the averages were 1.095 million cells/µl and
57.3 thousand cells/µl, respectively.
The leucocyte formula of the blood showed an increased share of
neutrophils, in particular, young forms (band cells), as compared to
lymphocytes before the feeding with the experimental feed started. This is
not a favourable indicator and may suggest the presence of some infection in
the organism of the fish. Eosinophils and basophils were absent in trout
fingerlings, which is normal.
After a 30-day feeding period, the total blood serum protein level
approached the norm (6.2 g%) both in the treated fish (6.03 g%) and the
control (5.80 g%).
The hemoglobin concentrations increased and exceeded the norm, with the
values of treated fish significantly higher than those of the control (treated –
110.7 g/l, control – 88.3 g/l). This is a favourable indicator. The increased
hemoglobin levels were probably connected to the more intensive growth and
the related increase in the metabolic rate due to the need of transporting more
oxigen to the tissues. The erythrocyte sedimentation rate (ESR), which is
another indicator of the physiological health of fish, was within the reference
range both in the treated and the control fish.
The content of the basic cellular elements – red and white blood cells – in
the blood was also within the reference range. Red blood cells were quite
large (which is characteristic of younger fish, generally they become smaller
with age); this explains why the hemoglobin concentration was above the
norm while the number of red blood cells did not exceed it. The leucocyte
formula was normal. There was no shift from lymphocytes toward
69
neutrophils as observed in the previous stage of the studies. Basophils and
eosinophils were absent, as it should normally be in rainbow trout
fingerlings.
Thus, the studies showed that most blood parameters of rainbow trout
fingerlings had changed within a wide range before the experiment started.
However, the mean values always stayed within the reference range, with the
only exception of the total blood serum protein, which was almost half of the
norm, indicating a bad physiological status of the fish. Abnormalities were
also observed in the leucocyte formula, i.e. a shift from lymphocytes toward
band cells, which might have indicated the presence of some infection in the
organism.
By the end of the experiment, the leucocyte formula had stabilized and
corresponded to the norm, both in the treated and the control fish. The red
blood parameters were also more stable and stayed within the reference
range, even the total serum protein practically reached the normative value.
Hemoglobin concentrations were significantly above the norm, both in the
treated (110.7 g/l) and the control fish (88.3 g/l); yet, this is not an
unfavourable indicator but a result of the intensive growth and the increased
metabolic rate.
Therefore, the use of experimental feed (with hormone) at the fingerling
stage did not affect the basic physiological functions of fish tissues.
Results of histological studies in rainbow trout fry treated with androgen.
At the beginning of the experiment, 20 treated trout samples were collected
weighing 0.31 g. As a result of the histological analysis of the sagittal
sections of trout fry, the following trout fry groups different in terms of the
structure of their gonads were identified (Table V).
Table V. Percentage share of trout fry groups with different gonad structure
Trout fry groups Percentage share of trout fry
1 (females) 25
2 (males) 40
3 (indeterminate sex) 35
It was concluded that the group of trout with indeterminate sex was the
most promising for obtaining sex-reversed fish. According to the
methodology (Metalnikova et al., 1989; Metalnikova and Privezentsev,
2010), the hormonal treatment of trout should start before the beginning of
the cytological differentiation of the gonads in order to get sex-reversed
70
individuals in large numbers (up to 100% of the quantity of hormone-treated
genetic females). The results of histological studies on the fry used for
experimental hormone treatment showed that the used fry, at the age of more
than three weeks after hatching, was already feeding externally, had a bigger
size and gonads with a cytologically determinate sex. For this reason, we
identified the third group, i.e. trout with an indeterminate sex, as the most
promising one (Table V).
Tables VI and VII show the changes in the share of treated trout with
different sexual characteristics in the anatomic structure of the gonads during
and after the methyltestosterone treatment.
Table VI. Percentage share of treated trout with different gonad structure, stages II and III.
Gonad types, %
Female with resorption Sterile Sterile
Stage Female Male Intersex Sterile
of over 70% of oocytes female male
II 7.1 14.4 42.9 7.1 7.1 7.1 14.3
III 0 33.3 25.0 33.3 8.33 0 0
By the end of the methyltestosterone treatment of the experimental trout,

the number of females with resorption of over 70% of oocytes in the sections
had increased significantly (Table VI). The number of intersex specimens
decreased in the sample, but the share of males increased (possibly because
of sex-reversed individuals) (Table VI).
71
Figure 16. Sagittal sections of gonads
in intersex trout, Bogushevskiy Fish
Hatchery. Age of trout: 0+, treated
with methyltestosterone. Fixed in
Chamberlain’s fixative, stained in
Gill’s Hematoxylin No. 2 and
counterstained in 1% alcoholic eosin.
Magnification: а) eyepiece 10х,
objective 40х; б) eyepiece 10х,
objective 20х; в, г) eyepiece 10х,
objective 40х.
Notes to Figure 16:

