Aquaculture 515 (2020) 734588
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Effects of lavender (Lavandula angustifolia) extract inclusion in diet on growth performance, innate immunity, immune-related gene expression, and stress response of common carp, Cyprinus carpio
T
Morteza Yousefia,∗, Sergey Viktorovich Shabuninb, Yury Anatolyevich Vatnikova, Evgeny Vladimirovich Kulikova, Hossein Adinehc, Mohammad Khademi Hamidic, Seyyed Morteza Hoseinid,∗∗ a
Department of Veterinary Medicine, Peoples' Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St, Moscow, 117198, Russian Federation Federal State Budget Scientific Institution All-Russian Veterinary Research Institute of Pathology, Pharmacology and Therapy, 114b Lomonosov St, Voronezh, 394087, Russian Federation c Department of Fisheries, Faculty of Agriculture and Natural Resources, Gonbad Kavous University, Gonbad Kavous, Golestan, Iran d 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 Nutrition Aquaculture Immunology
In the present study, the effects of dietary lavender (Lavandula angustifolia) extract (LE) on growth performance and immune, antioxidant, and stress responses in common carp, Cyprinus carpio were investigated. For this, four diets containing 0, 0.5, 1.0 and 1.5% LE were offered to triplicate groups of fish (25.8 ± 1.66 g) for 70 days, and then the fish were exposed to a 3-h crowding stress. Growth performance, blood leukocyte count, plasma proteins and innate immune parameters, and head kidney cytokines’ gene expression were studied after the 70-day feeding trial (before the crowding stress). Plasma cortisol, glucose, superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured before and after the crowding stress. Results showed that dietary LE had no significant effects on the fish growth performance, blood leukocyte differential count, plasma albumin and total protein, and head kidney interferon gamma-1 gene expression. Blood leukocyte and plasma globulin, alternative complement and total immunoglobulin significantly increased in the fish fed with 1.0–1.5% LE diets compared to the fish fed with the control diet (0% LE). The plasma lysozyme activity significantly increased along with dietary LE supplemental levels. The lowest expression of interlukin-1 beta gene was related to the 1.0% LE treatment. Tumor necrosis factor-alpha gene expression significantly decreased along with increase in dietary LE levels and the lowest expression was related to the fish fed with 1.5% LE diet. The LE administration led to significant increase in interlukine-10 gene expression and the highest expression was related to the fish fed with 1.0% and 1.5% LE diets. The fish fed with 1.5% LE diet had significantly higher transforming growth factorbeta gene expression compared to the control group. Liver SOD and CAT activities showed significant increases along with dietary LE supplementation levels. After the crowding stress, hepatic SOD decreased in the control group, but not the LE-supplemented groups. Stress led to significant decrease in hepatic CAT activities in all treatments, but CAT activity increased along with the LE levels with highest activity in the fish fed with 1.5% LE. The stress led to significant increase in hepatic MDA in the treatments control and 0.5% LE, but not 1.0–1.5% LE groups. The stress led to significant increase in plasma cortisol and glucose levels in all treatments but dietary LE levels significantly decreased these parameters. Dietary LE supplementation at levels of 1.0–1.5% is recommended for common carp to suppress stress, inflammation and oxidative conditions and augment immune responses in the fish.
∗
Corresponding author. Corresponding author. POB: 1419963111, Iran. E-mail addresses:
[email protected] (M. Yousefi),
[email protected] (S.M. Hoseini).
∗∗
https://doi.org/10.1016/j.aquaculture.2019.734588 Received 17 August 2019; Received in revised form 7 October 2019; Accepted 8 October 2019 Available online 09 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Aquaculture 515 (2020) 734588
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1. Introduction
Table 1 Ingredients and composition of experimental diets (% on dry matter basis) containing different levels of dietary lavender (L. angustifolia) extract (LE).
