Rosemary leaf powder improved growth performance, immune and antioxidant parameters, and crowding stress responses in common carp (Cyprinus carpio) fingerlings

Aquaculture 505 (2019) 473–480

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Rosemary leaf powder improved growth performance, immune and antioxidant parameters, and crowding stress responses in common carp (Cyprinus carpio) fingerlings

T

Morteza Yousefia, , Seyyed Morteza Hoseinib, , Yury Anatolyevich Vatnikova, Evgeny Vladimirovich Kulikova, Stanislav Gennadievich Drukovskya ⁎

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a

Department of Veterinary Medicine, Peoples' Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St, Moscow 117198, Russian Federation Inland Waters Aquatics Resources Research Center, Iranian Fisheries Sciences Research Institute, Agricultural Research, Education and Extension Organization, Gorgan, Iran

b

ARTICLE INFO

ABSTRACT

Keywords: Phytotherapy Health Rosmarinus officinalis Diet Aquaculture

The present study aimed to investigate the beneficial effects of dietary rosemary leaf powder (RLP) as growth promotor and immunostimulant on plasma immunity as well as stress responses in common carp (Cyprinus carpio) fingerlings. In a 65-day feeding trial, fish were fed on experimental diets containing RLP (0, 1, 2 and 3%). At the end of the experiment, fish performance, survival rate, and proximate composition were investigated. Thereafter, blood samples were collected from treatments for investigation of biochemical and humoral immune parameters. Then, fish were subjected to net-confinement crowding stress for 3 h. Blood samples were collected before and after stress pressure, to measure plasma cortisol and glucose levels. The obtained results showed that fish growth increased along with the increase of dietary RLP levels up to 3%. But proximate composition indices were not influenced by dietary RLP treatments. The RLP-supplemented fish had significantly higher plasma total protein, albumin, globulin, lysozyme, and ACH50 compared to the control group. WBC values were significantly higher in the 2 and 3% RLP-treatments compared to those of the control one. Whereas, fish treated with 3% RLP showed highest plasma total Ig levels. CAT and SOD activities were significantly higher in RLP-supplemented groups than in the control group. Cortisol and glucose levels in all groups were significantly affected by stress; nevertheless, the RLP administration significantly mitigated the stress-induced increase in plasma cortisol and glucose levels. The present results confirmed that the oral administration of RLP has the potential to promote growth performance, enhance antioxidant and immunological parameters, and mitigate the negative effects of crowding stress on common carp fingerlings.

1. Introduction

stressful condition and result in infectious diseases outbreaks (Hoseinifar et al., 2019). Moreover, similar to other aquaculture species, common carp may expose to some stressors, including handling, sampling, poor nutrition, sorting and transportation, which may cause stress in this species (Parodi et al., 2014; Faggio et al., 2015; Hoseini et al., 2018c; Yousefi et al., 2018; Abdel-Tawwab et al., 2019). Variety of fish body functions may affect by these stressors. Secretion of corticosteroids and catecholamines are the first, hyperglycemia, gluconeogenesis, lipolysis, hydromineral changes and immunosuppression are the second, and changes in fish whole body (growth, reproduction and diseases resistance) are known as the third stress responses in fish (Barton, 2002). Furthermore, stress deteriorates fish health and leading to immunosuppression and increasing infectious

Aquaculture products supply > 20 and 50% protein intake in developing and poorest countries, respectively (Awad and Awaad, 2017). According to this, over the last decades, the intensive and semi-intensive aquaculture practices have shown a rapid increase to meet these needs. Common carp is among the most important reared fish species in many parts of the world representing 71.9% of freshwater production (Abdel-Tawwab and Monier, 2018), and contributes 8% (ca. 4.6 million tons) of the world's total finfish aquaculture production (FAO, 2018). It has been an increasing desire to develop intensive culture as a promising approach to meet the growing demands for this species (Hoseinifar et al., 2019). However, high stocking densities may cause a

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Corresponding author. E-mail addresses: [email protected] (M. Yousefi), [email protected] (S.M. Hoseini).

