Iranian Journal of Fisheries Sciences 15(2) 886- 897 2016

The effects of different dietary vitamin C and iron levels on

the growth, hematological and immunological parameters of
rainbow trout Oncorhynchus mykiss fingerlings

Adel A.1; Khara H. 2*

Received: August 2013 Accepted: September 2014

Abstract
A 60-days growth trial was conducted in a flow-through culture system to examine the
effects of different dietary vitamin C and iron levels on the growth, hematological and
immunological parameters of rainbow trout (Oncorhynchus mykiss) fingerlings (with
average initial weight of 5.12 ± 0.02 g). Three levels of vitamin C (150, 200 and 250
mg kg-1 diet) and iron (5, 7 and 9 mg kg-1 diet) (as ferrous sulfate) and their
combination were used to prepare diets used in nine experimental treatments with three
replicates. The results revealed that the supplementation of basal diet with vitamin C
and iron significantly affected the final body weight (FBW), total length (TL), feed
conversion ratio (FCR) and specific growth rate (SGR). Among the haematological
parameters haematocrit and mean corpuscular volume (MCV) were significantly
influenced by vitamin C and iron. Results also showed that lysozyme and IgM
concentrations significantly changed among dietary treatments. Based on the results of
this study, it can be conclude that addition of vitamin C and iron to the basal diet of
cultured rainbow trout will improve the growth rate and well-being of this fish.

Keywords: Growth performance, Hematological and immunological parameters,

Vitamin C, Iron, Rainbow trout

1- Department of Fishery, Guilan Scince and Research Branch, Islamic Azad University, Rasht, Iran
2-Department of Fishery, Lahijan Branch, Islamic Azad University, lahijan, Iran, P.O.Box: 1616
*Corresponding author's Email: [email protected]
887 Adel and Khara, The effects of different dietary vitamin C and iron levels on the growth,…

Introduction sustained growth and health of many

The aquaculture industry is growing living organisms as well as fish.
rapidly worldwide, but continues to Nutrients do not function as
contend with some issues such as independent units and have some
disease, product quality, feed interactions with other nutrients in
contamination and environmental terms of function and metabolism. Thus
impacts. Fish nutrition and its impact on the requirement level of a particular
animal welfare is an important nutrient may be affected by the level of
aquaculture issue. Poor understanding another nutrient in either the diet or
of the fish dietary requirements in metabolically in the animal. There is
formulation and development of some evidence that iron may be
formulated artificial diets for intensive affecting the metabolism of ascorbic
aquaculture have resulted in mortality acid in the trout (Desjardins, 1985).
rates (Halver, 2002). Vitamins are However, it should be noted that this
important essential nutrients for aquatic apparent interaction between iron and
species. Several morphological and ascorbic acid metabolism most be due
functional abnormalities have been to the effects of iron supplementation
reported in various fish species on both the diet rancidity and on the
deprived of vitamins. In a research Lall stability of dietary ascorbic acid.
and Ollivier (1993) concluded that in However, there is still a paucity of
rainbow trout the disease resistance and information on the interaction of these
hormonal antibody production was two nutrients in fish. The objective of
directly and positively related to the the present study was to assess the
levels of vitamin C. It has been proven effects of different levels of dietary
that dietary levels of vitamins C vitamin C and iron on the growth,
enhance the antibody production and hematological and immunological
immune memory in juvenile of milk parameters of rainbow trout fingerlings.
fish (Azad et al., 2007). Additionally,
some previous research findings Materials and methods
indicate that dietary supplementation Experimental diets
with immunomodulatory vitamins such The basal diet formula applied in this
as vitamins Can improve the immune study is given in Table 1. Supplemental
response and disease resistance of a levels of vitamin C (L-ascorbyl-2-
variety of cultured fish species (Durve polyphosphate (LAPP) (STAY-C-35,
and Lovell, 1982; Verhlac et al., 1998), 350 g kg-1 ascorbic acid equivalent,
while some other studies have failed to DSM, Netherland) at 150, 200 and 250
show positive responses with over mg kg-1 and iron (ferrous sulfate,
fortification of such vitamins (Hardie et FeSO4H2O) at 5, 7 and 9 mg kg-1 were
al., 1990; Li et al., 1998). So, it is clear added to the basal diet to prepare nine
that the biological role played by experimental diets as follow: T1; 5 mg
vitamins C is very vital for the Fe, T2: 7 mg Fe, T3: 9 mg Fe , T4:150
888 Adel and Khara, The effectsIranian Journal
of different of Fisheries
dietary vitaminSciences 15(2)
C and iron 2016
levels on the growth,… 888

