Enzyme supplementation in nutritionally reduced diets for piglets in the early-nursery phase

Optimization of nutrient utilization in weaned piglets, aiming for greater feed efficiency and lower excretion, is a key focus in animal production. Among the strategies, enzyme supplementation stands out. The study evaluated the use of an enzyme complex in diets with reduced nutritional and energy levels for piglets in the nursery phase. Forty piglets weaned at 26 days old, with an initial average weight of 7.85 ± 1.27 kg, were used. The animals were distributed in a randomized block design, in a 2×2 factorial arrangement: with or without nutritional reduction and with or without enzyme supplementation. The complex contained phytase (1500 U/g) and xylanase (350 U/g). There was no interaction between factors for performance (P > 0.05). Nutritional reduction increased feed intake (P = 0.018) and worsened feed conversion (P = 0.024) in Period II, and in Period III it reduced weight gain (P = 0.032) and further worsened feed conversion (P = 0.026). Enzyme supplementation improved weight gain (P = 0.012) and feed conversion (P = 0.050) in Period III. The reduced diet resulted in a lower incidence of diarrhea (P < 0.002). Nutritional reduction decreased ash and phosphorus digestibility, whereas enzyme supplementation improved NDF (P = 0.021) and ADF digestibility (P = 0.014). The combination of nutritional reduction and enzyme supplementation reduced the relative weights of the liver (P = 0.027) and pancreas (P < 0.0001). Nutritional reduction impaired intestinal morphometry, reducing villus height (P = 0.002) and crypt depth (P = 0.037). Enzyme supplementation increased crypt depth (P = 0.018) and reduced the villus height-to-crypt depth ratio (P = 0.005). Additionally, it increased the expression of NaPi-IIb (P = 0.050), SGLT-1 (P = 0.015), and PEPT-1 (P = 0.018) transporters. It is concluded that nutritional reduction negatively affected the piglets, and although supplementation with the enzyme complex did not fully neutralize the nutritional challenge, it was effective in improving the digestibility of the fibrous fraction and increasing gene expression of nutrient transporters.

1 Introduction

In swine production cycle, weaning is a critical period characterized by pronounced social, environmental, and nutritional changes. These factors often lead to a temporary reduction in feed intake, particularly during the first week after weaning, which disrupts metabolic and physiological homeostasis and compromises growth performance. The stress experienced by piglets during this phase can induce morphological and functional alterations in the intestine, including reduced enzymatic activity, absorptive capacity, and secretory function (Campbell et al., 2013). In addition, the gastrointestinal tract (GIT) of young piglets remains immature, with limited endogenous enzyme secretion and an underdeveloped immune system (Inoue et al., 2015). This immaturity impairs the digestion and absorption of plant-based ingredients, such as corn, soybean meal, and wheat bran, which may contain antinutritional factors that interfere with nutrient digestibility and absorption.

Given these challenges, implementing nutritional strategies that enhance nutrient utilization is essential to mitigate the negative effects of the post-weaning period. The inclusion of exogenous enzymes has emerged as an effective approach to enhance feed efficiency by compensating for the limited digestive capacity of weaned piglets. Previous studies have shown that enzyme supplementation can compensate for the initial digestive immaturity of piglets and improve performance parameters (Torres-Pitarch et al., 2017). Enzymes such as phytase and xylanase target specific antinutritional components in plant-based feed ingredients, thereby improving the release, accessibility, and absorption of nutrients and potentially enhancing growth performance. Although enzyme efficacy has been widely documented, most available data originate from diets formulated to meet conventional nutritional requirements. Consequently, there is still less information regarding their functional contribution in diets intentionally formulated with reduced nutrient density, which represents a distinct practical scenario relevant to feed-cost optimization.

