Increased dietary Trp, Thr, and Met supplementation improves growth performance and protein deposition of salmonella-challenged growing pigs under poor housing conditions

Abstract

Highly intensified rearing conditions and precarious sanitary management predispose pigs to immune system activation, altered amino acid (AA) metabolism, and decreased growth performance. Thus, the main objective of this study was to evaluate the effects of increased dietary tryptophan (Trp), threonine (Thr), and methionine + cysteine (Met + Cys) supplementation on performance, body composition, metabolism, and immune responses of group-housed growing pigs under challenging sanitary conditions. A hundred and twenty pigs (25.4 ± 3.7 kg) were randomly assigned to a 2 × 2 factorial arrangement, consisting of two sanitary conditions (SC, good [GOOD] or salmonella-challenge and poor housing condition [Salmonella Typhimurium (ST) + POOR]) and two diets, control (CN) or supplemented with AA (Trp, Thr, and Met + Cys:Lys ratios 20% higher than those of the CN diet [AA>+]). Pigs were followed during the growing phase (25–50 kg) and the trial lasted 28 d. The ST + POOR SC pigs were challenged with Salmonella Typhimurium and raised in a poor housing condition. The ST + POOR SC increased rectal temperature, fecal score, serum haptoglobin, and urea concentration (P < 0.05) and decreased serum albumin concentration (P < 0.05) compared with GOOD SC. Body weight, average daily feed intake, average daily gain (ADG), feed efficiency (G:F), and protein deposition (PD) were greater in GOOD SC than in ST + POOR SC (P < 0.01). However, pigs housed in ST + POOR SC fed with AA+ diet had lower body temperature (P < 0.05), increased ADG (P < 0.05) and nitrogen efficiency (P < 0.05), and a tendency for improved PD and G:F (P < 0.10) compared with CN diet fed pigs. Regardless of the SC, pigs fed AA+ diet had lower serum albumin (P < 0.05) and tended to decrease serum urea levels (P < 0.10) compared with CN diet. The results of this study suggest that the ratio of Trp, Thr, and Met + Cys to Lys for pigs are modified by sanitary conditions. Furthermore, supplementation of diets with a blend of Trp, Thr, and Met + Cys improves performance, especially under salmonella-challenge and poor housing conditions. Dietary tryptophan, threonine, and methionine supplementation can modulate immune status and influence resilience to sanitary challenges.

The impaired growth performance and protein deposition of pigs under sanitary challenge can be attenuated by dietary tryptophan, threonine, and methionine supplementation. The amino acid supplementation mitigates immune system activation and improves the efficiency of nitrogen utilization, increasing pigs’ resilience with no in-feed antibiotics in a group-housed system.

Introduction

In commercial pig production, growing pigs often face nonideal housing conditions (e.g., increased animal density, batch mixing, and poor hygiene management) which facilitates disease transmission through increased microbial pressure in the environment (Campos et al., 2017) and affects pig health, and productivity (Jayaraman and Niachoti, 2017). The degradation of housing hygiene is known to induce chronic immune system overstimulation (Le Floc’h et al., 2014). For instance, pigs reared in conventional housing systems with high microbial loads grow 10%–20% more slowly than pigs kept in “clean” environments (Lee et al. 2005; Renaudeau 2009).

During exposure to a sanitary challenge, pigs respond with a cascade of metabolic alterations, including anorexia, increased breakdown and decreased synthesis of skeletal muscle proteins, and increased nutrient utilization for immune system functioning, such as hepatic synthesis of acute phase proteins and immune cells proliferation (Le Floc’h et al., 2004; Campos et al., 2014). Hence, growth and tissue accretion are reduced (Campos et al., 2019a), and amino acid (AA) requirements may change during a chronic immune system activation (van der Peet-Schwering et al., 2019). This raises the question of whether the current AA recommendations for growing-finishing pigs estimated to maximize the growth performance of healthy pigs (NRC, 2012) would be adequate for pigs under a sanitary challenge.

Some AA have been adopted as a nutritional strategy to attenuate the effects of sanitary challenges in pigs’ growth performance and health. These AA are called functional AA due to their functions beyond nutritive utilization (meeting growth requirements) during immune system activation, benefiting gastrointestinal integrity (Chalvon-Demersay et al., 2021), and enhancing immune responses (Rodrigues et al., 2021a,b). For example, in growing pigs with chronic immune system activation, increased dietary supplementation of tryptophan (Trp), threonine (Thr), and methionine + cysteine (Met + Cys) has improved protein deposition (van deer Meer et. al., 2016) and body weight gain (Rodrigues et al., 2021a). Furthermore, higher body nitrogen retention and feed efficiency have been observed with Trp supplementation for pigs under lipopolysaccharide challenge (de Ridder et al., 2012). In addition, sulfur AA (Met + Cys) supplementation increased protein deposition rate (+14%) in lipopolysaccharide-challenged pigs (Kim et al., 2012). Besides, higher levels of dietary Thr resulted in a linear increase in body protein deposition (+32%) and average daily gain (+12%) in immune system-stimulated pigs (McGilvray et al., 2019; Wellington et al., 2019).

Despite growing evidence that some AA requirements are affected by immune system activation, most results were obtained for post-weaning piglets housed in small groups supplemented with Trp, Thr, or Met alone for a short period of time (1–2 wk of challenge). Additionally, in these studies, pigs were repeatedly challenged with lipopolysaccharide (Kim et al., 2012; McGilvray et al., 2019) or Escherichia coli inoculation (Capozzalo et al., 2017a,b; Kahindi et al., 2018), which may lead to severe clinical responses and mortality (high dose) or immune tolerance over time (low doses). Thus, it can be questioned to what extent these results can be extrapolated to a commercial condition where pigs are constantly exposed to multiple antigens without severe clinical signs of disease or mortality (van der Meer et al., 2016), and which may require at the same time the supplementation of more than one of these AA.

Moreover, there is limited information about immune system overstimulation on AA requirements of growing pigs, especially for pigs housed in large groups and exposed to different challenges during a long period of time. Therefore, this study was performed to evaluate the effects of dietary Trp, Thr, and Met + Cys supplementation on growth and immune system activation of growing pigs challenged with Salmonella Typhimurium and group-housed under poor housing condition. It was hypothesized that increased dietary supplementation of Thr, Trp, and Met + Cys above requirement improves performance and reduces inflammation in growing pigs challenged with Salmonella Typhimurium and group-housed under poor housing condition.

