Animal Nutrition 6 (2020) 457e466 Contents lists available at ScienceDirect Animal Nutrition journal homepage: http://www.keaipublishing.com/en/journals/aninu/ Original Research Article Dietary xylanase and live yeast supplementation influence intestinal bacterial populations and growth performance of piglets fed a sorghum-based diet Gemma Gonza lez-Ortiz a, *, Marco A. Callegari b, Pete Wilcock a, Diego Melo-Duran c, Michael R. Bedford a, Hilario R.V. Oliveira d, Marcos A.A. da Silva d, Carlos R. Pierozan e, e Caio A. da Silva a AB Vista, Marlborough, Wiltshire, SN8 4AN, United Kingdom b AKEI Animal Research, Fartura, 18870 000, Brazil c Servei de Nutricio i Benestar Animal (SNiBA), Department de Ciencia Animal i dels Aliments, Universitat Autonoma de Barcelona (UAB), 08193, Spain d Universidade Estadual Norte do Parana , Bandeirantes, 86360 000, Brazil e Universidade Estadual de Londrina, Londrina, 86051 990, Brazil a r t i c l e i n f o a b s t r a c t Article history: This study was to evaluate the effect of xylanase supplementation and the addition of live yeast, Received 24 July 2019 Saccharomyces cerevisiae, on growth performance and intestinal microbiota in piglets. One hundred and Received in revised form eighty commercial crossbred 23-d-old piglets (PIC 417) were sorted by initial BW and allocated to 3 12 March 2020 treatments: control (CTR) diet, CTR diet supplemented with xylanase at 16,000 birch xylan units/kg (XYL) Accepted 17 May 2020 and XYL diet supplemented with live yeast (2  1010 CFU/g) at 1 kg/t (XYL þ LY). Each treatment had 10 Available online 5 September 2020 replicates, with 6 animals each. A sorghum-based diet and water were available ad libitum for 42 d of the study. Average daily gain (ADG) and average daily feed intake (ADFI) were measured from 0 to 42 d (23- Keywords: Sorghum to 65-d-old) and feed conversion ratio (FCR) calculated. At the end of the study, bacterial identification Nursery piglet through 16S rRNA (V3 to V4) sequencing of the ileal and caecal digesta from one piglet per replicate was Xylanase performed. No treatment effects were observed on ADFI. Pigs offered the live yeast in addition to the Live yeast xylanase had increased ADG compared with those supplemented with xylanase alone (XYL þ LY vs. XYL; Growth performance P ¼ 0.655). FCR was improved with XYL and XYL þ LY compared with CTR (P ¼ 0.018). Clostridiaceae Microbiota counts in the ileum tended to reduce by 10% with XYL and 14% with XYL þ LY compared to CTR (P ¼ 0.07). XYL and XYL þ LY increased the counts of Lactobacillaceae in the caecum compared with CTR (P < 0.0001). Dietary supplementation of live yeast combined with xylanase improved growth perfor- mance and microbial balance of piglets during the nursery phase. © 2020, Chinese Association of Animal Science and Veterinary Medicine. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Prior to 2006 the common practise worldwide in animal pro- duction was to include antibiotics in feedstuffs at sub-therapeutic levels as growth promoters. As of Jan. 1, 2006, the European * Corresponding author. Union (EU) was the first trading block to ban the use of antibiotic E-mail address: gemma.gonzalez@abvista.com (G. Gonza lez-Ortiz). Peer review under responsibility of Chinese Association of Animal Science and growth promoters, due to the potential link of antimicrobial Veterinary Medicine. resistance transference from animals to humans (Smith et al., 2002). Since that ban, there is a focus on looking for nutritional solutions to mitigating certain common illnesses that increased in the absence of antibiotic use as well as the use of different feed Production and Hosting by Elsevier on behalf of KeAi ingredients. https://doi.org/10.1016/j.aninu.2020.05.005 2405-6545/© 2020, Chinese Association of Animal Science and Veterinary Medicine. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 458 lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza Enzymes and probiotics have been proposed as antibiotic al- 2.2. Experimental diets ternatives in livestock (Gaggia et al., 2010; Thacker, 2013). Enzyme supplementation aims to decrease the resistant of feed components Sorghum, soybean meal, and soya oil were used as primary in- to endogenous enzymes and improve nutrient digestibility and gredients to formulate the experimental diets that met or exceeded animal performance. There are several exogenous enzyme types nutrient recommendations for weaned piglets fed in 2 phases: pre- available in the market, but with arabinoxylan making up the starter, from 23 to 44 d of age; and starter, from 44 to 65 d of age highest percentage of non-starch polysaccharide (NSP) in a typical (NRC, 2012). The composition of the experimental diets is shown in pig or poultry diet (Masey-O'Neill et al., 2014). Xylanase is the most Table 1. For each phase, one basal diet was made, then split equally important NSP degrading enzyme. The hydrolysis