Mycotoxins are toxic secondary metabolites produced by certain species of molds that commonly contaminate agricultural crops [1
]. The fungus Fusarium graminearum
has the ability to produce several mycotoxins including deoxynivalenol (DON) and zearalenone (ZEA). For both mycotoxins, swine are one of the most sensitive species [1
]. Deoxynivalenol can impact gut health, alter brain neurotransmitter concentrations, alter immunity, and cause organ damage. On the other hand, ZEA has a structure similar to estradiol-17β that allows binding to estrogen receptors which can result in embryonic death, smaller litters, and smaller offspring [2
Although not feeding animals mycotoxin contaminated grains is the ideal way to reduce the harmful effects of mycotoxins, contaminated feed may be unavoidable. Thus, to reduce toxic effects within the animal, feed additives with mycotoxin mitigation properties can play an important role [6
]. Products containing yeast materials have potential to adsorb mycotoxins due to the physical properties of the yeast cell wall, which has structures that allow for binding of mycotoxins [8
]. Some yeast materials may also improve the health of pigs through their prebiotic properties, which in turn can protect gut health, benefit the immune system, and improve performance [12
Deoxynivalenol and ZEA have been previously shown to be harmful mycotoxins for swine, further information is needed on how these mycotoxins impact pig organ health, immunity, and oxidative stress when these mycotoxins simultaneously contaminate feedstuffs. The objective of this study was to determine the effects of feeding corn naturally contaminated with DON and ZEA on pig performance and health status. Additionally, this study investigated the ability of two yeast based feed additives to help pigs to manage the mycotoxin challenge.
4. Experimental Section
4.1. Animals and Experimental Diets
Eighty four gilts (9.1 ± 0.1 kg, crossbred pigs, Smithfield Premium Genetics, Rose Hill, NC, USA) averaged six weeks of age, were used in this study. Pigs were housed in solid concrete floor indoor pens (1.42 × 3.86 m) at the North Carolina State University Swine Evaluation Station (Clayton, NC, USA). Pigs were grouped by body weight (BW) and randomly assigned to four treatments within a BW group. Each treatment had seven replicates and three pigs per pen.
Corn naturally contaminated with mycotoxins was identified and the mycotoxin concentrations were confirmed by Veterinary Diagnostic Laboratory at North Dakota State University (Fargo, ND, USA). Corn was analyzed for 19 mycotoxins including DON and ZEA. Quantification of DON and ZEA was conducted using GC-MS. Corn contained DON (25 mg/kg) and ZEA (3.4 mg/kg) and used to make experimental diets (Table 7
). This contaminated corn was blended with corn without mycotoxins in order to reach analyzed levels of 4.8 mg/kg DON and 0.3 mg/kg ZEA in the final diets. Non-contaminated corn was also used to formulate a control without mycotoxins. Mycotoxin analysis in corn and final diets was completed by collecting 10 samples from different locations to obtain a representative mixture [40
]. The 10 samples were combined and thoroughly blended together before two subsamples were collected for analysis of mycotoxin content. Mycotoxin contaminants were measured by the North Dakota State Veterinary Diagnostic Laboratory (Fargo, ND, USA). The level of DON in contaminated grains was determined using gas chromatography-mass spectrometry with a quantitation limit of 500 μg/kg.
Pigs were fed experimental diets based on their assigned treatment groups representing: CON (control); MT (4.8 mg DON/kg and 0.3 mg ZEA/kg); MT-YC (MT + 2 g/kg of a yeast cell wall product, Integral, Alltech Inc., Nicholasville, KY, USA); and MT-YF (MT + 2 g/kg of a yeast fermentation product, Original XPC, Diamond V, Cedar Rapids, IA, USA). The yeast cell wall based product in MT-YC is composed of hydrolyzed yeast which includes the cell wall fraction of the organism, whereas the yeast fermentation product in MT-YF is the dried anaerobic fermentation product from Saccharomyces cerevisiae. All experimental diets were fed for 42 days, and average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were determined. During the entire experimental period, all pigs had free access to feed and water. Concentrations of essential nutrients met requirements suggested by the National Research Council (1998). A protocol for the use of animals in this study was approved by North Carolina State University Animal Care and Use Committee.