Exploring enzyme supplementation in nutrient-reduced diets is scientifically relevant and economically attractive. From a practical perspective, such diets can reduce feed costs and environmental nutrient load, as the lower inclusion of highly digestible ingredients (e.g., soybean meal or inorganic phosphates) decreases the excretion of undigested nutrients, particularly nitrogen and phosphorus (Ruiz et al., 2008). When enzymes are incorporated into these formulations, they may compensate for the lower nutrient density by improving digestibility and nutrient availability. Phytic acid, non-starch polysaccharides (NSP), trypsin inhibitors, and allergenic proteins are among the main antinutritional factors present in corn and soybean meal (Baker et al., 2021). Phytase is one of the most widely used enzymes in swine production, and its inclusion in diets aims to reduce the antinutritional effects of phytate, increasing the availability of phosphorus (P), calcium (Ca), amino acids (AA), and energy (Jang et al., 2017; She et al., 2017; Baker et al., 2021). However, the extent to which these improvements translate into better performance under nutrient-restricted feeding conditions remains less clearly defined, supporting the relevance of the present study.

Xylan is a structural component of plant cell walls and may increase digesta viscosity, hindering nutrient absorption and reducing dietary energy utilization. Xylanase supplementation aids in the depolymerization of xylan structures, breaking down plant cell wall matrices into smaller chains (Choct, 2015; Petry et al., 2020). This process reduces digesta viscosity, promotes the release of encapsulated nutrients, and enhances the accessibility of endogenous digestive enzymes to their substrates within the short digestive transit time, thereby improving nutrient digestibility (De Lange et al., 2010; Kiarie et al., 2013; Tiwari et al., 2018). Thus, it is relevant to investigate whether xylanase activity is also reflected in broader physiological indicators, such as intestinal morphology and gene expression of transporters, aspects evaluated in the present study.

Evaluating enzyme supplementation under nutritionally challenging conditions allows for a better understanding of the true potential of these enzymes to enhance nutrient utilization and animal performance. This approach not only tests their compensatory effects but also supports the development of cost-effective and environmentally sustainable feeding strategies for nursery pigs.

Therefore, the objective of this study was to evaluate performance, diarrhea incidence, nutrient digestibility, metabolic organ weights, intestinal morphology, and gene expression of nutrient transporters in nursery piglets fed reduced-nutrient diets supplemented with an enzyme complex containing phytase and xylanase.

2 Methods

2.1 Animal ethics

All procedures in this study were approved by the Animal Ethics Committee of the Federal University of Paraíba (CEUA/UFPB), under protocol number: 1402180423.

2.2 Animals and experimental diets

A total of 40 weaned piglets (male and female) of the commercial line Biriba’s Swine Genetics were used, weaned at 26 days of age. The initial average body weight was 7.85 ± 1.27 kg. The animals were housed in suspended cages with perforated plastic floors, equipped with nipple drinkers and semi-automatic feeders.

The animals were distributed in a randomized block design to control for initial body weight and sex differences, using a 2×2 factorial arrangement, considering diets with or without nutritional and energy reduction, and with or without enzyme complex supplementation. The piglets were assigned to four treatments (experimental diets) with five replicates per treatment, and each experimental unit consisted of two animals (one castrated male and one female).

The nutritional matrix of the enzyme complex composed of phytase (1500 U/kg) and xylanase (350 U/kg) was used to reduce the following nutrients: 0.15% phosphorus, 0.15% calcium, 0.2% crude protein, 0.029% lysine, 0.011% methionine, 0.02% methionine + cysteine, 0.004% tryptophan, 0.014% threonine, 0.022% arginine, and 88 kcal/kg of metabolizable energy.

The experimental diets (Table 1) were formulated to meet the minimum nutritional requirements of the animals according to (Rostagno et al., 2017). The treatments were as follows: Positive Control Diet (DCP), formulated to meet 100% of the nutritional requirements; Positive Control Diet with Enzyme Complex (DCPE); Nutritionally Reduced Diet (DCN), formulated with reductions according to the enzyme complex matrix; and Nutritionally Reduced Diet with Enzyme Complex (DCNE). Diets were formulated for three phases: Phase I – from 26 to 32 days of age; Phase II – from 33 to 42 days of age; and Phase III – from 43 to 60 days of age.

