Effects of dietary organic, inorganic, and nanoparticulate selenium sources on growth, hemato-immunological, and serum biochemical parameters of common carp (Cyprinus carpio) Sadegh Saffari, Saeed Keyvanshokooh, Mohammad Zakeri, Seyed Ali Johari, Hossein Pasha-Zanoosi & Mansour Torfi Mozanzadeh Fish Physiology and Biochemistry ISSN 0920-1742 Fish Physiol Biochem DOI 10.1007/s10695-018-0496-y
1 23
Your article is protected by copyright and all rights are held exclusively by Springer Science+Business Media B.V., part of Springer Nature. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.
1 23
Author's personal copy Fish Physiol Biochem https://doi.org/10.1007/s10695-018-0496-y
Effects of dietary organic, inorganic, and nanoparticulate selenium sources on growth, hemato-immunological, and serum biochemical parameters of common carp (Cyprinus carpio) Sadegh Saffari & Saeed Keyvanshokooh & Mohammad Zakeri & Seyed Ali Johari & Hossein Pasha-Zanoosi & Mansour Torfi Mozanzadeh Received: 25 May 2017 / Accepted: 27 March 2018 # Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract An 8-week feeding trial was conducted to compare the effects of supplementing (0.7 mg kg−1) different dietary selenium (Se) sources including organic [selenomethionine (SeMet)], inorganic [sodium selenite (Na2SeO3)], and nanoparticulate Se (nanoSe) on physiological responses of common carp, Cyprinus carpio juveniles (9.7 ± 0.1 g). Basal diet without Se supplementation used as control. Fish fed nano-Se supplemented diet had the highest weight gain (97.2 ± 10.8%) and feed efficiency ratio (42.4 ± 0.8%). Intestinal villi height was significantly taller in fish fed nano-Se diet than in the control group in both foregut and midgut sections. Serum glutathione peroxidase and superoxide dismutase activities were significantly higher in nano-Se and SeMet groups than in control and sodium selenite
groups. Fish fed Se-supplemented diets had greater red blood cell counts and hematocrit and hemoglobin values than the control group (P < 0.05). Nano-Se and SeMet groups showed a significant increase in white blood cell counts, neutrophil percentage, and serum lysozyme activity than the other groups. Fish fed nano-Se diet had the highest serum hemolytic activity, total immunoglobulin, and total protein and albumin contents, as well as the lowest serum total cholesterol and low density lipoprotein levels (P < 0.05). Overall, significant improvements in growth performance, feed utilization, intestinal morphology, and hemato-immunological and serum biochemical parameters of common carp juveniles suggest nano-Se as an efficient source for providing dietary Se in this species.
S. Saffari : S. Keyvanshokooh (*) : M. Zakeri Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khouzestan, Khorramshahr, Iran e-mail:
[email protected]
Keywords Selenium . Antioxidant enzymes . Intestinal villus . Immune response . Serum biochemical parameters
S. A. Johari Department of Fisheries, Faculty of Natural Resources, University of Kurdistan, Sanandaj, Kurdistan, Iran
Introduction
H. Pasha-Zanoosi Department of Physical Oceanography, Faculty of Marine Sciences, Khorramshahr University of Marine Science and Technology, Khouzestan, Khorramshahr, Iran M. T. Mozanzadeh South Iran Aquaculture Research Centre, Ahwaz, Khuzestan, Iran
Selenium (Se) when available in sufficient amount is an essential trace element which is necessary for growth and physiological function in animals including fish (NRC 2011). Selenium plays a vital role in some important types of selenoproteins such as glutathione peroxidase (GPx) and provides protection against oxidative damage (Rotruck et al. 1973). A
Author's personal copy Fish Physiol Biochem
dietary Se deficiency results in reduced activity of GPx, oxidative stress, growth reduction, and immune response suppression in fish; thus, dietary Se supplementation is the best way for providing fish requirement (NRC 2011). The optimum Se requirements varied from 0.15 to 0.7 mg kg−1 in different cultured fish species (NRC 2011). It has been reported that the organic forms of Se is more effective in terms of bioavailability than the inorganic forms (Schrauzer 2000; Burk et al. 2006). Moreover, Se nanoparticles have attracted attention due to novel features like lower toxicity, higher chemical stability, biocompatibility, and ability to gradually release Se after ingestion (Skalickova et al. 2017). For example, it has been reported that administration of diet with nano-Se and vitamin E significantly improved growth performance, stress resistance, humoral immune responses, and serum biochemical parameters in rainbow trout (Oncorhynchus mykiss) and also altered the expression of liver proteins involved in metabolic status of this species (Naderi et al. 2017a,b,c). A plethora of studies have been conducted for determining optimum dietary Se requirements in various cultured aquatic species (NRC 2011). For example, the optimum dietary Se requirements in freshwater species such as crucian carp (Carassius auratus gibelio) and blunt snout bream (Megalobrama amblycephala) were determined as 0.5 and 0.2 mg kg−1, respectively (Zhou et al. 2009, Liu et al. 2016). For some marine fish species including grouper (Epinephelus malabaricus), cobia (Rachycentron canadum), and yellowtail king fish (Seriola lalandi), the optimum dietary Se levels were reported as 0.7, 0.8, and 2 mg kg−1, respectively (Lin and Shiau 2005; Liu et al. 2010; Le and Fotedar 2014). However, information about effects of different Se sources on growth performance and health status of cultured aquatic species is scarce. Recently, Ashouri et al. (2015) reported that supplementation of 1 mg nano-Se kg−1 diet improves growth performance, antioxidant defense system, and health status of common carp. However, according to the current literature, the effects of different dietary Se sources on physiological responses of common carp have not been elucidated. Thus, the present study was conducted to evaluate dietary supplementation of different Se sources including organic, inorganic, and nanoparticulate Se on growth performance and hemato-immunological and serum biochemical parameters of common carp, Cyprinus carpio juveniles.
