Enrichment of common carp (Cyprinus carpio) diet with medlar (Mespilus germanica) leaf extract: Effects on skin mucosal immunity and growth performance

Accepted Manuscript Enrichment of common carp (Cyprinus carpio) diet with medlar (Mespilus germanica) leaf extract: Effects on skin mucosal immunity and growth performance Seyed Hossein Hoseinifar, Hassan Khodadadian Zou, Hamed Kolangi Miandare, Hien Van Doan, Nicholas Romano, Maryam Dadar PII:

S1050-4648(17)30344-3

DOI:

10.1016/j.fsi.2017.06.023

Reference:

YFSIM 4642

To appear in:

Fish and Shellfish Immunology

Received Date: 20 April 2017 Revised Date:

4 June 2017

Accepted Date: 6 June 2017

Please cite this article as: Hoseinifar SH, Zou HK, Miandare HK, Van Doan H, Romano N, Dadar M, Enrichment of common carp (Cyprinus carpio) diet with medlar (Mespilus germanica) leaf extract: Effects on skin mucosal immunity and growth performance, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.06.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Enrichment of common carp (Cyprinus carpio) diet with Medlar (Mespilus germanica) leaf

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extract: Effects on skin mucosal immunity and growth performance

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Running head: Dietary Medlar on carp growth and health

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Seyed Hossein Hoseinifar a*, Hassan Khodadadian Zou a, Hamed Kolangi Miandare a, Hien Van

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Doan, Nicholas Romano c, Maryam Dadar b

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Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University

of Agricultural Sciences and Natural Resources, Gorgan, Iran b

Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension

Organization (AREEO), Karaj, Iran c

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a*

Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang,

Selangor, Malaysia

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*Author for correspondence:

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SH Hoseinifar, Department of Fisheries, Faculty of Fisheries and Environmental Sciences,

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Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.

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E-mail address: [email protected]

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Tel.: +98 1732427040; fax: +98 1732245886.

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Abstract

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A feeding trial was performed to assess the effects of dietary Medlar (Mespilus germanica) leaf

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extract (MLE) on the growth performance, skin mucus non-specific immune parameters as well

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as mRNA levels of immune and antioxidant related genes in the skin of common carp (Cyprinus

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carpio) fingerlings. Fish were fed diets supplemented with graded levels (0, 0.25, 0.50, and

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1.00%) of MLE for 49 days. The results revealed an improvement to the growth performance

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and feed conversion ratio in MLE fed carps (P < 0.05), regardless of the inclusion level. The

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immunoglobulin levels and interleukin 8 levels in the skin mucous and skin, respectively,

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revealed significant increment in fish fed 1% MLE (P < 0.05) in comparison with the other MLE

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treatments and control group. Also, feeding on 0.25% and 0.50% MLE remarkably increased

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skin mucus lysozyme activity (P < 0.05). However, there were no significant difference between

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MLE treated groups and control (P > 0.05) in case protease activity in the skin mucous or tumor

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necrosis factor alpha and interleukin 1 beta gene expression in the skin of carps (P > 0.05). The

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expression of genes encoding glutathione reductase and glutathione S-transferase alpha were

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remarkably increased in MLE fed carps compared to the control group (P < 0.05) while carp fed

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0.50% or 1.00% MLE had significantly increased glutathione peroxidase expression in their skin

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(P < 0.05). The present results revealed the potentially beneficial effects of MLE on the mucosal

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immune system and growth performance in common carp fingerlings.

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Keywords: Medlar; Common carp; mucosal immune response; Antioxidant enzymes; herb

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extract; Immunostimulant

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1. Introduction During the past several years, public awareness about the risks of antibiotic residue has

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subsequently increased demands for so called “green fish” (i.e fish raised in an antibiotic free

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environment) [1-3]. This trend, along with the establishment of stricter regulations on antibiotic

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use, have substantially decreased the applications of antibiotics in aquaculture [4]. It has been

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suggested that the use of natural immunostimulants can be an effective alternative approach to

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prevent infectious disease outbreaks in commercial aquaculture settings [5-9]. Immunostimulants

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are regarded as natural compounds that can increase the resistance of a host to diseases,

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particularly infectious diseases, by enhancing their immune system [10, 11]. Some of the most

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well-known immunostimulants include pre or/ probiotics as well as herbal plants, which show

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that great promise at not only improving the health status of cultured fish or shellfish, but also

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growth and nutrient utilization [3, 6, 7, 12-17].

