“Porcine Digestible Peptides (Pdp) in Weanling Diets Improves Gut Barrier Function, Immune Response and Nutrient Transport in Nursery Pigs”

Background: Porcine digestive peptides (PDP) have proven to be a valid substitute for other high-quality dietary protein sources for piglets, like spray-dried plasma (SDP), but knowledge about their in�uence on intestinal function is still scarce. This study was conducted to investigate the effects of PDP, SDP or a combination of both on growth performance and intestinal function of weaned pigs. Results: A total of 180 piglets (Trial 1) and 198 piglets (Trial 2) were used to evaluate the partial substitution of soybean ingredients with 2% SDP or 2% PDP (Trial 1), and with 3% SDP or the combination of 1% SDP and 2% PDP (SDP-PDP; Trial 2) during the pre-starter period (d0-14). A common commercial diet was offered up to day 35 in Trial 2. The effects on intestinal function were evaluated by quantifyingthe gene expression (56 genes in an Open Array Real-Time PCRPlatform) in jejunum and ileum samples obtained from piglets on d 14 after weaning (Trial 2).Piglets fed SDP, PDP and SDP-PDP had a higher body weight (BW), average daily gain (ADG) and feed e�ciency (G:F) than the soybean controlon day 14 (P< 0.05), but no differences were observed among groups at the end of starter phase (Trial 2). Ten genes in jejunum and �ve in ileum samples were differentially expressed among treatments (P< 0.05). The highest expression was found in the SDP-PDP group. These genes are involved in the barrier function of intestinal cells,the immune response, mucosal nutrient transport, digestion and metabolization of oxidation products. Conclusion: The combination of SDP and PDP upregulated genes related to the intestinal functionwithout affecting growth performance. The underlying mechanisms that produced these effects are still unknown; however, protein hydrolysates might contain bioactive peptides that could be the cause.


Background
Weaning is a stressful period for piglets as they have to deal with a change from sow milk to a less digestible, plant-based dry solid diet that contains many ingredients that the pig has not eaten before [1].The rst consequence of weaning is a reduction in feed intake, which in turn causes a reduction in the villi height [2] and a disruption of the gut microbiota ecosystem with a loss of diversity [3].After weaning, piglets are more susceptible to gut in ammatory problems and their intestinal function can be affected.This often leads to post-weaning diarrhea and increased mucosal permeability [4].A strategy for helping piglets to reduce intestinal disturbances during this period is to include high-quality digestible animal protein sources in their feed.Certain protein sources, like spray-dried plasma (SDP) contain biologically active components that give them physiological or regulatory functions beyond their nutritional value [5].
In particular, SDP has shown potential for modulating the immune response, reducing intestinal mucosa in ammation and maintaining its integrity [6][7][8].
Porcine digestible peptides (PDP) are a coproduct of the heparin industry and are obtained from the enzymatic hydrolysis of porcine intestinal mucosa.Currently, PDP can be used in postweaning diets as a substitute for SDP, sh meal and other sources of high-quality protein in terms of preference and digestibility [9,10].The ability of PDP to increase villous height suggests that they may improve gut health and nutrient uptake [11].Like other sources of protein hydrolysates, PDP may also have implications for intestinal physiology as speci c bioactive peptides have a key biological function in the barrier and immune function, and nutrient transport in the small intestine [5,12].However, there is no available literature exploring possible functional properties of PDP.In the present study, in a rst trial we hypothesize that including PDP and SDP in a high crude protein (CP) diet containing a high content of soybean ingredients might improve piglet performance after weaning (Trial 1).In a second trial we also hypothesize that partially substituting soybean meal (SBM) in weanling diets with SDP or a combination of SDP and PDP could improve piglet performance and intestinal function during the pre-starter period (Trial 2).Intestinal function was evaluated using the Open Array technology.The selected genes based on a literature review were representative target genes from different functional groups.