of soluble ara- into 3 batches, each of which were supplemented with or without binoxylan to reduce digesta viscosity (Choct et al., 2004) coupled different experimental products: 1) control (CTR) diet, without any with abrasion of feedstuff cell walls (Bedford, 2018) and the sub- added supplement; 2) xylanase diet, with xylanase (Econase XT sequent release of nutrients entrapped (Bedford, 2002) are the 25P, AB Vista, Marlborough, UK; 160,000 birch xylan unit/g) at main suggested mechanisms of xylanase activity. Moreover, the 100 g/t (XYL); and 3) xylanase diet supplemented additionally with release of xylo-oligosaccharides (XOS) as a result of xylan degra- live yeast (S. cerevisiae strain 1242; Vistacell, AB Vista, Marlborough, dation in the distal sections of the gastrointestinal tract (GIT) may UK; 2  1010 CFU/g) at 1 kg/t (XYL þ LY) resulting in 3 experimental additionally play an important role, especially in non-viscous diets treatments, each having 10 replicates and 6 pigs per pen. Feed providing a substrate for fermentation by the intestinal microbiota additives were added on top. All diets were supplemented with (De Maesschalck et al., 2015). phytase (Quantum Blue, AB Vista, Marlborough, UK; 10,000 phytase Probiotics are live cultures that, when administered to humans units [FTU]/g) at 500 FTU/kg. Experimental feeds were presented in or animals in adequate amounts, equilibrate the intestinal micro- a mash form and did not contain any antibiotic. ZnO and CuSO4 biota and benefit the host (Fuller, 1989). The most widely used were included at therapeutic doses (Zn at 1,500 mg/kg and Cu at probiotics are lactobacilli and bifidobacteria, but live yeast Saccha- 150 mg/kg) in the pre-starter diet only. romyces cerevisiae var. boulardii has long been known effective for treating gastroenteritis (Hatoum et al., 2012). The modification of the intestinal environment is the result of several mechanisms such as the synthesis of inhibitor compounds, increase of mucins secretion, adherence-site competition against pathogens and for Table 1 Ingredients and calculated composition of the experimental diets (as-fed, %). nutritional sources, bacterial toxin inhibition and improvement of nutrients absorption (Strompfova et al., 2004). All these mecha- Item Pre-Starter Starter nisms and others such as oxygen scavenging (Julien et al., 2015) and Ingredients pathogenic binding (Jiang et al., 2015; Kiros et al., 2018) are Sorghum 52.51 67.76 important and potentially play a role in live yeast functionality. Soybean meal 22.11 26.14 Soya oil 3.26 2.95 The hypothesis of the study was that the positive effects of Fish meal 5.00 0.00 xylanase on piglet performance through the in situ production of Whey (5.5% DM) 15.00 0.00 oligosaccharides in the intestine can be further improved with live Salt 0.05 0.50 yeast supplementation in low viscosity diets through modification Limestone 0.27 0.39 of the GIT environment and therefore the microbiota composition. Dicalcium phosphate (18% P) 0.54 0.86 L-Try 0.01 0.01 The objective of the study was to evaluate the effect of xylanase Lys$HCl 0.41 0.52 supplementation and the addition of live yeast (S. cerevisiae, strain Met 0.16 0.16 1242) on the performance and intestinal microbiota in piglets fed a Thr 0.17 0.21 sorghum-based diet. Vitamin and mineral premix1 0.50 0.50 Phytase2 0.005 0.005 Calculated nutrient composition CP 21.72 20.12 2. Materials and methods DM 87.57 87.50 Ca 0.80 0.70 The study was carried out following the recommendations from P 0.74 0.68 Fat 5.64 5.38 the Guide for the Care and Use of Laboratory Animals of the Na- Fibre 1.90 2.34 tional Animal Experimentation Control Council of Brazil (CEUA). Dig. Met þ Cys 0.75 0.68 The trial was approved by the Ethics Committee of Animal Exper- Dig. Lys 1.36 1.24 iments of Akei Animal Research (protocol number: 001.2017). Dig. His 0.48 0.46 Dig. Try 0.24 0.22 Dig. Thr 0.88 0.81 Dig. Arg 1.13 1.09 2.1. Animals and housing Dig. Iso 0.81 0.73 Dig. Leu 1.72 1.67 A total of 180 commercial crossbred 23-d-old piglets (PIC 417), Dig. Val 0.88 0.80 Dig. Gly 0.76 0.65 50% males and 50% females, were purchased from a local farm Dig. Ser 0.84 0.80 ~o Paulo, Brazil). Upon arrival, piglets (weaned at 23 d of (Taguaí, Sa Dig. Phe þ Tyr 1.53 1.48 age; BW was 6.84 ± 0.195 kg) were placed immediately in 30 pens Available P 0.45 0.37 in an environmentally controlled room, with 6 piglets per pen. Metabolizable energy, MJ/kg 14.23 14.02 Animals were distributed into 5 blocks by initial BW. Within each Dig. ¼ digestible. 1 block, pigs were distributed in pens for a balanced BW distribution. One kilogram of feed contains: 30,000 IU vitamin A; 7,500 IU vitamin D3; 75 mg Each pen was 1.7 m length and 1.5 m width (total area 2.55 m2) and vitamin E; 7.50 mg vitamin K3; 6.75 mg vitamin B1; 20 mg vitamin B2; 10 mg vitamin B6; 100 mg vitamin B12; 0.10 mg niacin; 47 mg pantothenic acid; 3 mg folic was equipped with a nipple water drinker, a lidded 20-cm hopper acid; 0.40 biotin; 1.50 mg selenium; 0.50 mg iron; 0.05 mg copper; 0.20 g manga- per piglet. Test diets and water were provided ad libitum nese; 5 mg cobalt. 