Materials and Methods

Animals, housing, and management

All experimental procedures applied in this trial followed the Brazilian National Council of the Control of Animal Experimentation (CONCEA) and were reviewed and approved [protocol no. 4784/20] by the Ethical Committee on Animal Use (CEUA) of Faculdade de Ciências Agrárias e Veterinárias (FCAV/UNESP – Jaboticabal, SP, Brazil).

One hundred and twenty female pigs [Pietrain × (Large White × Landrace)] with an initial body weight (BW) of 25.4 ± 3.7 kg were used in a 28 d trial. Pigs were housed in the facilities of the Swine Research Laboratory at São Paulo State University (Unesp; Jaboticabal, SP, Brazil). Animals were identified and assigned to one of two similar environmentally controlled growing-finishing rooms (0.9 m²/pig) with full concrete floors. Both rooms were cleaned and disinfected before the animals’ allocation. Fourteen days before the study started, rectal swabs were individually collected to screen Salmonella spp. presence and only negative pigs were included in the trial.

In each room, the ambient temperature was controlled through an automated evaporative pad cooling system (Big Dutchman, Araraquara, SP, Brazil) and exhaust fans. The temperature was set at 22 °C and recorded every 30 min using two data loggers (Hobo, Onset Computer Corporation, Bourne, MA, USA) located in the middle of the pen. Artificial lights were used to maintain a 12 h photoperiod (0700–1900 h). Additionally, each room was equipped with four Automatic and Intelligent Precision Feeders (University of Lleida, Lleida, Spain) and six ball bite drinkers to allow ad libitum access to feed and water, respectively. Pigs had an individual radio frequency ear tag (Allflex, Joinville, SC, Brazil) attached in the right ear to access the feeders.

Each feeder consisted of a single space feeder that delivers volumetric amounts of up to four diets stored in independent feed containers located on the top of the feeder. The feeder identified the pig as its head entered the feeder and then provided the assigned experimental diet (see section Experimental diets) in response to each animal request. This allowed the pigs to be housed in the same pen, and any pig could access any of the feeders and receive the prescribed diet according to its experimental group (Pomar et al., 2011).

At day 0, pigs were blocked by BW and were randomly assigned to one of four experimental groups in a 2 × 2 factorial arrangement. Pigs were allocated in two sanitary ­conditions (SC): good (GOOD) or challenged with Salmonella Typhimurium and raised under poor housing condition (ST + POOR); and were fed two diets (D): control diet (CN), with the AA profile according to NRC (2012) or supplemented diet (AA+), with the AA profile containing 20% higher standardized ileal digestible (SID) Trp:Lys, Thr:Lys, and Met + Cys:Lys ratios than the CN. Each room represented a SC. Thus, the study was composed of four experimental groups with 30 pigs each.

Sanitary challenge

A sanitary challenge was used to induce an immune response in pigs under the ST + POOR SC. At d 0, all the 60 pigs in the ST + POOR SC room were inoculated via oral gavage with 5 mL of brain heart infusion (CM 1135, Oxoid, Thermo Fisher Scientific, NH, England) broth solution with 2 × 10⁹ colony forming units (CFU) of Salmonella enterica subsp. enterica serovar Typhimurium (ST) selected for antibiotic resistance to nalidixic acid (25 μg/mL). The inoculum was prepared according to the recommendations of Wood et al. (1991) and Oliveira et al. (2010) from an original strain of Salmonella Typhimurium (RLO971/09) filed at the Laboratory of Ornitopathology, Department of Veterinary Pathology, Unesp, Jaboticabal, SP, Brazil. The oral inoculum was prepared in brain heart solution cultured in stationary conditions overnight at 37 °C, and adjusted by spectrophotometry (OD600) to reach the desired concentration of 2 × 10⁹ CFU. After this procedure 1 mL of the inoculum was added to 4 mL of brain heart infusion broth, to increase the inoculant volume for a better oral gavage procedure.

In both SC, the gilts were fasted for 6 h and had no water consumption for 1 h prior to inoculation. After the ST inoculation, fresh manure from a commercial pig farm was spread on the ST + POOR SC pen floor Besides, during the 28 d trial, the ST + POOR SC was not cleaned, and no hygiene protocol was applied as described by Li et al. (2017). The objective of combining ST inoculation, manure spreading, and no hygiene protocol in the ST + POOR SC was to enhance and recycle the pathogenic pressure in the barn, which may prolong the challenge through a chronic immune system activation of pigs and keep the deterioration in housing hygiene throughout the 28 d trial.

Otherwise, in the GOOD SC, pigs received a placebo inoculum via oral gavage with 5 mL of brain heart infusion broth solution without ST, and manure was not spread on the floor. The GOOD SC room was cleaned twice a day with a water jet stream, and potassium monopersulfate (1:200; Virkon; Lanxess, Colony, Germany) was diluted and applied in the facility once a week as part of the biosecurity protocol to improve the hygiene condition. The team members were also required to wear clean clothing and clean footwear with a bleach solution (1:10) when entering the building. Regardless of SC, pigs did not receive any medication or preventive treatment.

Experimental diets

Experimental corn-soybean meal-based diets were formulated using the reported nutrient content and analyzed AA content of ingredients to meet or exceed the nutrient requirements for 25–50 kg gilts according to NRC (2012) and AMINODat® 6.0 (2021; Tables 1 and 2, respectively). In the CN diet, AA profile met the SID AA requirements according to NRC (2012), while in the AA+ diet, AA profile contained 20% higher SID Trp, Thr, and Met + Cys to Lys ratios. No in-feed antibiotics as growth promoters were used before or throughout the trial. The diets were steam pelleted (2.5 mm) and provided ad libitum through the feeders.

Table 1.

