Feeding thermally processed spray-dried egg whites, singly or in combination with 15-acetyldeoxynivalenol or peroxidized soybean oil on growth performance, digestibility, intestinal morphology, and oxidative status in nursery pigs

Abstract

Two experiments (EXP) determined the susceptibility of spray-dried egg white (SDEW) to oxidation (heating at 100 °C for 72 h; thermally processed, TP) and whether feeding TP–SDEW, 15-acetyldeoxynivalenol (15-ADON), or peroxidized soybean oil (PSO), singularly or in combination, would affect pig performance, intestinal morphology, digestibility, and markers of oxidative stress in nursery pigs. In EXP 1, 32 pigs (7.14 kg body weight, BW) were placed individually into pens and fed diets containing either 12% SDEW, 6% TP–SDEW plus 6% SDEW, or 12% TP–SDEW. Performance was measured at the end of the 24-d feeding period with biological samples harvested following euthanasia. In EXP 2, 64 pigs (10.6 kg BW) were placed individually into pens and fed diets containing 7.5% soybean oil or PSO, 10% SDEW or TP–SDEW, and diets without or with 3 mg 15-ADON/kg diet in a 2 × 2 × 2 factorial arrangement. Performance was measured at the end of the 28-d feeding period with biological samples harvested following euthanasia. In EXP 1, dietary treatment did not affect pig performance, apparent ileal digestibility of amino acids (AAs), apparent total tract digestibility (ATTD) of gross energy (GE) or nitrogen (N), ileal crypt depth, or villi height:crypt depth ratio (P > 0.05). The effects of feeding TP–SDEW on protein damage in the plasma and liver (P < 0.05) were variable. In EXP 2, there were no three-way interactions and only one two-way interactions among dietary treatments on parameters evaluated. There was no effect of feeding TP–SDEW on ATTD of GE or N, intestinal morphology, or on oxidative markers in the plasma, liver, or ileum (P > 0.05). There was no effect of feeding diets containing added 15-ADON on ATTD of GE, ileal AA digestibility, intestinal morphology, oxidative markers in the plasma, liver, or ileum, or pig performance (P > 0.05). Feeding pigs diets containing PSO resulted in reduced ATTD of GE and N, plasma vitamin E concentration, and pig performance (P < 0.01) but did not affect intestinal morphology or oxidative markers in the liver or ileum (P > 0.05). In conclusion, it was difficult to induce protein oxidation in SDEW and when achieved there were limited effects on performance, digestibility, intestinal morphology, and oxidative status. Furthermore, singly adding 15-A-DON to a diet had no effect on the animal. At last, adding PSO reduces animal performance, but has limited effect on digestibility, intestinal morphology, and oxidative status in nursery pigs.

Feeding young pigs a heated spray-dried egg white or a laboratory-purified mycotoxin have limited, if any, effects on growth performance, digestibility, intestinal morphology, and oxidative status in nursery pigs. In contrast, feeding young pigs a thermally processed soybean oil will result in a dramatic reduction in performance, but has limited effects on digestibility, intestinal morphology, and oxidative status in nursery pigs.

Introduction

Swine can be exposed to a variety of chemical, environmental, and nutritional stressors that can affect their well-being and productivity. Enteric stressors such as mycotoxins, thermally processed proteins, and peroxidized lipids have been shown to reduce performance, intestinal morphology, energy and nutrient digestibility, and increase oxidative stress (Awad et al., 2014; Frame et al., 2020; Kerr et al., 2020b). Mycotoxins (MTXs) such as aflatoxin, deoxynivalenol (DON), fumonisin (FUM), and zearalenone (ZEA) are commonly found in grains or grain by-products fed to swine (D’Mello et al., 1999; AssunCao et al., 2016). One of the most common mycotoxins in corn and corn by-products is DON which is known to reduce pig performance (Bergsjo et al., 1993; Rotter et al., 1996; Li et al., 2011), intestinal function (Pinton et al., 2009, 2012; Pierron et al., 2016) and create a pro-inflammatory response (Kouadio et al., 2005; Sun et al., 2016), thereby increasing reactive oxygen species (ROSs) and oxidative stress (Frankič et al., 2008; Zhang et al., 2009; Strasser et al., 2013). Protein oxidation is a concern in muscle, meat, and food systems (Shacter, 2000; Estevez et al., 2017). In contrast, processed feedstuffs are considered relative stable ingredients, but proteins within a feedstuff may become vulnerable to oxidation by further processing or storage (Feng et al., 2015; Zhang et al., 2017; Frame et al., 2020, 2021). Even though all amino acids (AAs) are susceptible to heat damage (Papadopoulos, 1989; Shacter, 2000), proteins which have increased levels of cysteine and methionine are more reactive to oxidative species than other AAs because of their S content (Gruhlke and Slusarenko, 2012; Celi and Gabai, 2015). At last, it has also been reported that feeding oxidized soybean oil can significantly reduce growth performance (DeRouchey et al., 2004; Boler et al., 2012, Yuan et al., 2020) and increase serum and urine concentrations of lipid damage (Lindblom, 2017; Kerr et al., 2020a). Further research is needed to understand the potential of feeding oxidized protein on animal growth and oxidative status and it is not known if there are interactive effects between different proposed inducers of enteric oxidative stress on growth performance, intestinal morphology, digestibility, and oxidative stress biomarkers in nursery pigs.

Materials and methods

The Iowa State University Animal Care and Use Committee approved all experimental protocols (19-321 and 20-041).

Laboratory experiment

Prior to conducting the animal study, a laboratory experiment was conducted on thermally processing (TP) spray-dried egg white (SDEW) to determine its oxidation potential and the time of heating necessary to induce the formation of protein carbonyls (PCs) to use in subsequent animal experiments. In this study, SDEW were placed into 26.4 cm × 32.4 cm × 6.5 cm deep aluminum pans, (filled one-half full) and heated at 100 °C in a forced-air drying oven based on methods described by Frame et al. (2020, 2021) and sampled at 0, 48, 72, 96, and 120 h, Table 1, with samples stored at −20 °C until analysis for PC using a commercial kit, Table 2. A solubility assessment was also conducted to understand the best method to get SDEW and TP–SDEW into solution for PC analysis where it was determined that solubility was best achieved with a 24-h incubation at room temperature with concentration of 100 mg SDEW or TP–SDEW/mL Millipore water.

Table 1.

Protein carbonyl concentration of thermally processed spray-dried egg white¹

Time heated, h	0	48	72	96	120
PC, nmol/mg protein	7.3	6.5	8.2	18.9	19.6

¹Pasteurized spray-dried egg whites (Rembrandt Foods, Rembrandt, IA), 21.5 kg, were placed into a 26.4 cm × 32.4 × 6.5 cm deep aluminum pan, heated in a 100 °C forced-air drying oven, hand-mixed every 24 h to facilitate equal heating, and sampled at 0, 48, 72, 96 and 120 h. To analyze for protein carbonyls (PCs), samples were solubilized using 100 mg spray-dried egg whites/mL Millipore water and incubated for 24 h prior to analysis. Analysis completed using a commercial kit (Kit #100005020, Cayman Chemical Company, Ann Arbor, MI).

Table 2.

Analytical kits for oxidative stress biomarkers in plasma and tissue samples

Assay kit	Catalog number	Plasma dilution factor	Liver or ileal dilution factor
ISP1	516351	None	None
PC1	100005020	None	None
8-OH-2dG1	589320	1:75	None
ROM2	MC006	1:100	None
OXYa2	MC434	1:100	None

¹F2-isoprostanes (ISP), protein carbonyls (PCs), and 8-hydroxy-2'-deoxyguanosine (8-OH 2-dG) assay kits were purchased from Cayman Chemical Company (Ann Arbor, MI).

²Reactive oxygen metabolites (ROMs) and OXY-adsorbent (OXYa) assay kits were purchased from Diacron International (Grosseto, Italy).

Experiment 1

Dietary treatments

Three treatments were designed consisting of feeding diets to nursery pigs containing either 12% unprocessed SDEW, 6% TP–SDEW plus 6% unprocessed SDEW, or 12% TP–SDEW plus 0% unprocessed SDEW. Similar to the laboratory study, the SDEW were placed into 26.4 cm × 32.4 cm × 6.5 cm deep aluminum pans (filled one-half full), heated in a 100 °C forced-air drying oven, hand-mixed every 24 h to facilitate equal heating, and heated for 120 h. After thermal processing and before feed mixing, the TP–SDEW was stored at −20 °C to prevent further oxidation.