Materials and methods Diet preparation Ingredients and chemical composition of the basal diet used in this experiment were according to Ashouri et al. (2015) (Table 1). Basal diet without Se supplementation used as control. Inorganic Se (sodium selenite (selenite), Na2SeO3, 99% purity, Sigma Chemical Co., St Louis, MO, USA), organic Se (selenomethionine (SeMet), 98% purity, Sigma Chemical Co., St Louis, MO, USA), and nano-Se (99.95% purity, 30–45 nm particle size, 3.89 g cm3 true density, Iranian Nanomaterials Pioneers, Mashhad, Iran) were added to the basal diet at 0.7 mg kg−1 dry diet according to the findings of previous studies on optimum dietary Se requirement of common carp juveniles (Jovanovic et al. 1997; Elia et al. 2011; Ashouri et al. 2015). Nano-Se was the particles of red elemental Se in the redox state of zero and prepared by adding bovine serum albumin to the redox system of selenite and glutathione according to Zhang et al. (2001). The ingredients and different Se sources were mixed, extruded, and air dried at room temperature, and then were kept in the − 20 °C until used. The final actual concentration of Se in each diet was measured by an atomic absorption spectrophotometer equipped with transversely heated graphite atomizer system (Younglin AAS 8020, Korea) as described previously by Elia et al. (2011) (Table 2). Table 1 Formulation and proximate composition of the basal diet (mean ± SEM, n = 3) Ingredient
(g/kg) Proximate composition (g/kg)
Fish meal
300
Protein
320.0 ± 11.1
Soybean meal
160
Lipid
102.0 ± 3.1
Corn meal
200
Moisture
92.0 ± 2.1
Ash
11.1 ± 9.1
Wheat middling 180 Rice bran
80
Fish oil
20
Soybean oil
20
Se-free premixa
40
a Included: (as mg/kg of premix): vitamin A, 50000 (IU/kg); vitamin D3, 10,000 (IU/kg); vitamin E, 30; vitamin B1, 20; vitamin B2, 10; vitamin B6, 3; vitamin K3, 15; nicotinamide, 150; calcium pantothenate, 40; Copper (Cu++ ), 30; iron (Fe++ ), 100; zinc (Zn++ ), 150; manganese (Mn++ ), 200
Author's personal copy Fish Physiol Biochem Table 2 The final actual concentration of Se (mg kg-1) in experimental diets Control
0.43 ± 0.03
Sodium selenite
1.17 ± 0.08
SeMet
1.19 ± 0.06
Nano Se
1.16 ± 0.05
extracted blood was poured into heparinized vials (n = 9 fish per diet treatment, n = 3 fish per diet replicate). For serum antioxidant enzymes and immunological and biochemical parameters (n = 12 fish per diet treatment, n = 4 fish per diet replicate), blood was allowed to clot at 4 °C for 1 h and then centrifuged (4000×g, 10 min at room temperature). Sera were separated and frozen at − 80 °C until used.
Fish maintenance and feeding
Light microscopy study
Common carps obtained from a local hatchery (Ahvaz, Iran) were used in the study. Fish were randomly distributed into 12 cylindrical fiberglass tanks with a volume of 300 L and each tank stocked with 14 fish (mean body weight 9.7 ± 0.1, mean ± standard deviation). Each dietary treatment was assayed in triplicate. Fish were acclimated for 2 weeks before the onset of the nutritional trial and fed by basal diet without Se supplementation. Fish were fed three times a day at 8:00, 13:00, and 19:00 with the experimental diets for 8 weeks according to Ashouri et al. (2015). Daily feeding rate was ca. 3% of body weight according to batch weighing of fish every 2 weeks. Uneaten food was siphoned out 1 h after feeding and weighed to determine feed intake values. Once every 3 days, 50% of the rearing water was replaced. The mean water quality features were kept as follows: temperature 28.4 ± 0.2 °C, dissolved oxygen 6.4 ± 0.1 mg mL−1, pH 7.8 ± 0.2, and the photoperiod was 12L:12D (light/darkness).
At the end of the experiment, the visceral mass of three fish per dietary treatment was dissected and fixed in 4% buffered formaldehyde (pH = 7.4), dehydrated in a graded series of ethanol, cleared with xylene, embedded in paraffin, and cut in serial sections (3–5 μm thick). Hematoxylin and eosin-stained sections were then photographed and studied using a digital microscope (Dino-Eye AM4023X, Taiwan). A computerized microscopic image analyzer (Digimizer 4.1.1) was used to determine histomorphometric parameters including villus perimeter and villus at foregut and midgut sections on ten different villi per fish. The criteria for selection of histological sections for examination were based on the presence of an intact villus that was perpendicularly sectioned through the midline axis.
