A longer adaptation period to a functional amino acid-supplemented diet improves growth performance and immune status of Salmonella Typhimurium-challenged pigs

Abstract

We recently showed that dietary supplementation with key functional amino acids (FAA) improves growth performance and immune status of Salmonella Typhimurium (ST)-challenged pigs. It is not known if ST-challenged pigs will benefit from a longer adaptation period to FAA. The objective of this study was to evaluate the effects of different adaptation periods to diets containing FAA above requirements for growth on performance and immune response of weaned pigs subsequently challenged with ST. A total of 32 mixed-sex weanling pigs (11.6 ± 0.3 kg) were randomly assigned to 1 of 4 dietary treatments, being a basal amino acid (AA) profile fed throughout the experimental period (FAA−) or a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) fed only in the postinoculation (FAA+0), for 1 wk pre- and postinoculation (FAA+1), or throughout the experimental period (FAA+2). After a 14-d adaptation period, pigs were inoculated with ST (2.15 × 10⁹ CFU/mL). Growth performance, body temperature, fecal score, acute-phase proteins, oxidant/antioxidant balance, score for ST shedding in feces and intestinal colonization, and fecal and digesta myeloperoxidase (MPO) were measured pre- and postinoculation. Postinoculation body temperature and fecal score, serum haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), and fecal MPO were increased while serum albumin and plasma reduced glutathione (GSH):oxidized glutathione (GSSG) were reduced compared to pre-inoculation (P < 0.05). Average daily gain and G:F were greater in FAA+2 pigs compared to FAA− pigs (P < 0.05). Serum albumin was higher in FAA+2 and FAA+1 compared to FAA+0 and FAA− pigs (P < 0.05) while FAA+2 pigs had lower haptoglobin compared to FAA− (P < 0.05). Plasma SOD was increased and GSH:GSSG was decreased in FAA− pigs compared to the other treatments (P < 0.05). Score for ST shedding in feces was progressively lower from d 1 to 6 regardless of treatment (P < 0.05) and was lower in FAA+2 pigs compared to FAA− and FAA+0 (P < 0.05). Counts of ST in colon digesta were higher in FAA− and FAA+0 pigs compared to FAA+2 (P < 0.05). Fecal and colonic digesta MPO were lower in FAA+2 and FAA+1 pigs compared to FAA− (P < 0.05). These results demonstrate a positive effect of a longer adaptation period to FAA-supplemented diets on performance and immune status of weaned pigs challenged with Salmonella.

Introduction

Enteric pathogen exposure, including Salmonella and Escherichia coli infection, leads to a dramatic decrease in growth performance in pigs (Rodrigues et al., 2021a), which is partly due to the direct damage to the gastrointestinal tract (Heo et al., 2009; Boyer et al., 2015). The postweaned pig is particularly susceptible to these negative effects due to the gastrointestinal health issues and immune system disturbance associated with weaning transition (Jha and Berrocoso, 2016). Nutritional strategies aimed at attenuating the stressful conditions faced by the weanling pig will be at the forefront not only by increasing growth but also by supporting gastrointestinal and overall health (Kim et al., 2012).

The requirements for individual amino acids (AA) for growth, including Thr (Wellington et al., 2018), Met (Litvak et al., 2013), and Trp (de Ridder et al., 2012), are increased during situations of immune system stimulation. More recently, a higher demand for immune system support, maintenance of the intestinal mucosal barrier, and regulation of antioxidant defense have been linked with the increased requirements of AA, which characterize their “functional” roles beyond protein synthesis (Wu, 2010; Le Floc’h et al., 2018). For example, Wang et al. (2010) showed that a higher Thr intake is important for adequate maintenance of gut barrier function in healthy piglets. Likewise, a Trp-supplemented diet altered gut microbial composition and diversity, improving intestinal mucosal barrier function and lowering the expression of gut inflammatory cytokines in healthy weaned piglets (Liang et al., 2018). Finally, Chen et al. (2014) reported that an increased Met intake in postweaning healthy pigs was required for optimal protein synthesis and mucosal integrity in the small intestine. It may be expected, therefore, that supplementation with key functional AA (FAA) may improve gut health and support the immune system development of presumably healthy, weaned pigs and better prepare them for potential subsequent immune system stimulation.

We have recently reported that weaned pigs fed FAA-supplemented diets, including Thr, Trp, and Met, for 1 week pre- and post-Salmonella inoculation had greater performance and immune status compared to pigs fed a basal AA profile (Rodrigues et al., 2021b). The positive effects were mainly associated with attenuation of acute-phase response and improvements in the antioxidant balance. In the majority of studies, the effects of disease challenge on AA requirements and/or the effect of supplemental AA have been examined at the time of challenge or following a standard dietary adaptation. It is not known whether a longer adaptation period to FAA-supplemented diets will further improve growth performance, gut health, and immune status of Salmonella-challenged weaned pigs.

The objective of the present study was to determine if adaptation period to the supplementation of FAA will impact growth performance and immune status of weaned pigs subsequently challenged with Salmonella. It was hypothesized that a longer period of feeding FAA-supplemented diets to piglets would maximize the benefits on growth performance and immune status previously reported, therefore improving performance and immune status in pigs inoculated with Salmonella.

Materials and Methods

The experimental protocol was approved by the University of Saskatchewan’s Animal Research Ethics Board (AUP #20190003) and followed the Canadian Council on Animal Care guidelines (CCAC, 2009).