2 throughout the trial. Quantum Blue 10G, AB Vista, Marlborough, UK; 10,000 phytase units (FTU)/g. lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza 459 2.3. Experimental procedures equal to 20 (1%), the read was considered in downstream analysis. To increase the reliability of the read, excluding possible diversity Piglets were weighted individually on d 0, 21, and 42 of the trial generated by chimeric amplicons or erroneous nucleotide incor- (23, 44, and 65 d of age respectively), to measure mean BW and porated in PCR, 100% identical reads were clustered (Vieira et al., calculate average daily gain (ADG) for each period and cumula- 2019). If any cluster was represented by fewer than 5 reads, it tively. Feed consumption was determined by pen, and the mortality was not considered in further analysis. In the pipeline, each cluster was checked twice daily, and the weights of dead piglets were used gets a unique identification stablished as the operational taxonomic to adjust the feed conversion ratio (FCR). On 65 d of age, 30 piglets unit (OTU). Clustered sequences, i.e., OTU, were then subjected to (one per pen) were randomly selected and desensitised by using 2 taxonomic classification and compared using a 16S rRNA database electrodes at 350 V and 1.3 A during approximate 3 s (Petrovina O  IS (NeoRefdb, Neoprospecta Microbiome Technologies, Brazil). Se- 2000), then the piglets were exsanguinated by severing the jugular quences with at least 99% of identity in the reference database were vein in the neck. The total GIT was removed immediately from the taxonomically assigned and the samples were then evaluated based abdominal cavity. The digesta content from the ileum and caecum on their microbiome composition, focussing on bacterial profiles was immediately collected on an individual basis by gently and their quantity amounts. squeezing each section into a tube, rapidly frozen in dry ice and then stored at 80  C for subsequent analysis of microbiota profile. 2.5. Statistical analyses The faecal quality was evaluated twice a day. The absence of diar- rhoea was considered by normal, and diarrhoea occurrence was Animal performance were subjected to a one-way analysis of characterized by liquid or loose faecal appearance (Giang et al., variance using JMP Pro 12 (SAS). Pen was used as an experimental 2012). unit for all performance measurements, and the piglet was considered an experimental unit when the microbial populations 2.4. Sample analyses from the ileum and caecum were analysed. The initial BW was included as a covariate. Means were separated only when the Diets were analysed for xylanase activity by enzyme linked treatment P-value was at a significant level and then compared by immunosorbent assay (ELISA) method using Quantiplate Kits for using a protected least significant difference (LSD) test (Snedecor Econase XT (Enzyme Services & Consultancy, Innovation & Tech- and Cochran, 1967). Chi-square comparisons were used to eval- nology Centre, Ystrad Mynach, UK) and phytase activity by an ELISA uate the diarrhoea frequency. Biostatistical analysis for microbiota method, using Quantiplate Kits for Quantum Blue supplied by were performed in an open source software R-Studio v.3.6.1. Di- Envirologix (Enzyme Services & Consultancy, Innovation & Tech- versity was analysed at OTU level using the vegan package nology Centre, Ystrad Mynach, UK). Live yeast recovery in feeds was (Oksanen et al., 2019). Richness and alpha diversity were calculated analysed by viable yeast count using the pour plate method using with raw counts based on Simpson, Shannon and Inverse-Simpson malt extract agar and CFU reported per gram of feed (Enzyme estimators. Beta diversity was evaluated by multivariate ANOVA Services & Consultancy, Innovation & Technology Centre, Ystrad based on dissimilarities through envfit and adonis function. Finally, Mynach, UK). differential abundance analysis was performed with taxa relative Ileal and caecal samples from piglets were collected and bac- abundances under a zero-inflated log normal mixture model, and terial DNA was extracted using AMPureXP beads (Beckman Cou- P-values were corrected by false-discovery rate (FDR) with the leter, Brea, CA) after thermal lysis process of 96  C for 10 min. metagenomeseq package (Paulson et al., 2019). Statements of sig- Amplicon sequencing library preparation was performed for bac- nificance were based on P-value of equal to or less than 0.05, and a teria using the V3 to V4 16S rRNA gene primers 341F (50 - P-value between 0.05 and 0.10 was considered as a trend. The CCTACGGGRSGCAGCAG-30 , Wang and Qian (2009)) and 806R (50 - correlation plot was created using R package “corrplot” (Wei and GGACTACHVGGGTWTCTAAT-30 , Caporaso et al. (2012)), with the Simko, 2017). following conditions: the first PCR primers contain the Ilumina sequences based on TruSeq structure adapter (Ilumin, San Diego, 3. Results CA), allowing the second PCR with indexing sequences. The PCR were always carried out in triplicate using Platinum Taq (Invi- Analysed enzyme activities and live yeast recoveries in feed trogen, USA) with the following conditions: PCR1, 95  C for 5 min, samples were all close to our expected values (Table 2). 