Dietary mycotoxicosis prevention with modified zeolite (Clinoptilolite) feed additive in Nile tilapia (Oreochromis niloticus)

Journal Pre-proof Dietary mycotoxicosis prevention with modified zeolite (Clinoptilolite) feed additive in Nile tilapia (Oreochromis niloticus) Eman Zahran, Engy Risha, Mohamed Hamed, Tarek Ibrahim, Dušan Palić PII:

S0044-8486(19)31229-3

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734562

Reference:

AQUA 734562

To appear in:

Aquaculture

Received Date: 19 May 2019 Revised Date:

18 July 2019

Accepted Date: 1 October 2019

Please cite this article as: Zahran, E., Risha, E., Hamed, M., Ibrahim, T., Palić, Duš., Dietary mycotoxicosis prevention with modified zeolite (Clinoptilolite) feed additive in Nile tilapia (Oreochromis niloticus), Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734562. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.

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Dietary Mycotoxicosis Prevention with Modified Zeolite (Clinoptilolite) Feed Additive in

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Nile tilapia (Oreochromis niloticus)

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Eman Zahran1*, Engy Risha2, Mohamed Hamed3, Tarek Ibrahim4, and Dušan Palić5

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Department of Internal Medicine, Infectious and Fish Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt,

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Clinical Pathology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt,

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Pathology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt,

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Department of Nutrition and Nutritional Deficiency Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt,

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Chair for Fish Diseases and Fisheries Biology, Faculty of Veterinary Medicine, Ludwig-Maximilians-University Munich, Munich, Germany

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*Corresponding author: Tel: +201211100560

Fax: +20502200696

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E-mail address: [email protected]

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Department of Internal Medicine, Infectious and Fish Diseases, Faculty of Veterinary Medicine,

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Mansoura University, Mansoura 35516, Egypt

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Abstract

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Consumption of feed ingredients with a naturally low level of total aflatoxins

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contamination and possible consequences on fish health and seafood quality is a growing

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concern. To investigate effects of such dietary exposure on fish health parameters, Nile tilapia

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(Oreochromis niloticus) were fed basal diet (control), and diet naturally contaminated with a mix

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of aflatoxins (AFs; AFB1and 2, AFG1 and 2) at level of 16 µg/kg (AFs group). To investigate

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safety and efficacy of surface-modified clinoptilolite adsorber as part of both polar and non-polar

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mycotoxin control and prevention strategies, we supplemented control and aflatoxin

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contaminated diets with Minazel-plus® at level of 2 g/kg (MZ, AFsMZ, respectively) during 8

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weeks. Effects of AFs and the protective role of Minazel-plus® on fish health were evaluated

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using growth performance and hepatosomatic index (HSI), hematological parameters, innate

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immune and antioxidant responses, bioaccumulation of mycotoxins in liver and musculature, and

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histopathological assessment of liver and kidney tissues. Significant differences in production

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and health parameters were observed between AF vs. Control, and MZ supplemented groups,

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including a decrease of red blood cells/RBCs, hemoglobin/Hb, and blood packed cell volume

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(PCV), changes in dynamics of leukocyte counts (specifically neutrophils), and decrease in

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serum lysozyme and bactericidal activity. Further, the increase in malondialdehyde (MDA), and

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decrease in catalase (CAT), reduced glutathione (GSH) and superoxide dismutase (SOD) activity

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were observed in AFs group compared to other groups. Aflatoxin residues were detected in both

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liver and musculature at 1.292 and 0.263 µg/kg, respectively. Supplementation of Minazel-plus®

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decreased AFs residues to 0.9 (liver) and 0.022 (muscle) µg/kg. Histopathology showed marked

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changes in liver and kidney of fish from AFs group. Reported results strongly suggest that feed-

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mill monitoring strategies for aflatoxin levels in feed ingredients need to be implemented and

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strictly enforced especially due to possible illegal use of highly contaminated feed, as well as

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inadequate or missing regulations of the safety level of aflatoxins in fish feed.

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Keywords: Fish, Minazel-Plus®, Aflatoxicosis, Immune response, Oxidative damage

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1. Introduction

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Fungal contamination of feed ingredients is a significant entry point for mycotoxins to

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increase hazard to animals and human beings. Mycotoxicoses pose an increasing hazard to

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animal and human health with high potential to cause significant economic losses in food and

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feed industry (Patriarca & Pinto 2017). Contamination of crops with mycotoxins depends on

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physical and chemical factors affecting mycotoxin production and accumulation (Pitt 2008).

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Mycotoxins are secondary metabolite compounds that persist throughout the dietary chain due to

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their resistance to a wide range of environmental factors or process treatments (Patriarca & Pinto

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2017). Developing countries and rural areas depending on local food production are in a higher

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risk of mycotoxin contamination problems due to inadequate or missing implementation of food

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security and quality control measures. Use of mycotoxin contaminated feeds frequently occurs

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due to increased demand for less expensive feed ingredients to satisfy growing animal industry

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needs, including aquaculture (Marroquín-Cardona et al. 2014).

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Aflatoxins (AFs/ AFB1, AFB2, AFG1, and AFG2) and their metabolites are potent liver

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carcinogens. They are produced by Aspergillus flavus and A. parasiticus fungi under favorable

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environmental conditions. AFs have been reported to cause a variety of deleterious effects

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including growth reduction, hepatotoxicity, nephrotoxicity, carcinogenicity, mutagenicity,

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teratogenicity, and cytotoxicity (Santacroce et al. 2008, Han et al. 2010, Bbosa et al. 2013). AFs

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are found in various agriculture commodities, corn and peanut being most frequently

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contaminated and considered to have a crucial role in the development of mycotoxicosis in both

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human beings and animals (including fish) (de Oliveira & Corassin 2014). Presence of

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mycotoxins in pelleted/formulated fish feed can occur due to the use of contaminated feed

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ingredients, cross-contamination with other contaminated feeds, or when the growth of toxigenic

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fungi and production of mycotoxins is occurring under poor storage conditions such as high

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temperature and humidity (de Oliveira & Corassin 2014).

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In recent years small and medium-scale aquaculture has become a vital source of animal

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protein, as well as significant socio-economic activity in both rural and urban areas of the low

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and medium income countries. Egypt has emerged as the world’s 2nd largest Nile tilapia

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(Oreochromis niloticus) producer and is currently contributing to over 80% of total farmed

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tilapia production in Africa (Cai et al. 2017). Tilapia diet is based on predominantly plant-based

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feed ingredients, and multiple surveys have found that such ingredients frequently suffer from

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significant mycotoxin contamination (Pietsch et al. 2013). Therefore, tilapia (and other cultured

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fish) may be in a higher risk of exposure to mycotoxin contaminated feed, leading to higher

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economic losses and increased potential for bioaccumulation and biomagnification of

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mycotoxins in fish destined for human consumption (Leung et al. 2006, Pietsch et al. 2013).

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The maximum allowable total aflatoxin concentration in most feed ingredients and non-

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ruminant feeds according to the U.S. Food and Drug Administration regulations is 20 µg/kg

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(FDA, 1989), of which maximum allowable concentration of AFB1 is 5 µg/kg (Van Eijkeren et

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al. 2006). The maximum allowable concentration of AFB1 in human food is 2 µg/kg under the

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European Commission (EC) Directive No 1881/2006 (EC 2006). While the FDA and EU

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regulations also include aquatic animal feeds, some countries have no regulations or standards

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for determination of maximum aflatoxin levels in fish feed (Han et al. 2010).

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Natural zeolites were studied extensively to prevent and alleviate mycotoxicosis, but recent

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breakthroughs and use of 3rd generation of the modified mycotoxin adsorbents such as

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Minazel-Plus® have brought in technological advancements to further improve adsorption

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characteristics of clinoptilolite mycotoxin binders. This patented technology involves

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modification of clinoptilolite surface with organic molecules and changing its properties to

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increase modified product ability to bind not only polar, but also non-polar organic molecules

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(e.g. T-2 toxin) while maintaining all physico-chemical characteristics of the original material

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(Nešić et al. 2009). Such modified clinoptilolite materials have been used as feed additives to

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prevent mycotoxicosis in terrestrial animal production (Nedeljković-Trailović & Petrujkic 2013,

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Nedeljković-Trailović et al. 2013, Nedeljković-Trailović et al. 2015), but its use has not been

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evaluated in aquatic animals.

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Overall, use of mycotoxin binders in aquaculture to prevent and control health

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consequences of mycotoxicosis is limited, and the safety and efficacy of various marketed

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products are not fully understood (Selim et al. 2014). For example, majority of studies

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investigating mycotoxin effects were focused on higher exposure levels (Tuan et al. 2002, Deng

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et al. 2010, Huang et al. 2011, Selim et al. 2014). However, information about the effects of low

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level mycotoxin exposures, including synergistic effects of feed contaminated with multiple

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mycotoxins at concentrations below regulatory or industry concentration thresholds is very

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limited (Santos et al. 2010).

