Materials and methods

Research was conducted at the Iowa State University Swine Nutrition Research Farms (Ames, IA) from May to June 2025. All animal procedures were approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC protocol #24–069) and were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS 2010).

Animals, housing, experimental design and diets

A total of 112 mixed-sex pigs (9.1 ± 0.37 kg; Terminal Line 337 × Maternal Line 1050, PIC Inc., Hendersonville, TN), 10 d post-weaning, and sourced from a commercial sow farm, were utilized and housed at the Iowa State University Swine Nutrition Farm (Ames, IA). Pigs were allotted using a completely randomized design and placed into 56 pens containing one barrow and one gilt per pen. All pigs were individually tagged and weighed. Once placed, pens were randomly assigned to 1 of 7 dietary treatments (n = 8 pens/treatment) containing dose titrated concentrations of DON. Pigs were housed in partially concrete slatted pens (0.97 m × 1.83 m) equipped with one nipple drinker and a two-hole feeder. At all times, the pigs had ad libitum access to feed and water. Ventilation rates and temperature set points were evaluated and adjusted weekly by farm personnel to ensure consistent environmental control. Individual pig body weights (BW) and pen feeder weights were taken on day 0, 14, and 24 for the calculation of pen average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (Gain:Feed; G:F) for phase 1 (14 d), phase 2 (10 d), and overall (d 0–24) of the study.

Experimental dietary treatments were formulated and manufactured as standard corn-soybean meal mash diets equal in energy and SID amino acids (Table 1). High DON corn (20 mg/kg) sourced from Ohio was used at the expense of clean corn (0.20 mg/kg) in the formulation to achieve target DON concentrations ranging from 0.30 to 5.25 mg/kg using a seven-point dose titration methodology. Complete feed DON concentrations (mg/kg) were formulated at 0.75 mg/kg increments to targets of < 0.30, 0.75, 1.50, 2.25, 3.00, 4.50 and 5.25 mg/kg (Table 1). Each diet was sampled at mixing and submitted to the Iowa State Veterinary Diagnostic Laboratory (Ames, IA, USA) for complete mycotoxin profiling and DON verification using LC-MS/MS standard testing procedures before the beginning of the study. All diets were within 0.5 mg/kg of their formulated DON concentration (Table 2). Aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, ochratoxin A, T-2 toxin, HT-2 toxin, and alpha-zearalenol were not detectable or below detection limits. Diets were formulated based on NRC (2012) amino acid and energy recommendations for the size of pig used in this growth assay and were nutritionally equivalent across treatments, ensuring that observed performance responses could be attributed primarily to differences in dietary DON concentration rather than changes in nutrient composition.

 | DON mg/kg
Ingredient, % | 0.30 | 0.75 | 1.50 | 2.25 | 3.00 | 4.50 | 5.25
Corn | 63.13 | 60.13 | 56.12 | 52.11 | 48.10 | 40.08 | 36.07
High DON corna | – | 3.00 | 7.01 | 11.02 | 15.03 | 23.05 | 27.06
Soybean meal, 45% CP | 31.70 | 31.70 | 31.70 | 31.70 | 31.70 | 31.70 | 31.70
Soybean oil | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00
Limestone | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89 | 0.89
Monocalcium phosphate 21% | 1.09 | 1.09 | 1.09 | 1.09 | 1.09 | 1.09 | 1.09
Salt | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50 | 0.50
L-lysine HCl | 0.57 | 0.57 | 0.57 | 0.57 | 0.57 | 0.57 | 0.57
DL-methionine | 0.23 | 0.23 | 0.23 | 0.23 | 0.23 | 0.23 | 0.23
L-threonine | 0.16 | 0.16 | 0.16 | 0.16 | 0.16 | 0.16 | 0.16
L-tryptophan | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05
L-valine | 0.13 | 0.13 | 0.13 | 0.13 | 0.13 | 0.13 | 0.13
L-isoleucine | 0.09 | 0.09 | 0.09 | 0.09 | 0.09 | 0.09 | 0.09
Vitamin premixb | 0.23 | 0.23 | 0.23 | 0.23 | 0.23 | 0.23 | 0.23
Trace mineral premixc | 0.15 | 0.15 | 0.15 | 0.15 | 0.15 | 0.15 | 0.15
Zinc oxide, 72% Zn | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05 | 0.05
Phytased | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03
Calculated Composition |  |  |  |  |  |  | 
MEe, Mcal/kg | 3.37 | 3.37 | 3.37 | 3.37 | 3.37 | 3.37 | 3.37
Crude protein, % | 20.0 | 20.0 | 20.00 | 20.0 | 20.0 | 20.0 | 20.0
Total calcium, % | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65 | 0.65
Available phosphorus, % | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45 | 0.45
Calcium:available phosphorus | 1.44 | 1.44 | 1.44 | 1.44 | 1.44 | 1.44 | 1.44
SIDf Lysine, % | 1.33 | 1.33 | 1.33 | 1.33 | 1.33 | 1.33 | 1.33
g SID Lysine:ME | 3.95 | 3.95 | 3.95 | 3.95 | 3.95 | 3.95 | 3.95

 | DON mg/kga
 | 0.30 | 0.75 | 1.50 | 2.25 | 3.00 | 4.50 | 5.25
Deoxynivalenol, mg/kg | 0.2 | 0.9 | 1.7 | 2.4 | 3.1 | 4.0 | 5.1
3-Acetyl Deoxynivalenol, µg/kg | −b | 15.3 | 25.5 | 34.6 | 49.5 | 59.9 | 81.4
Zearalenone, mg/kg | – | – | – | – | 0.5 | 0.2 | 0.4

Statistical analysis

Data were analyzed in R (v4.3.3; (R Core Team 2024)) using linear models (lm function) with dietary treatment as a fixed effect for growth performance parameters including BW, ADG, ADFI, and G:F. Pen was the experimental unit for all analyses. Model assumptions were evaluated at the pen level using studentized residuals, and pens with an absolute studentized residual greater than 3 were considered outliers and removed on a per-variable basis prior to estimation of treatment means. Treatment means were evaluated using orthogonal polynomial contrasts to assess linear and quadratic trends across increasing dietary DON concentrations using the contrast function. To evaluate potential biological thresholds, linear, two-segment, and linear-plateau regression models were compared using Akaike’s Information Criterion (AIC) and Bayesian Information Criterion (BIC) to assess improvements in model fit for ADG. The 1.7 mg/kg treatment level did not follow the overall monotonic dose-response pattern and exerted disproportionate influence on regression model fit, being well outside 95% confidence interval (CI) range. Regression analyses were conducted both with and without this treatment level, with primary interpretation based on models excluding the 1.7 mg/kg treatment. Regression equations were derived for overall growth performance parameters. Treatment differences were considered significant at P ≤ 0.05 and a tendency at 0.05 < P ≤ 0.10.