1. Mitotic divisions of gonial cells in
a cluster
2. Resorbing oocytes at the 3rd stage
of the protoplasmic oocyte growth
period
3. Blood capillaries
4. Mitotic divisions of gonial cells in
a capsule
5. Glandular formation consisting of
glandular cells
Figures 16 and 17 show photographs of typical sections of the gonads of

treated and control fish in August 2012 at the end of the methyltestosterone
treatment at the Bogushevskiy Fish Hatchery.
Figure 16 shows sagittal sections of the gonads of intersex trout specimens
treated with methyltestosterone. The photograph in Figure 16г is of special
interest as it shows the development of clusters of epithelial cells in the
interovarial space, which form something resembling a gland of internal
secretion, suggesting intensive secretion processes in these cells (Figure 16г).
The abundance of small capillaries in the gonadal matrix also indicate an
intensive secretory activity therein.
Figure 17 shows sections of the gonads of a female and males fixed in
August. The female was in the 2nd maturity stage, while the males were in the
2nd and 4th maturity stages.
72
Figure 17. Sagittal sections of trout gonads,
Bogushevskiy Fish Hatchery, Vitebsk Region,
Belarus. Age of trout: 0+, no methyltestosterone
treatment. Fixed in Chamberlain’s fixative,
stained in Gill’s Hematoxylin No. 2 and
counterstained in 1% alcoholic eosin.
Magnification: а) eyepiece 10х, objective 40х;
б) eyepiece 10х, objective 100х; в) eyepiece
10х, objective 40х, г) eyepiece 10х, objective
100х.
Notes to Figure 17:

1. Oocytes with cytoplasm resorption at the 3rd
stage of the protoplasmic oocyte growth period
of the previtellogenetic phase.
2. Lysis of the oocyte cytoplasm at the 3rd stage
of the protoplasmic oocyte growth period
because of incorrect rearing biotechnology
3. Spermatozoa in seminal ampullae
4. Spermatids in seminal ampullae
5. Primary and secondary spermatocytes in
seminal ampullae
6. Spermatocytes and development of
spermatids
7. Red blood cell in the gonadal matrix
As a result of the performed studies on the gonad structure of trout during

their experimental methyltestosterone treatment, seven groups of trout
differing in terms of their gonad structure were identified: females, males,
intersexes, sterile intersexes, sterile females, sterile males and sterile trout
(Table VI). From the point of view of the perspectives of success of the work
towards our objective, two groups of trout are of the most interest: the
females and the intersex individuals, the latter being more promising for
obtaining sex-reversed fish. Females should develop into intersex fish, which
can also be used for obtaining monosex progeny mostly consisting of
females, but they should be sacrificed in the process. Intersexes were
predominant in the sample, with up to 42.9%, which confirmed our
prediction.
At the end of the methyltestosterone treatment of the trout, the sample taken
from the treated group included mostly males (up to 33%), intersex
individuals (25% of the sample) and females with resorbing oocytes (up to
33.3%), which may develop into intersexes later.
73
Results of genetic studies on rainbow trout fry. Simultaneously with the
collection of histological samples, biological samples for molecular genetic
studies were collected from the same individuals of fry. As a result of the
PCR amplification of the OmyY1 locus (see Material and Methods) (Figure
18), 5 genetic males and 10 genetic females were identified. It was
demonstrated that this metod allowed to identify potential males at early
development stages in a small sample of marked individuals and to remove
them already at the fingerling stage. In August, 16 and 6 trout specimens
were collected from the treatment tank and the control tank, respectively, for
histological and genetic studies. The results of our research on sexing males
and females are presented in Figures 19 and 20.
Thus, it was confirmed that the locus OmyY1 was amplified to 100% in
potential genetic males of rainbow trout, i.e. all genetic males can be safely
removed from the set of marked young individuals (with a 100% match).
Figure 18. Gel with amplification products of the Y-specific OmyY1 locus (792
bp) (samples of treated fish, June). М – GeneRullerTM 100bp Plus DNA Ladder
molecular weight marker (Fermentas);
м – genetic male, ж – genetic female
Figure 19. Gel with amplification products of the Y-specific OmyY1 locus (792 bp)
(samples of treated fish, August). М – GeneRullerTM 100bp Plus DNA Ladder molecular
weight marker (Fermentas);
74
Figure 20. Gel with amplification products of the Y-specific OmyY1 locus (792 bp)
(samples of control fish, August). М – GeneRullerTM 100bp Plus DNA Ladder molecular
weight marker (Fermentas);
Results of the comparative analysis of fish production parameters during