mg vitamin C, T5: 200 mg vitamin C, Following that, the diets were cold-
T6: 250 mg vitamin C, T7: 5 mg Fe + pelleted into 3-mm diameter size using
150 vitamin C, T8: 7 mg Fe + 200 mg a pasta maker and then were dried in
vitamin C and T9: 9 mg Fe + 250 mg cool drier for 24 h. After drying, the
vitamin C equivalent kg-1 diet. Also, a diets were broken up and sieved into
treatment without addition of vitamin C appropriate pellet sizes, and were stored
and iron was used as control. In in a cool place until used, and then from
preparing the diet, dry ingredients were each diet one sample was taken for
first ground to small particle sizes in a chemical analysis.
mill. Ingredients were thoroughly
mixed, and then fish oil and water were
added to obtain a 25% moisture level.

Table 1: Composition of the experimental diet (dry weight).

Ingredients g kg-1 dry weight
Fish meal 620
Meat powder 60
Wheat flour 100
Soybean cake 50
Fish oil 40
Soybean oil 40
Lecithin 30
Vitamin mixture (Vitamin E free)a 15
Mineral mixture b 10
Salt 2
Proximate composition %
Crude protein 49.18
Crude lipid 14.12
Moisture 14.25
Ash 20.70
Crude Energy (kcal kg-1) 3012

a
Vitamin mixture was manually provided according to feed requirements of the fish and
ingredients were obtained from Science Laboratories (Ghazvin, Iran); which each 1000 g
vitamin mixture provides vitamin A, 1,600,000 I.U; vitamin D3, 400 000 I.U; thiamin, 6 g;
riboflavin, 8 g; niacin, 12 g; pantothenic acid, 40 g; pyridoxine, 4 g; folic acid, 2 g;
cyanocobalamin, 8 mg; vitamin C, 60 g; vitamin K3, 2 g; biotin, 240 mg and inositol, 20 g.
b
Mineral premix (mg kg)1 diet): NaCl, 500; MgSO4.7H2O, 7500; NaH2PO4.2H2O, 12 500;
KH2PO4, 15 500; Ca(H2PO4)2‫ئ‬H2O, 10 000; (CH2CHCOO)2Ca.5H2O, 1650;
ZnSO4.7H2O, 176.5; MnSO4.4H2O, 81; CuSO4.5H2O, 16.5; CoCl2.6H2O, 0.53; KI, 1.59;
starch, 147.5.
889 Adel and Khara, The effects of different dietary vitamin C and iron levels on the growth,…

Experimental fish and feeding trial samples were collected via

The experiment was designed in nine venipuncture and aspirated into a
treatments with three replicates for 8 microcentrifuge tube. The first sample
weeks. A total of 810 Rainbow trout was transferred to an eppendorf tube
fingerlings (average weight: coated with heparin as anticoagulant
5.12 ± 0.02 g; mean ± SD) were stocked and was used for hematological indices
into 27 fiberglass tanks (200L) at the determination including hematocrit
Rainbow trout farm in Sari city (Ht), number of red blood cell (RBC)
(Mazandran, Iran). Fish were fed four and total leukocyte count (WBC). Red
times a day at 2% of body weight with blood cell (RBC, x106) and total
adjustments made in quantity of feed leukocyte counts (WBC) were
supplied every week. Total fish weight determined with a Neubauer using Rees
in each tank was measured every 2 diluting solution. To obtain the
weeks for more accurate feeding rate differential counts of leukocytes, that is,
adjustment. A diurnal 12-h l light: dark the number of lymphocyte, neutrophil,
cycle was provided by fluorescent light, eosinophil and monocytes, we followed
and dissolved oxygen, temperature and the following procedure. The prepared
pH of the water were monitored blood smears were first air dried, fixed
throughout the experiment. in 96% ethanol for 30 minutes, stained
by Giemsa staining solution for 30
Growth performance minutes, and were examined under light
At the end of the feeding trial, fish were microscope (Klontz, 1994).
fasted for 24 hours and then weight end Hemoglobin concentration (Hb) was
and total length (TL), weight gain determined with Drabkin's reagent and
(WG), final weight (FW), feed read at absorbance at 540 nm (Jain,
conversion ratio (FCR), specific growth 1993). According to procedure of
rate (SGR), and condition factor (CF) Rehulka (2000), haematocrit (Ht) was
and survival rate were calculated measured in microhaematocrit
according to Huang et al. (2003). heparinized capillaries, using a
FCR = dry feed intake (g)/wet WG (g) microhematocrit centrifuge (13,000 rpm
SGR (% day-1) = (Ln Wf - Ln Wi) × for 3 min). Mean corpuscular volume
100/t (MCV) and mean corpuscular
CF = 100 × [wet weht (g)/TL (cm)3] hemoglobin (MCH) were obtained
Where Wf and Wi were final and initial according to the method described by
fish weights, respectively; TL was total Haney et al. (1992).
length and t is the experimental
duration in day. Biochemical analysis
Blood was centrifuged at 3000 rpm for
Hematological parameters analysis 15 minutes in cooling centrifuge for
Fishes were anaesthetized in 100 ppm separation of plasma which was stored
clove powder solution, and then blood at -18ºC till used for biochemical
890 Adel and Khara, The effectsIranian Journal
of different of Fisheries
dietary vitaminSciences 15(2)
C and iron 2016
levels on the growth,… 890

