Vol. 6, No. 2
ORIGINAL RESEARCH
Age, reproduction and fecundity of the spined
loach Cobitis taenia L. (Pisces, Cobitidae) from
Lake Klawój (Poland)
Dorota Juchno1, Alicja Boroń
Department of Zoology, University of Warmia and Mazury in Olsztyn,
Olsztyn, Poland
Received: 15 March 2006; accepted: 16 June 2006
SUMMARY
This is the first study concerning the features of the reproduction process
of the karyologically identified spined loach C. taenia (2n=48). The
histology of 71 ovaries, and gonadosomatic index (GSI) of karyologically
identified spined loach Cobitis taenia L. from Lake Klawój (Northern
Poland) were examined. The absolute and relative fecundity of 25 females
was estimated by gravimetric method. The age of fish was determined
according to the annual increments of otholits. The spawning of C. taenia
from Lake Klawój took place from May to July, at a water temperature
exceeding 18.5°C. The GSI values at the beginning of the reproduction
period ranged from 7 to 19%. The average absolute fecundity of females
was 2078 eggs, with the number ranging from 869 to 3371 eggs. High
individual variability in the gonad histology and the GSI values during
the reproductive period was observed. Such variability could be the
result of beginning the reproduction process in the fish at various times
Corresponding author: Department of Zoology, University of Warmia and Mazury, ul. Oczapowskiego 5, 10-957 Olsztyn, Poland; e-mail: [email protected]
1
Copyright © 2006 by the Society for Biology of Reproduction
134
Reproduction and fecundity of spined loach
and, probably, due to the various numbers of batches laid and various
numbers of eggs per batch. Reproductive Biology 2006 6:133–148.
Key words: Cobitis taenia, reproduction, fecundity, ovary, age, karyology
INTRODUCTION
Morphologic and cytogenetic studies showed that separate bisexual species
and numerous polyploid forms (biotypes) of Cobitis are found within the
range of occurrence of what was thought by Berg [2] as a polytypic species
Cobitis taenia [7, 8, 24, 32]. In Poland, the diploid-polyploid Cobitis
complex including C. taenia and Danubian loach C. elongatoides Bacescu
& Maier as well as loach C. taenia and probably C. tanaitica Bacescu &
Maier have been found in several tributaries of the Vistula [6, 7] and the
Oder [7, 8, 9] Rivers. Polyploid fish are not distinguishable [7, 8, 32] by
their morphological features from diploid and other polyploid individuals.
One way to distinguish diploid and polyploid forms is by determining
the karyotype. The spined loach C. taenia karyotype consists of 2n=48
chromosomes [5, 31]. Among the sixteen Cobitis populations which have
been karyologically identified, those containing C. taenia have been found
only in Lakes Klawój, Wigry and Głębokie [7, 8].
Spined loach C. taenia inhabits the sandy bottom or silt bottom of rivers
and lakes, but not mountain brooks [3, 7, 10]. The fishes do not grow large,
usually about 12 cm. They lead nocturnal life and for most of the day they
remain buried in the sand or silt. The loaches achieve sexual maturity in
the first (males) or second (females) year of their life [10, 19].
The published research results which concern the reproduction and/
or fecundity of the taxa from genus Cobitis did not take into account
the level of ploidy [14, 16, 19, 20]. The only study which describes the
fecundity and histology of ovaries deals with the karyologically recognized
(diploid) female individuals of Danubian loach C. elongatoides [15]. The
loach currently belongs to the group of endangered species [32] and is
protected in Poland. It is endangered mainly because of the deterioration
of its environment. Another threat factor with growing occurrence is the
observed domination of polyploids of Cobitis, or rather that of unisexual
(female) populations of triploid hybrids [7, 8, 23].
Juchno & Boroń
135
In this study we examined the histology of ovaries and the gonadosomatic
index during the reproductive period of the spined loach Cobitis taenia
L. Moreover, we determined the individual fecundity and the reserve of
female reproductive cells and provided the characteristic features resulting
from the batch-spawning of karyologically identified loach C. taenia.
MATERIALS AND METHODS
The loach C. taenia is a protected species in Poland; the catching of the fish
was allowed by a permit from the Ministry of Environment DLOPiKog.
- 4201 - 04 - 43/01. The study was conducted upon the consent from the
local Commission of Ethics; individual consent no. 20/01.
