ARTICLE
Auteur(s) : Ewa
Brucka-Jastrzębska1, Dorota Kawczuga1,
Agnieszka Grzelak2, Grzegorz Bartosz2
1Department of Physiology, Faculty of Life
Sciences, University of Szczecin, Poland
2Institute of Biophysics, Faculty of Biology
and Environmental Protection, University of Lodz,
Poland
Rainbow trout is one of the most important freshwater fish
reared in the waters of America and Europe. Trout demands water
saturation with oxygen not lower than 80%. They are also poorly
resistant to elevated water temperature. The fish grow best at
water temperatures within 13-18°C and an oxygen content above
5 mg∙dm-3. They cannot survive prolonged periods of
temperatures above 21°C, and even short term occurrence of
temperatures above 25°C makes water useless for trout culture.
Rainbow trout are able to use various kinds of natural and
artificial feeds. For fish farmers, the most important fish
features are high rates of growth and adaptation to changeable
rearing conditions [1, 2].
Magnesium is one of the most abundant elements by mass in the
vertebrata body and is essential to all living cells, where it
plays a major role in manipulating important biological
polyphosphate compounds like ATP, DNA, and RNA. Many enzymes
require the presence of magnesium ions for their catalytic action,
including all enzymes utilizing or synthesizing ATP, or those which
use other nucleotides to synthesize DNA and RNA [3, 4]. Magnesium
plays a regulatory role in oxidative processes [4-7].
This study aimed to estimate the magnesium content, total
antioxidant capacity and lipid peroxidation in tissues of rainbow
trout and the relationship between these parameters during their
growth.
Materials and methods
Animals
The study was performed on rainbow trout (Oncorhynchus mykiss
Walbaum), which were grown in privately owned fish breeding pounds
in West-Pomeranian Province, Poland. The study involved
40 fish, from 4- to 8-months-old. The fish were grown in a
commercial trout farm (Goleniów, West-Pomeranian Province, Poland).
For this research we obtained the agreement of the Local Ethics
Committee (nr 9/05). The fish were collected three times, in spring
and summer, from April till August. Each time,
10-15 individuals were collected (table
1). During the growing period, the fish were fed an Aller
Aqua 576 pelleted feed (Aller Aqua Polska Co. Ltd., Nożynko-Czarna
Dąbrówka, Poland), containing 37% proteins and 12% lipids, the
detailed composition is presented in table
2. This feed is an intensified energy feed used in normal
trout breeding conditions. The daily food ration was 2.4 ±
0.2 g per fish. Fish were fed twice a day.
Table 1 Body weights and lengths of fish during the
experimental period.
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Rainbow trout
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Age of fish (month)
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Body weight (g)
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Length (cm)
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4th months
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Spring (April and May)
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182.4 ± 21.3
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24.7 ± 1.9
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6th months
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Spring/Summer (June and July)
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266.4 ± 19.2
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21.9 ± 1.3
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8th months
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Summer (July and August)
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377.5 ± 23.5
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38.4 ± 2.8
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Table 2 Composition of feed Aller Aqua for freshwater
fish.
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Parameters of pasture Aller 576
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Largeness of grain [mm]
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M [5-7]; L [6-10]
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Means ± SD
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Protein [%]
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42.0 ± 4.5
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Fat [%]
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30.0 ± 31
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Carbohydrates [%]
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14.0 ± 2.8
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Ash [%]
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7.5 ± 0.7
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Fiber [%]
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1.0 ± 0.2
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All-out energy [Kcal/MJ]
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5823 ± 39.8/2.3 ± 0.9
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Energy digestible [Kcal/MJ]
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4833 ± 32/20.2 ± 5.7
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Nitrogen (N) [d.m. %]
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7.1
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Phosphorus (P) [-d.m. %]
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7.0
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Energy in dry mass [Kcal/MJ]
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6162 ± 45/25.7 ± 3.8
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Water parameters
Water was monitored throughout the experiment (figure 1), including
temperature (12.83 ± 7.52%), dissolved oxygen content (7.81 ±
0.35 mg∙L-1), oxygen saturation (77.81 ± 2.55%), pH
(7.88 ± 0.55) and magnesium concentration (0.54 ±
0.8 mg∙L-1).
Tissue sampling
From each individual, samples of blood, liver, kidneys and gill
lamellae were collected for assays. Before examination, fish were
in 5 m x 20 m fish breeding ponds with a water
temperature of 12 - 16°C. Prior to tissue dissection, the fish
were transferred to a separate tank, which was gradually cooled to
induce fish hibernation. In order to hibernate the fish they were
transferred to a separate tank a the water temperature of 10°C.