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In terms of immunity, the skin and their associated immune compartments play a key role

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as the first line of defence against various pathogens in fish [18, 19]. In addition to having a

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protective role, the skin is also involved in respiration, ion regulation and thermal regulation [20,

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21]. In recent years, a modulation of the mucosal immune response by using immunostimulants

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has received increasing attention as reviewed by Caipang and Lazado [22]. In various studies, it

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has been revealed that dietary probiotics [23], prebiotics [24-27] as well as medicinal plants [18,

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28-30] can have beneficial effects on skin mucus immune parameters.

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In spite of the numerous studies on medicinal plants as immunostimulant in aquaculture,

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to the best of our knowledge there is no published data on Medlar (Mespilus germanica L.) as a

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potential medicinal herb. Medlar belongs to the Rosaceae family and mainly grows in the

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northern areas of Iran, temperate regions of Asia and the central forests of Europe [31]. It has

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been considered as a valuable herbal medicine with a long history in Eastern traditional medicine

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to treat fade fever, throat and mouth abscesses, Angina and thrush [32]. Bibalani and

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Mosazadeh-Sayadmahaleh [31] reported that different parts of Medlar (such as fruit, leaf and

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wood) can also be used as herbal medicine. Furthermore, Medlar fruit and leaves can inhibit

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reactive oxygen species (ROS), largely believed to be due to presence of flavonoids and phenolic

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acids [32, 33] as well as having antibacterial and antioxidant properties [31].

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Considering the beneficial effects of medlar reported in non-fish studies, as well as the

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importance of skin mucus immune responses against pathogens, the present study was performed

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to study the effects of different levels of a medlar leaf extract on the growth performance,

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mucosal immune parameters as well as the expression of immune and antioxidant related genes

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in the skin of common carp fingerlings.

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2. Material and Methods

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2.1. Medlar leaf extract preparation and experimental diets

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Medlar leaves were collected from a medlar plant (Golestan province, Iran) and identified

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by experts at the Department of Botany of Golestan University (Golestan, Iran). A Medlar leaf

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extract (MLE) was prepared following a method suggested by Sadeghi-Nejad et al. [34]. Briefly,

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the collected foliage were first dried and powdered and then 80% ethanol was added (10g/100ml)

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and left for 72 hours. The obtained extract was filtered using Whatman filter paper and dried by

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evaporating off the ethanol, which yielded the MLE. The experimental diets were prepared by

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supplementing MLE to a basal diet (Table 1) at 0% (control), 0.25%, 0.50% or 1.00%. These

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inclusion levels were selected according to previous studies on other plant extracts [16, 35]. The

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ingredients were blended thoroughly in a mixer and pelleted using a meat grinder as previously

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described [28]. The experimental diets were stored in sealed polythene bags at 4 ºC until further

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use.

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2.2. Fish and experimental design The experimental design was completely randomized to include three MLE treatments at

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0.25, 0.50 and 1.00% inclusion of MLE in the diets while a control group received no MLE

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additions. All treatments, including the control, were triplicated. The fish species used in the

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present study was common carp fingerling (8.31 ± 0.13 g) obtained from the Sijaval centre for

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propagation and restocking of teleost fish (Bandar-e Torkaman, Golestan Province, Iran). The

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fish were transported to the Aquaculture Laboratory at the Department of Fisheries (Gorgan

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University of Agricultural Sciences and Natural Resources, Gorgan, Iran). After 2-weeks of

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acclimatization, fifteen fish were stocked into each of the twelve 100-L fiberglass tanks and then

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randomly assigned one of the four treatments.

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Over this 49-day study, the fish were fed the experimental diets at rate of 3% body

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weight. This was done by measuring the fish every 2 weeks and adjusting the amount of feed

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accordingly. Each tank received constant aeration in the tank water as well as daily exchange of

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water at approximately 50%. The water quality parameters that included temperature, pH and

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dissolved oxygen were checked and were as follows 23 ± 1.2 ºC, 7.6 ± 0.4 and 7.0 ± 0.1

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monitored daily.

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One day prior to ending the experiment, the fish were fasted for 24h. On the final day of

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sampling, the fish were anesthetized by using clove powder (500 mg L-1) and then batch weighed

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to measure the growth performance and feed conversion ratio using the following equations,

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Weight gain = [(W2 (g) − W1 (g))/ W1] ×100

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W1 is the initial weight, W2 is the final weight

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Specific growth rate (SGR) = 100 × [(ln W2 − ln W1) / T]

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W1 is the initial weight, W2 is the final weight and T is the duration of the feeding trial

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Feed conversion ratio (FCR) = feed intake (g) / weight gain (g)

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Survival = (final number of fish / initial number of fish) ×100

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After measuring the fish weights, nine of the anesthetized were sampled from each

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treatment to measure some non-specific immune parameters in the skin mucus while another

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nine fish were sampled to measure immune and antioxidant related gene expression in the skin.