In aquaculture facilities, fish may experience stress due to handling, sorting, transportation, crowding and so on (Ashley, 2007). Stressors deteriorate fish health via different ways. Stressful conditions lead to cortisol elevation, which in turn increases energy expenditure (characterized by hyperglycemia) and growth suppression (Andrade et al., 2015; Yousefi et al., 2016). Besides, cortisol has been known as immunosuppressor by inhibiting inflammatory responses (Taheri Mirghaed et al., 2018; Ghelichpour et al., 2019; Hoseini et al., 2019d). Stressors, also, lead to disturbed antioxidant system and antioxidant: pro oxidant ratio balance, resulting in oxidative stress (Taheri Mirghaed et al., 2018). Stressors possess the fish to disease risks in aquaculture facilities. Diseases cause huge annual loss in aquaculture throughout the world and antibiotic therapy compels huge costs to aquaculture farms; moreover, antibiotic therapy rises great concerns regarding environment pollution and presence of new antibiotic-resistance bacteria (Alderman and Hastings, 1998). Studies on fish have demonstrated that antibiotics produce adverse effects on fish health including oxidative stress, immunosuppression and pathological damages (Rijkers et al., 1980; Guardiola et al., 2012; Yonar, 2012; Oliveira et al., 2013; Ko and Lee, 2015; Rodrigues et al., 2017; Nakano et al., 2018; Hoseini and Yousefi, 2019). Accordingly, fish-farmers are interested in methods to suppress stress, and augment fish health and immune function, so that prevent disease and negative effects of antibiotic therapy. Phytotherapy has recently gained great attention in aquaculture. Medicinal herbs are valuable feed additives because of their natural origin and environmental-friendly nature (Chakraborty and Hancz, 2011; Chakraborty et al., 2014; Abdel-Tawwab, 2016). Different types of herbal products including whole plant, extracts, essential oils, and active ingredients have been studied in different fish species for their positive health benefits. For example, dietary administration of whole rosemary leaf (Yousefi et al., 2019), Panax quinquefolium (AbdelTawwab, 2015), coffee bean (Abdel-Tawwab et al., 2015; AbdelTawwab et al., 2018) and clove (Adeshina et al., 2019) had growth promoter, hepatoprotective, antioxidant, immune booster and antistress effects in fish. dietary administration of palm fruit (Hoseinifar et al., 2015), thyme (Hoseini and Yousefi, 2019), Eriobotrya japonica (Hoseinifar et al., 2018) and Origanum heracleoticum (Zheng et al., 2009) extracts/essential oils led to improved fish growth performance, antioxidant, immune, and stress responses. Active herbal components such as cineole (Taheri Mirghaed et al., 2018), myrcene (Hoseini et al., 2019c), and eucalyptol (Hoseini et al., 2018a) were also beneficial in fish growth performance and health status. Lavender (Lavandula angustifolia) is considered as a medicinal herb for its anti-inflammatory (Cardia et al., 2018), antioxidant (Gülçin et al., 2004), and hepatoprotective (Selmi et al., 2015) effects. There is only one study on the effects of lavender extract on fish, showing it increases the number of phagocytic cells and improved phagocytic, respiratory burst and peroxidase activities of head kidney leukocyte in vitro (Fazio et al., 2017). However, lavender extract is rich in cineole and linalool; the compounds known for their anti-inflammatory, antioxidant, and anti-stress effects (Peana et al., 2002; Ciftci et al., 2011; Juergens, 2014; Taheri Mirghaed et al., 2018; Yousefi et al., 2018a). These effects make lavender as a potential health booster in fish. Common carp, Cyprinus carpio is an important aquaculture species with annual global production of 4.6 million tons (about 8% of total finfish production). As an aquaculture species, common carp at risk of stressors in aquaculture system, thus, it is necessary to study the effects of feed additives on this species growth and health. Therefore, the aim of the present study was to demonstrate if dietary lavender extract administration is beneficial in growth performance, and immune, antioxidant and stress responses in common carp (Cyprinus carpio).