Ingredient and nutrient composition of experimental diets¹ (%, as-fed basis)

Item	CN	AA+
Centesimal composition
Corn	74.32	74.32
Soybean meal	22.08	22.08
Limestone	0.73	0.73
Maltodextrin	0.50	0.50
Dicalcium phosphate	0.53	0.53
Salt	0.45	0.45
Kaolin	0.30	0.05
Soybean oil	0.10	0.10
l-Lysine (60%)2	0.480	0.480
dl-Methionine (99%)2	0.090	0.201
l-Threonine (98.5%)2	0.070	0.183
l-Tryptophan (98%)2	0.010	0.045
l-Valine (98%)2	0.0002	0.0002
Vit + min premix3	0.150	0.150
Antifungal	0.100	0.100
Choline chloride	0.060	0.060
Phytase4	0.005	0.005
Calculated chemical composition5
Net energy, kcal/kg	2,550	2,559
Crude protein, %	16.54	16.73
Total nitrogen, %	2.65	2.68
Lysine:crude protein	5.92	5.85
SID6 lysine, %	0.98	0.98
SID methionine + cysteine, %	0.55	0.66
SID methionine, %	0.32	0.43
SID threonine, %	0.59	0.70
SID tryptophan, %	0.17	0.20
SID valine, %	0.67	0.67
SID arginine, %	0.95	0.95
SID isoleucine, %	0.59	0.59
SID leucine, %	1.28	1.28
SID histidine, %	0.38	0.38
SID phenylalanine, %	0.70	0.70
Calcium, %	0.66	0.66
STTD7 phosphorus, %	0.31	0.31
Chloride, %	0.34	0.34
Potassium, %	0.64	0.64
Sodium, %	0.19	0.19

¹CN, control diet (basal AA profile); AA+, supplemented diet (supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio).

²Amino acids (BioLys, MetAmino, ThreAmino, TrypAmino, ValAmino, respectively) provided by Evonik Nutrition & Care GmbH (Hanau-Wolfgang, Germany).

³Mineral premix supplied (per kg of diet): Manganese (40 mg); copper (15 mg); iron (24.93 mg); cobalt (0.168 mg); iodine (1.416 mg); and zinc (74.971 mg). Vitamin premix supplied (per kg of diet): folic Acid (0.32 mg); D-pantothenic acid (14.8 mg); biotin (0.04 mg); niacin (28 mg); selenium (0.25 mg); Vit. A (6000 IU); Vit. B₁ (1.2 mg); Vit. B₁₂ (22 mcg); Vit. B₂ (4.4 mg); Vit. B₆ (1.4 mg); Vit. D₃ (1,400 IU); Vit. E (26 IU); and Vit. K₃ (2.16 mg).

⁴Phytase provided by Cargill and contained 500 FTU/ton.

⁵Nutrient content of diets based on estimated nutrient contents of ingredients, according to AMINODat® 6.0 (2021).

⁶Standardized ileal digestible.

⁷Standardized total tract digestible.

Table 2.

Analyzed crude protein and total amino acid contents of experimental diets¹ (as-fed basis)

Item	CN	AA+
Crude protein, %	16.00	17.00
Total amino acid2, %
Lysine	1.09 (1.07)	1.15 (1.07)
Methionine + cysteine	0.56 (0.61)	0.71 (0.72)
Methionine	0.32 (0.34)	0.46 (0.45)
Threonine	0.68 (0.67)	0.83 (0.78)
Valine	0.79 (0.75)	0.79 (0.75)
Arginine	1.01 (1.01)	1.04 (1.01)
Isoleucine	0.68 (0.65)	0.69 (0.66)
Leucine	1.50 (1.44)	1.49 (1.43)
Histidine	0.45 (0.43)	0.45 (0.43)
Phenylalanine	0.82 (0.78)	0.82 (0.78)

¹CN, control diet (basal AA profile); AA+, supplemented diet (supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio).

²Calculated values are given in parentheses.

Data collection

All animals were used for growth performance and fecal score evaluation and fecal ST quantification (n = 30 pigs/experimental group). Additionally, a group of 80 pigs (n = 20 pigs/experimental group) with the closest BW to the average of its experimental group were selected for the evaluation of body composition, nitrogen balance, rectal temperature, and blood sampling.

Growth performance, body composition, and nitrogen balance

Individual BW was recorded weekly, and individual daily feed intake (ADFI, kg/d) was measured by the feeders system. Average daily gain (ADG, kg/d) and ADFI were used to calculate feed efficiency (G:F, kg/kg). Total body lean, and fat mass were measured on d −1 (pre-challenge) and at 28 d post-challenge (dpc) by dual-energy X-ray absorptiometry (DXA; GE 205 Lunar Prodigy Advance; GE Healthcare, Madison, WI, USA). Animals were fasted for 6 h before being sedated by intramuscular injection of xylazine (1.5 mg/kg) and ketamine (15 mg/kg). Pigs were scanned in the prone position using the total body scanning mode in the manufacturer-provided software (Lunar enCORE software, version 8.10.027; GE Healthcare).

The DXA body lean and fat mass values were converted to its protein and lipid chemical equivalent, as proposed by Pomar and Rivest (1996). Total body protein (BP) and lipid content (BL) from each scanned pig were calculated as the difference between the respective body constituents estimated from DXA readings at the beginning and end of the experimental period. Afterwards, average daily protein (PD) and lipid deposition (LD) were calculated by dividing the corresponding variables by the duration of experimental period (28 d).

Nitrogen excretion (g/d) was obtained for each scanned pig by subtracting the nitrogen retention (PD divided by 6.25) from the nitrogen intake (estimated by [CP in the diet multiplied by the DFI] divided by 6.25). Nitrogen efficiency (%) was calculated by dividing the nitrogen retention by the nitrogen intake.

Rectal temperature, fecal score, and fecal Salmonella Typhimurium shedding

For the Salmonella spp. presence before the beginning of the trial, the rectal swabs were serially diluted in phosphate-buffered saline (1:10) until they reached the final concentration of 10⁻⁶. From each dilution, 0.1 mL was plated on the brilliant green agar (CM0263, Oxoid, Basingstoke, NH, England), and incubated at 37 °C for 24 h.

The rectal temperature was measured from 0 to 7 dpc with a digital thermometer (Accumed-Glicomed, RJ, Brazil) in both SC. In addition, the fecal score was performed individually in fecal samples collected from ST + POOR SC pigs at 5, 7, 14, and 21 dpc. Normal consistency feces received a score 0, semisolid feces a score 1, semisolid-watery feces score of 2, and watery feces given a score of 3. Furthermore, at 2, 5, 7, 14, 21, and 28 dpc, fresh fecal samples (10 g) were collected by rectal stimulation from individual pigs to evaluate fecal ST shedding. Fecal samples were serially diluted in phosphate-buffered saline (1:10) until they reached final concentration of 10⁻⁶. From each dilution, 0.1 mL was plated on brilliant green agar containing nalidixic acid. Plates were incubated at 37 °C for 24 h. The number of CFU per gram of fecal sample was transformed into log10 for further analysis. In the absence of ST growth, an equal volume of Rappaport-Vassiliadis broth double concentrated (CM0669, Oxoid, Basingstoke, NH, England) was added to tubes containing the respective homogenized sample in phosphate-buffered saline for ST enrichment. The samples were incubated at 37 °C for 24 h and plated in green brilliant agar containing nalidixic acid, and evaluated for the presence or absence of ST. In the case of positivity after enrichment, the sample was considered with a bacterial load of 2 log10, for calculation purposes.