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2.3. Evaluation of non-specific immune parameters in the skin mucus

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Skin mucus samples were collected at the end of experiment from nine fish in each

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treatment by using polyethylene bags according to Subramanian et al. [36] and slightly modified

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by Khodadadian Zou et al. [27]. The mucus samples were centrifuged (5810 R Eppendorf,

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Engelsdorf, Germany) (1500 × g for 10 min at 4ºC) and kept at -80 ºC until for further analysis

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for total immunoglobulin (Ig), lysozyme activity and protease activity.

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First, the soluble protein levels of mucus samples were measured according to the Lowry

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et al. [37] protocol and then the skin mucous total Ig levels were measured according to Siwicki

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and Anderson [38]. After the immunoglobulin molecules were precipitated down using 12%

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solution of polyethylene glycol (Sigma) and the protein content of mucus samples were re-

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measured as mentioned above. The total Ig levels were calculated as the difference between the

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two protein levels measured. The skin mucus lysozyme activity was calculated as we described

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in Khodadadian Zou et al. [27]. This method was a turbidimetric approach by using Micrococcus

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luteus (Sigma), which is a lysozyme-sensitive bacterium. Finally, the skin mucus protease

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activity was measured according to Ross et al. [39] and is based on using azocasein as described

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in detail in Hoseinifar et al. [29].

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2.4. Evaluation of immune and antioxidant related genes expression in skin

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2.4.1. Tissue sampling, RNA extraction and cDNA synthesis A total of nine fish in each treatment were randomly selected for gene expression studies

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using real time PCR. The skin tissue was sampled according to the protocol suggested by

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Esteban et al. [40] and snap-frozen in liquid nitrogen. The RNA was extracted from the skin

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samples according the manufacturer instructions (BIOZOL Reagent; Bioflux-Bioer, China). The

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isolated total RNA was treated with DNase I (Fermentas, Lithuania) to remove genomic DNA.

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The concentration and quality of RNA samples was evaluated with a Nanophotometer

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(Nanodrop technology, Wilmington, DE,USA) and agarose gel (1.5%), respectively, as

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previously described [27]. Thereafter, a cDNA synthesis kit (Fermentas, Lithuania) was used for

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cDNA synthesis from the isolated RNA.

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2.4.2. Primers and Real time PCR

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The sequences available in GenBank for common carp were used to design primers using

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the Oligo 7 program for a set of immune related genes that included tumor necrosis factor alpha

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(tnf-alpha), interleukin 1 beta (il1b) and interleukin 8 (il8) as well as antioxidant related genes

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that included glutathione reductase (gr), glutathione S-transferase alpha (gsta) and glutathione

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peroxidase (gpx). Table 2 represents the sequences of the primers used in the present study.

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Real-time PCR analysis was used to evaluate changes in the relative expression of the selected

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genes as described earlier [27]. The expression of the selected genes were corrected by the b-

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actin RNA content of each sample. The gene expression results were analyzed using the iQ5

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optical system software (BioRad) and ∆∆Ct method. In all cases, each PCR from each sample

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was performed in triplicate.

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2.5. Statistical analysis

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The data of the effects of MLE on mucosal immune parameters and expression of the

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selected immune and antioxidant related genes were analyzed using one-way analysis of variance

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(ANOVA) followed by a Tukey test. Prior to statistical analysis the normality of data and

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homogeneity of variances were checked and verified. In cases of P < 0.05, the differences were

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considered statistically significant. All of statistical analysis was performed using statistical

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package version 16.0 (SPSS Inc., Chicago, IL, USA).

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3. Results

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3.1. Growth parameters

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The growth performance parameters and survival of common carp fed different levels of

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dietary MLE after 49 days are shown in Table 3. Fish fed the MLE supplemented diets had

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significantly higher final weights, weight gain and specific growth rates (SGR) compared to the

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control group (P < 0.05). This increase in growth performance was not dose dependent and no

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significant difference was observed between the different dietary MLE treatments (P > 0.05).

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Meanwhile, carp in all the MLE treatments had significantly lower feed conversion ratios (FCR)

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compared to the control group (P < 0.05) and like the growth performance, the improvement in

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FCR was not dose dependent (Table 3). During the experiment, no mortality was observed in any

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of the treatments.

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3.2. Skin mucus immune parameters

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The effects of feeding on MLE supplemented diets on nonspecific immune parameters of

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common carp skin mucus are presented in Table 4. The total Ig level was only significantly

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higher in the dietary 1.00% MLE treatment compared to the other treatments. Fish fed dietary

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MLE at 0.25 and 0.50 % had significantly higher (P < 0.05) lysozyme activity than the control or

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1.00% dietary MLE treatment. No difference in lysozyme activity was detected between the

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control and 1.00% treatments (P > 0.05). Meanwhile, the skin mucus protease activity, was not

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significantly different between the MLE treated groups and control (P > 0.05).