Animals, Housing and Diet
Trial 1.The rst trial was conducted in the weanling unit of a commercial farm throughout the pre-starter period (d 0 to 14 post-weaning).
A total of 180 male and female weaned commercial crossing piglets ([Landrace x Large White] x Pietrain, weaned at d 28) with a body weight (BW) of 7.5 ± 1.15 kg without previous access to creep feed during lactation were moved to the nursery unit.Piglets were distributed into two blocks according to initial BW (heavy piglets: 8.6 ± 0.03 kg; light piglets: 6.4 ± 0.02 kg).Each block contained 9 pens of 10 animals to which the three experimental treatments were randomly assigned (6 pens or replicates/treatment).Each pen (3.2 m 2 in oor area) had a commercial non-lidded hopper (TR5, Rotecna, Agramunt, Spain) and a nipple waterer to ensure ad libitum feeding and free water access.
Treatments consisted of three different iso-protein pre-starter diets: a control diet (CON) with a high content of soybean ingredients and two extra diets with partial replacement of the extruded soybeans by 2% SDP (AP 820 P, APC Europe S.L., Granollers, Spain) or 2% PDP (Palbio 62SP, Bioiberica S.A.U., Palafolls, Spain).The basal pre-starter diet was formulated to contain 2,470 kcal of net energy (NE)/kg, 19.5% CP/kg and 1.28% dig Lys (Table 1) to meet the requirements for maintenance and growth of newly weaned piglets [13].Diets were presented in mash form and were fed ad libitum for fourteen consecutive days.No antimicrobials or ZnO were used in the experimental diets.A total of 198 male and female weaned commercial crossing piglets ([Landrace x Large White] x Pietrain, weaned at d 21) with a BW of 5.7 ± 0.60 kg that had access to creep feed during lactation, were moved to the nursery unit without transport to be used in the trial.
Animals were distributed into two blocks according to initial BW (heavy piglets: 6.3 ± 0.02 kg; light piglets: 5.1 ± 0.01 kg) and each block contained 9 pens of 11 animals to which three experimental treatments were randomly assigned (6 replicates for each treatment).Each pen (3 m 2 in oor area) had a commercial non-lidded hopper (TR5, Rotecna) and a nipple waterer to ensure ad libitum feeding and free water access.
Treatments consisted of three different iso-protein pre-starter diets: a CON diet with a high content of SBM and two extra diets with partial replacement of the SBM by 3% Spray-dried Plasma (SDP; AP 820 P, APC Europe S.L.) or a combination of 1% Spray-dried Plasma and 2% Porcine Digestible Peptides (SDP-PDP; Palbio 62SP, Bioiberica S.A.U.).
The basal pre-starter diet was formulated to contain 2,470 kcal NE/kg, 20.5% CP/kg and 1.35% dig Lys (Table 2) and to meet the requirements for maintenance and growth of newly weaned piglets [14].The pre-starter diets were fed ad libitum for fourteen consecutive days and a common starter diet was also fed ad libitum from d 15 until d 35 post-weaning (Table 2).No antibiotics, alternative antimicrobials or ZnO were included in the diets.

Data and Sample Collection
Piglets were ear tagged and individually weighed at weaning (d 0) and d 14 post weaning in trial 1 and at weaning (d 0), d 7, d 14 and d 35 post-weaning in trial 2. Feed disappearance from each hopper was measured throughout the experimental period.The average daily feed intake (ADFI), average daily gain (ADG) and feed e ciency (G:F) were calculated for the experimental period.
One piglet per pen was euthanized on d 14 in trial 2. A portion of 0.5 cm of jejunum and ileum tissues were collected at 30 cm and 1.3 cm from the ileo-cecal valve respectively and immediately snap frozen in 1 mL of RNA later (Deltalab, Rubí, Spain).Samples were stored at -80 ºC until use.

