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Effects of aflatoxin B1 combined with ochratoxin A and/or zearalenone on metabolism, immune function, and antioxidant status in lactating dairy goats

Objective This experiment investigated the effects of aflatoxin B1 (AFB1) alone or mixed with ochratoxin A (OTA) and/or zearalenone (ZEA) on the metabolism, immune function, and antioxidant status of dairy goats. Methods Fifty lactating Laoshan dairy goats were randomly assigned to one of five treatment groups (n = 10) for 14 days. Goats were fed no additive (control) or administered with 50 μg AFB1/kg dry matter (DM) (AFB1), 50 μg AFB1/kg DM+100 μg OTA/kg DM (AFB1+ OTA), 50 μg AFB1/kg DM+500 μg ZEA/kg DM (AFB1+ZEA), or 50 μg AFB1/kg DM+100 μg OTA/kg DM+500 μg ZEA/kg DM (AFB1+OTA+ZEA). Results Dry matter intake and milk production were lower in goats fed AFB1+OTA+ZEA than in controls. Supplementation with AFB1, OTA, and ZEA significantly decreased red blood cell count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean platelet volume, and significantly increased white blood cell count, when compared with the control group. Compared with control, the combination of AFB1, OTA, and ZEA significantly increased alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities, total bilirubin (TBIL), interleukin-6, and malondialdehyde (MDA), but significantly reduced immunoglobulin A concentration, the activities of superoxide dismutase (SOD) and glutathione peroxides (GSH-Px), and total antioxidant capacity (T-AOC) in serum. Administration of AFB1 combined with OTA led to higher ALP, ALT, TBIL, and MDA, as well as lower milk production, SOD and GSH-Px activities, and T-AOC, than administration of AFB1 combined with ZEA. Conclusion The mixture of AFB1, OTA, and ZEA exerted the greatest adverse effects on dairy goats, meanwhile the deleterious damage of the other mycotoxin combinations were in varying degrees. The findings of this study could provide guidance for the prevention and treatment of the consequences of contamination of animal feeds with combinations of mycotoxin.


INTRODUCTION
Mycotoxins, secondary metabolites produced by three important genera of fungi, Aspergillus, Penicillium, and Fusarium, are commonly present in animal feeds. Aflatoxin B1 (AFB1), ochratoxin A (OTA), and zearalenone (ZEA) are recognized as the principal mycotoxins, and these are usually present simultaneously in cow feed and feed materials [1]. The dele terious effects of single doses of AFB1, OTA, or ZEA on ruminants have been reported by many authors. Kourousekos et al [2] reported that 50 μg/kg AFB1 significantly decreased milk production in indigenous Greek goats. Battacone et al [3] found that, while 128 μg/kg AFB1 significantly increased serum alanine aminotransferase (ALT) activity and decreased alkaline phosphatase (ALP) activity in dairy ewes, no significant effects of 32 or 64 μg/kg AFB1 were observed. AFB1con taminated diets reduced feed ingestion, had adverse effects on the liver, and caused immunosuppression at both 350 μg/kg and 23.5 μg/kg dry matter (DM) in sheep [4,5]. However, a study by Weaver et al [6] showed no changes in serum hema tological parameters, clinical health, sexual behavior, or tissue damage after cows received 31.25, 62.5, 125, 250, or 500 mg/ cow pure ZEA. Finally, Höhler et al [7] reported that 20 mg/kg OTA in concentrate feed greatly reduces feed intake, while 2 or 5 mg/kg significantly reduces ALT activity, in sheep.
However, animal feeds are not typically contaminated with a single mycotoxin; instead, two or more mycotoxins tend to occur together. In China, a combination of AFB1, OTA, and/ or ZEA occurred in up to 100% of 34 feed raw materials and compound feed samples for cows from 18 provinces in 2009 [8]. Studies of the toxic effects of a combination of AFB1 and OTA in rats [9], poultry [10,11], rabbits [12], and pigs [13,14], as well as the toxic effects of a mixture of AFB1 and ZEA in gilts [15], have been reported, but the reports of the toxic ef fects of AFB1 combined with OTA and/or ZEA were rare in ruminants.
Nevertheless, many studies about the combination of two mycotoxins have investigated on ruminants. A study by Abeni et al [16] showed that, in replacement heifers, a diet contain ing aflatoxins (AF) (>10 μg/kg) and fumonisins B1 (FB1)+ fumonisins B2 (5,000 to 20,000 μg/kg) delayed growth, but did not affect the hematological profile or immunity. Further more, Winkler et al [17] found that cows fed a diet containing 0.5 mg/kg ZEA and 5 mg/kg deoxynivalenol (DON) did not suffer significant effects on their performance. However, in an experimental study of dairy cows to which a diet contaminated with 1 mg/kg ZEA, 600 μg/kg DON, and 10 μg/kg AFB1 was fed, negative effects on somatic cell count, blood parameters, and immunity were identified [18]. Therefore, we investigated the toxic effects of mixtures of AFB1, OTA, and ZEA on milk yield, dry matter intake (DMI), blood metabolic markers, im mune function, and antioxidant status in lactating dairy goats.

