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Research Article
 

Protective Effect of Urtica dioica Seeds’ Extract in Experimental Chronic Aflatoxicosis in Broiler Chickens



Erhan Ayna, Zübeyir Huyut, Omer Faruk Keleş, Zabit Yener and Ramazan Bal
 
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ABSTRACT

Background and Objective: Aflatoxins can lead to Aflatoxicosis in animals and humans. The aim of this study was to investigate hepatoprotective and antioxidant effect of Urtica dioica seeds’ extract (UDSE) on chronic aflatoxicosis in broiler chickens, which were evaluated by histopathologically liver degeneration and activities/levels of the oxidative stress and antioxidant parameters. Materials and Methods: Thirty-two broiler chickens were randomly divided into four experimental groups: control group served as a negative control and were fed with a normal diet; AFB1 group received aflatoxin B1 (AFB1) (1 mg kg1 diet) for 42 days; UDSE group received UDSE (30 mL kg1 diet) for 42 days and AFB1+UDSE group received AFB1 (1 mg kg1 diet) as well as UDS extract (30 mL kg1 diet) for 42 days. Results: In the AFB1 group, activities of SOD, GSH-Px and CAT and the levels of TAS and BcL-2 in liver and serum were significantly lower than that of the control group (p<0.001), TOS and Caspase-3 levels were also significantly high (p<0.001). Whereas, in the AFB1+UDSE group, the activity of GSH-Px in liver and serum and the activities of SOD and CAT and the level of TAS in serum were significantly higher than those in AFB1 group (p<0.001). In the AFB1+UDSE group, the levels of TOS and Caspase-3 were significantly lower (p<0.001) and the level of BcL-2 were higher in both liver and serum compared to those of the AFB1 group (p<0.001). Histopathologically, diffuse hydropic degenerations, periportal fibrosis and activation of perisinusoidal myofibroblastic (Ito) cells were observed in the AFB1 group. These morphological changes were reduced significantly in the AFB1+UDSE group. Conclusion: The UDSE has a protective effect on chronic aflatoxicosis in broiler, which appear to be attributable to its antioxidant and anti-apoptotic activity.

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  How to cite this article:

Erhan Ayna, Zübeyir Huyut, Omer Faruk Keleş, Zabit Yener and Ramazan Bal, 2021. Protective Effect of Urtica dioica Seeds’ Extract in Experimental Chronic Aflatoxicosis in Broiler Chickens. International Journal of Poultry Science, 20: 256-269.

DOI: 10.3923/ijps.2021.256.269

URL: https://scialert.net/abstract/?doi=ijps.2021.256.269
 
Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

 INTRODUCTION

Toxicity caused by contamination of mycotoxins in food is one of the main problems in the poultry industry1. The most dangerous mycotoxins are aflatoxins, especially aflatoxin B1 (AFB1), which are produced naturally by several species of Aspergillus in agricultural products. Aflatoxins are the most researched mycotoxin group in the sensitive laboratories due to their toxic and carcinogenic effects in farm animals, acute toxicological and chronic hepatocarcinogenic effects in humans2.

Aflatoxicosis causes damage to the liver, kidney and digestive system in the poultry, suppression of the immune system and, consequently, an increased sensitivity to many diseases. The liver, which performs the biotransformation and detoxification of various xenobiotics including steroids, eicosanoids, pharmaceuticals, pesticides, pollutants and carcinogens in the body, is the organ primarily affected by aflatoxicosis3. Toxication causes the enlargement, pallor and congestion of the liver and degeneration and necrosis in hepatocytes, proliferation in the bile duct4. Aflatoxicosis causes nephrosis in the kidney and impaired glomerular filtration and kidney function5,6. It impairs intestinal morphology, digestive ability, activities of digestive enzymes, intestinal microflora and intestinal natural immunity in the digestive system7-9. It causes damage to the lymphoid tissues such as thymus, bursa fabricus, spleen and bone marrow and suppression of phagocytic activity in the immune system, ultimately it reduces their disease resistance and humoral defence functions2,10. Additionally, it causes a decrease in feed consumption, growth rate, total body weight and egg production due to these systemic effects of AFB111.

Physical, chemical and biological methods are used for detoxification of AFB1 in feeds. Because AFB1 causes cell damage, free radical production and lipid peroxidation, it has been reported that the use of antioxidants known as superoxide anion cleansers may aid the overall detoxification process in the liver. For this reason, it has been suggested that the use of adsorbents and antioxidants in the diet may be effective, economical and practical to prevent aflatoxicosis in poultry12.