Sample collection At the end of 8-week feeding trial, all the fish were anesthetized with clove oil (150 ppm) and individually weighed. The weights of fish, liver, and viscera were measured to the nearest 0.1 g. Standard formulae were used to assess growth performance, feed utilization, and other parameters: weight gain (%): WG = (final weight − initial weight) / initial weight) × 100; feed efficiency ratio (%): FER = (weight gain (g) / feed intake (g)) × 100; protein efficiency ratio (PER) = weight gain (g) / protein intake (g); hepatosomatic index (%): HSI = (liver weight (g) / whole body weight (g)) × 100; viscerosomatic index (%): VSI = (visceral weight (g) / whole body weight (g)) × 100; condition factor (%): K = (body weight (g) / (body length (cm)) 3) × 100. Blood was collected randomly from the caudal vein of seven fish per tank. For hematological analyses the
Lipid peroxidation values and antioxidant enzyme parameters Glutathione peroxidase activity was assayed using the method described by Noguchi et al. (1973). GPx degrades H2O2 in the presence of reduced glutathione (GSH). The activity of GPx is expressed as 1 μmol L−1 of the substrate (GSH) depleted per minute per mg of protein or per mL serum. Activity of serum superoxide dismutase (SOD) was measured according to the method of McCord and Fridovich (1969). The assay mixture contained 1.2 ml of sodium pyrophosphate buffer, 100 μl of phenazine methosulfate, 300 μl of nitroblue tetrazolium, and 200 μl of the serum and water in a total volume of 2.8 ml. The reaction will be initiated by the addition of 200 μl of NADH. The mixture was incubated at 30 °C for 90 s and arrested by addition of 1.0 ml of glacial acetic acid. The reaction mixture will be then shaken with 4.0 ml of n-butanol, allowed to stand for 10 min and centrifuged. The intensity of the chromogen in the butanol layer will be measured at 560 nm in a
Author's personal copy Fish Physiol Biochem
spectrophotometer. One unit of enzyme activity is defined as the amount of enzyme that gave 50% inhibition of NBT reduction in 1 min. Serum catalase (CAT) activity was determined following the method of Abei (1984). Briefly, the activity was determined by measuring the decrease in absorbance at 240 nm (e = 40 M/cm) using 13.2 mM H2O2 in 50 mM phosphate buffer (pH 7.0) and 100 μl of serum. A mixture containing 50 mM phosphate buffer (pH 7.0) and 100 μl of serum was used as a control. Malondialdehyde (MDA) concentration, also known as thiobarbituric acid reactive substances, was measured colorimetrically using the method of Buege and Aust (1978). Briefly, 200 μl of serum was reacted with 2 ml of thiobarbituric acid (TBA) reagent containing 0.375% TBA, 15% trichloroacetic acid, and 0.25 N HCl. Samples were then boiled for 15 min, cooled and centrifuged. The absorbance of the supernatants was
measured spectrophotometrically at a wavelength of 532 nm. Lipid peroxidation was expressed as TBARS concentration using1,3,3,3 tetra-ethoxypropane as a standard.
Hematological analyses Hematocrit (%; Hct), hemoglobin concentration (Hb; g dL−1), and the number of red blood cells (RBCs) and white blood cells (WBCs) as well as differential WBC counts (lymphocyte, monocyte, neutrophil and basophil portions as WBC%) were assessed according to methods described by Blaxhall and Daisley (1973). Blood indices including the mean cell hemoglobin (MCH), the mean cell volume (MCV), and the mean cell hemoglobin concentration (MCHC) were calculated according to the following formulae (Lewis et al. 2001):
Mean cell volume ðMCVÞ ¼ Hct ð%Þ=RBC ð106 μLÞ 10: Mean cell hemoglobin ðMCHÞ ¼ Hb ðg dL−1Þ=RBC ð106 μLÞ 10: Mean cell hemoglobin concentration ðMCHCÞ ¼ ðg dL−1Þ ¼ Hb ðg dL−1Þ=Hct ð%Þ
Humoral immune parameters The alternative complement pathway hemolytic activity (ACP) was estimated as described by Tort et al. (1996) by 50% lysis of rabbit red blood cell (RARBC) as target cells in the presence of EGTA and Mg+2. Following washing of the RaRBC three times, the absolute lysis value was prepared by adding100 μl of the RaRBC to 3.4 ml distilled water. The lysate was then exposed to cold centrifugation, and the turbidity of the aqueous phase was determined at 414 nm. Subsequently, the serum specimens were diluted in the buffer and 250 μl of adjusted volume serum was added to 100 μl of RaRBC in test tubes. The prepared solution was kept at room temperature for 90 min with repeated mixing. Then, 3.15 ml of NaCl solution (0.85%) was added to all samples and the tubes were centrifuged for 10 min and the absorbance of the supernatant was quantified again. The level of cell lysis was determined, and hemolysis curve was drawn through plotting the hemolysis degree against the volume of serum added on a log/log-scaled graph.
The volume producing 50% hemolysis was considered for determining the hemolytic activity of the serum samples and was expressed as U ml−1. The lysozyme levels in the blood serum were determined using a turbidimetric assay according to the method of Ellis (1990) by measuring the lytic activity of the C. carpio juvenile serum against lyophilized Micrococcus lysodeikticus (Sigma, St Louis, MO, USA). A volume of 135 μl of M. lysodeikticus at a concentration of 0.2 mg ml−1 (w/v) in 0.02 M sodium citrate buffer (SCB), pH 5.8, was added to 15 μl of serum sample. As a negative control, SCB was replaced instead of serum. Results were expressed in milligram of lysozyme per milliliter of serum. Hen egg white lysozyme (Sigma) in phosphate-buffered saline was used as a standard. A unit of lysozyme activity was defined as the amount of serum causing a reduction of absorbance of 0.001 per minute at 450 nm at 22 °C. Serum total immunoglobulin (Ig) was measured using the method described by Siwicki et al. (1994). Primary separation of immunoglobulins from the serum was achieved by precipitation with polyethylene glycol (PEG), and the resulting supernatant was analyzed. To
Author's personal copy Fish Physiol Biochem
perform the assay, 100 μl of serum was combined with 100 μl 12% PEG and incubated at room temperature for 2 h in continuous agitation. Following the incubation time, the mixture was centrifuged (400×g, 10 min at room temperature), and total protein concentration in the supernatant was determined by the Biuret method. The total Ig levels were calculated considering total protein values less the quantity of protein in the supernatant. Serum biochemical analyses Serum biochemical parameters were analyzed by means of an autoanalyzer (Mindray BS-200, China) using commercial clinical investigation kits (Pars Azmoon and ZistChimi Kits, Tehran, Iran). Biochemical measurements were conducted for total protein (TP) and albumin (ALB), glucose (GLU), triglyceride (TG), total cholesterol (CHO), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). Moreover, the content of total globulin (GLO) was estimated by subtracting albumin from total protein (Kumar et al. 2005). Statistical analyses The results (mean ± SE, standard error) were evaluated by one-way analysis of variance (ANOVA) followed by Duncan to compare the means between each individual tested groups using SPSS (Version 16; SPSS Inc., Chicago, IL, USA) at P < 0.05 level.