For formulation purposes the SDEW was assumed to contain 3,000 kcal ME/kg, 84% crude protein, 0.10% calcium and 0.10% standardized total tract digestible phosphorus. Total Lys, Met, Cys, Thr, Trp, Ile, and Val were assumed to be 5.08, 3.20, 2.04, 3.67, 1.27, 5.02, and 6.17%, respectively. Because of no know AA digestibility estimates for SDEW, a 93% standardized ileal digestibility coefficient was used for all AAs. Diets were formulated to meet the NRC (2012) energy and nutrient requirements with diets formulated to contain 3,400 kcal ME/kg, 0.83% total calcium, 0.43 standardized total tract digestible phosphorus, 19.4% CP, 1.4% standardized ileal digestible Lys, and TSAA:Lys, Thr:Lys, Trp:Lys, Ile:Lys, and Val:Lys ratios of 0.55, 0.59, 0.165, 0.52, and 0.64, respectively, Table 3. Because vitamins have antioxidant abilities, vitamins were formulated as close to possible to meet the NRC (2012) requirements, specifically vitamin E was included at 17.5 IU/kg diet and vitamin A at 2,144 IU/kg diet. Titanium dioxide was added as an indigestible marker to diets to allow for determination of digestibility coefficients.

Table 3.

Composition of dietary treatments, Experiment 1

	TP–SDEW, %1
Ingredient, %	0%	6%	12%
Corn	60.07	60.07	60.07
Dairylac 802	15.00	15.00	15.00
Fish meal	5.00	5.00	5.00
Spray-dried egg white	12.00	6.00	0.00
TP–SDEW3	0.00	6.00	12.00
Soybean oil	3.83	3.83	3.83
Monocalcium phosphate	0.90	0.90	0.90
Limestone	0.85	0.85	0.85
Sodium chloride	0.40	0.40	0.40
L-Lysine·HCl	0.58	0.58	0.58
L-Threonine	0.14	0.14	0.14
L-Tryptophan	0.03	0.03	0.03
Zinc oxide	0.38	0.38	0.38
Copper sulfate	0.10	0.10	0.10
Mineral premix4	0.15	0.15	0.15
Vitamin premix5	0.07	0.07	0.07
Titanium dioxide6	0.50	0.50	0.50
Total	100.00	100.00	100.00

¹All diets were formulated to contain 3,400 kcal ME/kg, 0.90% total calcium, 0.50 standardized total tract digestible phosphorus, 19.4% CP, 1.4% standardized ileal digestible Lys, and TSAA:Lys, Thr:Lys, Trp:Lys, Ile:Lys, and Val:Lys ratios of 0.55, 0.59, 0.165, 0.52, and 0.64, respectively.

²Dairylac 80 is a sweet and dried whey soluble product containing approximately 3.2% CP, 0.06% Lys, and 80% lactose (International Ingredient Corporation, St. Louis, MO).

³TP–SDEW, thermally processed egg white; heated in a forced air oven at 100 °C for 120 h. The unheated SDEW was analyzed to contained 34 nmol PC/mg protein and the TP–SDEW contained 23 nmol PC/mg protein.

⁴Provided the following per kilogram of diet: 16.5 mg Cu (as CuSO₄), 165 mg Fe (as FeSO₄), 0.3 mg I (as Ca(IO₃)₂), 39 mg Mn (as MnSO₄), 165 mg Zn (as ZnSO₄), and 0.3 mg Se (Na₂SeO₃).

⁵Provided the following per kilogram of diet: 2,144 IU vitamin A, 245 IU vitamin D₃, 17.5 IU vitamin E, 1.1 mg vitamin K, 0.02 mg vitamin B₁₂, 3.9 mg riboflavin, 19.6 mg niacin, and 9.5 mg pantothenic acid.

⁶Used as an indigestible marker.

Animal experimentation

Thirty-two barrows were obtained from a commercial facility following weaning (between 19 and 21 d of age) and subsequently housed in the Swine Nutrition Farm at Iowa State University (Ames, IA) for the duration of the study. Upon arrival, pigs were held in a group pen for 7 d to facilitate feed intake and eliminate the use of unthrifty pigs. Following this 7-d adaptation, pigs were selected and randomly assigned to one of three dietary treatments resulting in 10 replications of the control diet and 11 replications of each TP–SDEW treatment. Pigs (initial BW, 7.14 ± 1.08 kg) were individually housed in pens measuring 121 cm × 42 cm and fed for 24 d with ad libitum access to feed and water.

Pigs and feeders were weighed on d 0 and d 24 to determine ADG, ADFI, and GF. On d 22 of the trial, approximately 8 mL of blood was collected from each pig via jugular venipuncture using a 10 mL vacuum tube containing sodium heparin as the anticoagulant. The blood was subsequently centrifuged at 2,500 × g for 10 min at 4 °C after which plasma was aliquoted into two microfuge tubes and immediately stored at −80 °C until assayed. Aliquots used for the measure of F2-isoprostanes (ISPs) contained 0.005% butylated hydroxytoluene per mL of plasma. On d 24, all pigs were euthanized by captive bolt followed by exsanguination for harvesting liver and ileal tissues, ileum contents, and fecal matter. Liver tissue was flash frozen in liquid nitrogen and kept on dry ice until being stored at −80 °C, ileal tissue was placed in 10% w/v formalin, ileal contents were initially chilled on dry ice stored at −80 °C and then dried at 65 °C for 24 h in a force air oven and ground prior to analysis (Kerr et al., 2020c), and fecal samples were initially chilled on dry ice and stored at −80 °C and then dried at 75 °C for 24 h and ground through a 2 mm screen prior to analysis (Jacobs et al., 2011).

Methodologies and calculations

Ileum samples that had been placed in 10% w/v formalin were stained with hematoxylin and eosin (Iowa State University Veterinary Diagnostic Laboratory, Ames, IA) and the resulting slides were analyzed for crypt depth and villus height (OLYMPUS BX 53/43 microscope with an attached DP80 Olympus camera, Waltham, MA). Ten well-defined villus and crypt pairs with proper orientation were measured per pig (OLYMPUS cellSens Dimension 1.16 software), averaged, and reported as 1 value per pig. Villus were selected equally around the entire cross-section, and for each villus height measured, the crypt depth to the right was also measured.

Analysis for markers of oxidative stress in the plasma and liver tissue were measured using commercially available assay kits (Table 2, Cayman Chemical Company, Ann Arbor, MI). All assays were performed according to the recommendations from the manufacturer, with assays performed in triplicate in 96-well microplates. Tissue assayed for ISP concentration as indication of lipid damage were processed by homogenizing100 mg of liver tissue per mL of 0.1 M phosphate buffer containing 1 mM ethylenediaminetetraacetic acid and 0.005% butylated hydroxytoluene and centrifuged at 8,000 × g for 10 min. The supernatant was collected and used as outlined in the kit booklet. No dilutions were necessary, and data were reported as free ISP in pg/mL. Protein damage in the liver was measured via PC by homogenizing 200 mg of tissue per mL of 50 mM phosphate buffer containing 1 mM ethylenediaminetetraacetic acid and centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant collected and plasma were used as outlined in the kit booklet to determine PC concentration, with no dilutions being necessary. Deoxyribose nucleic acid (DNA) damage was measured via 8-hydroxy-2'-deoxyguanosine (8-OH-2dG), because guanosine is the most susceptible nucleic acid base to oxidation (Wang et al., 2010). Tissue was assayed by extracting 25 mg of liver using a commercially available extraction kit (ZR Genomic DNA-Tissue MiniPrep, Zymo Research, Irvine, CA). Upon extraction DNA was digested from double stranded DNA to single stranded DNA via 15 uL of nuclease P1 (Sigma-Aldrich, St. Louis, MO) per sample. Nuclease P1 was prepared by adding 1.4 mL of 20 mM sodium acetate, 5 mM ZnCl, 50 mM NaCl, solution to 1.4 mg of lyophilized powder enzyme. The enzyme was added at the dilution of 1:20 to achieve a final dilution of 50 µg/mL. Following nuclease P1, samples were incubated at 50 °C for 60 min. Following incubation, each sample of digested single stranded DNA was converted from nucleotides to nucleosides via treatment of 1 unit of alkaline phosphatase (Roche Diagnostics, Mannheim, Germany) per 100 µg of DNA. Samples were then incubated at 37 °C for 30 min and then boiled for 10 min. After boiling, the supernatant was used to determine 8-OH-2dG concentration of each sample following the kit booklet procedure. Plasma was diluted 1:75, but no dilution was necessary for the liver tissue.

To determine apparent ileal digestible (AID) AA, analysis of diets and ileal digesta were analyzed for AA (AOAC, 2005; Official method 982.30E; a,b,c) and titanium (Myers et al., 2004) at a commercial laboratory (University of Missouri Agricultural Experiment Station Chemical Laboratories, Columbia, MO). To determine apparent total tract (ATTD) digestibility, diets and feces were analyzed for gross energy (GE) with an isoperibol bomb calorimeter (model 1281; Parr Instrument Co, Moline, IL) using benzoic acid as a standard, nitrogen (N) and sulfur (S) by thermo-combustion (VarioMAX CNS; Elementar Analysensteme GmbH, Hanau, Germany) where combustion gases were converted to individual gases and sorted into absorption column and measured using a thermal conductivity detector, and titanium by wet-ashing followed by colorimetric-spectrophotometry (Leone, 1973). After chemical analysis were completed on the diets, ileal, and fecal samples, digestibility coefficients were determined indirectly using the index method with titanium as the inorganic marker.