25 cycles of 95  C for 45 s, 55  C for 30 s and 72  C for 45 s and a final extension of 72  C for 2 min for; PCR2, 95  C for 5 min, 10 cycles of 3.1. Growth performance and diarrhoea 95  C for 45 s, 66  C for 30 s and 72  C for 45 s and a final extension of 72  C for 2 min; PCR3, the reaction was cleaned up using Overall mortality was 0.8% and no differences were observed AMPureXP beads (Beckman Coulter, Brea, CA) and samples were between the experimental treatments (P > 0.05) (data not shown). pooled in the sequencing libraries for quantification. The DNA The effects of experimental treatments on growth performance concentration of the pool amplicon was estimated with Picogreen from d 23 to 44, from d 44 to 65, and from d 23 to 65 are shown in drDNA assays (Invitrogen, USA), and then the pooled libraries were Table 3. No differences were observed on BW at 44 d of age diluted for accurate quantitative PCR quantification using KAPA (P ¼ 0.415) between experimental treatments, but a significant Library Quantification Kit for Ilumina Platforms (KAPA Byosystems, trend was observed on BW at 65 d of age (P ¼ 0.100). Piglets in Woburn, MA). The library pool was adjusted to a final concentration XYL þ LY treatment were 0.9 kg heavier compared to CTR (24.6 vs. of 11.5 pmol/L and sequenced in a MiSeq system by using the 23.7 kg; P ¼ 0.043). No differences were observed for average daily standard Ilumina primers provided in the kit. Single-end feed intake (ADFI) in any of the measured periods or the overall sequencing (300 nt) was performed using a V2  300 sequencing length of the study. ADG or FCR were not improved by XYL or kit. After sequencing, the bioinformatics pipeline performs XYL þ LY treatments during the pre-starter phase (P > 0.05). During sequence demultiplexing, adaptor and primer trimming. The reads the starter phase, a significant trend in experimental treatments were size normalized to 283 bp. Read quality filter (E) was per- was observed on ADG (P ¼ 0.098); piglets in XYL þ LY treatment formed converting each nucleotide Q score in error probability (ei), gained 37 g/d more than those in CTR treatment (P ¼ 0.040); FCR that was summed and divided by read length (L). If Q was minor or during this period tended to be improved with XYL (P ¼ 0.080) and 460 lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza Table 2 Analysed enzyme activities and live yeast recoveries in feed samples.1 Item Pre-Starter Starter CTR XYL XYL þ LY CTR XYL XYL þ LY 2 Phytase , FTU/kg 764 753 622 374 403 513 Xylanase3, BXU/kg <2,000 24,700 24,000 <2,000 13,900 16,000 Live yeast, CFU/g <1  104 <1  104 2.13  107 <1  104 <1  104 2.90  107 FTU ¼ phytase unit; BXU ¼ birch xylan unit. 1 CTR, control diet; XYL, CTR supplemented with xylanase at 16,000 BXU/kg; XYL þ LY, XYL supplemented with live yeast (2  1010 CFU/g) at 1 kg/t. 2 One FTU is defined as the amount of enzyme required to release 1 mmol of inorganic P/min from sodium phytate at 37  C and pH 5.5. 3 One BXU is defined as the amount of enzyme that produces 1 nmol of reducing sugars from birchwood xylan in 1 s at 50  C and pH 5.3. Table 3 Effects of xylanase and live yeast supplementation on growth performance of piglets fed a sorghum-based diet1. Item CTR XYL XYL þ LY Pooled SD P-values Treatment Initial BW BW, kg 44 d of age 13.4 13.3 13.6 0.513 0.415 <0.001 65 d of age 23.7 23.9 24.6 0.905 0.100 <0.001 23 to 44 d of age ADG, g/d 301 305 320 28 0.255 <0.001 ADFI, g/d 477 472 456 29 0.264 <0.001 FCR, g/g 1.576 1.501 1.487 0.114 0.195 0.801 44 to 65 d of age ADG, g/d 488 496 525 38 0.098 <0.001 ADFI, g/d 989 957 955 72 0.503 <0.001 FCR, g/g 2.036a 1.924ab 1.829b 0.137 0.009 0.610 23 to 65 d of age ADG, g/d 402 406 422 21 0.102 <0.001 Fig. 1. Effects of xylanase and live yeast supplementation on diarrhoea incidence of ADFI, g/d 730 706 716 47 0.516 <0.001 piglets fed a sorghum-based diet. CTR, control diet; XYL, CTR supplemented with FCR, g/g 1.819a 1.740 ab 1.698b 0.089 0.018 0.867 xylanase at 16,000 birch xylan units/kg; XYL þ LY, XYL supplemented with live yeast (2  1010 colony forming units/g) at 1 kg/t. Values in parentheses represent the SD ¼ standard deviation; ADG ¼ average daily gain; ADFI ¼ average daily feed number of animals with diarrhoea. Data columns with different letters differ at intake; FCR ¼ feed conversion ratio. P < 0.05. a, b Means in the same row with different superscripts differ at P < 0.05. 