Results During the feeding trial, the fish survival was 100% in all groups. Fish fed nano-Se diet had the highest WG (97.2 ± 10.8%), FI (21.9 ± 1.5 g fish−1), FER (42.4 ± 0.8%), and PER (1.3 ± 0.0%) (Table 3). Fish fed Sesupplemented diets had higher VSI values than the control group; however, other somatic parameters (HSI and K) did not change among experimental groups. Intestinal histomorphometric analyses of the foregut and midgut sections revealed that different dietary Se sources significantly affected perimeter and height of villi (Table 4). The perimeter of the villi was significantly increased in the foregut section of fish fed SeMet and nano-Se diets, compared with respective values for the control and sodium selenite groups. Fish fed Sesupplemented diets had greater villi perimeter in midgut section than the control group. Intestinal villi height was significantly taller for fish fed nano-Se diet than the control group in both intestinal sections, and other groups showed intermediate values. In this study, serum GPx (Fig. 1a) and SOD (Fig. 1b) activities were significantly higher in nano-Se and SeMet groups than the control and sodium selenite groups; however, serum CAT (Fig. 1c) activity did not differ among different dietary treatments. Moreover, serum MDA (Fig. 1d) was at highest (16.5 ± 1.1 nmol mL −1 ) and lowest (11.1 ± 0.8 nmol mL−1) levels in the control and nano-Se groups, respectively. In the present study, fish fed Se-supplemented diets had higher RBC, Hct, Hb, and MCV values than the
Table 3 Growth performance of common carp fed the diets containing different sources of Se for 56 days (mean ± SEM, n = 3) Se sources Growth parameters
Control
Selenite
SeMet
Nano-Se
WG (%)
63.2 ± 2.5a
75.5 ± 5.5a
77.8 ± 3.1a
97.2 ± 10.8b
FI (g fish−1)
16.0 ± 0.1a
18.7 ± 0.8b
19.4 ± 0.3b
21.9 ± 1.5c
a
a
a
42.4 ± 0.8b
FER (%)
38.3 ± 1.0
PER (%)
a
38.7 ± 1.1
39.0 ± 0.8
1.2 ± 0.0
1.2 ± 0.0a
a
1.2 ± 0.0
1.3 ± 0.0b
HSI (%)
2.2 ± 0.2
2.1 ± 0.2
2.6 ± 0.2
2.2 ± 0.1
VSI (%)
9.8 ± 0.2a
10.5 ± 0.3b
10.9 ± 0.3b
10.7 ± 0.1b
K (%)
1.4 ± 0.0
1.4 ± 0.0
1.4 ± 0.0
1.4 ± 0.0
WG weight gain, FI feed intake, FER feed efficiency ratio, PER protein efficiency ratio, HIS hepatosomatic index; VSI viscerosomatic index, K Fulton’s condition factor, Means in the same row with different superscripts are significantly different (P < 0.05)
Author's personal copy Fish Physiol Biochem Table 4 Morphological changes in intestinal sections of common carp fed different sources of Se for 56 days (mean ± SEM, n = 3) Se sources
Villus perimeter (μm) Villus height (μm)
Intestine parts
Control
Selenite
Foregut
519.4 ± 8.5a
554.9 ± 20.2a
Midgut
a
313.7 ± 7.9
ab
Foregut
a
325.8 ± 9.5
ab
59.7 ± 4.5
77.2 ± 2.5
a
Midgut
SeMet
ab
57.6 ± 3.2
71.3 ± 5.1
Nano-Se
594.6 ± 12.6b
656.3 ± 13.9b
bc
361.1 ± 14.7c
349.3 ± 11.9 ab
89.3 ± 9.1b
ab
75.6 ± 5.0b
76.1 ± 5.2 68.7 ± 5.3
Means in the same row with different superscripts are significantly different (P < 0.05)
control (Table 5). Moreover, fish in nano-Se and SeMet had higher WBC counts and neutrophil percentage than the other groups, but fish in the control had the highest lymphocyte percentage (P < 0.05). Serum lysozyme activities were higher in fish fed nano-Se and SeMet diets as compared to fish fed other experimental diets (Fig. 2a). On the other hand, fish fed nano-Se diet had the highest serum ACP activity (474.3 ± 17.1 U mL−1; Fig. 2b) and total Ig content (73.1 ± 7.0 mg L−1; Fig. 2c).
Regarding serum biochemical parameters, fish fed nano-Se diet had the highest serum TP (4.1 ± 0.1 g dL −1 ) and ALB (2.3 ± 0.1 g dL −1 ) levels (Table 6). In addition, CHO (94.3 ± 4.9 mg dL−1) and LDL (13.1 ± 2.3 mg dL−1) concentrations were lower in fish fed nano-Se diet as compared to other groups (P < 0.05). There were no significant differences in serum GLO, GLU, TG, and HDL levels among different treatments.