Gene Expression Study by qPCR
Gene expression was quanti ed by RT-qPCR to study the expression of 56 genes in two intestinal tissues in an Open Array Real-Time PCR Platform (Applied Biosystems, Waltham, MA, USA) by the Servei Veterinari de Genètica Molecular at the Veterinary Faculty of the Universitat Autònoma de Barcelona (Spain).
The pre-ampli ed product was diluted 1:10 with 0.1X Tris-EDTA pH 8.0 and 6 ul was transferred to 384well plates.These were analyzed in duplicate in Taqman Open Array gene expression plates customdesigned in a QuantStudio 12K Flex Real-Time PCR system (ThermoFisher Scienti c, Waltham, MA, USA).
One sample was used as an inter-plate control to check the replication of results from different plates.
Primers were designed spanning exon-exon boundaries or at different exons using the PrimerExpress 2.0 software (Applied Biosystems) for 55 genes.The IL8 gene primer was pre-designed by the company due to its complexity (see Additional le).Possible residual genomic DNA ampli cation and primer dimer formation were controlled.Finally, a customized Open Array panel containing 56 genes was obtained.

RNA Extraction and cDNA Preparation
Total RNA was obtained from 100 mg of frozen intestinal tissues with the RiboPure kit (Ambion, Austin, TX, USA) following the manufacturer's protocol.RNA concentration and purity was calculated with a NanoDrop ND-1000 spectrophotometer (NanoDrop products, Wilmington, DE, USA).RNA integrity was checked with Agilent Bioanalyzer-2100 equipment (Agilent Technologies, Santa Clara, CA, USA) following the producer's protocol.One microgram of total RNA was reverse-transcribed into cDNA in a nal volume of 20 µl.The High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) and random hexamer primers were used, and the following thermal pro le was applied: 25 ºC, 10 min; 37 ºC, 120 min; 85 ºC, 5 sec; 4 ºC hold.A total of 25 ng of cDNA sample was pre-ampli ed using a 2X TaqMan® PreAmp Master Mix and a 0.2X Pooled Taqman Gene Expression custom Assays in a nal volume of 10 µl.The thermal cycling conditions for the pre-ampli cation reactions were 10 min at 95 ºC; 14 cycles of 15 sec at 95 ºC and 4 min at 60 ºC; and a nal step of 10 min at 99 ºC.The pre-ampli ed cDNA product was stored at -20 ºC until use.

Gene Expression Data Analysis
Gene expression data were collected and analyzed using the ThermoFisher Cloud software 1.0 (Applied Biosystems) applying the 2 −ΔΔCt method for relative quanti cation (RQ) and using the sample with the lowest expression as a calibrator.Some parameters were adjusted: the maximum cycle relative threshold (Crt) allowed was 26, ampli cation score < 1.240, quanti cation cycle con dence > 0.8, and the maximum standard deviation allowed between duplicates was set at < 0.38.Samples that did not t these requirements or had an inconclusive ampli cation status were deleted.RQ values were checked for normalization by a log 10 transform, and all the statistical analyses were performed with the R 3.4.3software [33] and Bioconductor [34].We carried out a one-way ANOVA and calculated the Benjamini-Hochberg false discovery rate (FDR Q-value) to control multiple P-values [35], setting an upper bound for the expected proportion of false signi cant tests, that is, false signi cant treatment differences in mean expression levels between treatments.Pairwise post hoc treatment comparisons were carried out using Tukey's honest signi cant difference test [36].Statistical differences between results for the treatments were identi ed at P-values under 0.05 for the ANOVA and Tukey's analysis, and Q-values under 0.2 for the FDR.
A principal component analysis (PCA) was performed with samples as cases and gene log 10 -expressions as variables.The function PCA of the FactoMiner R-library [37] was used for dimension reduction and visualization in the rst two principal dimensions.The variables factor map was restricted to genes showing cos2-qualities over 0.45 and signi cant differences.This graph shows which of these genes are correlated along the samples.The sample identi er numbers are represented in the individual factor map, or factorial scores map, and are colored according to the diet assigned to each sample.Finally, the heatmap visualization method was used to obtain double clustering both for genes (with a correlationbased distance: , in which d is distance and r is the correlation coe cient, and the complete linkage hierarchical clustering) and for samples (with Euclidean distance and Ward's D2 method).These methods were chosen based on the following: the Euclidian distance between two samples adds all the squared differences in the log-expression level of them in each gene, and then the Ward's D2 linkage method uses the Euclidean squared-distance to cluster in a way that minimizes the increment of the variance into the resulting clusters.Correlation based distance is preferred for obtaining the gene clusters because the expression levels in different genes may not be comparable (see Murtagh and Legendre [38], for Ward's method and Everitt [39], for clustering methods).The function heatmap.2 in the R-library gplots was used [40].