Animals, experimental design, and diets
All animals involved in this study were cared for according to the principles of Chinese Academy of Agricultural Sciences Animal Care and Use Committee. Fifty lactating Laoshan dairy goats (193±14 d in milk, 1.36±0.4 kg milk/d yield, and 3.4±1.0 parity) were grouped according to parity (primiparous or multiparous), average daily milk production, and days in milk, and randomly assigned to one of five treatment groups (n = 10), according to a completely randomized block design. The goats received no additives (Control group), 50 μg AFB1/ kg DM (AFB1 group), 50 μg AFB1/kg DM + 100 μg OTA/kg DM (AFB1+OTA group), 50 μg AFB1/kg DM + 500 μg ZEA/ kg DM (AFB1+ZEA group), or 50 μg AFB1/kg DM + 100 μg OTA/kg DM + 500 μg ZEA/kg DM (AFB1+OTA+ZEA group). All the mycotoxins were administered in purified form, and the doses were chosen according to the Chinese National Standards for mycotoxins, which fixed the upper feed limits at 50 μg/kg for AFB1, 100 μg/kg for OTA, and 500 μg/kg for ZEA [19]. The experimental period consisted of 1 week of adaptation to the environment and 2 weeks' feeding with diet containing mycotoxins.
The basal diet (fed as a total mixed ration [TMR]; Table 1) was formulated to meet or exceed the nutrient requirements for energy, protein, minerals, and vitamins, according to the National Research Council [20]. During the experimental pe riod, all the goats were housed in a free stall barn with access to fresh water, and were fed four times daily, at 06:00, 10:00, 15:30, and 20:00, to ensure no more than a 5% refusal rate. Purified AFB1, OTA, and ZEA were separately dissolved in methanol, and pelleted concentrate was used as a carrier for the mycotoxins, which were topdressed in equal proportions onto the TMR at the morning feeding. The dose of methanol ad ministered to the control and treated groups was 1 mL.

Sampling and measurements
Feed consumption was measured on 2 consecutive days of each week. The DMI of individual goats was calculated by subtracting the mass of the feed remaining from that offered. The AFB1, OTA, and ZEA concentrations in the TMR were determined using the method of Ge et al [21]. The goats were milked twice daily (at 06:00 and 18:30), and the yield was recorded on each occasion. At the start and the end of the experiment, the goats were weighted on a platform scale prior to their morning feed.
Blood samples were collected from each goat before the morning feed by jugular venipuncture on days 2, 7, and 14. Nine milliliters of blood was collected in ethylenediamine tetraacetic acidtreated tubes and stored at 4°C until analysis of the hematological profiles. Nine milliliters of blood were also collected in vacuum tubes without anticoagulant, cen trifuged at 3,000×g for 15 min at 4°C to obtain serum, and stored at -80°C for later analysis of biochemical parameters, activities of antioxidant enzymes, immunoglobulin (Ig) A, IgM, and IgG levels, and concentrations of interleukin (IL)2, IL4, and IL6.

