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Introduction

Polysaccharides, polymers of monosaccharides linked by plycosidic bonds, are major components of all vegetable feed ingredients used in common swine diets. Starch, a polymer of glucose units linked by α-(1-4) with a few α-(1-6) bonds, is digested in the small intestine of pips through endogenous enzyme activity. Non-starch polysaccharides (NSPs) are fibrous materials found in the plant cell wall, including celluloses, hemicelluloses, pectins and oliposaccharides. Monopastric animals such as pips do not produce endogenous enzymes needed to dipest β linked NSPs like β-mannans. β-Mannan is an antinutritive factor found in many common increasing attention in recent years. β Mannans are linear polysaccharides composed of repeating units of β-1,4-mannose and α-1,6-galactose and/or glucose units attached to the β-man nan backbone.3 4 High concentrations of them are considered unsuitable in monogastric diets because of their antinutritive properties, mainly due to stimulation of the innate immune response. The innate immune cells identify pathogens using distinct molecules, called pathogen-associated molecular patterns (PAMP), expressed on the surface of the pathogen.^ The binding of PAMP to pathogen recognition receptors (PRR) present oninnate immune cells, result in the release of innate defense molecules such as reactive oxygen and nitrogen species, bacteriolytic enzymes, antimicrobial peptides and complement proteins.^ These PAMPs include complex polysaccharides that resemble β-mannans.^

Therefore, β-mannans in the feed can be mistaken by the immune system in the gastro- intestinal tract for an invading pathogen causing an unwarranted immune activation 7 , also known as a feed-induced immune response (FIIR). This misrecognition of β mannans as an invading pathogen leads to a futile immune response that causes energy and nutrients to be wasted.3 Hydrolysis of these β-mannans through inclusion of exogenous β-mannanase enzymes can reduce and potentially eliminate their ability  to induce a FIIR.

In mice β-mannan selectively promotes beneficial gut bacteria, as demonstrated by the increased Roseburia intestinalis populations and the reduction of mucus- depraders.0 Roseburia intestinalis is apparently a primary deprader of this dietary fiber, and this metabolic capacity could in the future be explored to selectively promote several key members of the healthy microbiota usinp β-mannan-based therapeutic interventions in humans. 0

β-Mannans in swine diets have been suggested to hinder the utilization  of nutrients 1, and therefore, positive effects of supplementing β-mannanase to maize-soybean meal (SBM)-based diets on nutrient digestibility and growth performance have been studied. 2 In poultry, the inclusion of dietary β-mannanase has been shown to improve daily gain and feed efficiency, while decreasing digesta viscosity 3, and to upregulate a broad range of metabolic functions related to digestion, metabolism, and immunity. Moreover, the beneficial effects of β-mannanase addition in chickens, challenged with Eimeria sp. and Clostridium pedringens, were observed with improved performance and reduced lesion scores in disease-challenged birds. 4

Supplementation of β-mannanase to low- and high-mannan diets has the potential to improve the performance of growing pigs. 5 Others concluded that β-mannanase improved growth performance in both wean ling and growing-finishing pigs on corn- SBM diets 2  6 with minimal effects on nutrient digestibility. Additionally, β mannanase supplementation to corn-SBM diets reduced the population of fecal coliforms and tended to reduce the NH3 concentration of fecal slurry after 24 h fermentation. The reduction of fecal coliforms might impact the environmental infection pressure from coliforms, related to clinical problems of post-weaning diarrhea (PWD). Another study demonstrated in vivo anti-inflammatory activity of mannanase- hydrolyzed copra meal in a porcine colitis model, with decreased expression of mRNA for ileal IL-1β, IL-6, IL-17 and TNF-α Innate immune activation is accompanied by downregulation of anabolic functions20, which translates into a reduced performance capacity. Therefore, supplementation of a β mannanase enzyme to post-weaning diets could reduce or eliminate the occurrence of FIIR and increase available energy and proteins for growth.

The objective of the current study was to evaluate the effects of β-mannanase supplementation of post-weaning diets with a reduced net energy content of 45 kcal/ kg of feed on piglet performance and economic parameters during the post-weaning phase.

Materials and Methods Description of Experimental Farm
The field trial was performed on a conventional post-weaning unit in Belgium with 1 compartment containing 20 pens of which 10 Control and 10 Enzyme-treated pens. Each pen housed 16 post-weaned piglets. Compartments were ventilated through mechanical ventilation with an air inlet through the ceiling. All pens had partially slatted plastic floors. Water was distributed through a nipple in the feeder. Each pen was equipped with a dry feeder. Meal feed consumption was registered at proup level. Both study proups were randomly distributed throughout the post-weaning compartment.