Composition of experimental diets (as-fed basis).
Composition of experimental diets (as-fed basis).
|CON (%)||MT (%)|
|Ground yellow corn 2||72.00||52.00|
|Ground yellow corn with mycotoxins 3||0.00||20.00|
|Soybean meal, dehulled||25.30||25.30|
|Vitamin premix 4||0.03||0.03|
|Trace mineral premix 5||0.15||0.15|
|Calculated composition|| || |
|Dry matter, %||89.6||89.6|
|Metabolizable energy, Mcal/kg||3.37||3.37|
|Crude protein, %||18.0||18.0|
|True ileal idgestible Lys, %||0.83||0.83|
|True ileal idgestible Cys + Met, %||0.54||0.54|
|True ileal idgestible Trp, %||0.18||0.18|
|True ileal idgestible Thr, %||0.58||0.58|
|Available P, %||0.23||0.23|
|Total P, %||0.54||0.54|
|Deoxynivalenol (DON), mg/kg||0.00||5.00|
|Zearalenone (ZEA), mg/kg||0.00||0.68|
|Analyzed composition|| || |
|Deoxynivalenol 6, mg/kg||0.36||4.82|
|Zearalenone 6, mg/kg||ND 7||0.33|
4.2. Blood Sampling
The pig with the median initial BW from each pen was bled on day 42 for immunological, hematological, and biochemical analysis. Blood was collected in Monovette tubes (Sarstedt, Newton, NC, USA) without anticoagulant to obtain serum for liver biochemistry, immunoglobulin, cytokine, and oxidative stress parameters. Blood was allowed to clot before centrifuging for 15 min at 3000 g (4 °C) to collect serum, and samples were stored at −80 °C until analyzed. Blood samples were also collected in tubes containing EDTA to obtain whole blood for hematological analysis.
4.3. Immunological and Oxidative Stress Parameters
The immunoglobulin subset immunoglobulin G (IgG) was measured via enzyme linked immunosorbent assay (ELISA), as described by the manufacturer (Bethyl, Montgomery, TX, USA). Goat anti-pig IgG was used as a capture antibody to coat wells. Serum samples were diluted to 1:100,000. Horseradish peroxidase goat anti-pig IgG was used as a detection antibody in combination with the tetramethylbenzidine enzyme substrate. A stop solution of 0.18 M sulfuric acid (H2SO4) was used to stop the enzyme-substrate reaction. Absorbance was read at 450 nm using a Synergy HT ELISA plate reader (BioTek Instruments, Inc., Winooski, VT, USA) and Gen5 data analysis software (BioTek Instruments, INC, Winooski, VT, USA). Samples were quantified relative to the standard curve constructed with known amounts of pig IgG. The ELISA IgG detection limit was 7.8–500 ng/mL.
The cytokine tumor necrosis factor alpha (TNFα) was measured in serum by ELISA following the manufactures procedure (R&D Systems, Minneapolis, MN, USA). A total of 50 μL assay dilute was added to microplate wells coated with a monoclonal antibody specific to porcine TNFα, followed by 50 μL of standard, control, or sample. Detection occurred by the use of a color reagent substrate and a stop solution of diluted hydrochloric acid, and absorbance was read at 450 nm and 540 nm. The detection limit range for TNFα was 2.8–5.0 pg/mL.
The parameters malondialdehyde (MDA) and 8-hydroxy-deoxyguanosine (8-OHdG) were measured in serum as indicators of oxidative stress. Lipid peroxidation was measured by MDA using TBARS assay following the manufactures protocol (Cell Biolabs, Inc., San Diego, CA, USA). Samples and standards were added to microcentrifuge tubes, followed by SDS lysis solution and thiobarbituric acid (TBA). All tubes were incubated at 95 °C for 50 min, and then placed on ice for 5 min to cool before being centrifuged at 3000 rpm for 15 min. The supernatant was removed to a 96 well microplate and absorbance was read at 532 nm using the Synergy HT ELISA plate reader. The MDA content was determined in samples by comparison with the MDA standard curve.
