Dietary Polygonum minus extract ameliorated the growth performance, humoral immune parameters, immune-related gene expression and resistance against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss)

Journal Pre-proof Dietary Polygonum minus extract ameliorated the growth performance, humoral immune parameters, immune-related gene expression and resistance against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss)

Milad Adel, Mahmoud A.O. Dawood, Shafigh Shafiei, Fahimeh Sakhaie, Seyed Pejman Hosseini Shekarabi PII:

S0044-8486(19)32779-6

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734738

Reference:

AQUA 734738

To appear in:

aquaculture

Received date:

19 October 2019

Revised date:

10 November 2019

Accepted date:

12 November 2019

Please cite this article as: M. Adel, M.A.O. Dawood, S. Shafiei, et al., Dietary Polygonum minus extract ameliorated the growth performance, humoral immune parameters, immunerelated gene expression and resistance against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss), aquaculture (2019), https://doi.org/10.1016/ j.aquaculture.2019.734738

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© 2019 Published by Elsevier.

Journal Pre-proof Dietary Polygonum minus extract ameliorated the growth performance, humoral immune parameters, immune-related gene expression and resistance against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss)

Milad Adela, Mahmoud A.O. Dawoodb,*, Shafigh Shafieic, Fahimeh Sakhaied, Seyed Pejman Hosseini Shekarabie

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Department of Aquatic Animal Health and Diseases, Iranian Fisheries Science Research

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a

Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO),

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Tehran, Iran. b

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Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University,

Department of Health and Food Quality Control, Faculty of Veterinary Medicine, Shahrekord

University, Shahrekord, Iran.

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c

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Kafrelsheikh, Egypt.

School of Pharmacy, Shahid Beheshti University, Tehran, Iran.

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Department of Fisheries Science, Bandar-Abas Branch, Islamic Azad University, Bandar-

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Abas, Iran

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d

*Corresponding author's Email: [email protected]

Journal Pre-proof Abstract The aim of this study was to investigate the effects of Polygonum minus extracts on growth performance, skin mucus and serum immune parameters as well as immune-related gene expressions in rainbow trout. For this purpose, rainbow trout (mean weight: 50 ± 2.2 g) were fed experimental diets containing different levels 0 (control), 5, 10 and 15 mg/kg diet of P. minus for 8 weeks. At the end of the trial, the results showed that final body weight, weight gain and specific growth rate were significantly increased in fish fed diets containing P. minus

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(P<0.05). In addition, the feed conversion ratio of fish treated with P. minus was significantly decreased (P<0.05) compared to the control group. The blood RBCs, WBCs, Hb, total protein,

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albumin, and globulin showed a significant increase with decreased alkaline phosphatase

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(ALP) and alanine aminotransferase (ALT) in fish treated with P. minus compared to the

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control treatment. The blood lysozyme activity and total Ig showed significantly higher values in fish fed P. minus at 10 or 15 mg/kg than the other groups, while the respiratory burst activity

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increased significantly in fish fed P. minus at 15 mg/kg diet than the other groups (P<0.05).

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The skin mucus total protein and esterase increased significantly in fish fed P. minus at 15

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mg/kg diet than the other groups (P<0.05), while the skin mucus alkaline phosphatase, protease and lysozyme activity increased significantly in fish fed P. minus at 10 or 15 mg/kg diet than the other groups (P<0.05). The relative immune gene expressions (IL-1β, IL-8, and lysozyme) upregulated relatively in fish fed P. minus at 15 mg/kg diet than the other groups. The relative expression of TNF-α upregulated in fish fed P. minus at 10 or 15 mg/kg diet. The cumulative mortality of rainbow trout subjected to Yersinia ruckeri infectious exhibited relatively low mortality levels in all supplemented groups with the lowest being in fish fed 15 mg/kg P. minus. Highest percentage survival (%) rate was found in the 15 mg P. minus per kg diet. The results of this study confirm the beneficial effects of dietary P. minus on growth performance and immune parameters of rainbow trout.

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Keywords: Polygonum minus; Rainbow trout; Immunity; Skin mucus; Immune-related gene expression; Growth performance.

1. Introduction Aquaculture produced finfish and shellfish account for one-sixth of the animal protein people consume globally (FAO, 2018). The need to produce large quantities and in short times requires

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the use of intensive aquaculture techniques and substances capable of growth stimulation (Adel et al., 2016b; Yan et al., 2017; Yılmaz et al., 2018). However, intensive conditions are stressful

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for the species due to the high density of individuals and the occurrence of diseases (Dawood

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et al., 2019; El Megid et al., 2020). The growing concern about the use of chemicals, such as

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antibiotics in animal farming, has been prevented in many countries around the globe (Dawood, 2016; Van Hai, 2015). Thus, the application of natural alternatives is highly encouraged to

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reduce chemicals and to meet the critical issues of the intensive practices with the aim of

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improving the general health status of humans and animals (Ahmadifar et al., 2019a; Saleh et