Table 1

Table 1. Ingredients and centesimal and nutritional composition of diets according to experimental treatments.

2.3 Performance and incidence of diarrhea

At the beginning and end of each phase, animals were individually weighed, the feed offered, and the feed leftovers were weighed to determine average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (FCR). Performance data were analyzed for the phases from 26 to 32 days of age, from 26 to 42 days of age, and from 26 to 60 days of age.

To evaluate the effect of the experimental diets on diarrhea incidence, fecal scores were recorded for the piglets from 26 to 60 days of age during the experimental period. Fecal consistency was assessed twice daily, at 08:00 a.m. and 04:00 p.m., through visual inspection using the following scoring system: 1 – normal feces, 2 – soft feces, and 3 – watery feces. The same observer carried out all evaluations. Scores of 1 and 2 were classified as non-diarrheic feces, while score 3 was classified as diarrheic according to Pascoal (Pascoal et al., 2012).

2.4 Dietary digestibility and mineral availability

To evaluate the digestibility of the experimental diets, a partial fecal collection method was used, with feces collected directly from the rectal ampulla of the animals twice daily. An indigestible marker, 1% acid-insoluble ash (Celite ^® 545), was added to the diet from 43 to 60 days of age.

Feed samples were also collected and stored in a freezer at -18°C for subsequent analysis. Feces and feed samples were thawed, homogenized, pre-dried at 55°C for 72 hours and ground for the determination of dry matter (DM), organic matter (OM), ash, crude protein (CP), gross energy (GE), neutral detergent fiber (NDF), and acid detergent fiber (ADF), according to the methods described by (AOAC, 2006). The gross energy of the feces and feed was determined using a bomb calorimeter (Parr 6100 model).

Phosphorus content was determined using a UV-VIS spectrophotometer (model UV-5100, Metash Instruments, Shanghai, China).

For calcium determination, the samples were analyzed using a flame atomic absorption spectrometer (iCE 3500, Thermo Scientific, Cambridge, UK). A hollow cathode lamp containing calcium (Photron, Victoria, Australia) was used as the primary radiation source, and background correction was performed using a deuterium lamp coupled to the equipment. The standard curve was prepared using a calcium standard solution (Specsol, São Paulo, Brazil). Instrument parameters followed the manufacturer’s recommendations, and the data were processed using SOLAAR software (Thermo Scientific, Cambridge, UK).

Based on the analytical results for DM, OM, CP, gross energy, neutral detergent fiber, acid detergent fiber, calcium, and phosphorus, the apparent digestibility coefficients of nutrients and energy were calculated according to Sakomoura (Sakomura and Rostagno, 2016).

$$ATTD(\%)(1-(\frac{AIAconcentrationinthedietXComponentinthefeces}{AIAconcentrationinthedietXComponentinthediet}\left)\right)X100$$

2.5 Relative organ weight and intestinal morphometry

At 60 days of age, the animals were subjected to a six-hour fasting period. Euthanasia was performed following the procedures outlined in Resolution No. 37/2018 of CONCEA and the Euthanasia Guide for Teaching and Research Animals of UNIFESP (2019).

The animals were rendered unconscious by intravenous injection of 9% sodium pentobarbital solution (90 mg/kg of body weight).

Immediately after euthanasia, the abdominal cavity was opened, and the viscera were removed. The liver, spleen, and pancreas were weighed separately, and the relative weights of the organs were calculated in relation to body weight.

For the analysis of the small intestine structure, samples approximately 25 to 35 mm in length were collected from the mid-jejunum. The intestinal segments were fixed in Metacarn solution (containing 60% methanol, 30% chloroform, and 10% acetic acid) for twelve hours under refrigeration. Subsequently, the Metacarn solution was replaced with 70% ethanol for preservation until morphometric analysis.

The samples were kept in 70% ethanol for 24 hours, then rinsed under running water for five minutes. They were dehydrated in a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections were then cut from the paraffin blocks to prepare the histological slides.