https://doi.org/10.1016/j.aquaculture.2019.02.070 Received 16 January 2019; Received in revised form 24 February 2019; Accepted 26 February 2019 Available online 27 February 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

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diseases (Awad and Awaad, 2017; Aliko et al., 2018; Gobi et al., 2018; Sehonova et al., 2018). Growth performance, stress resistance, antioxidant status, and immune responses can be enhanced by nutritional manipulation (Hoseini and Hosseini, 2010; Khodadadi et al., 2018). Medicinal plants and their bioactive compounds including alkaloids, phenolic compounds, and steroids have been successfully applied in aquatic organisms to promote appetite, growth performance, stress responses and immunity (Sivaram et al., 2004; Harikrishnan et al., 2011; Reverter et al., 2014; Faggio et al., 2015a, b; Guardiola et al., 2016; Abdel-Tawwab, 2016; Abdel-Tawwab et al., 2018b; Capillo et al., 2018; Reyes-Cerpa et al., 2018; Van Doan et al., 2018; Elumalai et al., 2019). For example, previous studies confirmed the positive effects of herbal plants such as Psidium guajava (Gobi et al., 2016), Curcuma longa (Abdel-Tawwab and Abbass, 2017), Toona sinensis (Wu et al., 2010), Ocimum gratissimum (Abdel-Tawwab et al., 2018a), Cynodon dactylon (Kaleeswaran et al., 2011), and Eriobotrya japonica (Hoseinifar et al., 2018) on fish growth, health, and immunity. Rosemary (Rosmarinus officinalis) is a medicinal herb species of the Labiatae family with active extracts mainly composed of 1,8-cineole, carnosol, carnosic acid, rosmarinic acid, β- and α-pinene, camphor, and camphene (Gao et al., 2014; Taheri Mirghaed et al., 2018; Zoral et al., 2017), and rich in antioxidants (Zoral et al., 2017). Several studies have shown that rosemary extract or its active ingredients have anti-inflammatory, hepatoprotective, antithrombotic, diuretic, antidiabetic, antinociceptive, anticancer and antioxidant activity in experimental animals and humans (Gao et al., 2014; Johnson, 2011; Ngo et al., 2011; Petiwala and Johnson, 2015; Takayama et al., 2016). Jiang et al. (2011) found that rosemary extract exhibited strong antibacterial activity against Gram-positive and Gram-negative bacteria. Beside these positive effects, rosemary did not change serum urea, uric acid, creatinine, and ammonia excretion rate of sea bass (Dicentrarchus labrax) after a 45-day feeding trial (Yılmaz et al., 2012). Also, Nile tilapia, Oreochromis niloticus (L.) fed diet supplemented with turmeric (Curcuma longa), rosemary (Rosmarinus officinalis), and thyme (Thymus vulgaris) showed significantly increasing of weight gain, specific growth rates, and protein efficiency ratio (PER) compared to those in the control group (Hassan et al., 2018). However, rosemary extract did not affect the growth and feed intake, but reduced the plasma levels of glucose and triglycerides after four weeks, and glucose, HDL/LDL cholesterol ratio and plasma alanine aminotransferase after 12 weeks administration to gilthead seabream (Sparus aurata) (Hernández et al., 2015). Blood leukocyte count and plasma total protein, albumin and globulin are the suitable indicators of fish health (Hoseini et al., 2019). Plasma lysozyme is an indicator of the innate immune system in fish and a crucial enzyme with bactericidal activity; the enzyme activity rises during stress and infection to enhance the host defence; whereas, a decrease in the enzyme activity might be an indicator of health deterioration (Saurabh and Sahoo, 2008). Serum alternative complement (ACH50) as an indicator of innate immune system involves in opsonization, inflammation and cell membrane attack (Holland and Lambris, 2002). Serum immunoglobulin (Ig) acts as an adaptive immune system component to recognize the antigen (Hoseini et al., 2016). Plasma cortisol and glucose levels are the most reliable stress responses in fish (Hoseini et al., 2019). The above-mentioned immune parameters are sensitive to stress (Tort, 2011). Superoxide dismutase (SOD) and catalase (CAT) are the important antioxidant enzymes, which are sensitive to oxidative conditions and stress (Taheri Mirghaed et al., 2018). Monitoring of the above-mentioned immunological, stress and antioxidant parameters may give a general insight into fish health under rearing conditions. Oddly enough, the feasibility of rosemary as a feed additive on humoral immunity of aquatic species has been scarcely investigated. However, according to the results obtained by the previous researches, it hepatizes that oral administration of rosemary can improve carp health and immunity. Therefore, in the present study, common carp were fed with different diets for 65 days, aiming to find the effects of