Because of its rich composition, nettle is one of the most researched natural antioxidants13. Previous studies showed that nettle has various pharmacological activities such as immunomodulator, antioxidant14-16, antibacterial15, anti-viral17, analgesic, anti-inflammatory18-20, antihypertensive, anti-hyperlipidemic and antidiabetic21-23, hepatoprotective24,25 and anticarcinogenic26. In addition, in a previous study27 with a trial period of 21 days, U. dioica extract was added to food against the aflatoxicosis and very positive results have been reported. In the present study, we aimed to investigate protective and antioxidant effects of Urtica dioica seeds’ extract on chronic aflatoxicosis in broiler chickens with a trial period of 42 days, which were evaluated by histopathologically liver degeneration, biochemically oxidative stress and antioxidant parameters, anti-apoptotic factor BcL-2 and Caspase-3 and immunohistochemically actin filaments in the liver tissue.

 MATERIALS AND METHODS

Chemicals: Sodium hydrogen phosphate (Na2HPO4) to prepare phosphate buffer as well as aflatoxin were purchased from Sigma Aldrich Company (St. Louis, MO, United States). ELISA kits for all biochemical parameters measured in liver and serum samples were purchased from YL Biotech Co., Ltd. (Shanghai, China).

Grouping of experimental animals and nutrition program: In this study, 32 Avian breed broiler chicks, aged one day and without vaccine, were used. The ethics committee approval for the study was obtained from the Adana Veterinary Control Institute Experimental Animals Local Ethics Committee (approval protocol number: 2019-2/390). The chicks having equal live body weight were randomly divided into 4 equal groups. The groups were given water with 5% sugar for the first 4-5 h and for the next 42 days, they were given ad libitum access to water and food. During this period, birds were housed in compartments at a constant ambient temperature of 22°C with a 12 h light/dark cycle.

The following were the four animal groups:

  • Control group (N = 8): This group served as a negative control and broiler chicks in this group were fed only standard diet during the whole study period.
  • AFB1 group (N = 8): The broiler chicks in this group were fed with feed containing AFB1 (1 mg of AF kg1 of food) for 42 days.
  • UDSE group (N = 8): Broiler chicks in this group were fed with feed containing UDSE (30 mL kg1 of food) for 42 days.
  • AFB1+UDSE group (N = 8): The broiler chicks in this group were fed with AFB1 (1 mg of AF kg1 of food) and UDSE (30 mL kg1 of food) for 42 days.

All the experimental chicks were weighed at the beginning of the experiment. Total feed consumptions were measured and recorded on days 7th, 14th and 21sd, 28th, 35th and 42th.

Creating feed with aflatoxin: One mg of AFB1 was added to one kilogram of commercial feed, which was found to have no aflatoxin by laboratory analysis (mg kg1).

Plant materials and extraction procedure: Urtica dioica seeds were used as plant material. The seeds were ground in an electric mill and their natural oil was extracted in a rotary evaporator using diethyl ether as solvent28. The extract (30 mL kg1) was added to the feed.

Collection of serum samples: At the end of the trial period blood samples were collected into yellow-capped biochemistry tubes and broilers were euthanized via cervical dislocation. For biochemical analysis, serum samples were obtained by centrifuging of blood at 3000 xg for 15 min. at +4°C.

Homogenization of liver tissues and supernatant production: Only 1.8 mL of phosphate buffer (50 mM) with a pH of 6.7 and was added to 200 mg of tissue and homogenized for about 15 s with a homogenizer. The mixture was then centrifuged at 3000 xg and +4°C for 30 min. The supernatant in upper phase was collected and then put in another tube and kept at -80°C for the following analysis.

Biochemical measurements in serum and liver samples: The activities of SOD, GSH-Px and CAT and the levels of TAS, TOS, BcL-2 and Caspase-3 in the serum and liver tissue homogenates were measured with commercially purchased enzyme-linked immunosorbent assay (ELISA) kits (ELISA, YL biont, Shanghai YL Biotech Co., Ltd.) in accordance with the package insert.