Statistical analysis

All data were analyzed as a completely randomized design with the individual pig as the experimental unit. There were 10 replications of control diet and 11 replications of each treatment diet. Analysis of variance was facilitated using the Proc GLM procedure of SAS (version 9.4; SAS, 2013) with means reported and separated using LSMEANS. Differences were considered significant at P ≤ 0.05 unless otherwise stated.

Experiment 2

Dietary treatments

Eight dietary treatments were arranged in a 2 × 2 × 2 factorial with main factors being the addition of 7.5% soybean oil that was either fresh (SO) or peroxidized (PSO), 10% SDEW that was either not processed or TP–SDEW, and diets without or with 3 mg 15-acetyldeoxynivalenol (15-ADON)/kg diet. Soybean oil was peroxidized using methods described by Yuan et al. (2020) in which SO was heated at 135 °C for 42 h with 30 L/min of continuous air infusion. SDEWs were thermally processed by heating multiple aluminum pans (26.4 cm × 32.4 cm × 6.5 cm, filled one-half full) of SDEW in a 100 °C forced-air drying oven, hand-mixed every 24 h to facilitate equal heating, and heating for 120 h. Diets containing 15-ADON were achieved by first mixing 0.48 g of 15-ADON (USDA-ARS, Peoria, IL) into 1 kg of ground corn, which was then added and mixed in to 10 kg of ground corn, which was then mixed into the complete diet with the remaining feed ingredients. 15ADON was isolated from a liquid yeast extract-peptone-dextrose mixture (1 g yeast extract, 1 g peptone, 50 g dextrose/liter) culture of Fusarium graminearum strain B4-1 (Chen et al., 2000). Cultures were grown at 28 °C in a rotating incubator (200 rotations per min) for 7 d after which the cultures were extracted with ethyl acetate. The extracts were then dried on a rotary evaporator and the concentrated extract was separated on a silica gel column eluted with hexane/ethyl acetate (3:1). Column fractions were monitored using gas chromatography–mass spectrometry. The 15-ADON was greater than 95% pure. Diets were formulated to meet the NRC (2012) energy and nutrient requirements with diets formulated to contain 3,600 kcal ME/kg, 0.83% total calcium, 0.43 standardized total tract digestible phosphorus, 17.8% CP, 1.40% standardized ileal digestible Lys, and TSAA:Lys, Thr:Lys, Trp:Lys, Ile:Lys, and Val:Lys ratios of 0.55, 0.59, 0.165, 0.52, and 0.64, respectively, Table 4. Similar to Experiment 1, vitamins were formulated as close to possible to meet the NRC (2012) requirements, specifically vitamin E was included at 17.5 IU/kg diet and vitamin A at 2,144 IU/kg diet. Titanium dioxide was added as an indigestible marker to diets to allow for determination of digestibility coefficients.

Table 4.

Composition of dietary treatments, Experiment 2

Ingredient	Percentage1
Corn	58.64
Dried whey	15.00
Fish meal	5.00
Monocalcium phosphate	0.91
Limestone	0.87
Sodium chloride	0.40
L-Lysine·HCl	0.70
L-Threonine	0.21
L-Tryptophan	0.05
Mineral premix2	0.15
Vitamin premix3	0.07
Titanium dioxide4	0.50
15-A-DON5	(0.0003)
Spray-dried egg white6	10.00
Soybean oil7	7.50
Total	100.00

¹All diets were formulated to contain 3,600 kcal ME/kg, 0.83% total calcium, 0.43% standardized total tract digestible phosphorus, 17.8% CP, 1.4% standardized ileal digestible Lys with TSAA:Lys, Thr:Lys, Trp:Lys, Ile:Lys, and Val:Lys ratios of 0.55, 0.59, 0.165, 0.52, and 0.64, respectively.

15-A-DON, 15-acetyldeoxynivalenol; Lys, lysine; Thr, threonine; Trp, tryptophan; Ile, isoleucine; Val, valine; CP, crude protein; ME, metabolizable energy; SDEW, spray-dried egg white; TP–SDEW, thermally processed spray-dried egg white; SO, soy oil; PSO, peroxidized soy oil.

²Provided the following per kilogram of diet: 16.5 mg Cu (as CuSO₄), 165 mg Fe (as FeSO₄), 0.3 mg I (as Ca(IO₃)₂), 39 mg Mn (as MnSO₄), 165 mg Zn (as ZnSO₄), and 0.3 mg Se (Na₂SeO₃).

³Provided the following per kilogram of diet: 2,144 IU vitamin A, 245 IU vitamin D₃, 17.5 IU vitamin E, 1.1 mg vitamin K, 0.02 mg vitamin B₁₂, 3.9 mg riboflavin, 19.6 mg niacin, and 9.5 mg pantothenic acid.

⁴Used as an indigestible marker.

⁵For diets with 15-ADON, 15-acetyldeoxynivalenol was included at 3 mg/kg diet.

⁶Diets either contained 10% SDEW or 10% TP–SDEW (100 °C for 120 h).

⁷Diets either contained 7.5% SO or 7.5% PSO (135 °C for 42 h, air bubbled at 30 L/min).

Animal experimentation

Newly weaned (between 19 and 21 d of age) barrows (32) and gilts (32) were obtained from a commercial facility and subsequently housed in the Swine Nutrition Farm at Iowa State University for the duration of the study. Pigs were held in a group pen for 14 d upon weaning then transferred to individual pens (42 cm × 121 cm) for 7 d for adaptation before beginning the dietary treatments. There were two rooms of 32 pens with an even distribution of 16 barrows and 16 gilts per room. The 64 pigs, initial BW of 10.6 kg, were randomly assigned to 1 of the 8 dietary treatments, resulting with 8 replications per diet, with pigs having ad libitum access to feed and water. Pigs and feeders were weighed on d 0 and d 28 to determine ADG, ADFI, and GF. From each pig, approximately 16 mL of blood was collected via jugular venipuncture on d 21, a fresh fecal sample was collected on d 26, and on d 28, following euthanasia and exsanguination, the liver and ileal tissues were harvested, and ileum contents obtained. Samples were harvested and processed as described in Experiment 1. In addition, plasma vitamin E was measured as well as non-specific biomarkers of oxidative status in the plasma by measuring derivatives of reactive oxygen metabolites (ROMs) and antioxidant adsorbent capacity and structural component made of mucopolysaccharides, AAs, and proteins (OXYa), and the ratio of ROM to OXYa, referred to as the oxidative stress index (OSi) which has been suggested to characterize an animal’s overall oxidative stress level (Abuelo et al., 2013). For the ROM and OXYa assays, plasma was diluted 1:100 along with a high and low calibration. Plasma vitamin E was analyzed by the Iowa State University Veterinary Diagnostic Laboratory (ISUVDL Method 9.833) using an established procedure and internally validated methods. To analyze plasma vitamin E using this method, a 0.5 mL aliquot of plasma was placed in a 15 mL screw-top tube, then 2 mL of 95% ethanol and 4 mL of 95/5 hexane/chloroform were added after which the sample was gently shaken to mix and then centrifuged for 5 min at 2,000 rpm. Following centrifugation, 2 mL of the hexane/chloroform solution was transferred to a 7 mL glass vial encased in foil. This solution was then dried using a nitrogen stream after which the extract was then dissolved in 250 µL high-performance liquid chromatography (HPLC)- grade methanol. The sample was then analyzed using ultra HPLC.

Statistical analysis

Data were analyzed as a 2 × 2 × 2 factorial arrangement of treatments with the individual pig as the experimental unit and the room as a blocking factor, equating to 8 replications per treatment combination. Analysis of variance was facilitated using the Proc GLM procedure of SAS (version 9.4; SAS, 2013) with means reported and separated using LSMEANS. Differences were considered significant at P ≤ 0.05 unless otherwise stated. There were no three-way interactions and only one two-way interaction (SO × SDEW for apparent ileal AA digestibility) such these terms were eliminated from the statistical model resulting in only the main effects being reported in tables and discussed in detail. Even though there were equal representation of gilts and barrows across treatments and within each room, gender was not considered in the model or for data interpretation because this was not the focus of the experiment.

Results

Laboratory experiment and SDEW analysis

The laboratory study determined that thermally processing SDEW at 100 °C for 120 h would increase protein carbonyls from 7.3 nmol/mg (non-thermally processed SDEW) to 19.6 nmol/mg when heated for 120 h, Table 1. It was based on these data that the animal experiments would use this temperature and length of time for heating SDEW for use in diet formulations. Using this same method to generate PC in SDEW in Experiment 1, the unheated SDEW was analyzed to contained 34 nmol PC/mg protein and the TP–SDEW contained 23 nmol PC/mg protein, indicating that the TP–SDEW did not generate additional PC above that analyzed in the unheated SDEW. In contrast, in Experiment 2 the TP–SDEW contained 52 nmol PC/mg protein. For the TP–SDEW used in Experiments 1 and 2, CP and all AA of the TP–SDEW were slightly higher than the unheated SDEW, Table 5.

Table 5.