Statistical analysis
Data were analyzed using general linear models followed by Duncan's multiple range tests, on Statistical Analysis System Institute [22]. Treatment, time, and the treatment×time inter action were the fixed effects, while goat identity was a random effect. Analysis of variance was used for analyses of body mass and average daily gain (ADG). Values are presented as least squares mean±standard error of mean. Significant differences were accepted when p<0.05.

Effects of mycotoxins on performance
The effects of mycotoxin ingestion on goat performance are reported in Table 2. Goats fed AFB1 alone and AFB1 com bined with ZEA showed no significant (p>0.05) differences in DMI, milk yield, body mass, or ADG compared with the control group. However, DMI in the AFB1+OTA+ZEA group was significantly lower (p<0.05), while there were no signifi cant differences (p>0.05) in DMI between the other groups. Compared with the control group, supplementation with AFB1+OTA or AFB1+OTA+ZEA significantly (p<0.05) de creased milk production by dairy goats, but there was no significant difference (p>0.05) between the AFB1 and AFB1+ ZEA groups. Thus, AFB1 combined with both OTA and ZEA had the greatest adverse effects on performance, while AFB1 combined with OTA alone had a greater effect on milk pro duction than AFB1 mixed with ZEA.

Effects of mycotoxins on hematological parameters
The hematological parameters of the goats are summarized in Table 3. Except for WBC, RBC, MCV, MCH, and MPV, none of the hematological parameters were significantly affected by mycotoxin intake (p>0.05). RBC, MCV, MCH, MPV were significantly (p<0.05) higher, and WBC was significantly (p< 0.05) lower in the AFB1+OTA+ZEA group than in the other groups, indicating that the combination of AFB1, OTA, and ZEA has a particularly deleterious effect on hematology. In addition, the time of sampling affected all these hematological parameters, apart from HGB.

Effects of mycotoxins on serum biochemical parameters
A diet supplemented with AFB1+OTA+ZEA significantly (p< 0.05) increased ALT and ALP activities, and TBIL concen tration in serum, but there were no significant differences between the other treatments and the control group (Table   4). Goats fed AFB1+OTA or AFB1+OTA+ZEA showed sig nificantly (p<0.05) higher AST activity than the control group. The AST, ALT, and ALP activities, and the TBIL concentra tion, in the AFB1+ZEA group were lower (p>0.05) than those in the AFB1+OTA group, but no significant differences were observed between the AFB1+OTA and AFB1+ZEA groups. The time of sampling affected the serum concentrations of ALP, CR, and TP, and tended to affect TC. There was a treat ment×time interaction for serum Glu and a tendency for such an interaction with ALP. These results suggested that there was a synergistic or additive effect of combining AFB1 with OTA and ZEA on serum biochemical parameters, and that the com bination of AFB1 and OTA exerted a greater effect than that of AFB1 and ZEA to increase ALP, AST, and ALP activities,  and TBIL concentration.

Effects of mycotoxins on immune function
There were no significant (p>0.05) differences in parameters indicative of immune function, except for IgA and IL6 levels ( Table 5). Serum IgA was significantly lower (p<0.05) and IL6 was significantly higher (p<0.05) after mycotoxin adminis tration, with the effects of AFB1+OTA+ZEA being most pronounced in each case. These findings suggest that AFB1, OTA, and ZEA had synergistic or additive effects to depress immune function. The time of sampling affected serum IgM and tended to affect IgG concentration. There was no treat ment×time interactions (p>0.05) with regard to immune function parameters, with the exception of IgM.

