Anti-nutritional Effects Of Fusarium Mycotoxins And Its Mechanism In Weanling Piglets

Four in vivo and in vitro experiments were conducted in this study to investigate the anti-nutritional effects of Fusarium mycotoxins such as deoxynivalenol(DON) and zearalenone(ZEN) on weanling piglets and the underlying mechanisms.Exp.1 Effects of Feeding Diets Contaminated by Naturally Moldy Maize on Performance and Nutrient Digestibility and Metabolism in Weanling PigletsAn experiment was conducted to investigate the effects of graded levels of naturally moldy maize containing Fusarium mycotoxins such as deoxynivalenol(DON) and zearalenone(ZEN) in diets on performance,organ weight,intestinal mucosal morphology,nutrient utilization and serum biochemical parameters in weanling piglets.A total of 75 castrated Yorkshire×Rongchang weanling piglets with an average initial weight of 8.04±0.64 kg(mean±SEM) were randomly allocated to three diets(five pens of five piglets per diet) for 28 d.Diets included control,mycotoxin-contaminated diet containing 25%moldy maize,and mycotoxin contaminated diet containing 50%moldy maize.The results were as follows:1.The average daily gain(ADG) and average daily feed intake(ADFI) of piglets decreased linearly or quadraticly with the inclusion of moldy maize in the diet during d1 to 14 and d15 to 28(P＜0.05).2.The kidney weights of piglets fed the contaminated diets quadraticly decreased(P＜0.05), whereas the spleen weights of piglets linearly increased(P＜0.05) as the level of moldy maize in the diet increased.However,no significant changes were observed for the weights of liver, thymus,and pancreas with increasing dietary moldy maize.The gallbladder weight was decreased for the diet contained 25%moldy maize.3.Feeding contaminated diets linearly or quadraticly decreased the villus height and mucosa thickness in proximal duodenum(P＜0.05) and proximal jejunum(P＜0.05) with increasing dietary moldy maize.The crypt depth in proximal duodenum linearly increased(P＜0.05),the crypt depth in proximal jejunum linearly or quadraticly increased(P＜0.01),moreover the villus widths were also significantly different in proximal duodenum and jejunum(P＜0.05 or P＜0.01) as increased amounts of moldy maize in diets. 4.The apparent digestibility of organic matter,protein,phosphorus and protein biological value linearly or quadraticly decreased(P＜0.05),whereas calcium apparent digestibility linearly decreased(P＜0.05).5.There were quadratic influences on serum activity of GPT(P＜0.05) and serum concentration of ALB(P＜0.05) in piglets with increased dietary moldy maize amounts on d 14 of the experiment.These changes,however,were not detectable on d 28(P＞0.05).Serum TP concentrations of piglets fed contaminated diets were not affected on d 14 of the experiment(P＞0.05),however it responded in a quadratic fashion(P＜0.05)on d 28.The serum concentrations of GLO,A/G and BUN were not affected by treatments on each stage(P＞0.05).The serum concentrations of the examined immune-globulins A,M and G were not affected by the experimental treatment(P＞0.05).It was concluded that the feeding diets contaminated by naturally moldy maize can reduce performance and nutrient apparent utilization,damaged small intestinal mucosal morphology, and influence serum biochemical parameters in weanling piglets.Exp.2 Effects of Fusarium Mycotoxins on Proliferation and Oxidative Damage of Intestinal Epithelial Cell of PigletsIn vitro model of primary culture porcine epithelial intestinal cell was used to study the effects of DON and ZEN alone or combination on the cell proliferation as measured by MTT and oxidation damage as reflected by malonedialdehyde(MDA) production,lactate dehydrogenase(LDH) release,glutathione content,activities of superoxide dismutase(SOD). Experimental treatments were arranged as a 4×4 factorial with two Fusarium