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3.3. Immune and antioxidant related gene expression Figure 1A, B and C shows the mRNA levels of the immune related genes of tnf-alpha,

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il1b and il8, respectively in the skin of common carp fingerlings after being fed increasing levels

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of MLE. The results of tnf-alpha and il1b gene expression levels revealed a non-significant

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increase in MLE treatments compared to control group (P > 0.05). The highest expression level

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of il8 was observed in fish fed 1% MLE treatment, which was significantly higher than the other

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treatments (P < 0.05). Meanwhile, carp fed dietary MLE of 0.25 or 0.50% was significantly

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higher than those in the control group.

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The mRNA levels of the antioxidant relates genes (gsta, gpx and gr) in the common carp

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skin after 49 days of being fed increasing levels of MLE are presented in Figure 2A, B & C,

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respectively. The expression of genes encoding gr in MLE fed carps was significantly increased

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(P < 0.05) in all MLE treatment groups compared control. No significant difference was found

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for gr gene expression among the MLE groups.

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significantly higher gsta gene expression, but the highest gsta expression was found in the

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dietary 1% MLE treatment, which was significantly higher than those in the 0.25% MLE

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treatment. Carp fed 0.5% or 1% MLE significantly increased gpx expression in the skin of carp

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(P < 0.05), while there were no significant difference between gpx expression of 0.25% MLE

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treatment and the control group (P > 0.05).

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Similarly, all the MLE fed groups had

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4. Discussion

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It is well-known that feeding comprises majority of variable cost (50-80% of production

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costs) in the intensive commercial production of aquatic animals. Therefore, improvements to

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growth performance and feed utilization are key criteria when assessing the success of using feed

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additives. The present results determined that supplementations of MLE in the diets of carp

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significantly improved both growth and feed conversion ratio and this finding is particularly

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noteworthy considering Medlar, and other herbs or plants, are readily available and inexpensive

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in many countries. The growth enhancing effects of medicinal herbs have often been attributed

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to stimulation of appetite, increased digestive enzyme activity and/or presence of certain

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bioactive compounds that can be unique to each plant or herb [16, 41]. In this study, however,

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the feeding rates were fixed which may point to the latter two causes and requires further

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investigations. There are many bioactive compounds found in various plant and herbs and one or

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several could be responsible for beneficial effects. For example, ursolic acid (UA), which is a

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pentacyclic triterpenoid commonly found in fruit peels and many herbs, has been found to

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stimulate muscle growth through elevation of protein kinase (Akt) activity in the skeletal muscle

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of mice Kunkel et al. [42]. Although to the best of our knowledge there is no information about

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MLE effects on aquatic animals, in accordance with present finding above mentioned medicinal

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herbs which contain UA showed similar growth enhancing effects on different species [43].

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Thus, it can be suggested as a possible mode of action which needs to be confirmed in future

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studies. It should be mentioned that there is limited information on complete profile of Medlar

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leaf extract and further studies would be helpful to see other bioactive compounds.

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The skin mucus and its components play a pivotal role as barrier against a wide range of

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pathogens and exto-parasites [44]. This essential part of fish non-specific immune system is

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reportedly affected by environment conditions [45] as well as nutrition and feed additives [22].

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The results of present study revealed an increase in skin mucus total Ig levels and lysozyme

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activity in carps treated with MLE. The Ig, or antibodies, are important components of immune

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system and help in fighting various pathogens including viruses, fungi, bacteria and parasites

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while lysozymes are antimicrobial enzymes and an increase to these activities indicate enhanced

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immunological function.

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improve innate immunity [16, 28, 46]. For example, Adel et al. [41] observed an elevation of

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non-specific immune parameters in the skin mucus of Caspian brown trout (Salmo trutta

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caspius) fed dietary peppermint (Mentha piperita) extracts in a dose dependent manner from 1 –

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4%. Similarly, increasing dietary Ferula (Ferula assafoetida) from 0.5 to 2.0% elevated

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lysozyme activity in the skin mucus of common carps fingerling [28]. Likewise, administration

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of date palm extract elevated immune parameters in common carp [47] and gilthead seabream

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(Sparus aurata) [18]. In contrast, Mansouri Taee et al. [48] reported that dietary Myrtle (Myrtus

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communis) had no significant effect on skin mucus lysozyme activity in rainbow trout

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(Oncorhynchus mykiss) fingerlings. Similar to potential reasons for improving growth, the

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beneficial effects of medicinal herbs on non-specific immune parameters is often attributed to

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presence of bioactive compounds [12, 16], which as previously mentioned could be due to the

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presence of UA. Previous studies on this natural compound have revealed it can increase the

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cellular immune system and pancreatic β-cell function [49] in mice as well as activating the

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intracellular killing effect of macrophages in cell line cultures [50]. However, the determination

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of such modes of action in aquatic organisms need further in depth studies.