Blood sampling and analysis

Blood samples were collected from pigs at 7 and 28 dpc from the jugular vein after 6 h of fasting. The pigs were contained with snout rope and the sampling procedure was performed in less than 2 min to avoid pain and interference on blood parameters. Per sampling day, two 8-mL serum tubes (BD Vacutainer; NJ, EUA) were collected per animal for haptoglobin, albumin, total protein, urea, and creatinine concentrations. Blood samples in serum tubes were allowed to clot for 1 h, after which serum was collected after centrifugation for 10 min at 3000 × g at 4 °C (Novatecnica, NT 835, Piracicaba, SP, Brazil) and stored at −80 °C for further analysis. Serum levels of haptoglobin and albumin were accessed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Weber and Osborn, 1969). The molecular weight and protein fraction concentrations were determined by computer densitometry (Shimadzu 9301 PC, Shimadzu Corp, Kyoto, Japan) using a simple scanner. Biomarkers were used for protein identification (Sigma Marker, Sigma-Aldrich Biotechnology LP, Germany). For the densitometric evaluation of protein bands, reference curves were made from the reading of the standard marker. Afterwards, serum haptoglobin and albumin concentrations were corrected by total serum total protein analysis. Serum concentrations of total protein, urea, and creatinine were determined by the biuret method with commercial reagents (Labtest; Labtest Diagnostica SA, Lagoa Santa, MG, Brazil) and performed by semi-automatic spectrophotometry (Labmax Plenno, Labtest Diagnostica SA; Lagoa Santa, MG, Brazil).

Statistical analyses

Data were tested for normality using the UNIVARIATE procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC) and the Shapiro-Wilk test, and the studentized residual was used to identify outliers (> 3 standard deviations from the mean). Data were analyzed using the GLIMMIX procedure of SAS and presented as least-squares means. Experimental SC, D, and their interactions (SC × D) were included as fixed effects, while the blocks of BW were included as a random variable effect in the statistical model. Additionally, the initial BW was used as a covariate for body composition analysis. The data collected over time were included in the analysis as a repeated measure in time, and each pig was considered as an experimental unit. As two different rooms were used to allocate each condition (GOOD and ST + POOR SC), it might result in pseudo-replicates. However, housing animals in two different barns were established to avoid cross-contamination between sanitary conditions and have different confounding factors. Differences between means were determined using the Tukey post hoc test, except to analyze the percentage of positive pigs for fecal ST shedding and fecal scores. For the percentage of positive pigs for fecal ST shedding, a chi-square test was used to compare days and diets. The Cochran-Mantel-Haenszel test was used to assess fecal score changes over time and D effect on fecal score severity at each day of measurement. Additionally, an ordinal logistic analysis was performed according the Proportional Odds Model with the LOGISTIC procedure of SAS to assess the D effect on fecal score severity. The categorical indicator (fecal score) was assigned as the response, and day and D were assigned as the categorical predictors. The significance level adopted for all analysis was 5% (P ≤ 0.05), and a trend towards significance was considered at 0.05 < P ≤ 0.10.

Results

Before the beginning of the trial (d −14 to 0), there was no effect of the rooms on the pigs’ growth. Pigs housed in both facilities (representing GOOD and ST + POOR SC) had similar BW (25.3 ± 0.21 kg vs. 25.5 ± 0.21 kg, respectively) and ADFI (0.96 ± 0.02 kg vs. 0.98 ± 0.02 kg, respectively), which allowed comparisons between SC during the experimental period. Throughout the experimental period, four animals were removed. In the GOOD SC, one pig died, and another pig showed dyspnea, which was treated with tulathromycin. In the ST + POOR SC (after the sanitary challenge), two pigs presented fever, excessive weight loss, and no voluntary feed intake for three consecutive days. They were treated with enrofloxacin to respect animal welfare principles and removed from the experiment. In addition, ST + POOR SC pigs were lethargic and showed reduced rates of feed intake after the challenge; however, signs of recovery (increased voluntary feed intake, and no visible lethargy) were observed after 5 dpc.

Rectal temperature and fecal score

After 24 h of challenge, rectal temperature increased in ST + POOR SC compared to GOOD SC (P < 0.05; Fig.1a). The rectal temperature in pigs of the ST + POOR SC reached its maximum (39.6 °C) at 3 dpc. From 4 to 6 dpc, rectal temperature of ST + POOR SC pigs decreased, remaining higher than pigs of the GOOD SC (P < 0.05). At 7 dpc, rectal temperature of pigs in ST + POOR SC was similar to pigs in GOOD SC and to the pre-challenged period (P > 0.10). Regarding AA supplementation, during 7 dpc, there was a diet effect for rectal temperature (P < 0.05). Pigs fed CN diet had higher rectal temperature than the ones fed AA+ at 3 dpc (39.7 ± 0.12 °C vs. 39.4 ± 0.12 °C) and 4 dpc (39.7 ± 0.11 °C vs. 39.5 ± 0.11 °C). However, no significant differences were observed afterwards (Fig. 1b).

Figure 1.

Rectal temperature (A) of pigs in good sanitary condition (dashed line) or challenge with Salmonella Typhimurium and raised under poor housing condition (solid line). Different lowercase letters indicate difference between sanitary conditions, and different uppercase letters indicate difference between days for the same sanitary condition by the Tukey’s test (P < 0.05). Rectal temperature (B) of pigs housed in POOR sanitary condition fed with control (CN: control diet with basal AA profile [dash-dot line]) or AA supplemented (AA+: supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio [solid line]). Different lowercase letters indicate differences between diets by the Tukey’s test (P < 0.05).

The ST + POOR SC negatively affected the fecal score and it resulted in transitory diarrhea, with higher mean fecal score at 5 dpc when compared to 7, 14, and 21 dpc (P < 0.05; Fig. 2a). Afterwards, a decrease in the mean fecal score was observed with the lowest mean fecal score at 21 dpc (Fig. 2a). Regarding dietary treatments, there was a difference in fecal score between diets only at 14 dpc, with higher mean fecal in pigs fed CN compared to AA+ diet (P < 0.05; Fig. 2b). Additionally, at 14 dpc, pigs fed AA+ diet had a higher probability (three times) to show a less severe score compared to pigs fed CN diet (P < 0.05; Table 3).