Statistical Analysis
Production performance data were analyzed with ANOVA using the generalized linear model procedure of the statistical package SAS® (version 9.4, SAS Inst.Inc., Cary, NC).Normality and homoscedasticity were checked with Shapiro-wilk's test using the Univariate procedure and Levene's test using the generalized linear model procedure respectively.Data were analyzed taking the experimental treatment and the block of weight as the main factors.Their corresponding interaction was also included in the model.The statistical unit was the pen of 10 pigs in trial 1 and the pen of 11 pigs in trial 2. The results are presented as least square means taking into account the Tukey adjust.The level of signi cance considered was α = 0.05.

Growth Performance
The productive performance of piglets during the rst two weeks after weaning (Trial 1) is summarized in Table 3. Animals fed SDP and PDP showed higher BW, ADG and G:F (P < 0.05) than piglets in the control group. 1 BW: body weight; ADFI: average daily feed intake; ADG: average daily gain; G:F: feed e ciency.
a,b Different letters in the same row indicate signi cant statistical mean differences in the two diets (Tukey's test P-value < 0.05).
For trial 2, the productive performance results are shown in Table 4. Piglets of the SDP and SDP-PDP group showed greater BW and ADG and G:F than CON piglets on d 14 (P < 0.05).Although a numeric difference in BW was observed at d 35 between the supplemented animals and the CON group, no signi cant differences in BW, ADG or G:F were observed. 1BW: body weight; ADFI: average daily feed intake; ADG: average daily gain; G:F: feed e ciency.
a,b Different letters in the same row indicate signi cant statistical mean differences in the two diets (Tukey's test P-value < 0.05).