Table 1 Dietary formulation and proximate composition analysis of experimental diets (% on dry matter basis) containing different levels of rosemary leaf powder. Ingredients (% total)

Rosemary leaf powder levels (%) 0.0 (Control)

1.0

2.0

3.0

Fishmeal Soybean meal Meat meal Wheat meal Fish oil Soybean oil Lysinea DL-methionineb Mineral mixc Vitamin mixd Rosemary

10 23 21 42.3 1 1 0.7 0.5 0.25 0.25 0

10 23 21 41.3 1 1 0.7 0.5 0.25 0.25 1

10 23 21 40.3 1 1 0.7 0.5 0.25 0.25 2

10 23 21 39.3 1 1 0.7 0.5 0.25 0.25 3

Proximate composition (%) Dry matter Crude protein Crude fat Crude ash Calculated gross energy (kJ/g)

90.6 38.9 8.65 6.11 21.5

91.1 39 8.55 6.05 21.4

90.3 39.1 8.71 6.12 21.6

90.8 38.8 8.84 6.04 21.4

a

Faravar Lysine Pars Co., Tehran, Iran. Mad Tiour Co., Sanandaj, Iran. c The premix provided following amounts per kg of diet: Mg: 350 mg; Fe: 13 mg; Co: 2.5 mg; Cu: 3 mg; Zn: 60 mg; NaCl: 3 g; dicalcium phosphate: 10 g. d The premix provided following amounts per kg of feed: A: 1000 IU; D3: 5000 IU; E: 20 mg; B5: 100 mg; B2: 20 mg; B6: 20 mg; B1: 20 mg; H: 1 mg; B9: 6 mg; B12: 1 mg; B4: 600 mg; C: 50 mg. b

oral rosemary leaf powder administration on growth performance, body composition, immunological, antioxidant, and stress responses. 2. Material and methods 2.1. Preparation of rosemary leaf powder (RLP) and experimental diets The fresh rosemary leaves were collected from Gorgan (Golestan Province, Iran) and transferred to the laboratory. Then, the leaves were oven dried (at 45 °C for 72 h) and powdered using mill, passed through an 80-mesh sieve and stored in zippack at 4 °C until use. A basal diet was formulated (Table 1) which served as a control diet and experimental diets were prepared by adding of 1, 2 and 3% RLP. 2.2. Fish and experimental conditions Ethics for procedures and laboratory animal use have been followed according to Naderi et al. (2012). Juvenile common carps were obtained from a local fish farm and transferred to the Fisheries Research Station of Gharesoo, Bandar-e-Torkman, Iran. After fish acclimatization for two weeks, a total number of 360 common carp (12.93 ± 0.09 g) were stocked into 12 80-L fibreglass tanks (70*30*30 cm) assigned to four treatments with three replicates at the density of 30 fish per tank. The fish were fed on one of the experimental diets twice a day at a rate of 2% of body weight for 65 days. Feeding rate was adjusted based on the data of biometry (every ten days). The tanks were continuously aerated, cleaned daily and 70% of water was replaced by aerated freshwater to maintain optimum water quality. Water temperature (21.1 ± 0.69 °C), dissolved oxygen (6.54 ± 0.82 mg/L), and pH (7.45 ± 0.45) were monitored daily during the experiment using Hach HQ40d portable apparatus (Loveland, Colorado, USA). Water total ammonia levels were measured using Le and Boyd (2012) method and converted into unionized ammonia according to water pH and temperature (Emerson et al., 1975). 474

