Weaning is a critical stage in a pigs’ life that causes morphological and functional changes in the intestine where most of the nutrients are being digested and absorbed (1). Furthermore, various other stressors induce post-weaned growth depression, such as separation from the sow and littler, transition from a liquid to a solid diet, and hierarchy formation. The endogenous secretion of several enzymes and hydrochloric acid is considered insufficient during this phase. Furthermore, microbial dysbiosis at weaning is one of the leading causes of post-weaning diarrhea, which results in significant economic losses. Therefore, most post-weaning diarrhea is caused by Escherichia coli, enterotoxigenic E. coli (ETEC) strains mostly, but also by other pathogens such as enteroadherent strains of E. coli, Salmonella enterica, or rotavirus (2).

Pig diets are typically mainly composed of corn and soybean meal. Despite their high nutritional value, these ingredients contain several anti-nutritional factors that can impair animal metabolism, such as fibers found in cell walls (3). Certain anti-nutritional factors can be reduced through the treatment of these ingredients, but not all are effectively managed by the typical processes employed in the industry. These components (arabinoxylans, xylose, phytate, and mannans) are difficult to manage during weaning when pigs face many other challenges to their gut health (4). Simple diets, which are formulated with a higher inclusion of soybean meal, may be particularly challenging, and for that reason, the use of complex diets is considered necessary during this phase (5). However, complex diets are more expensive because of the greater inclusion of highly digestible feed ingredients such as dried animal plasma, whey, and soy isolates. The literature on the use of simple or complex diets in the post-weaned period is limited and controversial, especially for processed soy protein (5, 6).

Among the various commercially available enzymes, three stand out for their complementary activities in energy utilization and their effect on pig gut health in the post-weaned period. β-mannanases are major mannan-degrading enzymes and are considered energy-saving because they reduce the cost of unnecessary immune response activation (7, 8). Mannans are present, especially in soybeans, and can cause intestinal inflammation, called the feed-induced immune response (FIIR) (7). Furthermore, arabinoxylans and xylose are anti-nutritional factors present in all vegetable ingredients, including corn, wheat bran, and soybean meal, which can reduce nutrient digestibility, increase endogenous losses, and increase digestive viscosity (9). In this context, and by different action mechanisms, the inclusion of β-mannanases, arabinofuranosidases, and xylanases in swine diets may improve energy utilization and gut health (9–11).

However, more information on this matter is important to help nutritionists choose between simple and complex diets because different formulations significantly alter the availability of substrates for enzyme activity and, consequently, can affect the potential benefit obtained from enzyme addition. Both enzyme additions are used with an energy matrix in the feed formulation, which leads some nutritionists to frequently ask questions regarding their potential use in combination. As these additives perform different functions, such as saving energy that would otherwise be spent on the immune response (β-mannanases) or releasing nutritional components for absorption (arabinofuranosidases and xylanases), their effects may be additive. Therefore, this study was conducted to evaluate the impact of adding β-mannanase alone or in combination with a multi-carbohydrase complex, such as xylanase, β-glucanase, and arabinofuranosidases, in simple or complex diets on diet digestibility, nutrient and energy metabolism, as well as gut health in post-weaned pigs.

The current study was performed to evaluate whether adding β-mannanase alone or in combination with arabinofuranosidases, β-glucanase, and xylanases to simple and complex diets could improve diet digestibility, nutrient metabolism on post-weaned pigs’ intestinal health. Although both enzymes are used with an energy matrix in feed formulation, we also hypothesized that these additives could have additive effects, as they perform different functions in the animal, such as saving energy that would otherwise be spent on the immune response (β-mannanases) or releasing nutritional components for absorption (arabinofuranosidases, β-glucanase, and xylanases). Hence, we hypothesized that these enzymes could produce greater effects on the intestinal health of post-weaned pigs. We also hypothesized that a simple diet could replace a complex diet, without compromising energy, nutrient metabolism, and intestinal health.