Histopathological examination: At the end of the experiment, necropsies of the subjects were performed and the liver tissue samples, which is considered as the main target organ for AFB1 toxicity, were collected. The macroscopic changes were observed and recorded. The liver tissue samples were fixed in a 10% formalin solution and were then embedded in paraffin blocks. After that 4 μm sections were taken with a rotary microtome. The sections were stained with hematoxylin-eosin (HE) and, when needed, some sections were stained with Masson's trichrome and Sudan black dyes and then examined under the microscope.

Immunohistochemical examination: In immunohisto-chemical examination, the expression of alpha-smooth muscle actin (ASTHMA) were determined using the streptavidin/biotin immunoperoxidase kit (Histostain-Plus Bulk Kit; Zymed, South San Francisco, CA, USA) according to the streptavidin-peroxidase method (ABC). After the sections were taken on adhesive slides, they were passed through xylene and alcohol series. The sections were washed with phosphate buffer solution (PBS) and incubated in 3% H2O2 for 20 minutes for inactivation of endogenous peroxidase. The sections were placed in the antigen retrieval solution (citrate buffer) and covered and then heat-treated twice for 20 min. After this, they were taken out of the oven, it was waited until reached at the room temperature and then the tissues were washed again with PBS, they were blocked by protein blocking (non-immune serum) for 20 minutes. Polyclonal antibodies of ASTHMA (Novocastra-NCL-L-CD8-4B11, 1/100 dilution) were added to the tissues and incubated overnight at +4°C. After that, the sections were washed with PBS and incubated for 20 min at room temperature with the biotinylated secondary antibody. The sections washed again with PBS and then kept in streptavidin-peroxidase for 20 min. After washing with PBS, diaminobenzidine (DAB) was dropped and incubated for 1-2 min. Then all tissues were kept in Mayer's hematoxylin for 1-2 min and washed in tap water. The sections were passed again through the alcohol and xylene series and were then covered with Entellan. Negative controls were used to confirm staining. These slides were reacted with PBS instead of primary antibodies. Sections were examined and photographed under a light microscope (Nikon 80i-DS-RI2).

Statistical analysis for biochemical data: All statistical analyses were performed using the Statistical Package for Social Science (SPSS) version 21.0 for windows (SPSS Inc., Chicago, IL, USA). Descriptive statistics of the groups were expressed as mean and standard deviation. Kruskal-Wallis non-parametric test was used to determine whether the differences between the groups were significant in the same parameter. Post-hoc test (multiple comparison testes) was performed to determine from which group the significant differences were originated. Values of p less than 0.05 were considered significant.

 RESULTS

Live weight data: The effects of AFB1-containing feed with and without UDSE on live body weight gain (BWG) of the broilers after feeding for up to 6 weeks are summarized in Table 1. The average live weight of the groups was similar at the beginning of the study (day 0). The body weight gain was significantly lower in the aflatoxin treated groups than that of the control group at all weekly measurements (p<0.05). However, improvement was observed in body weight gain of broiler chicks fed with AFB1 and UDSE; there were no significant difference in terms of live body weight between AFB1+UDSE group and the control group at any weekly measurements (p>0.05).

Biochemical data: In order to evaluate protective and antioxidant effects of Urtica dioica seeds’ extract on chronic aflatoxicosis in broiler chickens, activities of SOD, GSH-Px and CAT and the levels of TAS, TOS, BcL-2 and Caspase-3 were measured in the liver homogenates as well as the serum. Effect of aflatoxin B1 with and without Urtica dioica seeds’ extract on the activities of SOD, GSH-Px and CAT and the levels of TAS, TOS, BcL-2 and Caspase-3 in the serum and the liver tissue is shown in Table 2 and Table 3 respectively.

In the liver homogenate, the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and the levels of TAS and anti-apoptotic factor BcL-2 in AFB1 group were significantly lower than that of the control group (p<0.001) and the levels of TOS and caspase-3 were significantly higher in AFB1 group than that of the control group (p<0.001) (Table 2 and Fig. 1). In addition, the activities of SOD, GSH-Px and CAT and the level of TAS in the UDSE group were significantly higher than those of the control and AFB1 groups (p<0.001). However, as in AFB1+UDSE groups, the administration of Urtica dioica seeds’ extract as well as AFB1 significantly elevated activities of hepatic SOD and GSH-Px and CAT (p<0.001) compared to AFB1 group. Note that, the level of TOS in AFB1 group was higher than those in all other groups (p<0.001) and with the addition of UDSE, in AFB1+UDSE group the level of TOS was lower compared to AFB1 group (p>0.001).