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2.3. Growth performance

phosphate buffer (pH = 6.2). Alternative complement activity (ACH50) was measured using sheep RBC as target in EGTA-Mg++ − gelatinveronal buffer (Yano, 1992). Plasma level of total Ig was estimated after polyethylene glycol precipitation according to Siwicki and Anderson (1993). Total white blood cell count (WBC) (×103 μL) was calculated according to Ranzani-Paiva et al. (2004). Blood cell hemolysates were diluted in phosphate buffered saline (pH = 7.4) and centrifuged for 10 min (1200 × g). The resultant supernatants were used for determining activities of catalase (CAT) and superoxide dismutase (SOD). The CAT activity was assayed using hydrogen peroxide decomposition method according to previously-described method (Góth, 1991). One unit of the CAT activity was defined as the amount of enzyme required to decompose 1 μM of H2O2 per min. The SOD (conversion of superoxide anion to hydrogen peroxide method; ZellBio, GmbH, Veltinerweg, Germany) was measured using microplate reader and commercial kits. The amount of SOD required to inhibit the rate of reduction of cytochrome C by 50% (i.e. to a rate of 0.0125 absorbance unit per min) was defined as 1 unit of SOD activity (McCord and Fridovich, 1969).

After the 65-day feeding trial, parameters of growth performance and survival rate were determined as follows:

Weight gain (WG%) = 100 × (final body weight–initial body weight) /initial body weight; Specific growth rate (SGR,%g/day) = 100 × [(Ln final weight)–Ln (initial weight)]/days of the experiment; Feed efficiency ratio (FER) = wet weight gain (g)/dry feed consumed (g); Fish survival rate (%) = 100 × (fish number in each group remaining at the end of the experiment /initial number of fish).

2.4. Proximate composition At the end of the experiment, three fish per each tank were captured, killed (by sharp blow on head), and stored at −80 °C to future whole-fish body composition analyses. The procedures of the AOAC (1995) were used to perform fish proximate composition analysis. Moisture was estimated gravimetrically after oven (Heraeus, D-63450, Hanau, Germany) drying of homogenized samples to constant weight at 105 °C. Ash was determined by incineration of samples in a muffle furnace (Isuzu, Tokyo, Japan) at 600 °C for 3 h. Crude protein was measured by the Kjeldahl procedure (N × 6.25) using an automatic Kjeldahl system (230-Hjeltec Analyser; Foss Tecator, Hoganas, Sweden) and total fat of samples was estimated by extracting with chloroform using an automatic Soxtec system (Foss, SoxtecTM 2050).

2.6. Statistical analyses The normality of obtained data was confirmed by Shapiro-Wilk test. Effects of different levels of RLP in experimental diets were tested by one-way ANOVA followed by Duncan test as a post-hoc test. Analysis of the crowding stress data was conducted using two-way ANOVA (Crowding stress and RLP levels). The means difference was considered as significantly different at P < .05. All statistical analysis was done by SPSS 16.0 (SPSS Inc., Chicago, IL, USA).

2.5. Blood sampling and analysis

3. Results

At the end of the 65-day experiment, three fish were randomly sampled from each tank and immediately anesthetized with 100 mg/L clove oil (Hoseini et al., 2014). Blood samples were obtained from the caudal vein using heparinized syringes (5 mg heparin /ml blood), discharged into sterile vials and centrifuged at 1200 × g for 10 min. Plasma was poured into new tubes and stored at −70 °C until analysis. In order to induce stress condition, tanks water levels were decreased by tierce for three hours. Blood samples were withdrawn from all treatments before and after the crowding stress. Plasma levels of glucose, total protein, and albumin were measured spectrophotometerically using commercial kits (Pars Azmun Co. Ltd., Tehran, Iran) as reported previously (Mazandarani et al., 2017; Taheri Mirghaed et al., 2017). Plasma globulin level was calculated by subtracting plasma albumin from total protein levels. Cortisol level was estimated using ELISA method and commercial kit (IBL, Gesellschaft fur Immunchemieund Immunbiologie, Hamburg, Germany). Plasma lysozyme level was determined by turbidimetric assay according to previously-published method (Ellis, 1990) with Micrococcus lysodeikticus (Sigma, St Louis, MO, USA) as target in 0.05 M