the development of the methodology of sex reversion in rainbow trout fry.
The results of the harvest of trout fry from the tanks of the Bogushevskiy
Fish Hatchery showed that the overall survival rate (from stocked larvae) of
the treated fry was 11.5% higher than the control. The biomass of fish
harvested from the treatment raceways was 113 kg higher than that of the
control. The number of the harvested treated trout fry was significantly
higher than in the control (1294 fish more) (Table VII).
Thus, the addition of 7 mg/kg hormone to the feeds of trout fry reared in
raceways and tanks for 60 days in two farms did not increase their weight
gain and linear growth compared to the control, but the final biomass,
number and survival rate of the treated fry was significantly higher than the
control stock of rainbow trout.
Conclusions
The effect of androgen (methyltestosterone) on the growth, development
and sex reversal of RAS-reared rainbow trout fry and fingerlings was studied.
It was found that a 7 mg/kg feed dosage of the preparation with a 3-month
exposition resulted in sex-reversal-related changes in the gonads of trout fry
with occurrence frequencies of 25–42,9% of the studied specimens. The
androgen-treated trout showed no difference from the control in terms of their
morphometric indices. The survival of treated fry was 11.5% higher than that
of the control, even when infected with ichthyophthiriosis, and the frequency
75
of morphological abnormalities was also lower in the treated fish than in the
control, which indicates that the hormone had no negative impact on the trout
fry. No significant difference was found in the biochemical and
haematological parameters of the treated and control fish, either.
The results of molecular genetic studies on the Y-specific OmyY1 locus of
trout fry allow an effective removal of live males at the fingerling stage from
small samples of marked individuals.
The results of histological and genetic sexing studies performed on the
same trout fingerlings showed a 100% match.
New knowledge was obtained on the possibilities of modifying the genome
expression of salmonids using androgens in RAS conditions.
A methodology of live removal of males of salmonids at early development
stages using molecular genetic methods is being developed.
The role of androgens in the growth and development of coldwater fishes
and the sex reversal at fry and fingerling stages was evaluated on the example
of rainbow trout.
Table VII. The results of rearing of the treated and control stocks of rainbow trout on
experimental feeds at the Bogushevskiy Fish Hatchery
Stock Tank Area, Stocked Average weight of Harvested Survival Total
no. m2 fry, fry, g fingerlings, rate, % biomass of
ind. ind. fingerlings,
kg
initial final
Treated 6 28 11100 5.2+1.0 132.8+22.6 7727 69.6 1020.0
Control 7 28 11000 5.2+1.2 142.0+34.8 6387 58.1 907.0
References
Brunelli, J.P., Wertzler, K.J., Sundin, K., Thorgaard, G.H., 2008. Y-