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Several researchers have also found that plant or herbal extracts

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An evaluation of the mRNA levels of immune related genes (tnf-alpha, il1b and il8)

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showed an up-regulation in carp fed the MLE diets, and in case of il8 (cxcl80), this was

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significant. Both tnf and il1b are proinflammatory cytokines that are excreted by immune cells,

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and play a vital function in mediating the innate immune responses [51, 52]. il8 is an important

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chemokine produced by macrophages, which induces chemotaxis and phagocytosis [53]. To the

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best of our knowledge, there is no available data on the effects of MLE on cytokines gene

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expression in skin. Regarding other medicinal herbs, Cerezuela et al. [18] reported an

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upregulation of immune related genes in gilthead seabream fed date palm extract. More research

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should be conducted to determine whether a bioactive compound is responsible and, if so, which

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one. Such research would improve our knowledge on the immunomodulatory roles of natural

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products and better tailor diets to optimize immunological function in aquatic farmed animals.

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Antioxidant enzymes play an important role in protecting against oxidative damage by

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eliminating free radicals, which can be naturally produced in body metabolism process and/or

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from the pollutants in the environment [54-57]. Moreover, the free radical scavenging activity of

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antioxidant enzymes was demonstrated to be positively affected by the presence of dietary

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antioxidants [58-60]. Therefore, during last few decades progressive research attempts were

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made on the evaluation of different medicinal herbs as antioxidants in fish [16, 43]. Indeed,

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various herbs have been shown to have a higher antioxidant ability, which is largely due to their

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high content of phenolics and flavonoids, which are natural components that can scavenge free

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radicals (hydrogen donors, singlet oxygen quenchers) [31, 61]. This study revealed an up-

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regulation of antioxidant enzyme gene expression (gsta, gpx and gr) in common carp fingerlings

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fed with MLE. This finding is in agreement with a study on dietary Ferula assafoetida since this

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elevated the expression of antioxidant enzyme genes in common carp [28]. Contrasting findings

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were found with palm fruit extract since Esteban et al. [40] reported an upregulation of

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antioxidant enzyme gene expression in the mucosae of gilthead seabream after being fed with a

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palm fruit extract but the same ingredient had no effect on gr or gsta expression levels in

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common carp [25]. Determining the reasons behind such contradictory results should be

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determined in future studies. These results showed potentially beneficial effects of MLE for

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improvement of carp antioxidant defence. However, the present study was on gene expression

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level and confirmation of this hypothesis merits enzymatic studies as well.

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Overall, the use of a MLE had remarkable impacts on the growth performance and

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mucosal immune defence and can therefore be considered as an effective ingredient in the diets

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of carp fingerlings. Determining, the optimum inclusion levels needs to be investigated in future

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studies as well as measuring protein and enzyme levels for confirmation of results obtained at the

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gene expression level. Furthermore, the protective role of MLE against infectious diseases

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should be investigated under experimental challenge to see if the changes in mucosal parameters

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are effective in disease resistance. Such research would increase our knowledge regarding the

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mechanisms of natural products and reduce the reliance on antibiotic use, which will ultimately

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lead to a more eco-friendly aquaculture product.

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Acknowledgements

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The present study was funded by the research affairs of Gorgan University of Agricultural

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Sciences and Natural Resources. The authors would like to thanks the staff at Aquaculture Lab

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and Molecular Lab of GUASNR for their kind help during the experiment.

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References

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1. Carbone D, Faggio C. Importance of prebiotics in aquaculture as immunostimulants. Effects on immune system of Sparus aurata and Dicentrarchus labrax. Fish & Shellfish Immunology. 2016 54:172-8. 2. Dawood MAO, Koshio S. Recent advances in the role of probiotics and prebiotics in carp aquaculture: A review. Aquaculture. 2016 454:243-51. 3. Hai NV. Research findings from the use of probiotics in tilapia aquaculture: A review. Fish & Shellfish Immunology. 2015 45:592-7.