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The objective of this study was to evaluate effects of lower level aflatoxins mixture

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exposure on fish health and production parameters including growth performance;

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hematological, innate immune, and oxidative stress responses, quantification of aflatoxin

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residues in muscle and liver, and histopathological analysis. Clinoptilolite modified using

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modern technology (Minazel-Plus®) has been evaluated for its potential to prevent effects of low

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level dietary mycotoxin exposure in Nile tilapia.

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2. Materials and methods:

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2. 1. Diet preparation:

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Two batches of mycotoxin contaminated whole corn and soybean meal were obtained from

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a Feed Mill in Dakahlia province, Egypt and ground to a fine, homogeneous consistency

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using a plant grinder. Corn and soybean meal samples were analysed and were found to

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contain 32.8 µg/kg of total aflatoxins (AFs) (0.026 µM, corn as AFB1/16.8, B2/0.6, G1/14.6,

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and G2/0.8 µg/kg) and 23.35 µg/kg of total AFs (0.018 µM, soybean as AFB1/1.05, B2/0.2,

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G1/21.3, and G2/0.8 µg/kg) of total aflatoxins (AFs/ AFB1, B2, G1, and G2). The Minazel-

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plus® is a commercial product kindly provided from Patent Co., Serbia. It is a chemical

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modification of Zeolite (clinoptilolite E567/568; Minazel-Plus®) surface with the addition of

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organic cations with high selective adsorption of both polar and non-polar mycotoxins in the

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contaminated feed (European Patent No. 163854).

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A basal diet was formulated based on wheat, soybean meal, corn, fish oil, and a premix

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with vitamins, minerals amino acids, salt, and monocalcium phosphate (Table 1). Nutrient

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concentrations were formulated to meet requirements for Nile tilapia (National Research

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Council 1993). The control diet was prepared with corn and soybean meal without detectable

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aflatoxin contamination. The mycotoxin contaminated diet (AFs) was prepared by replacing

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uncontaminated control corn and soybean meal with AFs contaminated ones (AFs level 16

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µg/kg, 0.013 µM). A control diet mixed with 2 g/kg diet (9.5 µM) of Minazel-Plus® (MZ) and

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contaminated AFs diet mixed with 2 g/kg diet (9.5 µM) of Minazel-Plus® (AFsMZ) were also

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prepared as above. After preparing the diet, two samples of feed from each treatment were

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analyzed to confirm mycotoxin concentrations in the experimental diets. All ingredients were

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mixed with oil and water was added until stiff dough resulted. Each diet was then extruded

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through a mincer with care to avoid cross-contamination. The resulting strands were shadow-

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dried, broken up, sieved into pellets, and stored in plastic bags at 4 °C until use.

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Aflatoxin residues in diet, liver, and musculature were analyzed was performed in

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Central Reference labs (Central Unit for Analysis and Scientific Services, National

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Research center, Cairo, Egypt) according to (AOAC 2000) standards using High-

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performance liquid chromatography with fluorescence detection (HPLC-FLD). This is

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current standard methodology for quantification of AFs due to its accuracy and high

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sensitivity clean-up steps involving immune affinity columns (IAC) to remove interferences

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and allow for pre-concentration of AFs. This analytical method has been validated for use

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in analysis of AFs contamination in feed and feed ingredient samples, among others, and

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used on daily basis in the accredited National Research Center.

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2.2. Experimental design

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One hundred and sixty-eight Nile tilapia were obtained from a private fish farm in Kafr

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El-Sheikh Governorate, Egypt. The initial body weight of fish was 43.75 ± 2.25 g. All fish were

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acclimated for one week in stock tanks and then randomly distributed into 12 experimental

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aquarium tanks (80 x 40 x 30 cm) supplied with aerated and de-chlorinated tap water (14 fish per

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tank). Three tanks were randomly assigned to each of three experimental groups fed previously

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mentioned diets (MZ, AFs, and AFsMZ) and one control group. Water changes were done at

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50% daily to avoid metabolite accumulations (static-renewal system). Fish were fed to satiation

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twice per day for 8 weeks, except the day before sampling. Water quality parameters including

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pH, dissolved oxygen, temperature, and ammonia were measured and were maintained in the

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ranges of 25 ±1 °C, pH 7.1–7.3, dissolved oxygen 6.5–7.8 mg/L; NH3/NH4+ and nitrite 0.25

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mg/L and 12 h:12 h (light: dark) photoperiod. All experimental procedures were in compliance

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with the Animal Care and Use Guidelines at Mansoura University and approved by the local

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Administrative Panel on Laboratory Animal Care Committee.

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2.3. Sample collection

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Six fish from each group (2 fish /tank) were sampled during the experimental trial at

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weeks 1, 2, 4, and 8. Collected fish (one group at the time) were sedated with a low dose of

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buffered tricaine-methane-sulphonate; (MS-222, FINQUEL®, ARGENT) at 30 mg/L + 60 mg/L

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sodium bicarbonate), and euthanized (one fish at the time) in a separate container with a high

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dose of anesthetic (200 mg/L MS-222 + 400 mg/L sodium bicarbonate). Each fish was weighed

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and measured to calculate the mean body weight (Wt), weight gain, body weight index (BWI),

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specific growth rate (SGR), condition factor (K), and hepatosomatic index (HSI) according to

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the following formulae:

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Weight gain = ([FW(g) – IW (g)] / IW (g)) × 100

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BWI (%) = 100 × (weight gain (WG) / IW (g)), where FW and IW are the final and initial body

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weight, respectively.

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K= (W (g) × 102 /L3 (cm); SGR (% day) = 100 [(In FW- In IW) / T], where T is the duration of

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feeding (days).

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HSI (%) = [liver weight (g) / body weight (g)] × 100.

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Blood samples were collected from the caudal vein and divided into two aliquots. One

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aliquot was transferred into Eppendorf tubes containing dipotassium salt of EDTA as an

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anticoagulant (0.5 mg mL-1 blood) for hematological analysis. Another aliquot was used to

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collect serum; tubes were left to clot for 12 h (at 4 o C), prior to centrifugation at 1700 x g for 10

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min, and stored at -20 o C until use. Hematological parameters were analyzed at weeks 1, 2, 4,

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and 8, while serum lysozyme and bactericidal activity were measured at weeks 2, 4, and 8 of the

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feeding period. Liver and posterior kidney tissues were excised immediately after euthanasia.

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Liver was homogenized for antioxidant enzyme analysis at weeks 2, and 8 of the feeding

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period. At the end of the trial, liver and kidney were fixed in 10% buffered formalin for

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histopathology.

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2.4. Hematological parameters

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Total erythrocyte (RBCs) and leukocyte counts (WBCs) were performed manually

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(Hrubec et al. 1996) using Neubauer hemocytometer with Natt-Herrick’s solution (Natt &

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Herrick 1952) as a diluent stain. Estimation of hemoglobin (Hb) content (g/dL) was done

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spectrophotometrically using cyanomethaemoglobin method (Drabkin l964). Packed cell volume

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(PCV), MCV (fl), MCH (pg) and MCHC (%) were estimated, according to Jain (1986). Blood

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smears, using the EDTA treated blood, were stained with Wright’s Giemsa stain and used for the

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differential count (Hrubec et al. 1996).

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2.5. Immunological parameters

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2.5.1. Serum lysozyme activity

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Serum lysozyme activity was measured by the turbidometric assay (Ellis 1990) based on

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the lysis of Micrococcus lysodeikticus (Sigma Chemical Co), with some modification as

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described elsewhere (Zahran & Risha 2014).

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2.5.2. Bactericidal activity

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Bactericidal activity was measured according to Kampen et al. (2005) with some

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modifications (Zahran & Risha 2014). The bactericidal activity was calculated by subtracting

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the absorbance of samples from that of control and reported as absorbance units.

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2.6. Oxidative stress parameters in liver tissue homogenate:

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The liver tissues were individually washed in ice-cold fish saline, and 0.2 g of each

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sample in cold PBS solution (pH 7.4) was grounded in glass homogenizer tubes (pellet pestle

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motor). The homogenates were centrifuged at 1700 x g for 10 min at 4°C (Centrikon H-401

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centrifuge), and fresh supernatants were aliquoted and stored at -80oC. MDA levels were

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determined spectrophotometrically (Photometer 5010, Photometer, BM Co. Germany), and

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expressed as nmol/g tissue. The activity of CAT was determined according to Aebi (1984) by

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measuring the decrease of hydrogen peroxide concentration at 520 nm. The reduced glutathione

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(GSH) was determined using Elmann’s reagent (DTNB) (Beutler et al. 1963). The SOD activity

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was determined by an enzymatic colorimetric method (Nishikimi et al. 1972) using diagnostic

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kits (Biodiagnostics, Egypt) based on inhibition of photoreduction of nitroblue tetrazolium

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(NBT).