specific sequences and polymorphisms in rainbow trout and Chinook
salmon. Genome, 51: 739-748.
Brunelli, J.P., Steele, C.A., Thorgaard, G.H, 2010. Deep divergence and
apparent sex-biased dispersal revealed by a Y-linked marker in rainbow
trout. Molecular Phylogenetics and Evolution, 56: 983-990.
Council of Ministers of the Republic of Belarus, 2010. Resolution 1453 of
07.10.2010 of the Council of Ministers of the Republic of Belarus on the
approval of the State Programme for the Development of Fisheries
Activities in 2011-2015. http://poseidon.by/content/20101007-ob-
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utverzhdenii-gosudarstvennoi-programmy-razvitiya-rybokhozyaistvennoi-
deyatelnost; accessed: 20.01.2012. (In Russian)
Hisar, O., Yanik, T., Kocaman, E.M., Arslan, M., Slukvin, A.,
Goncharova, R., 2012.Effects of diludine supplementation on growth
performance, liver antioxidant enzyme activities and muscular trace
elements of rainbow trout (Oncorhynchus mykiss) juveniles at a low water
temperature. Aquaculture Nutrition 18 (2): 211-219.
Limanskiy, V.V., Yarzhombek, A.A., Bekina, E.N., Andronnikov, S.B.,
1984. Instructions on physiological and biochemical analyses in fish.
Мoscow, VNIIPRKH, pp. 58. (In Russian)
Metalnikova, K.V., Burtsev, I.А., Slizchenko, А.G., 1989.Methodological
recommendations on obtaining monosex female offspring in steelhead
trout. Мoscow, VNIRO, pp. 14. (In Russian)
Metalnikova, K.V., Privezentsev, Yu.A., 2010. Method of obtaining
multiply used sex-reversed fish. Patent 2402203 of the Russian Federation.
(In Russian)
Pausheva, Z.P., 1988. Practical methods in plant cytology. Moscow,
Agropromizdat, pp. 270. (In Russian)
Pravdin, I.F., 1966. Handbook on studying fish. Moscow, Pishchevaya
Promyshlennost’, pp. 376. (In Russian)
Roskin, G.I., Levinson, L.B., 1957. Microscopy techniques.Moscow,
Sovetskaya Nauka, pp. 489 (In Russian)
State Committee on Science and Technology of the Council of Ministers
of the USSR, 1978.Unified methods of water analysis in the USSR.
Leningrad, Gidrometeoizdat, pp. 144. (In Russian)
77
Recirculation aquaculture systems in Center of Aquaculture and
Environment Engineering, Warmia and Mazury University in Olsztyn,
Poland
Dariusz Kucharczyk, Daniel Żarski, Sławomir Krejszeff
Department of Lake and River Fisheries, University of Warmia and Mazury

in Olsztyn, Poland
Abstract
The Faculty of Fisheries was established in 1951 as one of four major faculties at The
School of Agriculture in Olsztyn. From the very beginning, the Department of Fisheries was
incorporated in the structure of the Faculty. In recent years perch has become one of the most
promising species for diversification of intensive freshwater aquaculture. This has led to the
development of basic protocols allowing artificial reproduction, rearing of larvae and general
culture techniques. However, there is still a lack of fully effective procedures of production
cycle and those aspects are still being developed. The research activity in our department,
since the mid-1990s has been mainly focused on improvement of reproductive protocols with
special emphasis put on synchronization of ovulation (where a new classification of oocyte
maturation stages for percids was developed by our team), hormonal treatment, gametes
quality as well as hatchery techniques, including gamete management and optimization of in
vitro fertilization procedures. However, very recently, due to the new possibilities which
arose in our new research facilities, we started to focus on larviculture of this species and this
field of research will be a very important part of further experiments on this species.
Figure 1. The view of the new building with the new facilities. Officially it was opened on
1 February 2012.
78
The facilities of the Department of Lake and River Fisheries are divided
into several parts. The most important of them are described below.
Laboratory of cold-water hatchery

The room includes two tanks with working capacity of app. 1000 dm 3
equipped with thermoregulation, UV sterilization and electronically
controlled lighting. In the tanks, it is possible to cool water to app. 4ºC with
chilled water. They will be used as short-term accommodation for spawners
before controlled reproduction. In the central area of the room there is
hatching equipment with standard (7 dm3) Weiss jars and long-stream
hatching apparatus (4 boxes) for salmonid eggs. The set also includes a large
shallow tank serving as receiver in which larvae can absorb the content of
yolk sac. In addition, there is one set of mini-hatchery in which small
amounts of spawn may be incubated in 0.3 and 0.7 dm3 jars.
The Laboratory of Aquaculture 1

The laboratory is equipped with a circulation system for the rearing of fry,
juveniles and breeders. The circulation system consists of storage tanks
(upper and lower) with a capacity of 1 m3 and 6 tanks for fish rearing
(3 located at the top and 3 situated at the bottom), each with a capacity of
0.4 m3 (Figure 2). Each tank may be detached from the circulation system
and supplied with a separate source of water. If necessary, water may be
cooled to the optimal temperature with a cooler powered with glycol or
heated with heaters with a total power of 6000 W. The heaters and cooler are
located in the lower storage tank together with two Syncra 12.0 pumps with
capacity of 12500l/h. Each circulation is equipped with two UV 80W lamps
and PolyGeyser model DF-6 for water filtering. The laboratory has a special
circulation system that consists of two storage tanks (lower and upper) with a
capacity of 1 m3 and two 2.7 m3 tanks and four 0.4 m3 tanks. In the
circulation system, water may be heated with heaters with a total power of
6000 W located in the lower storage tank. In this tank, there are also two
Syncra 12.0 pumps with a capacity of 12500 l/h. Each circulation system is
equipped with two UV 80 W lamps and a PolyGeyser model DF-6 for water
filtering. In the laboratory there is also a large handling table and a
refrigerator. The laboratory is used for accommodating selects and fry
rearing.
79
Figure 2. The view of a 2.7 m3 tank with Eurasian perch (Perca fluviatilis) spawners.
The Laboratory of Larviculture 2