Effects of mycotoxins on antioxidant status
The activities of SOD and GSHPx in serum were significantly lower and the MDA concentration was higher (p<0.05) when goats were fed mycotoxins, compared with those fed the con trol diet ( Table 6). The animals receiving AFB1+OTA+ZEA had the lowest SOD, GSHPx, and TAOC, and the highest MDA concentration (p<0.05) in serum, indicating that all three mycotoxins together had a synergistic or additive effect to impair antioxidant status in goats. The serum activities of SOD and GSHPx, and TAOC, were also lower (p<0.05), and the MDA concentration was higher (p<0.05), when goats were fed AFB1+OTA, than when they were fed the control diet, but no significant effects were observed (p>0.05) in goats fed AFB1 alone or AFB1 combined with ZEA, suggesting that AFB1 combined with OTA has a more severe effect on antioxidant status in dairy goats than AFB1 combined with ZEA. The time of sampling affected all the serum antioxidant parameters, apart from TAOC, and each also displayed treatment×time of sam pling interaction.

Effects of mycotoxins on performance
The dairy industry plays an important role in human nutri tion worldwide. The feeding of dietary mycotoxins to dairy animals can harm them or impair milk production, leading to heavy economic losses. Thus, the control of toxin con tamination of food and feedstuffs is an urgent requirement for this industry. Because animal feed can naturally contain multiple mycotoxins, it is important to study the specific ef fects of the presence of combinations of mycotoxins on the  health and productivity of dairy animals. In this study, the effects of AFB1 combined with OTA and/or ZEA on dairy goat performance were investigated. In the present study, a significant decrease in DMI was observed in the AFB1+OTA+ZEA group compared with the other groups, suggesting that the combination of all three my cotoxins had greater adverse effects on the DMI of dairy goats than either AFB1 alone, or when combined with just OTA or ZEA. To our knowledge, the combined effects of these three toxins on DMI have not been investigated in ruminants. The study by Battacone et al [3] showed no significant difference in DMI because of feeding lactating dairy ewes with 32, 64, or 128 μg/kg AFB1. Similarly, no effects were observed when cows were fed a diet containing 500 mg/kg ZEA [6], or when sheep were administered 3.5 mg/kg OTA [7]. However, Kiyothong et al [23] found that a contaminated TMR (38 μg/kg AFB1, 541 μg/kg ZEA, 501 μg/kg OTA, 270 μg/kg T2 toxin (T2), 720 μg/kg DON, 701 μg/kg FB1) significantly decreased DMI in lactating dairy cows, suggesting that multiple mycotoxins were more effective than single contaminants at reducing DMI, despite the dose of each mycotoxin being relatively low. The data from the present study indicated that additive effects of AFB1, OTA, and ZEA combination on DMI was operated compared with the previous study.
Many authors have demonstrated that milk production decreases following a reduction in food consumption [24,25]. Consistent with this, in the present study there was a decrease in milk production alongside the decrease in DMI in goats in the AFB1+OTA and AFB1+OTA+ZEA groups. No previous studies have investigated how dairy goat milk production might be influenced by feeding of AFB1 combined with OTA and/or ZEA. However, Applebaum et al [26] observed a significant decrease in milk production by dairy cows receiving 13 mg/kg impure AF compared with cows receiving purified AF, indi cating that multiple mycotoxins might have had a greater effect on milk production than a single mycotoxin. There is grow ing evidence that reductions in milk production could be attributed to the consumption of multiple mycotoxins. For example, Kiyothong et al [23] observed that a contaminated TMR containing 38 μg/kg AFB1, 541 μg/kg ZEA, 501 μg/kg OTA, 270 μg/kg T2, 720 μg/kg DON, and 701 μg/kg FB1 de creased milk production in cows. The results of Ogido et al [27] also suggested that the toxicity of some individual my cotoxins could be enhanced in a synergistic or additive manner when they are simultaneously present in animal feeds. The lower milk production by goats fed a diet supplemented with AFB1 and OTA, rather than AFB1 and ZEA, may relate to the production of AFB1 and OTA by the same species. Myco toxins of similar structure that are produced by the same species or the same family are likely to exert additive effects [28]. Our findings suggest that further investigations should be conducted to clarify the interactions between AFB1 and OTA, and between AFB1 and ZEA.
The lack of effect of mycotoxins on body mass and ADG in the present study agrees with studies performed in heifers fed a mixture of AF and fumonisin as natural feed contaminants [16]. This is likely to be because the dietary protein and energy levels were the same in each diet in the present study.