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2.7. Histopathological examination

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At the end of the feeding trial, six fish from each group were euthanized in buffered MS-

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222 as previously mentioned. Liver and kidney tissues were fixed in 10 % neutral buffered

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formalin. The fixed specimens were processed through the conventional paraffin embedding

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technique. From the prepared paraffin blocks, 5-µm thick sections were cut and stained with

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hematoxylin and eosin (Bancroft & Gamble 2008).

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2.8. Statistical analysis

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Data were checked for normality and homogeneity of variances using Kolmogorov-

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Smirnov and Levene's tests, respectively. All values were analyzed using GraphPad Prism

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version 7.4 (GraphPad Software Inc.). A two-way ANOVA was used to examine the possible

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effect of AFs and MZ levels on each measured parameter. Two-way ANOVA was followed by

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Tukey's, and Bonferroni's multiple comparisons test to assess the differences between means of

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groups at the same time point and means of the same group at different time points. Differences

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were considered statistically significant when P < 0.05. All parameters are displayed as mean ±

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standard error and graphically presented using GraphPad Prism and Microsoft Excel 2016.

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3. Results

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3.1. Growth performances and HSI

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The results of growth indices and HSI are presented in Table 3. On day 56, AFs group

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weighed significantly higher (P < 0.05) than the control group. An increase in mean body weight

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was observed in all groups at day 56; compared to day 14 in control group (P < 0.05); day 7 and

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28 in MZ group (P < 0.05); day 7 (P < 0.05), 14, and 28 (P < 0.001) in AFsMZ group; and day 0,

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28 (P < 0.05) and day 7, 14 (P < 0.001) in AFs group. Condition factor (K) was only increased in

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AFs group at day 14 compared to control (P < 0.05) and to MZ groups (P < 0.001), while in the

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control group it was significantly increased (P < 0.05) at day 56 compared to day 14. Thus, mean

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body weight and K factor were affected significantly due to different treatment, time, and

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time*treatment interaction. No significant differences were observed in WG, BWI, SGR, and

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HSI related to the treatment and/or time effect.

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3.2. Hematological parameters

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The effects of AFs and/or MZ dietary treatment on hematological parameters are presented

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in (Fig. 1A-G). AFs group showed a significant decrease in RBCs count compared to control (P

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< 0.05) and MZ (P < 0.01) groups at week 2. Additionally, MZ group showed a significant

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increase (P < 0.05) in RBCs count compared to AFsMZ group. RBCs were also significantly

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reduced in AFs group compared to control, MZ (P < 0.001), and AFsMZ (P < 0.01) at week 8

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(Fig. 1A).

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AFs group exhibited a significant decrease (P < 0.01) in Hb content compared to control

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group at week 4. At week 8, AFs group Hb level was significantly lower compared to control,

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MZ (P < 0.001), and AFsMZ (P < 0.05), similarly to RBC counts (Fig. 1B). PCV was

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significantly decreased (P < 0.05) in AFs group at week 2 compared to both control and MZ

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groups. At week 4, PCV sustained its decrease in AFs group compared to control (P < 0.01) and

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AFsMZ (P < 0.05). Regarding time effect within the same group; PCV showed a significant

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increase (P < 0.05) in AFsMZ group at week 4 compared to other time points. Only PCV showed

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a significant time*treatment interaction (two-way ANOVA, P < 0.05), while no significant

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interactions were observed for other parameters measured. Similar to RBCs and Hb results at

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week 8, PCV was significantly decreased in AFs group compared to control, MZ (P < 0.001),

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and AFsMZ (P < 0.05) (Fig. 1C).

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Total leukocyte count (TLC) was significantly lower in AFs group compared to control,

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and MZ supplemented groups at week 8 and also was decreased significantly within AFs group

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(P < 0.001) at week 8, when compared to earlier time points (Fig. 1D). Neutrophils were

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significantly increased in AFs group compared to other groups at week 4 and to other time points

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within the same group. In AFsMZ group, neutrophil counts were significantly lower in week 8

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compared to week 1 (Fig. 1E). Lymphocytic counts were significantly decreased in AFs group

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compared to other groups at weeks 8 and to other time points within the same group. AFsMZ

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group exhibited a significant decrease in the lymphocytic count at week 8 compared to week 1

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and 4. The monocytic count was significantly lower in AFs group compared to MZ at week 2,

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while, it was increased significantly in AFs group at week 4 compared to week 2 and 8 (Fig. 1F).

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Monocytic count in AFsMZ was significantly decreased at week 2 and 8 compared to week

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1(Fig. 1G). Neutrophils and lymphocytes showed significant time*treatment interaction (two-

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way ANOVA, P < 0.01, <0.05), respectively. MCV, MCH, and MCHC exhibited no significant

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differences between groups at any time point of the experiment.

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3.3. Innate immune parameters

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Bactericidal activity showed a significant reduction at week 8 in AFs group compared to

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control, AFsMZ (P < 0.01), and MZ (P < 0.001) groups. Within the same group, a significant

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decrease (P < 0.01) was observed in AFsMZ group at week 4 compared to other time points.

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Lysozyme activity was significantly lower at week 4 (P < 0.0001) in AFs group compared to

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both MZ and AFsMZ groups (Fig. 2A), additionally, lysozyme activity in MZ and AFsMZ

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groups was significantly higher (P < 0.05) compared to control. At week 8, lysozyme activity

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sustained significantly lower level in AFs group compared to MZ (P < 0.0001) and AFsMZ (P <

287

0.001), while MZ group maintained higher lysozyme activity (P < 0.05) compared to control

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group (Fig. 2B). Decreased lysozyme activity (P < 0.01) in AFs group was noted at weeks 4 and

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8 when compared to week 2. Significant differences in innate immune parameters were not seen

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in week 2, and no significant interaction of time*treatment was observed.

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3.4. Oxidative stress analysis

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The treatment, time factors, and time*group interaction significantly affected the levels of

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oxidant-antioxidant enzyme activities in liver tissues (Fig. 3). The effects of AFs on enzyme

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activity analysis in MDA level in liver showed similar pattern at weeks 2 and 8 when in AFs

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group a significant increase (P < 0.0001) in MDA levels was observed (Fig 3A). MZ and

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AFsMZ groups showed no statistical changes compared to control. Within AFs group, a

297

significant decrease in MDA level (P < 0.01) was observed in week 8.

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CAT level in liver was significantly decreased at week 8 in AFs group compared to control

299

(P < 0.0001), AFsMZ (P < 0.0001), and MZ (P < 0.001), groups; while MZ and AFsMZ groups

300

showed no statistical changes compared to control. CAT level was significantly lower (P <

301

0.0001) in AFs group at week 8 compared to week 2 (Fig 3B).

302

Similarly, GSH level in liver showed significant changes at week 8, where AFs group

303

exhibited lower GSH level (P < 0.001) compared to MZ and AFsMZ groups; however, no

304

statistical changes were noticed when compared to control.

305

increased (P < 0.05) in AFsMZ group at week 8 compared to week 2 (Fig 3C).

Furthermore, GSH level was

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SOD level in liver showed a similar pattern at week 2 and 8, where its level was decreased

307

significantly (P < 0.0001) in AFs group compared to other groups. However, MZ and AFsMZ

308

groups showed an increase (P < 0.05; P < 0.001) in SOD level compared to control at week 2

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and similarly at week 8 at a significance level of P < 0.01 and P < 0.05, respectively (Fig 3D).

310

Time*treatment interaction effect on SOD levels was not found significant.

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3.5. Aflatoxin residues in fish liver and musculatures

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Detectable levels of aflatoxins were observed in livers from fish in AF, and AFsMZ groups

313

[1.292 µg/kg (0.001 µM), 0.959 µg/kg (0.0007 µM); respectively], while no residues were

314

detected in control or MZ groups. Total AFs residues of 0.263 µg/kg (0.0002 µM) were detected

315

in the musculature of fish from AFs group, and significantly lower concentration was observed

316

in AFsMZ group (0.022 µg/kg, 0.00002 µM). No residues were found in control or MZ groups

317

(Table. 2).

318

3.6. Histopathological analysis of liver and kidney tissues

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Liver and kidney from AFs group revealed marked histopathological changes. Liver from

320

AFs treated fish exhibited severe hemorrhage, severe congestion in hepatic sinusoids,

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perivascular edema, marked vacuolation and signet ring appearance of hepatocytes, severe

322

hepatocellular necrosis and eosinophilic granular cells infiltration in hepatic parenchyma (Fig. 4

323

A, B). Kidney revealed massive coagulative necrosis of renal tubules and massive hemorrhage in

324

renal parenchyma with marked congestion of interstitial blood capillaries (Fig. 5A, B)

325

Minor histopathological changes have been detected in AFsMZ group. Liver revealed

326

some histopathological changes in the form of focal hepatocellular necrosis, mild perivascular

327

eosinophilic granular cells infiltration, and vacuolization of hepatocytes along with normally

328

central located nucleus, normal hepatic parenchyma and normal hepatic sinusoids (Fig. 4C).