Composition of unprocessed and thermally processed SDEW, DM basis

Nutrient, %	SDEW1	TP–SDEW2	TP–SDEW2
Crude protein	81.55	85.33	84.34
Amino acid
Alanine	5.00	5.19	5.11
Arginine	4.74	4.95	4.93
Aspartic acid	8.67	8.97	8.84
Cysteine	2.41	2.49	2.42
Glutamine	11.28	11.70	11.32
Glycine	2.99	3.10	3.06
Histidine	2.01	2.09	2.03
Isoleucine	4.63	4.71	4.54
Leucine	7.04	7.29	7.24
Lysine	5.80	5.96	5.91
Methionine	3.03	3.10	3.08
Phenylalanine	5.16	5.32	5.39
Proline	2.97	3.07	3.03
Serine	4.99	5.13	4.99
Threonine	3.73	3.84	3.85
Tryptophan	1.57	1.59	1.69
Tyrosine	2.95	3.38	3.47
Valine	6.29	6.52	6.49
Protein carbonyls, nmol PC/mg protein	34	23	52

¹The unheated spray-dried egg white (SDEW) was analyzed to contained 34 nmol PC/mg protein. For formulation purposes the spray-dried egg white was assumed to contain 3,000 kcal ME/kg, 84% crude protein, 0.10% calcium, and 0.10% standardized total tract digestible phosphorus.

²Thermally processing (TP) of SDEW was accomplished by heating the SDEW in a forced air oven at 100 °C for 120 h.

Experiment 1

Apparent ileal digestibility of AA and ATTD of GE and N of the complete diet was unaffected (P > 0.05) by increasing the inclusion of TP–SDEW into the diet. Pigs fed diets containing 6% or 12% TP–SDEW had greater digestibility of S compared to pigs fed diets containing no TP–SDEW (P < 0.05), Table 6. Pigs fed diets containing 6% or 12% TP–SDEW had shorter villi heights compared to pigs fed diets containing no TP–SDEW (P < 0.01), but the level of dietary TP–SDEW had no effect on crypt depths or the resultant villi height:crypt depth ratio (P > 0.05), Table 7.

Table 6.

Dietary amino acid, energy, and nutrient digestibility of pigs fed diets containing varied levels of dietary thermally processed spray dried egg white, Experiment 1¹

	Dietary treatment2			Statistics3
Digestibility	0	6	12	SEM	P-value
AID, %4
Alanine	70.8	68.4	67.3	2.1	0.52
Arginine	76.2	73.6	74.8	1.8	0.61
Aspartic acid	69.4	68.3	66.2	1.9	0.56
Cysteine	62.5	63.4	56.7	2.2	0.09
Glutamine	71.7	71.0	71.2	2.2	0.97
Glycine	45.0	51.5	42.4	4.6	0.34
Histidine	63.7	65.0	62.3	2.1	0.62
Isoleucine	69.7	68.9	68.5	2.2	0.94
Leucine	70.2	70.6	70.7	2.2	0.99
Lysine	83.8	82.3	81.5	1.2	0.43
Methionine	79.7	79.1	78.8	1.7	0.94
Phenylalanine	75.4	74.6	75.3	1.9	0.96
Proline	55.2	55.3	52.1	3.1	0.72
Serine	72.6	72.9	71.2	1.7	0.79
Threonine	65.9	67.9	63.2	1.7	0.19
Tryptophan	82.4	79.9	80.3	1.1	0.24
Tyrosine	72.1	72.3	72.4	1.8	0.99
Valine	66.3	66.5	64.8	2.1	0.83
ATTD, %5
Gross energy	81.1	81.8	82.2	0.9	0.67
Nitrogen	69.5	71.2	74.9	1.7	0.11
Sulfur	49.2b	56.6a	57.5a	2.1	0.03

¹There were 10 pigs fed the 0% thermally processed spray-dried egg white (TP–SDEW) treatment and 11 pigs feed for both the 6% and 12% TP–SDEW treatments. Treatments were applied to individually fed pigs following a 7-d adaptation period post weaning (21 d of age), being fed for 24 d.

²Dietary treatments were created by the inclusion of unprocessed SDEW with TP–SDEW (heated in a forced air oven at 100 °C for 120 h). All diets contained a total of 12% spray dried egg whites. The SDEW contained 34 nmol PC/mg protein and the TP–SDEW contained 23 nmol PC/mg protein.

³Difference in subscripts signifies P < 0.05.

⁴Apparent ileal digestibility, AID.

⁵Apparent total tract digestibility, ATTD.

Table 7.

Ileal morphology of pigs fed diets containing varied levels of dietary thermally processed spray-dried egg white, Experiment 1¹

	Dietary treatment2			Statistics3
Ileal histology4	0	6	12	SEM	P-value
Villi height, um	396a	337b	352b	12	< 0.01
Crypt depth, um	90	89	87	5	0.92
Villi:Crypt	4.8	4.1	4.4	0.4	0.41

¹There were 10 pigs fed the 0% thermally processed spray-dried egg white (TP–SDEW) treatment and 11 pigs feed for both the 6% and 12% TP–SDEW treatments. Treatments were applied to individually fed pigs following a 7-d adaptation period post weaning (21 d of age), being fed for 24 d.

²Dietary treatments were created by the inclusion of unprocessed SDEW with TP–SDEW (heated in a forced air oven at 100 °C for 120 h). All diets contained a total of 12% spray-dried egg whites. The SDEW contained 34 nmol PC/mg protein and the TP–SDEW contained 23 nmol PC/mg protein.

³Difference in subscripts signifies P < 0.05.

⁴Ileum samples were stained with hematoxylin and eosin and analyzed for crypt depth and villi height where 10 villus and crypt pairs with proper orientation were averaged by pig and reported as 1 value per pig.

Feeding pigs TP–SDEW had variable effects on protein damage measured in the plasma and liver. Pigs fed diets containing 6% TP–SDEW had lower plasma PC compared to pigs fed diets containing 12% TP–SDEW, with pigs fed no TP–SDEW being intermediate (P < 0.05). In contrast, pigs fed diets containing 6% TP–SDEW had lower liver PC compared to pigs fed diets not containing any TP–SDEW, with pigs fed diets containing 12% TP–SDEW being intermediate (P < 0.05). There was no observable effect of feeding pigs TP–SDEW on plasma or liver 8-OH-2dG or ISP (P > 0.05), Table 8. No effect of dietary TP–SDEW was noted on pig performance (P > 0.05), Table 9.

Table 8.

Measures of oxidative stress in plasma and liver of pigs fed diets containing varied levels of dietary thermally processed spray-dried egg white, Experiment 1¹

	Dietary treatment2			Statistics3
Oxidative measure	0	6	12	SEM	P-value
Plasma4
PC, nmol/mL	60ab	43b	97a	14	0.02
8-OH-2dG, pg/mL	2,998	2,207	2,512	334	0.28
ISP, pg/mL	55	56	55	3	0.97
Liver5
PC, nmol/mg	194a	112b	180ab	23	0.04
8-OH-2dG, pg/mg	472	434	471	45	0.80
ISP, pg/mg	203	215	225	23	0.80

¹There were 10 pigs fed the 0% thermally processed spray-dried egg white (TP–SDEW) treatment and 11 pigs feed for both the 6% and 12% TP–SDEW treatments. Treatments were applied to individually fed pigs following a 7-d adaptation period post weaning (21 d of age), being fed for 24 d. PC, protein carbonyls; 8-OH-2dG, 8-hydroxy-2'-deoxyguanosine; ISP, F2-isoprostanes.

²Dietary treatments were created by the inclusion of unprocessed SDEW with TP–SDEW (heated in a forced air oven at 100 °C for 120 h). All diets contained a total of 12% spray-dried egg whites. The SDEW contained 34 nmol PC/mg protein and the TP–SDEW contained 23 nmol PC/mg protein.

³Difference in subscripts signifies P < 0.05.

⁴Plasma obtained from individual pigs on d 21.

⁵Liver tissue obtained from individual pigs following harvest on d 24.

Table 9.

Growth performance of pigs fed diets containing varied levels of dietary thermally processed spray-dried egg white, Experiment 1¹

	Dietary treatment2			Statistics3
Performance criterion	0	6	12	SEM	P—value
ADG, kg	0.203	0.193	0.183	0.19	0.76
ADFI, kg	0.403	0.392	0.369	0.02	0.57
GF	0.504	0.486	0.481	0.02	0.83

¹There were 10 pigs fed the 0% thermally processed spray-dried egg white (TP–SDEW) treatment and 11 pigs feed for both the 6% and 12% TP–SDEW treatments. Treatments were applied to individually fed pigs following a 7-d adaptation period post weaning (21 d of age), being fed for 24 d. ADG, averaged daily gain; ADFI, average daily feed intake; GF, gain to feed ratio; SEM, standard error of the mean.

²Dietary treatments were created by the inclusion of unprocessed SDEW with TP–SDEW (heated in a forced air oven at 100 °C for 120 h). All diets contained a total of 12% SDEWs. The SDEW contained 34 nmol PC/mg protein and the TPSDEW contained 23 nmol PC/mg protein.