mycotoxins DON and ZEN.The final concentrations of DON were 0,2,4,and 6μg/mL,and the final concentrations of ZEN were 0,1,5,and 10μg/mL.Cell suspension(2.98×10~5 cells/mL) were cultured in 96-well plates at a density of 5.4×10~4 cells/well for cell proliferation assay by MTT and LDH release assay.Cells were cultured in 24-well plates at a density of 1.2×10~5 cells/mL for oxidative stress assay.After 6-day incubation of five wells,the medium was eliminated, cells were incubated at the presence of toxins for 3,6,12,24,and 48 h respectively for cell proliferation assay by MTT.After 10-day incubation of another six wells,the medium was eliminated;cells were incubated at the presence of toxins for 24 h for LDH release assay and oxidative stress assay.The results were as follows:1.DON and ZEN alone or combination reduced the porcine intestinal epithelial cell proliferation at 2μg/mL and 5μg/mL or over,respectively.Combinations of DON and ZEN displayed interactive effects in reducing cell proliferation.DON reduced cell proliferation to a greater extent than ZEN.2.DON and ZEN alone or combination increased LDH release of intestinal epithelial cell at 2μg DON/mL and 10μg ZEN/mL or over,respectively.DON damaged cell membrane to a greater extent than ZEN.3.The comparison revealed that DON and ZEN alone or combination increased MDA production of intestinal epithelial cell at 2μg DON/mL and 5μg ZEN/mL or over,respectively. When combination of DON and ZEN,the lowest observed adverse effect level(LOAEL) of DON for the significant increase of MDA was 4μg/mL.Combination of DON and ZEN displayed significant interactive effects in MDA production.DON displayed a greater effect than ZEN.4.The LOAEL of DON alone or combination with ZEN for significant reduction of GSH concentration and T-SOD activity in intestinal epithelial cell was 2μg DON/mL.The LOAEL of ZEN alone for reducing GSH concentration and T-SOD activity were 1μg/mL and 10μg/mL, respectively.When combination of DON and ZEN,the LOAEL of ZEN for significantly reducing GSH and T-SOD were 1μg/mL and 5μg/mL,respectively.5.Combination of DON and ZEN reduced GSH concentration and T-SOD activity in intestinal epithelial cell to a greater extent than individual effect.DON had greater damage than ZEN on cell antioxidant system.Combination of DON and ZEN displayed less than additive effects in reducing GSH concentration.The above findings indicated that DON and ZEN alone or combinations are able to induce oxidative damage and inhibit proliferation of porcine intestinal epithelial cell.DON had more toxic effects than ZEN.Exp.3 Effects of Fusarium Mycotoxins on Nutrients Absorption of Intestinal Epithelial Cell of PigletsOur findings indicated that DON and ZEN could cause the oxidative damage of pIEC and damage to the integrity of cell membrane and then influence digestion and absorption of nutrients,in exp 2.The primary culture porcine epithelial intestinal cell was used to study the effects of DON and ZEN alone or combination on the activity of sucrase,isomaltose,Na~+-K~+ ATPase,and the absorption of sucrose and dipeptide.Experimental treatments were arranged as a 3×3 factorial with two Fusarium mycotoxins DON and ZEN.The final concentrations of DON were 0,2 and 4μg/mL,and the final concentrations of ZEN were 0,5 and 10μg/mL.There were 9 treatments,in which 10 repeats in each treatment.Cells were cultured in 24-well plates at a density of 1.2×10~5 cells/mL for cell proliferation.After 10-day incubation,the medium was eliminated,cells were incubated at the presence of toxins for 24 h for mucosa enzymatic activity assay and nutrients uptake assay.The results were as follows:1.The LOAEL of DON alone for reducing sucrase activity and uptake of glucose and cephalexin in intestinal epithelial cell was 2μg DON/mL(P＜0.05).The LOAEL of DON alone reduce maltase