Feedstuffs
Control
0.5E
1.0E
1.5E
Fishmeal Soybean meal Meat meal Wheat meal Fish oil Soybean oil Lysine Methionine Vitamin mix Mineral mix LE
10 23 21 42.3 1 1 0.7 0.5 0.25 0.25 0
10 23 21 41.8 1 1 0.7 0.5 0.25 0.25 0.5
10 23 21 41.3 1 1 0.7 0.5 0.25 0.25 1
10 23 21 40.8 1 1 0.7 0.5 0.25 0.25 1.5
Dry matter (%) Crude protein (%) Crude fat (%) Crude ash (%) Energy (mj/kg)
90.6 38.9 8.65 6.11 15.6
91.1 39 8.55 6.05 15.7
90.3 39.1 8.71 6.12 15.6
90.8 38.8 8.84 6.04 15.6
2. Materials and methods 2.1. LE preparation and composition Dry lavender leaves were purchased from a local market. Hot water extraction was conducted according to Wu et al. (2010). Briefly, the leaves were washed with deionized water and dried under constant air blow before being milled. 100 g of the milled product were added to 1000 mL water. The mixture were placed on flame and allowed to boil for 2 h. Then, the suspension was filtered by nylon mesh and vacuum pump. According to the a previous study Hadipour et al. (2013), the extract contains 420 mg/L linalool and 196 mg/L 1,8-cineole. 2.2. Diets’ preparation Four experimental diets (Table 1) were prepared with varying levels of LE [0% (control), 0.5% (0.5E), 1.0% (1.0E) and 1.5% (1.5E)]. The diet ingredients were mixed, thereafter; 300 ml of water (for the control diet) or water + LE (for the LE-supplemented diets) were added to each kilogram of the mixture. To prepare the 0.5E, 1E and 1.5E diets, 5, 10 and 15 ml of LE were mixed with 295, 290 and 285 ml water, before mixing with one kg of the dietary mixture. The dough was then passed through a meat grinder and the resultant strings were dried over night before being crushed in appropriate size. The feeds were kept at 4 °C until use. 2.3. Animal care ethics During this study, ethics of animal care were followed as described by Naderi et al. (2012). 2.4. Experimental protocol A total number of 240 common carp juveniles (25.8 ± 1.66 g) were stocked in 12 aquaria (100 L water volume) supplied with sponge aerator. The fish were fed the control diet for 10 days to acclimatize with the experimental conditions. After the acclimation, the aquaria divided into 4 treatments (with three replicates), each was fed with either of the control, 0.5, 1.0 and 1.5% LE for 70 days. The fish were fed based on 2% of biomass daily, divided into two meals. The aquaria biomasses were determined every other week to adjust feed amount. During the acclimation and afterward, the aquaria water were daily replaced with clean water (75%). The aquaria were siphoned and
2
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continuously-aerated during the experiment. Water temperature, dissolved oxygen, pH and unionized ammonia were checked every week (by Hach multi-parameter meter HQ40d, Loveland, Colorado, USA and Wagtech photometer 7100, Berkshire, UK) being 20.2 ± 1.24 °C, 6.57 ± 0.91 mg/L, 7.62 ± 0.68 and 0.08 ± 0.02 mg/L, respectively. After 70 days, weight gain percentage, feed conversion ratio (FCR) and specific growth rate (SGR) were determined according to Hoseini et al. (2018b): At the end of the experiment, the fish were weighed and growth performance data were determined according to the following equations:
Weight gain percentage = 100×
SGR (%/d) = 100×
FCR =
medium and sheep RBC as target. Serum total immunoglobulin (total Ig) were determined by precipitation of Ig with polyethylene glycol and subtraction of initial and final total protein according to Siwicki and Anderson (1993). 2.6.3. Hepatic antioxidant condition analyses The liver samples (1 g) were homogenized with 10 ml of buffer (100 mM-Tris-HCl, 0.1 mM-EDTA and 0.1% triton X-100 (v/v), pH 7.8) on ice. The mixture was centrifuged for 30 min at −4 °C and the supernatant was used for malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) assay. The MDA (thiobarbituric acid method; ZellBio, GmbH, Veltinerweg, Germany) was determined using commercial kits in microplate reader (Hoseini and Yousefi, 2019). The CAT activity was measured according to Goth (1991). One unit of CAT activity was defined as the amount of enzyme required to decompose 1 μM of hydrogen peroxide per min. SOD activities were measured using microplate reader and commercial kits (conversion of superoxide anion to hydrogen peroxide method; ZellBio, GmbH, Veltinerweg, Germany) (Hoseini and Yousefi, 2019). The amount of SOD required for 50% reduction in the rate of reduction of cytochrome C (i.e. to a rate of 0.0125 absorbance unit per min) was defined as 1 unit of SOD activity.