Experiment 2

Analyzed concentrations of DON, ZEN, and FUM in the complete diets differed little, Table 10. While 3 mg of 15-ADON was added per kg diet for half of the experimental diets, analysis of the diets shows that diets with no added 15-ADON contained 0.38 mg 15-ADON/kg diet compared to diets with added 15-ADON which contained 0.80 mg 15-ADON/kg diet. As shown in Table 5, the TP–SDEW contained 52 nmol PC/mg protein compared to 34 nmol PC/mg protein in the SDEW, with CP and all AA being slightly higher in the TP–SDEW compared to the unheated SDEW. Thermally processing of SO (i.e., PSO) increased the concentrations of palmitic, stearic, and oleic fatty acids while concentrations of linoleic and linolenic fatty acids were decreased. In addition, PSO had greater concentrations of aldehydes compared to unprocessed SO, Table 11.

Table 10.

Mycotoxin analysis of complete diets, mg/kg diet, Experiment 2

MTX1	(-) 15-ADON				(+)15-ADON
SO2	SO		PSO		SO		PSO
SDEW3	SDEW	TP–SDEW	SDEW	TP–SDEW	SDEW	TP–SDEW	SDEW	TP–SDEW
DON4	0.5	0.8	0.5	0.4	0.4	0.5	0.5	0.4
ZEN5	0.3	0.6	0.4	0.2	0.2	0.2	0.3	0.1
FUM6	< 0.2	< 0.2	< 0.2	< 0.2	< 0.2	0.6	< 0.2	< 0.2
15-ADON7	0.4	0.4	0.4	0.3	0.7	0.7	0.9	0.9

¹Mycotoxin (MTX) treatments. Diets contained either no MTX addition (-) or the addition (+) of 3 mg of 15-acetyldeoxynivalenol (15-ADON)/kg diet.

²Soybean oil (SO) treatments. Diets contained 7.5% of either unprocessed SO or peroxidized SO (PSO) achieved by heating SO at 135 °C for 42 h with constant air bubbling at 30 L/min.

³Spray-dried egg white (SDEW) treatments. Diets contained 10% of either unprocessed SDEW or thermally processed (TP) SDEW by heating SDEW in a forced air oven at 100 °C for 120 h.

⁴Deoxynivalenol (DON), mg/kg diet.

⁵Fumonisin (FUM), mg/kg diet.

⁶Zearalenone (ZEN), mg/kg diet.

⁷15-acetyldeoxynivalenol (15-ADON), mg/kg diet.

Table 11.

Composition of soybean oil and peroxidized soybean oil, Experiment 2

Item	SO	PSO
Fatty acid, % of total fat2
C8:0, Caprylic	-	0.43
C16:0, Palmitic	10.95	15.13
C18:0, Stearic	3.90	5.50
C18:1, Oleic	22.26	27.95
C18:2, Linoleic	54.86	45.81
C18:3, Linolenic	6.97	3.87
C19:1, Nonadecanoic	0.20	0.13
C20:0, Arachidic	0.29	0.41
C20:1, Gadoleic	0.17	0.25
C22:0, Behenic	0.28	0.38
Other fatty acids	0.13	0.15
Aldehydes, mg/kg3
2,4-Decadienal	0.1	534.3
Hexanal	2.5	110.6
4-Hydroxynonenal	0.2	176.5
Total aldehydes	7.2	1,349

¹Either unprocessed soybean oil (SO) or peroxidized SO (PSO) achieved by heating SO at 135 °C for 42 h with constant air bubbling at 30 L/min.

² Analyzed by Barrow-Agee, Memphis, TN.

³ Analyzed by University of Minnesota, St. Paul, MN.

There were no three-way interactions and only one two-way interaction such that detail on the main effects and the one two-way interaction between SO and SDEW relative to AID of AA will only be presented in detail. There was no effect of feeding TP–SDEW on ATTD of GE, N, or S (P > 0.05, Table 12), on villus height, crypt depth, or villus height:crypt depth ratio (P > 0.05, Table 13), or on oxidative markers in the plasma, liver, or ileum (P > 0.05, Table 14). There was an increase in ADG and GF in pigs fed TP–SDEW compared to pigs fed unprocessed SDEW (P < 0.01, Table 15).

Table 12.

Impact of thermally processed spray-dried egg white, 15-acetyldeoxynivalenol, or peroxidized soybean oil on apparent total tract digestibility of energy and nutrients, Experiment 2¹

	Main effect2		Statistics
ATTD, %3	(-) TP–SDEW	(+) TP–SDEW	SEM	P-value
Gross energy	85.53	86.42	0.36	0.09
Nitrogen	80.77	81.08	0.44	0.61
Sulfur	79.42	78.84	0.44	0.36
	Main effect2		Statistics
ATTD, %3	(-) 15-ADON	(+) 15-ADON	SEM	P-value
Gross energy	85.50	86.41	0.36	0.10
Nitrogen	80.25	81.60	0.44	0.04
Sulfur	79.08	79.18	0.44	0.87
	Main effect2		Statistics
ATTD, %3	(-) PSO	(+) PSO	SEM	P-value
Gross energy	88.32	83.63	0.36	< 0.01
Nitrogen	82.34	79.52	0.44	< 0.01
Sulfur	81.19	77.06	0.44	< 0.01

¹Pigs were fed common diet 14 d postwean and then adapted to individual pens for 7 d. On d 21, postwean pigs (initial BW 10.6 kg) were put on trial for 28 d (final BW 18.5 kg). TP–SDEW, thermally processed spray-dried egg whites (achieved by heating SDEW in a forced air oven at 100 °C for 120 h); 15-ADON, 15-acetyldeoxynivalenol (, achieved by adding 3 mg 15-ADON/kg diet); PSO, peroxidized soy oil (chieved by heating SO at 135 °C for 42 h with constant air bubbling at 30 L/min); SEM, pooled standard error of the mean.

²Dietary treatments were factorially arranged among TP–SDEW (either 7.5% unprocessed SDEW or 7.5% TP–SDEW), 15-ADON (either no 15-ADON added or 3 mg/kg diet of 15-ADON), and PSO (either 7.5% unprocessed soybean oil or 7.5% PSO). There were no three- or two-way interactions observed (P > 0.01) such that only the main effects are shown representing 32 pigs per main effect mean.

³Apparent total tract digestibility (ATTD) using feces collected on d 26.

Table 13.

Impact of thermally processed spray dried egg white, 15-acetyldeoxynivalenol, or peroxidized soybean oil on ileal intestinal morphology, Experiment 2¹

	Main effect2		Statistics
Morphology3	(-) TP–SDEW	(+) TP–SDEW	SEM	P-value
Villi height, µm	333	334	7.5	0.31
Crypt depth, µm	82	83	4.0	0.87
Villus:crypt	4.8	4.9	0.2	0.57
	Main effect2		Statistics
Morphology3	(-) 15-ADON	(+) 15-ADON	SEM	P-value
Villi height, µm	341	335	7.5	0.58
Crypt depth, µm	85	80	4.0	0.36
Villus:crypt	4.8	4.9	0.2	0.73
	Main effect2		Statistics
Morphology3	(-) PSO	(+) PSO	SEM	P-value
Villi height, µm	346	331	7.5	0.15
Crypt depth, µm	83	81	4.0	0.78
Villus:crypt	4.9	4.7	0.2	0.55

¹Pigs were fed common diet 14 d postwean and then adapted to individual pens for 7 d. On d 21, postwean pigs (initial BW 10.6 kg) were put on trial for 28 d (final BW 18.5 kg). TP–SDEW, thermally processed spray-dried egg whites (achieved by heating SDEW in a forced air oven at 100 °C for 120 h); 15-ADON, 15-acetyldeoxynivalenol (achieved by adding 3 mg 15-ADON/kg diet); PSO, peroxidized soy oil (achieved by heating SO at 135 °C for 42 h with constant air bubbling at 30 L/min); SEM, pooled standard error of the mean.

²Dietary treatments were factorially arranged among TP–SDEW (either 7.5% unprocessed SDEW or 7.5% TP–SDEW), 15-ADON (either no 15-ADON added or 3 mg/kg diet of 15-ADON), and PSO (either 7.5% unprocessed soybean oil or 7.5% PSO). There were no three- or two-way interactions observed (P > 0.01) such that only the main effects are shown representing 32 pigs per main effect mean.

³Ileum tissue samples were taken at harvest (d 28) and submerged in 10% formalin until cross sectioned, put on a slide and stained with hematoxylin and eosin and analyzed for villi height and crypt depth where 10 villus and crypt pairs with proper orientation were averaged by pig and reporter as 1 value per pig.

Table 14.