activity and Na~+-K~+ ATPase activity was 4μg/mL(P＜0.05).2.The LOAEL of ZEN alone for reducing sucrase activity,Na~+-K~+ ATPase activity and uptake of glucose in intestinal epithelial cell was 10μg DON/mL(P＜0.05).The LOAEL of ZEN alone for reducing the uptake of cephalexin was 5μg/mL(P＜0.05).However,ZEN had no effect on the maltase activity at all levels alone(P＞0.05).3.Combination of DON and ZEN in certain concentration inhibited sucrase,maltase and Na~+-K~+ ATPase(P＜0.01) by the means of additive effects or less than additive effects. Interestingly,DON combined with lower concentration of ZEN synergistically decreased uptake of glucose,while combined with higher concentration of ZEN had a less than additive on uptaking of glucose(P＜0.05).Combination of DON and ZEN had no interactive effect on uptake of cefalexin(P＞0.05).Therefore,these results suggested that DON and ZEN had significantly effect on activities of sucrase,maltase,and Na~+-K~+ ATPase in intestinal cell.The uptake of glucose and cefalexin was affected also.The sucrase activity,Na~+-K~+ ATPase activity and the uptake of glucose and cefalexin were more sensitive towards DON than towards ZEN,however,maltase activity was more sensitive towards ZEN than towards DON.Exp.4 Effects of Fusarium Mycotoxins on Genes Expression of Digestive Enzyme and Transporter-Related in Porcine Intestinal Epithelial CellOur studies had indicated that DON and ZEN influenced mucosa enzymatic activity and uptake of nutrients in porcine intestinal epithelial cell.For further investigating the underlying mechanism of DON and ZEN influence the mucosa enzymatic activity and nutrients absorption, an 3×3 factorial design with two Fusarium mycotoxins DON and ZEN was conducted to investigate the effects of DON and ZEN on the gene expression of sucrase-isomaltase(SI), leueine aminopeptidase(LAP),sodium/glucose eotransporter 1(SGLT1) and dipeptide transporter 1(PepT 1) in primary culture porcine intestinal epithelial.There are 9 treatments,in which 3 repeats in each treatment.The final concentrations of DON were 0,2 and 4μg/mL,and the final concentrations of ZEN were 0,5 and 10μg/mL.The expression of nutrient uptake-related enzyme and transporter mRNA in pIEC was assessed by SYBR Green I real-time quantitative RT-PCR.The results were as follows:1.The LOAEL of DON down-regulate expression of SI,LAP and PepT1 mRNA in pIEC was 2μg/mL(P＜0.05).DON down-regulated expression of SGLT1 mRNA at lower concentration(2μg/mL)(P＜0.05),while had no effect at higher concentration(4μg/mL).2.ZEN alone had no effect on the expression of SI and PepT1 mRNA at all levels(P＞0.05).ZEN down-regulated expression of LAP and SGLT1 mRNA at the concentration of 10μg ZEN/mL(P＜0.05).3.DON combination with ZEN,the LOAEL of DON down-regulate expression of SI,LAP, SGLT1 and PepT1 mRNA in pIEC was 2μg/mL(P＜0.05),however,ZEN did not affect on them (P＞0.05).DON combination with ZEN synergistically down-regulated the expression of SI and PepT1 mRNA,further,there were no significant interactive effects on the expression of LAP and SGLT1 mRNA.Therefore,these results suggested that DON down-regulate expression of SI,LAP,SGLT1 and PepT1 mRNA and ZEN down-regulate expression of LAP and SGLT1 with Slight of next adjust a function.DON combination with ZEN synergistically down-regulated the expression of SI and PepT1 mRNA,and the effects of DON on related digestive enzyme activity and transporter mRNA expression was further more than ZEN.ImplicationsThese results of in vitro and in vivo studies indicated that Fusarium mycotoxins DON and ZEN can damage pIEC,inhibite cell proliferation and down-regulate gene expression of digestive enzymes and transporter.Fusarium mycotoxins DON and ZEN can impair antioxidant system in cell,reduce nutrients uptake,decrease animal performance,and then show anti-nutritional effects.