Figure 2.

Post-challenge mean fecal score of pigs challenged with Salmonella Typhimurium raised under poor housing conditions (A) fed with control (CN: control diet with basal AA profile [filled box]) or AA supplemented diet (AA+: supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio [open box]) (B). Normal, semisolid, semisolid-watery, and watery feces were given the scores 0, 1, 2 and 3, respectively.

Table 3.

Logistical regression analysis of the fecal score¹ probability (%) of growing pigs challenged with Salmonella Typhimurium and raised under poor housing condition

Days post-challenge	CN2			AA+			Estimate	Odds ratio3	RSD4	P-value
	0	1	2	0	1	2
5	11.3	39.9	48.8	9.7	37.2	53.1	−0.17	0.84	0.51	0.74
7	42.4	33.3	24.4	41.9	33.4	24.8	−0.02	0.98	0.49	0.96
14	31.4	34.0	34.5	57.6	30.3	15.1	1.08	2.96	0.51	0.03
21	57.8	33.7	8.5	68.5	26.0	5.5	0.46	1.59	0.54	0.39

¹As few fecal samples were categorized liquid feces (score 3) after 7 dpc, fecal samples categorized as scores 2 and 3 were grouped, and categorized as score 2.

²CN, control diet (basal AA profile); AA+, supplemented diet (supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio).

³Odds ratio = AA + CN.

⁴Residual SD.

Fecal Salmonella Typhimurium shedding

Throughout the trial, pigs raised in the GOOD SC remained negative for ST shedding, having no cross-contamination between rooms. In ST + POOR SC, ST colony detection persisted until the end of the trial (24.4%; 14 out of 58 pigs), however the percentage of positive pigs and fecal ST ­shedding continuously reduced from 2 to 28 dpc, regardless of the dietary treatment (P < 0.05; Fig. 3a). From 2 to 14 dpc, there was no difference between dietary treatment on the percentage of positive pigs and fecal ST shedding (P > 0.10). Meanwhile, there was a significant decrease in the percentage of positive pigs for ST shedding at 21 and 28 d (22% and 24%, respectively), with a higher percentage of positive pigs when fed CN (31%; 14 out of 28 pigs) compared to AA+ diet (14%; 4 out of 28 pigs; P < 0.05). The same pattern was observed for the ST shedding. Pigs fed CN diet kept higher ST colony counts (from 2 to 4.3 log CFU/g) than pigs fed with AA+ at 21 and 28 dpc (from 0.5 to 4 log CFU/g; P < 0.05; Fig. 3b).

Figure 3.

Fecal Salmonella Typhimurium shedding percentage (A) of pigs challenged with Salmonella Typhimurium and raised under poor housing conditions, fed with control (CN, control diet with basal AA profile [filled box]) or AA supplemented diet (AA+, supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio [open box]). Fecal Salmonella Typhimurium shedding quantification (B) of pigs challenged with Salmonella Typhimurium and raised poor housing conditions, fed with control (CN, control diet with basal AA profile [filled box]) or AA supplemented diet (AA+, supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio [open box]). Different uppercase and lowercase letters indicate the difference between days and between dietary treatments by the Tukey’s test (P < 0.05), respectively.

Blood parameters

No interactions between SC × D were observed for any blood parameter (P > 0.10; Table 4) at 7 and 28 dpc. However, at 7 dpc, serum total protein, and urea concentrations were affected by SC, with pigs in the ST + POOR SC presenting higher urea levels, and lower total protein concentrations than GOOD SC pigs (P < 0.05). Additionally, at 7 and 28 dpc, the ST + POOR SC pigs had increased haptoglobin and decreased albumin serum concentrations compared to pigs housed in GOOD SC (P < 0.05). No D effect (P > 0.10) was observed for haptoglobin or total protein serum ­concentrations at 7 and 28 dpc. Meanwhile, serum albumin and urea were modified by D at 7 and 28 dpc. Pigs fed AA+ diet had higher (P < 0.05) serum albumin concentration, and tended to reduce urea levels (P < 0.10) than those fed the CN diet. No SC or D effect was observed on serum creatinine concentration at 7 and 28 dpc (P > 0.10).

Table 4.

Blood parameters of growing pigs fed control or supplemented diet above the requirement raised under good sanitary condition or challenged with Salmonella Typhimurium and raised under poor housing conditions for 28 d

Item	GOOD1		ST + POOR		RSD2	P-value
	CN	AA+	CN	AA+		SC	D	SC × D
7 d post-challenge
Haptoglobin, g/L	0.27	0.40	0.86	0.89	0.13	<0.01	0.31	0.50
Total protein, g/dL	6.60	6.65	6.15	6.31	0.26	<0.01	0.48	0.70
Albumin, g/L	41.63	43.13	35.77	38.44	1.87	<0.01	0.05	0.57
Urea, mg/dL	17.05	17.11	21.63	18.23	2.03	0.03	0.08	0.20
Creatinine, mg/dL	0.83	0.88	0.95	0.88	0.18	0.56	0.90	0.52
28 d post-challenge
Haptoglobin, g/L	0.22	0.24	0.35	0.39	0.07	<0.01	0.50	0.78
Total protein, g/dL	5.76	5.71	5.73	5.85	0.14	0.47	0.63	0.31
Albumin, g/L	34.54	35.70	31.36	34.49	1.54	0.01	0.01	0.26
Urea, mg/dL	17.03	16.78	20.08	16.63	1.83	0.15	0.09	0.12
Creatinine, mg/dL	1.28	1.31	1.29	1.32	0.05	0.80	0.28	0.83

¹GOOD, good sanitary condition; ST + POOR, salmonella challenge and poor housing condition; CN, control diet (basal AA profile); AA+, supplemented diet (supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio).

²Residual SD.

Growth performance and body composition

In the first week post-challenge, ST + POOR SC pigs had lower (P < 0.05) performance than those in GOOD SC (Table 5). Pigs in the ST + POOR SC had a reduction of 22%, 37%, 28%, and 4% on ADFI, ADG, G:F, and BW, respectively, compared to pigs in GOOD SC (P < 0.05). There were no D effects on growth performance from 0 to 7 dpc (P > 0.10).

