The use of Avena sativa extract against Aeromonas hydrophila and its effect on growth performance, hematological and immunological parameters in common carp (Cyprinus carpio)

Abstract

In this research the effects of oat Avena sativa extract on the non-specific immune system of common carp (Cyprinus carpio) was examined. For this purpose, the fishes (average weight 9.91 ± 1.52 g) were fed with 5 g kg ⁻¹, 10 g kg ⁻¹ and 20 g kg ⁻¹ oat extract supplemented diets for 60 days. Following 60 days of feeding, the fishes were injected with Aeromonas hydrophila and mortalities were recorded. Lysozyme and myeloperoxidase activity, improved in all groups that received feed supplemented with oat extract. Serum glucose and cholesterol decreased while total protein and albumin in fish increased with the use of the diet supplement with oat extract. Haemoglobin (Hb), mean cell hemoglobin (MCH) and mean cell haemoglobin concentration (MCHC) increased with diet supplemented with oat extract. Oat extract at the concentration of 10 g kg⁻¹ showed significantly higher relative percentage survival (67%) when compared with the control against A. hydrophila injection. Also the dietary supplementation with oat extract caused a significant increase in growth parameters (final weight (FW), weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR), when compared to non-supplemented control basal diet. The results suggest that A. sativa extract can be used as a feed supplement to enhance fish immune response and disease resistance against A. hydrophila.

Keywords:

• Aeromonas hydrophila
• Avena sativa
• Cyprinus carpio
• immune system
• oat extract

Introduction

Common carp (Cyprinus carpio), is a freshwater fish inhabiting several freshwater ecosystems such as lakes, ponds and dams and it is widely distributed thoughout the world especially in Asia, Europe and the Middle East (Eddy & Underhill 1974). Moreover, common carp is the third most frequently introduced species world-wide (Saikia & Das 2009). However, the diseases caused by pathogens bacterial in common carp culture are becoming severe resulting in significant morbidity and mortality. The culture of C. carpio in fresh water aquaculture has suffered due to bacterial infections particularly by the occurrence of “motile aeromonas septicemia” caused by Aeromonas hydrophila, which results in heavy losses and causes economic loss to fish farmers (Austin & Adams 1996). In several fish species, including carps, A. hydrophila is regarded as an opportunistic pathogen both in the farm and field. The disease is frequently associated with haemorrhagic septicemia (Kuge et al. 1992; Roberts et al. 1992; Zhang et al. 2014). In order to control the proliferation of these bacteria, antibiotics are widely used in intensive aquaculture. Nevertheless prolonged use of antibiotics could lead to many negative effects such as antibiotic resistance in bacteria or antibiotics residues in environment and fish products (Cabello 2006). Therefore, the research for new methods to prevent infectious diseases has become crucial in common carp culture. Immunostimulants increase resistance to infectious diseases, not only stimulating the acquired immune response, but also enhancing innate immune mechanisms (Galindo-Villegas & Hosokawa 2004). The immunostimulants also have additional advantages, such as growth enhancement and increase in the survival rates of the fish under stress (Heo et al. 2004). Many immunostimulants have been found to be effective in common carp (Harikrishnan et al. 2003; Yin et al. 2009; Maqsood et al. 2010; Anbazahan et al. 2014; Jagruthi et al. 2014; Wang et al. 2015).

Avena sativa L. (Gramineae) commonly known as oat, groats, haber, hafer, avena, straw, oatmeal is an edible plant and it is a species of grain cultivated for its seed (Coffman 1977). The bran of this plant has also been used as a traditional folk medicine for the treatment of rheumatism, gout, and liver and skin diseases on the benefit of its diuretic and sedative effects (Wenzig et al. 2005). Up till now, saponins (Waldemar 1982), flavonoids (Popovici et al. 1977; Peterson 2001), and beta-D-glucan (Ahmad et al. 2010) have been found in the plant. Also the oat is rich in protein, has lots of beneficial minerals such as iron, calcium, potassium, magnesium, copper, zinc, silicon, selenium and contains several vitamins like Vitamin B₁, B₂, B₆, B₁₂, Niacin, Vitamin C, Vitamin A, Vitamin E. Different chemical constituents like carbohydrates, proteins, avenanthramides, lipids (9 glycolipids and 11 phospholipids), an indole alkaloid-gramine, number of flavonoids, 3 flavonolignans, saponins and sterols have been reported from A. sativa. Oats and its constituents are reported to possess varied pharmacological activities like lowering blood cholesterol and blood sugar, as well as being immunomodulatory, anticancer, antioxidant, antiatherogenic, and topical anti-inflammatory (Singh et al. 2013).