The addition of BM and BM + MCC improved the dry matter, protein, and energy digestibility coefficients. The effects were greater when BM + MCC was added, suggesting an additive effect between the enzymes. The increase in the nutrient digestibility coefficient may occur because β-mannanase reduces the viscosity of digesta promoted by β-mannans, allowing better contact of the digesta with the gut (21). In agreement, Jeon et al. (22) reported that dietary addition of 0.05% β-mannanase resulted in greater standardized ileal digestibility of amino acids, total tract apparent digestibility, and crude protein for growing pigs. Lu et al. (4) observed that the addition of xylanase to post-weaned pigs improved dry matter and crude energy digestibility. These findings may be associated with xylanase releasing nutrients trapped inside the cell wall of cereal grains, primarily arabinoxylans, which are found in corn’s aleurone and the starchy endosperm. Furthermore, increased digestibility is usually associated with increased energy digestibility (23). Digestible energy, on the other hand, is not a nutrient per se but rather the sum of improved digestibility of starch, fat, protein, and fiber (9). Duarte et al. (11) found that adding 45,000 U/kg xylanase to the diet of post-weaned pigs reduced digesta viscosity in the jejunum. As a result, by reducing viscosity, enzymes’ access to substrates can be increased.

Moreover, enzyme addition increased the protein and energy metabolizability coefficients. On average, BM provides 64 kcal/kg of metabolizable energy, while BM + MCC provides 100 kcal/kg. BM addition showed a very similar metabolizable energy matrix in simple (63 kcal/kg) or complex diets (64 kcal/kg). However, the matrix of metabolizable energy calculated for combined enzymes was greater in the simple diet (120 kcal/kg) than in the complex diet (78 kcal/kg), which is probably related to the greater amount of substrate for the enzyme blend to act on. These results support the hypothesis that these enzymes can have complementary effects, despite having energy matrices.

Complex diets are defined by the higher inclusion of more palatable and digestible ingredients, such as whey, plasma, viscera meal, and soy isolate (24). While simple diets include more soybean meal is the main protein source in swine diets because of its high protein and amino acid content, consistent quality, and availability in the market. However, soybeans contain anti-nutritional factors such as protease inhibitors and mannans, which interfere with the digestion and absorption of nutrients. In this trial, the simple diet showed greater protein digestibility and metabolizability coefficients than the complex diet. It is important to highlight that this was probably a consequence of the ingredient composition. Both diets were formulated considering metabolizable energy and digestible amino acid values, and the crude contents were similar between simple and complex diets. The simple diet had a higher inclusion of soybean oil compared to the complex diet. It is known that the inclusion of lipids improves the digestibility of nutrients because it increases the secretion of cholecystokinin in the intestine, pancreatic juice, and enzymatic activity. This could explain our finding of greater protein digestibility in the simple diet.

Although this is a methodological limitation, no other option was available once different ingredients were used to generate simple and complex diets. As a direct comparison between simple and complex diets is not a direct objective of the trial, these results need to be interpreted with caution. Apart from the formulation procedure, this difference may be also related to the faster passage rate of the complex diet as the feed retention rate increases (25, 26). Ratanpaul et al. (27) reported that feed intake decreases as the feed retention rate increases. This is consistent with the findings of the present study. Furthermore, a higher retention rate allows endogenous enzymes to act for a longer period in the digesta (27). Other possible mechanisms that could explain our findings include gastric distension and feedback effects on the hypothalamus, which reduce the gastric emptying rate (28).

Zhao et al. (29) reported that soluble dietary fiber has a high ability to bind water and thereby increase viscosity, thus increasing fecal mass and moisture content. One possible explanation could be that the addition of BM and BM + MCC reduced the amount of non-starch polysaccharides in the digesta, resulting in less fiber available to bind with water, thus explaining the lower moisture in the feces of the pigs that were fed the exogenous enzymes. Even after adjusting fecal production to a dry matter basis, the addition of BM + MCC reduced fecal production by 17%. This is possibly due to the feed retention rate and better digestibility coefficients, as previously mentioned, and is a positive result when considering the potential mitigation of the environmental impact of pig production.