Impact of thermally processed spray dried egg white, 15-acetyldeoxynivalenol, or peroxidized soybean oil on oxidative markers in the plasma, liver, and ileum, Experiment 2¹

	Main effect2		Statistics
Criterion	(-) TP–SDEW	(+) TP–SDEW	SEM	P-value
Plasma3
PC, nmol/mL	127	127	9.1	0.96
ISP, pg/mL	151	102	32	0.27
8 OH 2dG, ng/mL	13	13	0.44	0.72
Vitamin E, ppm	0.6	0.6	0.03	0.53
ROM, UCARR	575	572	29	0.94
AXC, µmol HClO/mL	400	437	45	0.56
OSi	4.9	1.8	1.7	0.21
Liver4
PC, nmol/mg	203	186	20	0.56
ISP, pg/mg	164	177	13	0.48
8 OH 2dG, pg/mg	209	199	13	0.72
Ileal4
PC, nmol/mg	369	347	29	0.59
ISP, pg/mg	401	355	24	0.17
8 OH 2dG, pg/mg	341	342	25	0.97
	Main effect2		Statistics
	(-) 15-ADON	(+) 15-ADON	SEM	P-value
Plasma3
PC, nmol/mL	134	120	9.1	0.27
ISP, pg/mL	125	128	32	0.95
8 OH 2dG, ng/mL	12.9	12.7	0.44	0.70
Vitamin E, ppm	0.6	0.6	0.03	0.66
ROM, UCARR	581	567	29	0.78
AXC, µmol HClO/mL	430	407	45	0.71
OSi	1.71	4.99	1.7	0.17
Liver4
PC, nmol/mg	213	176	20	0.21
ISP, pg/mg	177	165	13	0.50
8 OH 2dG, pg/mg	207	199	13	0.66
Ileal4
PC, nmol/mg	364	352	29	0.78
ISP, pg/mg	394	361	24	0.31
8 OH 2dG, pg/mg	322	362	25	0.27
	Main effect2		Statistics
Criterion	(-) PSO	(+) PSO	SEM	P-value
Plasma3
PC, nmol/mL	126	128	9.1	0.83
ISP, pg/mL	100	153	32	0.24
8 OH 2dG, ng/mL	12	14	0.44	0.02
Vitamin E, ppm	1.1	0.2	0.03	< 0.01
ROM, UCARR	589	559	29	0.47
AXC, µmol HClO/mL	414	423	45	0.88
OSi	4.1	2.6	1.7	0.55
Liver4
PC, nmol/mg	197	192	20	0.88
ISP, pg/mg	174	167	13	0.69
8 OH 2dG, pg/mg	200	206	13	0.73
Ileal4
PC, nmol/mg	386	330	29	0.17
ISP, pg/mg	375	381	24	0.84
8 OH 2dG, pg/mg	355	329	25	0.47

¹Pigs were fed common diet 14 d postwean and then adapted to individual pens for 7 d. On d 21, postwean pigs (initial BW 10.6 kg) were put on trial for 28 d (final BW 18.5 kg). TP-SDEW, thermally processed spray-dried egg whites (achieved by heating SDEW in a forced air oven at 100 °C for 120 h); 15-ADON, 15-acetyldeoxynivalenol (achieved by adding 3 mg 15-ADON/kg diet); PSO, peroxidized soy oil (achieved by heating SO at 135 °C for 42 h with constant air bubbling at 30 L/min); SEM, pooled standard error of the mean; PC, protein carbonyl; ISP, F2-isoprostanes; 8 OH 2dG, 8 hydroxy 2' deoxyguanosine; ROM, reactive oxygen metabolites; OXYa, antioxidant adsorbent capacity; OSi, oxidative stress index (ROM:OXYa ratio).

²Dietary treatments were factorially arranged among TP–SDEW (either 7.5% unprocessed SDEW or 7.5% TP–SDEW), 15-ADON (either no 15-ADON added or 3 mg/kg diet of 15-ADON), and PSO (either 7.5% unprocessed soybean oil or 7.5% PSO). There were no three- or two-way interactions observed (P > 0.01) such that only the main effects are shown representing 32 pigs per main effect mean.

³Blood samples were collected on d 21 with sodium heparin as the clotting agent, samples were spun down and plasma was aliquoted and stored at −80 °C until assayed for PC, ISP, 8-OH-2dG, Vitamin E, ROM, and AXC.

⁴Tissues were collected at harvest on d 28 and snap frozen in liquid nitrogen until assayed. Tissues were homogenized and assayed for PC, ISP, and 8-OH-2dG.

Table 15.

Impact of thermally processed spray-dried egg white, 15-acetyldeoxynivalenol, or peroxidized soybean oil on pig performance, Experiment 2¹

	Main effect2		Statistics
Criterion	(-) TP–SDEW	(+) TP–SDEW	SEM	P-value
ADG, kg/d	0.25	0.30	0.01	< 0.01
ADFI, kg/d	0.59	0.60	0.01	0.63
GF	0.41	0.50	0.01	< 0.01
	Main effect2		Statistics
Criterion	(-) 15-ADON	(+) 15-ADON	SEM	P-value
ADG, kg/d	0.28	0.26	0.01	0.31
ADFI, kg/d	0.61	0.59	0.01	0.35
GF	0.46	0.45	0.01	0.51
	Main effect2		Statistics
Criterion	(-) PSO	(+) PSO	SEM	P-value
ADG, kg	0.31	0.23	0.01	< 0.01
ADFI, kg	0.63	0.56	0.01	< 0.01
GF	0.49	0.41	0.01	< 0.01

¹Pigs were fed common diet 14 d postwean and then adapted to individual pens for 7 d. On d 21, postwean pigs (initial BW 10.6 kg) were put on trial for 28 d (final BW 18.5 kg). TP-SDEW, thermally processed spray-dried egg whites (achieved by heating SDEW in a forced air oven at 100 °C for 120 h); 15-ADON, 15-acetyldeoxynivalenol (achieved by adding 3 mg 15-ADON/kg diet); PSO, peroxidized soy oil (achieved by heating SO at 135 °C for 42 h with constant air bubbling at 30 L/min), SEM, pooled standard error of the mean; ADG, average daily gain; ADFI, average daily feed intake; GF, gain-to-feed ratio.

²Dietary treatments were factorially arranged among TP–SDEW (either 7.5% unprocessed SDEW or 7.5% TP–SDEW), 15-ADON (either no 15-ADON added or 3 mg/kg diet of 15-ADON), and PSO (either 7.5% unprocessed soybean oil or 7.5% PSO). There were no three- or two-way interactions observed (P > 0.01) such that only the main effects are shown representing 32 pigs per main effect mean.

Table 16.

Impact of thermally processed spray dried egg white, 15-acetyldeoxynivalenol, or peroxidized soybean oil on apparent ileal amino acid digestibility, Experiment 2¹

	SO		PSO		Statistics
AID, %3	SDEW	TP–SDEW	SDEW	TP–SDEW	SEM	P × T	PSO	TP
Alanine	61.4	64.0	57.0	64.7	1.8	0.16	0.31	0.01
Arginine	77.5	78.0	81.0	83.7	1.8	0.56	0.02	0.39
Aspartic acid	62.9ab	60.8ab	59.0b	63.5a	1.6	0.04	0.69	0.44
Cysteine	67.2	65.2	61.4	65.4	1.6	0.07	0.09	0.56
Glutamic acid	68.7ab	68.7ab	64.8b	71.9a	1.5	0.02	0.83	0.02
Glycine	53.4a	49.1a	39.3b	52.1a	2.4	0.01	0.03	0.08
Histidine	71.3	71.7	70.9	76.7	1.7	0.12	0.19	0.08
Isoleucine	63.0b	64.6b	60.9b	69.4a	1.5	0.03	0.38	0.01
Leucine	69.4b	70.5b	68.0b	74.6a	1.3	0.05	0.32	0.01
Lysine	76.7	77.5	72.8	77.3	1.4	0.19	0.16	0.07
Methionine	73.7b	73.8b	71.9b	79.5a	1.4	0.01	0.18	0.01
Phenylalanine	70.5b	71.2b	69.2b	76.3a	1.4	0.03	0.17	0.01
Proline	62.7a	61.4a	52.4b	63.4a	2.1	0.01	0.06	0.03
Serine	73.1ab	70.7ab	69.3b	74.8a	1.9	0.04	0.94	0.41
Threonine	67.1a	68.0a	60.5b	70.6a	2.1	0.03	0.35	0.01
Tryptophan	76.9	79.9	76.7	82.8	1.2	0.21	0.27	0.01
Tyrosine	70.5ab	70.4b	67.4b	75.4a	1.8	0.03	0.61	0.03
Valine	59.3ab	59.7ab	55.4b	64.1a	1.7	0.02	0.89	0.01

¹Pigs were fed common diet 14 d postwean and then adapted to individual pens for 7 d. On d 21, postwean pigs (initial BW 10.6 kg) were put on trial for 28 d (final BW 18.5 kg). TP–SDEW, thermally processed spray-dried egg whites (achieved by heating SDEW in a forced air oven at 100 °C for 120 h); 15-ADON, 15-acetyldeoxynivalenol (achieved by adding 3 mg 15-ADON/kg diet); PSO, peroxidized soy oil (achieved by heating SO at 135 °C for 42 h with constant air bubbling at 30 L/min); SEM, pooled standard error of the mean.