The aim of the study was to assess oral administration of three different concentrations of oat extract (OE) derived from Avena sativa on biometrical, haematological, biochemical and immunological indices of common carp (Cyprinus carpio) in experimental conditions. A further aim was to investigate the effects of supplemental dietary oat extract on disease resistance of C. carpio against A. hydrophila.

Materials and methods

Preparation of oat extract

Samples of A. sativa were attained from their natural environment from Balıkesir region of Turkey. One hundred grams of dried ground of oat were extracted with 100 mL water in 1000 mL conical flasks at 60 °C in a water bath incubated for 24 h in a water bath and then filtered (0.45 μ Whatman filter). The same process was repeated three times for the complete extraction. Water was evaporated using a lyophilizator for the complete extraction. The extract were stored in a refrigerator at 4 °C for future use (Lee et al. 2000; Tanker & Tanker 2003).

Fish and experimental design

Healty common carp, Cyprinus carpio (9.91 ± 1.52 g) were obtained from Antalya-Kepez (Antalya, Turkey). The study was carried out in triplicate (three aquarium per experimental groups) with 216 fish allocated into 50 L aquariums (18 fish/aquarium). The oat extract was added to the feed at 0 g kg⁻¹ (OE0) as control, 5 g kg⁻¹ (OE5), 10 g kg⁻¹ (OE10) and 20 g kg⁻¹ (OE20). The control diet contained no supplementation (OE0) (Table 1). The fishes were fed with 2% of body weight during the trial. Water was changed daily at a rate of ∼10% of the total volume. After 60 days of feeding, nine fishes from each group were randomly chosen and their blood samples were collected. Moreover, at the end of the feeding period, all the groups were injected intraperitoneally (i.p.) with 100 μl PBS containing A. hydrophila at 1.5 × 10⁶ CFU mL⁻¹. During the experimental period the main parameters of water were measured as: temperature 25.2 ± 0.6 °C, pH 7.6 ± 0.6 and dissolved oxygen 5.63 ± 0.68 mg L⁻¹.

Table 1. Percentage and proximate composition of the experimental diets containing supplement of different oat extract (OE) rate.

	Experimental diets
	OE0	OE5	OE10	OE20
Ingredients, %
Fish meal	23.00	23.00	23.00	23.00
Soybean meal	37.00	37.00	37.00	37.00
Wheat flour	12.00	12.00	12.00	12.00
Fish oil	5.00	5.00	5.00	5.00
Vitamin–mineral mix^a^,^b	4.00	4.00	4.00	4.00
Starch	19.00	18.50	18.00	17.00
Oat extract	0	0.50	1.00	2.00
Total	100	100	100	100
Chemical analyses
Protein, %DM	35.10	35.54	35.11	35.42
Fat, %DM	7.72	7.53	7.75	7.62
Ash, %DM	5.94	5.32	5.45	5.92
NFE^c, %DM	47.16	47.17	47.18	47.17
Energy^d, kj/g	19.35	19.38	19.36	19.39

a Vitamin Mix: Vit. A, 18,000 IU; Vit. D3, 2500 U; Vit. E, 250 mg/kg; Vit. K3, 12 mg/kg; Vit. B1, 25 mg; Vit. B2, 50 mg; Vit. B3, 270 mg; Vit. B6, 20 mg; Vit. B12, 0.06 mg; Vit. C, 200 mg; Folic acid, 10 mg; Calcium d–pantothenate, 50 mg; Biotin, 1 mg; Inositol, 120 mg; Choline chloride, 2000 mg.

b Mineral Mix: Fe, 75.3 mg; Cu, 12.2 mg; Mn, 206 mg; Zn, 85 mg; I, 3 mg; Se, 0.350 mg; Co, 1 mg.

c Nitrogen–free extracts (NFE) = dry matter – (crude lipid + crude ash + crude protein + crude fiber).

d Energy calculated according to 23.6 kJ g ^− 1 protein, 39.5 kJ g ^− 1 lipid, and 17.0 kJ g ^− 1 NFE.