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al., 2019; Van Doan et al., 2019c; Wang et al., 2019). The oral administration of substances, such as plant extracts, rich in bioactive molecules or phytochemicals ,capable of conferring to the farmed species the disease resistance and accelerating growth is a strategy that has attracted much attention in recent years (Awad et al., 2017). Several herbal plants are reported to have beneficial effects when administered to farmed fish. Their overall effects are indeed antioxidants,

anti-inflammatory,

immunostimulatory,

antimicrobials,

and

microbiota

regulators (Ahmadifar et al., 2019c; El-Deep et al., 2019). Dietary herbal products contain compounds with different chemical properties that produce immunostimulant effects .The levels of these compounds depend on different factors such as the plant age and geographical region, and extraction method (Hoseini et al., 2019) . Therefore,

Journal Pre-proof some researchers tried to use pure active ingredients of the plants, instead of their extracts. For example, dietary Polygonum minus was found to be effective medicinal plant with antioxidant and immunomodulatory effects (Ahmad et al., 2018; Christapher et al., 2015; Hassim et al., 2015). The supplementation of P. minus resulted in improving the growth performance, immune and antioxidant system of Anabas testudineus and African catfish (Panase et al., 2018; Veerasamy et al., 2014). Beside these studies that investigated immunomodulation of herbal products under normal conditions, some researchers showed beneficial effects of herbal

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products against infectious diseases in fish (Srichaiyo et al., 2020; Van Doan et al., 2019a; Van Doan et al., 2019b; Van Doan et al., 2019c).

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Rainbow trout (Oncorhynchus mykiss) remains one of the most commonly cultured fish species

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worldwide, due to their easy breeding, tolerance to varied environments and diseases, fast

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growth, and high market demand (Saeidi asl et al., 2017). However, it faces great challenges due to the infection of Yersinia ruckeri, threatens the health of fish by affecting cultured fishes,

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including rainbow trout (Adel et al., 2016a; Baba et al., 2018). Y. ruckeri has developed in the

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most damaging impediment to the expansion of the rainbow trout industry worldwide. Head-

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kidney organ can play an important role against infectious bacteria by the production of macrophages (Press et al., 1999; Yılmaz et al., 2018). Furthermore, it has a functional immuneendocrine interactions and even neuro-immuno-endocrine connections that may improve the immune response of fish during disease outbreaks (Yang et al., 2013). It has been reported that medicinal herbs and immunostimulants can activate the head-kidney cytokines in rainbow trout infected with Y. ruckeri (Altunoglu et al., 2017; Awad et al., 2011; Ji et al., 2017; Skov et al., 2012; Yılmaz et al., 2018). Some reports have been published on the use and effects of various dietary additives for rainbow trout (O. mykiss) exposed to Y. ruckeri pathogen. Among these additives, transcinnamic acid, olive leaf (Olea europea L.), Mentha piperita, pomegranate seed oil, Ducrosia

Journal Pre-proof anethifolia essential oil, rosehip (Rosa canina), macroalga Sargassum angustifolium hot water extract, Coriandrum sativum extract, and Panax ginseng extract are the most salient ones (Acar et al., 2018; Adel et al., 2016a; Baba et al., 2018; Bulfon et al., 2017; Dehghan et al., 2016; Farsani et al., 2019; Şahan et al., 2017; Yılmaz et al., 2018; Zeraatpisheh et al., 2018), with reference to resistance of rainbow trout against Y. ruckeri. However, different than the present study, none of these earlier reports presented the effects of P. minus extracts on the growth performances, humoral immune parameters, immune-related gene expressions and resistance

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against Y. ruckeri infection.

Therefore, the present study was established to evaluate the effects of using P. minus extracts

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on the growth performances, humoral immune parameters, immune-related gene expressions

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and resistance against Y. ruckeri infection in rainbow trout (O. mykiss).

2.1. Experimental Fish

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2. Materials and methods

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Healthy rainbow trout (n =480) had the mean weight of 50.0±2.2 g was obtained from a

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commercial fish farm at Mazandaran province, Iran. They were carefully transported to Caspian Sea Ecology Research Center using portable aerators and reasonable tanks and acclimatized for 2 weeks in fiber glass tanks (2000 L) provided with Spongy filters with the water flow rate of 100 liters h-1. Fish were distributed randomly into 12 fiber glass tanks, each with 20 fish before fed with four prepared diets for 8 weeks. The water quality was monitored daily at the temperature of 15.2±1.1°C; dissolved oxygen of 7.8 ± 0.5 mg l-1, pH of 7.4 ± 0.1 and electrical conductivity of 5231.7 ± 216.4 MM cm-1. The photoperiod was maintained of 14 h light and 10 h dark cycle. Fish were distributed randomly into 4 groups (n= 30×4= 120) and fed with commercial diets (Abzian diet, Mazandaran, Iran) for two weeks. The fishes were fed

Journal Pre-proof three times daily at a ratio of 3% of their body weight per day and partial water exchange was done daily to remove waste feed and fecal materials.

2.2. Preparations of extract and experimental diets P. minus plants were collected from natural habitat in Kerman province of central Iran and its identification was done according to standard methods by Shahrekord University Botany section. One kg of P. minus leaf was shade dried in a well aired semi dark room and then the

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dried plants were ground into fine powder using a grinder. Acquired powders were mixed in 1 L volumetric flask by 1:5 proportions with 80 % ethanol for 48 h by using a shaker. The extract

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was then filtered by Büchner funnel and filter paper. Primary extracts were distilled in rotary

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distillation unit at 37 °C for 4 h. The extracts were condensed and concentrated by using

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lyophilizer and stored at -20 ºC for further use. Components of the basal diet (Table 1) were mixed with the obtained P. minus extracts in an appropriate concentration, to get four different

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experimental diets: with 0 (control group, 0), 5, 10, 15 mg/kg of P. minus extracts (Veerasamy

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et al., 2014). The diets were made into pellets, allowed to dry and stored at 4 °C until use.

for 8 weeks.