1 Mean values for 10 replicate pens with 6 piglets per replicate pen. CTR, control diet; XYL, CTR supplemented with xylanase at 16,000 birch xylan units/kg; XYL þ LY, Table 4 XYL supplemented with live yeast (2  1010 CFU/g) at 1 kg/t. Effects of xylanase and live yeast supplementation on alpha diversity in the ileal and caecal microbiota of piglets fed a sorghum-based diet1. improved with XYL þ LY (P ¼ 0.002) compared to CTR treatment. Item CTR XYL XYL þ LY Pooled SD P-value When the overall period was considered, pigs in XYL þ LY treat- Ileum ment had increased ADG compared with those in XYL treatment Shannon 1.09 1.14 0.73 0.501 0.237 Simpson 0.60a 0.55a 0.32b 0.214 0.044 (XYL þ LY vs. XYL, P ¼ 0.655). Effect of treatment on FCR was sig- Inverse Simpson 2.56 2.55 2.31 1.349 0.925 nificant from 23 to 65 d (P ¼ 0.018). FCR tended to be improved Cecum with XYL by 0.08 (P ¼ 0.058) and XYL þ LY by 0.12 (P ¼ 0.010) Shannon 1.89 1.60 1.66 0.354 0.184 compared to CTR treatment. Simpson 0.77 0.66 0.69 0.124 0.134 Experimental treatments influenced the incidence of diarrhoea Inverse Simpson 4.82 3.48 3.81 1.444 0.114 (P < 0.05) (Fig. 1). Diarrhoea occurred in 40% of the piglets in non- SD ¼ standard deviation. a, b supplemented CTR treatment, 35% of the pigs in XYL treatment, and Means in the same row with different superscripts differ at P < 0.05. 1 Mean values from 10 piglets per treatment. CTR, control diet; XYL, CTR sup- only 20% of the pigs in XYL þ LY treatment. plemented with xylanase at 16,000 birch xylan units/kg; XYL þ LY, XYL supple- mented with live yeast (2  1010 CFU/g) at 1 kg/t. 3.2. Microbial profile Three alpha-diversity estimators (Simpson, Shannon and in- (Table 5 and Fig. 3). The relative proportion of Clostridiaceae was verse Simpson) were performed in the ileal and caecal digesta of reduced by 9.8% in the ileum with XYL and 14.2% with XYL þ LY piglets at 65 d of age (Table 4). No differences were observed in any compared to CTR (P ¼ 0.068). The relative proportions of Bifido- of the estimators in the caecum (P > 0.05). Shannon and inverse bacteriaceae, Clostridiaceae and Enterobacteriaceae in the caecal Simpson estimators in the ileum diversity were not influenced by digesta were also influenced with the experimental treatments the experimental treatments, but XYL þ LY treatment reduced (P ¼ 0.011, P ¼ 0.076 and P ¼ 0.021, respectively). No differences Simpson estimator when compared to CTR and XYL treatments were observed in the natural logarithm (ln) values for the ileal (P < 0.05). XYL and XYL þ LY treatments tended to influence beta microbiota (data not shown). In contrast, the ln changes promoted diversity in the ileum (Fig. 2A) and the caecal digesta (Fig. 2B) by treatments at family level in the caecal microbiota are presented (P < 0.10). in Fig. 3. The count of Clostridiaceae in CTR treatment was 196% In both intestinal regions, 5 phyla were detected being Firmi- higher than that in XYL treatment (P < 0.0001), but lower counts cutes the predominant but some family members in the ileal and of Lactobacillaceae (P < 0.0001) and Streptococcaceae (P < 0.0001) caecal digesta were influenced by XYL and XYL þ LY treatments were observed in CTR treatment. Ruminococcaceae ln value in CTR lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza 461 Fig. 2. Comparison of beta-diversity of ileum (A) and caecum (B) microbiota between CTR, XYL and XYL þ LY treatments. CTR, control diet; XYL, CTR supplemented with xylanase at 16,000 birch xylan units/kg; XYL þ LY, XYL supplemented with live yeast (2  1010 colony forming units/g) at 1 kg/t. The nonmetric-multidimensional scaling (NMDS) plots was generated using BrayeCurtis distances. P-value was obtained from analysis of similarities (ANOSIM). Table 5 Relative proportion of microbial populations in the ileal and caecal digesta based on the operational taxonomic units1. Item Ileum Cecum 2 CTR XYL XYL þ LY Pooled SD P-value Adjusted CTR XYL XYL þ LY Pooled SD P-value2 Adjusted P-value 3 P-value 3 Bacteroidaceae 0.0 0.0 0.2 0.22 1.000 1.000 n.d n.d n.d Bifidobacteriaceae 0.0 0.0 0.2 0.24 1.000 1.000 0.5 0.3 0.1 0.58 0.011 0.115 Clostridiaceae 20.0 10.2 5.8 11.92 0.068 0.163 4.5 1.6 4.5 5.75 0.076 0.241 Enterobacteriaceae 7.8 3.2 0.7 9.54 0.129 0.257 2.2 0.0 3.4 6.89 0.021 0.115 Enterococcaceae n.d n.d n.d 0.0 0.0 0.1 0.22 1.000 1.000 Eubacteriaceae 0.0 0.2 1.6 2.24 1.000 1.000 8.8 7.9 3.6 5.18 0.218 0.399 Lachnospiraceae 0.0 0.7 0.6 1.52 1.000 1.000 5.6 3.6 5.4 3.29 0.128 0.282 Lactobacillaceae 53.9 64.3 78.7 20.44 0.468 0.702 41.2 56.4 49.2 14.70 0.654 0.720 Mycoplasmataceae 0.0 0.2 0.0 0.41 1.000 1.000 n.d n.d n.d Prevotellaceae 0.0 0.0 0.9 1.29 1.000 1.000 1.6 1.5 0.3 1.91 0.420 0.513 Ruminococcaceae 0.0 3.6 2.9 7.82 1.000 1.000 17.5 13.7 14.0 8.15 0.088 0.241 Streptococcaceae 18.4 17.7 7.4 15.38 0.318 0.545 5.3 6.5 6.0 4.26 0.358 0.492 Veillonellaceae 0.0 0.0 1.0 1.37 1.000 1.000 12.9 8.6 13.3 8.76 0.281 0.442 SD ¼ standard deviation; n.d ¼ non-determined. 