a
c 450
b
14
b CAT (mU mg protein -1)
400
GPx (mU mg protein -1)
350 300
a
a
250 200
150
Selenite
SeMet
Nano se
Control
Selenite
SeMet
Nano se
d 20
b 300
18
b
c b
16
250 a
b
14
MDA (nmol mL-1)
SOD (mU mg protein -1)
6
0 Control
350
200
8
2
50
b
12 10
4
100
0
16
a
150 100
a
12 10 8 6 4
50
2 0
0
Control
Selenite
SeMet
Nano se
Control
Selenite
SeMet
Nano se
Fig. 1 Serum antioxidant enzymes activities including GPx (a), SOD (b), and CAT (c) as well as MDA level (d) in common carp fed different dietary Se sources
Author's personal copy Fish Physiol Biochem Table 5 Hematological parameters of common carp fed the diets containing different sources of Se for 56 days (mean ± SEM, n = 3) Se sources Hematological parameters
Control
Selenite
RBC (×106 cell μL−1)
1.2 ± 0.1a a
Hct (%) −1
SeMet
Nano-Se
1.4 ± 0.1ab
1.3 ± 0.1ab
b
b
36.2 ± 1.7b
b
27.7 ± 0.7
38.8 ± 1.1
a
ab
1.4 ± 0.1b
38.4 ± 2.0
Hb (g L )
7.5 ± 0.7
8.9 ± 0.6
10.1 ± 0.6
9.7 ± 0.7b
MCV (fL)
245.0 ± 16.1a
291.1 ± 22.5ab
302.9 ± 15.1b
262.4 ± 17.8ab
MCH (pg)
65.9 ± 7.2
67.2 ± 7.3
81.3 ± 7.0
69.5 ± 5.0
MCHC (g dL−1)
26.8 ± 2.3
22.8 ± 1.3
27.2 ± 2.5
27.3 ± 2.5
WBC (×103 cell μL−1)
7.3 ± 0.4a
7.6 ± 0.8a
10.4 ± 0.6b
09.7 ± 0.8b
a
b
a
Lymphocyte (%)
90.5 ± 1.3
85.9 ± 2.1
85.1 ± 0.7
86.1 ± 1.8a
Monocyte (%)
6.4 ± 0.9
8.9 ± 1.0
8.4 ± 0.6
6.9 ± 0.9
a
a
b
Neutrophil (%)
2.1 ± 0.5
4.0 ± 0.9
5.4 ± 0.7
5.6 ± 1.1b
Basophil (%)
1.0 ± 0.4
1.2 ± 0.3
1.1 ± 0.4
1.4 ± 0.4
RBC red blood cell, Hct hematocrit, Hb hemoglobin, MCV mean cell volume, MCH mean cell hemoglobin, MCHC mean cell hemoglobin concentration, WBC white blood cell, Means in the same row with different superscripts are significantly different (P < 0.05)
a
c
120
120
100 a
60 40
80
a
a
Selenite
SeMet
a
60 40
20
20
0 Control
b
100
-1
80
b
a
Ig (mg mL )
Lyzozyme activity (U mL-1)
b
b
Selenite
SeMet
Nano se
0
Control
Nano se
600 b
500 a
ACP (U mL-1)
400
a
a
300
200
100
0
Control
Selenite
SeMet
Nano se
Fig. 2 Serum immunological parameters including lysozyme (a) and ACP (b) activities as well as Ig content (c) in common carp fed different dietary Se sources
Author's personal copy Fish Physiol Biochem Table 6 Blood biochemical parameters of common carp fed the diets containing different sources of selenium for 56 days (mean ± SEM, n = 3) Se sources Biochemical parameters
Control
Selenite
SeMet
Nano-Se
TP (g dL−1)
3.3 ± 0.1a
3.7 ± 0.1b
3.5 ± 0.1ab
4.1 ± 0.1c
ALB (g dL−1)
1.8 ± 0.2a
1.9 ± 0.2a
1.9 ± 0.2a
2.3 ± 0.1b
−1
GLO (g dL )
1.5 ± 0.1
1.8 ± 0.2
1.7 ± 0.2
1.8 ± 0.2
GLU (mg dL−1)
61.8 ± 4.1
58.3 ± 3.3
64.2 ± 3.9
55.7 ± 2.8
TG (mg dL−1)
253.3 ± 10.0
258.8 ± 9.6
237.0 ± 7.4
244.5 ± 8.0
CHO(mg dL−1)
110.5 ± 5.4b
108.2 ± 4.9b
104.5 ± 4.8b
94.3 ± 4.9a
HDL (mg dL−1)
27.5 ± 2.8
27.8 ± 3.2
31.5 ± 2.6
32.3 ± 3.9
32.3 ± 5.6 b
28.6 ± 4.7b
25.6 ± 5.9b
13.1 ± 2.3a
−1
LDL (mg dL )
TP total protein, ALB albumin, GLO globulin, GLU glucose, TG triglyceride, CHO cholesterol, HDL high-density lipoprotein, LDL lowdensity lipoprotein, Means in the same row with different superscripts are significantly different (P < 0.05)
Discussion The results of the present study showed that fish fed nano-Se-supplemented diet had the highest WG and FE, indicating that nano-Se was the most bioavailable Se source to common carp juveniles. In contrast, Lorentzen et al. (1994) demonstrated that dietary supplementation of inorganic (sodium selenite) and organic Se (SeMet) (1 and 2 mg kg−1 diet, respectively) could not enhance WG in Atlantic salmon (Salmo salar). The speciesspecific variations in the intestinal Se absorption rate; the Se concentrations in rearing water, bioavailability, and different metabolic pathways for different Se sources; and the amount of dietary vitamin E (AbdelTawwab et al. 2007; Hao et al. 2014) may result in discrepancies of optimal dietary Se level in various fish species. In the current study, fish fed nano-Se and SeMet diets had greater perimeter and height of intestinal villi than control and sodium selenite groups. These results suggest that nano-Se and SeMet could maintain the integrity of the intestinal tract more efficiently as indicated by taller villi, and also protect the intestinal epithelial cells covering the villi. This maintenance effect may possibly be attributed to improved antioxidant enzyme activities and redox status in the intestine, which could result in less oxidative stress. On the other hand, the improved WG in nano-Se group could partly be related to increased ability of fish to assimilate the nutrients because of greater intestinal surface area, increasing the brush boarder enzyme secretion as well as reduction in enterocyte cell death and/or enterocyte turnover rates
associated with oxidative stress (Read-Snyder et al. 2009; Nugroho and Fotedar 2015). In this context, it has been reported that the protein content and GPx activity of the intestinal epithelial cells of crucian carp increased with increasing concentrations of nano-Se in the in vitro culture medium (Wang et al. 2013). Moreover, the authors of the previous study showed that nano-Se supplementation resulted in more consistency and integrity of the intestinal epithelial cells. Thus, better WG and FE of the common carp fed on nano-Se diet in our study may be explained by higher protein contents of the intestinal cells that could result in better metabolism of absorbed nutrients. In addition, Nugroho and Fotedar (2015) demonstrated higher numbers of longer microvilli with more integrity in crayfish (Cherax cainii) fed on organic Se-supplemented diet. The antioxidant effect of Se is accounted for its incorporation in selenocysteine, which is part of the active center of the GPx (Kohrle et al. 2000). Glutathione peroxidase has a protective role against oxidative radicals in body cells and enhances the body’s cell resistance such as immune cells against peroxidative damage (Burk et al. 2003). The results of this study showed higher serum GPx and SOD activities in nanoSe and SeMet groups as compared to the sodium selenite and control groups, indicating that nano and organic forms of Se could efficiently strengthen the antioxidant system against oxidative stress. A serum concentration of MDA is an index of lipid peroxidation and oxidative stress, and its levels depend upon the antioxidants. In the present study, fish fed on Se-supplemented diets had lower serum MDA levels compared to the control. Also,