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During the experimental period, fish groups were fed three times in a day at 3 % of body weight

2.3. Growth performance

All fish were deprived of food for 24 h before weighing and sampling, and the following parameters were measured at the end of feeding trial after eight weeks. Weight gain = W2 (g) - W1 (g) Specific growth rate (SGR) = 100 (Ln W2 - Ln W1)/T Feed conversion ratio (FCR) = feed intake (g) /weight gain (g) Survival rate (SR) = (final amount of fish/initial amount of fish)×100

Journal Pre-proof Where W1 is the initial weight, W2 is the final weight and T is the number of days in the feeding period (Tacon, 1990).

2.4. Blood sampling and hematological analysis At the end of the feeding trial, 8 fish were selected from each individual tank and anesthetized with clove oil (20 mg l-1). Blood samples were collected from the caudal vein of individual fish and immediately divided into two half parts. One half was transferred to a tube containing anti-

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coagulant (heparin) for studying the respiratory burst assay and make the hematological analysis, while the other half was transferred to non-heparinized tubes for biochemical and

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immunological studies. Sera samples were obtained by blood centrifugation (3500 X g, 15 min)

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and stored at -20 ºC for further analysis that conducted within 1 week.

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The total red blood cells (RBC: 106 mm-3) and white blood cells (WBC: 103 mm-3) were enumerated in an improved Neubaeur hemocytometer using Hayem and Turck diluting fluids

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(Blaxhall et al., 1973). Haematocrit (Ht %) was determined by the standard microhematocrit

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method and expressed as percentage. The haemoglobin (Hb, g dl-1) level was determined

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according to cyanomethemoglobin procedure. Furthermore, differential leukocyte cells were measured by preparing Giemsa stained smears. Blood smears were studied by light microcopy in order to make blood cell counts.

2.5. Biochemical analysis Glocoes, total protein, albumin and globulin level and three enzymatic activities of liver including; alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were determined in fish sera by using commercial kits (Pars Azmoon Company, Tehran, Iran) and a biochemical auto analyzer (Eurolyser, Belgium) (Adel et al., 2016a).

Journal Pre-proof 2.6. Mucosal immune parameters The mucus samples were scraped from the anterior to posterior direction on dorsal body surface using a sterile spatula following the method described by Balasubramanian et al. (2013). The collected mucus were thoroughly mixed with equal quantity of sterilized Tris buffered saline (TBS, 50 mM Tris HCl, pH 8.0, 150 mM NaCl) and centrifuged at 30,000 X g at 4 oC for 15 minutes (Beckman coulter, Avanti J-26 XPI, Brea, CA, USA). Supernatant was then collected

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-80 oC to avoid bacterial growth and degradation until used.

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and filtered with Whatman no.1 filter paper. The filtrate was then collected and kept frozen at

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2.7. The skin mucus protein levels (mg/ml) and enzyme activities determination

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The total protein concentration of mucus was measured according to the method that was

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described by Lowry et al. (1951). Alkaline phosphatase activity was estimated using Pars Azmoon kit (Tehran Company, Iran) and absorption was read at 405 nm by spectrophotometer

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(Sanchooli et al., 2012). Also, lysozyme activity in mucus of rainbow trout was determined

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using method that described by Ellis et al. (1990) with more modifications. Briefly, 50 μl of

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sera was added to 2 ml of a suspension of Micrococcus lysodeikticus (0.2 mg ml-1 in a 0.05 M sodium phosphate buffer (pH 6.2) and absorbance was measured at 450 nm after 0.5 min and 3 min by spectrophotometer (Biophotometer, Eppendorf). Finally, protease and esterase activity were determined using the methods that described by Sheikhzadeh et al. (2012).

2.8. Immunological activities in serum and blood leukocytes Serum Ig level was quantified by microprotein determination method (C-690; Sigma). Precipitation was carried out by 12 % polyethylene glycol solution and the difference in protein content before and after precipitation was considered as the total Ig level (Siwicki et al., 2000). The activity of serum lysozyme was measured similarly to the protocol used for skin mucus as

Journal Pre-proof described in section 2.7. Respiratory burst activity of blood leukocytes was measured by chemiluminescent assay (LUMI Skan Ascent T392, Finland) employing the strategy of Binaii et al. (2014). Finally, complement components (C3 and C4) were estimated by using commercial kits (Pars Azmoon Company, Tehran, Iran) by immuno turbidometry assay (Adel et al., 2016a).

2.9. RNA isolation and cDNA synthesis

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At the end of the feeding trial, 15 fish/group (5 fish from each tank) were selected and euthanized with clove oil (200 mg l-1). Then, the head kidney was obtained from selected fish

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for RNA isolation. RNA was extracted from homogenized tissue suspensions by GeneJET

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RNA Purification Kit based on the manufacturer's protocol (Sinaclon Co, Tehran, Iran). RNA

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quantification and RNA integrity analysis were quantified by using a Multiskan Go (Thermo Fisher Scientific, USA). Purified RNA was treated with RNase-free DNase I (Thermo Fisher

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Scientific, USA) and subjected to Real time PCR using specific pairs of primers to amplify IL-

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1β, IL-8, TNF-α and lysozyme. cDNA synthesis for each one of the samples 1μg of total RNA

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was reverse transcribed utilizing the Thermo ScientificTM Rivert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Incorporation, USA). Then, the cDNA was kept at -80 °C for next step.