Blood samples and analyses

Nine fishes from each groups (three fishes from per aquarium) were selected and anesthetized by 0.01 mg L⁻¹ of phenoxyethanol. After that blood samples were obtained from caudal vein using syringe at the end of the 60 days feding trial. Newly collected blood samples were used to determine the hematological parameters. Blood was centrifuged for 15 min. at 3500 g. After centrifugation, serums were stored at −20 °C for future analysis.

Hematological assay

The hematological indices Red blood cell (RBC) count (×10⁶ per mm3), hematocrit (Hct; %), and hemoglobin (Hb) concentrations (g/dL) were designated Blaxhall and Daisley (1973) method. Mean corpuscular volume (MCV), mean corpuscular Hb (MCH), and mean corpuscular Hb concentrations (MCHCs) were calculated according to Bain et al. (2006).

Biochemical assay

The determination of plasma glucose (GLU), total protein (TPROT), albumin (ALB), globulin (GLO), triglyceride (TRIG), and cholesterol (CHO) were determined using commercially diagnostic kit (Bioanalytic Diagnostic Industry, Co.).

Lysozyme activity

Serum lysozyme (Lyso) was assessed using the turbidometric assay. A Micrococcus lysodeikticus suspension of 875 μl (Sigma, ATCC 4698) at a concentration of 0.2 mg/ml (in PBS) was added to 25 μl of serum samples and were measured spectrophotometrically at 530 nm after 0.5 and 4.5 minutes at 25 °C, with a spectrophotometer. A unit of lysozyme activity was defined as the amount of serum caused reduction in absorbance of 0.001 min⁻¹.

Myeloperoxidase activity

Total myeloperoxidase (MPO) content in blood serum was measured according to Quade and Roth (1997) with minor modifications. Thirty microliters serum was diluted with 370 ml of HBSS without Ca2 + or Mg2 + in eppendorf tubes. Hundred microliters of 0.1 mg/ml (w/v) 3,3′,5,5′-tetramethylbenzidine dihydrochloride and 0.06% fresh hydrogen peroxide were added. The reaction was followed kinetically by measuring the increase of absorbance. Reaction velocities were determined as IU, defined as the amount of enzyme required to produce an 0.001 increase in absorbance per minute 0.5 ml of reaction mixture (ΔA 450/min/ml).

Challenge study

To study the resistance of the common carp to A. hydrophila, 45 fish from each experimental and control groups were used. After 60 days of feeding and blood samplings, the fish were injected intraperitoneally with 0.1 mL of a 1.5 × 10⁶ CFU mL⁻¹ A. hydrophila was suspended in phosphate buffered saline. The fish were checked regularly with eyes for any overt signs of disease including behavioural abnormalities and dead fish taken slowly from aquariums without creating stress factors. Mortality was noted in all the groups for 6 days of post infection. The confirmation of the infection was accomplished after re-isolating the bacteria from the dead fishes. Reisolated bacteria identification was obtained by using classical biochemical (Austin & Austin, 2007) and API 20 Strep kit (Biomerieux, France).

Relative percentage survival

Recorded mortality data was used for calculating Relative Percentage Survival (RPS) following Amend (1981).

Growth performance

At the end of 60 days, fishes in each aquarium were individually weighed. Growth performance was calculated as following formulae:

Crude protein, crude lipid, moisture and ash in feed ingredients and diets were determined following standard methods (AOAC 1998).

Statistical analysis

Statistical analysis of the data involved one-way analysis of variance (ANOVA) followed by Tukey’s pairwise multiple comparison test. The data were expressed as arithmetic means and standard error (SE). Differences were considered significant at p < 0.05.

Results

There was no mortality at the end of the study in all the groups. Growth performance parameters of common carp feed with oat extracted suplemented diets shown in Table 2. WG of carps fed with the OE10 diet was tended to increase than fish fed with other diets (p < 0.05). The oat extract influenced FCR. The FCR value was significantly different other groups were vary depending on the OE10 diet. Best specific growth ratio (SGR) was obtained for common carp fed with the OE10 diet (p < 0.05).

Table 2. Weight gain, specific growth rate (SGR) and feed conversion rate (FCR) in carp fed the experimental diets.

