Research Article

Ameliorative Effect of GSPE Against AFB1 Induced Immunotoxicity and Hepatotoxicity in Japanese Quail

Maha Khalil1, Gamal Shams2, Hosny Abdel Fadil2, Nagah Edrees2, Mostafa Abonorag1, Nasser El-Sabbagh3, Eman A. Ahmed4*

1Animal Health Research Institute, Agriculture Research Center, Ismailia 41511, Egypt; 2Pharmacology department, Faculty of Veterinary Medicine, Zagazig University, Egypt; 3Pharmacology department, Faculty of Veterinary Medicine, Alexandria University, Egypt; 4Pharmacology department, Faculty of Veterinary Medicine, Suez Canal University, 41522 Ismailia, Egypt.

Received | October 03, 2021; Accepted | December 30, 2021; Published | March 24, 2022

*Correspondence | Eman Ahmed, Pharmacology department, Faculty of Veterinary Medicine, Suez Canal University, 41522 Ismailia, Egypt; Email: eman_ahmed@vet.suez.edu.eg

Citation | Khalil M, Shams G, Fadil HA, Edrees N, Abonorag M, El-Sabbagh E, Ahmed EA (2022). Ameliorative effect of GSPE against AFB1-induced immunotoxicity and hepatotoxicity in Japanese quail. Adv. Anim. Vet. Sci. 10(4):898-904.

DOI | https://dx.doi.org/10.17582/journal.aavs/2022/10.4.898.904

ISSN (Online) | 2307-8316

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Mycotoxins, mostly aflatoxins (AF), immensely threaten human and animal health alike because of their teratogenic, carcinogenic, mutagenic, and immunosuppressive effects resulting from consumption of foods of animal origin such as eggs, milk, meat, cheese, with toxic residues (Nazhand et al., 2020). Moreover, AF exposure has been reported to elicit impaired productive performance, hepato-renal injury, or even mortality, paving the way for a substantial economic loss (Guo et al., 2021; Saleemi et al., 2020). Several studies thus far have linked AFB1 with liver damage via oxidative stress, cell apoptosis, and inflammatory response (Limaye et al., 2018; Wang et al., 2019). Recently, researchers have evinced growing interest in plant-derived natural agents as low toxic and potential therapeutic approaches. GSPE has appropriate antioxidant efficacy and free radical capturing amplitude (Abdou et al., 2021; Hussein et al., 2020). GSPE has a prophylactic effect on the cellular membrane versus oxidative damage, and as a consequence, it prohibits hepatic lipid peroxidation (Deng et al., 2020). Not to mention anti-microbial activity, GSPE has been indicated to have anti-cancer, a cardio-protective, anti-inflammatory, and hepatoprotective effect, antidiabetic effect, neuroprotective effect, anti-obesity, as well as lipid-lowering activities (Liu et al., 2016; Othman et al., 2020). Noteworthy, GSPE has proven to ameliorate oxidative stress and liver injury in the zearalenone-intoxicated liver (Long et al., 2016a; Taranu et al., 2020). Furthermore, it was reported that the AFB1-triggered toxicity in pigs could be mitigated by dietary grape seed meal through retrieving the inflammatory markers and oxidative status of AFB1-treated animals (Taranu et al., 2019). It prevents ROS-induced DNA damage and provides a potential protective effect against oxidative stress and free radical-induced pathological conditions (Liu et al., 2016; Sheng et al., 2020). Therefore, this study evaluate the protective impact of GSPE as an active additive in counteracting the deleterious effects of AFB1 in quails that are exposed to AFB1-intoxicated diets regarding biochemical, lipid peroxidation, pro-inflammatory cytokines, and histopathological parameters.

Materials and Methods

Ethical approval

Study experimental protocol was conducted according to Local Experimental Animal Care Committee and approved by the ethics of the institutional committee of the Faculty of Veterinary medicine, Zagazig University, Egypt.

Chemicals

Aflatoxin B1 is obtained from Animal Health Research Institute and added to the diet to get a concentration of 1 mg/kg diet (Rajput et al., 2017). GSPE is purchased from Green and healthy company at a dose of (500 mg/kg) (Rajput et al., 2019).