Animals, housing, and diets

A total of 32 mixed-sex weanling pigs (Camborough Plus × C3378; PIC Canada Ltd.) of 11.6 ± 0.3 kg initial body weight (BW) were obtained from the Prairie Swine Centre, Inc. (Saskatoon, SK) and transported to the Animal Care Unit of the Western College of Veterinary Medicine (Saskatoon, SK). The pigs were placed on trial in two blocks using two experimental rooms. In each experimental room (25°C ambient temperature), pigs were housed individually on solid floors lined with rubber mats. Pigs were randomly assigned to 1 of 4 treatments in a randomized complete block design (RCBD, n = 8 pigs/treatment) for 21 d, which consisted of a 14-d pre-inoculation/adaptation period and 7-d postinoculation period. Dietary treatments consisted of a basal AA profile fed throughout the experimental period (FAA−) or a functional AA profile (FAA+) fed either postinoculation (FAA+0), for 1 wk preinoculation and postinoculation (FAA+1), or throughout the experimental period (FAA+2). Diets were corn–wheat–barley–soybean meal-based and were formulated using the reported nutrient content and analyzed AA content of ingredients to meet or exceed nutrient requirements for 11–25 kg pigs according to NRC (2012) and AMINODat 5.0 (Evonik, 2016; Tables 1 and 2). The FAA− profile met the standardized ileal digestible (SID) AA requirements according to NRC (2012) and the FAA+ profile contained Thr, Met, and Trp at 120% of requirements as previously reported (Rodrigues et al., 2021b). As in our previous study (Rodrigues et al., 2021b), only FAA content was adjusted in the diets in order to minimize the confounding effects of altering dietary ingredients and nonessential AA content across the two diets. Pigs were fed ad libitum and had unrestricted access to water.

Table 1.

Ingredient and nutrient composition of experimental diets (as-fed basis)¹

	Diets
Ingredients, %	FAA−	FAA+
Corn	50.34	50.02
Wheat	13.00	13.00
Barley	17.50	17.50
Soybean meal, 47% CP	13.00	13.00
L-Lys HCl2	0.97	0.97
DL-Met2	0.32	0.46
L-Trp2	0.07	0.10
L-Thr2	0.40	0.55
L-Leu2	0.22	0.22
L-Iso2	0.20	0.20
L-Val2	0.29	0.29
L-His2	0.13	0.13
L-Phe2	0.25	0.25
Salt	0.35	0.35
Vitamin/mineral premix3	0.40	0.40
Limestone	1.38	1.38
Monocalcium phosphate	1.18	1.18
Calculated nutrient content4
DM, %	87.64	87.68
CP, %	16.06	16.26
ME, kcal/kg	3,221	3,225
NE, kcal/kg	2,452	2,455
Amino acids, % SID
Arg	0.73	0.73
His	0.43	0.43
Ile	0.66	0.66
Leu	1.28	1.28
Lys	1.28	1.28
Met+Cys	0.72	0.86
Phe+Tyr	1.20	1.20
Thr	0.80	0.96
Trp	0.21	0.25
Val	0.83	0.83
Ala	0.58	0.58
Asp	0.97	0.97
Glu	2.43	2.43
Gly	0.46	0.46
Pro	1.04	1.04
Ser	0.57	0.57

¹FAA−, basal amino acid profile; FAA+, functional amino acid profile (Thr, Met, and Trp at 120% of requirements for growth); SID, standardized ileal digestible.

²L-Lys HCl, Archer Daniels Midland Company (Decatur IL); DL-Met, Evonik Operations GmbH (Mobile AL); L-Trp, L-Thr, and L-Val, Jefo Nutrition Inc. (Saint-Hyacinthe, QC, Canada); all other AA, ACP Chemicals, Inc. (St. Leonard, QC, Canada).

³Supplied per kg of complete diet: vitamin A, 6,000 IU; vitamin D, 9.3 mg; vitamin E, 35 IU; menadione, 2.5 mg; vitamin B12, 0.02 mg; thiamine, 1.00 mg; biotin, 0.10 mg; niacin, 20 mg; riboflavin, 4 mg; pantothenate, 12 mg; folic acid, 0.50 mg; pyridoxine, 5.0 mg; Fe,75 mg; Zn, 75 mg; Mg, 20 mg; Cu, 10 mg; Se, 0.15 mg, and I, 0.50 mg.

⁴Nutrient content of diets based on estimated nutrient contents of ingredients according to NRC (2012) and analyzed AA content according to Evonik Operations GmbH.

Table 2.

Analyzed nutrient content of experimental diets (as-fed basis)¹

	Diets
Nutrients, %	FAA−	FAA+
Dry matter	88.19	88.39
Crude protein	16.3	16.6
Total amino acids2
Arg	0.78 (0.80)	0.78 (0.80)
His	0.45 (0.48)	0.48 (0.48)
Ile	0.71 (0.74)	0.74 (0.74)
Leu	1.42 (1.43)	1.44 (1.43)
Lys	1.31 (1.37)	1.39 (1.37)
Met+Cys	0.73 (0.79)	0.91 (0.94)
Phe	0.91 (0.93)	0.93 (0.93)
Thr	0.82 (0.89)	0.98 (1.04)
Trp	0.24 (0.23)	0.27 (0.27)
Val	0.92 (0.93)	0.94 (0.93)
Ala	0.72 (0.71)	0.72 (0.71)
Asp	1.16 (1.15)	1.17 (1.15)
Glu	2.82 (2.76)	2.76 (2.76)
Gly	0.55 (0.55)	0.55 (0.55)
Pro	1.09 (1.04)	1.06 (1.04)
Ser	0.67 (0.66)	0.66 (0.66)

¹FAA−, basal amino acid profile; FAA+, functional amino acid profile (Thr, Met, and Trp at 120% of requirements for growth).