329

Kidney tissue displayed normal renal glomeruli and mild vacuolar degeneration of renal tubular

330

epithelium lining renal tubules (Fig. 4C). Liver and kidney in MZ (Fig. 4D, 5D) and control

331

groups (liver, Fig. 4E, F; kidney, Fig. 5E, F) showed normal tissue architecture.

332 333

4. Discussion

334

Mycotoxin contaminated diets can cause changes in production parameters in different

335

organisms (Mohapatra et al. 2011, Mahfouz & Sherif 2015). In the present study, we found that

336

low level aflatoxin exposed group weighed significantly higher than the control group at day 56.

337

K factor was increased only at day 14 in AFs group compared to control and MZ. No significant

338

changes were observed in other measured parameters (WG, BWI, SGR, and HSI). It has been

339

demonstrated that some dietary toxins at low doses could display hormesis effects, possibly

340

explaining weight gain observed in AFs fish group (Imani et al. 2017). Our findings were also

341

similar to (Mahfouz & Sherif 2015), who observed an increase in body weight without

342

significant changes in HSI in Nile tilapia at 12 weeks compared to 6 weeks when fed

343

contaminated aflatoxin diet at 20 µg kg-1. Huang et al. (2011) reported changes in body weight in

344

gibel carp (Carassius gibelio) fed basal diets supplemented with 50 and 100 µg/kg of aflatoxins

345

after 16 weeks. Similar results were also reported by Han et al. (2010) for gibel carp fed seven

346

different levels of dietary aflatoxin in the range from 3.20 to 28.60 µg/kg of AFB1. In Deng et al.

347

(2010), tilapia fed with aflatoxin contaminated diets (at 19, 85, 245, 638, 793, and 1641 µg/kg)

348

showed no significant differences in body weight, HSI and condition factor for 10 weeks. Our

349

results were in agreement with Alinezhad et al. (2017), who reported that there were no

350

significant differences in growth indices and HSI in Nile tilapia fed 5 µg/kg of AFB1 for 56

351

days.

352

Findings from our study are in disagreement with several studies that showed the negative

353

impact of aflatoxin on growth performance in fish, however, such effect was only evident when

354

higher dietary levels of AFs were applied for a longer time (up to 16 weeks in some studies)

355

(Jantrarotai & Lovell 1990, Chavez-Sanchez et al. 1994, Boonyaratpalin et al. 2001, Huang et al.

356

2011, Mohapatra et al. 2011). Nile tilapia had been reported to show a reduction in growth only

357

at a diet containing more than 10 mg/kg of AFB1 (Tuan et al. 2002). These discrepancies could

358

be due to the variable fish sensitivity to mycotoxin dose and duration of exposure (Tuan et al.

359

2002, Amany et al. 2009, Selim et al. 2014, Mahfouz & Sherif 2015). Tilapia fed MZ

360

supplemented diet exhibited a significant increase from starting weight; however, no

361

difference between groups was observed. This result was supported by previous studies which

362

recorded an enhancement of the growth performances in broiler chickens fed 2000 µg/kg diet of

363

ochratoxin A (OTA) alone or combined with MZ at 0.2 % (Nedeljković-Trailović & Petrujkic

364

2013, Nedeljković-Trailović et al. 2015), and in Nile tilapia fed AFs contaminated diet with or

365

without 0.5 % hydrated sodium calcium aluminosilicate (HSCAS) (Selim et al. 2014).

366

Furthermore, feed supplemented with 0.2 % HSCAS was shown to increase the growth

367

performances in ducks fed diets contaminated with AFs (Rattanasinthuphong et al. 2017).

368

Hematological parameters in fish are good indicators for the presence of multiple

369

environmental and toxic stressors that can induce significant variations (Kumar et al. 2011). As

370

observed in our study, the erythrogram showed normocytic normochromic anemia in AFs group.

371

Diet supplementation with MZ mitigated adverse effects of AFs on the hematological parameters

372

measured at week 4 and 8 (Fig. 1A-C), suggesting a protective role of MZ against AFs response.

373

Reduction in the hematological parameters might be attributed to the immunosuppressive

374

effect of aflatoxin on fishes, possibly through inhibition of enzyme activity involved in heme

375

biosynthesis, or increasing the rate of erythrocyte destruction in hematopoietic organs, leading to

376

erythropoiesis, hemosynthesis, and osmoregulatory dysfunction (Jenkins et al. 2003, ATSDR

377

2005). This finding has been supported by the presented histopathological results showing

378

massive necrosis. Reduction of WBCs, neutrophils, and lymphocytic counts could also be related

379

to epinephrine release during stressful conditions leading to spleen contraction and fewer

380

leukocytes production, and impairment of the immune system (Witeska 2003). Marked

381

improvement of these parameters (Fig. 1D-G) again suggest the beneficial effect of the Minazel-

382

Plus® supplementation in the contaminated diet.

383

Our results are consistent with previous studies in which low dietary level of AFs at 10 and

384

20 µg/kg affected hematological parameters significantly in rohu, Labeo rohita after 2 months,

385

where RBCs, Hb, and WBCs counts were decreased compared to the control. Also, after

386

application of mold inhibitor combination (0.25% clove oil and 0.32% of sodium propionate),

387

the parameters restored to control levels (Mohapatra et al. 2011). In another study, Mahfouz and

388

Sherif (2015) used the approved level of AFB1 as their control (20 µg/kg) and compared its

389

effect against higher level of AFB1 (100 µg/kg) in Nile tilapia, and found that RBCs, Hb, MCV,

390

MCH, and MCHC were significantly decreased in the group exposed to high AFB1 compared to

391

the low exposure level after 6 weeks and further decrease after 12 weeks. Additionally, they

392

found that only neutrophils were significantly increased after 6 and 12 weeks, but lymphocytes

393

were significantly decreased in high AFB1 exposed group. Similar to the current results, El-

394

Boshy et al. (2008) reported normocytic normochromic anemia in Nile tilapia fed AFB1 even at

395

a higher dose of 200 µg/kg for 3 weeks. When gibel carp was exposed to series of dietary AFB1

396

concentrations (0, 10, 20, 50, 100, 200, 1000 µg/kg), it was found to be less susceptible to

397

aflatoxicosis, as no significant changes were observed in hematocrit levels (Huang et al. 2011).

398

In a study conducted by Pradeepkiran (2015) on common carp, AFB1 at 200 µg/kg showed

399

a similar decrease in RBCs and Hb that was restored near to the control levels by diet

400

supplementation with probiotics (garlic and/or cheese). Using adsorbents appears to be a

401

practical way for mycotoxin elimination (Garcia et al. 2003, Nedeljković-Trailović & Petrujkic

402

2013). Based on the presented results, Minazel-Plus® supplemented diets protected tilapia

403

against a decrease of hematological parameters caused by AFs. Our findings are consistent with

404

previous studies where mycotoxin adsorbents showed the potential to protect against the harmful

405

effects of mycotoxins, such as using HSCAS, Saccharomyces cerevisiae and an esterified

406

glucomannan in tilapia and duck (Selim et al. 2014, Rattanasinthuphong et al. 2017).

407

Bactericidal activity was significantly reduced in AFs groups at week 8 compared to all

408

other groups, and lysozyme activity was significantly reduced in AFs group at weeks 4 and 8.

409

Addition of Minazel-Plus® to aflatoxin diet (AFsMZ) rescued both bactericidal and lysozyme

410

activities from being similar to the control levels. The reduction in lysozyme activity in AFs

411

group could have contributed to reduced bactericidal activity. Additionally, the reduction in both

412

bactericidal and lysozyme activity was consistent with the observed leukopenia in this study.

413

Collectively, the observed changes suggest immunosuppressive effects of AFs mixture at a

414

low dose, albeit mechanisms are not yet completely understood. Possible suppression of the

415

release of antimicrobial factors in serum via lysozyme, as well as interfering with release and

416

function of antiproteases, were suggested as possible causes of specific and non-specific immune

417

response dysfunction in aquatic organisms exposed to mycotoxins (Sahoo & Mukherjee 2002,

418

Rodríguez-Cervantes et al., 2010, Selim et al. 2014) (). Our findings were similar to that

419

observed in Nile tilapia fed AFB1 at a higher dose of 200 µg/kg for 3 weeks (El-Boshy et al.

420

2008) with noticed improvement after β-glucan supplementation to AFs treated group. Sahoo

421

and Mukherjee (2002) observed the similar negative effect of AFs on bactericidal and lysozyme

422

activities in rohu, Labeo rohita, and parenteral administration of both β-glucan and α-tocopherol

423

brought back the activity level to that of the control group. Red drum (Sciaenops ocellatus) fed

424

AFB1 spiked diets revealed a significant decrease in lysozyme activity although it was detected

425

only at high dose (3000 and 5000 µg/kg), and rescued by NovaSil diet supplementation at 5 %

426

combined with 5000 µg/kg AFB1 with same effect on innate immune response as in control

427

group (Zychowski et al. 2013).