Table 5.

Growth performance, body composition, and nitrogen balance of growing pigs fed control or supplemented diet raised under good sanitary condition or challenged with Salmonella Typhimurium and raised under poor housing conditions for 28 d

Item	GOOD1		ST + POOR		RSD2	P-value
	CN	AA+	CN	AA+		SC	D	SC × D
Initial conditions
BW, kg	25.42	25.36	25.36	25.48	0.58	0.92	0.91	0.77
Body protein, kg	3.38	3.37	3.38	3.39	0.02	0.32	0.87	0.48
Body lipid, kg	4.92	4.95	4.88	4.86	0.08	0.18	0.93	0.58
0 to 7 d post-challenge
Final BW, kg	29.45	29.19	27.60	28.22	0.95	<0.01	0.72	0.36
ADG, kg	0.58	0.56	0.32	0.39	0.09	<0.01	0.65	0.26
ADFI, kg	1.20x	0.94x	0.74y	0.79y	0.06	<0.01	0.62	0.08
G:F, kg/kg	0.54	0.57	0.37	0.44	0.09	0.01	0.32	0.65
0 to 14 d post-challenge
Final BW, kg	34.02	33.92	31.06	32.58	1.08	<0.01	0.20	0.14
ADG, kg	0.61x	0.61x	0.41z	0.51y	0.05	<0.01	0.08	0.07
ADFI, kg	1.16x	1.09xy	0.96z	1.00yz	0.02	<0.01	0.59	0.06
G:F, kg/kg	0.52	0.56	0.40	0.49	0.03	<0.01	<0.01	0.20
0 to 21 d post-challenge
Final BW, kg	40.19x	40.02x	35.91y	38.30x	1.37	<0.01	0.11	0.07
ADG, kg	0.70a	0.69a	0.50c	0.61b	0.05	<0.01	0.05	0.03
ADFI, kg	1.34a	1.26ab	1.12c	1.17bc	0.02	<0.01	0.69	0.05
G:F, kg/kg	0.52x	0.55x	0.43y	0.51x	0.03	<0.01	<0.01	0.08
0 to 28 d post-challenge
Final BW, kg	47.98a	47.84a	42.18c	45.12b	1.60	<0.01	0.08	0.06
ADG, kg	0.81a	0.80a	0.60c	0.70b	0.04	<0.01	0.04	0.03
ADFI, kg	1.45x	1.38xy	1.24z	1.29yz	0.07	<0.01	0.80	0.08
G:F, kg/kg	0.55y	0.58x	0.47z	0.54y	0.01	<0.01	<0.01	0.06
Final body protein, kg	7.24x	7.27x	6.46y	6.95x	0.22	<0.01	0.03	0.06
Final body lipid, kg	7.38	7.32	6.51	6.84	0.24	<0.01	0.31	0.16
Protein deposition, g/d	137x	139x	109y	127x	7.71	<0.01	0.03	0.07
Lipid deposition, g/d	87	84	57	70	8.60	<0.01	0.32	0.11
Nitrogen intake, g/d	37	36	32	35	0.52	<0.01	0.43	0.20
Nitrogen excretion, g/d	15	14	15	14	0.39	0.48	0.75	0.73
Nitrogen efficiency, %	60a	60a	55b	59a	0.02	<0.01	0.03	0.03

¹GOOD, good sanitary condition; ST + POOR, salmonella challenge and poor housing condition; CN, control diet (basal AA profile); AA+, supplemented diet (supplemented AA profile containing 20% above Trp, Thr, and Met + Cys:Lys ratio).

²Residual SD.

For body composition, initial BW as a covariate was significant for all variables, P ≤ 0.05.

^a,bDifferent lowercase letters indicate the difference between experimental groups by the Tukey’s test (P ≤ 0.05).

^x,yDifferent lowercase letters indicate the tendency between experimental group by the Tukey’s test (0.05 < P < 0.10).

From 0 to 14 dpc, there was a tendency for interaction between SC × D for ADFI and ADG (P < 0.10), and a SC effect on BW (P < 0.05). Pigs housed in GOOD SC fed with CN diet had higher consumption (P < 0.10) than pigs in ST + POOR SC, regardless dietary treatment. On the other hand, ST + POOR SC pigs fed AA+ diet had greater ADG (+24%) than those fed CN diet (P < 0.10), whereas GOOD SC pigs had similar ADG irrespective of diet (P > 0.10). At the same time, ST + POOR SC pigs had a lower BW (−6%) compared to GOOD SC (P < 0.05).

Interactions between SC × D for ADFI and ADG (P < 0.05), and trends for BW and G:F (P < 0.10) were observed until the third-week post-challenge (0 to 21 dpc). Pigs housed in GOOD SC fed CN diet had greater ADFI (P < 0.05) than ST + POOR SC fed CN or AA+ diet. Nevertheless, ST + POOR SC pigs fed AA+ diet had a greater ADG (+22%), G:F (+19%) and higher BW (+7%) compared to pigs fed CN diet.

Considering the entire experimental period (0 to 28 dpc), interaction between SC × D was observed for ADG (P < 0.05), and a trend for BW, ADFI, and G:F (P < 0.10). Pigs housed under ST + POOR SC fed AA+ diet had higher ADG (+17%) and a trend for higher final BW (+7%) compared to those fed CN diet. Pigs housed in GOOD SC fed CN diet had higher ADFI (P < 0.05) than pigs in ST + POOR SC. In the meantime, pigs fed AA+ diet showed improved G:F compared to the CN diet in both conditions (P < 0.10). However, pigs under ST + POOR SC had a greater difference in G:F compared to GOOD SC pigs due to AA supplementation (+14% vs. +5%, respectively).

Regarding body composition, a tendency of SC × D interaction (P < 0.10) was noticed (Table 5) for BP and PD. At 28 dpc, AA+ affected BP content (P < 0.10) of pigs under a sanitary challenge. Under ST + POOR SC, pigs fed AA+ diet had higher BP content compared to pigs fed CN diet (+7%) and were similar to pigs housed in GOOD SC. Besides, for the overall growing period (0 to 28 dpc), ST + POOR SC pigs fed AA+ had higher PD (+17%) compared to ST + POOR SC pigs fed CN diet (+7%) and similar PD to GOOD SC pigs (P < 0.10). At the same time, no SC × D interaction (P > 0.10) for BL content was observed. There was a significant effect of SC (P < 0.05), with GOOD pigs showing higher BL mass (+9%) and LD (+25%) compared to ST + POOR SC pigs.