3.1. Growth performance and body proximate composition Growth performances of different treatments are shown in Table 2. A percentage of 3% RLP showed significantly higher final weight, WG%, and SGR compared with the other treatments. However, FER significantly increased in the fish of 3% RLP treatment compared to that of the control treatment. According to the body composition results presented in Table 3, there was not observed any significant differences among the experimental groups. 3.2. Blood parameters According to Table 4, highest total protein and albumin levels were noticed in fish fed with 2% RLP followed by 1% RLP (P < .05). Significant differences were observed in plasma globulin at any level of RLP inclusion when compared with fish fed with the control diet. The RLP-treated fish had similar plasma lysozyme and alternative complement activities, which were significantly higher than the control fish. Fish were fed with 3% RLP showed highest total Ig levels compared to

Table 2 Growth performance of common carp fingerlings fed on diets with different levels of rosemary leaf powder (RLP) for 65 days. Growth index

Initial weight (g) Final weight (g) WG % FER SGR (%g/day) Survival rate (%)

Experimental diets Control

1% RLP

2% RLP

3% RLP

12.88 ± 0.14 26.60 ± 0.42a 106.58 ± 1.12a 0.79 ± 0.02a 1.12 ± 0.01a 100

12.85 ± 0.26 28.87 ± 0.57b 124.23 ± 1.74b 0.84 ± 0.02ab 1.24 ± 0.01b 100

13.00 ± 0.19 29.00 ± 0.58b 123.05 ± 1.74b 0.84 ± 0.03ab 1.23 ± 0.01b 100

12.96 ± 0.22 33.47 ± 0.37c 158.32 ± 1.66c 0.89 ± 0.02b 1.46 ± 0.01c 100

Values (mean ± SEM for three replicate groups) in the same row not sharing a same superscript are significantly different (P < .05). WG, Weight gain; FER, feed efficiency ratio; SGR, specific growth rate. 475

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lead to a decrease in blood SOD activity (Fig. 2). Crowding stress and RLP supplementation significantly affected plasma cortisol and glucose levels, but not their interaction. Crowding stress significantly increased plasma cortisol and glucose levels. Whereas, RLP-supplemented groups significantly decreased their levels and the highest levels of plasma cortisol and glucose were observed in control compared to RLP-treated groups (Fig. 3).

Table 3 Proximate composition of the whole-body of common carp fingerlings fed on diets with different levels of rosemary leaf powder (RLP) for 65 days. Proximate composition (%)

Experimental diets Control

1% RLP

2% RLP

3% RLP

Moisture Protein Lipid Ash

73.59 ± 1.2 14.31 ± 0.7 5.47 ± 0.50 4.11 ± 0.30

72.93 ± 1.2 14.03 ± 0.7 6.57 ± 0.50 4.50 ± 0.32

73.10 ± 1.35 13.90 ± 1.00 6.67 ± 0.25 4.41 ± 0.42

73.90 ± 0.43 14.63 ± 0.35 5.76 ± 0.22 4.90 ± 0.35

4. Discussion

Values (mean ± SEM for three replicate groups) in the same row not sharing a same superscript are significantly different (P < .05).

Various plants, their ingredients, oils, and active compounds have been used for years as functional foods, due to their advantageous effects on fish such as growth promotion, immunostimulation, antioxidant modulation and lipid lowering effects (Van Hai, 2015; AbdelTawwab, 2016; Awad and Awaad, 2017; Adeshina et al., 2018; Dawood et al., 2018; Reyes-Cerpa et al., 2018). Recently the considerable interest to use of rosemary has increased because of its effects to control diseases in human and aquatic animals (Abutbul et al., 2004; Van Hai, 2015; Zoral et al., 2017), and to keep fish fillet quality during storage (Álvarez et al., 2012; Gao et al., 2014). However, evidences of rosemary application in fish culture are still scarce and according to the literatures, there is no study on the effects of rosemary on fish blood immunity, antioxidant activity and stress responses. In line with the present study, Hassan et al. (2018) showed that the 1% rosemary supplementation significantly increased weight gain, SGR, and protein efficiency ratio (PER) but had insignificant effect on carcass composition of Nile tilapia. In sea bass, 1% rosemary had no significant effect on fish growth performance and body composition (Yılmaz et al., 2012). Also, gilthead seabream growth and feed intake were not affected by the diet supplemented with rosemary extract (Hernández et al., 2015). This difference could be explained by differences of fish