Gained biomass (g ) Initial biomass (g )
Ln (final biomass ) Ln (initial biomass ) dys of rearing
Consumed feed (g ) Gained biomass (g )
2.5. Stress and sample collection
2.6.4. Kidney gene expression The RNX-plus extraction kit (Sinagene, Iran) was used for total RNA extraction from the kidney samples according to the kit instructions. The quality and purity of extracted RNA were determined by spectrophotometry as the 260:280 ratios were 1.8–2.0 as well as using agarose gel (1.5%). Then, DNase I (Fermentas, Lithuania) was used to avoid DNA contamination. Afterward, Complementary DNA (cDNA) was synthesized using cDNA synthesis kit (Fermentas, Lithuania) according to the manufacturer's protocol. The primer sets for quantification of mRNA levels of selected genes were designed based on the common carp sequences found in Gen Bank (Table 2). The Oligo 7 program was used for designing the primers. The SYBR green method was followed for determination of relative expression of the selected genes using Real-time PCR analysis as described previously (Hoseinifar et al., 2018). For each sample, the PCR reactions were performed in duplicates. The expression levels of intelukin-1 beta (IL-1b), intelukin-10 (IL-10), tumor necrosis factors-alpha (TNF-a), transforming growth factor beta (TGF-b) and interferon gamma-1 (IFN) were normalized based on expression level of housekeeping gene (beta actin) (Hoseinifar et al., 2018). After verification of PCR efficiency to be around 100%, the gene expression data were analysed based on DDCt method.
After termination of the growth trial, blood and liver samples were taken from each treatment. Nine fish were sampled per treatments (three per aquarium). The fish were captured by dip net and placed in eugenol bath (100 mg/L) for 60 s. Then, blood samples were taken from caudal vein and collected in heparinized tubes. After the blood sampling, the fish were euthanized by spinal cord cutting and liver and kidney were dissected. Immediately after the blood collection, the aquaria water levels were decreased by 90% for 3 h for stress induction. After the stress, blood samples were taken from each treatment as mentioned above. Blood samples were divided into two portions; one was used for leukocyte count and the other for plasma separation. The blood samples were centrifuged for 10 min (1200 g) for plasma separation. The plasma samples were kept at −70 °C until analysis. Liver and kidney samples were immediately frozen in liquid nitrogen and kept in a −70 °C freezer for further analyses. 2.6. Analysis 2.6.1. Leukocyte count The leukocytes were counted using an improved Neubaeur hemocytometer. Blood smears were prepared, air-dried, fixed in methanol and stained using May–Giemsa solution for differential leukocyte count. Leukocytes in blood smears were categorized into lymphocytes, monocytes, neutrophils and eosinophils (Blaxhall, 1972).