Animals and dietary treatments

A total of sixty (14 days-old) Japanese quail (Coturnix japonica) were obtained from a local hatchery. With free access to feed and water. Post 7 days of acclimatization, birds (average body weight = 128.56 g) were housed in stainless steel cages and randomly allotted to four treatment groups; 15 birds each. The 15 birds were alocated into three replicates of five birds per replica. A basal diet was formulated in order to meet the nutritional needs of Japanese quails (NRC, 1994; Table 1). The treatments were as the follows: The control group; diet free from feed additive or AFB1, AFB1 group; 1 mg/kg diet, GSPE group; 500 mg/kg diet and the 4th group fed diet containing aflatoxin and GSPE (500 mg/kg). Supplementation was continued for 5 weeks and the parameters for growth performance were recorded for all groups, taking into account body feed intake, weight gain, and weekly weight gain and feed conversion ratio (FCR).

Collection of samples and measurements

At the end of the trial, nine quails per group (3 from each replicate) were randomly selected, and blood samples were collected in a serum-separating tube. Samples were centrifuged for 15 min at 3000 ×g and kept for biochemical analyses. The liver was washed with normal saline and directed for oxidative biomarkers estimation and histological examination.

Serum biochemical and pro-inflammatory cytokines analysis

Serum samples were used for assessment of Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities. In addition, uric acid and creatinine analyses were performed to assess renal function. Tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were also analyzed using Enzyme-linked Immunosorbent Assay (ELISA) kits. All procedures were performed using commercial kits according to the manufacturer’s instructions (CUSABIO BIOTECH CO. Ltd., Houston, TX 77054, USA)

 

Table 1: Ingredients and nutrient composition of the experimental diets for quail.

Ingredients	%
Yellow corn (8.9%)*	56.81
Soybean meal (44.1%)*	33.20
Corn gluten meal (62%)*	6.90
Dicap	0.86
Limestone	1.35
Lysine	0.19
Methionine	0.09
Salt	0.30
Premix	0.30
Calculated composition
Crude protein (%)	24
ME (Kcal kg-1)	2900
Calorie/protein	120.8

 

*Determined values of crude protein %. #High mix premix, each 3 kg provide: Vitamin A - 12 mIU, vitamin D3-2 mIU, vitamin E - 1000 mg, vitamin K3-1000 mg, vitamin B1-1000 mg, vitamin B2-5000 mg, vitamin B6-1500 mg, vitamin - B12 10 mg, biotin - 50 mg, pantothenic acid - 10,00 mg, nicotinic acid - 30,000 mg, folic acid - 1000 mg, manganese - 60,000 mg, zinc - 50,000 mg, iron - 30,000 mg, copper - 4000 mg, iodine - 300 mg, selenium - 100 mg, cobalt - 100 mg, carrier (CaCO3) to 3 kg.

 

Oxidative stress markers evaluation

Malondialdehyde concentration (MDA) and Superoxide Dismutase activities (SOD), and GSH-Px, and Catalase content (CAT) were measured for estimation of antioxidant activity in liver tissues, using commercial kits (CUSABIO BIOTECH CO. Ltd., Houston, TX 77054, USA) following the assay kits instructions.

Histopathological analysis

Dissected and cleaned liver samples fixed in 10% neutralized buffered formalin, dehydrated in different alcohol percentages, cleared in xylene, and finally made as paraffin blocks. Tissue section (5 μm) stained with hematoxylin-eosin (H and E) for demonstration of histopathological alterations.

Statistical analysis

The statistical analysis was performed using the GraphPad Prism software (version 9, San Diego, USA). Kruskal Wallis was used to evaluate the difference between groups. Dunn’s multiple comparisons test as a Post hoc test was carried. The differences were significant when adjusted p < 0.05. The results are expressed as mean ± standard deviation.

Results and Discussion

Growth performance

The results of productive performance are summarized in Table 2. During the experiment, the birds that received AFB1 diet (1 mg/kg) showed significantly lower body weight and weight gain than other birds. The adverse signs of AFB1 were mitigated by including 500 mg/kg of GSPE into AFB1-contaminated diets compared with the birds fed AFB1 alone. Besides, FCR of birds was adversely affected by AFB1 along the trial (p < 0.05). Adding 500 mg/kg of GSPE caused marked improvement in FCR (p < 0.05) in AFB1 fed group. Remarkably, GSPE alone has no effect on the performance of quails.