1 Mean values from 10 piglets per treatment. CTR, control diet; XYL, CTR supplemented with xylanase at 16,000 birch xylan units/kg; XYL þ LY, XYL supplemented with live yeast (2  1010 CFU/g) at 1 kg/t. 2 Obtained using a zero-inflated Gaussian mixture model. 3 P-values corrected by false-discovery rate. 462 lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza ln change Different mechanism of action through xylanase supplementation could explain the positive results observed in the present study: 1) CTR / XXYL P -value viscosity reduction, although it is unlikely to be the main reason as Family the soluble NSP content is very low (Choct et al., 2004); 2) abrasion Clostridiaceae 1.96 <0.0001 of feedstuff cell walls and the subsequent release of nutrients Lactobacillaceae -0.36 <0.0001 entrapped (Bedford, 2002) and 3) arabinoxylo-oligosaccharides Streptococcaceae -00.41 -0 41 <0.0001 (AXOS) production in the GIT not only from the endogenous CTR / XYL+LY CT enzyme action by the microbial populations but also from the ac- Family tion of exogenous xylanase (Morgan et al., 2017, 2019). Therefore, these AXOS could be acting as prebiotic or signalling compounds in Lachnospiraceae -0.35 0.0024 the GIT to stimulate fibre degrading microbiota as reviewed by G. Lactobacillaceae -0.61 1 <0.0001 lez-Ortiz et al. (2019). The physicochemical properties of Gonza Ruminococcaceae 0.16 0.0003 digesta, nutrient or energy digestibility have not been evaluated in Streptococcaceae -0.47 0.0002 the present study, but if the previous hypotheses are right, the XYL / XYL+LY XY fibre-degrading enzymes produced in situ by the microbial com- Family munities in the GIT due to the stimulation of the xylo-oligomers Eubacteriaceae 0.49 0.0049 produced by the xylanase would potentially increase further the Prevotellaceae 1.78 0.0052 breakdown of cell walls with greater efficiency than supplemented enzyme. However, the efficiency of arabinoxylan breakdown by Ruminococcaceae -0.72 0.0004 endogenous or exogenous xylanase has not been studied and is a potential area of future research. In the case of sorghum, the release Fig. 3. Logarithm (ln) value changes promoted in treatment contrasts (adjusted of entrapping nutrients is of great interest as the protein structure P < 0.05) in family taxa in the caecal microbiota. CTR, control diet; XYL, CTR supple- in the grain and its distribution in the endosperm is proportionally mented with xylanase at 16,000 birch xylan units/kg; XYL þ LY, XYL supplemented greater and has a denser surface and higher water penetration with live yeast (2  1010 CFU/g) at 1 kg/t. Positive values and negative values indicate greater and lower abundance based on the absolute operational taxonomic units, resistance compared to corn and other grains (Selle et al., 2018). respectively. Differences presented are based on all taxa detected in samples per Starch granules are surrounded by kafirin protein bodies and cell treatment. wall fibre in the sorghum endosperm. This complex matrix can be cross-linked, forming structures that limit enzymatic action, especially compromising amino acid and starch digestibility (Liu treatment was 16% higher than that in XYL þ LY treatment et al., 2016; Pan et al., 2017). Therefore, xylanase supplementation (P ¼ 0.0003), but the ln values of Lachnospiraceae (P ¼ 0.0024), in sorghum-fed pigs may be a feasible strategy to improve nutrient Lactobacillaceae (P < 0.0001) and Streptococcaceae (P ¼ 0.0002) in and energy profitability and the release of AXOS to have a better XYL þ LY treatment were higher than those in CTR treatment. animal performance. Finally, differences were also observed due to live yeast supple- Further to the evaluation of xylanase, it was considered to study mentation. The ln values of Eubacteriaceae and Prevotellaceae in if the supplementation of live yeast in addition to the xylanase XYL treatment were 49% and 178% higher than those in XYL þ LY provided any extra benefit. Piglets in XYL þ LY treatment were treatment (P ¼ 0.0049 and P ¼ 0.0052, respectively), and the ln 0.7 kg heavier at 65 d of age compared to those supplemented with value of Ruminococcaceae in XYL þ LY treatment was 72% lower xylanase. Overall, live yeast supplementation combined with than that in XYL treatment (P ¼ 0.0004). xylanase during the nursery phase increased ADG and improved The correlations between animal performance and the relative FCR with no effects on ADFI. The enhancement of FCR of piglets abundance of bacterial families in the ileum and the caecal digesta observed in the present study was in agreement with previously are presented in Figs. 4 and 5, respectively. A positive and signifi- published reports on live yeast (Jiang et al., 2015). During the cant correlation between BW gain and the relative abundance of weaning phase, piglets have to undergo many stressors (environ- Lactobacillaceae in the