Altogether, 138 females, were caught from Lake Klawój (Northern
Poland), from May 2001 to September 2002 with a special fry net, with
mesh size of 0.2 cm (tab. 1). The water temperature at the fish catching
Table 1. Standard lenght (Sl), total lenght (Tl), body weight (W) and gutted weight
(Wo) (mean±SD) of the spined loach C. taenia (2n=48) females from Lake Klawój
age
N
1+
2+
3+
4+
5+
5
16
40
75
2
Sl
(mm)
51.9±2.96
61.0±3.31
73.0±4.94
82.0±3.52
89.2±3.61
Tl
(mm)
59.4±3.15
69.6±4.00
83.2±5.89
93.4±4.08
101.4±4.17
W
(g)
1.1±0.15
1.7±0.57
3.2±0.69
4.5±0.60
6.3±1.19
Wo
(g)
1.0±0.14
1.4±0.40
2.6±0.51
3.5±0.47
4.6±0.31
N: number of specimens, SD: standard deviation
site was measured each time. The chromosome preparations were made
by the ‘splash’ technique, described by Ráb and Roth [24] and modified
by Boroń [6]. All the fish caught in Lake Klawój had 2n=48 chromosomes
and belonged to the C. taenia species. The fish were measured (Tl: total
length, Sl: body length to the nearest 0.1 mm) and weighed (W: body
weight, Wo: gutted weight, to the nearest 0.1 g). In order to determine the
gonadosomatic index (GSI=Wg/W × 100), ovaries were weighed (Wg)
136
Reproduction and fecundity of spined loach
with 0.01 g accuracy. The fish’s age was assessed based on the annual
growth of otholits [22, Jeleń et al., unpublished].
Seventy-one ovaries taken for the histological examinations were
preserved in buffered formalin or in Bouin’s solution. Hematoxylin and
eosin (HE), as well as Mallory’s method, were applied to stain 7 μmthick histological slices [34]. The preliminary examinations revealed the
absence of any significant statistical differences (Student’s test, p>0.05)
between the sizes of oocytes in the posterior and anterior part of an ovary.
The histological slides were used to determine the development stages of
reproductive cells and gonads against the maturity scale of the ovaries
of bony fishes, developed by Sakun & Buckaja [28] and Rinchard &
Kestemont [25]. The oocyte development stages, marked with letters from
A to E, were adopted after Pimpicka [21], introducing some alterations
to facilitate the presentation of results. The developmental stages of
oogonia (A), previtellogenic (B) and vitellogenic oocytes (CD1, D2 and
E) were distinguished; the latter according to the level of accumulation
of reserve substance. The CD1 and D2 stages represent the oocytes of
endogenous vitellogenesis (during the vacuolization stage). These stages
were distinguished by the degree of cytoplasm filled with cortical follicles
(vacuoles): in CD1 oocytes, vacuoles filled up to 50% of the cytoplasm,
and in the D2 oocytes, 50-100% of the cytoplasm was filled with vacuoles.
Oocytes during exogenous vitellogenesis, i.e. the ones which were filling
with yolk were considered to be in the E stage.
The fecundity of female loach from Lake Klawój was estimated in
the fish caught on 2 and 15 May and 1 June 2001 and those caught on
26 April and 31 May 2002. Altogether, the absolute fecundity (Fa) was
estimated in 25 ovaries by the gravimetric method [11]. The histological
slides confirmed that the females were in the prespawning period and the
ovaries were in the III or IV stage of maturity. The histological analysis
of the ovaries specified that oocytes of 0.20 mm in diameter which begin
the stage of vitellogenesis (vacuolization) and can be laid during a given
reproductive season, were counted. The relative fecundity (Fr) was
expressed by dividing the absolute fecundity (Fa) by the fish body weight.
The result was the number of eggs per 1 gram of body weight.
Juchno & Boroń
137
The reserve of previtellogenic (B) oocytes was evaluated for future
reproductive seasons of C. taenia taking into account the estimated values
of Fa and the percentage of previtellogenic (B) and vitellogenic (CDE)
oocytes (Juchno et al., unpublished). The average fecundity was adopted
as Fa=2078 and the average share of 60% of B oocytes and 40% of CDE
oocytes (average values for the fish for which the Fa was calculated).
