Alleviation of mycotoxin biodegradation agent on zearalenone and deoxynivalenol toxicosis in immature gilts

Experimental animals, diets and management

Feed samples were taken at the beginning and end of the experiment for ZEA, aflatoxin (AF), DON, and ochratoxin A (OTA) analyses. The concentrations of ZEA and DON in the moldy corn were 2.65 and 4.01 mg/kg, respectively. The concentrations of ZEA + DON in CO, COA, MO and MOA diets were 90.68 μg/kg + 33 μg/kg, 89.88 μg/kg + 30 μg/kg, 596.86 μg/kg + 796 μg/kg and 600.99 μg/kg + 782 μg/kg, respectively. AF and OTA were not detected in any of the diets. The MBA was composed of 40% B. subtilis ANSB01G, 40% Devosia sp. ANSB714 and 20% carrier (rice husk meal) by industrial fermentation and dry-processing technologies. The B. subtilis ANSB01G was from one batch-fermented in a Luria-Bertani medium at 37 °C for 24 h and dried at 65 °C. The total viable counts of fermented-dried B. subtilis ANSB01G and fermented-dried Devosia sp. ANSB714 were both 1 × 10⁹ colony forming unit (CFU)/g.

Experimental procedures and swine care used in this study were in accordance with guidelines of the care and use of laboratory animals issued by the National Institute of Health [23] and by China’s Ministry of Agriculture. Gilts were penned (pen size, 0.55 m × 2.2 m) individually with ad libitum access to water. All gilts were fed individually diet of 3 kg daily provided in two equal portions (07:00 and 14:00 h). Body weights were measured at 0, 7, 14, 21 and 28 d. Feeds intakes and refusals were recorded daily with automatic feed system (Nedap Velos, Nedap China Ltd.). Average daily gains (ADG), average daily feed intake (ADFI) and feed: gain (F:G) were calculated.

HPLC analysis

The concentration of ZEA, DON, AF and OTA in the diets were measured using HPLC method according to Chinese certification GB/T 23504–2009, GB/T 23503–2009, GB/T 18979–2003 and GB/T 23502–2009, respectively, with some small improvements [24]. The detection limits for these mycotoxins were 1.5 μg/kg for the ZEA, 0.1 μg/kg for the AF (AFB₁, AFB₂, AFG₁, and AFG₂), 0.02 mg/kg for the DON and 0.5 μg/kg for the OTA, respectively.

Vulva size and organ weight determination

The length, width and height of vulva of pigs were measured at 0, 7, 14, 21 and 28 d for calculating the vulva volume as an approximately cylindroid shape (π × vulva length × vulva width × vulva height/4) according to the method described by Zhao et al. [24] with a slight modifications. Liver, heart, kidney, spleen and reproductive organs (ovary + cornua uteri + vagina) were examined macroscopically for an evaluation of their general appearances and then weighed separately. The weights were expressed on the basis of relative body weight (g/kg).

Plasma immunology, antioxidant and hormone parameters

Samples of blood were obtained by venipuncture of the jugular vein after a 12-h fast at the end of the study. Blood was collected into vacutainers containing (Heparin sodium) as anticoagulant. Plasma was obtained by centrifugation at 3,000×g at 4 °C for 15 min and the immunology, antioxidant and hormone parameters were examined in the plasma samples.

The immunoglobulins including IgA, IgG and IgM were processed using the enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions and determined using a microplate reader (DNM-9202G, Beijing Perlang Co., Ltd., Beijing, China). The cytokines including ininterleukin-1β (IL-1β), interleukin-8 (IL-8) and interleukin-10 (IL-10) were measured using R-911 automatic radioimmunoassay counter (USTC Holdings Co., Ltd., China).

The activities of nitric oxide synthase (NOS), hydroxyl free radical (•OH), glutathione peroxidase (GSH-Px) and total superoxide dismutase (SOD) as well as the concentrations of malondialdehyde (MDA) in the plasma were measured using the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the kit instructions.

The plasma estradiol (E2), luteotrophic hormone (LH), prolactin (PRL) and follicle-stimulating hormone (FSH) were measured via the commercial radioimmunoassay kits (Beijing Chemical in Biotech Co., Ltd., Beijing, China) following the manufacturer’s recommended procedure. Briefly, the samples were incubated with iodine (¹²⁵I) in a monoclonal antibody solution. The incubated samples were then aspirated and the binding activity was measured using a gamma counter (γ-counter) (GC-1200, USTC Chuangxin Co., Ltd., Zonkia Branch, China). Hormone concentrations were measured according to each sample’s level of radioactivity.