The slides were stained with hematoxylin and eosin (H&E) to evaluate the following parameters: villus height (VH), crypt depth (CD), and villus width (VW). From these measurements, the villus height-to-crypt depth ratio (VH/CD), mucosal thickness (MT), and absorptive area (AA) were calculated, following a modified methodology described by (Moreira Filho et al., 2015).

A light microscope (Olympus BX53) equipped with a Zeiss Axion camera and Cellsens Dimension image capture software was used to analyze the histological slides.

2.6 Relative gene expression of TNF-α and jejunal nutrient transporters

Jejunum fragments of approximately 1 cm were collected, rinsed in saline solution (0.9% NaCl), and finely chopped using a scalpel and scissors. The samples were placed into 2 mL microtubes and stored at -80°C until mRNA isolation.

Total mRNA was extracted using the Qiagen RNeasy^® Mini Kit (Cat. No. 74106), and complementary DNA (cDNA) synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following the manufacturers’ protocols.

Primers were designed for the expression analysis of the following genes: tumor necrosis factor-alpha (TNF-α), sodium-glucose cotransporter 1 (SGLT-1), peptide transporter 1 (PEPT-1), mucin 2 (MUC-2), sodium-dependent phosphate transporter type 2 (NaPi-IIb), and the reference genes β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 2).

Table 2

Table 2. Primers used for quantitative real-time PCR (qPCR).

Relative gene expression of mRNA was determined by quantitative real-time polymerase chain reaction (qPCR) using Power SYBR^® Green Master Mix (Thermo Fisher Scientific, Applied Biosystems) and gene-specific primers. After confirming that the primers amplified with an efficiency of approximately 100%, qPCR assays were performed.

The final reaction volume was 20 μL, containing 10 μL of SYBR Green Master Mix, 4 μL of each primer, 0.3 μL of reference dye, 0.7 μL of distilled and deionized water, and 5 μL of cDNA template. The qPCR thermal cycling conditions were as follows: an initial denaturation at 95°C for 3 minutes (1 cycle), followed by 40 cycles of 95°C for 15 seconds and 60°C for 20 seconds, and a final melt curve stage of 95°C for 1 minute, 55°C for 30 seconds, and 95°C for 30 seconds (1 cycle).

Following each run, a melt curve analysis was performed to verify the specificity and purity of the qPCR products. β-actin (ACTB) and GAPDH were used as internal reference genes to normalize cDNA input. Relative gene expression was calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001), with reference genes used for normalization.

2.7 Statistical analysis

The data were evaluated using a 2x2 factorial design, with or without nutritional and energy reduction, and with or without enzymatic complex supplementation. The assumptions of error normality and homogeneity of variances were verified using the Cramer-von Mises and Levene’s tests, respectively. The observed values were subjected to analysis of variance using the GLM (General Linear Models) procedure in the SAS^® statistical software (OnDemand for Academics). Mean comparisons and interactions between factors were performed using Tukey’s test at a 5% probability level.

3 Results

There was no interaction between nutritional reduction and enzymatic supplementation (P > 0.05) for performance. However, in Period II (0 to 17 days), nutritional reduction resulted in higher average daily feed intake (ADFI) (P = 0.018) and higher feed conversion ratio (FCR) (P = 0.024), without affecting average daily gain (ADG). Pigs supplemented with enzymes showed a lower FCR (P = 0.040) Table 3.

Table 3

Table 3. Daily feed intake (DFI), daily weight gain (DWG), feed conversion (FC) of piglets fed diets with and without nutritional reduction and supplementation with enzyme complex (phytase and xylanase).

In Period III (0 to 34 days), nutritional reduction resulted in lower ADFI (P = 0.030) and ADG (P = 0.032), leading to a higher FCR (P = 0.026). Regarding enzymatic supplementation, animals fed diets without enzymes had lower feed intake (P = 0.051) and exhibited lower ADG (P = 0.012) compared to those receiving enzyme-supplemented diets. Likewise, piglets fed enzyme-supplemented diets showed improvements in FCR (P = 0.050) Table 3.