Statistical analysis
The average lengths and weights of the bodies of fish of various ages were
compared by Student’s t-test (in relation to a grouping variable; p<0.05). In
order to compare the significance of differences in the GSI index between
samples taken on various days, the analysis of variance and Tukey’s test were
applied for samples of various sizes (p<0.05). The strength and significance of
the relationship between the absolute fecundity (Fa) and selected individual
features of the females included in the study (body length and weight, the
gonad weight and the fishes’ age) was analyzed by determining Pearson’s
correlation coefficient r (p<0.05) and regression equations. The data were
analysed with the STATISTICA 6.0 and Microsoft Excel 2000 software.
RESULTS
Age and size of fish
The body length and body weight of sexually mature females from Lake
Klawój fluctuated from 53.3 to 91.7 mm and from 1.2 to 5.4 g, respectively.
Their age ranged from 1+ to 5+. The largest number of females were in the
fourth (3+) and fifth (4+) year of their lives, and the smallest number – in
the second (1+) and the sixth (5+) year (tab. 1). The body length increased
proportionally to the age. The older loaches were significantly (p=0.000005)
longer than those which had just reached sexual maturity (in the second (1+)
and third (2+) year of life). The body weight of the youngest females, at the
age of 1+ and 2+ did not differ significantly (p=0.0526). However, the older
fishes were significantly (p=0.000001) heavier than the young ones.
138
Reproduction and fecundity of spined loach
Reproduction
At the beginning of the reproductive period (April, May), oocytes began
or continued their exogenous vitellogenesis (intensive yolk accumulation).
The ovaries were in the IV (fig. 1a), and in some females – in the III stage
of maturity. The mean value of the GSI index did not exceed 10%.
Figure. 1. Cross section of ovaries of C. taenia from Klawój Lake during reproductive
period: (a) 15 May: IV maturity stage, (b) 31 May: IV2 maturity stage, (c) 4 July: IV2
maturity stage, (d) 17 July: IV2 maturity stage, (e) 17 July: VI/III maturity stage, (f)
13 August: VI/III maturity stage. B, CD1, D2, E: stages of oocyte development; α, β:
atretic oocytes; f: postovulatory follicles; scale bar=150 µm
Juchno & Boroń
139
During the spawning period, the ovaries of the females contained
previtellogenic oocytes (B) and oocytes in various stages of vitellogenesis
(D, E) as well as postovulatory follicles (f) – an indication that a batch
of eggs had been laid. Such histology of gonads is typical of stage IV2
(fig. 1b). In the first year of study, the first postovulatory follicles were
observed in some females as early as in mid-June, when the temperature
of water reached 18.5°C. In the second year of study, first postovulatory
follicles were observed at the end of May and the beginning of June,
when the temperature of water was higher – 21°C. During that time, some
irregularities (asynchronicity) were observed in the maturing of the germ
cells. Apart from previtellogenic (B) and vacuolized (D) oocytes, some
others filled with various amounts of yolk (E; fig. 1c). The ovaries of those
females also contained oogonia (A).
At the beginning of June 2001, the GSI reached the statistically
significant average value of about 16%, with the values ranging from
13 to 18%. In July, the ovaries became clearly different in terms of
their histological features. Some loaches were preparing to lay another
batch of eggs (their ovaries were in maturity stage IV2 – fig. 1d), and
the gonadosomatic index ranged from 10 to 15%. The process of
vitellogenesis was favored by the high temperature of water (~24°C) in
Lake Klawój. However, the ovaries of females in mid-July indicated the
end of the spawning period (VI stage of maturity – fig. 1e) and showed
the GSI ranged from 1 to 3%.
During the post-spawning period (August and September), the ovaries
of all the loaches were at the post-spawning stages. They were filled
mainly with previtellogenic oocytes but also with many postovulatory
follicles and oocytes at various stages of atresion (fig. 1f) and a small
number of D and E oocytes. There were also oogonia and oocytes in
early prophase. The GSI index did not vary widely and ranged from 1
to 6%. The histological examination revealed that during the winter and
spring the ovaries of the loach C. taenia from Lake Klawój must have
remained in the III stage of maturity. No age dependent differences were
found in reaching the maturity stages by ovaries and in the values of
gonadosomatic index.