Protein extraction and western blotting in the ovary and uteri

The right parts of ovaries and uterus carefully dissected from 16 pigs (four pigs for each treatment) were snap frozen in liquid nitrogen and stored at − 80 °C until the analysis. The expressions of caspase-8, Bax, Bcl-2, and caspase-3 in the ovary and uteri were estimated using Western blotting assays. Total protein was extracted via a P1250 kit (Apply gen Technologies Inc., Beijing, China) and the equal amounts of the extracted protein (50 μg) were estimated using 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after centrifugation at 1,000×g at 4 °C for 15 min. The estimated protein then was transferred to the nitrocellulose membranes (Millipore Co., Billerica, MA, USA) at 80 V for 1 h. Membranes were blocked with blocking solution (5%, w/v, non-fat dried milk in the Phosphate Buffered Saline with Tween-20 (PBST)). Each membrane was incubated with a specific primary antibody caspase-8 (1:1,000 dilution), Bax (1:500 dilution), Bcl-2 (1:1,000 dilution) and caspase-3 (1:500 dilution) at 4 °C overnight. After three washes with PBST (15 min), the membrane was incubated with the diluted HRP-conjugated secondary antibody (diluted 1:2,500 in PBST) at room temperature for 1.5 h. The immunoblots were visualized by enhanced chemiluminescence reagent (ECL) following exposure of the filters to X-O mat Kodak autoradiography films. The bands were visualized and quantified using Image J 1.42 software. The signal intensity was normalized to β-actin (Boster Biological Technology Co., Ltd., Wuhan, China). All experiments were conducted in duplicate sets.

Determination of zearalenone and its metabolites and deoxynivalenol in the liver

Zearalenone and its metabolites in the liver were analyzed using the method of Duca et al. [25] with a slight modification. Briefly, a quantity of 5.0 g of a grounded liver sample was mixed with 10 mL of a buffered solution of acetic acid-ammonium acetate (pH 4.8). This mixture was incubated for 15 h at 37 °C with 80 μL of a solution of β-glucuronidase/arylsulfatase with a pH 4.0 adjusted with glacial acetic acid. The mixture was then extracted with 10 mL of acetonitrile and 200 μL of NaOH solution (1 mol/L) while being stirred at 200 r/min for 60 min. The sample was centrifuged for 10 min at 5,000 r/min, and 10 mL of the supernatant was collected. The collected supernatant mixed with 40 mL of a phosphate-buffered saline solution (pH 7.4) and then filtered using glass fiber filter paper. The filtrate (40 mL) was passed through an immunoaffinity column (Clover Immuno Clean CF ZER, Clover, China) for the extraction and purification of ZEA and ZEA metabolites following the manufacturer’s instructions. The sample-loaded column was then washed with 10 mL of distilled water at 3 mL/min before the retained ZEA and ZEA metabolites were eluted using 2 mL of methanol. Then the methanol was evaporated to dryness under a gentle stream of nitrogen. The dried eluate was diluted in 200 μL of the mobile phase solution and 20 μL was injected into the HPLC system (Shimadzu LC-10 AT) for the separation and determination of the concentrations of ZEA and its metabolites.

Deoxynivalenol residues in the liver were analyzed using the method described by Zhao et al. [22]. Briefly, freeze-dried liver samples (0.5 g) were added with 60 μL of β-glucuronidase (EC 3.2.1.31, type H-2, 85,000 IU/mL, Sigma) and 3.0 mL of sodium acetate buffer (pH 5.5). The samples were extracted with a mixture of acetonitrile and water, followed by centrifugation at 5,000 r/min for 30 min after incubation at 37 °C for 12 h. After filtration, 2 mL of the extract was collected, with 8 mL of phosphate buffered saline (PBS, pH 7.4) containing 0.1% Tween-20 and centrifuged at 5,000 r/min for 30 min. The supernatant (8 mL) was passed through an immune-affinity column at a flow rate of 1 mL/min by gravity and washed with10 mL of PBS and 10 mL of double distilled water respectively, at a flow rate of 3 mL/min by gravity. DON was eluted using 2 mL of methanol, the eluent was evaporated by nitrogen flow stream at 40 °C, and the residue was dissolved in 100 μL of the mobile phase. Then, 20 μL of the eluent was injected into the HPLC system for the determination of the concentrations of DON.