Parameters	OE0	OE5	OE10	OE20
WG, %	71.91 ± 9.04^b	77.42 ± 7.50^ab	93.09 ± 5.51^a	75.81 ± 0.69^b
SGR	0.90 ± 0.09^b	0.95 ± 0.07 ^ab	1.10 ± 0.10^a	0.94 ± 0.01^ab
FCR	1.92 ± 0.15^a	1.78 ± 0.08^a	1.47 ± 0.10^b	1.70 ± 0.04^ab

Data were presented as mean ± SE (n = 3/group). Values within the same row having different superscripts are significantly different (p < 0.05).

Hematological variables

The effects of oat extract on common carp hematological variables are presented in Table 3. There were no significant differences among the Hct, RBC and MCV levels in any of the experimental groups or the control group (p > 0.05). Besides the Hb, MCH and MCHC were also found to be significantly higher in the experimental groups (p < 0.05). Among the groups, the OE10 showed the highest level of Hb.

Table 3. Serum biochemical and hemato-immunological parameters of C. carpio juveniles fed diets containing oat extract for 60 days.

Paremeters	Control	OE5	OE10	OE20
Myeloperoxidase, U/L	33.65 ± 0.75^c	38.97 ± 0.52^b	48.40 ± 0.76^a	49.78 ± 0.29^a
Lysozyme activity, U/L	533.3 ± 6.66^d	933.3 ± 6.66^c	1200.00 ± 8.64^b	1770.77 ± 9.68^a
GLU, mg/dL	54.25 ± 6.42^b	53.14 ± 3.15^b	50.84 ± 9.65^a	65.90 ± 12.19^a
TRİG, mg/dL	56.41 ± 8.03^a	64.10 ± 13.54^a	66.41 ± 13.09^a	68.72 ± 10.75^a
CHOL, mg/dL	65.64 ± 13.33^a	62.93 ± 18.01^a	64.48 ± 9.73^a	48.39 ± 14.15^b
TPROT, g/dL	1.96 ± 0.18^b	2.07 ± 0.26^b	2.96 ± 0.39^a	2.24 ± 0.29^b
ALB, g/dL	1.36 ± 0.15^b	1.43 ± 0.33^b	1.99 ± 0.35^a	1.40 ± 0.22^b
GLO, d/dL	0.60 ± 0.27^a	0.64 ± 0.39^a	0.97 ± 0.24^a	0.85 ± 0.46^a
Hct, %	24.11 ± 0.48^a	25.33 ± 0.52^a	24.11 ± 0.42^a	24.00 ± 0.28^a
Hb, g/dL	8.88 ± 0.21^c	13.20 ± 0.72^b	15.90 ± 0.59^a	14.49 ± 0.79^ab
RBC, ×10^6 mm^3	4.66 ± 0.24^a	5.27 ± 0.15^a	4.97 ± 0.21^a	5.17 ± 0.35^a
MCV, μm^3	53.33 ± 4.08^a	48.44 ± 1.89^a	49.46 ± 2.92^a	48.33 ± 3.57^a
MCH, pg cell^−1	19.58 ± 1.36^c	25.23 ± 1.61^b	32.57 ± 2.29^a	28.52 ± 1.55^ab
MCHC, %	37.02 ± 1.28^c	52.02 ± 2.34^b	66.00 ± 2.34^a	60.42 ± 3.37^a

Values are mean (n = 9). Mean ± SE with common superscripts in the same line are not significantly different (p > 0.05).

GLU: glucose; Trıg: triglyceride; CHOL: cholesterol; TPROT: total protein; ALB: albumin; Hct: hematocrit; Hb: hemoglobin; MCV: mean cell volume; MCH: mean cell hemoglobin; MCHC: mean cell hemoglobin concentration.

Biochemical variables

The effects of different concentrations of oat extract on serum biochemical parameters of common carp are summarized in Table 3. Serum GLU value was tended to increase in carp fed with OE20 diet (p < 0.05). Other serum parameters, which are TPROT, ALB, GLO values were fed with OE10 diet were significantly higher than the control values (p < 0.05). The CHOL level was significantly higher in control group (p < 0.05). The addition of OE reduced the CHOL levels. There were no significant differences in TRIG levels among the groups (p > 0.05).

Lysozyme activity

As it is shown in Table 3, serum lysozyme activity, significantly increased in the oat extract supplemented diet fed groups at all concentrations in C. carpio. The differences were significant (p < 0.05) throughout the trial period. In all concentrations of oat extract-added diet fed fish serum showed higher lysozyme activity compared to the control. Among the concentrations, the OE20 group showed the highest lysozyme activity.