2.7. Statistical analysis Data of growth performance, leukocyte count, plasma proteins, plasma immune parameters and kidney gene expression were analysed by one-way ANOVA and Duncan test to find the effects of dietary LE
2.6.2. Plasma biochemical analyses Plasma total protein, albumin, and glucose levels were determined by commercial kits provided by Pars Azmun Co. (Tehran, Iran) as reported by Hoseini and Tarkhani (2013). The kits were designed based on Biuret, bromocresol green and glucose oxidase methods, respectively. Plasma cortisol levels were determined by ELISA method using commercial kit (IBL, Gesellschaft für Immunchemieund Immunbiologie, Germany) following Hoseini et al. (2018a). The kit was designed based on competitive ELISA method. Cortisol antigen was coated on the internal surface of a 96-well plate, which reacted with the sample cortisol. Inter- and intra-assay coefficients of variation were 8.69 and 10.2%, respectively. Plasma lysozyme was determined turbidimetrically with Micrococcus luteus as target according to Ellis (1990). One unit of lysozyme activity was defined as the amount of enzyme producing a decrease in absorbance of 0.001/min in 1 mL plasma. Serum alternative complement activity (ACH50) was determined according to Yano (1992) using magnesium containing gelatin-veronal buffer as reaction
Table 2 Primers sequences and amplification efficiencies. Gene
Sequences of primers
Accession no
Beta actin
F: AGACATCAGGGTGTCATGGTTGGT R: CTCAAACATGATCTGTGTCAT F: ACCAGCTGGATTTGTCAGAAG R: ACATACTGAATTGAACTTTG F: TGATGACATGGAACCATTACTGG R: CACCTTTTTCCTTCATCTTTTCA F: GGTGATGGTGTCGAGGAGGAA R: TGGAAAGACACCTGGCTGTA F: ACGCTTTATTCCCAACCAAA R: GAAATCCTTGCTCTGCCTCA F: GTCGCTGCTGCTTGATA GAA R: CTGAAGCTCCCTCCATACTT
M24113.1
IL-1b IL-10 TNF-a TGF-b IFN
3
AB010701.1 AB110780 AJ311800.1 DQ411314.1 AM261214
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Table 3 Effects of different dietary lavender (L. angustifolia) extract (LE) levels on growth performance of common carp after 70 days. Treatments
Initial weight (g)
Final weight (g)
Feed intake (g)
Weight gain (%)
FCR
Control 0.5E 1.0E 1.5E P F
25.7 ± 25.7 ± 26.0 ± 25.9 ± 0.964 0.090
58.5 ± 57.2 ± 59.8 ± 60.0 ± 0.628 0.512
52.6 ± 51.5 ± 55.0 ± 54.0 ± 0.742 0.795
127 ± 122 ± 130 ± 131 ± 0.597 0.665
1.60 ± 1.63 ± 1.63 ± 1.58 ± 0.817 0.311
1.38 1.69 1.85 1.66
2.67 2.80 3.00 2.98
levels. Data of liver oxidative condition markers, and plasma cortisol and glucose were subjected to two-way ANOVA to find the effects of dietary LE and stress. Significant interaction effects of dietary LE and stress were found on these parameters; thus, one-way ANOVA and Duncan tests were used to analyse these data. P < 0.05 was considered as significance. Data were presented as mean ± SEM. Statistical analyses were performed using SPSS v.22.
2.88 2.70 3.11 3.05
5.23 5.65 5.47 5.29
SGR (%/d) 0.07 0.08 0.09 0.06
1.17 ± 1.14 ± 1.19 ± 1.19 ± 0.586 0.685
0.06 0.05 0.07 0.05
Table 5 Plasma proteins levels of common carp fed different levels of dietary lavender (L. angustifolia) extract (LE) for 70 days. Different letters in the same column show significant difference at P < 0.05 (Duncan’ test; n = 9).