A trend toward lower haptoglobin concentrations was found when BM and BM + MCC treatments were compared to the control. Haptoglobin is a major acute-phase protein in pigs and an increase in its plasma concentration is an indicator of systemic inflammation (30). Huntley et al. (31) observed a decrease in serum haptoglobin concentration after administering β-mannanase to post-weaned pigs, indicating that β-mannanase reduced the immune response caused by β-mannans. Furthermore, McAfee et al. (32) reported that it is related to the control of inflammatory responses.

Biomarkers are useful for identifying various gastrointestinal conditions, particularly non-invasive biomarkers that can avoid animal euthanasia. Calprotectin is a cytosolic protein composed of S100A8 and S100A9 (33). This protein binds to calcium and zinc and is found in neutrophil granulocytes (34). Calprotectin is a useful biomarker because when neutrophils congregate at sites of inflammation, they produce more calprotectin (35). Calprotectin is already used in human health, but it is thought to be a promising biomarker of pig intestinal inflammation (36). The literature on the effects of additives on pig calprotectin concentrations still very limited. Xiao et al. (37) reduced calprotectin concentrations by adding chitosan to post-weaning pig diets. To the best of our knowledge, the present study is the first to test calprotectin in post-weaned pigs in the context of enzyme addition. The addition of both enzymes greatly reduced calprotectin concentrations. Thus, it is possible to infer that enzyme addition reduced inflammation caused by dietary anti-nutritional factors such as β-mannans, arabinoxylans, xylose, trypsin, and others.

Gut morphology is also used as an indicator of gut health (30). Increased villus height is usually interpreted as a positive trait of gut morphology and is also used as an indicator of gut health (30). In the current study, both responses (villus height and villus area) improved in pigs fed a complex diet. Some physical properties of a simple feed (e.g., more abrasive) may be important to understand this difference. The enzymatic addition of BM + MCC also improved the same responses, which correlated with an improved nutrient digestibility coefficient.

Simple diets are a suitable alternative for the nursery phase if well-formulated. The use of simple feeds is a way to further explore the potential of BM + MCC enzyme addition since its effects were more pronounced in simple diets for some responses, such as metabolizable energy. The effects of the addition of both enzymes can be greater when working with the enzyme combination since both have an energy matrix. It was observed that both enzymes collaborated to the lower inflammation status assessed in this study through the fecal calprotectin concentrations. To the best of our knowledge, no studies are available focusing on dietary complexity and correlating this promising biomarker for gut health with animal performance. Exploring growth performance was not feasible in our trial due to the limitation imposed by housing conditions (metabolic crates), but it would be highly significant for future research. Additionally, our trial focused on pigs that were weaned at 28 days. It is advisable to interpret these results cautiously when considering scenarios with younger weaning ages.

Feeding strategies using simple diets reduce feed costs in the swine industry because of the reduced inclusion of expensive special ingredients (i.e., spray-dried spray, whey powder, and protein isolates). Furthermore, the addition of exogenous enzymes reduced the amount of soybean oil in the feed formulas. This reduction could potentially mitigate environmental impacts, as soybean oil is generally associated with a high potential for global warming. The environmental and economic implications of simplifying the diets are highly dependent on the scenario in which the modification is performed; however, this feeding strategy could be explored by swine nutritionists as a viable alternative for nursery pigs.

The simple diet had greater protein digestibility and metabolizability coefficients than a complex diet. A complex diet had greater villus height and villus area than a simple diet. Both enzyme additions increased energy digestibility, with BM addition releasing 64 kcal and BM + MCC releasing 100 kcal of metabolizable energy per kg of feed. The addition of BM and BM + MCC also reduced the calprotectin and haptoglobin concentrations and improved the absorption area.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal studies were approved by The experimental protocol was approved by the institutional ethics committee on the use of animals (CEUA/UFRGS – Protocol 39,604). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

GG: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. IA: Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Software, Supervision, Validation, Visualization, Writing – review & editing. CC: Data curation, Formal analysis, Methodology, Writing – review & editing. TS: Data curation, Formal analysis, Methodology, Writing – review & editing. MM: Data curation, Formal analysis, Methodology, Writing – review & editing. RM: Formal analysis, Methodology, Writing – review & editing. VH: Formal analysis, Methodology, Writing – review & editing. MK: Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing – review & editing.