2.10. Gene expression Primers of RT-qPCR used in this study were showed in Table 2. Real-time PCR was executed by using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, USA) based on the manufacturer's instructions and 1μg cDNA. Thermal cycling conditions: 94 °C for 10 min, followed by 40 cycles of 94°C for 30s, 62 (lysozyme, IL-1β, IL-8, TNF-α) for 30s and 72° C for 45s, and a final extension step of 72 °C for 5 min. The PCR and RNA product

Journal Pre-proof were inspected by electrophoresis using 1% w/v agarose gel electrophoresis. The results were evaluated in real time PCR (CFX Connect™ Real-Time PCR Detection System, USA) (Baba et al., 2018). Triplicate amplification reactions were carried out for each sample. The expression of the target genes was normalized using the reference gene (ef1α). The relative expression ratio of a target gene was calculated using the 2 -ΔΔCt method, where the mean value was obtained when normalized against the expression of the reference gene (Pfaffl, 2001).

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2.11. Challenge test

For the challenge test, a lyophilized stock of Y. ruckeri (KC291153) was obtained from the

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Faculty of Veterinary Medicine, Tehran University, Tehran, Iran and cultivated in tryptic soy

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broth (TSB, Merck, Darmstadt, Germany) at 25 oC for 48 h. At the end of the 8 week-feeding

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trial, fish from all treatments tank (10 fish from each tank in triplicate) were challenged by intraperitoneal (IP) injection with 0.1 mL of Y. ruckeri in 0.9 % (w/v) saline containing 1.5 x

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108 colony forming units (CFU)/ml. In the control group, instead, 0.1 ml of PBS was injected.

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All fish were fed with non-supplemented diet and kept under observation for 14 days. Finally,

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relative percent survival (RPS) values were calculated using the following formula described by Baba et al. (2018).

2.12. Statistical analysis

All the tests were performed in triplicate. The data were subjected to statistical analysis using the SPSS software version no. 21 (SPSS Inc., Chicago, IL, USA). The statistical analysis was done by using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests. P-value of <0.05 was considered significant. The survival of fish in each challenge treatment group was estimated using Kaplan-Meier analysis, whilst the differences amongst the groups were assessed using the log-rank (Mantel-Cox) test for pairwise comparisons.

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3. Results 3.1. Growth performance Table 3 showed significant enhancement in the final body weight (FBW), weight gain (WG) and specific growth rate (SGR) of rainbow trout fed P. minus at 15 mg/kg diet (P < 0.05). On the other hand, P. minus significantly decreased the feed conversion ratio (FCR) 15 mg/kg diet (P < 0.05). However, no significant differences were observed on WG, SGR, and FCR in fish

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fed the control diet and 5 or 10 mg/kg diet (P > 0.05).

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3.2. Blood hematology and biochemistry

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Feeding rainbow trout fingerlings with P. minus incorporated diet significantly (P < 0.05)

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increased the WBCs and Hb in fish fed 15 mg/kg with insignificant (P > 0.05) difference with fish fed 10 mg/kg (Table 4). Similarly, fish fed P. minus at 10 or 15 mg/kg diet showed

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significantly higher RBCs and HCT than the other groups (P < 0.05).

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ALP and ALT of fish fed P. minus at 10 or 15 mg/kg diet showed significantly lower values

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than the other groups (P < 0.05) (Table 5). The blood total protein was also increased significantly in fish fed P. minus at all levels than the control group (P < 0.05) (Table 5). The blood albumin and globulin increased significantly in fish fed P. minus at 15 mg/kg diet than the other groups (P < 0.05) (Table 5).

3.3. Blood and skin mucus immunity The blood lysozyme activity and total Ig showed significantly higher values in fish fed P. minus at 10 or 15 mg/kg than the other groups (Table 6). While the respiratory burst activity increased significantly in fish fed P. minus at 15 mg/kg diet than the other groups (P < 0.05) (Table 6).

Journal Pre-proof The skin mucus total protein and esterase increased significantly in fish fed P. minus at 15 mg/kg diet than the other groups (P < 0.05) (Table 7). While the skin mucus alkaline phosphatase, protease and lysozyme activity increased significantly in fish fed P. minus at 10 or 15 mg/kg diet than the other groups (P < 0.05) (Table 7). The highest blood and skin mucus immunity variables were observed in fish fed 15 mg/kg.

3.4. Immune related gene expressions

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The relative immune gene expressions (IL-1β, IL-8, and lysozyme) were relatively upregulated in fish fed P. minus at 15 mg/kg diet (Figures 1, 2 and 3). The relative expression of TNF-α

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was relatively upregulated in fish fed P. minus at 10 or 15 mg/kg diet (Figure 4).

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3.5. The challenge against Y. ruckeri infection

Figure 5 showed that fish mortalities began day 2 post challenge in fish fed the control or P.