3. Results The fish growth performance and feed efficiency are presented in Table 3. There was no fish mortality in any treatments during the experiment. Statistical analyses showed that there was no significant difference in the fish weight gain, FCR, and SGR among the treatments. Leukocyte counts are presented in Table 4. The treatments 1.0E and 1.5E had significantly higher total leukocytes count than the control, but there was no significant difference in leukocytes differential count among treatments. The results of plasma proteins’ levels are presented in Table 5. There was no significant difference in plasma total protein and albumin among treatments. However, plasma globulin levels in the treatments of 1.0E and 1.5E were significantly higher than the treatments of 0.5E and control. There was a tendency of increase plasma immune parameters along with increase in dietary LE supplementation (Fig. 1). Plasma lysozyme activities increased along with the increase in dietary LE levels and the highest value was related to the treatment 1.5E. Plasma ACH50 activity and total Ig levels of the treatments 1.0E and 1.5E were significantly higher than the treatments of 0.5E and control. Dietary LE supplementation significantly affected expression of IL1b, IL-10, TNF-a and TGF-b, but not IFN genes (Fig. 2). The lowest expression of IL-1b gene was related to the treatment 1E; moreover, the treatment 1.5E had lower gene expression than the treatments of 0.5E and control. The TNF-a gene expression significantly decreased along with the increase in dietary LE levels and the lowest expression was related to the treatment 1.5E. The highest expression of IL-10 gene was related to the treatments of 1.0E and 1.5E; moreover, the treatment 0.5E had higher gene expression than the control treatment. The treatment 1.5E had significantly higher TGF-b gene expression compared to the control and 0.5E groups. Dietary LE significantly affected liver SOD, CAT and MDA, and serum glucose and cortisol; stress affected all the mentioned parameters
Treatments
Total protein (g/dL)
Albumin (g/dL)
Globulin (g/dL)
Control 0.5E 1.0E 1.5E P F
2.79 ± 2.94 ± 3.26 ± 3.15 ± 0.102 2.35
1.49 ± 1.54 ± 1.51 ± 1.42 ± 0.853 0.26
1.31 ± 1.39 ± 1.75 ± 1.73 ± 0.005 8.71
0.35 0.40 0.42 0.39
0.15 0.21 0.22 0.18
0.16a 0.18a 0.18b 0.17b
Fig. 1. Activities of plasma lysozyme and ACH50 and levels of plasma total Ig (mean ± SEM) of common carp fed different levels of dietary lavender (L. angustifolia) extract (LE) for 70 days. Different letters above the bars show significant difference at P < 0.05 (Duncan’ test; n = 9).
Table 4 Total and differential leukocyte count of common carp fed different levels of dietary lavender (L. angustifolia) extract (LE) for 70 days. Different letters in the same column show significant difference at P < 0.05 (Duncan’ test; n = 9). Treatments
Total leukocyte (cell/mm3)
Lymphocyte (%)
Neutrophil (%)
Eosinophil (%)
Monocyte (%)
Control 0.5E 1.0E 1.5E P F
15541 17593 19135 18963 0.024 3.91
79.0 ± 79.3 ± 79.8 ± 78.8 ± 0.977 0.07
18.5 ± 18.8 ± 18.2 ± 18.3 ± 0.972 0.06
1.00 ± 0.83 ± 0.83 ± 1.00 ± 0.962 0.08
1.00 ± 1.00 ± 0.83 ± 1.00 ± 0.978 0.06
± ± ± ±
145a 166ab 154b 144b
5.88 4.59 5.02 5.00
4
1.05 1.12 1.01 1.00
0.11 0.13 0.14 0.14
0.18 0.18 0.16 0.18
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Fig. 2. The expression of immune-related genes (mean ± SEM) in the kidney of common carp fed different levels of dietary lavender (L. angustifolia) extract (LE) for 70 days. Different letters above the bars show significant difference in each gene, separately at P < 0.05 (Duncan’ test; n = 9).