²Analyzed values of total AAs with calculated values in parenthesis.

Inoculation, rectal swab protocol, and fecal sampling

On d 0 of the inoculation period (d 15 of the experiment), after being confirmed negative for the inoculated pathogen, all pigs (n = 32) were orally inoculated twice within 4 h, each time with 1 mL of a solution containing 2.15 × 10⁹ CFU/mL of Salmonella enterica subsp. enterica (S.) serovar Typhimurium var. Copenhagen (ST) selected for antibiotic resistance to Nalidixic acid and Novobiocin (Nal+/Nov+) (Wellington et al., 2019). On d −2 pre-inoculation and d 1, 2, 4, and 6 postinoculation, rectal swabs were obtained from individual pigs, diluted 1:10 in buffered peptone water (BPW), and cultured on brilliant green (BG) agar plates containing 30 μg/mL Nalidixic acid and 50 μg/mL Novobiocin (Nal+/Nov+). Further, 1 mL of the dilution was enriched in 4 mL of selenite–cysteine broth (30 μg/mL Nal+/50 μg/mL Nov+) and incubated overnight at 37°C and later 200 μL was cultured on BG agar plates (30 μg/mL Nal+/50 μg/mL Nov+). Colony counts were recorded on all plates after incubation for 24 h at 37°C. A scoring system was used for each plate to assign fecal shedding scores (Wellington et al., 2019). Plates prepared from swabs with colony counts >30 were given a score of 3. Plates positive for antibiotic-resistant ST but with colony counts <30 were given a score of 2. A score of 1 was assigned to swabs that were negative for antibiotic resistant ST after direct plating but positive after enrichment. Swabs negative for antibiotic resistant ST after direct plating and on enrichment were given a score of zero. On d 0 pre-inoculation and d 4 postinoculation, fecal samples were obtained from individual pigs for analysis of myeloperoxidase (MPO) activity as a pro-inflammatory biomarker according to adapted methodology from Bloomer (2018). Briefly, fecal samples were stored at −80°C, allowed to thaw and diluted (1:1) before centrifugation twice at 1,150 × g for 10 min at 4°C. For each sample, 750 μL of the resulting supernatant was transferred to 2 mL polypropylene microcentrifuge tubes and centrifuged again at 7,000 × g for 10 min at room temperature. The samples were then assayed for determination of MPO activity using a colorimetric assay kit according to the manufacturer’s instruction (ab105136; Abcam, Cambridge, MA).

Growth performance

Individual pig BW and feed intake were obtained on d −14 and −7 pre-inoculation and on d 0 and 7 postinoculation for calculation of pre- (wk 1 and 2) and postinoculation average daily gain (ADG), average daily feed intake (ADFI), and gain:feed (G:F).

Rectal temperature and fecal score

Rectal temperature and fecal score were obtained from all pigs on d −1 pre-inoculation and daily postinoculation until d 6. Rectal temperatures were obtained using a digital thermometer (Life Brand, ON, Canada). A scoring system was used to assign fecal scores, with normal consistency feces given a score of 0, semisolid feces without blood given a score of 1, watery feces without blood given a score of 2, and blood-tinged feces given a score of 3.

Blood sampling and analysis

Blood samples were obtained at 0900 hours from all pigs before inoculation (d 0) and on d 4 and 7 postinoculation via jugular vein puncture into 10 mL heparin coated vacutainer tubes (BD, Mississauga, ON, Canada) or tubes containing no additive. Blood samples collected into additive-free tubes were allowed to clot. Serum and plasma were obtained by centrifugation at 2,500 × g at 4°C for 15 min and stored at −20°C for subsequent analysis. Serum albumin was analyzed by bromocresol green method using a Cobas C 311 (Roche Diagnostics, Laval, QC, Canada) according to Doumas et al. (1971). Serum haptoglobin was analyzed in the Animal Health Laboratory at the University of Guelph (Guelph, ON) on a Roche Cobas 6000 c501 analyzer according to the method described by Makimura and Suzuki (1982). Plasma content of superoxide dismutase (SOD, ab65354), malondialdehyde (MDA, ab118970), and reduced glutathione:oxidized glutathione (GSH:GSSG, ab138881) were determined according to the manufacturer’s instructions (Abcam) of the respective kits.