The laboratory is equipped with a rearing circulation system for fry. The
circulation system consists of upper and lower storage 1m3 tanks and six
0.4 m3 rearing tanks (3 located at the top and 3 situated at the bottom)
(Figure 3). Each tank may be detached from the circulation system and
supplied with a separate source of water. If necessary, water may be cooled to
the optimal temperature with a cooler powered with glycol or heated with
heaters with a total power of 6000 W. The heaters and cooler are located in
the lower storage tank together with two Syncra 12.0 pumps with a capacity
of 12500 l/h. Each circulation is equipped with two UV 80 W lamps and a
PolyGeyser model DF-6 for water filtering. The laboratory includes a
hatchery which consists of upper and lower 1m3 storage tanks. The lower
storage tank is surrounded by two batteries constructed of 5 large Weiss jars
and one battery of 12 small Weiss jars. The hatchery is equipped with an
80 W UV lamp and two Syncra 12.0 pumps with a capacity of 12500 l/h.
Water is heated with a heater with a total power of 2000 W. If necessary,
water may be cooled to optimal temperature with a cooler powered with
glycol. In the room there is also a circulation system for the rearing of
larvae. This circulation consists of upper and lower storage tanks and two
rearing apparatuses (lower and upper) with a capacity of 0.3 m3. Two 500 W
heaters and two Syncra 4.0 pumps with a capacity of 3500 l/h are located in
the lower storage tank. Each circulation has a 55 W UV lamp. The lower
storage tank also includes a filtration chamber filled with 45 litres of filtration
80
medium. The outlets of the rearing apparatuses are protected with a mesh in
order to prevent larvae from escaping. In addition, the laboratory has two
water baths for incubation of Artemia nauplii. Water in the baths is heated
with a 500 W heater. Each water bath has 15 incubators, each powered with a
separate source of air. The lighting is installed above the baths. There is also
a sink and a refrigerator in the room. This laboratory provides space for the
rearing of fry and larvae as well as for incubation of spawn of species which
prefer low temperature.
Figure 3. The view of a part of the closed system used for rearing juveniles and
keeping breeders.
The Laboratory for Hatching Techniques