Table 4 Plasma total protein, albumin and globulin levels of common carp fingerlings fed on diets with different levels of rosemary leaf powder (RLP) for 65 days. Plasma index

Experimental diets Control

Total protein (g/dL) Albumin (g/dL) Globulin (g/dL)

1% RLP a

2% RLP bc

2.82 ± 0.15

4.27 ± 0.20

1.12 ± 0.05a 1.70 ± 0.12a

1.75 ± 0.09bc 2.51 ± 0.15b

3% RLP c

4.73 ± 0.22

3.96 ± 0.17b

1.85 ± 0.11c 2.88 ± 0.15b

1.53 ± 0.07b 2.47 ± 0.18b

Values (mean ± SEM for three replicate groups) in the same row not sharing a common superscript are significantly different (P < .05).

other groups (P < .05). The treatments 2 and 3% RLP had significantly higher blood WBC compared to the 1% RLP and control groups (Fig. 1). All RLP-treated fish had significantly higher blood CAT activity compared to the control group. The increase in dietary rosemary levels

Fig. 1. Plasma lysozyme and alternative complement activities, total immunoglobulin levels, and blood WBC of common carp fingerlings fed on diets with different levels of rosemary leaf powder (RLP) for 65 days. Different letters show significant difference among the treatments (Duncan test). 476

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Fig. 2. Blood CAT and SOD activities of common carp fingerlings fed on diets with different levels of rosemary leaf powder (RLP) for 65 days. Different letters show significant difference among the treatments (Duncan test).

Fig. 3. Plasma cortisol and glucose levels of common carp fingerlings fed on diets with different levels of rosemary leaf powder (RLP) for 65 days and subjected to stress. Different letters show significant difference among the treatments (Duncan test).

species, feeding period, and source of the rosemary. Rosemary affected the blood parameters, especially at the highest levels (2 and 3% RLP) and it supports the hypothesis that the enhanced growth performance is related to the beneficial effects of rosemary on fish health. In line with the obtained results, Hoseini et al. (2018b) found that the 1,8-cineole administration significantly improved Oncorhynchus mykiss growth performance and serum biochemical parameters, but not affected on body composition. Awad et al. (2012) showed that rainbow trout growth performance, especially weight gain, SGR and digestive

enzymes increased by the diets supplemented with lupine, mango and nettle. Mahdavi et al. (2013) reported that dietary administration of Aloe vera extract was effective as a growth promotor and appetite stimulator in common carp. Pancreatic enzymes as the important factors in nutrient digestion and assimilation can be stimulated by herbs (Frankic et al., 2009). This trend can be related to the improved growth performances by increasing RLP percentage in this study. Plasma total protein, albumin and globulin are the suitable indicators of fish health (Hoseini et al., 2019). In line with these results, 477