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minus at 5 mg/kg. The cumulative mortality rate 14 days post challenge were the lowest in fish

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fed P. minus at 15 mg/kg (30.31 %), while the highest one was recorded in the control group

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(60.33 %). The highest survival rates and RPS were found in fish fed P. minus at 5, 10 or 15 mg/kg compared to the control with highest being in fish fed P. minus at 15 mg/kg (P < 0.05) (Table 4).

4. Discussion In this study, we determined the effect of P. minus extract supplementation of rainbow trout fingerling diet on growth performance, hematological parameters and immune response (blood, mucus and immune-related genes). The use of plant extracts as growth promoters, immunity stimulators and antioxidants in fish farming has been long known (El-Deep et al., 2019; Saleh

Journal Pre-proof et al., 2019). More recently, their use has been applied to aquaculture activity with positive effects on fish health (Amin et al., 2019; Moustafa et al., 2019; Van Hai, 2015). In our study, the supplementation with P. minus extract, significantly increased final body weight, weight gain, specific growth rate (SGR), and decreased feed conversion ratio (FCR). It has been demonstrated that the medicinal plants can act as growth promoters by boosting the digestive enzymes which in turn can increase the growth rates and survival of aquatic animals (Faggio et al., 2015; Van Hai, 2015). Growth performance and feed conversion ratio are

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essential parameters need to judge the potential use of feed additives in aquafeed. P. minus is rich with many essential elements including, crude protein, essential amino acids, minerals,

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vitamins antioxidant and flavonoids which are vital for the growth and feed utilization in

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animals (Ahmad et al., 2018; Christapher et al., 2015; Hassim et al., 2015). Likewise, Panase

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et al. (2018) indicated that P. minus improved the feed efficiency and weight gain of A. testudineus. In line with our results, Ghasemi Pirbalouti et al. (2011) demonstrated that herbal

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plants had a beneficial effect on rainbow trout growth performance. One of the most critical

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aspects of medicinal herbs administration is related to the concentration used. In general,

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indications on the best concentration of medicinal herbs usually come from in vivo experiments, based on “trial and error” approach. In the current study, supplementation of P. minus extract at 15 mg per kg diet presented the highest growth performance among the tested groups.

Hematological parameters are simple but important tools to determine the health status of the fish (Burgos-Aceves et al., 2019; Dawood et al., 2015; Faggio et al., 2014; Fazio et al., 2013). Plant extracts have been reported to positively affect hematological parameters in fish (Adel et al., 2016b; Saeidi asl et al., 2017; Van Doan et al., 2019d; Yılmaz et al., 2018). In the current study, RBCs, WBCs, Hb and HCT were significantly increased by P. minus extract feeding in rainbow trout. Similarly, in the A. testudineus fed P. minus showed improved WBCs (Panase

Journal Pre-proof et al., 2018). In a study carried out in African catfish fed diets enriched with P. minus, there was no statistically significant difference between the RBCs, WBCs, Hb and HCT in all experimental groups. The observed changes in haematological parameters might be because of relatively long-term influence of the P. minus supplementation. The increased RBC, therefore, intensifies the concentration of Hb and eventually leads to high oxygen-carrying capacity in fish. Such fishes, hence, may be more capable of supplying oxygen to tissues in situations where oxygen is highly required. The emerging picture is compatible to a safe administration

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of such herbs also sustained by the significant increase in the immunity in the rainbow trout fed with P. minus enriched diets with respect to the control group.

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The blood aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are

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responsible for the catalysis of interconversion of non-essential amino acids such as alanine,

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aspartate and glutamate (Racicot et al., 1975). Therefore, higher activity of these enzymes implies an enhanced amino acid interconversion for protein synthesis or elevated levels of

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catabolism to meet increased energy demand during stress or pathogen invasion. In present

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study we found that AST and ALT activities decreased with the increasing inclusion level of

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P. minus extract showed lowest ALT and AST activities in fish fed P. minus at 10 and 15 mg/kg. Thus, lower transaminase activities could be a consequence of improved liver condition, due to functional substances in the P. minus extract. P. minus has been found to possess immunomodulatory properties, which have been attributed to the monomer polyphenolic compounds which may help in improving the health status of the organism (Ahmad et al., 2018; Panase et al., 2018; Veerasamy et al., 2014). The findings of the present study also showed the potential of P. minus to be used as a dietary phyto immunostimulant for rainbow trout. Fish received diets supplemented with P. minus elicited considerable modulated immune responses including remarkable serum and skin mucus immune responses. Lysozyme activity, plasma and mucus total proteins and immunoglobulins

Journal Pre-proof are suitable indicators of fish health and among the most frequently tested immune parameters in herbal supplemented diets in fish (Harikrishnan et al., 2011). The results presented here are in line with the literature studies and show that rainbow trout fingerlings treated with P. minus enriched diets experience an increase in mucus lysozyme activity, total proteins and total Ig, at any level of supplementation, with respect to the controls, confirming the suitability of such indicators. Fish defense system, in which the integument provides the first line of defense, is rather

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complicated. As Ángeles Esteban (2012) points out skin mucus and its components have a significant effect as hindering various sorts of pathogens and exo-parasites. Meanwhile, skin

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mucus is reportedly affected by environmental conditions, nutrition and feed additives (Lazado

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et al., 2014). The results of this study for rainbow trout skin mucus showed a rise in non-

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specific immune parameters (total Ig, lysozyme and protease activity) which are dose dependent. In fact, fish fed P. minus demonstrated the highest level of the measured parameters.