During the first period, animals that received nutritionally reduced diets (P < 0.002) presented lower fecal scores than those on non-reduced diets, indicating a lower incidence of diarrhea. No differences were observed regarding enzymatic supplementation on fecal scores (Figure 1).

Figure 1

Figure 1. Effect of nutritional reduction in diets with or without enzyme complex (Phytase and Xylanase) on the incidence of diarrhea in weaned piglets. The fecal score indicates the tendency to diarrhea: 1 – normal; 2 – pasty; 3 – liquid. DCP- Control diet; DCPE- Control diet with enzyme; DCN- Diet with energy reduction according to the values ​​of the product matrix); DCNE - Diet with energy reduction and Enzyme complex. Capital letters compare diets differ by Tukey’s test at the 5% probability level.

The digestibility coefficients of NDF (P = 0.021) and ADF (P = 0.014) were higher with enzymatic supplementation (Table 4).

Table 4

Table 4. Nutrient and energy digestibility coefficients of diets with and without nutritional reduction and supplementation with complex (phytase and xylanase).

An interaction effect was observed for the relative weights of the liver (P = 0.027) and pancreas (P < 0.0001) (Table 5). Animals that received nutritionally reduced diets supplemented with enzymes had lower relative weights of the liver and pancreas. Spleen weight decreased when animals were fed enzyme-supplemented diets, regardless of nutritional reduction.

Table 5

Table 5. Relative weight of liver, pancreas and spleen of weaned piglets fed diets with reduced nutritional value and with or without addition of enzyme complex (phytase and xylanase).

Nutritional reduction resulted in lower villus height (VH) (P = 0.002) and crypt depth (CD) (P = 0.037). Pigs supplemented with enzymes showed greater crypt depth (P = 0.018) and a lower villus height-to-crypt depth ratio (VH/CD) (P = 0.005) (Table 6).

Table 6

Table 6. Morphology of the jejunal epithelium of weaned piglets fed diets with reduced nutritional value and with or without addition of enzyme complex (phytase and xylanase).

Regarding gene expression, there was an effect of enzymatic supplementation resulting in higher mRNA expression of NaPi-IIb, SGLT-1, and PEPT-1 (Figure 2). The enzyme factor influenced the sodium-dependent phosphate transporter type 2 (NaPi-IIb), which was less expressed in animals consuming diets without enzymes (P = 0.050). A significant interaction was observed for SGLT-1 and PEPT-1 (P = 0.015 and P = 0.018, respectively), both showing higher expression in animals receiving the DCNE diet compared to those fed the DCPE diet.

Figure 2

Figure 2. Effect of nutritional reduction of diets with or without addition of enzyme complex (phytase and xylanase) on gene expression in the jejunum of weaned piglets. Tumor necrosis factor (TNF-α), mucin type 2 (MUC-2), sodium-dependent phosphate transporter type 2 (NaPi-IIb), sodium-glucose cotransporter type 1 (SGLT-1), enterocyte dipeptide and tripeptide transporter (PEPT-1). DCP- Control diet; DCPE- Control diet with enzyme; DCN- Diet with reduced energy according to the values ​​of the product matrix); DCNE - Diet with reduced energy and enzyme complex. ² Capital letters compare Diets (DCP vs DCN and DCPE vs DCNE), lowercase letters compare Enzyme factor (DCP vs DCPE and DCN vs DCNE). Within each main factor, means followed by distinct letters differ from each other by Tukey’s test at the 5% probability level.

4 Discussion

Diets with nutritional reduction, when associated with the inclusion of exogenous enzymes, have been proposed to improve the utilization of ingredients through enhanced digestive efficiency and nutrient absorption. This aids in more cost-effective production and, consequently, reduces environmental issues related to excessive nutrient excretion through feces.