Intestinal Mucosa Gene Expression Values
After performing qPCR of the intestinal tissue, 37 genes were detected as expressed in jejunum tissue and 27 genes in ileum tissue from the 54 target genes initially included in the Open Array panel.The conservation of one tissue sample from the jejunum and seven from the ileum was affected and ampli cation was not possible.
The results of the analysis of gene expression in the jejunum intestinal tissue (Trial 2) are shown in Table 5.Ten genes showed differences between groups (P < 0.05, Q < 0.2).In fact, all the Q-values in Table 6 are below 0.169, which is the upper bound of the false positive proportion.The CLDN15 and TFF3 genes from the barrier function group and the SLC11A2/DMT1 gene from the nutrient transport group showed a higher expression for the SDP-PDP treatment compared to the CON treatment.From the immune response functional group, the SDP-PDP group showed a higher expression level in three genes: GBP1 compared to the SDP group, IFNGR1 compared to the CON group, and TLR4 compared to the other two groups.Finally, the SDP-PDP group had a higher expression level than the CON and SDP groups in two enzyme-coding genes (HNMT and APN), and in the gene coding for a protein that metabolizes oxidation products, SOD2. 2 CON: Control diet; SDP: Diet with 3% spray-dried plasma inclusion; SDP-PDP: Diet supplemented with a combination of 1% spray-dried plasma and 2% porcine digestible peptides (2%).
a,b Different letters in the same row indicate signi cant statistical mean differences in the two diets (Tukey's test P-value < 0.05).
The results of the analysis of gene expression in the ileum are shown in Table 6.Five genes showed signi cant differences among treatment means according to the ANOVA results (P < 0.05) but not according to the FDR (Q > 0.2).Three of the genes that were included in the barrier function group coded for mucins (MUC2 and MUC13) and occludin (OCLN) and had a higher mean expression in the SDP-PDP group than the CON group.From the immune response group, the GBP1 gene showed a higher expression level in the SDP-PDP treatment than in the SDP treatment.The gene coding the hormone glucagon (GCG) had a greater expression in the SDP-PDP group than in the SDP and CON groups. 1 MUC13: Mucin 13; MUC 2: Mucin 2; OCLN: Occludin; GBP1: Guanylate binding protein; GCG: Glucagon.
a,b Different letters in the same row indicate signi cant statistical mean differences in the two diets (Tukey's test P-value < 0.05).
A PCA was carried out to determine the correlation in the gene expression values among samples distributed in the three diet groups (Fig. 1).The 2D representation preserves 60% of the total variance (61.27% in the jejunum and 59.36% in the ileum).The results for the jejunum show a correlation in the gene expression pattern.In this tissue, eight of the nine signi cant genes (APN, HNMT, SLC11A2, CLCN15, IFNGR1, TLR4, GBP1 and SOD2) are well represented in 2D (long arrows) and all of them fall in the rst quadrant.Furthermore, the blue colored samples in the SDP-PDP group fall in the same quadrant or near to it, which indicates medium to high expression levels.A similar conclusion can be drawn from the PCA plots for the ileum tissue.Here, four out of the ve signi cant genes (OCLN, MUC13, GCG and MUC) show long arrows on the right-hand side of axis one, together with three of the four samples in the SDP-PDP group.Therefore, these plots clearly show a relationship between the highest expression levels in these A heatmap was also made in order to observe overall similarities among the gene expression pro les of the animals (Fig. 2).On the jejunum and ileum heatmaps, part of the samples from the PDP-SDP treatment are clustered together but genes do not show any evidence of an association by functional group.Heatmaps and PCAs are exploratory techniques that both indicate that samples in the PDP-SDP group tend to cluster separated from the other two groups; however, PCA is more speci c for indicating more clearly which gene expressions determine this clustering.All the results agree with the ANOVA and Tukey's test, but these analyses deal with mean treatment values while heatmaps and PCA-plots re ect individual performance.CON = control diet; SDP = diet with 3% spray-dried plasma inclusion; SDP-PDP = diet supplemented with a combination of 1% spray-dried plasma and 2% porcine digestible peptides.Genes are organized in rows and samples in columns.Samples are labeled with different colors representing every treatment and genes depending on its functional group.