Reproduction and fecundity of spined loach
140
Absolute (Fa) and relative (Fr) fecundity
The fish used to estimate fecundity were 3+ or 4+ years old. Individual
values of the Fa varied in a wide range from 869 to 3371 eggs in 2001 and
from 1415 to 3150 eggs in 2002. The average Fr of the female loaches
ranged from 254 to 1062 eggs per 1 g of body weight in the first year
and from 378 to 782 eggs per 1 g of body weight in the second year of
the study. As no statistically significant difference (p>0.05) was shown to
exist between the absolute Fa in the first and second year, in subsequent
analyses these samples were examined collectively. The average Fa and Fr
values for the females in Lake Klawój were 2078 eggs (SD±684) and 629
eggs (SD±208) per 1 g of body weight, respectively (tab. 2).
It was observed that only a slight increase in the absolute fecundity
accompanied the growth of age, weight and length of the female loaches. The
correlation analysis of Fa and selected individual features of the females (Sl,
W, Wo, Wg) confirmed the absence of any relationship between Fa and the
Table 2. Absolute (Fa) and relative (Fr) fecundity in particular ranges of body
lenght (Sl), body weight (Wo) and age of C. taenia females from Lake Klawój
Sl
(mm)
70.1 - 80.0
80.1 - 90.0
N
10
15
Wo
(g)
2.1 - 3.0
3.1 - 4.0
4.1 - 5.0
6
17
2
age
N
3+
4+
5
20
N
range
968-3345
869-3371
range
968-2209
869-3371
2237-3150
range
968-2147
869-3371
Fa
mean±SD
2054±637
2094±735
range
347-968
254-1062
mean±SD
1789±459
2108±728
2694±646
range
347-888
254-1062
543-745
mean±SD
1725±466
2166±710
range
347-752
254-1062
Fa
Fa
N: number of specimens, SD: standard deviation
Fr
Fr
Fr
mean±SD
696±186
584±216
mean±SD
665±180
614±230
644±143
mean±SD
617±160
632±222
Juchno & Boroń
141
body size in the fish (tab. 3). Only the relationship between the Fa and Wg
was statistically significant and linear. The value of the r coefficient for this
relationship, with the level of significance 0.05 equalled 0.5379 (tab. 3).
Table 3. Correlation coefficients r and regression equations for relationships
between absolute fecundity (Fa) and: body lenght (Sl), body weights (W, Wo),
weight of ovary (Wg) and age
relationship
Fa – Sl
Fa – Wo
Fa – W
Fa – Wg*
Fa – age
N
25
25
25
25
25
r
0,0603
0,1608
0,2768
0,5379
0,2635
linear regression
y=10,497x + 1231,2
y=228,96x + 1311,3
y=0,0003x + 3,8356
y=1505,7x + 1304,6
y=441,39x + 400,81
*p<0.05.
The reserve of previtellogenic oocytes B for future reproductive seasons
of C. taenia was about 3117, which constitutes the oocyte pool for nearly
two years.
DISCUSSION
Age and size of fish
Females of C. taenia achieve maturity later than the males; at the age of
2+, Sl is about 60 mm and body weight about 2 g [10, 26]. The loach is a
short-lived species, most of the caught fish were 3+ years old, some were
4+ and only a few of them were 5+ years old [26]. The oldest females (1%)
among speciments in this study were 5+ years old but most of them (54%)
were in the fifth year of their lives.
Reproduction
This is the first study concerning the features of the reproduction process
of the karyologically identified spined loach C. taenia. Spawning of C.
142
Reproduction and fecundity of spined loach
taenia takes place from May to July, usually in shallow littoral, among the
submerged vegetation where the water temperature is at least 16-18°C [3,
10]. A similar time of spawning was observed for C. bilineata Canestrini
from northern Italy [19]. A later spawning period (June-July) was observed
for C. taenia from the United Kingdom [26]. Ekmekçi and Erk’akan [13]
showed that the C. simplicispina from Turkey begins to spawn in April,
when the water temperature is 15°C and finishes in May. The spawning
period of C. elongatoides from the Czech Republic takes place from May
to July [15].
