Diets from each experimental and within each phase were sampled from 9 different locations, a total of 2 kg per diet. Sub-samples of 200 g of each diet and phase were sent to the North Dakota State University Veterinary Diagnostic Laboratory (Fargo, ND, USA) for mycotoxin analysis, and 300 g of each diet were sent to the North Carolina Department of Agriculture (Raleigh, NC, USA) for proximate analysis. The CON had detectable deoxynivalenol levels because corn DDGS used in CON formulation had deoxynivalenol contamination. Nevertheless, MT had 1.9 mg/kg more deoxynivalenol than CON, close to the planned concentration of 2 mg/kg (Table 3 ).
Serum samples were submitted for a biochemical profile at Antech Diagnostic Laboratory (Cary, NC, USA). Antioxidant status and immune markers were evaluated in mid-jejunal mucosa by quantifying protein carbonyls (STA-310, Cell Biolabs, Inc., San Diego, CA, USA), malondialdehydes (STA-330, Cell Biolabs, Inc., USA), total glutathione (STA-312, Cell Biolabs, Inc., USA), tumor necrosis factor-alpha (PTA00, R&D Systems, Inc., Minneapolis, MN, USA), and interleukin-8 (P8000, R&D Systems, Inc., USA). The manufacturer’s manual for each kit was followed in the laboratory assays of protein carbonyls, malondialdehydes, and total protein according to procedures described by Zhao and Kim [26 (link)]. Measurements of total glutathione, tumor necrosis factor-α, and interleukin-8 followed the procedures as described by Holanda et al [24 (link)].
Transversal sections of 0.5 cm were transferred to 70% ethanol after 14 days and sent to the North Carolina State University Histopathology Laboratory (College of Veterinary Medicine, Raleigh, NC, USA), where samples were included in paraffin, microtomed, and stained for Ki-67 antigen by immunohistochemistry before assembling of histological slides [8 (link)]. Histological evaluation of gut morphology was performed by one single evaluator recording villus height and width, crypt depth, and for calculating villus height:crypt depth ratio, and the proportion of proliferating cells to the total number of cells in the crypt using the Image JS tool [27 (link)] in ten pictures for each experimental unit (pig) according to the measurements described by Holanda and Kim [12 (link)]. Measurements of villus height and width were used to calculate the average mid-jejunal surface area for each villus by using the following formula [28 (link)]:
Serum samples were submitted for a biochemical profile at Antech Diagnostic Laboratory (Cary, NC, USA). Antioxidant status and immune markers were evaluated in mid-jejunal mucosa by quantifying protein carbonyls (STA-310, Cell Biolabs, Inc., San Diego, CA, USA), malondialdehydes (STA-330, Cell Biolabs, Inc., USA), total glutathione (STA-312, Cell Biolabs, Inc., USA), tumor necrosis factor-alpha (PTA00, R&D Systems, Inc., Minneapolis, MN, USA), and interleukin-8 (P8000, R&D Systems, Inc., USA). The manufacturer’s manual for each kit was followed in the laboratory assays of protein carbonyls, malondialdehydes, and total protein according to procedures described by Zhao and Kim [26 (link)]. Measurements of total glutathione, tumor necrosis factor-α, and interleukin-8 followed the procedures as described by Holanda et al [24 (link)].
Transversal sections of 0.5 cm were transferred to 70% ethanol after 14 days and sent to the North Carolina State University Histopathology Laboratory (College of Veterinary Medicine, Raleigh, NC, USA), where samples were included in paraffin, microtomed, and stained for Ki-67 antigen by immunohistochemistry before assembling of histological slides [8 (link)]. Histological evaluation of gut morphology was performed by one single evaluator recording villus height and width, crypt depth, and for calculating villus height:crypt depth ratio, and the proportion of proliferating cells to the total number of cells in the crypt using the Image JS tool [27 (link)] in ten pictures for each experimental unit (pig) according to the measurements described by Holanda and Kim [12 (link)]. Measurements of villus height and width were used to calculate the average mid-jejunal surface area for each villus by using the following formula [28 (link)]:
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