²Dietary treatments were factorially arranged among TP–SDEW (either 10% unprocessed SDEW or 10% TP–SDEW), 15-ADON (either no 15-ADON added or 3 mg/kg diet of 15-ADON), and PSO (either 7.5% unprocessed soybean oil or 7.5% PSO). There were no three- or two-way interactions observed (P > 0.01) such that only the main effects are shown representing 32 pigs per main effect mean. There was no effect of dietary 15-ADON on AID of any AA (P > 0.05).

³Apparent ileal digestibility (AID) using ileal contents collected on d 26.

Compared to pigs fed diets without added 15-ADON, pigs fed diets containing added 15-ADON had greater ATTD of N (P < 0.05), but there was no effect of feeding diets containing added 15-ADON on ATTD of GE or S (P > 0.05, Table 12), on ileal AA digestibility (P < 0.05, data not shown), on villus height, crypt depth, or villus height:crypt depth ratio (P > 0.05, Table 13), on oxidative markers in the plasma, liver, or ileum (P > 0.05, Table 14), or on ADG, ADFI, or GF (P > 0.05, Table 15) compared to pigs fed diets without added 15-ADON.

Feeding pigs diets containing PSO compared to pigs fed diets fed unprocessed SO resulted in reduced ATTD of GE, N, and S (P < 0.01), did not affect villus height, crypt depth, or villus height:crypt depth ratio (P > 0.05, Table 13), increased plasma 8-OH-2dG (P < 0.05) and decreased plasma vitamin E (P < 0.01) with no impact observed on the other oxidative markers in the plasma, liver, or ileum (P > 0.05, Table 14), and decreased ADG, ADFI, or GF (P < 0.01, Table 15). The two-way interaction between PSO and SDEW for apparent ileal digestibility of AA is difficult to explain. In reviewing AID of AA as a whole, the interaction between PSO and SDEW appears to be attributed no differences in AID of AA between in pigs fed diets containing SDEW and TP–SDEW when SO was included in the diet, but if the diet contained PSO, then pigs fed diets containing TP–SDEW had a greater AID of Asp, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Ser, Tyr, and Val compared to pigs fed diets containing SDEW that has not been thermally processed.

Discussion

Protein oxidation is a commonly known concern in muscle, meat, and food systems (Shacter, 2000; Estevez, 2011; Lund et al., 2011; Zhang et al., 2013; Davies, 2016; Estevez et al., 2017). While not often considered in the livestock feeding industry, proteins may be vulnerable to oxidative modifications during processing and subsequent storage. The processing of oilseeds and animal by-products by thermal heating is important for reduction in water content, extraction of oil, inactivation of anti-nutritional factors, and control of microbial and pathogen growth; all along making feedstuffs that can be handled, stored, and used in animal feeding programs more efficiently. Overheating can, however, lead to potential AA oxidation, racemization, cross-linking, and Maillard reactions (Rojas and Stein, 2013; Drulyte and Orlien, 2019; Jahanbin, 2021). While the mechanisms of heat damage in proteins are known to affect all AA (Papadopoulos, 1989; Shacter, 2000; Weber et al., 2015), most studies have focused on Lys damage as evaluated in blood meal (Waibel et al., 1977), meat and bone meal (Batterham et al., 1986), field peas (Stein and Bohlke, 2007); distillers dried grains with solubles (Almeida et al., 2013), and soybean meal (González-Vega et al., 2011; Oliveira et al., 2020). While there have been numerous studies evaluating heating or over processing of feedstuffs relative to AA digestibility (Rojas and Stein, 2013; Drulyte and Orlien, 2019) few studies have tried to relate formation of PC during this processing to intestinal morphology or oxidative status within an animal.

In the current experiments, SDEW were selected as the protein source because they contain a high level of sulfur AA which are known to be more reactive to oxidation compared to other AA (Gruhlke and Slusarenko, 2012; Celi and Gabai, 2015). While SDEW are not often used in swine feed formulations, they were specifically selected to test the concept that oxidized proteins may affect energy and nutrient digestibility, intestinal morphology, oxidative status, and performance in nursery pigs. Previous work evaluating oxidized protein is limited, having been conducted by heating soy protein isolate (Zhang et al., 2017), spray-dried bovine plasma (Frame et al., 2020), or chicken by-product meal (Frame et al., 2021). Zhang et al. (2017) increased the PC content of soy-protein isolate by heating at 100 °C for 8 h (1.4 vs. 2.5 nmol PC/mg protein) and when fed to broiler chicks for 21 d, observed an increase in serum malondialdehyde (MDA) and PC, but no increase in liver MDA or PC, and a decrease in ADG but no change in ADFI for feed efficiency. Frame et al. (2020) increased the PC content of spray-dried bovine plasma by heating at 100 °C for 72 h (2.7 vs. 8.1 nmol PC/mg protein) and observed no effect on intestinal morphology, energy or nutrient digestibility, or measures of oxidative stress, but reported an increase in ADG and ADFI when fed to 12.5 kg gilts for 19 d. At last, Frame et al. (2021) increased the PC content of chicken by-product meal by heating at 100 °C for 72 h (44.9 vs. 60.7 nmol PC/mg protein) and observed no effects on intestinal morphology, ATTD of GE, measures of oxidative stress, or pig performance when fed to 5.5 kg pigs for 35 d. Because these studies show inconsistency between oxidizing different proteins and their effects when fed to swine and poultry, the current studies were designed to clarify the effects of feeding TP–SDEW as a potentially oxidized protein source on growth performance, apparent energy and nutrient digestibility, intestinal morphology, and specific measures of oxidative stress.

Laboratory

In this study, it was determined that thermally processing SDEW at 100 °C for 120 h would increase protein carbonyls from 7.3 nmol/mg (non-thermally processed SDEW) to 19.6 nmol/mg Table 1. These results were promising given they agreed with others (Zhang et al., 2017; Frame et al., 2020, 2021) that previously processed proteins could be oxidized, whereupon it was decided that this method of heating SDEW would be used for the subsequent animal experimentations. When using this exact same method to generate PC in SDEW in Experiment 1, however, the TP–SDEW contained only 23 nmol PC/mg protein compared to the unheated SDEW which was analyzed to contained 34 nmol PC/mg protein, thus indicating that the TP–SDEW did not have additional PC generated. In contrast, in Experiment 2 the TP–SDEW contained 52 nmol PC/mg protein, thereby representing a substantial increase in PC generation by the heating process. Taken together these data suggests that heating a dried protein product, in this case SDEW, may induce protein oxidation as indicated by PC analysis, but the result is inconsistent.

Experiment 1

Given the lack of an increase in PC in the TP–SDEW used in Experiment 1, the results of Experiment 1 must be viewed not as a thermal process that induced an increase in PC in TP–SDEW, but only as a dry heating process of a protein source. As such, an important distinction that must be made is that feedstuffs evaluated by others (Waibel et al., 1977; Batterham et al., 1986; Stein and Bohlke, 2007; González-Vega et al., 2011; Almeida et al., 2013; Oliveira et al., 2020) were subjected to heating in the presence of greater moisture content (e.g., drying of whole blood in a drum or ring dryer, drying wet distillers grains with solubles in a drum dryer) and by potentially using processing pressure (e.g., extrusion or autoclaving) during the processing event. It is noteworthy that in the current experiment the SDEW obtained was approximately 92% dry matter such that in its natural state it had little water (e.g., ≤ 8%) available for generation of ROSs, it was heated using only ambient air (oxygen = 21% oxygen), and it was thermally processed in a forced air oven where air was passed over, not through, the sample during heating. Both of which likely limited the potential for the proteins in the SDEW to be oxidized. In light of these facts, is not surprising that the majority of the data obtained in Experiment 1 showed no effect due to increasing the inclusion level of TP–SDEW in diets fed to nursery pigs. This is supported by little change in the AA composition of the TP–SDEW as presented in Table 5. Of the differences that were observed (i.e., ATTD of S, villi height, and PC in the plasma and liver) among the experimental treatments, the direction of change appeared to be random events and not explained on a consistent biological basis. For example, neither the increase in ATTD of dietary S with increased inclusion level of TP–SDEW or the PC in the plasma and liver being lowest in diets containing 6% TP–SDEW compared to pigs fed diets containing 0% and 12% TP–SDEW having the highest plasma and liver PC concentrations, make biological sense if it is assumed that the thermal process either generated PC in the SDEW or produced proteins that were irreversible damaged.