Effects of mycotoxins on hematological parameters
The most striking effects of mycotoxin combinations on hema tological parameters were the lower WBC and higher RBC, MCV, MCH, and MPV. Such adverse effects have been report ed by many authors. Abeni et al [16] showed that a combination of AFB1 and fumonisin could reduce RBC and MCH, and increase MCV and HGB, while Dänicke et al [29] observed that ZEA and DON together also had a negative effect on hematology. However, the effects of combinations of AFB1 with OTA and/or ZEA on ruminant hematological parame ters have not been reported to date. In the present study, we observed higher WBC and lower RBC, MCV, and MCHC in goats fed AFB1 combined with ZEA than in those fed AFB1 alone, while there were no significant differences between ad ministration of AFB1 alone and AFB1+OTA. Consistent with these data, Battacone et al [3] observed no effects of AFB1 on hematological parameters in dairy goats that had received 32, 64, or 128 μg purified AFB1/kg DM, while Müller [30] found that ruminants are able to degrade up to 5 to 12 mg/kg ochra toxin in feed. Therefore, low levels of AFB1 combined with low concentrations of OTA do not affect the hematology of dairy goats.

Effects of mycotoxins on serum biochemical parameters
When liver cells are damaged or their cell membrane perme ability is increased, ALT, AST, ALP, and TBIL are released into the blood in larger quantities, and consequently higher enzyme activity can be detected [31]. In the present study, there were no significant differences in serum biochemical parameters between goats fed with AFB1 and the control group. This is in accordance with the study by Battacone et al [3], which found that 32 and 64 μg/kg AFB1 in the diet had no signifi cant effect on serum biochemical parameters in dairy goats, implying that these quantities of AFB1 alone did not damage hepatocytes.
In this study, AST activity was significantly higher in the AFB1+OTA group than the control group, while the AFB1 and AFB1+ZEA groups showed intermediate values. No sig nificant differences were observed in the activities of the other hepatic enzymes (ALT and AST) or between AFB1+ZEA and the control group, suggesting that AFB1+OTA caused more liver damage than AFB1+ZEA. However, the serum ALT, AST, ALP, and TBIL values of the AFB1+OTA group were higher than those of the AFB1+ZEA group, suggesting that AFB1 combined with OTA has more deleterious effects on the liver than AFB1 combined with ZEA. An explanation for these re sults may be that AFB1 and OTA are produced by the same species. Speijers et al [28] reported that mycotoxins produced by the same species are likely to have additive effects on the liver. In addition, Prabu et al [12] found an additive effect of AFB1 and OTA on liver damage in rabbits. By contrast, Sun et al [32] found that 2.5 mg AFB1/kg body mass and 5.0 mg ZEA/kg body mass had antagonistic effects on ALT, AST, and ALP activities in mice.
The increases in serum ALT, AST, ALP, and TBIL induced by supplementation with mixtures of mycotoxins, indicating an adverse effect in the liver, have not been demonstrated in many previous studies. However, the findings of Shreeve et al [33] were in accordance with our results. They observed that the mean tissue concentration of aflatoxin M1 was at least twice as high in animals fed an AFB1+OTAcontaminated diet than in those exposed to AFB1+ZEA, and therefore had more del eterious effects on the kidney. In addition, Prabu et al [12] reported that AFB1 combined with OTA caused more liver damage in rabbits than either AFB1 or OTA alone. Thus, a combination of AFB1, OTA, and ZEA may have had additive or synergistic effects on the liver, leading to significant increases in AST, ALT, and ALP activities, and TBIL concentration in serum.