428

Aflatoxins are known as potent inducers of oxidative stress response with subsequent

429

adverse cellular effects (Abbès et al. 2008). It was observed in this study that AFs exposure

430

increased MDA levels significantly at the 2nd and 8th weeks, while in Minazel-Plus® and AFs

431

supplemented group the MDA levels were not different from the control, suggesting that

432

mycotoxin binder managed to prevent the toxic effects of AFs. Increased MDA levels may also

433

be related to observed immunosuppressive effects of the AFs on lysozyme and bactericidal

434

activities, possibly due to excessive reactive oxygen species (ROS) production during the

435

metabolic processing of AFs in the liver, and subsequent engagement of various mechanisms to

436

reduce oxidative stress damage through multiple pathways, including regulation of innate

437

immune responses (Wu et al. 2013). Several previous studies had reported similar findings where

438

MDA level was increased due to AFs toxicity (Souza et al. 2018) and has improved near the

439

control level after supplementation with herbal plants (El-Barbary 2016), algal extract (Abdel-

440

Wahhab et al. 2006), natural or modified clinoptilolite (Wu et al. 2013) and montmorillonite

441

(Abbès et al. 2008).

442

Mechanisms of detoxification of reactive oxygen species include several enzymes (SOD,

443

CAT, GSH, and GST) and their activity can be affected by oxidative damage (Sharbidre et al.

444

2011). Excess of hydrogen peroxide may diminish SOD activity, while the superoxide anion may

445

be in charge of decreased CAT activity (Modesto & Martinez 2010). In the present study, levels

446

of SOD and GSH were nominally decreased in aflatoxins group at week 2 and 8 (SOD), and in

447

week 8 (GSH). In contrast, the levels of SOD and GSH were significantly increased in the group

448

fed MZ alone, or MZ combined with AFs (Fig 3. C, D). These results indicate that although AFs

449

did not appear to affect the SOD and GSH levels, the supplementation with MZ enhanced the

450

overall antioxidant enzyme levels and could potentially protect the organs against excessive ROS

451

production. Similarly, CAT level was significantly reduced at week 8 in AFs group, and the

452

addition of MZ was able to keep the CAT level again at par with the control group.

453

The observed pattern of enzyme activity levels could be attributed to the ability of the SOD

454

system to manage a lower dose of AFs and successfully neutralize an increase in superoxide

455

ions. Simultaneously, CAT activity was possibly reduced due to its role in scavenging excess

456

amount of superoxide ions produced by the SOD. Studies where AFs interfere with oxidant-

457

antioxidant defense mechanisms but are rescued when combined with other natural compounds

458

support our findings (El-Boshy et al. 2008, Mahfouz 2015, Mahfouz & Sherif 2015, El-Barbary

459

2016, Souza et al. 2018).

460

The liver is known as the primary target organ for aflatoxicosis. Therefore, feeding an

461

aflatoxin contaminated diet would affect the liver with a specific pathophysiological alteration

462

associated with aflatoxicosis (Meissonnier et al. 2008). In our results, aflatoxin residues of 1.292

463

and 0.263 µg/kg were detected in liver and musculature of AFs group, respectively, which is

464

lower than safe/ permissible limit for human consumption (Rajeev Raghavan et al. 2011).

465

Supplementation with Minazel-Plus® was able to reduce aflatoxin levels in liver and musculature

466

to 0.959 and 0.022 µg/kg, respectively. This could be due to tissue preferences of AFs

467

accumulation in different species, since it was reported that higher concentrations of AFB1 were

468

found in the bile, liver, kidney and pyloric caeca of rainbow trout, but in channel catfish AFB1 or

469

its metabolites were found to also accumulate in the muscle (Han et al. 2010). Moreover, the rate

470

of biotransformation and tissue depletion might have been associated with the differences in

471

tissue residue levels of AFs (Huang et al. 2011).

472

Our results are consistent with a previous study of Mahfouz and Sherif (2015), where

473

detectable AFB1 residues (5 and 8 µg/kg) were found in the liver of Nile tilapia fed diet

474

artificially contaminated with AFB1 at 20 µg/kg, and no detectable residues were found in

475

musculature. Consistently with our findings, El-Sayed and Khalil (2009) recorded AFB1

476

residues of 4.25 µg/kg in the musculature of sea bass fed diet artificially contaminated with

477

AFB1 at 18 µg/kg. AFB1 was accumulated in muscles tissue (~3 µg/kg), of gibel carp fed

478

artificially contaminated AFB1 diet at 16 µg/kg (Han et al. 2010).

479

As shown here, MZ supplementation reduced the aflatoxin levels in liver and musculature.

480

Such effect is supported by other studies showing potential of different adsorbents to ameliorate

481

aflatoxicosis (HSCAS, Saccharomyces cerevisiae, or esterified glucomannan in Nile tilapia fed

482

artificially contaminated AFB1 diet) (Arana et al. 2011, Selim et al. 2014), and calcium bentonite

483

clay at 1 % supplemented diet was able to alleviate AFB1 toxicity in muscles tissue of Nile

484

tilapia fed AFB1 spiked diet at 4000 µg/kg (Hussain et al. 2017). Further, this finding supports

485

our results regarding the oxidative damage biomarkers.

486

Liver is the most sensitive organ to toxicants as the leading site of detoxification (Crestani

487

et al. 2007). In the present study, the feeding of AFs contaminated diet resulted in significant

488

histopathological changes in liver and kidney. Our results were similar to previous studies that

489

reported pathological lesions in fish under low AFs toxicity, particularly in the liver (Hendricks,

490

1993). In rainbow trout, concentration as low as 0.5 µg/kg AFB1 in diet resulted in liver tumors

491

after 6 months feeding (Halver 1969).

492

On the contrary, several studies detected no histopathological lesions in liver and kidney

493

during low AFs exposure such as Tuan et al. (2002) in Nile tilapia (Oreochromis niloticus)

494

exposed to 0-250 µg/kg of dietary AFB1, in hybrid tilapia (Oreochromis niloticus × O. aureus)

495

exposed to 19, 85, and 245 µg/kg of dietary AFB1 (Deng et al. 2010), and in gibel carp fed with

496

0-50 µg/kg of dietary AFB1 (Huang et al. 2011). The controversy could be due to variable

497

factors that can modulate the responsiveness of fish to carcinogen exposure including: water

498

temperature, nutritional status, genetic variation, route and duration of exposure, unmonitored

499

activation or presence of inhibitors or promoters of gene transcription, and fish species-specific

500

sensitivity could all impact the extent of liver damage in fishes (Han et al. 2010, Mahfouz &

501

Sherif 2015).

502

Interestingly, our results not only proved that Minazel-Plus® diet supplementation has no

503

deleterious effects on fish liver and kidney but also showed that it could partially ameliorate the

504

AFs effects and reduce the intensity of histopathological changes in both liver and kidney of fish.

505

Our findings are consistent with previous studies describing safe and significant protective

506

effects of MZ in broiler fed 2000 µg/kg diet of ochratoxin A (OTA) (Nedeljković-Trailović &

507

Petrujkic 2013, Nedeljković-Trailović et al. 2015).

508

5. Conclusion

509

To conclude, aflatoxin levels in the feed below allowable concentrations can affect the

510

immune and oxidative stress responses, induce histopathological changes, and bio-accumulate in

511

liver and musculature of Nile tilapia. Supplementation of surface-modified clinoptilolite

512

(Minazel-Plus®) to aflatoxins contaminated diet was successful in mitigating overall adverse

513

aflatoxin effects on fish health. Further, Minazel-Plus® supplementation alone enhanced

514

antioxidant and immune responses in fish. Presented findings provide valuable insights into the

515

potential of low aflatoxin levels to cause adverse health effects in Nile tilapia. Our results

516

strongly support the development and implementation of good practices in prevention and

517

monitoring strategies of mycotoxin levels in dietary ingredients. Such approaches are of

518

particular importance in countries without regulated, safe aflatoxin levels in fish feed leading to a

519

higher risk of using the contaminated feed in aquaculture, followed with possible consequences

520

to fish health and seafood safety, and increased public health concerns.

521 522

Acknowledgments

523

This research received no specific grant from any funding agency in the public,

524

commercial, or not-for-profit sectors. The study was supported in-kind by Mansoura

525

University Faculty of Veterinary Medicine and Chair for Fish Diseases, Faculty of

526

Veterinary Medicine, Ludwig-Maximilians-University Munich. We thank the PatentCo,

527

Misicevo, Serbia for providing modified clinoptilolite (Minazel Plus®) for this study.

528

References

529

Abbès, S., Salah-Abbès, J.B., Hetta, M.M., Ibrahim, M., Abdel-Wahhab, M.A., Bacha, H.,

530

Oueslati, R., 2008. Efficacy of Tunisian montmorillonite for in vitro aflatoxin binding and

531

in vivo amelioration of physiological alterations. Applied Clay Science 42, 151-157.