Experiment 2

15-ADON

Experiment 2 was designed to use three different enteric stressors, either singularly or in combination, each which have been shown to reduce performance, intestinal integrity, energy and nutrient digestibility, and induce oxidative stress. Initially, naturally contaminated corn containing several MTX (e.g., aflatoxin, DON, FUM, ZEA) was considered for use in Experiment 2, but a corn with naturally occurring MTX could not be obtained for research purposes. It was thus theorized that DON would be a suitable substitute because DON is one of the most common MTX in corn and corn by-products and is known to reduce pig performance (Bergsjo et al., 1993; Rotter et al., 1996; Li et al., 2011), impair intestinal absorption and destruct barrier function (Pinton et al., 2009, 2012), and create a pro-inflammatory response (Kouadio et al., 2005; Li et al., 2011; Sun et al., 2016) thereby increasing ROS and oxidative stress in the form of DNA fragmentation (Frankic et al., 2008; Zhang et al., 2009) and protein carbonyl content (Strasser et al., 2013). It has also been suggested that DON may have specificity to oxidative DNA damage (Awad et al., 2014). Because a natural source of DON was not found, a major derivative of naturally occurring DON, 15-ADON, was obtained (USDA-ARS, Peoria, IL) for research purposes. While the derivative toxicity of 15-ADON compared to naturally contaminated DON is unclear in swine, based on Forsell et al. (1987), Pestka et al. (1987), and Prelusky et al. (1988), a similar toxicity between DON and 15-ADON was assumed whereupon 3 mg 15-ADON/kg diet was used which is similar to the level of naturally occurring DON known to have negative effects on animal productivity. Even though meticulous effort was taken to ensure proper mixing of the 15-ADON into the diets, analysis of the final diets did not confirm that the levels 15-ADON were achieved as planned, Table 10. In addition, naturally occurring MTX can be more toxic than a singularly added MTX or MTX-derivative due to multiple MTX being present at the same time (D’Mello et al., 1999; Wan et al., 2013; Assunção et al., 2016). Given these concerns it was not surprising that no interactive or main effects due to the addition of 15-ADON were observed, and therefore no additional discussion is warranted.

Peroxidized soybean oil

Feeding PSO was selected because it has been clearly established that feeding thermally processed SO typically reduces growth and energy digestibility, even though the effect on measures of oxidative stress is variable (Lindblom et al., 2018a, 2018b; Overholt et al., 2018; Kerr et al., 2020a, 2020b; Yuan et al., 2020). In addition, it was hypothesized that feeding PSO may interact with dietary TP–SDEW or 15-ADON on one or more of the animal-based measures being taken. Soybean oil was processed as previously described (Yuan et al., 2020) but with an inclusion level of 7.5% instead of 10% to improve feed flowability in the self-feeders. As expected, thermally processing SO at 135 °C for 42 h with constant air bubbling at 30 L/min greatly increased aldehyde concentration in the PSO compared with the SO (Kerr et al., 2020b; Yuan et al., 2020).

The reduction in ATTD of GE in pigs fed diets containing PSO is supported by others (DeRouchey et al., 2004; Yuan et al., 2007; Rosero et al., 2015; Lindblom et al., 2018a; Overholt et al., 2018; Kerr et al., 2020b) who reported a decrease in lipid digestibility due to feeding pigs peroxidized lipids; suggesting that at least port of the reduction in animal performance is due to the reduction in digestible energy intake, and consequently on ADG. In the current study, ATTD of N was reduced which is supported by Yuan et al. (2007) but this is not supported by others (DeRouchey et al., 2004; Lindblom et al., 2018a; Overholt et al., 2018; Kerr et al., 2020a). The differences in digestibility results may be due to processing SO at 135 °C for 42 h in the current trial which has been shown to be most detrimental to pig performance compared to processing SO at 90 °C for 84 h or 180 °C for 21 h (Yuan et al., 2020).

Measures on the impact of feeding PSO on intestinal morphology is limited. Rosero et al. (2015) reported small increases in villus height, crypts depth, and villus height:crypt depth ratio due to feeding nursery pigs 6% PSO, but no effect was observed herein in feeding nursery pigs diets containing 7.5% PSO. As summarized in Kerr et al. (2020b), the effects of feeding peroxidized lipids on measures of oxidative stress in pigs is inconclusive. In the current experiment, feeding pigs diets containing PSO had essentially no effect on indicators of oxidative stress as measured in the plasma, liver, or ileal tissue. One effect that was noted, however, was the dramatic reduction in plasma vitamin concentration in pigs fed SO vs. PSO (1.1 vs. 0.2 ppm; respectively). While no other indicators of oxidative stress were altered, this precipitous drop in vitamin E is of critical importance given that vitamin E is a key component in ROS management in the body (Sies et al., 1992; Hensley et al., 2004; Finaud et al., 2006). Our results are supported by others (Boler et al., 2012; Liu et al., 2014a; Hanson et al., 2016; Chang et al., 2019; Silva-Guillen et al., 2020) who have reported decreases in serum vitamin E in pigs consuming peroxidized lipids. Such a precipitous drop in plasma vitamin E warrants future research. The decrease in pig performance was dramatic and similar in magnitude using a similar processing temperature as reported by Yuan et al. (2020). A large part of this reduction is due to the large reduction in ADFI, but also in part due to a decrease in ATTD of GE in the diet.

Thermally processed spray-dried egg white

Unlike that observed for analyzed PC concentrations in the TP–SDEW used in Experiment 1, the TP–SDEW used in Experiment 2 had an increase in PC concentration comparted to SDEW (52 vs. 34 nmol PC/mg protein, respectively). Nonetheless, pigs fed diets containing the TP–SDEW did not exhibit any differences in ATTD of GE, N, or S, no changes in intestinal morphology, and no alterations for markers of oxidative stress. In contrast, there was an increase in ADG and GF of pigs fed diets containing TP–SDEW compared to pigs fed SDEW. In comparison, Zhang et al. (2017) reported some alterations in oxidative stress measures (e.g., increased serum MDA and PC, decreased serum GSH, decreased liver catalase activity, increased liver advanced oxidation protein products, increased jejunal mucosa PC concentration), but the reduction in ADG (approximately 5%) appeared to be largely due to a decrease in ADFI (approximately 3%), with feed efficiency decreasing only 2%, as no indices of energy or nutrient digestibility were reported. In contrast, Frame et al. (2020) reported an increase in ADG and ADFI when spray-dried plasma was thermally processed, with inconsistent effects on energy and nutrient digestibility, no change in intestinal morphology, and no observed changes in measures of oxidative stress in the plasma, jejunum, colon, or liver. At last, while caution must be taken when comparing the current data to Frame et al. (2021) because their use of chicken by-product meal is confounded in that this protein feedstuff also contains approximately 14% fat (range 9% to 24%, Kerr et al., 2017) and can be peroxidized under thermal conditions (Liu et al., 2014b). Even when this is taken into consideration, heating of chicken by-product meal only decreased ATTD of N with no effect on ATTD of GE, no change in ileal morphology, no change in measures of oxidative stress in the plasma, jejunum, colon, liver, or urine, and no effect on pig performance (Frame et al., 2021).

TP-SDEW × PSO

In reviewing AID of AA in as a whole, the interaction between PSO and SDEW appears to be attributed to no differences in AID of AA in pigs between diets containing SDEW and TP–SDEW when SO was contained in the diet, but if the diet contained PSO, then pigs fed diets containing TP–SDEW had greater AID of Asp, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Ser, Tyr, and Val compared to pigs fed diets containing SDEW that has not been thermally processed. No biological explanation could be put forth for this effect. It is noteworthy that in pigs fed SO, there was no difference in AID of dietary AA between feeding pigs SDEW and TP–SDEW, which is supported by the lack of AID of dietary AA among diets fed to pigs in Experiment 1.

In summary, the results indicating that it is difficult to induce protein oxidation in previously processed SDEW using dry heat in a forced air oven (100 °C for 72 h), and even if some protein oxidation occurs (e.g., increased concentration of PC), there is limited, if any, effects on growth performance, digestibility, intestinal morphology, and oxidative status in nursery pigs. The data also indicates that adding 3 mg/kg of a laboratory purified MTX derivative (e.g., 15-ADON) is difficult to ensure proper mixing from which to subsequently analyze, and even if proper mixing was achieved, that a singly added MTX may have no effects on the animal. At last, the data show that adding SO that has been thermally processed to contain high concentrations of aldehydes will result in a dramatic reduction in animal performance, but has limited effects on digestibility, intestinal morphology, and oxidative status in nursery pigs.

Glossary

Abbreviations

AA

amino acids

ADG

average daily gain

ADFI

average daily feed intake

AID

apparent ileal digestibility

ATTD

apparent total tract digestibility

BW

body weight

DNA

deoxyribose nucleic acid

DON

deoxynivalenol

EXP

experiment

FUM

fumonisin

GE

gross energy

GF

gain to feed ratio

ISP

F2-isoprostanes

MDA

malondialdehyde

MTX

mycotoxins; N, nitrogen

OXYa

antioxidant adsorbent capacity

OSi

oxidative stress index

PSO

peroxidized soybean oil

PC

protein carbonyls

ROM

reactive oxygen metabolites

ROS

reactive oxygen species

SDEW

spray dried egg white; S, sulfur

SO

soybean oil

TP–SDEW

thermally processed spray dried egg white

ZEN

zearalenone; 8-OH-2dG, 8-hydroxy-2-deoxyguanosine

15-ADON

15-acetyldeoxynivalenol

Conflict of Interest Statement

All the authors have no conflicts of interest.

Author Contributions

VCW and BJK were responsible for experimental design and conduct, and analyzing the data; SPM: synthesized and analyzed all aspects related to 15-acetyldeoxynivalenol; VCW, BJK, and SPM: assisted in writing, editing, and approval of the final manuscript.