 

Serum biochemical indices

As shown in Figure 1, birds fed the AFB1 diet recorded a significant increase in serum ALP and AST activities (p < 0.05). GSPE supplementation could mitigate AFB1-triggered liver damage by decreasing the ALT, AST, and ALP levels significantly. Creatinine level was elevated in AFB1 group in comparison to the control group (p < 0.05), whereas they were reserved in (GSPE+ AFB1) birds concerning the AFB1group (p < 0.05) (Figure 1).

Serum inflammatory cytokines

The inflammatory activity is estimated by the production of pro-inflammatory cytokine in serum, as shown in Figure 2. Notably, AFB1 treatment significantly increased serum IL- 6 and TNF-α levels (P < 0.05), whereas GSPE supplementation decreased the levels of these two inflammatory markers in comparison with the AFB1 birds (P < 0.05). The results proved that GSPE is able to mitigate the inflammatory response induced by AFB1.

 

Liver oxidative stress biomarkers

The effects of GSPE on hepatic antioxidant parameters of quails challenged with aflatoxin are presented in Figure 2. Data showed that AFB1 administration increased liver Malondialdehyde (MDA) content (p < 0.05), this content of MDA was almost restored in AFB1+GSPE group in comparison to the control level. The scavenging ability of antioxidant enzymes (SOD, GPx, and CAT) were decreased following exposure of quails to AFB1 (P > 0.05). Whereas GSPE counteracts this effect in the liver of AFB1

 

Table 2: Effects of grape seed proanthocyanidin extract (GSPE) on growth performance of quails fed AFB1contaminated diet.

FCR (%)	Feed intake (g)	Weight gain (g)	Final weight (g)	Initial weight (g)	Groups
1.49± 0.821	273.92±12.40	184.67±10.13	307.56±9.19	122.89±5.40	G1 (CON)
1.71±0.100*	270.35±5.91	156.67± 5.37*	287. 33±7.58 *	130.67±5.08	G2 (AFB1)
1.34±0.095	271.51±4.93	204.89±6.87	329.56±5.56	124.67±3.28	G3 (GSPE)
1.41±0.945	280.1± 6.13	199.78±7.55	330.67±4.56	130.89 ± 5.70	G4 (AFB1+GSPE)

 

Values are represented as the mean ± SD (n = 9). * Mean values within a column were significantly different (p < 0.05). AFB1, aflatoxin B1; GSPE, grape seed proanthocyanidin extract; FCR, feed conversion ratio.

 

treated group (P > 0.05). Co-administration of GSPE and AFB1 relieved AFB1-induced oxidative stress and lowered the MDA level.

Histopathological changes in liver

Microscopic examination showed no histopathological alterations in liver sections of control and GSPE group as presented in Figure 3. In contrast, AFB1 treatment-induced noticeable pathological alterations in hepatic tissues, such as marked fibrosis of portal triad area associated with focal dilatation of hepatic sinusoids, compared to the control group. Markedly, results clarified that the addition of GSPE (500 mg/kg) to AFB1-challenged diets alleviated liver injury elicited by AFB1. Whereas slight hydropic/fatty degeneration was noticed in the liver of GSPE + AFB1 group. Therefore, administration of GSPE (500mg/kg) can partially adverse AFB1 effect.

 

The present study was designed with the aim of assessing the ability of GSPE to alleviate the negative effects of AFB1 on growth performance, antioxidant capacity, biochemical

parameters, and liver injury evoked by aflatoxin. In this study, after five weeks, AFB1 administration (1mg/kg) significantly decreased the AWG and increase the FCR (p < 0.05). On the other hand, AFB1did not influence the daily feed intake along with the experiment. Overall, the results obtained regarding reduced final body weight and weight gain observed in quail chicks fed on AFB1 alone also accords with the earlier reported findings by (Mahrose et al., 2021) Previously, broilers exposure to AFB1 (0.5 mg/kg) significantly reduced weight gain and increase FCR and in turn economic losses (Rashidi et al., 2020). In another research, ducks fed an AFB1-contaminated diet revealed a significant diminished average daily gain and FCR (Han et al., 2008). The possible explanation for these results might be ascribed to the adverse effects of AFB1 on the metabolism of protein, lipid, carbohydraten, and the pancreatic enzymes activity, as well as bile acid concentration (Tessari et al., 2010). Previous results are in contrast to earlier findings reported by Manafi and Khosravinia (2013), who found that broilers fed diet contaminated by AFB1 (500 μg of /Kg) had no significant impact on weight gain along with the whole experiment (8 weeks) and no mortality was observed. This negative impact of AFB1 on growth performance could be alleviated by the addition of the GSPE. These results align with previous studies that revealed that supplementation of GSPE to AFB1-fed birds could improve growth performance compared to the AFB1-fed birds (Long et al., 2016c; Rajput et al., 2017).