532

Abdel-Wahhab, M.A., Ahmed, H.H., Hagazi, M.M., 2006. Prevention of aflatoxin B1-initiated

533

hepatotoxicity in rat by marine algae extracts. J Appl Toxicol 26, 229-238.

534

Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121-126.

535

Alinezhad, S., Faridi, M., Falahatkar, B., Nabizadeh, R., Davoodi, D., 2017. Effects of

536

nanostructured zeolite and aflatoxin B1 in growth performance, immune parameters and

537

pathological conditions of rainbow trout Oncorhynchus mykiss. J Fish Shellfish

538

Immunology 70, 648-655.

539

Amany, M.K., Hala, M., Mohammad, M., Abdel-Wahab, M., 2009. Pathological studies on

540

effects of aflatoxin on Oreochromis niloticus with application of different trials of control.

541

J Egypt. J. Comp. Pathol. Clin. Pathol 22, 175-193.

542 543

AOAC, 2000. Official Method 991.31. Aflatoxins in Corn, Raw Peanuts and Peanut Butter. Immunoaffinity Column (Aflatest) Method AOAC International, p. 49.42.18.

544

Arana, S., Dagli, M.L.Z., Sabino, M., Tabata, Y.A., Rigolino, M.G., Hernandez-Blazquez, F.J.,

545

2011. Evaluation of the efficacy of hydrated sodium aluminosilicate in the prevention of

546

aflatoxin-induced hepatic cancer in rainbow trout. J Pesquisa Veterinária Brasileira 31,

547

751-755.

548 549

550 551

552 553

554 555

ATSDR, U., 2005. Toxicological profile for lead. (Draft for Public Comment). J service., USD ohahs P. h.. Agency for Toxic substances Disease Registry. Alanta, USA. Bancroft, J.D., Gamble, M., 2008. Theory and practice of histological techniques, in: ed., t. (Ed.). London: Churchill Livingstone. Bbosa, G.S., Kitya, D., Odda, J., Ogwal-Okeng, J., 2013. Aflatoxins metabolism, effects on epigenetic mechanisms and their role in carcinogenesis. J Health 5, 14. Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the determination of blood glutathione. The Journal of Laboratory and Clinical Medicine 61, 882.

556

Boonyaratpalin, M., Supamattaya, K., Verakunpiriya, V., Suprasert, D., 2001. Effects of

557

aflatoxin B1 on growth performance, blood components, immune function and

558

histopathological changes in black tiger shrimp (Penaeus monodon Fabricius). J

559

Aquaculture Research 32, 388-398.

560 561

Cai, J., Quagrainie, K., Hishamunda, N., 2017. Social and Economic Performance of Tilapia Farming in Africa. FAO Fisheries and Aquaculture Circular.

562

Chavez-Sanchez, M.C., Palacios, C.M., Moreno, I.O., 1994. Pathological effects of feeding

563

young Oreochromis niloticus diets supplemented with different levels of aflatoxin B1. J

564

Aquaculture 127, 49-60.

565

Crestani, M., Menezes, C., Glusczak, L., dos Santos Miron, D., Spanevello, R., Silveira, A.,

566

Gonçalves, F.F., Zanella, R., Loro, V.L., 2007. Effect of clomazone herbicide on

567

biochemical and histological aspects of silver catfish (Rhamdia quelen) and recovery

568

pattern. Chemosphere 67, 2305-2311.

569 570

de Oliveira, C.A., Corassin, C.H., 2014. Aflatoxins, Mycotoxins and their implications in food safety (1st ed.), 1st ed. ed. Future Medicine, London, UK, pp. 6-19.

571

Deng, S.-X., Tian, L.-X., Liu, F.-J., Jin, S.-J., Liang, G.-Y., Yang, H.-J., Du, Z.-Y., Liu, Y.-J.,

572

2010. Toxic effects and residue of aflatoxin B1 in tilapia (Oreochromis niloticus × O.

573

aureus) during long-term dietary exposure. Aquaculture 307, 233-240.

574

Drabkin, D., l964. Bio Chem. 164, 703.

575

EC, 2006. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum

576

levels for certain contaminants in foodstuffs. Off J Eur Union 364.

577

El-Barbary, M.I., 2016. Detoxification and antioxidant effects of garlic and curcumin in

578

Oreochromis niloticus injected with aflatoxin B1 with reference to gene expression of

579

glutathione peroxidase (GPx) by RT-PCR. J Fish Physiology Biochemistry 42, 617-629.

580

El-Boshy, M., El-Ashram, A., El-Ghany, N.A.A., 2008. Effect of dietary beta-1, 3 glucan on

581

immunomodulation on diseased Oreochromis niloticus experimentally infected with

582

aflatoxin B1, 8th international symposium on Tilapia in aquaculture, pp. 1109-1127.

583

El-Sayed, Y.S., Khalil, R.H., 2009. Toxicity, biochemical effects and residue of aflatoxin B1 in

584

marine water-reared sea bass (Dicentrarchus labrax L.). Food and Chemical Toxicology

585

47, 1606-1609.

586

Ellis, A.E., 1990. Lysozyme assays. Techniques in fish immunology 1, 101-103.

587

Garcia, A.R., Avila, E., Rosiles, R., Petrone, V.M., 2003. Evaluation of two mycotoxin binders

588

to reduce toxicity of broiler diets containing ochratoxin A and T-2 toxin contaminated

589

grain. Avian diseases 47, 691-699.

590 591

Halver, J.E., 1969. Chapter X Aflatoxicosis and Trout Hepatoma. Aflatoxin: scientific background, control, and implications 7, 265.

592

Han, D., Xie, S., Zhu, X., Yang, Y., Guo, Z., 2010. Growth and hepatopancreas performances of

593

gibel carp fed diets containing low levels of aflatoxin B1. J Aquaculture Nutrition 16, 335-

594

342.

595

Hendricks, J.D., 1993. Carcinogenicity of aflatoxins in nonmammalian organisms. The

596

toxicology of aflatoxins: human health, veterinary and agricultural significance., 103-

597

136.

598

Hrubec, T., Smith, S., Robertson, J., Feldman, B., Veit, H., Libey, G., Tinker, M., 1996.

599

Comparison of hematologic reference intervals between culture system and type of hybrid

600

striped bass. J American Journal of Veterinary Research 57, 618-623.

601 602

Huang, Y., Han, D., Zhu, X., Yang, Y., Jin, J., Chen, Y., Xie, S., 2011. Response and recovery of gibel carp from subchronic oral administration of aflatoxin B1. J Aquaculture 319, 89-97.

603

Hussain, D., Mateen, A., Gatlin, D.M., 2017. Alleviation of aflatoxin B1 (AFB1) toxicity by

604

calcium bentonite clay: Effects on growth performance, condition indices and

605

bioaccumulation of AFB1 residues in Nile tilapia (Oreochromis niloticus). Aquaculture

606

475, 8-15.

607

Imani, A., Salimi Bani, M., Noori, F., Farzaneh, M., Moghanlou, K.S., 2017. The effect of

608

bentonite and yeast cell wall along with cinnamon oil on aflatoxicosis in rainbow trout

609

(Oncorhynchus mykiss): Digestive enzymes, growth indices, nutritional performance and

610

proximate body composition. Aquaculture 476, 160-167.

611 612

613 614

Jain, N.C., 1986. Schalman's Veterinary Haematology, 4th edition ed, Lea and Babings, Philadelphia. P.A, U.S.A. Jantrarotai, W., Lovell, R.T., 1990. Subchronic toxicity of dietary aflatoxin B1 to channel catfish. J Journal of Aquatic Animal Health 2, 248-254.

615

Jenkins, F., Smith, J., Rajanna, B., Shameem, U., Umadevi, K., Sandhya, V., Madhavi, R., 2003.

616

Effect of sub-lethal concentrations of endosulfan on hematological and serum biochemical

617

parameters in the carp Cyprinus carpio. J Bulletin of Environmental Contamination

618

Toxicology 70, 0993-0997.

619 620

Kampen, A.H., Tollersrud, T., Lund, A., 2005. Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro. Infect. Immun. 73, 1578-1583.

621

Kumar, N., Prabhu, P.A.J., Pal, A., Remya, S., Aklakur, M., Rana, R., Gupta, S., Raman, R.,

622

Jadhao, S., 2011. Anti-oxidative and immuno-hematological status of Tilapia

623

(Oreochromis mossambicus) during acute toxicity test of endosulfan. J Pesticide

624

Biochemistry Physiology 99, 45-52.

625

Leung, M.C.K., Díaz-Llano, G., Smith, T.K., 2006. Mycotoxins in pet food: a review on

626

worldwide prevalence and preventative strategies. Journal of Agricultural and Food

627

Chemistry 54, 9623-9635.

628 629

Mahfouz, M.E., 2015. Ameliorative effect of curcumin on aflatoxin B1-induced changes in liver gene expression of Oreochromis niloticus. J Molecular Biology 49, 275-286.