Digesta and tissue collection and analysis

On d 7 postinoculation, all pigs (n = 32) were euthanized by penetrating captive bolt followed by exsanguination. Subsequently, liver, mesenteric lymph nodes (MLN), and spleen were sampled under aseptic conditions into sterile tubes containing 20 mL BPW, weighed, and homogenized followed by plating of 200 μL of the dilution on Nal+/Nov+ BG agar plates. Further, 1 mL was diluted in 4 mL selenite-cysteine broth (Nal+/Nov+) for enrichment overnight at 37°C with shaking, after which 200 μL was plated and incubated. Intestinal digesta samples (~1 g) were obtained from the ileum, cecum, and colon and each diluted in 4 mL BPW and kept at 4°C. The digesta samples were serially diluted to 10^–7 and 200 μL of each dilution was plated on BG agar (Nal+/Nov+) and cultured at 37°C for 24 h after which colonies were counted on each plate, with colony counts of 30–300 used in the calculation of colony forming units per gram digesta (CFU/g). Ileal, cecal, and colonic digesta MPO activity was measured using a colorimetric kit (ab105136, Abcam) according to the same methodology described above for fecal samples.

Statistical analysis

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 MIXED procedure of SAS as a RCBD. Treatments (FAA−, FAA+0, FAA+1, and FAA+2) were included as fixed effects and block as a random effect variable. For data collected over time, day (pre- and postinoculation) was included in the analysis as a repeated effect (i.e., acute-phase response and oxidant/antioxidant balance [d 4 and 7 postinoculation]; Salmonella shedding score in feces [d 1, 2, 4, and 6 postinoculation]; and fecal MPO [d 4 postinoculation]). Differences between means were determined using the Tukey post hoc test and considered significant at P ≤ 0.05. A trend toward significance was considered at P < 0.10.

Results

Rectal temperature and fecal score

Rectal temperature and fecal score are shown in Figure 1. Inoculation with ST increased rectal temperature within 24 h, which remained higher during the first 4 d postinoculation (P < 0.05). Likewise, inoculation with ST negatively affected fecal score within 24 h, which remained worse for the duration of the study compared to the pre-inoculation score (P < 0.05). There was no effect of dietary treatment on rectal temperature or fecal score (P > 0.10).

Figure 1.

Rectal temperature and fecal score of pigs inoculated with Salmonella Typhimurium (indicated by arrow). Normal consistency feces were given a score of 0, semisolid feces without blood were given a score of 1, watery feces without blood were given a score of 2, and blood-tinged feces were given a score of 3. No significant (P > 0.10) effects of treatments on rectal temperature and fecal score were observed. Values are least squares means; n = 32 pigs.

Salmonella scoring for shedding in feces and colonization

Pigs were negative for Salmonella prior to inoculation. Salmonella shedding score in feces was quantified on d 1, 2, 4, and 6 postinoculation (Figure 2). Overall shedding was lower in FAA+2 pigs, higher in FAA− and FAA+0 pigs, and intermediate in FAA+1 pigs (P < 0.05). Shedding was progressively lower from d 1 to 6, regardless of treatment (P < 0.05). Table 3 shows ST quantification in ileum, cecum, and colon digesta. Counts of ST in colon were reduced in FAA+2 pigs, increased in both FAA− and FAA+0 pigs, and intermediate in FAA+1 pigs (P < 0.05). Ileal and cecal counts of ST were not affected by dietary treatment (P > 0.10). Figure 3 shows the presence of the inoculated ST in the liver, MLN, and spleen. There was no effect of dietary treatment on the presence of the inoculated ST in the liver, spleen, and MLN (P > 0.10).

Figure 2.

Postinoculation fecal shedding of Salmonella Typhimurium in pigs fed a basal AA profile throughout the experimental period (FAA−) or a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) postinoculation (FAA+0), for 1 wk pre- and postinoculation (FAA+1), or throughout the experimental period (FAA+2). A shedding score of 3 was assigned to plates positive for the inoculated ST with counts >30 and plates positive but with counts <30 were given shedding score of 2. A shedding score of 1 was assigned to plates that were only positive after enrichment and plates negative after enrichment were scored zero. Overall shedding was lower in FAA+2 pigs, higher in FAA− and FAA+0 pigs, and intermediate in FAA+1 pigs (P < 0.01). Shedding was progressively lower from d 1 to d 6, regardless of treatments (P < 0.01). Values are least squares means; n = 8 pigs/treatment.

Table 3.

Salmonella Typhimurium quantification in intestinal contents (log10 CFU/g; d 7 postinoculation) of Salmonella-inoculated pigs¹

Item	FAA−	FAA+0	FAA+1	FAA+2	SEM	P-value
Ileum	5.47	4.61	4.95	3.92	1.273	0.78
Cecum	4.55	4.09	3.98	3.79	0.434	0.63
Colon	6.28a	6.03a	4.42ab	3.32b	0.517	<0.01

¹Values are least squares means; n = 8 pigs/treatment. FAA−, pigs fed a basal AA profile throughout the experimental period. FAA+0, pigs fed a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) postinoculation. FAA+1, pigs fed FAA+ for 1 wk pre- and postinoculation. FAA+2, pigs fed FAA+ throughout the experimental period. SEM, Standard error of the mean.

^a–bMeans within a row with different superscripts differ (P ≤ 0.05).

Figure 3.

Salmonella Typhimurium translocation to the MLN, spleen and liver in pigs fed a basal AA profile throughout the experimental period (FAA−) or a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) postinoculation (FAA+0), for 1 wk pre- and postinoculation (FAA+1), or throughout the experimental period (FAA+2). A score of 3 was assigned to plates positive for the inoculated ST with counts >30 and plates positive but with counts <30 were given shedding score of 2. A score of 1 was assigned to plates that were only positive after enrichment and plates negative after enrichment were scored zero. No significant (P > 0.10) effects of treatments on bacteria translocation in either MLN, spleen or liver were observed. Values are least squares means; n = 8 pigs/treatment.