Author's personal copy Fish Physiol Biochem
the lowest serum MDA level was observed in fish fed on nano-Se diet. In agreement, other studies demonstrated that nano-Se and organic sources of Se could enhance GPx activity, antioxidant capacity, and oxidative stress resistance in different fish species (Lin and Shiau 2005; Zhou et al. 2009; Liu et al. 2010; Le and Fotedar 2014; Ashouri et al. 2015; Liu et al. 2016). In contrast, Cotter et al. (2008) demonstrated that sodium selenite (0.4 mg kg−1) resulted in higher GPx activity in hybrid striped bass than Se-yeast when supplemented. The potent antioxidant capacity of the Se (Molnár et al. 2011; Ashouri et al. 2015; Khan et al. 2016) might increase stability of the RBCs membranes and their survivability by protecting them against oxygen free radicals, causing membrane damage, cell hemolysis, and anemia. In this context, Le et al. (2014) reported that increasing dietary Se (Se-yeast) levels (from 0 to 2 mg kg−1) led to an increase in RBC’s GPx activity in yellowtail king fish, which could protect RBCs from oxygen free radicals. In this study, different sources of Se increased the hematological parameters (RBCs count, Hb level, Hct%, and MCV) in juvenile common carp in comparison with control diet, indicating healthier status of fish fed on Se-supplemented diets. Similar to the finding of the present study, several studies have proved the role of Se in improving the hematological indices in different fish species such as Hct% in hybrid tilapia (El-Hammady et al. 2007); RBC counts in Nile tilapia (Oreochromis niluticus; Molnár et al. 2011); and RBC counts, Hb, and Hct% values in African catfish (Clarias gariepinus; Abdel-Tawwab et al. 2007) and golden mahseer (Tor putitora; Khan et al. 2016, 2017). Selenoproteins, especially GPx, protect neutrophils and macrophages from superoxide radicals derived from respiratory burst activity (Hodgson et al. 2006). In this study, WBCs counts and neutrophil percentage were increased in nano-Se and SeMet groups which could be due to the enhancement of fish health status. In fish, it has been reported that nano-Se or SeMet is more readily absorbed, and more potent in terms of bioavailability and effects on health, than sodium selenite (Wang et al. 1997; Le and Fotedar 2014). Similar to our results, Zhou et al. (2009) reported that a diet supplemented with nano-Se or SeMet significantly increased WBC counts in crucian carp as a consequence of increasing GPx activity in plasma and tissue. On the other hand, in the current study, lymphocyte percentage decreased in fish fed on Se-supplemented diets. In this context, it should be mentioned that the leukogram changes and immune
responses would be different depending on species, individual differences, nutritional condition, and animal welfare (Nandra 1997). Se can affect fish immune system via enhancing the activation of antioxidant enzymes (i.e., GPx, SOD and CAT) (Han et al. 2011; Le and Fotedar 2014; Naderi et al. 2017a), regulation of cell signaling molecules (i.e., nuclear factor kappa B and interleukin 2 (IL-2)), regulation of the function of immune cells (i.e., lymphocytes, natural killer cells, and neutrophils) by activating highaffinity IL-2 receptor (Rayman 2004), and anti-stress effects (Naderi et al. 2017b), which can finally lead to immune competence promotion. The results of this study showed that lysozyme activity was higher in nano-Se and SeMet groups compared to other groups, indicating that nano-Se and organic Se had higher bioavailability than inorganic Se for lysozyme activity. Enhanced lysozyme activity in nano-Se and SeMet groups could be associated with increased WBC counts, especially neutrophil percentage, which is the main producer of the lysozyme in blood. Similarly, dietary Se supplementation led to an increase in serum lysozyme activity in yellowtail king fish (Le et al. 2014; Le and Fotedar 2014), rainbow trout (Naderi et al. 2017a), and gold mahseer (Khan et al. 2017). In contrast, Cotter et al. (2008) showed that different Se sources had no effects on lysozyme activity in hybrid striped bass. Moreover, the increased serum SOD activity in nanoSe and SeMet groups may represent higher non-specific cellular immune responses in these groups, since the activity of SOD for detoxifying superoxide anion is associated with the respiratory burst activity of neutrophils and macrophages (Lin et al. 2011). In addition, fish fed nano-Se-supplemented diet had relatively higher serum ACP activity and Ig level as compared to other groups, suggesting that nano-Se was more effective than other Se sources in improvement of immune responses in common carp juveniles. In this context, it has been reported that dietary Se supplementation alone (Le and Fotedar 2014) or in combination with vitamin E (Le et al. 2014) led to an increase in serum antibody titration and bactericidal activity in yellowtail king fish. Plasma or serum total protein, which is mainly synthesized by liver parenchymal cells, has been used as a broad clinical indicator of health, immune competence, stress, and nutritional condition in fish (Riche 2007). In addition, it is believed that the absorbed Se is bound to albumin and transported to the liver, where it could be used for selenoprotein synthesis (Suzuki et al. 2010).