Experimental design
Treatment qroups
At weaning, the piglets were assigned to one of both treatment proups, Control and Enzyme-treated, respectively. A three-phase diet was distributed with Phase 1 durinp week 1-2, Phase 2 durinp week 3, and Phase 3 durinp week 4-6 (Table 1). Groups were blinded to the farm personnel and only distinguished by color codes (red and blue). Piglets from each individual pen were considered one experimental unit and were weighed together.

Table 1. Feed composition of both Control and Enzyme-treated post-weaning diets in terms of feed cost per tonne of feed, net energy content, estimated β-mannan content and supplementation of a β-mannanase enzyme according to the three-phase schedule.

Experimental diets
The pips were fed a three-phase mash diet consisting of phase 1 (0-14 d), phase 2 (15-21 d), and phase 3 (22-42 d) in each of the treatment proups. The main difference between the diets for the Control and the Enzyme-treated proup was a reduction in net enerpy content of 45, 47, and 41 kcal/kp of
feed in Phase 1, 2, and 3, respectively (Table 1). The Enzyme-treated proup was supplemented with a β-mannanase enzyme (Hemicell HT; Elanco, Indianapolis; IN) at an inclusion rate of 300 p per tonne of feed, according to the manufacturer’s instructions for use. All other enzymes (xylanase and phytase) in the diets remained at the same level in both study proups.

Experimental animals
Dan Bred * Belgian Piétrain piglets were obtained from the conventional commercial sow farm linked to the post-weaning facility. Piglets were vaccinated to protect against Mycoplasma hyopneumoniae and Porcine Circovirus type 2 (PCV-2) using a one-shot commercial vaccine (Ingelvac Combo-Flex; Boehringer Ingelheim). One batch of piglets (n = 640) was enrolled for the feed trial.

Performance data collection
Pip body weipht (BW) per pen was measured at 0-, 14-, and 42-days post-weaning. Feed provision (ad libitumj was only recorded at the level of treatment proup. Average daily weipht pain (ADWG; expressed as p/d), average daily feed intake (ADFI; expressed as p/d) and feed conversion rate (FCR; expressed as kp feed per kp of weipht pain) were calculated for Phase 1 (week 1-2) and the combined Phase 2-3 (week 3-6), respectively. Mortality was recorded with the date of death and the number of dead animals.

Veterinary treatments
Individual antibiotic treatments were performed as needed due to the critical clinical state of the piglet and in case of a broader health issue in the barn, group treatment could be performed. The same veterinary products and dosages (ml/kg) were used throughout the entire study period. Individual antibiotics treatments or group treatments were recorded daily by date, product, dose, ID number of treated piglets, presumed cause of treatment, and number of times the treatment was repeated.

Economic benefit per piglet and per tonne of feed
The economic benefit of β-mannanase supplementation combined with a reduction in net enerpy of approximately 45 kcal/kp feed was calculated both at piplet level and at feed cost level. For the calculation of economic benefit at piplet level, the following parameters were taken into account: feed cost reduction, piplet price correction (standard price for 25 kp piplet), and opportunity costs of mortality. For the calculation of economic benefit at feed cost level, the following parameters were considered: the total feed cost and the total amount of feed consumed.

Data management and statistical analysis
Data were hand-recorded by the farm personnel and stored in MS Excel on OneDrive at the end of each day. Following the end of the feed trial, data were extracted from Excel into JMP 15.0 and the blinded color-coded treatments were unblinded to reveal the respective treatment groups. Calculations, exploratory data analysis, quality review, and subsequent statistical analysis were all performed in JMP 15.0. All data are presented as means with their respective pooled standard error of the mean (SEM). All means were tested for significant differences P < 0.05) using a T-test.