Growth performance

Growth performance data for pre- and postinoculation is presented in Table 4. There were no dietary treatment effects on growth performance during the first or second pre-inoculation period (P > 0.10). In the postinoculation period, pigs fed FAA+2 diets had greater ADG and G:F (P < 0.05) compared to pigs fed FAA− diets, with FAA+0 and FAA+1 pigs being intermediate (P < 0.05).

Table 4.

Pre- and postinoculation growth performance of Salmonella-inoculated pigs¹

Item	FAA−	FAA+0	FAA+1	FAA+2	SEM	P-value
Pre-inoculation wk 1 BW (d −14), kg	11.74	11.63	11.63	11.73	0.346	0.98
Pre-inoculation wk 2 BW (d −7), kg	13.77	13.37	13.65	13.70	0.465	0.87
Inoculation BW (d 0), kg	16.85	16.34	16.74	17.40	0.701	0.71
Final BW (d 7), kg	18.33	18.62	19.55	20.59	1.093	0.26
Pre-inoculation wk 1 period (day −14 to −7)
Average daily gain, kg	0.290	0.248	0.289	0.281	0.032	0.79
Average daily feed intake, kg	0.446	0.381	0.405	0.407	0.049	0.75
Gain:feed, kg/kg	0.65	0.65	0.71	0.69	0.089	0.73
Pre-inoculation wk 2 period (day −7 to 0)
Average daily gain, kg	0.440	0.424	0.441	0.529	0.061	0.59
Average daily feed intake, kg	0.778	0.750	0.740	0.801	0.042	0.92
Gain:feed, kg/kg	0.57	0.57	0.60	0.66	0.088	0.49
Postinoculation period (day 0 to 7)
Average daily gain, kg	0.211b	0.326ab	0.401ab	0.456a	0.059	0.01
Average daily feed intake, kg	0.720	0.705	0.763	0.727	0.052	0.87
Gain:feed, kg/kg	0.29b	0.46ab	0.53ab	0.63a	0.099	0.02

¹Values are least squares means; n = 8 pigs/treatment. FAA−, pigs fed a basal AA profile throughout the experimental period. FAA+0, pigs fed a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) postinoculation. FAA+1, pigs fed FAA+ for 1 wk pre- and postinoculation. FAA+2, pigs fed FAA+ throughout the experimental period. SEM, Standard error of the mean.

^a–bMeans within a row with different superscripts differ (P ≤ 0.05).

Blood parameters

Serum indicators of acute-phase response (albumin and haptoglobin) and plasma indicators of oxidant/antioxidant balance (SOD, MDA, GSH, GSSG, and GSH:GSSG) are shown in Table 5. Albumin decreased at 4 d postinoculation and remained decreased at d 7 postinoculation (P < 0.05). Haptoglobin increased at d 4 postinoculation and decreased at d 7 postinoculation (P < 0.05). Overall, albumin was higher in FAA+2 and FAA+1 pigs compared to FAA+0 and FAA− pigs (P < 0.05). Furthermore, FAA+2 pigs had the lowest overall haptoglobin, FAA− the highest, with FAA+0 and FAA+1 being intermediate (P < 0.05). SOD increased at d 4 postinoculation and decreased at d 7 postinoculation (P < 0.05). Overall, SOD was increased in FAA− pigs compared to the other treatments (P < 0.05). MDA increased at d 4 postinoculation returning to baseline content at d 7 postinoculation (P < 0.05). There was no effect of dietary treatments on MDA content (P > 0.10). Plasma GSH:GSSG was reduced by d 4 postinoculation, which remained lower at d 7 postinoculation, as a result of reduced plasma GSH and increased plasma GSSG (P < 0.05). Overall plasma GSH:GSSG was decreased in pigs fed FAA− compared to the other treatments, mainly due to lower GSH (P < 0.05).

Table 5.