Discussion
The diets in the present study were formulated to contain a high inclusion of soybean ingredients.The βsheet structures of raw legume proteins and the intermolecular β-sheet aggregates derived from heating are negatively correlated with feed digestibility values [41].The β-sheet structures represent more than 30% of the secondary structure of soybean seeds, while they represent less than 10% in the ingredients of animal origin [42].Consequently, the ow of protein into the distal parts of the GIT tends to be larger when more soybean ingredients are included, which promotes protein fermentation and selective growth of proteolytic bacteria [42].Furthermore, soybeans contain anti-nutritional factors, such as antigens, trypsin inhibitors and lectins, which can produce digestive disorders and reduce nutrient availability [43,44].The consequences of high inclusion of soybean ingredients, aggravated by the situation of post-weaning anorexia, can lead to intestinal disorders, such as post-weaning diarrhea [2,45].This can negatively affect the animal growth performance.In addition, proteins of animal origin as well as animal protein hydrolysates have higher palatability than vegetal proteins [10,46,47], which can translate into an increased feed intake after weaning.The results of these trials demonstrate that including high-quality animal proteins, such as spray-dried plasma (SDP) and porcine digestible peptides (PDP), promotes a positive effect on growth performance of weanling piglets, increasing BW, ADG and G:F at the end of the pre-starter phase (d14).
Spray-dried plasma improved the productive performance in all these trials, evidencing that it is a clear positive control.The results reported herein are similar to other studies that showed that SDP improved performance, especially when piglets were challenged with experimental infection and did not contain in feed medication [48].SDP is commonly used because it can stimulate feed intake due to its palatability [49], as observed in Trial 1.Its bene cial effects are explained by the preservation of blood immunoglobulins, growth factors, and bioactive peptides or compounds during the spray-drying process.These components can interact with the gut-associated lymphoid tissue [50], therefore preserving the small intestinal barrier function and reducing intestinal in ammation and damage [6].
Consumption of feed containing the hydrolysates, PDP or SDP-PDP, enhanced the productive performance of piglets compared to their corresponding control group, and these groups were equivalent to the SDP group.This result is in accordance with other authors who also obtained similar growth performance by feeding PDP and SDP in early-weaned piglets [51].Some research data have shown improved growth performance, feed intake and e ciency of animals fed PDP compared to other highquality protein sources like sh meal [46].Other studies indicate that PDP might improve gut health and nutrient uptake, based on the improved villus height of the small intestine compared to some sources of intact protein like SDP [52].The bene cial effects of PDP on growth performance could be due to its content of short-chain peptides that are more easily absorbed by pigs than intact proteins [53] or even free amino acids [54].
It has been reported that PDP might improve gut health and nutrient uptake based on the improved villus height of the small intestine of animals that were fed this hydrolysate compared to animals that were fed a source of intact protein like SDP [52] or sh meal [11].This is the rst study performed using gene expression to determine the effects of PDP on intestinal function.
The outcome of the PCA individual factor map of the gene expression shows that the factorial scores of the SDP-PDP group tended to be closer to each other and more separate from the CON and SDP group scores in the jejunum and ileum.In addition, the correspondence of most of the arrows representing the signi cant genes in the PCA variables map and most of the samples in the SDP-PDP group in a similar position in both tissues indicates that there is a relationship between the higher expression levels in these genes and the differences that the SDP-PDP group showed from the CON and SDP groups.Heatmap representation also helped to visualize a partial clustering of the samples from the SDP-PDP treatment in the jejunum and ileum.In line with this, the statistical ANOVA showed a stronger effect of the SDP-PDP diet than SDP with respect to the CON diet.Considering the similarities between the results of the two tissues, although the statistical differences in gene expression found in the ileum according to the ANOVA (P < 0.05) did not occur in the FDR (Q > 0.2), we believe it was necessary to discuss these results.The lack of signi cance found in the FDR test in the ileum is probably due to the low number of samples that were ampli ed compared to the jejunum.The combination of inferential treatment comparison techniques (ANOVA and Tukey, essentially) and powerful exploratory methods (PCA and heatmap) provides a clearer and dual (individuals and genes) idea of the differences in gene expression.
Proteins coded by these genes participate in the barrier function of gut cells, in nutrient transport in the mucosa, in digestion, in the immune response and in the metabolization of oxidation products.
The results from the barrier function showed that the expression of TFF3 and CLDN15 in the jejunum and MUC2, MUC13 and OCLN in the ileum was upregulated in the SDP-PDP group compared to the CON group.