Myeloperoxidase activity (MPO)

The effect of oat extract supplemented diet fed on fish on the leukocyte myeloperoxidase activity in serum is depicted in Table 3. The highest significant myeloperoxidase values (p < 0.05) were recorded in fish fed with the OE10 and OE20 concentration of oat extract. All concentrations of oat extract led to significantly higher myeloperoxidase than the control group.

Challenge test with A. hydrophila

After 60 days of feeding, fishes were challenged with A. hydrophila and cumulative mortality was recorded for 6 days (Figure 1). The OE10 and OE20 groups showed reduced mortality compared to the control group (p < 0.05). The relative percentage survival (RPS) and survival rate of groups challenged with A. hydrophila are presented in Table 4. A significantly higher survival rate was determined in fish fed with diets supplemented with both 10 g kg⁻¹ and 20 g kg⁻¹ concentrations of oat extract following exposure to A. hydrophila. More specifically, the highest protection was recorded in fishes of OE10 group followed by the OE20group. The mortality percentage was highest (80%) in the control group and lowest (26.66%) in OE10 group. The relative percentage survival was highest (67%) in OE10 group and lowest in OE5 (5%) group.

Figure 1. Kaplan–Meier survivorship curves (cumulative survival [%] over time [h]) for common carp after challenge with Aeromonas hydrophila; the fish were fed with oat extract supplemented diets (0, 5, 10 or 20 g of OE/kg of feed; control diets, OE5, OE10, and OE20, respectively) prior to bacterial challenge.

Table 4. Relative Percentage Survival (RPS) (%) of challenged Cyprinus carpio fed oat extract supplemented diet and the control diet.

Groups	Survival (%)	Mortality	RPS (%)
Control	20^c	80	–
OE5	23.33^c	76.66	5
OE10	73.33^a	26.66	67
OE20	66.66^b	33.33	59

Data are represented as mean ± SE (n = 30). Different letters represent the significantly different (p < 0.05).

Discussion

The use of chemicals as growth promoter and resistance of bacterial disease in fish culture reason to many problems like resistance to antibiotics while the utilization of chemicals can be harmful to fish health, consumers and environment (Alderman & Hastings 1998). There from, most of the attention has been paid to natural products in order to substitute antibiotics in aquaculture. The most important of the possibilities of natural products is the use of herb extracts. They are obtained from many plant materials such as flowers, buds, seeds, leaves, fruits (Rattanachaikunsopon & Phumkhachorn 2009). This study evaluated the effect of oat extract on growth performance, non-specific immune response and disease resistance of common carp against Aeromonas hydrophila.

In fishes, blood is a patho-physiological reflector of the entire body and the counts of hematological parameters in blood give an indication of the health statue by determining any abnormality occurring owing to the use of immunostimulants (Tewary & Patra 2011). The hematocrit value is an important tool of health status of fish in aquaculture (Mulero et al. 1998). In the present study no significant changes were observed in hematocrit level. Many authors reported that there was no enhancement of hematocrit level after using immunostimulant compounds in fishes (Eslamloo et al. 2012; Binaii et al. 2014). In our study the Hb, RBC, MCH and MCHC levels significantly increased in the group fed with especially 10 g kg⁻¹ oat extract supplemented diet. Also parallel works have been documented in different fish species such as common carp, C. carpio, (Harikrishnan et al. 2005), juvenile beluga, Huso huso, (Binaii et al. 2014). The increase in the levels of serum protein, albumin and globulins in fish is thought to be associated with a stronger innate immunity response (Wiegertjes et al. 1996). The present study showed an enhancement of total protein in group fed with 10 g kg⁻¹ oat extract supplemented diet that recorded the highest values compared to the other groups. This is in agreement with previous studies conducted using Astragalus membranaceus, Polygonum multiflorum, Isatis tinctoria and Glycyrrhiza glabra (Yuan et al. 2007), garlic (Nya & Austin 2011), Nigella sativa and quercetin (Awad et al. 2013) in the sense that they have all enhanced serum total protein level in different fishes. Also Binaii et al. (2014) recorded increases in total protein level in juvenile beluga fed with nettle. These studies suggested that high concentration of total protein in fish serum was likely to be a result of the enhancement of non-specific immune response. Albumin and globulin are the main plasma proteins in fishes (Gunter et al. 1961). The present results indicate that the albumin and globulin values increased along with the use of oat extract enriched diets. Similar results in globulin were reported in rainbow trout fed with garlic enriched diets (Nya & Austin 2009). Increasing albumin level was reported by Jagruthi et al. (2014) in carp fed with astaxanthin supplemented diet for 4 weeks. An increase in glucose level was one of the stress indicators in fishes (Morgan & Iwama 1997). In this study oat extract supplemented diet decreased glucose values in common carp compared to the control group. As the value of oat extract increased in diet, the level of glucose decreased. This might be due to the capability of the plant extract to reduce the effects of stressors. This is in agreement with the reports of Citarasu et al. (2006), Sahu et al. (2007) and Abasali & Mohamad (2010) that glucose levels were reduced in different fish fed with herbal immunostimulant diets. In the present study, cholesterol level had significantly decreased in the 20 g kg⁻¹ group compared to the control group. The triglyceride levels were slightly higher in oat extract groups but there was no significant difference. Similar results were obtained for various fish species fed with herbal enhanced diets (Immanuel et al. 2009; Metwally 2009).