Effects of mycotoxins on immune function
The decrease in serum IgA and the increase in IL6 were most pronounced in the AFB1+OTA+ZEA group, indicating that AFB1 combined with OTA and ZEA had additive or synergis tic effects to depress immune function. Previous work shows that mycotoxins induce liver toxicity, and that liver disease may result in an increase in serum IgA [34]. The current results agree with those of Whitlow and Hagler [35], who found that mycotoxins suppress the immunity of dairy cows. Korosteleva et al [36] also found a reduction in serum IgA in cows fed a diet containing Fusarium mycotoxins (fumonisins and DON), while Kiyothong et al [23] demonstrated negative effects of mycotoxins on rumen and immune function in dairy cows receiving a naturally contaminated TMR (containing 38 μg/kg AFB1, 270 μg/kg T2, 720 mg/kg DON, 701 mg/kg FB1, 541 mg/kg ZEA, and 501 mg/kg OTA).
Rats fed a combination of AFB1 and OTA showed increases in serum IL4 and H 2 O 2 , and a decrease in IL10, implying an impairment in immune function [37]. By contrast, we ob served a lower concentration of serum IgA and higher IL6 in goats fed an AFB1+ZEA diet than in those fed an AFB1+ OTAcontaining diet, suggesting that AFB1+OTA had a great er immunosuppressive effect than AFB1+ZEA. In agreement with this, Verma et al [38] reported that a combination of AF and OTA at higher dietary concentrations (2 mg/kg and 4 mg/kg) significantly decreased cellmediated immunity and hemagglutination titer, and caused severe reductions in the immune response of broilers. Other consistent data were re ported by Gao et al [39], who demonstrated in the Caco2 cell line that AFM1 and OTA exhibited synergistic or antagonistic effects, depending on whether low or high concentrations were administered, while an antagonistic interaction between AFB1 and ZEA occurred at all the concentrations tested, suggesting that the former combination is more cytotoxic.

Effects of mycotoxins on antioxidant status
The mechanism of mycotoxin toxicity involves oxidative stress and liver damage induced by lipid peroxidation, and the pro duction of free radicals, which attack the unsaturated bonds of membrane phospholipids and damage liver cell membranes [40]. The generation of reactive free radicals after mycotoxin administration can be sufficient to overwhelm the antioxi dant defense in the liver and cause cell damage, resulting in reductions in serum SOD and GSHPx activities and an in crease in MDA concentration [40]. Shen et al [41] found that 1 mg/kg body mass AFB1 can cause lipid peroxidation in rat liver, accompanied by an increase in MDA. In addition, Soyöz et al [40] reported that the level of lipid peroxidation and the serum SOD activity were higher and lower than control, re spectively, after 289 μg/kg per day OTA administration. In the present study, mycotoxin administration decreased SOD and GSHPx activities, and TAOC, and increased MDA concen tration in serum. These results are consistent with mycotoxin induced defects in antioxidant defense being induced via greater lipid peroxidation and liver damage. The AFB1+OTA and AFB1+OTA+ZEAinduced increases in ALT, AST, ALP, and TBIL were consistent with the reductions in SOD, GSHPx, and TAOC activities, and the increases in MDA concentra tion, also observed in these groups, together implying that these combinations of mycotoxins caused both lipid peroxi dation and liver damage.
In the present study, goats administered with AFB1+OTA+ ZEA lead to lower SOD and GSHPx activities, and TAOC, and higher MDA concentration significantly than any of the other groups. It suggest that AFB1, OTA, and ZEA have ad ditive or synergistic effects on the antioxidant status of dairy goats. In accordance with this finding, previous studies showed that the combination of AFM1, OTA, and ZEA exhibited syn ergistic or additive cytotoxic effects in the Caco2 cell line [39]. Additionally, Girish and Smith [42] found that the complex interactions between multiple mycotoxins can cause severe oxidative stress. Finally, Jiang et al [43] found that diets natu rally contaminated with mycotoxins (AF, 102.08 mg/kg; ZEA, 281.92 mg/kg; fumonisin, 5,874.38 mg/kg; DON, 2,038.96 mg/kg) induced oxidative stress in broiler chickens.
The serum activities of SOD and GSHPx, and TAOC, were lower, and the MDA concentration was higher, when goats were fed AFB1+OTA, compared with those fed the con