Providing diets with lower nutritional density, especially in energy, requires increased feed intake to meet the animal’s needs (Gonçalves et al., 2015). With the increase in daily feed intake during the second evaluation period, feed conversion worsened, which may be related to the fact that the diet contained fewer nutrients and energy, requiring greater feed consumption by the animals to meet their nutritional needs. These results are consistent with previous studies in which pigs fed diets deficient in nutrients such as amino acids, phosphorus, calcium, and energy showed inferior performance results (Olukosi et al., 2007; Woyengo et al., 2015; Lu et al., 2016; Lee et al., 2019). In this study, it was observed that animals fed diets supplemented with the enzyme complex showed improved feed conversion. This effect may be related to the action of the enzyme complex on antinutritional factors, releasing previously unavailable nutrients and thus increasing their absorption. Our results are consistent with those of Chen. et al (Chen et al., 2023), who evaluated diets with and without nutritional reduction, including versions supplemented with a multienzyme complex (xylanase, beta-glucanase, α-arabinofuranosidase, and phytase), and observed that diets supplemented with the multienzyme complex improved feed intake, weight gain, and feed conversion.

From 26 to 32 days of age, there was a higher incidence of diarrhea in animals fed diets without nutritional reduction. This occurs due to physiological changes that affect intestinal structure and function, reducing nutrient absorption and increasing the availability of substrates for the growth of pathogenic bacteria such as E. coli, as well as elevating osmolarity factors that contribute to diarrhea (Pluske et al., 1996). This condition can compromise gastrointestinal and immune development and growth in piglets (Lallès et al., 2007; Campbell et al., 2013), impairing their performance, damaging microvilli and crypts, and compromising nutrient absorption and intestinal health (Duarte and Kim, 2021).

Studies such as Yue and Qiao (2008), who reduced crude protein from 23.1% to 18.9% with amino acid supplementation, also indicate a lower incidence of post-weaning diarrhea and improved fecal consistency, reinforcing the benefits of this nutritional strategy.

Enzyme supplementation, such as phytase and carbohydrases (endo-β-xylanase, β-glucanase), is a widely used strategy to prevent diarrhea in weaned piglets, although our study did not confirm its efficacy (Brandão Melo et al., 2020). These enzymes can help improve digestibility by increasing nutrient availability and reducing the amount of undigested feed in the post-weaning phase (Nortey et al., 2007).

Exogenous enzyme supplementation can improve nutrient digestibility, being directly related to metabolic processes and consequently to the performance results of piglets (Huang et al., 2021; Moita et al., 2022). Changes in the digestibility of phytic acid or fibrous diet components can release nutrients previously bound to molecules that would otherwise be unavailable for absorption in the intestinal epithelium. Additionally, non-starch polysaccharides (NSPs) can increase digesta viscosity (Selle and Ravindran, 2008; Lei et al., 2013; Passos et al., 2015; Lee et al., 2019), reducing nutrient digestibility in non-ruminant animals.

In this study, we observed that the digestibility coefficients of neutral detergent fiber and acid detergent fiber were higher in diets with enzyme supplementation, highlighting the effect of the enzyme complex in releasing nutrients in the digestive tract and increasing digestibility in pigs (Lei et al., 2013; Lu et al., 2016).

In the present study, when evaluating nutrient digestibility in relation to nutritional reduction, it was observed that the phosphorus digestibility coefficient was lower in diets with nutritional reduction. These results corroborate those found by Huang et al (Huang et al., 2021), who, using diets with nutritional reduction, observed lower phosphorus digestibility. In contrast, Lu H (Lu et al., 2016) obtained different results, where diets with nutritional reduction but with the addition of an enzyme complex containing phytase, xylanase, and β-glucanase showed an average increase of 14% in phosphorus digestibility. The absence of improvement in phosphorus digestibility in our study, despite enzyme supplementation, may be explained by several factors. The dose of phytase used (1500 U/kg) may not have been sufficient to fully compensate for the 0.15% reduction in dietary phosphorus and calcium. Furthermore, the effectiveness of phytase depends on the phytate content of the ingredients, and the corn–soybean meal base of our diets may have provided limited substrate for enzyme action. It is also possible that changes in the Ca:P ratio resulting from the nutritional reduction impaired phosphorus solubility and absorption, thereby reducing the enzyme’s effectiveness. Additionally, although greater expression of the NaPi-IIb phosphate transporter was observed, morphological impairments in the intestinal mucosa (e.g., reduced villus height) may have limited the efficiency of absorption. Taken together, these factors help explain why phosphorus digestibility decreased in reduced diets and why enzyme supplementation did not reverse this effect.

