The diets in the present study were formulated to contain a high inclusion of soybean ingredients. The β-sheet structures of raw legume proteins and the intermolecular β-sheet aggregates derived from heating are negatively correlated with feed digestibility values . 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 . Consequently, the flow 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 . 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 . SDP is commonly used because it can stimulate feed intake due to its palatability , as observed in Trial 1. Its beneficial 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 , therefore preserving the small intestinal barrier function and reducing intestinal inflammation and damage .
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 . Some research data have shown improved growth performance, feed intake and efficiency of animals fed PDP compared to other high-quality protein sources like fish meal . 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 . The beneficial 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  or even free amino acids .
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  or fish meal . This is the first 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 significant 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 significance found in the FDR test in the ileum is probably due to the low number of samples that were amplified 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 . However, the CLDN15 gene codes for a pore-forming protein , which is important for the normal-sized morphogenesis of the small intestine . 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 . 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 first barrier against invading bacteria, safeguarding the intestine and its structural integrity . This barrier positive effect is in line with a study carried out by Smith et al. , in which animals infected with Lawsonia intracelullaris had impaired ileum gene expression of TFF3, CLDN15 and MUC2. In addition, Zhou et al.  also found that infection with different strains of enterotoxigenic E. coli F4 caused MUC13 downregulation in IPEC-J2 cells. Liu et al.  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 , and IFNGR1 is part of the receptor that mediates the biological effects of IFN-γ . 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 fight 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 inflammatory cytokines and subsequent loss of the barrier function . Upregulation of GBP1 by the SDP-PDP diet is probably related to IFNGR1 upregulation because GBP1 expression is strongly induced by IFN‐γ , 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 significantly 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 . SLC11A2/DMT1 gene expression is upregulated in circumstances of low Fe intake  and hyperglycemia conditions . 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.  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 inflammatory effects of histamine more efficiently than other groups. On the other hand, APN is a Zn-dependent enzyme that takes part in the final digestion of peptides . Its upregulation in the small intestine has been documented with products considered beneficial for intestinal health, such as the probiotic Lactococcus lactis . 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 , 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  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 first defense against reactive oxygen species (ROS) formed during normal cell metabolism . Elimination of ROS by SOD2 can be considered as an anti-inflammatory effect due to the important role that ROS plays in triggering and promoting inflammation . As well as the TLR4 gene, expression of SOD2 is stimulated by lipopolysaccharides but cytokines or ROS can also upregulate it . 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 [5,12,65–68]. The possibility that PDP contains bioactive peptides that modify the gene expression of the intestine and influence intestinal health deserves to be explored.