Postovulatory follicles, which indicated that the first batch of eggs had
been laid, were observed in mid-June in the first year of the study, when the
water temperature reached 18.5°C. In the second year they were observed
at the end of May and the beginning of June, when the water temperature
was 21°C. A higher water temperature is believed to be the main factor
which sped up the beginning of the spawning process in the second year of
the study. The spawning period in Lake Klawój lasted until the middle (in
some fish even to the end) of July. As late as in mid-July, in some females
were observed ovaries filled with oocytes ready to be laid. However, in
August, the oocytes of all the loaches under study indicated the end of the
reproductive period and the transition to stage VI (rest) of gonad maturity.
In Lake Zegrze, female loaches’ ovaries were filled with mature eggs in
August [10]. The development stages of gonads did not correspond with
the age and/or size of the examined females.
The loaches of the genus Cobitis spawn in batches [10, 14, 15, 19, 26].
This type of spawning is indicated by the asynchronous ovary maturation
and the presence of the postovulatory follicles appearing during the
reproductive period [10, 15, 19]. The annual cycle of ovary development
of the fish spawn during spring and summer, can run follow two paths. In
the first path, ovaries start accumulating the reserve material, including
yolk, soon after laying eggs in autumn and the process lasts throughout
winter. In other species, in autumn and winter, ovaries are filled with
previtellogenic oocytes and those during the endogenous vitellogenesis,
but they do not accumulate yolk. Exogenous vitellogenesis is very short
and usually takes place in early spring [25]. The histological analysis of
Juchno & Boroń
143
fish gonads, presented in this paper, reveals that ovaries of the loach from
Lake Klawój were in stage III (rest) of maturity from late autumn to early
spring. Similarly, in C. paludica (de Buen) [20] and C. simplicispina Hankó
[13] in the resting period in autumn and winter, no oocytes with yolk were
observed in gonads.
The type of spawning and the period of the reproductive season are
determined by the histological structure of an ovary during the reproductive
period [25]. In all the fish with batch spawning, the maturing oocytes,
which constitute a portion of the eggs laid at a spawning site, are gradually
replenished from the reserve of oocytes at the stages of endogenous
vitellogenesis [25], or previtellogenic oocytes, reaching the final stage of
maturity very quickly. From April to September, oocytes in various stages
of vitellogenesis and previtellogenic oocytes were found in the ovaries
of the fish under study. Similarly, in the ovaries of C. paludica [20] and
C. simplicispina [13] translucent stages, i.e. previtellogenic oocytes,
were observed throughout the year. However, Soriguer et al. [30], did not
find translucent oocytes in June, when oocytes with yolk dominated in
the ovaries of C. paludica. These oocytes appeared in early autumn and
dominated until vitellogenesis started [30].
Determining the number of egg batches laid by fish which spawn in
batches is difficult. It is usually suggested that the fish which belong to the
genus Cobitis lay two or three batches [19, 20]. However, some authors
stress the differences in the number of batches laid by females from one
population (under laboratory conditions) [4, 5] and the difference in the
number of eggs per batch [4, 5, 20]. Different numbers of batches depends
on the weather conditions [27].
The process of accumulating reserve substances in the ovaries of the
females can be obtained partly by tracing the changes in the gonadosomatic
index. In species which spawn in late spring and in summer such as loach,
the index remains low in winter and then rises sharply just before the
spawn [19, 25, 33]. A rapid increase in the weight of ovaries takes place
when the temperature rises and increasing amounts of food are consumed
[33]. Changes in the gonadosomatic index, calculated for the population
in which females spawn in batches, must not be used as the only credible
144
Reproduction and fecundity of spined loach
indicator of the number of batches laid. The highest GSI values from 18%
for the fish from Lake Lucień [16] to 26% in the loach from Italy [19]
were observed during the reproduction period. At the time, the values of
GSI vary widely – from 2 to 20% for C. elongatoides from the Czech
Republic [15], from 12 to 26% for C. taenia from Italy [19], but also
lower: from 7 to 17% for C. simplicispina from Turkey [13] or from 5 to
18% for Cobitis from Lake Lucień [16] and from 5.2% to 12.2% for C.
elongatoides from Hungary [14]. In the population of C. taenia from Lake
Klawój, the highest, average value of GSI (calculated at the beginning of
June, before spawning) was about 16%. However, it seems that only the
range in which the index varies reflects the typical individual variability
which is typical of the species. The GSI values for the loach from Lake
Klawój at the beginning of the reproduction period (May, June) ranged
from 3 to 18%.