In this study, different percentage values in organ weights were observed among animals that consumed diets meeting nutritional requirements (DCP) and diets with a reduced nutritional matrix supplemented with an enzyme complex (DCNE). A difference of 16.5% was observed in liver relative weight, 35.53% in the pancreas, and 17.13% in the spleen. The addition of the enzyme complex reduced the relative spleen weight in piglets. This effect may be related to improved nutrient digestibility and the reduction of antinutritional factors in the diet, resulting in lower antigenic stimulation and, consequently, a reduced need for systemic immune response. In contrast, the study cited (Jia and Pamer, 2009) reported an increase in spleen weight when xylanase was added to diets containing soybean meal and canola meal. These divergent results may be due to differences in ingredient composition, dietary antinutritional load, enzyme source and activity, or animal physiological status. While in that study xylanase potentially enhanced immune stimulation through changes in fiber fermentation or antigen exposure, in the present experiment the enzyme complex appears to have reduced immune activation, thereby decreasing spleen weight. Therefore, the effect of xylanase on spleen development seems to be context-dependent, varying according to diet composition and the metabolic and immune challenges faced by the animals.

Intestinal morphological characteristics, such as villus height and crypt depth, indicate the absorptive capacity of the intestinal mucosa. Taller villi increase the contact area of enterocytes, improving nutrient absorption (Catalan et al., 2016). Shallower crypts indicate a healthy intestine with lower cell turnover, allowing nutrients to be directed toward productive gain (Lemos et al., 2013). A high villus/crypt ratio suggests more efficient digestion and absorption (Montagne et al., 2003).In our study, it was observed that the diet with nutritional reduction resulted in the worst outcomes regarding epithelial structure. In the jejunum, the negative control diet promoted a smaller villus area (VA), but this was accompanied by a shallower crypt depth (CD).

The structure of the intestinal epithelium is related to performance results since, in the third experimental phase, close to the slaughter period and histological collections, piglets had higher average daily feed intake (ADFI), but lower average daily gain (ADG) and feed conversion ratio (FCR) in diets with nutritional reduction, indicating that nutrients from higher ADFI may have been used for cellular turnover rather than muscle deposition, as suggested by Pluske (Pluske et al., 1996). The results found in our study corroborate those found by Zuo et al (Zuo et al., 2015), who found that a diet without nutritional reduction, compared to a diet with nutritional reduction, contributed to greater VH and a higher VH/CD ratio.

When observing the effect of phytase and xylanase supplementation in the diets, it was possible to verify that, in the jejunal epithelium, enzyme-supplemented diets increased crypt depth (CD) and consequently reduced the VH/CD ratio, a response generally considered less favorable. This differs from previous studies, such as Liu et al (Liu et al., 2023), who reported positive effects on duodenal morphology, with increased villus height (VH) and VH/CD ratio and reduced CD, a result similar to that found by Luise (Luise et al., 2020) with an enzyme complex. Moita, Duarte, and Kim (Moita et al., 2022) also observed that increasing levels of xylanase progressively increased VH. The discrepancies observed in the present study may be related to differences in diet composition, nutrient reduction, enzyme type or dose, and the physiological response of the jejunal mucosa. Furthermore, the degradation of non-starch polysaccharides by xylanase may have stimulated epithelial renewal, resulting in deeper crypts without necessarily impairing absorptive function.