For nitrogen balance, no SC × D interaction was observed for nitrogen intake (P > 0.10) (Table 5); however, a SC effect was observed (P < 0.05). Pigs raised under ST + POOR SC presented a reduced nitrogen intake compared to GOOD SC. Nevertheless, there was a SC × D interaction (P < 0.05) for nitrogen efficiency. The ST + POOR SC pigs fed AA+ diet had greater nitrogen efficiency than ST + POOR pigs fed CN (+9%) and it was similar to GOOD SC pigs. Meanwhile, no differences between experimental groups were found for nitrogen excretion (P > 0.10).

Discussion

This study aimed to evaluate whether increasing dietary Trp, Thr, and Met to Lys ratio would attenuate the negative impacts of sanitary challenge (induced by Salmonella Typhimurium challenge and poor housing condition) on growth performance and mitigate the chronic immune system activation of group-housed pigs. Our major and original finding is that increasing dietary Trp, Thr, and Met + Cys to Lys ratio improves protein deposition and nitrogen efficiency of group-housed growing pigs under a sanitary challenge.

These results go in line with the hypothesis that enteric challenges modify animal metabolism and limit the growth potential expression of pigs by inducing an immune response. Under such conditions, functional AA are good candidates for feeding adjustments because they can regulate key metabolic pathways to improve animals’ health, and growth (Wu, 2007). Accordingly, Trp, Thr, and Met have been applied as functional AA due to their key roles in modulating immune responses (Wu, 2007). Hence, increased requirements of Trp (Le Floch’h et al., 2008), Thr (Ren et al., 2014), and Met (Kim et al., 2012) have been observed in pigs under sanitary challenges. Recent findings have shown that dietary supplementation of Trp, Thr, and Met can mitigate the negative effects of chronic system activation on growth performance (van der Meer et al., 2016; Rodrigues et al., 2021a). However, long-term studies are scarce, and there is limited information about the impact of immune system overstimulation and AA dietary supplementation on body protein deposition (Litvak et al., 2013), especially when pigs are housed in large groups.

In this context, threonine, an important AA for intestinal mucosal integrity (Ruth and Field, 2013), may benefits pigs by reducing the gut inflammatory response (Trevisi et al., 2009) through mucus synthesis, and bacteria binding to the mucosal surface. Methionine may contribute to antioxidant capacity, as a substrate to glutathione synthesis (Sierżant et al., 2019), to neutralize the reactive oxygen species produced by some immune cells to exert their cytotoxic functions during an immune response. Tryptophan may also attenuate gut inflammation (Rodrigues et al., 2021a) and modulate gut microbiota through indole components, which have an anti-inflammatory function and bacteriostatic properties on gram-negative enterobacteria, especially against Salmonella and Shigella genus (Gao et al., 2018).

Response of pigs to Salmonella Typhimurium challenge and poor housing condition

The presence of feces with watery consistency observed in ST + POOR SC pigs demonstrates that the degradation of sanitary conditions affects gut integrity. Pathogens’ attachment and colonization of the gut mucosa stimulate the innate immune system and increase cell permeability, which results in leaky gut and diarrhea (Campbell et al., 2013; Wang et al., 2015). In addition, the febrile response in ST + POOR SC pigs points out that the sanitary challenge applied was sufficient to overwhelm intestinal immunity resulting in a systemic inflammatory response. When an infection occurs, a series of innate immune cells are activated (e.g., monocytes and macrophages), triggering the synthesis of pro-inflammatory cytokines (e.g., IL-1β and TNF-α) (Netea et al., 2000), which are translated to the brain where fever and sickness behavior (e.g., feed intake reduction) are elicited. Thus, lower ADFI and higher body temperature in ST + POOR SC pigs observed in the first week could have been a mechanism to reduce the pathogenicity caused by infections.

Different from fever, acute-phase proteins remain for a longer period in the bloodstream during infections (Asai et al., 1999), and their decrease is correlated with the resolution of the inflammatory response (Eckersall, 2000). Serum haptoglobin and albumin are positive and negative acute-phase proteins, respectively, and have been directly associated with pigs’ health status (van der Meer et al., 2016; Kampman-van de Hoek et al., 2016). Indeed, higher haptoglobin and reduced albumin serum concentrations observed in ST + POOR SC pigs until 28 dpc, confirm an immune system overstimulation throughout the experimental period. Collectively, these results indicate that the sanitary challenge model successfully infected the pigs, which can be characterized by good indicators such as diarrhea (Wessels et al., 2021), fever (Rodrigues et al., 2021a), and higher serum levels of haptoglobin (Parois et al., 2017).

Under a sanitary challenge, alteration in nutrient utilization and protein metabolism occurs, with the redirection of dietary and body nutrient reserves to support the immune system (Campos et al., 2014, 2019a). Since the muscle AA profile differs from the immune component’s profile, an AA imbalance may occur in immune system-stimulated pigs (Reeds et al., 1994). Therefore, a proportion of the mobilized AA not used for immune response and protein synthesis is converted into urea and excreted, negatively affecting nitrogen efficiency. Although no significant differences in nitrogen excretion were observed between experimental groups, there was a reduction in ADFI and nitrogen intake, which may have contributed to this result. If there was no such reduction in nitrogen intake, the nitrogen excretion might be higher for ST + POOR SC pigs. Nevertheless, pigs under ST + POOR SC had higher urea serum concentrations (at 7 and 28 dpc) and lower nitrogen efficiency utilization than GOOD SC pigs, which may indicate an AA imbalance (Heo et al., 2009).

Moreover, the sanitary challenge affected the growth rate. The reduced ADG and BW observed in ST + POOR SC pigs are partially due to the lower ADFI, which agrees with ­previous studies (Le Floc’h et al., 2009; van der Meer et al., 2016; Rodrigues et al., 2021a). On the other hand, the G:F was also affected. At 28 dpc, the reduction in G:F of ST + POOR SC pigs shows that the ADG (−19%) was influenced more than just by the reduction in ADFI (−10%) when compared to GOOD SC. This can be explained by the redirection of AA and energy from growth to the immune system (Le Floc’h et al., 2004). Under enteric infections (such as caused by Salmonella Typhimurium) and poor housing conditions, intestinal integrity is impaired, which may result in increased endogenous losses and poor digestion (Coop and Kyriazakis, 1999), leading to reduced AA and energy availability. In addition, the reduction in ADG may also result from the metabolic cost associated with fever (heat production; Campos et al., 2019b) and cells and immune component synthesis. This finding is similar to what was reported in growing challenged pigs’ studies (Pastorelli et al., 2012a; van der Meer et al., 2016) and in meta-analytical approaches (Pastorelli et al., 2012b; Rodrigues et al., 2021b), suggesting that the major percentage of the ADG and BW loss were due to a disturbance in metabolism and nutrient utilization or greater maintenance requirement.