ileum was observed. Looking at the impli- mental, social and dietary) with consequences in animal perfor- cations of microbiota on FCR, the relative abundance of Lactoba- mance. Together, with the immaturity of their intestinal and cillaceae was negatively correlated with FCR whereas the relative immune systems, it may lead the animals to develop anorexia, in- abundance of Clostridiaceae was positively correlated with FCR. A testinal stasis, a low rate of feed digestion and the risk of diarrhoea negative and significant correlation between BW gain and the s et al., 2007). This post-weaning lag may increase mortality, (Lalle relative abundance of Eubacteriaceae in the caecal content was reduce growth in animals from wean to finish and increase het- observed. The relative abundance of Lactobacillaceae in the caecal erogeneity of weight lots. Nutritional strategies such as yeast sup- content was negatively correlated with feed intake. plementation are used for improving intestinal development and immune function in pigs. It has been reported that some strains of 4. Discussion live yeast and components of yeasts can improve growth perfor- mance, the small intestine architecture and immune function in The literature for the use of sorghum as the sole cereal source in weaned piglets (Bontempo et al., 2006; Jiang et al., 2015; Li et al., nursery diet of piglets is limited and therefore the effects of xyla- 2006; Trevisi et al., 2015, 2017). A variety of components make up nase and live yeast on piglets fed sorghum-based diets are scarce. yeasts such as b-glucans, mannans, nucleotides and peptides which The effects of xylanase supplemented in sorghum-based diets have have been related with the positive effects observed when live attracted less attention because sorghum has less soluble NSP yeast, cell wall extracts or dead yeasts have been supplemented compared to wheat and corn, and the viscosity is likely irrelevant in (Kollar et al., 1997). However, the main differential feature of live this grain (Bach Knudsen, 2014). Nevertheless, arabinoxylan is the yeast compared to other types of yeast by-products is the oxygen main cell wall component in cereals, and in the present study, scavenging capacity. A fully functional hindgut has to offer a strictly xylanase supplementation over a 42-d period improved animal anaerobically environment to allow fermentation of the undigested performance in sorghum-based diets justifying that sorghum has nutrients, mainly NSP, coming from the small intestine (Rinttila € and sufficient arabinoxylan content to elicit a response to xylanase. Apajalahti, 2013). Presence of oxygen in samples obtained from the lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza 463 Fig. 4. Correlations between performance parameters and relative abundances of bacterial family taxa in the ileum content. BWG ¼ BW gain; FI ¼ feed intake; FCR ¼ feed con- version ratio. The *(P < 0.05), **(P < 0.01) and ***(P < 0.001) symbols indicates a statistically significant correlation. The scale colours (Spearman's r from 1 to þ1) indicate whether the correlation is positive (blue coloured circles) or negative (red coloured circles) between factors. hindgut of humans and rats clearly demonstrated that changes in consequently less diarrhoea episodes. In the present study, diar- bacterial composition reduce the presence of fibre-degrading rhoea indices were high in the CTR treatment, suggesting some members (Albenberg et al., 2014). Ruminal digesta from animals kind of postweaning disturbance. Xylanase supplementation receiving live yeast had lower prevailing concentrations of oxygen, marginally reduced the incidence, but it is noteworthy the further but improved fibre digestion due to the protection of highly reduction of the diarrhoea occurrence in piglets supplemented oxygen-sensitive fibre-degrading bacteria like Fibrobacter succino- with xylanase in combination with live yeast. Many studies have genes (Chaucheyras-Durand and Fonty, 2001). If the same findings demonstrated that supplementation with live yeast or poly- can be found in monogastric animals, live yeast supplemented to saccharides from its cell walls may improve disease resistance and piglets may also reduce the presence of oxygen in the hindgut, and enhance performance through immunostimulation and mainte- increase the survival and presence of fibrolytic bacteria. As Jha and nance of a favourable intestinal environment of animals (Badia Berrocoso (2016) also discussed, the reduction on dissolved oxygen et al., 2012b). Yeast cells are natural mannose-rich products that stimulated fermentation and production of short-chain fatty acids can be used as a substrate for adhesion of Gram-negative bacteria (SCFA). Moreover, increasing fermentation and production of SCFA (Gedek, 1999). Administration of live yeast helped reduce post- may reduce the opportunities for pathogens to colonize the intes- weaning