analysis. Lysozyme level was cell (WBC), hemoglobin (Hb),

determined by turbidometric assay lymphocyte, monocyte, mean
according to the method of Sankaran corpuscular hemoglobin (MCH), and
and Gurnani, (1972) with slight mean corpuscular hemoglobin
modifications. Aliquots (1.75 mL-1) of concentration (MCHC) were not
Micrococcus lysodeikticus suspension affected by the supplemental vitamin C
(Sigma) (0.375 mg mL-1, 0.05 M PBS, and iron levels (Tables 3 and 4).
pH 6.2) were mixed with 250 ml-1 of However, hematocrit (Hct) and mean
each sample and the optical density was corpuscular volume (MCV) were
measured after 15 and 180 s by significantly influenced by different
spectrophotometer (Biophotometer dietary vitamin C and iron levels. At the
Eppendorf) at 670 nm. PBS was used as end of the experiment, significant
the blank and results were expressed in differences were also observed in IgM
amounts of lysozyme (mg) per 1 mg of (Fig. 1) and lysozyme (Fig. 2) in
sample calibrated using a standard rainbow trout fed with diets containing
curve determined with hens egg white different levels of vitamin C and iron.
lysozyme (Sigma) in sterile sodium
phosphate buffer. IgM content was Discussion
determined following the method of The results of the present study showed
Puangkaew et al., 2004. significant differences on FBW, TL,
FCR and SGR among treatments with
Data analysis different dietary vitamin C and iron
All data were subjected to a one-way levels. Similarly, studies with common
analysis of variance (ANOVA) after carp, (Gouillou-Coustans et al., 1998)
confirmation of normality and hybrid tilapia, Oreochromis niloticus
homogeneity of variance. Significance (Shiau and Hsu, 2002), Japanese
of the differences between means was seabass, Lateolabrax japonicus (Ai et
tested using Duncan’s multiple range al., 2004), juvenile grouper,
test (p<0.05). All assays were carried Epinephelus malabaricus (Lin et al.,
out in triplicates and data are shown as 2005) and juvenile cobia, Rachycentron
mean ± SD for each dietary group. canadum (Zhou et al., 2012) indicate
positive effect of vitamin C on the
Results growth. Our results indicated that
The addition of different dietary dietary vitamin C could improve the
vitamin C and iron levels to the basal growth performance of juvenile
diets significantly affected the FBW, rainbow trout. However, Ai et al.,
TL, FCR and SGR; as these values (2006) reported that dietary vitamin C
were higher in fish fed diet containing levels didn’t influence the growth
5 mg Fe + 150 vitamin C (T7) than performance of the juveniles of large
those fish fed the other treatment (Table yellow croaker.
2). Red blood cells (RBC), White blood
891 Adel and Khara, The effects of different dietary vitamin C and iron levels on the growth,…

Table 2: Final body weight (FBW), weight gain (WG) (g/fish), total length (TL), feed conversion
ratio (FCR), specific growth rate (SGR) and condition factor (CF) of rainbow trout fed the
experimental diets for 8 weeks.
Initial Final
WG SGR (% Condition Survival
Treatments weight weight TL (cm) FCR
(%) day-1) factor rate (%)
(g) (g)
Control 5.12 ± 0.02 24.6 ± 5.3 c 27.6 ± 0.71 13.5 ± 1.08 1.5 ± 0.04 ab 2.06 ± 0.25 de 1.04 ± 0.31 100