The laboratory is equipped with one circulation system for spawners that
consists of upper and lower storage tanks with a capacity of 1 m 3 and four
1m3 tanks (two located at the bottom and two situated at the top). Each tank
may be detached from the circulation system and supplied with a separate
source of water. If necessary, water may be cooled to the optimal temperature
with a cooler powered with glycol or heated with heaters with a total power
of 6000 W. The heaters and cooler are located in the lower storage tank
together with two Syncra 12.0 pumps with a capacity of 12500 l/h (Figures 4
and 5). The circulation system is equipped with two 80W UV lamps and a
PolyGeyser model DF-6 for water filtering. Apart from the spawner
circulation, there are also two hatcheries which consist of upper and lower
storage tanks, each with a capacity of 1 m3. The lower storage tank is
surrounded by two batteries consisting of 5 large Weiss jars and one battery
81
of 12 small Weiss jars. Each hatchery is equipped with an 80 W UV lamp
and two Syncra 12.0 pumps. Water is heated with a heater with a total power
of 2000 W. Each hatchery is supplied with a separate source of water. The
laboratory also includes two rearing circulation systems for larvae. Each
circulation system consists of upper and lower storage tanks and two 0.3m3
rearing apparatuses (lower and upper). Two 500 W heaters and two Syncra
4.0 pumps with a capacity of 3500 l/h are located in the lower storage tank.
Each circulation has a 55 W UV lamp. The lower storage tank also includes a
filtration chamber filled with 45 litres of filtration medium (Figure 6). The
outlets of the rearing apparatuses are protected with a mesh in order to
prevent larvae from escaping. In addition, the laboratory has two water baths
for incubation of Artemia nauplii. Water in the baths is heated with a 500 W
heater. Each water bath has 15 incubators, each powered with a separate
source of air. The lighting is installed above the baths. There is also a large
handling table, a sink and a cabinet for laboratory tools in the room. The
laboratory is used for collecting male and female gametes from spawners and
incubation of spawn as well as rearing, hatching and incubation of Artemia
nauplii.
Figure 4. The hatchery unit equipped with Weiss jars.
82
Figure 5. Incubation of Eurasian perch eggs.
Figure 6. Mass rearing of asp (Aspius aspius) larvae.
83
The Laboratory for Biotechniques of Fish Reproduction
In this laboratory there are two circulations for spawners which consist of
upper and lower storage tanks with a capacity of 1 m3 and four 1 m3 tanks
(two located at the bottom and two situated at the top) for keeping spawners
(Figure 7). Each tank may be detached from the circulation and supplied
with a separate source of water. If necessary, water may be cooled to the
optimal temperature with a cooler powered with glycol or heated with heaters
with a total power of 6000 W. The heaters and cooler are located in the lower
storage tank together with two Syncra 12.0 pumps with a capacity of
12500 l/h. The circulation is equipped with two 80 W UV lamps and a
PolyGeyser model DF-6 for water filtering. There is also a handling table, a
sink and a cabinet for laboratory tools in the room. The laboratory is used for
collecting male and female gametes from spawners (Figures 8 and 9). Many
different species are reproduced in this laboratory, e.g. perch (Figure 8),
pikeperch, pike, African catfish, eel (Figures 9-11) and many cyprinids:
common carp, crucian carp, goldfish, asp, barbel, nase, dace, ide and others.
Figure 7. The unit used for fish reproduction
84
.
Figure 8. Eurasian perch females before artificial spawning.
Figure 9. Female of European eel (Anguilla anguilla) before stripping of eggs.
Figure 10. Stripping the eggs of a European eel female.
85
Figure 11. Observation of the first cleavage of European eel eggs.
Quarantine
Because, in many cases, the spawners were collected from the wild or pond
culture, it was necessary to keep the fish before further research. For this
reason, a separate “Quarantine Room” was established (Figure 12). In this
room there are three separate circulations for spawners which consist of
upper and lower storage tanks with a capacity of 1 m3 each. Each tank was
supplied with a separate source of water. If necessary, water may be cooled to
the optimal temperature with a cooler powered with glycol or heated with
heaters with a total power of 6000 W. The circulation units are equipped with
two 50W UV lamps and separate outside biofilters.
Figure 12. The tanks in the “Quarantine Room”.
86
Live food production units
For developing the protocol of larvalrearing, live food is necessary. For this
reason,a special unit for its production was established (Figures 13 and 14).
The production was done in optimal photo-thermal conditions.
Figure 13. The unit for production of live food: algae, rotifers and artemia.
Figure 14. Small-scale rotifer culture.
87
“Emergency system”
The reliability of closed systems is guaranteed by safety systems. The first
of them will be an external power generator (Figure 15). Additionally, the
most important equipment such as pumps are included in a specific grid,
supplied in the event of failure of the UPS devices (Figure 16). They allow
continuous operation of the pump for at least several hours. At the same time,
the alarm system is passed on to employees. This allows for safe and reliable
operation of the equipment used by us.
Figure 15. The external power generator.
Figure 16. The pump switch in the UPS emergency system.
88
Actual issues in recirculating aquaculture –
Summary of the roundtable discussions at the AQUAREDPOT
workshop
During the AQUAREDPOT workshop, two roundtable discussions were held

to evaluate the application possibilities of recirculating aquaculture systems
in research and production, as well as to identify the most important issues
faced by the industry.
Roundtable 1 – General issues and research applications
At the session moderated by Mr. Ep Eding, the participants discussed the

most important problems and issues with recirculating aquaculture and its
possible uses in research.
Water costs and water quality issues were extensively discussed and the
situation was compared in the countries of the participants. Possibilities of
reducing the costs by finding alternative uses to the produced wastes were
proposed, e.g. sludge from treatment facilities could be sold as fertilizers or
used in biogas plants. It was also noted that, thanks to the rich water
resources of CEE countries, they could efficiently treat their effluents in
natural systems instead of applying highly technological and costly artificial
systems.
The importance of careful planning, applying customized solutions, cost-

benefit analyses and market analysis during the establishment of new RAS
systems was underscored during the discussion. The viability of a fish farm
strongly depends on its size and the chosen solutions. While small or very
large farms tend to be more successful than medium ones, innovative
solutions can even ensure the viability of farms that, based on their
production parameters, would not be expected to be sustainable. In particular,
the example of Tropenhaus (Switzerland) was mentioned, which, while only
producing 20 tonnes of fish, manages to be sustainable through using thermal
water, integrated production of fish and tropical fruits and receiving visitors.
Economic issues were also touched upon as an important limiting factor of