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several studies demonstrated that different herbal materials simultaneously increased growth performance, and plasma total protein, albumin and globulin levels (Akrami et al., 2015; Bilen et al., 2011; Dügenci et al., 2003; Gabriel et al., 2015; Hoseini et al., 2018b; Immanuel et al., 2009). These finding confirmed the hypothesis of combination effects of health and growth performance in fish treated with herbal supplementation. innate immune parameters such as immunoglobulins, complement, and lysozyme as the key elements in the genesis of the immune response (Awad and Awaad, 2017; Caipang and Lazado, 2015; Magnadóttir, 2006), are among the most frequently tested immune parameters in herbal supplemented diets in fish. Therefore, the evaluation of these immune parameters in the circulating blood is an important sign of evaluation of the immune response. The present results are in agreement with data from previous experiments suggesting that the powerful effects of herbal and their extracts/active components to stimulate the immune system in fish facing stress and diseases. ReyesCerpa et al. (2018) reported that the combination of plant extracts (common Saint John's wort, lemon balm, and rosemary) improved the expression of immune genes associated to humoral-like response (CD4 and GATA-3) and secreted IgM. Dietary supplement with onion bulbs, rose hip seeds, and safflower showed enhancement of lysozyme, total protein, complement and immunoglobulin in beluga fish (Huso huso) (Akrami et al., 2015; Dadras et al., 2016). Taheri Mirghaed et al. (2018) showed that increase in dietary cineole levels significantly increased serum lysozyme and ACH50 activities in rainbow trout. Moreover, Catla catla (Indian carp) showed an enhancement in immune parameters (lysozyme and complement) after administration of a diet supplemented with Bermuda grass leaf for 60 days (Kaleeswaran et al., 2011). Therefore, elevation of total Ig, ACH50 and lysozyme hint at possible immunomodulatory effects of RLP in common carp. White blood cells including lymphocytes (T cells, B cells), phagocytes (monocytes and neutrophils) are considered as back-bone of all immune responses (Awad and Awaad, 2017). In agreement with the obtained results, previous studies revealed elevation of the blood WBC in fish fed with herbals and their active compounds. Taheri Mirghaed et al. (2018) found that an administration of a diet supplemented with cineole increased blood WBC and mitigated WBC decrease after crowding stress in rainbow trout. The oral administration of eucalyptol for common carp markedly mitigated copper-induced decrease in WBC, lysozyme, ACH50 and total Ig (Hoseini et al., 2018a). Moreover, in vitro evaluation of dihydroquercetin (a flavonoid compound found in many plants like nettle and deodar) effects on leucocytes demonstrated its immunomodulatory activity to stimulate the leucocytes and initiate the phagocytic activity (Awad et al., 2015). Superoxide dismutase and catalase are key antioxidant/sequential enzymes to convert superoxide ion to water (Hoseini and Yousefi, 2018). Thus, enhancement in the activity of these enzymes prove stimulation of antioxidant defence (Yousefi et al., 2018). Previous study showed that the oral administration of oregano extract improved SOD and CAT activity in channel catfish (Ictalurus punctatus) (Zheng et al., 2009). Additionally, Taheri Mirghaed et al. (2018) showed that different concentrations of cineole significantly increased serum CAT and SOD activities in rainbow trout. It has been demonstrated that flavonoid and phenolic compounds in herbals are responsible for their high antioxidant capacity (Reyes-Cerpa et al., 2018). Thus, our results suggest that RLP supplementation could stimulate antioxidant protection to convert reactive oxygen species into safe compounds to defend biological materials from oxidation (Hoseini and Yousefi, 2018). Various stress factors such as ambient temperature, stocking density, handling, water physicochemical parameters, transport, and sorting consistently affected the fish in aquaculture system (Xie et al., 2008; Yousefi et al., 2018). As a result, the axis of hypothalamus–pituitary–interrenal axis (HPI) of animal will be stimulated and lead to elevation of circulating levels of catecholamines and corticosteroids, and hyperglycemia (Xie et al., 2008; Yousefi et al., 2016). Elevation of