Lysozyme activity is another ingredient in the first line of barrier in innate immune system (Magnadóttir 2006). Biological and syntetic immunostimulant products are considered to increase serum lysozyme activity (Engstad et al. 1992). In the present study, fish fed diets supplemented with different levels of oat extract showed significantly higher lysozyme activities when compared to the control group. Similar results have also been reported in common carp fed with herbal immunostimulant diets (Abasali & Mohamad 2010; Anbazahan et al. 2014; Jagruthi et al. 2014; Wang et al. 2015). The increased lysozyme activity observed in this study supported a higher non-specific immune response in the common carp fed with oat extract supplemented diets. Myeloperoxidase (MPO) is another important enzyme which plays a role in the killing of microorganisms (Johnston 1978). In this study, MPO activity of serum in the experimental groups showed an increase compared to the control, especially after feeding with 2% oat extract supplemented diet. Similarly, MPO activity increased significantly in common carp fed with diets supplemented with different levels of extract of caretenoids (Sowmya & Sachindra 2015). Many authors reported an enhancement of MPO activity after using immunostimulant compounds in fishes (Awad et al. 2013; Kumar et al. 2013; Wu et al. 2013).

This study demonstrated that fishes fed with oat extract supplemented diet remarkably increased the survival rate of C. carpio against A. hydrophila pathogen. This result indicated that oat, A. sativa, extract had a positive effect on the survival rate of common carp and this could be due to the cooperative effects of the active compounds in the extract. According to several studies (Nya & Austin 2009; Awad & Austin 2010; Nya & Austin 2011), immunostimulants can enhance resistance of fish to several bacterial pathogens including A. hydrophila. In other studies, a lower mortality on A. hydrophila challenge was reported in C. carpio fed Azadiracta indica (Harikrishnan et al. 2003), astaxanthin (Jagruthi et al. 2014), carotenoids (Anbazahan et al. 2014; Wang et al. 2015). The results showed that 10 g kg⁻¹ oat extract supplemented diets increased the weight gain.

Several herbs were tested for their growth promoting activity in aquatic animals (Citarasu et al. 2002). Wang et al. (2015) observed that dietary supplementation of Rehmannia glutinosa increased the growth rate C. carpio. Positive effects of herbal extracts on growth performance of different fish have been reported by other authors (MacLennan et al. 2002; Immanuel et al. 2009; Talpur & Ikhwanuddin 2013; Kanani et al. 2014). The results shown in the present study also indicate that oat extract included in the diet is useful for improving the growth performance of common carp. Especially the feed conversion ratio and specific growth rate of the study showed that fishes fed with OE10 was significantly superior when compared to other the groups.

The present results showed that the oat extract has a potential value for aquaculture both in terms of increased growth, immune response and resistance to A. hydrophila when diet is supplemented with 10 g kg⁻¹ oat extract. Further studies focusing on the potential application of oat extract in other fishes and pathogens as an immunostimulant for the use in aquaculture are strongly recommended.

The authors wish to thank Süleyman BABA, Dr. Murat YABANLI and Aykut YOZUKMAZ for their help.

Funding information

This study was approved by the local ethics committee for animal experiments of Adnan Menderes University, Aydın, Turkey (Approval Number: 64583101/2015/141).

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.