RAS development. Considering food miles, carbon footprint and logistics, it
is important to organize fish production close to the markets. As the example
of Greece shows, even marine fish such as seabass can be economically and
competitively grown inland. It was also stressed that economic investment
should be mainly directed towards growing fry and fingerlings for sale, as
89
younger age groups grow better, cost more and allow to maximize profits.
Thus, small fish should be reared in RAS, while bigger fish could be grown
in ponds. The example of the existing 4 large trout-producing RAS in Russia
was cited, all of which are specialized on producing seed.
It was recognized that RAS required a significant initial investment, which is

an important obstacle to the use of this technology by CEE farmers. The role
of governments in facilitating this process was discussed. It was noted that
EU regulations prevented providing direct subsidies for production, but
innovations could be subsidized creating an environment favouring the
movement of the industry into a new direction. Particular reference was made
to Denmark, where the transformation of flow-through farms to RAS was
supported by the government by providing 50% cofinancing for the
establishment of model farms producing 500-1000 t of fish in RAS, which
started the process of transforming the whole industry.
The use of RAS in research was extensively discussed and several directions
to be studied in a research RAS were proposed. These included fish health
and disease prevention, gene banking and molecular genetic research on elite
broodstocks, breeding technology including fingerling rearing and table fish
production, testing commercial feeds in different environments, technology
development (energy efficiency, for new species, tank polyculture, water
filtering, etc.).
The importance of the cooperation between the research sector and the
industry in determining research priorities was highlighted and the situation
in different countries was discussed. The good example of Norway and
Poland was recognized. In Norway, researchers closely cooperate with the
industry. There is a research fund financed from industry contributions,
supporting research needed by the industry. In Poland, there are several
strong producer associations, e.g. for trout, carp, intensive farmers or lake
fisheries, whose management also includes researchers. Annual practical
conferences are held on topics that are of interest for the farmers, their
thematics is also developed in cooperation of researchers and producers.
Roundtable 2 – RAS and aquaculture production
During the session chaired by Dr. Laszlo Varadi, the main focus was on the
use of recirculation technologies in commercial aquaculture production. It
was recognized that recirculating aquaculture has been used in the CEE
region for a long time for different purposes and different species, both in
research and in production. Its further development is supported politically,
90
there are government projects available and there is cooperation between
scientists and the industry. There was a consensus that there was a big scope
for RAS development in the region and that we are only at the beginning of
the process.
However, a number of problems were also noted, among others, the difficulty
of finding investors and reliable partners for the technology transfer. Several
examples of failures due to suppliers were mentioned. NACEE was proposed
to help investors by listing reliable/blacklisting unreliable partners. While this
was recognized to be out of the scope of NACEE, workshops applied in the
Netherlands or the United States were quoted as a possible solution, where
planners are invited to present their designs for a system with the same given
parameters, which are then evaluated by scientists and producers. Such an
exercise allows comparing the designs and choosing the best expert.
The importance of taking local factors – such as the availability of land and
water, less stringent regulations, etc. – into account was highlighted. These
allow to develop simpler systems than in the EU, as they – for example – do
not need to target zero discharge.
Technological factors contributing to the sustainability of commercial

systems were discussed. Among these, the importance of continuously
maintaining an optimum stocking density was noted, not only for economic
reasons, but also from the point of view of biofilter capacity, which was also
recognized as one of the crucial technological bottlenecks in RAS.
A significant part of the discussion focused on economic issues. It was noted

that, as RAS was a high input – high output technology, it was profitable only
with more expensive fish, e.g. sturgeons, in particular for caviar production,
or high-value stocking material. The importance of careful cash-flow
forecasting and financial management was also strongly emphasized.
The participants recognized that RAS was not a universal solution and
needed to be customized to the actual situation, species and life stage to be
profitable.Failures are frequently attributed to the expensiveness of the
system, while often the reason is poor economic management and excessive
expectations. It is important to be aware of the technology’s limitations and
embark on it only after careful economic and market analyses, otherwise the
resulting failures will not only cause economic loss but may also harm the
public image of recirculating aquaculture.
91
Existing systems for rearing different species (sturgeons, pikeperch, eel,
African catfish, turbot, yellowtail, seabass, sea bream) were reviewed. It was
concluded, that there was no universal recipe for success. While high demand
and high price may justify production of a certain species in RAS, it will also
increase competition, gradually pushing down the prices, as happened with
African catfish. Marketing is essential, the most important is to find one’s
own market niche.
The possible role of NACEE and the AQUAREDPOT project in the