4. Discussion
Table 6 Liver activities of SOD and CAT and levels of MDA, and plasma levels of cortisol and glucose of common carp fed with LE-supplemented diets for 70 days and exposed to 1 h crowding stress. Different letters show significant difference at P < 0.05 (Duncan’ test; n = 9). Two way ANOVA
SOD
LE Stress LE × Stress
0.0001 > 0.175 0.0001 >
CAT
MDA
Glucose
Cortisol
0.0001 > 0.0001 > 0.0001 >
0.0001 > 0.0001 > 0.0001 >
0.0001 > 0.0001 > 0.0001 >
0.0001 > 0.0001 > 0.0001 >
23.8a 40.6b 50.4c 39.5b 22.9a 16.2a
50.5a 94.3b 101b 74.1a 61.5a 52.5a
38.2a 102b 116b 85.8b 47.3a 32.4a
The immune system is a target of different herbal products (Chakraborty and Hancz, 2011). Blood leukocytes and proteins are important components of the fish immune system, which change with administration of immunostimulants (Chakraborty and Hancz, 2011). In the present study, dietary LE significantly increased soluble immune (plasma globulin, total Ig, lysozyme, and ACH50) and cellular innate immune components (leukocytes). Such results are in line with those previously reported in different fish species. For example, Hoseinifar et al. (2018) reported that dietary loquat (Eriobotrya japonica) extract supplementation significantly improved serum lysozyme, ACH50, and total Ig of common carp after a 7-week feeding period. Taheri Mirghaed et al. (2018) and Hoseini et al. (2018b) found improvement in serum total protein, globulin, lysozyme, ACH50, and total Ig, and blood leukocytes count of rainbow trout (Oncorhynchus mykiss) fed with cineolesupplemented diets for 50 days. Dietary eucalyptol supplementation significantly improved common carp blood leukocytes count and serum globulin, lysozyme, and total Ig after a 14-day feeding trial (Hoseini et al., 2018a). There are no data on the effects of lavender on innate immune system of fish, but it has been shown that extract from lavender species (Lavandula multifida and L. angustifolia) increased the number of phagocytic cells and improved phagocytic, respiratory burst, and peroxidase activities of gilthead sea bream, Sparus aurata head kidney leukocyte in vitro (Fazio et al., 2017). The exact mechanism by which dietary LE (and other herbal extract) improves the fish innate immune system is not clear, but it might be due to increased overall health and well-being (see below), as it has been demonstrated that unfavourable conditions (e.g. stress) decrease overall health and immune function in fish (Tort, 2011). Likewise, increased plasma proteins and ACH50 might be due to improved fish liver health and function, as this organ is the site for many protein synthesis (Hoseini and Tarkhani, 2013), and lavender extract was found to be hepatoprotective (Selmi et al., 2015). Such a hepatoprotective effect has been reported for other herbal extract in common carp (Jia et al., 2012; Liu et al., 2015). The increase in lysozyme activity might be due to the increase in neutrophil number and/or lysozyme production (Costas et al., 2011), but it needs further investigations. Cytokines are indicators of inflammation in animal and stressful conditions led to inflammation in fish (Yarahmadi et al., 2016; Hoseini et al., 2019b). Cytokines IL-1b and TNF-a are pro-inflammatory proteins showing up-regulation during stressful conditions in fish, such as high stocking density (Yarahmadi et al., 2016; Hoseini et al., 2019b) and toxicant exposure (Begam and Sengupta, 2015). IL-10 and TGF-b are anti-inflammatory cytokines showing down-regulation during stressful conditions (Begam and Sengupta, 2015; Hoseinifar et al., 2018; Hoseini et al., 2019b). The present results clearly suggest that dietary
P-values
Comparison of pooled means Before stress After stress Control 0.5E 1E 1.5E
34.1 33.0 25.2a 29.5b 38.2c 41.4d
45.9b 32.8a 25.1a 31.5b 46.6c 54.5d
Comparison of means Control-Before stress 0.5E-Before stress 1E-Before stress 1.5E-Before stress Control-After stress 0.5E-After stress 1E-After stress 1.5E-After stress Pooled SEM
28.2b
34.4c
31.5d
58.1bc
46.4bc
31.1b
39.6d
26.3c
51.0abc
40.8b
36.4c 41.0d
52.6e 57.1f
22.0b 15.5a
47.5ab 45.6a
37.8ab 27.2a
22.4a
15.6a
69.3f
145f
185e
28.0b 40.1d 41.8d 1.04
23.1b 40.5d 51.9e 2.02
52.6e 23.8bc 18.9a 2.62
97.1e 75.7d 59.3c 4.77
130d 56.7c 37.8a 7.85