After 20 minutes the fish were transferred to a new tank with
a water temperature of 4-5°C. Directly after the blood collection,
the fish were decapitated and dissected. No anaesthetics were used,
as they affect biochemical parameters [8]. Blood was sampled from
the caudal vessel (a. et v. caudalis) into a heparinised syringe
(50 IU sodium heparin per 1 mL blood). Directly after the
blood collection, the fish were decapitated and dissected. When
dissecting the fish, anatomical observations of the organs and
tissues were recorded. Fish behaviour was observed throughout the
study period. The fish showed no changes in behaviour and external
appearance, and neither did their food consumption change.
Biochemical parameters
For the ferric reducing antioxidant power (FRAP) and
malondialdehyde (MDA) measurements the samples were frozen and kept
at -80°C until analysed. Tissue homogenates and erythrocyte lysates
were prepared according to Rice-Evans et al. [9]. The FRAP
method is based on the reduction of iron (III) ions [10]. The
determination involved a mixture of acetate buffer (300 mM)
with solutions of 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) and
FeCl3 × 6H2O (20 mM). Absorbance was
measured at 593 nm. The results have been converted to nmol of
Trolox Eq·mg-1 of proteins and haemoglobin. The FRAP
values in tissues were calculated from relevant calibration curves
after correcting the data with blank results. Lipid peroxidation
was measured by determination of MDA concentrations and expressed
per milligram of protein (nmol∙mg-1 protein). The
absorbance was measured at 595 nm. The MDA method is based on
the reaction of a chromogenic reagent, N-methyl-2-phenylindole,
with MDA at 45°C.
All reagents used were from Sigma-Aldrich® Chemie
GmbH (Steinheim, Germany). Total protein content was determined
with Bradford reagent containing Brilliant Blue G in phosphoric
acid and methanol (POCH, Warsaw, Poland).
For magnesium assay, the tissues were frozen and kept at the
temperature of - 20°C until analysed. Prior to the assay,
1 g tissue samples (weighed to the nearest 0.001 g) were
mineralised wet in 3 mL concentrated HNO3 in a CEM
MDS 2000 microwave oven (CEM Corporation Matthews, NC, USA).
Magnesium content in all tissues was reported as mg∙kg-1
wet weight (mg∙kg-1 wt/wt). Magnesium was determined by
inductively coupled plasma atomic emission spectrometry (ICP-MS) in
Elan 9000 Perkin Elmer Sciex apparatus (Perkin Elmer
BioSignal, Inc. Montreal, Canada). The optimized experimental
parameters for magnesium measurements are based on the method
obtained by Becker et al. [11].
The tissue content of magnesium was calculated from relevant
calibration curves after correcting the data with blank results.
The following concentrations were used in order to outline the
standard curve: 10.0 ± 0.8 mg∙kg-1 wt/wt., 80.0 ±
3.5 mg∙kg-1 wt/wt., 160.5 ±
9.5 mg∙kg-1 wt/wt. Magnesium concentration in the
tissues was converted to wet weight and given in mg per kg of body
weight.
Metal recoveries from the Crab Paste (LGC 7160) certified
reference material (Promochem GmbH, Wesel, Germany) were
quantitative (104.4 ± 10.3%). The relative standard deviations were
smaller than 10%. Experimental values averaged 366.5 ±
39.5 mg∙kg-1 wt/wt, while the certified value was
348.5 ± 21.3 mg∙kg-1 wt/wt.
Haematology
Erythrocyte counts (RBC) in 1 μL blood were counted in the
Bürker chamber, following 200 x dilutions in the Hayem fluid
[12]. The erythrocyte counts are expressed as
T∙l-1. Leukocyte count (WBC) in 1 μL blood
was counted in the Bürker chamber, following 20 x dilutions in
the Türck’s fluid. The leukocyte counts are expressed as
G∙L-1 [12].
Statistics
The results are given as arithmetic mean values (mean) and standard
deviations (SD). The data obtained were subjected to statistical
treatment involving analysis of variance (ANOVA) at the
significance level of p ≤ 0.01, and comparison of correlation
coefficients (R2). Statistical analysis was performed
using Statistica® 6.0 software.
Results
All the examined fish significantly gained body weight and length
during the study period, which indicated good ingestion of the feed
(table 1). A positive correlation
was observed between fish growth rate and the experiment duration.
Body weight increased by 14.4% (R2 = 0.898), and body
length by 25.3% (R2 = 0.868). Anatomical and
histological examination was conducted in order to eliminate
potentially sick individuals. Autopsy revealed no disorders or
disease symptoms. Moreover, the good health condition of the fish
was evidenced by basic haematological tests (RBC, WBC) [13, 14].
Haematological parameters in rainbow trout collected from both
rearing sites examined were within the reference limits for the
fish species, and did not differ significantly between the sites.
Levels of erythrocytes in the blood of rainbow trout were within
the range 3.2-5.2 T∙L-1 wt/wt. Levels of leukocytes
in the blood of rainbow trout were within the range
3.1-8.2 G∙L-1 wt/wt. Between 4th and
8th month of life erythrocyte counts decreased by 29.57%
(R2 = -0.982), while leukocyte counts increased by
12.88% (R2 = 1.000).