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Lysozyme, as one constituent of the innate immunity system, contributes to non-specific

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immunity of animal by their anti-inflammatory and bactericidal properties (Ángeles Esteban,

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2012). Moreover, the higher lysozyme activity correlated with more anti-bacterial activity versus gram-positive and gram-negative bacteria. The obtained results exhibited a significant increase in levels of skin mucus lysozyme in fish fed P. minus. As protective function, skin mucus proteases have a pivotal role in hindering the pathogenic invasion both by hindering their invasion mechanisms and colonization and cleaving their proteins (Subramanian et al., 2007). Dietary containing P. minus could significantly increase skin mucus proteases activity and the highest values were recorded at inclusion level of 15 mg/kg P. minus. This impact could be regarded as helpful for the protective role of skin mucus. In fact, it should be noted that protective role of skin mucus is very useful, since they influence the production of complement,

Journal Pre-proof immunoglobulins or antibacterial peptides which existed in fish mucus as innate immune components (Hjelmeland et al., 1983; Kennedy et al., 2009). Beneficial effects of P. minus inclusion on lysozyme activity, alternative complement pathway and total IgM were revealed by evaluation of serum immune parameters. A similar study on African catfish revealed increased lysozyme activity by P. minus feeding (Veerasamy et al., 2014). However, no study was found on the effect of P. minus on mucus immune response of fish. The main difference between these natural additives and vaccines is that the former kill or

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eliminate nonspecific pathogens, whereas the latter kill or eradicate specific ones (Ahmadifar et al., 2019b; Amin et al., 2019; Dawood et al., 2018; Wang et al., 2019). Protection against

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pathogen challenge is dependent on many factors, including injected pathogen concentration,

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fish immunological status with body weight, aquatic environmental parameters, and the types

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of additives ingested. Interestingly, our results showed increased resistance against Y. ruckeri

diets.

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infection, which might be related to the immunomodulatory role of P. minus in rainbow trout

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The use of herbal medicine for improving the immune status of fish takes a great interest from

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researchers to prove its effectiveness in different fish species (Aragona et al., 2018; Awad et al., 2017; Hoseinifar et al., 2018). But little is known about the role of these herbs in alleviating the damaging effect of pathogenic bacterial infection at either genes or cell levels. In the present work, the inclusion of P. minus extract in rainbow trout feed relatively up-regulated IL-1β, IL8, TNF-α, and lysozyme genes expression. At the same respect, dietary ginger relatively upregulated lysozyme gene expression in zebra fish (Ahmadifar et al., 2019a). In the present study, the serum lysozyme activity significantly increased in P. minus fed groups compared to the control. In a previous study, Lie (1989) linked the increase in the lysozyme activity in fish spleen and kidney to the abundance of cells related to the immune system, such as monocytes, macrophages and polymorphonucleated granulocytes; which are the main source for such

Journal Pre-proof protiolytic enzyme. Besides, based on the study of Engstad et al. (1992), the increase in lysozyme in the blood of stimulated fish is associated either with the proliferating phagocytes or the increased the amounts of lysozymes produced from the lysosomes, rendering lysozyme activity to be one of the best markers for evaluating the bactericidal effect of the feed additives. The increase in the lysozyme activity in our work could be due to the flavonoids which stimulates leucocytes and phagocytosis (Awad et al., 2017). Fish diseases and infections are emerging issues in the intensive aquaculture industry (Awad

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et al., 2017; Dawood et al., 2016). The enhancement of the immune response of fish farmed under intensive conditions seems to be an advantageous strategy to control the spreading of

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diseases and infections and the consequent mortality (Mehana et al., 2015). In a recent study,

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polyphenols extracted from chestnut (Castanea sativa) shell showed immunomodulatory

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activity on rainbow trout leukocytes (Coccia et al., 2019), with an overall effect of polyphenols on pro- and anti-inflammatory cytokines that seemed to suggest an alert effect of the immune

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system against a possible infectious agent. Labeo rohita fingerlings fed tannin containing diets

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showed a significant increase in immunological parameters, including the total leukocyte count

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and respiratory burst activity compared with fish fed a control diet (Prusty et al., 2007), confirming that tannins have an immunostimulant effect on fish. The larval stage is a very critical phase in aquaculture, when the application of immunostimulants could be particularly effective.

The present study showed that P. minus fed fish exhibited tolerance to Y. ruckeri challenge which resulted in a significant reduced mortality compared to the infected control group. It has been reported that the addition of Ducrosia anethifolia essence oil to rainbow trout feed slightly increases survival rate of fish against Y. ruckeri (Dehghan et al., 2016). Similarly, in rainbow trout fed diets with trans-cinnamic acid increased survival rate against Y. ruckeri (Yılmaz et al., 2018). It was claimed by Adel et al. (2016a) and Baba et al. (2018) that Y. ruckeri

Journal Pre-proof pathogenesis is highly correlated with the immune response, from that concept the protective effect of P. minus may be associated with richness in flavonoids and polyphenol in addition to the high concentrations of ascorbic acids which neutralize the excessive free radicals and contribute to enhancing the immune system (Ahmad et al., 2018; Christapher et al., 2015; Hassim et al., 2015). It is worth to note that, P. minus was efficient in the treatment of various human and animal diseases (Ahmad et al., 2018; Christapher et al., 2015; Hassim et al., 2015).