diarrhoea after enterotoxigenic Escherichia coli (ETEC) tinal environment. Taken together with the increased quantities of infection (Che et al., 2017; Trckova et al., 2014; Trevisi et al., 2015), acid glycoconjugates in the mucous cells of live yeast- possibly through the interaction of live yeast with ETEC binding to supplemented piglets (Bontempo et al., 2006), the new environ- the intestinal receptors (Badia et al., 2012a, 2012b). Animals from ment may confer a greater resistance to bacterial infection and the present study were not diagnosed for the aetiology agent 464 lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza Fig. 5. Correlations between performance parameters and relative abundances of bacterial family taxa in the caecal contents. BWG ¼ BW gain; FI ¼ feed intake; FCR ¼ feed conversion ratio. The *(P < 0.05), **(P < 0.01) and ***(P < 0.001) symbols indicates a statistically significant correlation. The scale colours (Spearman's r from 1 to þ1) indicate whether the correlation is positive (blue coloured circles) or negative (red coloured circles) between factors. causing the faecal consistency lost, but based in other studies, it can and animal health (Gibbs, 2009), but others like Faecalibacterium be speculated that live yeast had an effect on the piglet immune prausnitzi have been identified as butyrate producers (Rios-Covian response, fermentation activity, epithelial integrity and possibly et al., 2015). The identification of the microbiota at the genus and also impairing the attachment of pathogenic bacteria to the intes- specie levels in the Clostridiaceae family did not reveal any influ- tinal receptors, improving disease resistance, nutrient digestibility, ence of the experimental treatments (data not shown), but it animal performance and the faecal consistency (Badia et al., 2012b; cannot be discarded an inhibitory effect of xylanase and the com- Trevisi et al., 2015; Zanello et al., 2009). bination with live yeast in other members of the family not The ileal and caecal microbiota diversity was analysed as both detected in the current study. In contrast, the presence of Lacto- xylanase and live yeast supplementation are related with microbial bacillaceae tended to increase with xylanase and even further shifts in the GIT (Bedford and Cowieson, 2012; Gemma Gonza lez- when xylanase was combined with live yeast. Lactobacillus amylo- Ortiz et al., 2017; Le Bon et al., 2010). Clostridiaceae from the vorus was the most influenced strain by the experimental treat- ileum and caecum were reduced with the experimental treatments, ments. L. amylovorus has demonstrated to be an anti-inflammatory and in the caecum, XYL and XYL þ LY treatments increased the strain inhibiting Toll-like receptors when exposed to ETEC presence of Lactobacillaceae, Streptococcaceae and Lachnospir- (Finamore et al., 2014). Although there was no difference in the aceae. Some members belonging to the Clostridiaceae family can be relative proportion of Enterobacteriace levels between treatments, pathogenic. For example, Clostridium perfringens, Clostridium bot- it is interesting to highlight the numerically improvement on Lac- ulinum, Clostridium tetani, Clostridium septicum and Clostridium tobacillaceae to Enterobacteriaceae ratio in XYL and XYL þ LY difficile are some of the most known species threatening human treatments compared to CTR (20 and 112 vs. 7, respectively, in the lez-Ortiz et al. / Animal Nutrition 6 (2020) 457e466 G. Gonza 465 ileum, and 35 and 14 vs. 9, respectively, in the caecum). This ratio is Bach Knudsen KE. Fiber and nonstarch polysaccharide content and variation in  et al., common crops used in broiler diets. Poultry Sci 2014;93(9):2380e93. generally considered an indicator of gut health (Demeckova Badia R, Lizardo R, Martinez P, Badiola I, Brufau J. The influence of dietary locust 2002). 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Poultry feedstuffs: supply, composition and nutritive value. Oxon, in XYL compared to XYL þ LY. The shifts in microbial diversity United Kingdom: CABI Publishing; 2002. p. 319e36. observed in the present study may be the consequence of several Bedford MR. The evolution and application of enzymes in the animal feed industry: the role of data interpretation. Br Poultry Sci 2018;59(5):486e93. factors. On the one hand, xylanase, through XOS production, may Bedford MR, Cowieson AJ. Exogenous enzymes and their effects on intestinal stimulate lactate and butyrate producing bacteria, and xylanase microbiology. Anim Feed Sci Technol 2012;173(1):76e85. often has cross-feeding interactions with butyrate producing bac- Bontempo V, Di Giancamillo A, Savoini G, Dell' Orto V, Domeneghini C. Live yeast dietary supplementation acts upon intestinal morpho-functional aspects and teria (De Maesschalck et al., 2015); on the other hand, the resistance growth in weanling piglets. 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