Changes in the jejunum indicate a potentiation by SDP-PDP of the epithelial structure because the TFF3 gene participates in epithelial restitution and maintenance of intestinal mucosa integrity [25].However, the CLDN15 gene codes for a pore-forming protein [23], which is important for the normal-sized morphogenesis of the small intestine [26].In the ileum, the barrier promoting effect of the combination of SDP and PDP is also found at the epithelial integrity level, modulating the gene expression of OCLN, a tight junction involved in this process [55].However, this functional diet also acts at different levels, and in this case, the SDP-PDP diet strengthens the protective mucus layer by upregulating the expression of the oligomeric mucus gel-forming genes, here represented by MUC2 and MUC13.Therefore, this effect represents an enhancement of the rst barrier against invading bacteria, safeguarding the intestine and its structural integrity [56].This barrier positive effect is in line with a study carried out by Smith et al. [22], in which animals infected with Lawsonia intracelullaris had impaired ileum gene expression of TFF3, CLDN15 and MUC2.In addition, Zhou et al. [30] also found that infection with different strains of enterotoxigenic E. coli F4 caused MUC13 downregulation in IPEC-J2 cells.Liu et al. [21] performed a study in which pigs that were infected with E. coli showed that supplementation with Capsicum oleoresin upregulated the expression level of genes related to the integrity of membranes in the ileal mucosa at d5 post infection, including the OCLN and MUC2 genes.Overall, the SDP-PDP diet could be a potential intervention for counteracting some problems that occur in this particular period of life.
As a consequence, diets also had a clear effect on the expression of genes related to the immune response and metabolization of oxidation products.Again, the SDP-PDP diet showed more changes than the SDP diet alone.Expression of immune response genes TLR4, IFNGR1 and GBP1 was increased in the SDP-PDP group; TLR4 compared to the CON and SDP groups in the jejunum; IFNGR1 compared to the CON group in the jejunum; and GBP1 compared to the SDP group in the jejunum and ileum.
TLR4 is a receptor involved in the recognition of lipopolysaccharide, a major cell wall component of Gram-negative bacteria [20], and IFNGR1 is part of the receptor that mediates the biological effects of IFN-γ [57].The TLR4 and IFNGR1 genes have been reported to be upregulated in animals under stress and infection conditions in order to activate the innate immune response and ght against pathogens properly [21,29,58].Thus, upregulation of TLR4 and IFNGR1 might suggest that piglets fed with SDP-PDP seem to be more prepared for controlling infective processes and other intestinal challenges that can occur during the weaning period.In addition, GBP1 is a GTPase that regulates the inhibition of proliferation and invasion of endothelial cells.It protects against epithelial apoptosis induced by in ammatory cytokines and subsequent loss of the barrier function [28].Upregulation of GBP1 by the SDP-PDP diet is probably related to IFNGR1 upregulation because GBP1 expression is strongly induced by IFN-γ [28], although an upregulation of IFN-γ was not observed in this study.
Focusing on nutrient transport, only the expression of the SLC11A2/DMT1 gene was signi cantly upregulated in animals fed with the SDP-PDP combination compared to the CON group in the jejunum.
The divalent metal transporter (SCL11A2/DMT1) is located on the apical surface of the enterocyte and is involved in the intestinal Fe uptake [19].SLC11A2/DMT1 gene expression is upregulated in circumstances of low Fe intake [27] and hyperglycemia conditions [59].We have no evidence of differences in the Fe content or glycemic levels among experimental diets; therefore, the reason why the SDP-PDP treatment increased the expression of SLC11A2/DMT1 should be researched further.
Enzymes and hormones were also differentially expressed due to the SDP-PDP functional diet.On the one hand, the enzyme-coding genes HNMT and APN were upregulated in the jejunum.Kröger et al. [32] reported that high dietary inclusion of fermentable CP increased the HNMT expression in the colon, which is a histamine-degrading enzyme.They determined that the histamine catabolism activity of HNMT counter-regulated the increased production of this biogenic amine, reducing the fecal score of the piglets fed with a high fermentable CP diet.Considering that all diets had an elevated CP level, an increase in this enzyme could show that SDP-PDP was attenuating the in ammatory effects of histamine more e ciently than other groups.On the other hand, APN is a Zn-dependent enzyme that takes part in the nal digestion of peptides [60].Its upregulation in the small intestine has been documented with products considered bene cial for intestinal health, such as the probiotic Lactococcus lactis [61].Therefore, its increase due to the SDP-PDP diet may also be considered a positive physiological change.In the ileum, the GCG glucagon-encoding gene was more expressed in the SDP-PDP group than in the other groups.It is the precursor of the glucagon-like peptide GLP-2.As GLP-2 stimulates intestinal epithelial proliferation [62], its increased expression may be involved in the nutrient absorption surface.Accordingly, in this study the growth performance was higher for pigs fed PDP or SDP-PDP compared to CON.This is also in agreement with previous studies [52] in which PDP was associated with improvements in intestinal mucosa development measured by histometric indices.
Regarding the antioxidant defense mechanisms, we observed here that the expression of the SOD2 gene was also increased in the SDP-PDP group compared to both the SDP and CON groups.This mitochondrial enzyme is considered the rst defense against reactive oxygen species (ROS) formed during normal cell metabolism [31].Elimination of ROS by SOD2 can be considered as an antiin ammatory effect due to the important role that ROS plays in triggering and promoting in ammation [63].As well as the TLR4 gene, expression of SOD2 is stimulated by lipopolysaccharides but cytokines or ROS can also upregulate it [64].Thus, changes in SOD2 gene expression can be derived from the TLR4 upregulation also induced by the SDP-PDP intervention.
The underlying mechanisms that produced these effects are still unknown; however, ingredients generated from the hydrolysis of animal proteins include free amino acids, small and large peptides and they might also contain bioactive peptides with biological functions beyond their nutritional value that could cause these effects.Numerous bioactive substances have been studied, but increasing interest is currently focused on bioactive peptides of animal origin.For instance, enzymatic hydrolysis of milk, egg or animal red blood cell fractions produces bioactive peptides with antioxidative, antimicrobial, antihypertensive and immunomodulatory functions, among others [5,12,[65][66][67][68].The possibility that PDP contains bioactive peptides that modify the gene expression of the intestine and in uence intestinal health deserves to be explored.