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5. Conclusion

In conclusion, P. minus extracts, administered in a feeding trial of 8 weeks, show the efficacy

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of combining P. minus with different levels to positively affect growth performances, immune

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and hematological parameters as well as immune related gene expression of the rainbow trout.

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P. minus extracts can be regarded as a novel strategy for the benefit of fish tolerance against Y.

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Sargassum angustifolium hot water extract on hematological parameters and immune responses in rainbow trout (Oncohrynchus mykiss) infected with Yersinia rukeri. 30,

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rn

al

Pr

e-

pr

2029-2037.

Journal Pre-proof

Relative expression of IL-8

3 2.5 2 1.5

1 0.5 0

f

5 mg/kg extract 10 mg/kg extract 15 mg/kg extract

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Control

Figure 1. Relative expression of IL-8 in rainbow trout fed diets enriched with different levels

e-

pr

of Polygonum minus extract for 8 weeks.

Pr

2

rn

al

1.5 1 0.5 0

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Relative expression of IL-1β

2.5

Control

5 mg/kg extract 10 mg/kg extract 15 mg/kg extract

Figure 2. Relative expression of IL-1β in rainbow trout fed diets enriched with different levels of Polygonum minus extract for 8 weeks.

Relative expression of lysozyme

Journal Pre-proof 1.8 1.6 1.4 1.2

1 0.8 0.6 0.4 0.2 0

f

5 mg/kg extract 10 mg/kg extract 15 mg/kg extract

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Control

Figure 3. Relative expression of lysozyme in rainbow trout fed diets enriched with different

pr

levels of Polygonum minus extract for 8 weeks.

e-

1.6

Pr

1.4 1.2

al

1 0.8

rn

0.6

0.4 0.2 0

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Relative expression of TNF-α

1.8

Control

5 mg/kg extract 10 mg/kg extract 15 mg/kg extract

Figure 4. Relative expression of TNF-α in rainbow trout fed diets enriched with different levels of Polygonum minus extract for 8 weeks.

Journal Pre-proof

0 mg/kg

5 mg/kg

10 mg/kg

15 mg/kg

100 90

Survival rate (%)

80 70

60 50 40 30

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f

20 10 0

3

4

5

6 7 8 9 10 Days post challenge

pr

2

11

12

13

14

15

e-

1

Figure 5. Kaplan–Meier survivorship curves (cumulative survival [%] over time [Days 1 to

Pr

15]) for rainbow trout after challenge with Yersinia ruckeri; the fish were fed with Polygonum minus extract supplemented diets (0, 5, 10 or 15 mg P. minus per kg feed) prior to bacterial

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rn

al

challenge.

Journal Pre-proof Table 1. Proximate composition of the basal diet of rainbow trout used in this study. (%)

Crude protein

44.5

Crude lipid

12.9

Ash

9.8

Moisture

4.76

NFEa

15.3

Nitrogen-free extracts (NFE) ¼ dry matter - (crude protein +crude lipid+ ash fibre).

Table 2: Primers of RT-qPCR used in this study

pr

oo

f

a

Proximate composition

Gene

Accession number

Pr

forward/reverse

e-

qPCR primers, Amplicon(bp)

F: ACATTGCCAACCTCATCATCG

IL-1

al

R: TTGAGCAGGTCCTTGTCCTTG

AJ223954

F: AGAATGTCAGCCAGCCTTGT

rn

IL-8

R: TCTCAGACTCATCCCCTCAGT F: GGAGGGGTATGCGATGACACCTG

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TNF-α

AJ279069

Lysozyme

R: GAGGCCTTTCTCTCAGCGACAGC F: GTGTCTGATGTGGCTGTGCT R: TTCCCCAGGTATCCCATGAT

AJ249755.1 AB027305

Table 3. Growth performance of rainbow trout fed with diets supplemented with different levels Polygonum minus (PM) extract for 8 weeks. Index

Control

PM5

PM10

PM15

Journal Pre-proof IBW

50.2±2.0

50.3±2.1

50.5±2.2

50.5±1.9

FBW

93.6±1.4a

95.8±1.0a

94.8±1.6a

97.2±1.1b

WG (g)

43.4±3.2a

45.5±5.6a

44.3±5.4a

46.7±3.9b

SGR

1.48±0.08a

1.51±0.13a 1.49±0.10a 1.53±0.15b

FCR

1.52±0.04b

1.47±0.05b 1.45±0.07b 1.41±0.03a

SR (%)

92.5±1.5

93.00±1.5

96.5±1.75

97.5±1.5

Data are presented as mean ± S.D. Data in the same row with different superscript are significantly different (P<0.05). IBW, initial body weight; FBW, final body weight; WG,

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weight gain; SGR, specific growth rate; FCR, fed conversion ratio; SR, Survival rate.