except for liver SOD. LE levels and stress had interaction effects on liver SOD, CAT and MDA, and serum glucose and cortisol (Table 6). Liver SOD and CAT showed significant increase along with dietary LE supplementation levels and their highest values were related to the treatment 1.5E. After the stress, hepatic SOD decreased in the control group, but not the LE-supplemented groups. Stress led to significant decrease in hepatic CAT activities in all treatments, but CAT activity increased along with the LE levels with highest activity in the treatment 1.5E. The stress led to significant increase in hepatic MDA in the treatments of control and 0.5E, but not 1.0E and 1.5E. The stress led to significant increase in plasma glucose levels in all treatments; however, dietary LE levels significantly decreased plasma glucose levels. The stress led to significant increase in plasma cortisol levels in all treatments, except for the 1.5E; dietary LE levels significantly decreased plasma cortisol levels. 5
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LE had anti-inflammatory effects in common carp, which is indicator of boosted health. Dietary LE was reported to have anti-inflammatory properties (Silva et al., 2015; Cardia et al., 2018), which seems to be due to its major constitutes such as cineole (Juergens, 2014) and linalool (Peana et al., 2002). Although there is no study on the effects of dietary LE on cytokine responses in fish, Hoseinifar et al. (2015) showed that palm fruit extract had anti-inflammatory effects on common carp, down-regulating TNF-a and IL-1b gene expression. Stressors, such as over-crowding, may lead to oxidative stress, leading to decrease in SOD and CAT activities; these two enzymes are the first line of antioxidant defense that decompose pro-oxidant molecules (superoxide and hydrogen peroxide). Decreases in these enzymes activity lead to lipid peroxidation and MDA formation (Yousefi et al., 2018b). Similar to the present study, Taheri Mirghaed et al. (2018) reported oxidative stress in rainbow trout, O. mykiss exposed to over-crowding stress. Dietary LE showed strong antioxidant properties characterized by elevated antioxidant enzyme activities and declined MDA content in the fish. There is no study on the antioxidant effects of LE on fish, but in vitro studies clearly demonstrated such antioxidant effects (Gülçin et al., 2004; Kıvrak, 2018). The antioxidant effects might be due to the presence of high amounts of antioxidant compounds, cineole (Ciftci et al., 2011; Taheri Mirghaed et al., 2018) and linalool (Yousefi et al., 2018a). Such an antioxidant effect has been reported in studies on other herbal extracts/compounds (Zheng et al., 2009; Hoseini et al., 2019c; Yousefi et al., 2019). Interestingly, 1.5% LE was capable to completely inhibit oxidative stress caused by over-crowding stress, as it prevented significant changes in SOD activity and MDA content after the crowding stress. The results are in line with Taheri Mirghaed et al. (2018) reporting that dietary cineole administration significantly inhibited oxidative stress after over-crowding in rainbow trout, O. mykiss. The increase in circulatory levels of cortisol and glucose levels in the present study was in line with previous studies on common carp during stress (Hosseini and Hoseini, 2012; Hoseini et al., 2019a, 2019d). Such changes in blood cortisol and glucose are adaptive responses to provide demanded energy during the stress (Barton, 2002), however, cortisol elevation has negative effects on fish immune system (Tort, 2011). Dietary LE was able to suppress blood cortisol and glucose; thus, it has anti-stress effects. There are no study on this topic, however, previous studies have shown other herbal compounds, such as rosemary leaf (Yousefi et al., 2019), Rheum officinale extract (Xie et al., 2008), eucalyptol (Hoseini et al., 2018a), suppressed stress in different species. The anti-stress effects of dietary LE might be due to high amount of cineole, as previous studies demonstrated that cineole suppressed stress in different fish species (Hoseini et al., 2018a; Taheri Mirghaed et al., 2018). The decrease in cortisol levels and improved antioxidant defense may explain boosted fish immune responses in the LE-fed fish (particularly 1 and 1.5%) as there are strong crosslinks between immune responses and cortisol (Tort, 2011; Hoseini et al., 2016; Yousefi et al., 2016) as well as antioxidant system (Hoseinifar et al., 2017). In conclusion, dietary LE has anti-inflammatory, anti-stress and antioxidant effects in common carp. Suppressed cortisol levels and augmented antioxidant defense by dietary LE may explain boosted immune responses in the fish. Dietary LE supplementation at 1–1.5% is recommended for common carp to suppress stress, inflammation and oxidative conditions and augment immune responses in the fish.
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