The expression of the intestinal transporters evaluated in this study also helps to explain the observed results in performance and digestibility. NaPi-IIb is a key transporter for phosphate absorption (Xu et al., 2002), and dietary phosphate acts as an important physiological regulator of this process (Quamme, 1985). Vigors et al (Vigors et al., 2014). reported that pigs fed phytase-supplemented diets showed increased NaPi-IIb expression, which is consistent with our findings, where enzyme supplementation increased the expression of this transporter compared with diets without enzymes. This effect may be associated with the release of phosphorus from phytate by phytase, favoring intestinal absorption and contributing to the maintenance of mineral homeostasis.

Regarding carbohydrate and peptide transport, SGLT-1 is the main transporter responsible for glucose and other sugar absorption (Moran et al., 2010). Previous studies have indicated that antinutritional factors such as phytic acid can reduce its expression (Yoon et al., 1983), whereas Clarke et al (Clarke et al., 2018). observed that pigs fed high-quality barley diets supplemented with β-glucanase and β-xylanase showed increased expression of both SGLT-1 and PEPT-1. Consistently, our results demonstrated that enzyme supplementation enhanced the expression of SGLT-1 and PEPT-1, particularly when combined with the reduced-nutrient diet. This finding suggests that the enzymatic release of glucose and small peptides previously bound within plant cell wall structures is directly linked to the positive regulation of these transporters. Moreover, this regulation may be connected to the improvements observed in fiber digestibility and the trend toward better feed conversion in pigs receiving enzymes, indicating that enzyme supplementation not only increases nutrient availability but also enhances the intestinal absorption mechanisms.

The nutritional reduction applied in this study was sufficient to impair the animals’ development as observed. However, supplementation with the enzyme complex reduced some negative impacts, although it was not effective for the level of reduction applied. In this sense, new studies should be carried out combining nutritional reduction and enzyme supplementation, since the need to reduce some nutrients such as protein and phosphorus is essential to mitigate nitrogen and phosphorus excretion, and may also contribute to intestinal health by reducing undigested nutrient content in the gut.

5 Conclusion

It is concluded that the nutritional reduction of the diets impaired the piglets’ performance, and supplementation with an enzymatic complex containing phytase and xylanase activities was insufficient to overcome this challenge. However, supplementation with the enzymatic complex, regardless of the diet, resulted in performance benefits, greater fiber digestibility, and stimulated higher gene expression of nutrient transporters.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Ethics statement

The animal study was approved by Animal Ethics Committee of the Federal University of Paraíba (CEUA/UFPB) under protocol number 1402180423. The study was conducted in accordance with local legislation and institutional requirements.

Author contributions

CM: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. LP: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing, Funding acquisition, Project administration, Supervision. LC: Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, Validation, Visualization, Writing – review & editing. RG: Conceptualization, Formal Analysis, Methodology, Visualization, Writing – review & editing. PW: Project administration, Supervision, Validation, Visualization, Writing – review & editing. PG: Formal Analysis, Methodology, Supervision, Visualization, Writing – review & editing. JA: Data curation, Formal Analysis, Investigation, Methodology, Visualization, Writing – review & editing. WS: Formal Analysis, Investigation, Methodology, Visualization, Writing – review & editing. MS: Data curation, Formal Analysis, Investigation, Methodology, Writing – review & editing. MA: Formal Analysis, Investigation, Writing – review & editing. GR: Formal Analysis, Investigation, Writing – review & editing. PS: Data curation, Formal Analysis, Methodology, Visualization, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. Alltech Agroindustrial Ltd., Maringá, Paraná, Brazil.

Acknowledgments

Gratitude and recognition to the people who contributed to the author’s research and writing, to the Federal University of Paraíba, to the Coordination for the Improvement of Higher Education Personnel (CAPES) and to Alltech do Brasil.

Conflict of interest

Author LC was employed by company Alltech Agroindustrial Ltd.

The author(s) declared that this work received funding from Alltech Agroindustrial Ltd. The funder had the following involvement in the study: participated in the experimental design, funding of the study, manuscript review, and approval of the submission for publication. The company was not involved in data collection, statistical analysis, interpretation of the results, or experimental procedures.

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