Table 4. Hematological indices of rainbow trout fed diets enriched with different levels of

Parameter

Control

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Polygonum minus (PM) extract for 8 weeks PM5

PM10

PM15

1.29±0.02a 1.32±0.02a 1.46±0.03ab 1.77±0.04b

WBC (103/ mm3)

12.7±0.1a

12.8±0.6a

13.0±0.7b

13.7±0.5b

HCT (%)

30.8±0.9a

31.6±0.7a

32.1±0.6 b

33.9±0.5b

Hb (g dL-1)

8.68±0.12a 8.74±0.10a 8.86±0.15ab 9.47±0.13b

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RBC (106/ mm3)

Lymphocytes (%)

79.7±1.5

79.1±1.7

77.4±1.6

75.9±1.5

Neutrophils (%)

16.9±0.6

17.38±1.2

18.3±1.1

20.1±1.5

2.54±0.10

2.91±0.10

3.62±0.18

3.34±0.09

0.68±0.03

0.58±0.05

0.61±0.04

0.66±0.02

Monocyte (%) Eosinophils (%)

Data are presented as mean ± S.D. Data in the same row with different superscript are significantly different (P<0.05). RBC, red blood cells; WBC, white blood cells; HCT, hematocrit; Hb, hemoglobin concentration. Table 5. Biochemical parameters of rainbow trout after feeding with different levels of Polygonum minus (PM) extract incorporated diet. Parameter

Control

PM5

PM10

PM15

ALP (IU dl-1)

160.1 ± 12.2b

157.37 ± 16.4b

158.6 ± 9.6a

152.3 ± 16.1a

ALT (IU dl-1)

47.5 ± 3.1b

48.2± 1.7b

45.6 ± 2.3a

44.8± 4.1a

Journal Pre-proof Glucose (mg dl-1)

124.4 ± 2.4

123.0 ± 8.5

127.2 ± 4.3

132.6± 5.1

Total protein (g dl-1)

2.69 ± 0.3a

2.80 ± 0.1b

2.79 ± 0.2b

3.12 ± 0.1b

Albumin (g dl-1)

1.22 ± 0.1a

1.24 ± 0.2a

1.28 ± 0.1a

1.41 ± 0.1b

Globulin (g dl-1)

1.47 ± 0.03a

1.56 ± 0.02a

1.51 ± 0.05a

1.71 ± 0.04b

Cholesterol (mg dl-1)

345.9 ± 6.3

331.7 ± 5.0

329.4 ± 4.7

322.5 ± 5.3

Data are presented as mean ± S.D. Data in the same row with different superscript are

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significantly different (P<0.05).

of Polygonum minus (PM) extract for 8 weeks.

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Table 6. Immune parameters in sera of rainbow trout fed diets enriched with different levels

Control

Lysozyme activity (µg/mg)

25.7±1.5a

IgM (mg/dl)

PM10

PM15

27.5±1.6a

30.0±1.0b

34.6±1.2c

17.1±0.7a

18.8±0.8a

22.5±1.1b

23.2±1.0c

Respiratory burst activity (RLU/S) 642.1±54a

687.2±48a

753.1±68ab

912.5±37b

C3 (mg/dl)

15.2±1.7

17.8±1.0

19.6±1.3

21.8±0.9

22.6±2.2

22.3±1.6

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14.5±0.5

C4 (mg/dl)

PM5

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Parameter

21.4±1.2

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Data are presented as mean ± S.D. (n=8). Values in each row with different superscripts shows

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significant difference (P < 0.05).

Table 7. Total protein level and enzyme activities in mucus of rainbow trout fed diet containing different levels of Polygonum minus (PM) extract. Parameter

Control

PM5

PM10

PM15

The protein levels (mg/ml)

1.06±0.09a

1.15±0.1a

1.34±0.18ab

1.46±0.20b

Alkaline phosphatase (IU/l)

7.76±0.7a

7.92±0.5ab

8.15±0.8b

8.32±1.0b

Lysozyme (IU/mg)

37.85±1.2a

38.97±0.62a

41.05±0.71b

47.59±2.1b

Protease (IU/mg)

21.38±1.0a

21.59±0.5a

22.43±0.8b

22.01±0.5b

Esterase (IU/mg)

1.68±0.12a

1.75±0.11a

1.89±0.13ab

2.16±0.18b

Journal Pre-proof Data presented as mean ± S.D. of individual fish (n =5). Values in a row with different superscripts show significant difference (P< 0.05).

Table 8. Mortality rate, survival, and relative percentage survival (RPS) of infected rainbow

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trout fed with Polygonum minus (PM) extract at different levels. Survival rate (%)

RPS

Control

60.33

39.67a

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PM5

51.21

48.79b

PM10

42.66

57.34b

PM15

30.31

69.69c

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Mortality rate (%)

84.88

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70.71 50.24

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Data presented as mean ± S.D. Values in a column with different superscripts show significant

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difference (P< 0.05).

Journal Pre-proof Highlights 

Dietary Polygonum minus extracts improved growth performance and feeding efficiency in rainbow trout



The skin mucus immune responses were increased in P. minus extracts fed rainbow trout Serum immune parameters affected by feeding with dietary P. minus



Dietary P. minus upregulated the relative expressions of IL-1β, IL-8, TNF-α, and lysozyme gene expressions in rainbow trout

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Dietary P. minus increased the resistance of rainbow trout against Yersinia ruckeri

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infection

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

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

Journal Pre-proof Conflicts of interest

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The authors declare no conflicts of interest.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5