T1 5.13 ± 0.02 25.8 ± 7.1 d 28.7 ± 1.1 14.1 ± 1.17 1.4 ± 0.03 bc 2.12 ± 0.33 d 1.04 ± 0.05 100

T2 5.14 ± 0.01 27.1 ± 3.5 e 29.7 ± 1.3 14.2 ± 1.10 1.3 ± 0.01 bcd 2.18 ± 0.15 cd 0.96 ± 0.02 100

T3 5.12 ± 0.06 28.2 ± 7.5 f 30.7 ± 1.8 14.9 ± 0.77 1.2 ± 0.02 cd 2.23 ± 0.32 c 1.04 ± 0.09 100

T4 5.15 ± 0.02 21.6 ± 4.9 a 25.3 ± 1.4 13.6 ± 0.17 1.6 ± 0.04 a 1.91 ± 0.25 f 1.03 ± 0.11 100

T5 5.10 ± 0.05 23 ± 9.9 b g 26.3 ± 1.6 13.4 ± 1.53 1.4 ± 0.04 bc 1.99 ± 0.49 e 1.06 ± 0.16 100

T6 5.11 ± 0.04 31± 7.9 b ef 33 ± 1.2 15.3 ± 0.60 1.1 ± 0.02 e 2.36 ± 0.32 a 1.12 ± 0.06 100

T7 5.12 ± 0.03 29.4 ± 4.1 d 31.5 ± 1.09 14.6 ± 0.78 1.2 ± 0.01 cd
2.28 ±0.17 b 1.13 ± 0.02 100

T8 5.13 ± 0.02 27.6 ± 1.9 d 30.1 ± 0.61 14.5 ± 0.96 1.3 ± 0.02 bcd 2.21± 0.08 c 0.93 ± 0.03 100

T9 5.13 ± 0.01 25.8 ± 3.3 c 28.7 ± 1.1 13.8 ± 0.96 1.3 ± 0.01 bcd 2.12 ± 0.15 d 1.04 ± 0.07 100

Within a column, means with different superscripts are significantly different (p<0.05).

Table 3: Hematological parameters of rainbow rout fingerling fed 8 weeks with diets containing
different levels of vitamin C and iron.
WBC Hematocrit Hemoglobi MCH MCHC
Treatments RBC (×106) MCV (fl)
(×103) (%) n (gr/dl) (pg) (gr/dl)

Control 101 ± 21 67.6 ± 15 36 ± 2.6 e 6.3 ± 0.49 299 ± 21.1 bcd 52.6 ± 6.3 17.5 ± 1.6

T1 97 ± 16.2 69.6 ± 8.9 33.3 ± 2.8 cde 6.5 ± 0.42 289 ± 38.2 bcd 56.4 ± 7.1 19.5 ± 0.2
cd d
T2 102 ± 21.5 69.3 ± 14 31.6 ± 1.1 5.7 ± 1.2 303.8 ± 18.7 59.1 ± 6.2 17.9 ± 2.2

T3 101 ± 10.3 64 ± 15 28.3 ± 1.5 ab 5.9 ± 0.13 316 ± 49.5 cd 59.5 ± 10.3 18.8 ± 0.36
cd ab
T4 103 ± 12.7 64 ± 16.2 32 ± 1 5.9 ± 0.81 244 ± 11.5 51.1 ± 6.7 20.9 ± 2.01

T5 101 ± 21.7 64.6 ± 15.5 30.6 ± 1.1 bcd 6.1 ± 0.11 300 ± 25.7 bcd 56.1 ± 8.7 18.6 ± 1.8
abc abc
T6 100 ± 13.9 61 ± 12.2 29 ± 4 5.8 ± 0.49 273 ± 10.2 54.2 ± 0.09 19.9 ± 0.76
a bcd
T7 102 ± 16 62.6 ± 9.2 28 ± 1 5.7 ± 0.04 298 ± 51.6 61.1 ± 15.8 20.2 ± 2.02

T8 103 ± 10.3 66 ± 7.5 33.3 ± 2.3 cde 6.2 ± 1.4 232 ± 20.2 a 47.9 ± 2.4 20.6 ± 0.75
e ab
T9 102 ± 17.3 64 ± 13.7 36 ± 2.8 6.3 ± 0.49 254 ± 14.1 46.9 ± 9.1 18.6 ± 4.7

Within a column, means with different superscripts are significantly different (p<0.05).
892 Adel and Khara, The effects of different dietary vitamin C and iron levels on the growth,…
Iranian Journal of Fisheries Sciences 15(2) 2016 892

Table 4: Differential count of leukocyteof of rainbow rout fingerling fed 8

weeks with diets containing different levels of vitamin C and iron.