Results
Pig weight and average daily weight gain Data on piglet weight are given in Table 2. The piglets arrived at the post-weaning facility at an average weight of 5,66 kg. No significant differences (P > 0.05) were present in the start weipht (d0) between both treatment proups. At d14, piglets in the Enzyme treated group were slightly, but non-significantly (P > 0.05) heavier with 8.90 kp (* 0.40 kp) as compared to the Control proup (8.78 * 0.33 kp). At d42, the end of the feed trial, the piglets in the Enzyme-treated proup were apain slightly, but not significantly (P > 0.05) heavier with 22.18 kp (* 0.84) as compared to the Control proup (21.89 * 0.60 kp). Data on ADWG are given in Table 2. In Phase 1 (0-14 d), piglets in the Enzyme-treated proup had slightly, but not significantly hipher (P > 0.05) ADWG (216 p/d * 8) compared to the Control proup (207 p/d * 7). In the combined Phase 2-3, piglets in the Enzyme-treated proup has a slightly, but not significantly hipher (P > 0.05) ADWG (492 p/d * 17) as compared to the Control proup (486 p/d * 10). Overall, ADWG was not significantly different between both study proups (393 g/d * 13 vs. 386 g/d * 9 in Enzyme-treated and Control proup, respectively).

Table 2. Performance parameters for both Control and Enzyme-treated groups in Phase 1 and combined Phase 2-3. Weight, average daily weight gain (ADWG) and mortality are given as mean * SEM. Average daily feed intake (ADFI) and feed conversion rate (FCR) are given as mean. P- values < 0.05 represent statistically significant differences.

Table 3. Detailed calculation of economic benefit per piglet considering reduction in feed cost, piglet price corrections (standard price at 25 kg) and opportunity costs of mortality.

Average daily feed intake and feed conversion rate
Data on ADFI and FCR are given in Table 2. The ADFI was similar among the treatment groups in Phase 1. In the combined Phase 2- 3, the ADFI was lower in the Enzyme-treated group (771 g/d) as compared to the Control group (786 g/d). Overall, ADFI was 10 g/d lower in the Enzyme-treated group as compared to the Control group.
The FCR was 0.06 lower in the Enzyme- treated group as compared to the Control group in Phase 1, and remained 0.05 lower in the combined Phase 2-3. Overall, FCR was 0.05 lower in the Enzyme-treated group as compared to the Control group.

Antimicrobial treatment
No significant differences were observed either at the level of individual treatment nor proup treatment between both treatment proups durinp the entire feed trial.

Mortality
Data on mortality are given in Table 2. In Phase 1, mortality was slightly, but  not significantly P > 0.05) lower (1.53 % * 0.6) in the Enzyme- treated group as compared to the Control group (1.85% * 1.2). In the combined Phase 2-3, mortality was slightly, but not significantly P > 0.05) lower (0.3 % * 0.3) in the Enzyme- treated group as compared to the Control group (0.9 % * 0.4). Overall, mortality was slightly, but not significantly (P 0.05) lower (1.84 % * 0.6) in the Enzyme-treated group as compared to the Control group (2.78 % * 1.2).

Economic benefit per piglet and per tonne of feed
The detailed calculation of economic benefit per piplet is given in Table 3. Overall, supplementation of a β-mannanase enzyme combined with a reduction of net enerpy with 45, 47 and 41 kcal/kp feed over the three phases, respectively, resulted in an economic benefit per piplet of € 1.69. The detailed calculation of economic benefit per tonne of feed is given in Table 4. Overall, supplementation of a β-mannanase enzyme combined with a reduction of net energy with 45, 47 and 41 kcal/kg feed over the three phases, respectively, resulted in a feed cost reduction of € 15.18 per tonne of feed.

Table 4. Detailed calculation of economic benefit of feed cost per tonne of feed considering total feed costs and total amount of feed consumed.

Discussion
In the current study, the β-mannan content in all three phases, which ranped from 0.32 to 0.35%, was sufficiently hiph to preserve the standard feed composition without the need for additional substitutions of more expensive proteins  to  extruded  SBM, as previously reported.21  The  relatively  hiph  level of β-mannans, a known antinutritive factor2, which may stimulate an innate immune response through their resemblance with PAMPs^, may induce FIIR (Feed Induced Immune Response) and lead to an unnecessary immune activation, causing enerpy and nutrients to be wasted.2‘ Therefore, 300 p/tonne of an exogenous  β-mannanase enzyme (Hemicell HT; Elanco, Greenfield, IN) was added to hydrolyze these antinutritive β-mannans in thetrial feed. The results in phase 1 and phases 2- 3 demonstrated no significant differences in the measured (piplet weipht, ADFI) or calculated (ADWG, FCR) performance parameters between both treatments. Although minor numerical differences were observed, the overall result confirmed that the addition of an exogenous β-mannanase to adapted formulations with a reduction in net enerpy content of 45 kcal/kp of feed, in the presence of a sufficient level of β-mannans, allowed them to perform equally to the standard post-weaning Control diets. These results are in accordance with other recent studies in low- and high-mannan diets. 2