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27. Khodadadian Zou H, Hoseinifar SH, Kolangi Miandare H, Hajimoradloo A. Agaricus bisporus powder improved cutaneous mucosal and serum immune parameters and up-regulated intestinal cytokines gene expression in common carp (Cyprinus carpio) fingerlings. Fish & Shellfish Immunology. 2016 58:380-6. 28. Safari R, Hoseinifar SH, Nejadmoghadam S, Jafar A. Transciptomic study of mucosal immune, antioxidant and growth related genes and non-specific immune response of common carp (Cyprinus carpio) fed dietary Ferula (Ferula assafoetida). Fish & Shellfish Immunology. 2016 55:242-8. 29. Hoseinifar SH, Zoheiri F, Lazado CC. Dietary phytoimmunostimulant Persian hogweed (Heracleum persicum) has more remarkable impacts on skin mucus than on serum in common carp (Cyprinus carpio). Fish & Shellfish Immunology. 2016 59:77-82. 30. Hoseinifar SH, Khalili M, Rufchaei R, Raeisi M, Attar M, Cordero H, et al. Effects of date palm fruit extracts on skin mucosal immunity, immune related genes expression and growth performance of common carp (Cyprinus carpio) fry. Fish & Shellfish Immunology. 2015 47:706-11. 31. Bibalani GH, Mosazadeh-Sayadmahaleh F. Medicinal benefits and usage of medlar (Mespilus germanica) in Gilan Province (Roudsar District), Iran. Journal of Medicinal Plants Research. 2012 6:1155-9. 32. Nabavi SF, Nabavi SM, Ebrahimzadeh MA, Asgarirad H. The antioxidant activity of wild medlar (Mespilus germanica L.) fruit, stem bark and leaf. African Journal of Biotechnology. 2011 10:283-9. 33. Gruz J, Ayaz FA, Torun H, Strnad M. Phenolic acid content and radical scavenging activity of extracts from medlar (Mespilus germanica L.) fruit at different stages of ripening. Food Chemistry. 2011 124:271-7. 34. Sadeghi-Nejad B, Shiravi F, Ghanbari S, Alinejadi M. Antifungal activity of Satureja khuzestanica (Jamzad) leaves extracts. Jundishapur Journal of Microbiology. 2010 3:36-40. 35. Awad E, Austin D, Lyndon AR. Effect of black cumin seed oil (Nigella sativa) and nettle extract (Quercetin) on enhancement of immunity in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquaculture. 2013 388:193-7. 36. Subramanian S, MacKinnon SL, Ross NW. A comparative study on innate immune parameters in the epidermal mucus of various fish species. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2007 148:256-63. 37. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J biol Chem. 1951 193:265-75. 38. Siwicki AK, Anderson DP. Nonspecific defense mechanisms assay in fish: II. Potential killing activity of neutrophils and macrophages, lysozyme activity in serum and organs and total immunoglobulin level in serum. Fish Disease Diagnosis and Prevention Methods Olsztyn, Poland. 1993:105-12. 39. Ross NW, Firth KJ, Wang A, Burka JF, Johnson SC. Changes in hydrolytic enzyme activities of naive Atlantic salmon Salmo salar skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation. Diseases of aquatic organisms. 2000 41:43. 40. Esteban M, Cordero H, Martínez-Tomé M, Jiménez-Monreal A, Bakhrouf A, Mahdhi A. Effect of dietary supplementation of probiotics and palm fruits extracts on the antioxidant enzyme gene expression in the mucosae of gilthead seabream (Sparus aurata L.). Fish & shellfish immunology. 2014 39:532-40. 41. Adel M, Safari R, Pourgholam R, Zorriehzahra J, Esteban MÁ. Dietary peppermint (Mentha piperita) extracts promote growth performance and increase the main humoral immune parameters (both at mucosal and systemic level) of Caspian brown trout (Salmo trutta caspius Kessler, 1877). Fish & Shellfish Immunology. 2015 47:623-9. 42. Kunkel SD, Elmore CJ, Bongers KS, Ebert SM, Fox DK, Dyle MC, et al. Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity, glucose intolerance and fatty liver disease. PloS one. 2012 7:e39332. 43. Jeney G, Wet LD, Jeney Z, Yin G. Plant Extracts. Dietary Nutrients, Additives, and Fish Health: John Wiley & Sons, Inc; 2015, p. 321-32. 44. Ángeles Esteban M. An overview of the immunological defenses in fish skin. ISRN Immunology. 2012 2012. 45. Ogawa T, Ishii C, Kagawa D, Muramoto K, Kamiya H. Accelerated evolution in the protein-coding region of galectin cDNAs, congerin I and congerin II, from skin mucus of conger eel (Conger myriaster). Bioscience, biotechnology, and biochemistry. 1999 63:1203-8. 46. Burgos-Aceves MA, Cohen A, Smith Y, Faggio C. Estrogen regulation of gene expression in the teleost fish immune system. Fish & Shellfish Immunology. 2016 58:42-9. 47. Hoseinifar SH, Dadar M, Khalili M, Cerezuela R, Esteban MÁ. Effect of dietary supplementation of palm fruit extracts on the transcriptomes of growth, antioxidant enzyme and immune-related genes in common carp (Cyprinus carpio) fingerlings. Aquaculture Research. 2016:doi:10.1111/are.13192.