Conclusions
The present study suggests that PDP can substitute or complement SDP in post-weaning diets because it showed the same capacity to increase growth performance in a soy-based diet in high-CP challenged piglets.In addition, the SDP-PDP diet demonstrated it could immunostimulate piglets without affecting growth performance, upregulating bene cial genes for the gut barrier function, the nutrient transport and immune response.These results give rise to research the underlying mechanisms beyond the PDP potential to improve intestinal function, which could be related to bioactive peptides due to its nature of protein hydrolysate.

Trial 2 .
The second trial was conducted in a different commercial farm throughout the pre-starter (d 0 to 14 post-weaning) and starter period (d 14 to 35 post-weaning).
signi cant genes and the SDP-PDP diet group.The CON and SDP groups are mixed and do not show high expression values.CON = control diet; SDP = diet with 3% spray-dried plasma inclusion; SDP-PDP = diet supplemented with a combination of 1% spray-dried plasma and 2% porcine digestible peptides.For each tissue, the samples' picture is on the left, labeled with different colors depending on the treatment, and the gene expressions arrows diagram on the right.Only those genes that are statistically signi cant and have a long arrow have been depicted.Each arrow color indicates a different functional group.Trial 2.

1
AbbreviationsADFI Average daily feed intake ADG Average daily gain BW Body weight CP Crude protein CON Control Crt Cycle relative treshold FDR Benjamini-Hochberg false discovery rate G F:Feed e ciency NE Net energy PCA Principal component analysis PDP Porcine digestible peptides

Table 1
Composition of the experimental diets used ontrial 1, % as feed basis.

Table 2 .
Composition of the experimental diets used on trial 2 (%, as feed basis).

Table 3
Effect of the experimental treatments on growth performance of piglets in trial 1.
P-values come from the ANOVA test.

Table 4
Effect of the experimental treatments on growth performance of piglets in trial 2.
P-values come from the ANOVA test.

Table 5
Effect of the experimental treatments on relative gene expression on jejunum mucosa after pre-starter period.

Table 6
Effect of the experimental treatments on relative gene expression on ileum mucosa after pre-starter period.