Author's personal copy Fish Physiol Biochem
Based on the results of this study, the highest concentrations of TP and ALB were observed in fish fed on nano-Se, suggesting better health and nutritional status in this group. Similar results were reported in African catfish (optimum Se requirement = 0.3–0.5 mg kg−1 organic Se; Abdel-Tawwab et al. 2007) and common carp (Ashouri et al. 2015) as a consequence of increasing Se concentration in the liver. Based on our data, dietary nano-Se supplementation caused significant decrease in serum CHO and LDL concentrations, as reported previously in common carp (Ashouri et al. 2015) and rainbow trout (Naderi et al. 2017a). Previous studies have demonstrated that dietary Se supplementation increased LDL receptor activity (Dhingra and Bansal 2006a) but decreased 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase expression in rat (Dhingra and Bansal 2006b), which can lead to decrease in serum LDL and CHO levels (Yang et al. 2010). In conclusion, the results of this study showed that dietary nano-se supplementation increased growth performance and feed efficiency in common carp juveniles, possibly as a consequence of improving integrity and consistency of intestinal epithelial cells, antioxidant capacity, immunological responses, and serum biochemical health indices. Funding information This work was funded by Khorramshahr University of Marine Science and Technology (Grant No. 222945).
References Abdel-Tawwab M, Mousa MA, Abbass FE (2007) Growth performance and physiological response of African catfish, Clarias gariepinus (B.) fed organic selenium prior to the exposure to environmental copper toxicity. Aquaculture 272:335–345 Abei H (1984) Catalase in vitro. Methods Enzymol 272:121–126 Ashouri S, Keyvanshokooh S, Salati AP, Johari SA, PashaZanoosi H (2015) Effects of different levels of dietary selenium nanoparticles on growth performance, muscle composition, blood biochemical profiles and antioxidant status of common carp (Cyprinus carpio). Aquaculture 446:25–29 Blaxhall P, Daisley K (1973) Routine haematological methods for use with fish blood. J Fish Biol 5:771–781 Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302–310 Burk RF, Hill KE, Motley AK (2003) Selenoprotein metabolism and function: evidence for more than one function for selenoprotein. J Nutr 133:1517–1520 Burk RF, Norsworthy BK, Hill KE, Motley AK, Byrne DW (2006) Effects of chemical form of selenium on plasma
biomarkers in a high-dose human supplementation trial. Cancer Epidemiol Biomark Prev 15:804–810 Cotter PA, Craig SR, McLean E (2008) Hyperaccumulation of selenium in hybrid striped bass: a functional food for aquaculture? Aquac Nutr 14:215–222 Dhingra S, Bansal MP (2006a) Attenuation of LDL receptor gene expression by selenium deficiency during hypercholesterolemia. Mol Cell Biochem 282:75–82 Dhingra S, Bansal MP (2006b) Modulation of hypercholesterolemia-induced alterations in apolipoprotein B and HMG-CoA reductase expression by selenium supplementation. Chem Biol Interact 161:49–56 El-Hammady AKI, Ibrahim SA, El-Kasheif MA (2007) Synergistic reactions between vitamin E and selenium in diets of hybrid tilapia (Oreochromis niloticus × Oreochromis aureus) and their effect on the growth and liver histological structure. Egypt J Aquat Biol Fish 11:53–58 Elia AC, Prearo M, Pacini N, Dörr AJM, Abete MC (2011) Effects of selenium diets on growth, accumulation and antioxidant response in juvenile carp. Ecotoxicol Environ Saf 74:166–173 Ellis AE (1990) Serum antiproteases in fish and lysozyme assays. In: Stolen JS, Fletcher TC, Anderson DP, Roberson BS, Van Muiswinkel WB (eds) Techniques in fish immunology. SOS Publications, Fair Haven, pp 95–103 Han D, Xie S, Liu M, Xiao X, Liu H, Zhu X, Yang Y (2011) The effects of dietary selenium on growth performances, oxidative stress and tissue selenium concentration of gibel carp (Carassius auratus gibelio). Aquac Nutr 17:741–749 Hao X, Ling Q, Hong F (2014) Effects of dietary selenium on the pathological changes and oxidative stress in loach (Paramisgurnus dabryanus). Fish Physiol Biochem 40: 1313–1323 Hodgson JC, Watkins CA, Bayne CW (2006) Contribution of respiratory burst activity to innate immune function and the effect of disease status and agent on chemiluminescence responses by ruminant phagocytes in vitro. Vet Immunol Immunopathol 112:12–23 Jovanovic A, Grubor-Lajsic G, Djukic N, Gardinovacki G, Matic A, Spasic M, Pritsos CA (1997) The effect of selenium on antioxidant system in erythrocytes and liver of the carp (Cyprinus carpio L.). Crit Rev Food Sci Nutr 37:443–448 Khan KU, Zuberi A, Nazir S, Fernandes JBK, Jamil Z, Sarwar H (2016) Effects of dietary selenium nanoparticles on physiological and biochemical aspects of juvenile Tor putitora. Turk J Zool 40:704–712 Khan KU, Zuberi A, Nazir S, Ullah I, Jamil Z, Sarwar H (2017) Synergistic effects of dietary nano selenium and vitamin C on growth, feeding, and physiological parameters of mahseer fish (Tor putitora). Aquac Rep 5:70–75 Kohrle J, Brigelius-Flohé R, Bock A, Gartner R, Meyer O, Flohé L (2000) Selenium in biology: facts and medical perspectives. Biol Chem 381:849–864 Kumar S, Sahu NP, Pal AK, Choudhury D, Yengkokpam S, Mukherjee SC (2005) Effect of dietary carbohydrate on haematology, respiratory burst activity and histological changes in L. rohita juveniles. Fish Shellfish Immunol 19: 331–344 Le KT, Fotedar R (2014) Immune responses to Vibrio anguillarumin yellowtail kingfish, Seriola lalandi, fed selenium supplementation. J World Aquac Soc 45:138–148