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48. Mansouri Taee H, Hajimoradloo A, Hoseinifar SH, Ahmadvand H. Dietary Myrtle (Myrtus communis L.) improved non-specific immune parameters and bactericidal activity of skin mucus in rainbow trout (Oncorhynchus mykiss) fingerlings. Fish & Shellfish Immunology. 2017 64:320-4. 49. Jang S-M, Yee S-T, Choi J, Choi M-S, Do G-M, Jeon S-M, et al. Ursolic acid enhances the cellular immune system and pancreatic β-cell function in streptozotocin-induced diabetic mice fed a high-fat diet. International Immunopharmacology. 2009 9:113-9. 50. Podder B, Jang WS, Nam K-W, Lee B-E, Song H-Y. Ursolic acid activates intracellular killing effect of macrophages during Mycobacterium tuberculosis infection. J Microbiol Biotechnol. 2015 25:738-44. 51. Balkwill F, Burke F. The cytokine network. Immunology today. 1989 10:299-304. 52. Fehrenbacher JC, Burkey TH, Nicol GD, Vasko MR. Tumor necrosis factor α and interleukin-1β stimulate the expression of cyclooxygenase II but do not alter prostaglandin E 2 receptor mRNA levels in cultured dorsal root ganglia cells. Pain. 2005 113:113-22. 53. Secombes C, Hardie L, Daniels G. Cytokines in fish: an update. Fish & Shellfish Immunology. 1996 6:291304. 54. Halliwell B, Gutteridge JM. Free radicals in biology and medicine: Oxford University Press, USA; 2015. 55. Faggio C, Pagano M, Alampi R, Vazzana I, Felice MR. Cytotoxicity, haemolymphatic parameters, and oxidative stress following exposure to sub-lethal concentrations of quaternium-15 in Mytilus galloprovincialis. Aquatic Toxicology. 2016 180:258-65. 56. Bartoskova M, Dobsikova R, Stancova V, Zivna D, Blahova J, Marsalek P, et al. Evaluation of ibuprofen toxicity for zebrafish (Danio rerio) targeting on selected biomarkers of oxidative stress. Neuroendocrinology Letters. 2013 34:102-8. 57. Messina CM, Faggio C, Laudicella VA, Sanfilippo M, Trischitta F, Santulli A. Effect of sodium dodecyl sulfate (SDS) on stress response in the Mediterranean mussel (Mytilus Galloprovincialis): Regulatory volume decrease (Rvd) and modulation of biochemical markers related to oxidative stress. Aquatic Toxicology. 2014 157:94-100. 58. Dandapat J, Chainy GB, Rao KJ. Dietary vitamin-E modulates antioxidant defence system in giant freshwater prawn, Macrobrachium rosenbergii. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology. 2000 127:101-15. 59. Amar EC, Kiron V, Satoh S, Watanabe T. Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products. Fish & shellfish immunology. 2004 16:527-37. 60. Martínez-Álvarez RM, Morales AE, Sanz A. Antioxidant defenses in fish: biotic and abiotic factors. Reviews in Fish Biology and fisheries. 2005 15:75-88.

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Table 1. Dietary formulation and proximate composition of the basal diet (%)

40.0

Wheat flour

21.0

Soybean meal

13.5

Gluten

5.5

Soybean oil Fish oil Mineral premix*

6.0 6.0 3.0

Vitamin premix * Binder †

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Fish meal

2.0

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2.0

Anti fungi ‡

Antioxidant §

0.5

0.5

Proximate composition (% dry matter basis)

Dry matter

89.5

38.2

Crude lipid

10.2

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Crude protein Ash

Premix detailed by [25]

†

Amet binder ™, Mehr Taban-e- Yazd, Iran

‡

ToxiBan antifungal (Vet-A-Mix, Shenan- doah, IA)

§

Butylated hydroxytoluene (BHT) (Merck, Germany)

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Ingredients

3.4

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Table 2. The sequence and accession number of primers for evaluation of selected immune and antioxidant defence related genes expression in common carp skin Sequences of primers

Accession no

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Gene name

Forward: AGACATCAGGGTGTCATGGTTGGT b-actin

M24113.1

Reverse: CTCAAACATGATCTGTGTCAT Forward: ACCAGCTGGATTTGTCAGAAG il1b

AB010701.1

SC

Reverse: ACATACTGAATTGAACTTTG

Forward: GTCTTAGAGGACTGGGTGTA il8

AB470924.1

M AN U

Reverse: ACAGTGTGAGCTTGGAGGGA

Forward: GGTGATGGTGTCGAGGAGGAA tnf-alpha

AJ311800.1

Reverse: TGGAAAGACACCTGGCTGTA Forward: ACTCGTGCAGGTGTCTATGC gsr

HQ174244.1

Reverse: TTTGGAGTCTGCTTTGCCCT

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Forward: TACAATACTTTCACGCTTTCCC gsta