Pre- and postinoculation blood parameters of Salmonella-inoculated pigs¹

	Treatments						P-value
Item	FAA−	FAA+0	FAA+1	FAA+2	Mean (d)	SEM	Trt	Day
Serum albumin, g/L
Pre-inoculation (d 0)	34.88	34.00	35.87	36.25	35.25ª	1.242	<0.01	<0.01
Postinoculation (d 4)	28.63	30.00	35.00	36.12	32.44b
Postinoculation (d 7)	25.50	27.13	33.00	33.38	29.75b
Mean (trt)	29.67b	30.38b	34.62a	35.25a
Serum haptoglobin, g/L
Pre-inoculation (d 0)	0.96	0.71	0.80	0.50	0.74b	0.191	<0.01	<0.01
Postinoculation (d 4)	2.09	1.89	1.30	1.17	1.61ª
Postinoculation (d 7)	1.57	1.35	1.09	1.02	1.26b
Mean (trt)	1.54a	1.32ab	1.06ab	0.90b
Plasma SOD, mU/mL
Pre-inoculation (d 0)	30.92	24.67	28.47	27.83	27.97c	6.953	0.01	<0.01
Postinoculation (d 4)	71.11	56.54	54.46	55.73	59.46ª
Postinoculation (d 7)	60.51	40.62	39.80	39.51	45.11b
Mean (trt)	54.18a	40.61b	40.91b	41.02b
Plasma MDA, nmol/mL
Pre-inoculation (d 0)	0.41	0.43	0.49	0.46	0.45b	0.072	0.63	<0.01
Postinoculation (d 4)	0.57	0.62	0.58	0.49	0.57ª
Postinoculation (d 7)	0.41	0.41	0.49	0.37	0.42b
Mean (trt)	0.46	0.49	0.52	0.44
Reduced glutathione (GSH), Μm
Pre-inoculation (d 0)	4.83	5.28	5.40	5.87	5.35ª	0.745	0.02	<0.01
Postinoculation (d 4)	1.39	3.62	3.62	3.88	3.13b
Postinoculation (d 7)	0.71	3.76	3.73	3.58	2.95b
Mean (trt)	2.31b	4.22a	4.25a	4.44a
Oxidized glutathione (GSSG), μM
Pre-inoculation (d 0)	0.85	1.03	1.06	1.10	1.01b	0.066	0.56	0.04
Postinoculation (d 4)	1.63	1.42	1.48	1.67	1.55ª
Postinoculation (d 7)	1.02	0.98	0.83	0.91	0.94b
Mean (trt)	1.17	1.14	1.12	1.23
GSH:GSSG
Pre-inoculation (d 0)	5.65	5.13	5.10	5.34	5.31ª	1.097	0.03	<0.01
Postinoculation (d 4)	0.85	2.55	2.44	2.33	2.04b
Postinoculation (d 7)	0.70	3.83	4.49	3.93	3.24b
Mean (trt)	2.40b	3.84a	4.01a	3.87a

¹Values are least squares means; n = 8 pigs/treatment. FAA−, pigs fed a basal AA profile throughout the experimental period. FAA+0, pigs fed a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) postinoculation. FAA+1, pigs fed FAA+ for 1 wk pre- and postinoculation. FAA+2, pigs fed FAA+ throughout the experimental period. SEM, Standard error of the mean.

^a–bMeans within a row (trt comparison) or column (d comparison) with different superscripts differ (P ≤ 0.05).

Fecal and digesta MPO

Fecal MPO of pigs is shown in Figure 4. Fecal MPO was increased postinoculation compared to preinoculation (P < 0.05). Overall fecal MPO was increased in FAA− pigs, decreased in FAA+1 and FAA+2 pigs, being intermediate in FAA+0 pigs (P < 0.05). MPO was also analyzed in digesta samples (Table 6). Pigs fed FAA+2 diets tended to show reduced MPO in cecal digesta compared to FAA− (P < 0.10). Pigs fed FAA+2 and FAA−1 diets showed reduced MPO in colon digesta compared to FAA− and FAA+0 pigs (P < 0.05).

Figure 4.

Pre- (d 0) and postinoculation (d 4) fecal MPO in pigs fed a basal AA profile throughout the experimental period (FAA−), or a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) postinoculation (FAA+0), for 1 wk pre- and postinoculation (FAA+1), or throughout the experimental period (FAA+2). Overall MPO was lower in FAA+2 and FAA+1 pigs, higher in FAA−, and intermediate in FAA+0 pigs (P = 0.01). MPO was increased postinoculation compared to pre-inoculation, regardless of treatment (P = 0.01). Values are least squares means; n = 8 pigs/treatment.

Table 6.

MPO concentration in intestinal contents (µU/mL; d 7 postinoculation) of Salmonella-inoculated pigs¹

Item	FAA−	FAA+0	FAA+1	FAA+2	SEM	P-value
Ileum	4.51	4.88	5.01	4.52	1.03	0.98
Cecum	6.15	3.13	3.07	2.71	1.08	0.07
Colon	5.04a	3.83a	1.95b	1.93b	0.92	0.04

¹Values are least squares means; n = 8 pigs/treatment. FAA−, pigs fed a basal AA profile throughout the experimental period. FAA+0, pigs fed a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) postinoculation. FAA+1, pigs fed FAA+ for 1 wk pre- and postinoculation. FAA+2, pigs fed FAA+ throughout the experimental period. SEM, Standard error of the mean.

^a–bMeans within a row with different superscripts differ (P ≤ 0.05).

Discussion

We reported previously that feeding growing pigs a Thr-supplemented diet (20% above requirements for growth) improved performance during ST challenge (Wellington et al., 2019). More recently, we observed an attenuation of acute-phase response, improvement of oxidant/antioxidant balance, and enhancement in growth response of weanling pigs fed a mixture of Thr, Trp, and Met under Salmonella challenge (Rodrigues et al., 2021b). These studies confirmed previous evidence that immune stimulation increases AA requirements for growth, including Met and Cys (Litvak et al., 2013; Rakhshandeh et al., 2014), Thr (Jayaraman et al., 2015; Wellington et al., 2018), and Trp (Le Floc’h et al., 2009; de Ridder et al., 2012). However, previous studies have generally supplemented AA after or around challenge initiation. It may be expected that exposing pigs to FAA-supplemented diets for a longer period will better prepare them to cope with an enteric challenge. This may be hypothesized due to the functional roles performed by AA, as substrates for immune proteins, maintaining the intestinal barrier, and regulating the antioxidant defense (Wu, 2010; Le Floc’h et al., 2018). Therefore, in the current study we supplied Met, Thr, and Trp at 120% of NRC (2012) requirements for different periods of time prior to inoculation with an enteric pathogen and examined the impact on performance measures and key indicators of immune status and gut health in pigs.