The concentration of magnesium in various tissues of rainbow
trout was within the range
38.7-255.2 mg∙kg-1 wt/wt (figure 2A-D). Most
magnesium was found in the gills
(201.4-255.2 mg∙kg-1 wt/wt; figure 2D), while there was
less in the blood (38.7-45.5 mg∙kg-1 wt/wt; figure 2A).
The highest FRAP values were detected in the kidney (2.7 ±
0.8 nmol Trolox Eq·mg∙protein-1), and
the lowest in the gills (1.17 - 1.60 nmol Trolox
Eq·mg∙protein-1; figure 3C, D). In the
blood and the gills, no significant changes in FRAP values were
observed with fish age. MDA concentrations in the rainbow trout
tissue homogenates averaged from 4.50 to
11.36 nmol∙mg∙protein-1 (figure 4A-D). The highest
MDA concentration was found in the gills - 5.34 to
11.52 nmol∙mg∙protein-1 (figure 4D), and the lowest
in the blood - 2.04 to
7.12 nmol∙mg∙protein-1 (0.75 to
5.23 nmol∙mg∙haemoglobin-1) (figure 4A). MDA
concentrations increased along with fish age (figure 4A, C, D).
Discussion
In this experiment, fish had been kept in pools from April till
August, when water temperatures ranged from 6 to 14°C, which
was slightly below the optimum temperature range for trout culture.
A gradual increase in water temperature by 4°C during
5 months did not induce any adverse symptoms in the fish,
whereas a decrease in oxygen levels during the last two months of
the experiment (July, August) reduced the rate of body weight gain
by 5-8%. During the experiment, an increased rate of body weight
gain was observed, similar to the data from the literature [2, 15,
16]. This increased growth rate can probably be attributed to
a favourable combination of the environmental conditions and
adequate feed. Blood physiological values (RBC, WBC) in fish are
highly dependent on individual variability, age, rearing method,
diet and season of the year [14, 15]. Erythrocyte counts in rainbow
trout fluctuate during the year, around 1.4 ±
0.8 T∙L-1 [15]. Blood parameters typical for
healthy fish may vary in a wide range, therefore determination of
adequate physiological reference values is much more difficult than
in case of warm-blooded animals [14, 15]. The blood parameters
examined were within the reference values [16, 17].
The results of our study indicate that the magnesium content in
the blood of rainbow trout remained relatively constant for the
observed four months, while, in the organs examined, magnesium
levels increased along with age. The highest concentration of
magnesium was in the gills and the least in the kidney. Knox
et al. [15] studied how diets with different magnesium
contents affected the growth of the rainbow trout. These results
show that the magnesium requirement of rainbow trout is met by a
diet containing 0.5 g magnesium /kg diet [18]. Oikari
et al. [18] have shown that infusion of magnesium salt into
the body cavity of rainbow trout affects the magnesium
concentration in the plasma.
Magnesium plays a regulatory role in oxidative processes [3, 7,
17, 19]. A decrease in magnesium levels in the body leads to a
reduction in glutathione levels, especially in the erythrocytes
[20]. This mechanism has never been fully explained; however
magnesium is considered an essential cofactor of GSH synthesis.
Magnesium deficiency also intensifies production of ROS by
phagocytic cells [19, 21, 22]. Thus, it is of interest to correlate
Mg status and oxidative stress parameters [3, 4, 22]. A wide
range of oxygen tolerance was observed among fish. Cold-adapted
fish, like rainbow trout, usually need high oxygen levels, while
cyprinid species can survive from nearly full anoxia to hyperoxia
[22, 23]. We observed that 8-month-old rainbow trout had higher
FRAP values than 4- and 6-month-old specimens. FRAP levels were
lower when the dissolved oxygen content was higher. Also the degree
of lipid peroxidation increased between the 4th and 8th months of
life. However, in contrast to our results, data from the literature
indicate that FRAP in rainbow trout decreases with age [16, 17, 23,
24]. Riotola et al. [23, 25] showed that water over saturated
with oxygen boosted antioxidant enzyme activities in the liver and
gills of rainbow trout. According to Wdzieczak et al. [17]
that fish have fluctuations in antioxidant enzyme activities, which
could be due to high rates of free-radical generation. Thus, this
discrepancy between our results and those from the literature could
be explained by differences in water temperature and dissolved
oxygen content.
In conclusion, our work provides interesting observations of the
evolution with age of Mg status and oxidative stress parameters in
trout. It could be hypothesized that an adequate Mg status may help
to prevent oxidative damage during specific periods of trout life.
This needs to be evaluated in future works.
Acknowledgments
This study was financially supported by the State Committee for
Scientific Research (KBN) (grant No. N 304 026 31/0814).
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