DQ411314.1

Reverse: GGCTCAACACCTCCTTCAC Forward: AGGAGAATGCCAAGAATG gpx

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Reverse: GGGAGACAAGCACAAGG

GQ376155.1

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Table 3. Growth performance, feed utilisation and survival rate of common carp fingerling fed different levels of dietary medlar (Mespilus germanica) leaves extract (MLE) after 7 weeks. MLE (%) 0.25

0.50

1.00

Initial weight (g)

8.30 ± 0.10

8.39 ± 0.05

8.29 ± 0.10

8.26 ± 0.07

Final weight (g)

15.12 ± 0.46 b

16.98 ± 1.02 a

17.16 ± 0.62 a

16.81 ± 0.57 a

WG (%)

82.16 ± 5.06 b

102.23 ± 5.42 a

107.10 ± 3.58 a

103.36 ± 5.36 a

SGR (% day-1)

1.07 ± 0.03 b

1.25 ± 0.06 a

1.29 ± 0.09 a

1.26 ± 0.04 a

FCR

2.30 ± 0.10 b

1.87 ± 0.44 a

1.84 ± 0.25 a

1.89 ± 0.14 a

100

100

100

SC

M AN U

Survival (%)

RI PT

0.00 (control)

100

Values are mean ± SE and in each row with different letters denote significant differences (P <

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0.05).

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Table 4. The total Ig (mg ml-1), lysozyme activity (U mg-1 protein) and protease activity (%) in

germanica) leaves extract (MLE) after 7 weeks.

MLE (%)

Total Ig

13.05 ± 0.49 b

15.10 ± 0.84 b

Lysozyme activity

13.20 ± 0.28 b

18.75 ± 0.35 a

Protease activity

0.730 ± 0.028 a

0.50

1.00

13.75 ± 1.06 b

18.05 ± 1.41 a

17.50 ± 0.70 a

14.35 ± 0.49 b

0.745 ± 0.200 a

0.720 ± 0.042 a

SC

0.25

M AN U

0.00 (control)

RI PT

the skin mucus of common carp (Cyprinus carpio) fed increasing levels of medlar (Mespilus

0.735 ± 0.021 a

Values are mean ± SE (n=9) and in each row with different letters denote significant differences

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(P < 0.05).

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3

A

B a

2.5

a

1.5 a 1

a a

2 1.5 a 1 0.5

0.5

0

0

Control

MLE 0.25% MLE 0.50% MLE 1.00%

6

a

C 4

M AN U

Fold increase

5

b

3 2 c

b

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1 0

MLE 0.25% MLE 0.50% MLE 1.00%

SC

Control

a

RI PT

Fold increase

a

2

Control

MLE 0.25% MLE 0.50% MLE 1.00%

EP

Figure 1. Fold change (mean ± SD, n=9) on the relative expression of tumor necrosis factor alpha (tnf-alpha) [A], interleukin 1 beta (il1b) [B] and Interleukin 8 (il8) [C] in the skin of common carp (Cyprinus carpio) after 49 days of being fed increasing levels of medlar leaf (Mespilus

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Fold increase

2.5

germanica) extract (MLE). The fold change was obtained by dividing each sampling value by the mean control value at the same sampling time. The bars assigned with different letter denote significant difference between treatments (P < 0.05).

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5

5

A

B 4

3.5

a

a

3

Fold increase

a

2.5 2 1.5

a 3 ab

b

2 c

b 1

1 0.5

0 Control

MLE 0.25%

MLE 0.50%

SC

0

Control

MLE 1.00%

5

Fold increase

4

M AN U

C

MLE 0.25%

MLE 0.50%

MLE 1.00%

a

a

3

2

b

b

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1

0

Control

MLE 0.25%

MLE 0.50%

MLE 1.00%

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Figure 2. Fold change (mean ± SD, n=9) on the relative expression of Glutathione reductase (gr) [A], Glutathione S-Transferase Alpha (gsta) [B] and Glutathione peroxidase (gpx) [C] genes in the liver of common carp (Cyprinus carpio) after 49 days of being fed with different levels of

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Fold increase

4

RI PT

4.5

medlar leaf (Mespilus germanica) extract (MLE). The fold change was obtained by dividing each sampling value by the mean control value at the same sampling time. The bars assigned with different letter denote significant difference between treatments (P < 0.05).

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Medlar leaf extract (MLE) improved growth performance in fingerlings MLE intake altered immune related genes expression in carp skin MLE intake elevated the expression of antioxidant enzymes gene expression in skin.

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