Statistical analysis

The data were analyzed as a 2 × 2 factorial design using the MIXED procedure of SAS 9.1 (SAS Inst., Inc., Cary, NC, USA); toxins level, MBA level, and their interaction were fixed factors, and experimental period and animal were random factors. When there was an interaction, post hoc Duncan’s multiple range tests were used to analyze differences among treatment means. A value of P < 0.05 was considered as statistically significant.

Growing performance

The results clearly demonstrated that a diet contaminated with ZEA (596.86 μg/kg) and DON (796 μg/kg) had obviously negative effects on ADG and ADFI in the immature gilts. Similarly, Williams and Blaney [26] found that grower pigs fed a diet containing maize naturally contaminated with 11.5 mg/kg nivalenol (NIV) and 3 mg/kg ZEA had a deterioration of productive performance, such as decreased feed intake and ADG, in a 14-d experiment. The impact of ZEA + DON on eating behavior was also observed in our study and the ADFI of gilts fed with MO diet was decreased by 12% compared to the gilts fed with CO diet, indicating that combined DON and ZEA could induce anorexia of pigs. Clinical signs of hyperestrogenism and partial feed refusal were observed in gilts exposure to ZEA (64 mg/kg feed) and DON (4.5 mg/kg feed) [27]. It was reported that B. subtilis ANSB01G and Devosia sp. ANSB714 showed an effective ability to degrade ZEA and DON in vitro, respectively. Moreover, they displayed a high tolerance to simulated gastric fluid and intestinal fluid [21, 22]. In this study, B. subtilis ANSB01G and Devosia sp. ANSB714 could ameliorate the toxic effects of ZEA and DON in contaminated diet at low levels and improved the performance of gilts. It could be attributed to the ability of MBA to degrade ZEA and DON in the animal intestinal gut, consequently decreasing the toxicity of mycotoxin, which was consistent with the results of Zhao et al. [22, 24]. Different from previous study [24], chlortetracycline was added in all treatment diets in the current study; however, MBA could still alleviate the toxicosis caused by ZEA and DON, revealing that antibiotic would not influence the effectiveness of MBA.

Vulva size and organ weight

In vivo, 22.09 mg/kg ZEA in the diet caused negative alterations in the reproductive tract such as the uterus of pig, meanwhile this concentration of ZEA affected follicular and embryo development [28]. The clinical symptoms of ZEA toxicity in pigs consist of swelling of the vulva and prolapse of the vagina [24]. DON can also induce toxic effects on the ovaries, affecting follicular development and interfering with embryonic development in pigs [29, 30]. Vulva size increased linearly with the duration of feeding in gilts with diets containing 1.1 mg/kg of ZEA or greater [31]. In the present study, the vulva size measured on the day of 14, 21 and 28 were significantly smaller in the group MOA than that in MO group, supporting the fact that MBA can effectively mitigate the estrogenic swelling of the vulvas of immature female pigs caused by combined ZEA and DON.

Plasma immunological parameters

Our results demonstrated that plasma immunoglobulin concentrations were significantly influenced by dietary ZEA and DON. Similar results were also noticed in previous reports, in which the level of the plasma IgA was increased in rats due to ZEA exposure [33, 34]. The reason for this outcome was presumably that the increasing cytokines production promoted the generation of IgA and IgG in plasma of pigs exposed to the combined ZEA and DON. Cytokines exert an important function in the regulation of the immune response. In this study, toxins in diets caused obvious elevation of both pro- and anti-inflammatory cytokines (IL-1β, IL-8 and IL-10) in plasma of gilts. Any of cytokines IL-10 and IL-1β could directly or indirectly enhance differentiation of IgA-secreting B cells. It was reported that plasma IL-10 and IL-1β were increased when exposed to ZEA [33, 35]. The expression and synthesis of IL-1β and IL-8 were increased when exposure to ZEA-contaminated feed [34, 36]. Our observation gains strong support from these above reports. In this trial, MBA could reduce the elevated levels of IgG and IL-8, thus protecting the immune system of gilts from damage caused by combined ZEA and DON.