The individual variability of C. taenia during the reproductive period,
observed in the histological analysis of an ovary and the GSI values is a
result of beginning the reproduction process in the fish at various times
and, probably, the various numbers of batches laid and various numbers of
eggs per batch.
Fecundity
It is difficult to determine the fecundity in females which spawn in batches and
in which oocytes develop asynchronously, i.e. such as the fish under study.
The absolute fecundity is described as the number of, usually mature, eggs,
filled with yolk or all vitellogenic oocytes found in the ovary immediately
before the reproduction process, which can be laid at spawning sites during
the season under study [1, 12]. The oogonia and previtellogenic oocytes
which remain in an ovary are the reserve for subsequent years.
In order to estimate the absolute fecundity of the loach from Lake
Klawój, all the vitellogenic oocytes with the limiting diameter of 0.2 mm
were counted. A similar limiting value was adopted by Kostrzewa et al.
[16] for Cobitis genus from Lake Lucień. Some authors included larger
oocytes – over 0.3 mm [10] or over 1 mm [3]. Other did not determine
Juchno & Boroń
145
the size of the counted eggs [17, 30] but only remarked that the estimated
number refers to all the mature eggs found in an ovary. In this study, the
average absolute fecundity of female loach from Lake Klawój was 2078
eggs, with the number ranging from 869 to 3371 eggs. No statistically
significant relationship was found between the absolute fecundity and
body size. Comparing the results of C. taenia fecundity with the available
literature data is difficult as various methods were applied. The results
concerning the absolute fecundity of the taxa within the genus Cobitis
do not deviate significantly from the results of this study. They confirm
the wide individual variability of the females in this study, which did not
always depend on the size or age of the fish.
The absolute fecundity of species from the genus Cobitis caught in
Lake Lucień [16] was comparable to the estimated value for the fish in
Lake Klawój and equaled 2180 eggs on average. However, the values
ranged widely from 418 to 6800 eggs. When only the biggest oocytes (with
diameters over 0.6 mm) were counted in order to estimate the fecundity, this
ranged from 208 to 975 (501 on average). This latter value was probably
the number of eggs in the nearest (first) batch.
The absolute fecundity of the loach from Lake Dgał Wielki was low and
ranged from 175 to 452 eggs, as only the oocytes larger than 1 mm were
counted [3]. The number of oocytes obtained in this way referred probably
only to the first batch. Only the largest mature eggs were counted in order
to estimate the fecundity of the loach in the northern Italy [19]. In this
case, the number of eggs equalled 1012. According to Lobon-Cervia and
Zabala [17], the fecundity of the largest female C. taenia equalled 1400
eggs, whereas according to Bohlen [4] the fecundity of the females of this
species was much higher and ranged from 2905 to 4258 eggs (3618 on
average). The absolute fecundity of the fish from Lake Zegrze ranged from
112 to 1520 eggs and positively correlated with the body size, length and
the age of the females [10]. The relative fecundity ranged from 28 to 204
eggs per 1 g of the body weight, 108 on average. The relative fecundity in
a related species of C. elongatoides ranged from 35 to 105 eggs [14]. The
relative fecundity in the loach from Lake Klawój was higher and equalled
629 eggs per 1 g of a female body weight.
Reproduction and fecundity of spined loach
146
A separate discussion should be devoted to the issue of the reserve of
previtellogenic oocytes in an ovary, which is a source of egg batches laid in
subsequent years. The reserve of oocytes estimated for the loach from Lake
Klawój “covered the needs” for two future reproductive seasons, which
seems sufficient or nearly sufficient for the short-living loach. However,
it cannot be ruled out that the reserve of oocytes is replenished from
multiplying oogonia, the more so that the current reserve of previtellogenic
oocytes give rise to the eggs laid in the current reproductive period.
Clusters of oogonia were present in the histological picture of ovaries of
C. taenia, particularly after a batch of eggs had been laid and after the end
of the reproductive season. According to Makeeva and Emelanova [18],
mitotic divisions of oogonia produce a reserve of germ cells. In adult fish,
oogonia were observed on the borders of egg-bearing lobes. The presence
of oogonia in the ovaries of adult fish and a possibility of replenishing the
reserve of oocytes has been confirmed by numerous authors [1, 21, 29].
ACKNOWLEDGEMENTS
The study was financed by the University of Warmia and Mazury grant
0208.0213 and statutory research project 0208.805.
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