cortisol and glucose levels is known as the primary and secondary responses to stress (Barton, 2002). In agreement with the obtained results, previous studies demonstrated that stocking density cause the elevating of cortisol and glucose levels of blood (Leatherland, 1993; Ruane et al., 2002; Xie et al., 2008; Abdel-Tawwab, 2012; Abdel-Tawwab et al., 2014; Yarahmadi et al., 2016; Taheri Mirghaed et al., 2018). Elevating of these factors is the sign of adaptive strategies to supply demanded energy to cope with the stress (Taheri Mirghaed et al., 2018). The results herein suggested that the oral administration of RLP has the potential to prevent cortisol and glucose elevation and thus mitigate the negative effects of crowding stress. In line with these results, Xie et al. (2008) reported that diet supplemented with 2.0% anthraquinone extract suppress the crowding stress effects on common carp. Similarly, dietary cineole mitigated increasing of serum cortisol levels in rainbow trout subjected to 14-day crowding stress. Moreover, oral administration of eucalyptol markedly decreased serum cortisol and glucose levels in common carp, after copper intoxication (Hoseini et al., 2018a). In conclusion, it can be observed that all rosemary leaf powder levels had benefit, but 2 and 3% levels affected beneficially most of the tested factors. The results of the present study activity confirm the potential of RLP as the growth promotor and stimulatory agent of the immune system. The benefits of different levels of rosemary on the fish might be due to lower stress that boosts fish immune and overall health, characterized by improved fish growth performance. Moreover, it indicated that RLP could mitigate the negative effects of short-term crowding stress in common carp fingerlings. Further studies are needed to evaluate the effects of RLP under intensive stocking density and experimental infectious bacterial challenge for supporting our knowledge regarding the mode of action and optimum inclusion level. Acknowledgment There was no conflict of interest about this manuscript. The publication has been prepared with the support of the “RUDN University program 5-100”. References Abdel-Tawwab, M., 2012. Effects of dietary protein levels and rearing density on growth performance and stress response of Nile tilapia, Oreochromis niloticus (L.). Int. Aquat. Res. 4, 3. https://doi.org/10.1186/2008-6970-4-3. Abdel-Tawwab, M., 2016. Feed Supplementation to Freshwater Fish: Experimental Approaches. Lambert Academic Publishing, Berlin, Germany. Abdel-Tawwab, M., Abbass, F.E., 2017. Turmeric powder, Curcuma longa L., in common carp, Cyprinus carpio L., diets: growth performance, innate immunity, and challenge against pathogenic Aeromonas hydrophila infection. J. World Aquacult. Soc. 48, 303–312. https://doi.org/10.1111/jwas.12349. Abdel-Tawwab, M., Monier, M.N., 2018. Stimulatory effect of dietary taurine on growth performance, digestive enzymes activity, antioxidant capacity, and tolerance of common carp, Cyprinus carpio L., fry to salinity stress. Fish Physiol. Biochem. 44, 639–649. https://doi.org/10.1007/s10695-017-0459-8. Abdel-Tawwab, M., Hagras, A.E., Elbaghdady, H.A.M., Monier, M.N., 2014. Dissolved oxygen level and stocking density effects on growth, feed utilization, physiology, and innate immunity of Nile Tilapia, Oreochromis niloticus. J. Appl. Aquac. 26, 340–355. https://doi.org/10.1080/10454438.2014.959830. Abdel-Tawwab, M., Adeshina, I., Jenyo-Oni, A., Ajani, E.K., Emikpe, B.O., 2018a. Growth, physiological, antioxidants, and immune response of African catfish, Clarias gariepinus (B.), to dietary clove basil, Ocimum gratissimum, leaf extract and its susceptibility to listeria monocytogenes infection. Fish Shellfish Immunol. 78, 346–354. https://doi.org/10.1016/j.fsi.2018.04.057. Abdel-Tawwab, M., Samir, F., Abd El-Naby, A.S., Monier, M.N., 2018b. Antioxidative and immunostimulatory effect of dietary cinnamon nanoparticles on the performance of Nile tilapia, Oreochromis niloticus (L.) and its susceptibility to hypoxia stress and Aeromonas hydrophila infection. Fish Shellfish Immunol. 74, 19–25. https://doi.org/ 10.1016/j.fsi.2017.12.033. Abdel-Tawwab, M., Monier, M.N., Hoseinifar, S.H., Faggio, C., 2019. Fish response to hypoxia stress: growth, physiological, and immunological biomarkers. Fish Physiol. Biochem. 1–17. https://doi.org/10.1007/s10695-019-00614-9. Abutbul, S., Golan-Goldhirsh, A., Barazani, O., Zilberg, D., 2004. Use of Rosmarinus officinalis as a treatment against Streptococcus iniae in tilapia (Oreochromis sp.). Aquaculture 238, 97–105. https://doi.org/10.1016/j.aquaculture.2004.05.016. Adeshina, I., Jenyo-Oni, A., Emikpe, B.O., Ajani, E.K., Abdel-Tawwab, M., 2018. Stimulatory effect of dietary clove, Eugenia caryophyllata, bud extract on growth performance, nutrient utilization, antioxidant capacity, and tolerance of African

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