development of RAS technology in CEE was highlighted. It was proposed
that NACEE should organize workshops dedicated to more specific topics,
e.g. RAS in sturgeon breeding. NACEE could also help investors in
identifying reliable suppliers, as well as establish an expert group or working
party on RAS within the network.
92
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Development of industrial ﬁsh culture in Belarus

Technical Report · January 2013

Sex identification of sturgeon View project

The user has requested enhancement of the downloaded file.

Based on the presentations of the AQUAREDPOT workshop held

Research Institute for Fisheries, Aquaculture and Irrigation

Published by HAKI, Szarvas, 2013

Ideas behind the AQUAREDPOT project

Research projects and demonstration activity planned in the RAS to be

Production of stocking material in RAS as unity of selective breeding and

Nitrite-induced methemoglobinemia of freshwater fishes reared in

Application of recirculating aquaculture systems (RAS) in Polish sturgeon

Development of industrial fish culture in Belarus

Recirculation aquaculture systems in the Center of Aquaculture and

Actual issues in recirculating aquaculture –

HAKI is a regional centre of excellence in aquaculture research

Dénes Gál, Gergő Gyalog, László Váradi

Research Institute for Fisheries, Aquaculture and Irrigation, Szarvas,

Aquaculture is a recognised future option to provide animal protein to the

Research Instutute for Fisheries, Aquaculture and Irrigation, Szarvas,

Fish production and development options

Establishment of technical facilities for fulfilling the above tasks

Additionally, a RAS comprising of 27 small tanks will be constructed for the

Program for the utilization of these facilities

Golod, V. М.,Terentyeva, Е. G., Krupkin,V. Z.

Federal Centre of Fish Genetics and Selection, Ropsha, Russia

Keywords: Rainbow trout, fish seed, RAS, selection

Table I. Characteristics of 3-year-old females of Family 12

Table II. Characteristics of 3-year-old females of Family 14

Table III. Characteristics of the families of the first inbred generation

Table IV.Characteristics of 2-year-old Rostal trout females

Thus, the combining of the technical possibilities of RAS and targeted

Yuriy Fedkovych Chernivtsi National University, Chernivtsi, Ukraine

Keywords: nitrites, methemoglobin, glutathione, ascorbic acid.

Material and methods

Results and discussion

Figure 1. Methemoglobin content in the erythrocytes of freshwater fishes at different NaNO2

Alexander, J., Benford, D., Cookburn, A., 2009. Nitrites as undesirable

Kolman, R., Zdanowski, B.

The Stanisław Sakowicz Inland Fisheries Institute, Olsztyn-Kortowo, Poland

The development of sturgeon farming in Poland is closely related to recirculating

Keywords: RAS, sturgeons, artificial propagation, rearing, egg collection

Fields of RAS application in Polish sturgeon culture

Figure 2. Growth rate of Siberian sturgeon fry in RAS tanks

The composition of individual technological lines for seed production

Figure 3. Studying the maturity stage of a sturgeon female by ultrasound diagnostics.

Figure 4. Studying the maturity stage of a sturgeon female using a probe.

Figure 5. Rotationally moulded tanks in the broodfish keeping unit.

As adult sturgeon are characterized by a relatively low intensity of

Figure 6. Egg collection with the Podushka (1999) method.

Egg incubation unit

Figure 7. Russian sturgeon eggs in McDonald jars

Larval and fry rearing unit

Figure 8. RAS in the sturgeon larval and fry rearing unit

The juvenile development stages of fishes and, in particular, sturgeons, are

Weight Average oxygen Average ammonia

Development and use of all-female stocks for caviar production

Kolman R., 1978. Podchów wylęgu pstrąga jesiennego tarła w układzie

Kostousov, V.G.1, Barulin, N.V.2

Keywords: fish, industrial fish culture, recirculating aquaculture systems

Figure 1. Dynamics of fish production in RAS, 1987–2000.

Figure 2. Growth prospects of RAS production