Plasma oxidant stress parameters

Oxidant stress can cause damage to all components in cells including cleavage of proteins, endogenous DNA lesions and lipid peroxidation. Reactive oxygen species (ROS) such as hydroxyl radical (•OH) and hydrogen peroxide (H₂O₂) are the primary products generated from these damages. A great attention has been paid to the oxidative stress induced by Fusaria mycotoxins in the last decades. For example, dietary ZEA induced serious oxidative injuries in both renal and hepatic tissues in mice via adverse impact upon the level of the MDA, and the activities of CAT and SOD [37]. GSH-Px and SOD are the key antioxidant enzymes that can scavenge ROS generated from oxidant stress. Consequently, the activities of GSH-Px and SOD have been recognized as the leading parameters of anti-oxidative stress. Ren et al. [38] reported that the concentration of ZEA + DON (2.5 mg/kg BW and 30 mg/kg BW) showed more obvious effects on the dysregulation of splenic antioxidant functions, like as •OH inhibition capacities. However, there was no significant difference in the plasma antioxidant indices among treatment groups in our research, which probably related to the low dose of ZEA and DON.

Plasma hormone parameters

Numerous researches demonstrated that ZEA binds to estrogen receptors, generating an estrogen-like response and causing serious hyperestrogenism in several animal species, particularly in pigs [39]. The immature gilts appeared to be more predisposed to ZEA insult during immature than other growth stage. This is in agreement with Chen et al. [40] who observed that serum levels of prolactin in gilts fed a diet with 2.0 mg/kg ZEA were significantly increased. Another study also reported a significant increase in the serum level of PRL in pigs consuming a diet containing 238.57 μg/kg of ZEA in comparison to diet without ZEA [24].

Apoptotic examination

The results of western blotting assay for caspase-8, Bcl-2, Bax and caspase-3 proteins in ovaries of gilts are depicted in Fig. 1. The expressions of caspase-3 protein in ovaries of gilts fed MO diet were higher, while the expressions of Bcl-2 protein were lower than those fed CO diet (P < 0.05). The incorporation of MBA into MO diet had alleviated the negative effects of ZEA and DON on these protein expressions in ovaries of immature gilts. There were similar effects of mycotoxin and MBA on the expression of these proteins in the uterus of pigs, displayed in Fig. 2.

The initiator caspase-8 can directly activate pro-caspase-3 without any accelerator [41]. Caspase-3 is considered to be the key protein in the execution of apoptosis [42], as well as both pro-apoptotic (Bax) and anti-apoptosis (Bcl-2) are decisive factor and play a pivotal role in determining whether cells survival or death. Bcl-2 can inhibit cytochrome C translocation, thereby block caspase activation and the apoptotic process [43]. In the present study, ZEA and DON have up-regulated the expression of apoptotic protein caspase-3 in ovaries and uteri, as well as down-regulated the expression of anti-apoptotic protein Bcl-2 in ovaries of gilts. DON can significantly increase the protein levels of p53 and Bax/Bcl-2 ratio in mouse thymic epithelial cell line 1 and induce mitochondrial dysfunction [44]. ZEA can induce apoptosis of porcine granulosa cells in a dose-dependent manner by a caspase-3- and caspase-9-dependent mitochondrial pathway [45]. The result of our study is reinforced by the reports above. In the present analysis, the up-regulated protein caspase-3 and down-regulated protein Bcl-2 may accelerate the apoptotic process in ovaries and uteri of gilts. It is determined that apoptosis in the ovary and the uterus is linked to hormonal secretion, which was potentially related to ZEA in diets which binds to oestrogen receptors and activates the transcription of oestrogen-responsive genes, thereby affects endocrine. Furthermore, there was a significant mutual interaction between toxin and MBA on Bcl-2 in ovaries and caspase-3 in uteri of immature gilts. It indicated that adding MBA in moldy diet contaminated with low levels of ZEA and DON may be an effective approach to recover the expression levels of these apoptosis-related proteins in ovaries and uteri of gilts.

Determination of ZEA and its metabolites and DON in the liver

Neither ZEA and its metabolites (α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL)) nor DON were detected in liver samples collected in this study, in agreement with the result of Zhao et al. [24]. It was difficult to find the metabolites of these two kinds of mycotoxin in the liver, serum or muscle tissues of the animal due to the low concentration (less than 1 mg/kg) in the diet.