Author's personal copy Fish Physiol Biochem Le KT, Dao TT, Fotedar R, Partrigde GJ (2014) Selenium and vitamin E interaction in the nutrition of yellow kingfish (Seriola lalandi): physiological and immune responses. Aquac Nutr 20:303–313 Lewis S, Bain B, Bates ID (2001) Lewis practical haematology. Churchill Livingstone, New York Lin YH, Shiau SY (2005) Dietary selenium requirements of juvenile grouper, Epinephelus malabaricus. Aquaculture 250: 356–363 Lin S, Pan Y, Luo L, Luo L (2011) Effects of dietaryb-1, 3-glucan, chitosan or raffinose on the growth, innate immunity and resistance of koi (Cyprinus carpio koi). Fish Shellfish Immunol 31:788–794 Liu K, Wang XJ, Ai QH, Mai KS, Zhang WB (2010) Dietary selenium requirement for juvenile cobia, Rachycentron canadum L. Aquac Res 41:594–601 Liu GX, Jiang GZ, Lu KL, Li XF, Zhou M, Zhang DD, Liu WB (2016) Effects of dietary selenium on the growth, selenium status, antioxidant activities, muscle composition and meat quality of blunt snout bream, Megalobrama amblycephala. Aquacu Nutr 23:777–787. https://doi.org/10.1111/anu.12444 Lorentzen M, Maage A, Julshamn K (1994) Effects of dietary selenite or selenomethionine on tissue selenium levels of Atlantic salmon (Salmo salar). Aquaculture 121:359–367 McCord JM, Fridovich I (1969) Superoxide dismutase an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244: 6049–6055 Molnár T, Biró J, Balogh K, Mézes M, Hancz C (2011) Improving the nutritional value of Nile tilapia fillet by dietary selenium supplementation. Israel J Aquacult Bamidgeh 64:1–6 Naderi M, Keyvanshokooh S, Salati AP, Ghaedi A (2017a) Combined or individual effects of dietary vitamin E and selenium nanoparticles on humoral immune status and serum parameters of rainbow trout (Oncorhynchus mykiss) under high stocking density. Aquaculture 474:40–47 Naderi M, Keyvanshokooh S, Salati AP, Ghaedi A (2017b) Effects of dietary vitamin E and selenium nanoparticles supplementation on acute stress responses in rainbow trout (Oncorhynchus mykiss) previously subjected to chronic stress. Aquaculture 473:215–222 Naderi M, Keyvanshokooh S, Salati AP, Ghaedi A (2017c) Proteomic analysis of liver tissue from rainbow trout (Oncorhynchus mykiss) under high rearing density after administration of dietary vitamin E and selenium nanoparticles. Comp Biochem Physiol Part D Genom Proteom 22:10–19 Nandra RK (1997) Nutrition and the immune system: an introduction. Am J Clin Nutr 66:460–463 National Research Council of the National Academies (NRC) (2011) Nutrient requirements of fish and shrimp. The National Academic Press, Washington, DC Noguchi T, Cantor AH, Scott ML (1973) Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J Nutr 103:1502–1511
Nugroho RA, Fotedar R (2015) Effects of dietary organic selenium on immune responses, total selenium accumulation and digestive system health of marron, Cherax cainii (Austin, 2002). Aquac Res 46:1657–1667 Rayman MP (2004) The use of high-selenium yeast to raise selenium status, how does it measure up? Br J Nutr 92:57– 573 Read-Snyder J, Edens FW, Cantor AH, Pescatore AG, Pierce JL (2009) Effect of dietary selenium on small intestine villus integrity in Reovirus-challenged broilers. Int J Poult Sci 8: 829–835 Riche M (2007) Analysis of refractometry for determining total plasma protein in hybrid striped bass (Morone chrysops × M. saxatilis) at various salinities. Aquaculture 264:279–284 Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra W (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588–590 Schrauzer GN (2000) Selenomethionine: a review of its nutritional significance, metabolism and toxicity. J Nutr 130:1653–1656 Siwicki AK, Anderson DP, Rumsey GL (1994) Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis. Vet Immunol Immunopathol 41:125–139 Skalickova S, Milosavljevic V, Cihalova K, Horky P, Richtera L, Adam V (2017) Selenium nanoparticles as a nutritional supplement. Nutrition 33:83–90 Suzuki Y, Hashiura Y, Matsumura K, Matsukawa T, Shinohara A, Furuta N (2010) Dynamic pathways of selenium metabolism and excretion in mice under different selenium nutritional statuses. Metallomics 2:126–132 Tort L, Gomez E, Montero D, Sunyer JO (1996) Serum hemolytic and agglutinating activity as indicators of fish immunocompetence: their suitability in stress and dietary studies. Aquaculture 4:31–41 Wang C, Lovell RT, Klesius PH (1997) Response to Edwardsiella ictaluri challenge by channel catfish fed organic and inorganic sources of selenium. J Aquat Anim Health 9P:172–179 Wang Y, Yan X, Fu L (2013) Effect of selenium nanoparticles with different sizes in primary cultured intestinal epithelial cells of crucian carp, Carassius auratus gibelio. Int J Nanomedicine 8:4007–4013 Yang KC, Lee LT, Lee YS, Huang HY, Chen CY, Huang KC (2010) Serum selenium concentration is associated with metabolic factors in the elderly: a cross-sectional study. Nutr Metabol 7:38 Zhang J, Gao X, Zhang L, Bao YP (2001) Biological effects of a nano red elemental selenium. Biofactors 15:27–38 Zhou X, Wang Y, Gu Q, Li W (2009) Effects of different dietary selenium sources (selenium nanoparticle and selenomethionine) on growth performance, muscle composition and glutathione peroxidase enzyme activity of crucian carp (Carassius auratus gibelio). Aquaculture 291:78–81