Response to Salmonella inoculation

After ST inoculation, there was a rise in rectal temperature and deterioration in fecal score compared to pre-inoculation measurements. Moreover, ST inoculation decreased serum albumin and plasma GSH:GSSG while increasing serum haptoglobin and plasma SOD and MDA. Further, postinoculation fecal MPO was increased compared to the preinoculation measurement. Bacterial shedding in feces decreased progressively after inoculation but was still present after 6 d, which correlates with activation of acute-phase response and disturbance to the oxidant/antioxidant balance. Finally, presence of ST in digesta samples (ileum, cecum, and colon) and translocation to lymphoid tissues (MLN, spleen, and liver) were detected on d 7 postinoculation. Our findings emphasize clear responses to ST inoculation, including diarrhea (Correa-Matos et al., 2003) and fever (Gebru et al., 2010), activation of acute-phase response (Wellington et al., 2019), disturbance of the antioxidant balance (Lv et al., 2020), and poor gut health (Barba-Vidal et al., 2017). Taken together, they are in agreement with our previous findings (Rodrigues et al., 2021b) and confirm a successful and uniform stimulation of immune system of pigs by ST.

Effects of FAA adaptation period on parameters of growth performance, immune system stimulation, and intestinal inflammation in Salmonella-challenged pigs

Prior to ST inoculation, there was no effect of treatments on pig growth performance. This confirms that diets were properly formulated to meet or exceed nutrient requirements for this age and weight range of pigs and confirms the lack of effect of FAA supplementation above requirements on pre-Salmonella inoculation growth performance (Rodrigues et al., 2021b).

We observed an increased ADG and improved feed efficiency in FAA+2 pigs after inoculation with ST compared to FAA− pigs, with FAA+1 and FAA+0 showing intermediate results. Here, we confirmed our hypothesis that a longer adaptation period to FAA potentiated the previously reported benefits of key FAA on growth and nutrient utilization in pigs exposed to an enteric pathogen challenge. This is in line with reduced overall MPO content in FAA+2 and FAA+1 pigs, which indicates that FAA supplementation may have supported gastrointestinal development and health in the present study, improving the ability of pigs to cope with the subsequent Salmonella challenge. Indeed, it has been shown that increased intake of Thr (Koo et al., 2020), Met (Shen et al., 2014), or Trp (Liang et al., 2019) improved gut health in unchallenged pigs. For example, adequate maintenance of gut barrier function was associated with supplemental Thr intake (Wang et al., 2010). Likewise, intestinal mucosal barrier function was improved in Trp-supplemented pigs, mainly through changes in gut microbial composition and diversity, lowering the expression of gut inflammatory cytokines (Liang et al., 2018). Moreover, intestinal protein synthesis and mucosal integrity were enhanced by a higher Met intake in postweaning pigs (Chen et al., 2014). Reduced fecal and colonic MPO also correlate with FAA+2 pigs showing reduced ST score for shedding and ST counts in colon compared to FAA+0 and FAA− pigs. Further support for an extended adaptation to FAA-supplemented diets prior to disease challenge is provided by the positive effects of a longer adaptation period to AA+ diets on ADG and feed efficiency of ST-inoculated pigs, which were achieved without a concurrent increase in ADFI. This agrees with our previous study (Rodrigues et al., 2021b) and with a meta-analysis performed by our research group (Rodrigues et al., 2021a) and by Pastorelli et al. (2012) in which it was shown that the major portion of the reduction in growth due to enteric pathogen challenge was due to feed efficiency (i.e., nutrient utilization) and not due to the decrease in feed intake.

Serum haptoglobin and albumin, which are a positive and negative acute-phase proteins, respectively, and have been directly associated with the health status of pigs (Le Floc’h et al., 2009; Kampman-van de Hoek et al., 2016). The synthesis of acute-phase proteins is generally increased under immune stimulation and the altered nutrient utilization and metabolism lead to a higher demand for AA as substrates (Reeds et al., 1994). In the current study, postinoculation haptoglobin was increased while albumin was decreased compared to the preinoculation measurements, which is in line with previous findings (Turner et al., 2000; Wellington et al., 2019; Rodrigues et al., 2021b). The overall increase in albumin in FAA+2 and FAA+1 and decrease in haptoglobin in FAA+2 compared to the other treatments strengthens the evidence that a longer adaptation period to FAA is necessary to ameliorate the inflammatory response in these pigs. Attenuation of acute-phase response was accompanied by reduced colonization of the inoculated Salmonella in the distal gut (i.e., colon) in FAA+2 and FAA+1 pigs and reduced overall score for ST shedding in FAA+2 pigs compared to FAA− and FAA+0 pigs. This may be explained by the reduced fecal and colonic digesta MPO in FAA+2 and FAA+1 pigs compared to FAA−, indicating decreased intestinal inflammation (Kansagra et al., 2003; Young et al., 2012) with longer adaptation period to FAA supplementation. The positive effects of a longer adaptation period to FAA on performance, ST shedding and colonization, and acute-phase response, without any effects on translocation of ST to lymphoid tissues (MLN, spleen, and liver), agrees with our previous findings (Rodrigues et al., 2021b). This suggests a certain degree of containment of ST infection in the gut on one hand, with ST remaining in a latent state in surrounding lymphoid tissues on the other hand (Bellido-Carreras et al., 2019).