Different sanitary challenge models were used to assess their effects on the pigs’ growth performance and metabolism. Le Floc’h et al. (2006) observed an increase of 259% in the haptoglobin serum concentration and a decrease of 8% in the ADG when evaluating the impact of poor housing conditions. Furthermore, upon evaluating the effect of Salmonella Typhimurium inoculation in weaned pigs, Rodrigues et al. (2021a) observed an increase of 28% in haptoglobin, increased rectal temperature, and a reduction of 35% in the ADG in the first week post-challenge. These findings are similar to the results observed in this trial. However, we are not aware of any other sanitary challenge model which has evaluated the effect of poor cleaning routine and ST inoculation in growing group-housed pigs concomitantly. Taken together, the results observed herein demonstrate that the sanitary challenge model was able to impact the pig’ physiological (such as body temperature, fecal score, and body weight) and protein metabolic parameters, proving to be a reliable method to validate the pigs’ response during immune system overstimulation.

Effect of Trp, Thr, and Met supplementation on rectal temperature, fecal score and fecal Salmonella Typhimurium quantification

In the current study, increasing dietary supplementation of Trp, Thr, and Met + Cys reduced the rectal temperature of ST + POOR SC pigs fed AA+ diet when compared to pigs fed CN diet. Interestingly, this result was not observed in previous studies (Jayaraman et al., 2017; Wellington et al., 2019; Rodrigues et al., 2021a, 2021b), which may be due to the sanitary challenge model applied and the higher number of experimental units used for this analysis (n = 20 pigs/experimental group). The faster rectal temperature decrease in ST + POOR SC pigs fed AA+ compared to the CN diet suggests that Trp, Thr, and Met + Cys supplementation may have attenuated inflammation by increasing gut integrity (Ruth and Field, 2013). Indeed, the lower mean fecal score, the reduced number of positive pigs for fecal ST shedding, and the low ST quantification in ST + POOR SC pigs fed an AA+ diet indicate that these AA may have contributed to improving intestinal health by reducing bacterial establishment and colonization in the gut lumen (Faure et al., 2006; Gao et al., 2018).

Effect of Trp, Thr, and Met supplementation on serum metabolites

Regardless of the SC, pigs fed AA+ diet had higher albumin and a trend for reduction in urea serum concentration at 7 and 28 dpc. Albumin is the major blood protein and has high cysteine and threonine content in its composition (Reeds and Jahoor, 2001; Remus et al., 2019). Therefore, increasing AA dietary levels may have modulated albumin synthesis in the liver. Additionally, albumin is an important protein carrier for steroids, fatty acids, and hormones and it can serve as an anti-oxidant by preventing irreversible oxidative losses (Roche et al., 2008) by capturing free AA and transporting them to peripheral tissues for protein synthesis (De Feo et al., 1992). Thus, the higher albumin content in pigs fed AA+ may have contributed to maintaining AA supply for peripheral tissues, and pigs’ efficiency of AA utilization. Indeed, this was observed in pigs fed AA+, as a tendency for lower serum urea concentration, which is an indicator of AA utilization.

Effect of Trp, Thr, and Met supplementation on growth performance, body composition, and nitrogen balance

The increased ADG and nitrogen efficiency and the tendency to increase in BW, G:F, and PD in ST + POOR SC fed AA+ pigs compared to ST + POOR SC pigs fed CN diet confirms the hypothesis that the dietary AA+ profile better matches the AA requirements for growth and nutrient utilization in pigs under sanitary challenge. The AA+ diet may have reduced body nutrient mobilization in pigs for immune system activation resulting in more nutrients available for growth performance. This may be confirmed by the fact that ST + POOR SC pigs fed AA+ diet had a better performance than ST + POOR SC pigs fed CN diet, and at the same time, they had similar BW, BP, and PD and nitrogen efficiency compared to pigs housed in GOOD SC at 28 dpc. Overall, these positive results of Trp, Thr, and Met + Cys supplementation on ADG, BW, G:F and nitrogen efficiency is in agreement with previous studies (van der Meer et al., 2016; Rodrigues et al., 2021a). However, the magnitude of the AA supplementation effect on performance varies between studies, which might be related to the type of sanitary challenge model applied, animal density per pen, age/phase, and challenge duration.

In addition, it should be noticed that the improved ADG response without a significant increase in ADFI also suggests that AA supplementation improved pigs’ capacity to maintain productivity in a challenging environment. Although the ADG of ST + POOR SC pigs was lower than those in GOOD SC, pigs fed AA+ showed greater ADG compared with pigs fed CN diet in ST + POOR SC (from 14 to 28 dpc), which demonstrates that Trp, Thr, and Met + Cys supplementation supports pigs’ recovery after a sanitary challenge.

Conclusion

The Trp, Thr, and Met + Cys supplementation, 20% above NRC (2012) requirements, for growth mitigates the immune system activation and increases growth performance, protein deposition, and nitrogen efficiency of Salmonella Typhimurium-challenged growing pigs under poor housing conditions.

Glossary

Abbreviations:

AA

amino acids;

AA+

supplemented diet

ADFI

average daily feed intake

ADG

average daily gain

BL

body lipid

BP

body protein

BW

body weight

CFU

colony forming units

CN

control diet

D

diet

DXA

Dual-energy X-ray absorptiometry

G:F

gain:feed ratio

GOOD

good sanitary condition

LD

lipid deposition

Met + Cys

methionine + cystine

PD

protein deposition

SC

sanitary condition

SID

standardized ileal digestive

ST

Salmonella Typhimurium

ST + POOR

Salmonella Typhimurium challenge and poor housing condition

Thr

threonine

Trp

tryptophan

Conflict of Interest Statement

J.K.H. and H. G. B are employees of Evonik Operations GmbH and Evonik Brasil Ltda. All other authors declare no real or perceived conflicts of interest."