The outcomes of determination of biochemical indices showed an elevation in serum ALT, AST, ALP, uric acid, and creatinine after the exposure of quails to AFB1. Increased AST, ALT, and ALP activities serve as diagnostic biomarkers of liver injury (Hussain et al., 2016). Additionally, elevated levels of uric acid and creatinine could indicate impairment of protein catabolism and renal impairment (Abdel-Wahhab and Aly, 2005). In sum, these findings support the idea of (Ismail et al., 2020), who reported that AFB1 feeding exhibited deleterious influences on the liver and kidney function in rabbits and broiler chickens. Conversely, quails received AFB1+GSPE diet (500 mg/kg) showed marked decreased in serum AST, ALT, and ALP as compared to the AFB1 birds. These findings match those observed in earlier studies by Rajput et al. (2017), who reported that AFB1 provoked hepatic damage while GSPE administration alleviated these adverse effects. Besides, (Deng et al., 2020) reported that ProcyanidinB2 treatment could partially mitigate the acute liver injury triggered by AFB1.

MDA regarded as a key marker that measured for determining the level of lipid peroxidation and cellular damage (Li et al., 2021). In addition, SOD, GSH-PX, and CAT act as pivotal endogenous elements in the antioxidant defense system. These markers play a crucial role in intracellular redox balance through scavenging the free radicals (Deng et al., 2020). In our experiment, AFB1 inducing oxidative stress in the liver, expressed as increased level of MDA and decreased antioxidant markers, including SOD, GSH-PX and CAT. These findings are in line with previous investigations of Li et al. (2021) and Wang et al. (2018), who reported that exposure to AFB1 might result in oxidative stress by reducing levels of antioxidant enzymes. Nevertheless, GSPE suppressed lipid peroxidation and enhanced the activity of antioxidant enzymes in quail liver via effective removal of ROS. Likewise, previous studies have demonstrated that GSPE could overcome the oxidative damage triggered by AFB1 (Rajput et al., 2018; Yousef et al., 2018).

Serum levels of pro-inflammatory cytokines are considered markers of cellular immunity. Currently, AFB1 group showed a significant elevation in serum pro-inflammatory cytokines such as TNF-α and IL-6. These results align with recent research indicating that AFB1 might lead to inflammation and alter the immune response (Hassan et al., 2020; Li et al., 2014; Long et al., 2016d). Nevertheless, GSPE supplementation significantly mitigated increased levels of TNF-α and IL-6 in the serum of AFB1-fed quails. In accordance, Rajput et al. (2017) and Rajput et al. (2019) reported that GSPE could significantly modify the elevated levels of inflammatory cytokines in serum broiler following ingestion of AFB1 diet. All these results also confirmed the anti-inflammatory and antioxidant action of GSPE that could be responsible for its decisiveness effect.

Aflatoxins adversely affect birds, referring to alterations of relative edible organ weights and histopathological changes (Fan et al., 2015; Liu et al., 2016; Rashidi et al., 2020). Histological results proved the protective role of GSPE against injuries provoked by AFB1. Interestingly, GSPE feed additives alleviated AFB1-histopathological changes in the liver. These results match those observed in earlier studies that found a strong protective effect of GSPE against AFB1-induced liver damage (Rajput et al., 2017; Long et al., 2016b). Our findings also accord with earlier observations, which showed that supplementation of grape seed meal (GSM) into the AFB1-contaminated diet mitigated liver injuries and reversed liver oxidative stress and inflammation of intoxicated piglets (Taranu et al., 2020).

Conclusions and Recommendations

The present study, GSPE inhibited AFB1-induced oxidative stress by reducing lipid peroxidation and up-regulating the activity of the antioxidant enzymes.

Novelty Statement

This study has demonstrated for the first time, that adverse effects of aflatoxin in Japanese quail can be alleviated by dietary supplementation of GSPE

Author’s Contribution

The authors confirm contribution to the paper as follows: study conception and design: EA, MK, and HA; experiment: MK, GS, and HA; data collection: NE and MA; analysis and interpretation of results: MA; All authors draft manuscript Preparation, reviewed the results and approved the final version of the manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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