One of the main mechanisms through which Salmonella exploits the enterocytes, is by compromising their antioxidant capacity subsequently increasing their susceptibility to the resulting oxidative stress of infection (Mehta et al., 1998). Oxidative stress is mainly triggered by the disruption of the intestinal barrier and overall immune system stimulation (Circu and Aw, 2012), with reactive oxygen species being released due to the inflammatory reaction (Oz et al., 2007). Our findings showing increased postinoculation SOD and MDA, which are enzymatic antioxidants, are in line with our previous findings using the same inoculation model (Rodrigues et al., 2021b). Furthermore, they suggest an activation of intestinal mucosa to oxidative stress (increased plasma SOD; Dincer et al., 2007) and an increased intestinal neutrophil activity, atrophy, and metaplasia (increased plasma MDA; Siregar et al., 2018) due to ST inoculation. Glutathione (GSH) is a non-enzymatic cellular antioxidant which is converted to its oxidized (disulfide) form (GSSG) when removing peroxides of oxidative stress. The increased proportion of GSH relative to GSSG pre-inoculation and the switch to decreased GSH and increased GSSG postinoculation, resulting in a reduced GSH:GSSG, are in line with our previous findings (Rodrigues et al., 2021b) and indicate a higher demand of GSH to mitigate the negative effects of infection (Jones, 2002). In the present study, plasma SOD was increased while GSH:GSSG was decreased in FAA− pigs compared to the other treatments. This suggests that antioxidant balance was positively affected by FAA supplementation but not by a longer adaptation period. Indeed, positive effects in antioxidant defense systems have been associated with a higher Trp (Mao et al., 2014) and Met (Wu et al., 2019) intake. Furthermore, immune system stimulation is known to increase demands for GSH production for the maintenance of cell redox status Shoveller et al. (2003), which leads to higher sulfur amino acid (SAA) requirements in pigs (Rakhshandeh and de Lange, 2010; Rakhshandeh et al., 2010). It has been also shown that Cys flux associated with glutathione synthesis and Cys catabolism was dramatically increased during infection in rats (Malmezat et al., 2000). Moreover, it is known that the gut possesses the apparatus for significant transmethylation (recovery of Met from homocysteine) and transsulfuration (interconversion of Cys and homocysteine; Mudd et al., 1965; Finkelstein, 2000). This is in agreement with the evidence showing that the gut represents approximately 25% of whole-body transsulfuration and that a third of all Met used by the gut was used for Cys synthesis (Riedijk et al., 2007). Therefore, despite not being measured in intestinal tissue, it is reasonable to infer that, during the pre-inoculation period, the Met pool was conserved for protein synthesis, while, during postinoculation oxidative stress there was an increase in Met transsulfuration to meet the increased Cys demand for glutathione synthesis (Malmezat et al., 2000; Vitvitsky et al., 2003). This might explain the positive effect of FAA supplementation on antioxidant parameters without positive effects of a longer adaptation and suggests a metabolic requirement for, specially, Met and Cys by the gut under enteric infection. Given the positive effect of a longer adaptation to FAA on the attenuation of acute-phase response accompanied by reduced Salmonella colonization and shedding, our results indicate that the unchallenged pig utilizes supplemental AAs for gastrointestinal development as previously reported (Shen et al., 2014; Liang et al., 2019; Koo et al., 2020), and that a higher Met intake may be more advantageous under oxidative stress compared to a presumably healthy state (Luo and Levine, 2009) as evidenced by the lack of effects of a longer adaptation to FAA.

Conclusions

Collectively, our results suggest that a longer adaptation to diets supplemented with key FAA, specifically Thr, Met, and Trp, above estimated requirements for growth improves growth performance and immune status of pigs, mainly through improvements in gut health and reduction in ST colonization, despite the lack of effect on Salmonella presence in lymphoid tissues. We further show that the antioxidant defense systems are improved by FAA intake, which may be attributed to the dynamism of SAA metabolism. Supplementation of FAA above requirements for growth may be a valuable tool to attenuate the negative effects of windows of high enteric pathogen exposure.

Glossary

Abbreviations

AA

amino acid

ADFI

average daily feed intake

ADG

average daily gain

BG agar

brilliant green agar

BPW

buffered peptone water

BW

body weight

DM

dry matter

FAA

functional amino acids

G:F

gain:feed

GSH

reduced glutathione

GSSG

oxidized glutathione

MDA

malondialdehyde

ME

metabolizable energy

MLN

mesenteric lymph nodes

MPO

myeloperoxidase

NE

net energy

RCBD

randomized complete block design

SID

standardized ileal digestible

SOD

superoxide dismutase

ST

Salmonella Typhimurium

Author contributions

L.A.R., J.C.G.V., J.K.H., A.G.V.K., and D.A.C. designed the study; L.A.R. and M.O.W. conducted the study; L.A.R. and M.O.W. performed lab and data analysis; L.A.R. and D.A.C. wrote the manuscript. All the authors contributed to the interpretation of the results throughout the study and have read and approved the manuscript. D.A.C. was responsible for content of the final manuscript.

Conflict of interest statement

J. C. González-Vega and J. K. Htoo are employees of Evonik Operations GmbH. All other authors have no financial or personal conflicts of interest.

Data availability

Data available upon reasonable request to the corresponding author.