630

Mahfouz, M.E., Sherif, A.H., 2015. A multiparameter investigation into adverse effects of

631

aflatoxin on Oreochromis niloticus health status. J The Journal of Basic Applied Zoology

632

71, 48-59.

633 634

Marroquín-Cardona, A., Johnson, N., Phillips, T., Hayes, A., 2014. Mycotoxins in a changing global environment–a review. J Food Chemical Toxicology 69, 220-230.

635

Meissonnier, G.M., Pinton, P., Laffitte, J., Cossalter, A.-M., Gong, Y.Y., Wild, C.P., Bertin, G.,

636

Galtier, P., Oswald, I.P., 2008. Immunotoxicity of aflatoxin B1: Impairment of the cell-

637

mediated response to vaccine antigen and modulation of cytokine expression. Toxicology

638

and Applied Pharmacology 231, 142-149.

639

Modesto, K.A., Martinez, C.B.R., 2010. Roundup® causes oxidative stress in liver and inhibits

640

acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus. Chemosphere 78,

641

294-299.

642

Mohapatra, S., Sahu, N., Pal, A., Prusty, A., Kumar, V., Kumar, S., 2011. Haemato-immunology

643

and histo-architectural changes in Labeo rohita fingerlings: effect of dietary aflatoxin and

644

mould inhibitor. J Fish Physiology Biochemistry 37, 177-186.

645 646

647 648

National research Council, N.R.C., 1993. Nutrient requirements of fish. Nutrient Requirements of Domestic Animal Series. Natt, M.P., Herrick, C.A., 1952. A New Blood Diluent for Counting the Erythrocytes and Leucocytes of the Chicken. Poultry Science 31, 735-738.

649 650

651 652

Nedeljković-Trailović, J., Petrujkic, B., 2013. Efficacy of Minazel Plus ® In Reducing Detrimental Effects of Ochratoxin A, in Broilers. Engormix. Nedeljković-Trailović, J., Stefanović, S., Trailovic, S., 2013. In vitro investigation three different adsorbents against ochratoxin A in broilers. Br. Poult. Sci 54, 515-523.

653

Nedeljković-Trailović, J., Trailović, S., Resanović, R., Milićević, D., Jovanovic, M., Vasiljevic,

654

M., 2015. Comparative investigation of the efficacy of three different adsorbents against

655

OTA-induced toxicity in broiler chickens. Toxins 7, 1174-1191.

656

Nešić, V.D., Ostojin, M.V., Nešić, K.D., Resanović, R.D., 2009. Evaluation of the efficacy of

657

different feed additives to adsorbe T-2 toxin in vitro. Zbornik Matice srpske za prirodne

658

nauke, 55-59.

659

Nishikimi, M., Rao, N.A., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of

660

reduced phenazine methosulfate and molecular oxygen. J Biochemical Biophysical

661

Research Communications 46, 849-854.

662 663

Patriarca, A., Pinto, V.F., 2017. Prevalence of mycotoxins in foods and decontamination. J Current Opinion in Food Science 14, 50-60.

664

Pietsch, C., Kersten, S., Burkhardt-Holm, P., Valenta, H., Dänicke, S., 2013. Occurrence of

665

deoxynivalenol and zearalenone in commercial fish feed: an initial study. Toxins 5, 184-

666

192.

667 668

Pitt, J., 2008. Understanding Plant-Fungus Associations as a Key to Mycotoxin Control. Food Contaminants: Mycotoxins and Food Allergies 1001, 96-108

669

Rajeev Raghavan, P., Zhu, X., Lei, W., Han, D., YANG, Y., Xie, S., 2011. Low levels of

670

Aflatoxin B1 could cause mortalities in juvenile hybrid sturgeon, Acipenser ruthenus ♂×A.

671

baeri♀. Aquaculture 17, e39- e47.

672

Rattanasinthuphong, K., Tengjaroenkul, B., Tengjaroenkul, U., Pakdee, P., 2017. Efficacy of

673

mycosorbents to ameliorate the adverse effects of natural aflatoxin contamination in the

674

diets of Cherry Valley ducks. Livestock Research for Rural Development 29.

675

Rodríguez-Cervantes, C., Girón-Pérez, M., Robledo-Marenco, M., Marin, S., Velázquez-

676

Fernández, J., Medina-Díaz, I., Rojas-García, A., Ramos, A., 2010. Aflatoxin B1 and its

677

toxic effects on immune response of teleost fishes: a review. World Mycotoxin Journal 3,

678

193-199.

679

Sahoo, P.K., Mukherjee, S.C., 2002. Influence of high dietary α-tocopherol intakes on specific

680

immune response, nonspecific resistance factors and disease resistance of healthy and

681

aflatoxin B1-induced immunocompromised Indian major carp, Labeo rohita (Hamilton).

682

Aquaculture Nutrition. 8,159-167.

683

Santacroce, M.P., Conversano, M., Casalino, E., Lai, O., Zizzadoro, C., Centoducati, G.,

684

Crescenzo, G., 2008. Aflatoxins in aquatic species: metabolism, toxicity and perspectives.

685

J Reviews in Fish Biology Fisheries Research 18, 99-130.

686

Santos, G., Rodrigues, I., Naehrer, K., Encarnacao, P., 2010. Mycotoxins in aquaculture:

687

occurrence in feed components and impact on animal performance. J Aquac. Eur 35.

688

Selim, K.M., El-hofy, H., Khalil, R.H., 2014. The efficacy of three mycotoxin adsorbents to

689

alleviate aflatoxin B 1-induced toxicity in Oreochromis niloticus. J Aquaculture

690

International 22, 523-540.

691

Sharbidre, A.A., Metkari, V., Patode, P., 2011. Effect of methyl parathion and chlorpyrifos on

692

certain biomarkers in various tissues of guppy fish, (Poecilia reticulata). Pesticide

693

Biochemistry and Physiology 101, 132-141.

694

Souza, C.F., Baldissera, M.D., Descovi, S.N., Zeppenfeld, C.C., Garzon, L.R., da Silva, A.S.,

695

Stefani, L.M., Baldisserotto, B., 2018. Serum and hepatic oxidative damage induced

696

by a diet contaminated with fungal mycotoxin in freshwater silver catfish Rhamdia

697

quelen: Involvement on disease pathogenesis. Microbial pathogenesis 124, 82-88.

698

Tuan, N.A., Grizzle, J.M., Lovell, R.T., Manning, B.B., Rottinghaus, G.E., 2002. Growth and

699

hepatic lesions of Nile tilapia (Oreochromis niloticus) fed diets containing aflatoxin B1. J

700

Aquaculture 212, 311-319.

701

Van Eijkeren, J.C.H., Bakker, M.I., Zeilmaker, M.J., 2006. A simple steady-state model for

702

carry-over of aflatoxins from feed to cow's milk. Food Additives and Contaminants 23,

703

833-838.

704 705

Witeska, M., 2003. The effects of metals (Pb, Cu, Cd, and Zn) on hematological parameters and blood cell morphology of common carp. J Rozprawa Naukowa 72.

706

Wu, Y., Wu, Q., Zhou, Y., Ahmad, H., Wang, T., 2013. Effects of clinoptilolite on growth

707

performance and antioxidant status in broilers. J Biological Trace Element Research 155,

708

228-235.

709

Zahran, E., Risha, E., 2014. Modulatory role of dietary Chlorella vulgaris powder against

710

arsenic-induced immunotoxicity and oxidative stress in Nile tilapia (Oreochromis

711

niloticus). Fish & Shellfish Immunology 41, 654-662.

712

Zychowski, K.E., Hoffmann, A.R., Ly, H.J., Pohlenz, C., Buentello, A., Romoser, A., Gatlin,

713

D.M., Phillips, T.D., 2013. The Effect of Aflatoxin-B1 on Red Drum (Sciaenops ocellatus)

714

and Assessment of Dietary Supplementation of NovaSil for the Prevention of

715

Aflatoxicosis. 5, 1555-1573.

716 717

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Table legends:

719

Table 1. Ingredient composition of the diets (%) and levels of detected AFs.

720

Table 2. Residual levels of AFs (AFB1, 2, AFG1, 2) (µg/kg - µM) in liver and musculature of

721

Nile tilapia fed naturally contaminated AFs diet with (16 µg/kg – 0.013 µM) or without

722

MZ (2 g/kg - 9.5 µM) supplementation for 8 weeks.

723 724

Table 3. Growth performance and HSI of Nile tilapia fed naturally contaminated AFs diet (16 µg/kg – 0.013 µM) with or without MZ supplementation (2 g/kg - 9.5 µM) for 8 weeks.

725

Figure captions:

726

Fig. 1. Hematological parameters in Nile tilapia fed naturally contaminated AFs diet (16 µg/kg

727

- 0.013 µM) with or without MZ supplementation (2 g/kg – 9.5 µM) for 8 weeks. Data

728

were presented as the mean of six fish ± SEM. Values with a different letter superscript

729

indicate a significant difference between groups at the same time. Values with different

730

dagger superscript are significantly different between time points within the same group

731

(P< 0.05).