Production of 8-OHdG was determined by ELISA (Cell Biolabs, Inc., San Diego, CA, USA) following protocol to determine oxidative DNA damage. Undiluted samples were added to an 8-OHdG conjugate coated microplate, followed by diluted anti-8-OHdG antibody, and finally diluted secondary antibody enzyme conjugate. After incubation, the provided stop solution was added to each well, and allowed to incubate for 8–10 min before being stopped with a stop solution in order to achieve a color change which was not over saturated. Samples were then measured at 450 nm and concentration determined based on the standard curve.
4.4. Hematological and Biochemical Assays
Whole blood with EDTA was sent to Antech Diagnostics (Cary, NC, USA) for complete blood counting (CBC). Measurements included hematocrit, hemoglobin, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), platelet number, red blood cell (RBC) count, white blood cell (WBC) count, basophils, eosinophils, lymphocytes, monocytes, and neutrophils.
Concentrations of serum alanine aminotransferase (ALT), albumin, alkaline phosphatase, aspartate aminotransferase (AST), bilirubin, BUN to creatinine ratio (BUN:creatinine), calcium, chloride, cholesterol, creatinine, creatine phosphokinase (CPK), globulin, glucose, phosphorus, potassium, sodium, and urea nitrogen were measured (Antech Diagnostics, Cary, NC, USA) for determination of liver biochemistry.
4.5. Organ Collection and Analysis
Measurements of vulva height and width were taken from one pig per pen (median initial BW pig) on day 42, before being anesthetized for tissue collection. Samples of the liver, left kidney, spleen, jejunum, and uterus were collected and weighed before being fixed in either 10% buffered formalin or liquid nitrogen. Tissues in formalin were sent to the North Carolina State University Histopathology Laboratory (College of Veterinary Medicine, Raleigh, NC, USA) for hematoxylin and eosin (H&E) staining and slide preparation. Samples in liquid nitrogen were stored in at −80 °C until further analysis.
Microscopic examination of tissue damage for the liver and kidney were measured by a histopathologist blinded to treatment (College of Veterinary Medicine, Raleigh, NC, USA). Damages were based on the degree of change observed with values of 1: normal to minimal damage (0%–5%); 2: mild (5%–15%); 3: moderate (15%–40%); 4: severe (higher than 40%). Liver damage measurement included bile ductule hyperplasia, fibrosis, hydropic degeneration, inflammation, karyomegaly, necrosis, and vacuolation. Kidney damage measurement included fibrosis, inflammation, necrosis, protein casts, regeneration, and vacuolation. Jejunum villi length and crypt depths were measured using an Olympus Vanox microscope (Olympus Corporation, Center Valley, PA, USA) and Spot Advanced software program (SPOT Imaging Solutions, Sterling Heights, MI, USA). Jejunum tissue was also measured for MDA concentration. This analysis was completed by homogenizing tissue in PBS and resulting supernatant was analyzed for MDA by TBARS assay (Cell Biolabs, INC., San Diego, CA, USA) as previously described. Tissue samples were also analyzed for protein content (BCA Protein Assay Kit, Pierce Biotechnology, Rockford, IL, USA) after a 1:10 dilution for determination of MDA per mg protein. Measurements of uterus longitudinal muscle, circular muscle, submucosa, and mucosa thicknesses were collected using the Olympus Vanox microscope and Spot Advanced software.
4.6. Statistical Analysis
Data was analyzed using the GLM procedures of SAS (SAS Inst. Inc., Cary, NC, USA) following a completely randomized block design with pigs blocked by initial BW. A pen was considered as the experimental unit. Separation of means was completed using the PDIFF option of SAS. Probability values less than 0.05 were considered statistically significant and between 0.05 and 0.10 as trends.