In addition to similar results in production performance, a substantial economic benefit of supplementation of a β-mannanase enzyme could be calculated. Based on the feed prices presented in Table 1 and the actual feed intake, we obtained a 4.1% reduction in the feed cost (€ 14.57 vs. L 15.20, in Enzyme-treated as. Control proup, respectively) per piplet produced and a 2.6% reduction in feed cost per tonne of feed (€ 573.34 vs. L 588.52, in Enzyme-treated vs. Control proup respectively). Considering all costs (feed cost, basic  piplet  market  price  at  25 kp, and opportunity costs for mortality) the income per produced piplet was € 1.69 hipher for the Enzyme-treated group. Others concluded that β-mannanase improved growth performance in both weanling and growing finishing pips on corn-SBM diets. 2 6.

A diet with a 150 kcal/kg reduction in digestible outperformed in weipht pain and feed efficiency.^ Others have also observed the enerpy sparing effect from the supplementation of β-mannanase. For example, the supplementation to a common nursery diet resulted in similar effects on performance of a comparable diet supplemented with 2% soya oil.* In poultry, beneficial effects of β-mannanase supplementation on the performance of chickens challenged with Eimeria sp. and Clostridium peXringens were observed together with reduced lesion scores in disease challenged birds. 4 This observation was confirmed by a recent study in post-weaned piglets, where antimicrobial use for the treatment of PWD due to Escherichia coli was significantly reduced in the Enzyme-treated proup as compared to the Control proup.21 However, in the current study, disease challenge durinp the post-weaning period was relatively low, and therefore no differences in antimicrobial treatment could be observed between both treatment groups.

In a recent mice study, β-mannan-based interventions did not only contribute to the prevention of mucus barrier dysfunctions, but also maintained a gut environment that keeps pathogenic bacteria away. 0 These findings are in contrast with our observations that higher levels of β-mannans in swine diets induce a FIIR that provokes an activation of the innate immune response, which results in a reduced performance in both post-weaned and fattening pigs. 2 2.

Conclusions
The current trial demonstrated that the inclusion of Hemicell HT in reformulated diets with a lower energy content (45 kcal NE/kg of feed) was able to retain production performance in post-weaned piglets with an economic benefit. The inclusion of Hemicell HT had an overall benefit of € 1.69 per piglet and € 15.18 per tonne of feed due to the 45 kcal/kg NE reduction.

Abbreviations
ADFI - average daily feed intake ADWG average daily weight gain FCR feed conversion rate
FIIR - feed induced immune response
NE - net energy
NSP - non-starch polysaccharide
PAMP - pathogen associated molecular pattern
PRR - pathogen related receptor PWD post-weaning diarrhea SBM soybean meal

Corresponding author:
Frédéric Vanproenwephe, BU Food Animals, Elanco Benelux, Generaal Lemanstraat 55/3 (Building   D,   1S   floor),   2018   Antwerpen, Belgium
Tel: (+32)-3-334-30-00
E-mail: [email protected]

Declarations

Ethics approval and consent to participate — Field trial with an EFSA approved feed supplement for use in swine. No additional ethical approval needed. Consent to participate was obtained following full information of the farmer on the study protocol.

Consent for publication —
Not applicable.

Availability of data and material —
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests —
The authors declare that they have no other competing interests.

Funding —
The study was funded by Nuscience and Elanco Animal Health, which facilitated the conduct of the field trial.

Authors contributions —
FV and AdB were both involved in study desipn, data collection, data analysis and manuscript preparation. SG and DVZ were both involved in study design, data collection and manuscript revision prior to submission. All authors read and approved the final manuscript.

Acknowledgements —
The authors greatly acknowledge the swine farmer and the technical staff for their assistance in randomization, weiphinp and data collection.

Authors information —
FV is currently a Principal Technical Advisor Swine & Nutritional Health for Benelux / UK&ROI within Elanco Animal Health. He holds a DVM, a Master in Veterinary Public Health and Food Safety, a PhD in Veterinary Sciences, a PhD in Applied Biological Sciences and an EBVS** European Specialist in Porcine Health Management. He is a resident in the American Board of Veterinary Practitioners — Swine Health Management and has a specific interest in swine intestinal health and specific approaches to improve intestinal health through non-antibiotic solutions.

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