732 733

Fig. 2. Bactericidal activity(A), Serum Lysozyme activity(B) in Nile tilapia fed naturally

734

contaminated AFs diet (16 µg/kg - 0.013 µM) with or without MZ supplementation (2

735

g/kg – 9.5 µM) for 8 weeks. Data were presented as the mean of six fish ± SEM. Values

736

with a different letter superscript indicate a significant difference between groups at the

737

same time. Values with different asterisk superscript indicate the significant level and

738

differences between time points within the same group (*P < 0.05, 0.01, **P < 0.001,

739

*** P < 0.0001).

740

Fig. 3. MDA (A), CAT (B), GSH (C), and SOD (D) activity in liver of Nile tilapia fed

741

naturally contaminated AFs diet (16 µg/kg

- 0.013 µM) with or without MZ

742

supplementation (2 g/kg – 9.5 µM) for 8 weeks. Data were presented as the mean of six

743

fish ± SEM. Values with a different letter superscript indicate a significant difference

744

between groups at the same time. Values with different asterisk superscript indicate the

745

significant level and differences between time points within the same group (*P < 0.05,

746

0.01, **P < 0.001, *** P < 0.0001).

747

Fig. 4. Liver of Nile tilapia in AFs group (A) showing severe congestion and hemorrhage

748

replacing hepatic parenchyma (arrow). (HE, 100x). (B) necrosis of hepatocytes,

749

congestion of blood vessels, and heterophilic infiltration into hepatic tissue (arrow). (HE,

750

400x). In AFsMZ group (C) showing normal hepatocytes (arrow) and heterophilic

751

recruitment in perivascular connective tissue (arrowhead). (HE, 400x). In MZ group (D)

752

showing normal hepatic architecture with normal hepatocytes (arrow) and normal

753

hepatopancreas (arrowhead). (HE, 400x). In the control group showing normal hepatic

754

architecture with normal hepatocytes (arrow) and normal hepatopancreas (arrowhead). (E

755

= HE, 100x). Normal hepatic architecture with normal hepatocytes (arrow) and normal

756

hepatopancreas (arrowhead). (F = HE, 400x).

757

Fig. 5. Kidney of Nile tilapia in AFs group (A) displaying local extensive necrosis in renal

758

tubules (arrow). (HE, 100x). (B) necrosis in renal tubules and desquamation of necrotic

759

renal tubular epithelium into the lumen of renal tubules (arrow). (HE, 400x). In AFsMZ

760

group (C) displaying normal renal glomeruli (arrow) and normal renal tubules lined by

761

normal renal tubular epithelium (arrowhead). (HE, 400x). In MZ group (D) showing

762

normal renal architecture with normal renal glomeruli (arrow) and normal renal tubules

763

(arrowhead). (HE, 400x). In the control group showing normal renal glomeruli (arrow)

764

and normal renal tubules lined by normal renal tubular epithelium (arrowhead). (E = HE,

765

100x, F = 400x).

Table 1. Ingredient composition of the diets (%) and levels of detected AFs Ingredient

Control

Fish meal, Herring 72% CP Yellow Corn Soybean meal, 47% CP Wheat bran Corn gluten meal Fish Oil Vitamins and mineral premixa Salt (NaCl) Minazel-Plus®

MZ

AFs

AFsMZ

20.00 30.10 24.30 20.10 1.70 2.30 1.00 0.50 0.00

20.00 30.10 24.30 19.9 1.70 2.30 1.00 0.50 0.2

20.00 30.10 24.30 20.10 1.70 2.30 1.00 0.50 0.00

20.00 30.10 24.30 19.90 1.70 2.30 1.00 0.50 0.2

91.85 32.06 5.96 0.94 0.87 1.86 0.62 3007

91.82 32.01 5.90 0.93 0.88 1.86 0.62 3004

91.82 32.05 5.87 0.95 0.87 1.86 0.62 3007

91.80 32.03 5.93 0.94 0.87 1.86

Proximate analysis (% dry matter basis) Dry matter (%)* Crude protein (%)* Lipid (%) * Ca* P* Lysine ** Methionine** DE (Digestable Energy)** (kcal/kg)

3004

Aflatoxins concentrations in AFs and AFsMZ (µg /kg diet)

Yellow Corn Soybean meal a

9.87 (0.008 µM) 5.67 (0.004 µM)

Trace minerals and vitamins premixes were prepared to cover the levels of the micro minerals &vitamins for tilapia fish as recommended by (NRC, 1993). Vitamins premix (IU or mg kg-1 diet); vit. A 5000, vit. D3 1000, vit. E 20, vit. K3 2, vit. B1 2, vit. B2 5, vit. B6 1.5, vit. B12 0.02, Pantothenic acid 10, Folic acid 1, Biotin 0.15, Niacin 30. Mineral mixture (mg/kg diet); Fe 40, Mn 80, Cu 4, Zn 50, I 0.5, Co 0.2 & Se 0.2.

*analysed ** calculated value

Table 2. Residual levels of AFs (AFB1, 2, AFG1, 2) in liver and musculature of Nile tilapia fed naturally contaminated AFs diet (16 µg/kg-- 0.013 µM) with or without MZ supplementation (2 g /kg - 9.5 µM) for 8 weeks. Groups

Liver (µg/kg)

Musculature (µg/kg)

Control

ND

ND

MZ

ND

ND

AFs

1.292(0.001 µM)

0.263 (0.0002 µM)

AFsMZ

0.959 (0.0007 µM)

0.022 (0.00002 µM).

*ND = Not detected

Table 3. Growth performance and (HSI) of Nile tilapia fed naturally contaminated AFs diet (16 µg/kg) with or without MZ supplementation (2 g /kg) for 8 weeks. Group Time (Mean BWt)

Control

39.17 ±2.54

Day 0

MZ

AFs

44.67 ± 2.47

45.67 ± 2.82‡

‡

‡

AFsMZ

45.50 ± 2.69

Day 7

41.00 ±3.36

40.17 ± 2.40

44.50 ± 3.32

39.33 ± 3.80‡

Day 14

29.50 ± 1.91‡

43.00 ± 3.43

43.00 ± 2.54‡

33.17 ± 2.50‡

Day 28

35.33 ± 5.75

42.17 ± 3.86‡

46.33 ± 2.26‡

35.67 ± 3.80‡

Day 56

46.17 ± 6.19b†

56.83 ± 5.62ab†

62.83 ± 5.17a†

54.17 ± 3.84ab†

Group Time (K- factor)

Control

Day 0

1.68 ± 0.08

Day 7

1.57 ± 0.08

Day 14

1.45 ± 0.05

Day 28

1.73 ± 0.11 1.84 ± 0.08

Day 56

MZ

1.72 ± 0.18

1.65 ± 0.13

1.63 ± 0.08 b‡

†

1.42 ± 0.04

AFs

1.60 ± 0.06

1.65 ± 0.08 b

1.85 ± 0.08

AFsMZ

1.70 ± 0.11 a

1.66 ± 0.06ab

1.76 ± 0.12

1.89 ± 0.09

1.94 ± 0.15

1.69 ± 0.02

1.70 ± 0.06

1.73 ± 0.05

Group Control

MZ

AFs

AFsMZ

Time (HSI) Day 7

2.60 ± 0.33

2.27 ± 0.22

2.31 ± 0.20

3.14 ± 0.23

Day 14

3.12 ± 0.41

2.89 ± 0.30

2.81 ± 0.21

2.63 ± 0.21

Day 28

2.85 ± 0.30

2.77 ± 0.14

2.54 ± 0.13

2.98 ± 0.43

Day 56

2.60 ± 0.28

2.56 ± 0.10

2.85 ± 0.37

2.41 ± 0.19

Groups

WG (g)

BWI %

SGR %

control

7.00 ± 5.85

18.52 ± 14.80

0.10 ± 0.10

MZ

12.17 ± 4.51

27.08 ± 9.80

0.17 ± 0.06

MAFs

17.17 ± 4.39

38.44 ± 9.12

0.24 ± 0.05

AFsMZ

8.67 ± 4.85

21.72 ± 13.21

0.13 ± 0.08

Data were presented as the mean of six fish ± SEM. Values with a different letter superscript indicate significant difference between groups at same time. Values with different dagger superscript indicate significant differences between time points within the same group (P < 0.05). BWt/ body weighy, K factor/ condition factor, WG/weight gain, BWI/ body weight index, SGR/ specific growth rate, HSI/ hepatosomatic index

Highlights: • Aflatoxin levels below allowable concentrations in feed can induce immunotoxicity in Nile tilapia and significant histopathological changes in liver and kidney. • Aflatoxins in low concentration in fish diet have potential to bioaccumulate in liver and musculature of Nile tilapia. • Supplementation of Minazel-Plus® to aflatoxins contaminated diet was successful in mitigating overall adverse aflatoxin effects on fish health. • Minazel-Plus® supplementation alone enhanced antioxidant and immune responses in fish.