Treatments Monocyte (%) Neutrophil (%)

Control 99 ± 1 1±1
T1 99.5 ± 0.58 0.67 ± 0.57

T2 98 ± 1.03 1±1

T3 98.6 ± 1.5 1.3 ± 0.53

T4 99.6 ± 0.58 0.33 ± 0.05

T5 98.3 ± 2.08 2 ± 0.73

T6 99.3 ± 1.1 0.67 ± 0.16

T7 98.3 ± 1.5 1.67 ± 0.53

T8 99 ± 1 1±1
T9 99.6 ± 0.58 0.33 ± 0.58

Within a column, means with different superscripts are significantly different (p < 0.05).

160 f
140
IgM concentration (mg ml-1)

e e
120
100 d
d
80 d
c
60 bc
ab
40
20 a
0

Figure 1: Total IgM concentration of concentration of rainbow rout fingerling

fed 8 weeks with diets containing different levels of vitamin C and iron.
893 Adel and Khara, The effects of different dietary vitamin C and iron levels on the growth,…

50
45 f

Lysozym activity (units ml-1)

40 e
35
30 d
cd bc
25 b a bc
20 a a
15
10
5
0

Figure 2: Lysozyme activity of rainbow rout fingerling fed 8 weeks with diets
containing different levels of vitamin C and iron.

Studies with common carp (Sakamoto (2009) also have reported that addition
and Yone, 1978a), red sea bream of iron to the basal diet significantly
(Sakamoto and Yone, 1978b), Atlantic affected the Hct in juvenile gibel carp,
salmon (Andersen et al., 1996), channel C. auratus gibelio. Vitamin C is a
catfish (Barros et al., 2002) and powerful antioxidant protecting against
juvenile gibel carp, Carassius auratus oxidative damage to various tissues of
gibelio (Pan et al., 2009) did not fish including red blood cells (Sahoo
indicate an adverse effect of iron and Mukherjee, 2003). Lower red blood
deficiency on the growth performance. cells were regarded as the sun of
However, Shiau and Su, (2002) anemia which has been reported in most
reported that weight gain of Nile tilapia studies with fish where vitamin C
increased with dietary iron while deficiency was observed (NRC, 2011).
significantly decreasing at iron levels. Moreover, Adham et al. (2000)
These discrepancies might be due to demonstrated that feeds with
differences in individual size, insufficient vitamin C cause anemia,
development stage, and cultivation characterized by a decrease in the
environment. There were significant hemoglobin, reduction in the number of
differences in Hct and MCV values erythrocytes and hematocrit. Many
among our treatments. Similar results researchers have shown that ascorbic
were observed in pirarucu (Arapaima acid is involved in the metabolism of
gas) and cobia juvenile’s R. canadum iron in fish and a deficiency of ascorbic
(Zhou et al., 2012) fed with diet not acid has been observed to cause a
supplemented with vitamin C. In reduction in serum iron levels and a
agreement with our results Pan et al., redistribution of tissue iron stores in
 Iranian Journal of Fisheries Sciences 15(2) 2016 894

rainbow trout (Hilton et al., 1989) and a examine the effect of dietary vitamins
reduction in both hemoglobin and and minerals on juvenile rainbow trout
hematocrit levels in catfish, trout and growth performance and
snakehead fish (Lim and Lovell, 1978; haematological parameters.
Agrawal and Mahajan, 1980). There are
also some as that may indicate the Acknowledgements
effect of iron on ascorbic acid "Financial support by Lahijan Branch,
metabolism in trout. Despite the Islamic Azad University Grant No.
interaction of ascorbic acid and iron 17.20.5.3511 is gratefully
metabolism in fish, it is interesting to acknowledged. "
note that increasing the levels of
ascorbic acid do not appear to affect the References
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