Animal 16 (2022) 100660

Contents lists available at ScienceDirect

Animal
The international journal of animal biosciences

Dietary autolysed yeast modulates blood profiles, small intestinal

morphology and caecal microbiota of weaning pigs
S. Namted, K. Poungpong, W. Loongyai, C. Rakangthong, C. Bunchasak ⇑
Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Yeast products are potential feed additives due to their beneficial effects on gut health. Thus, we verified
Received 18 March 2022 the potential impacts of autolysed yeast (AY) on growth performance, blood profiles, gut morphology and
Revised 19 September 2022 microbiota in weaning pigs. In total, 72 castrated, commercial, crossbred, weaning pigs were divided into
Accepted 20 September 2022
three groups, with each group consisting of eight replicates with three piglets each. The experimental
Available online 21 October 2022
diets were as follows: 1) control diet (0% AY); 2) diet with 1.0% AY; 3) diet with 3.0% AY. For the overall
period, using 1.0% AY in the diet seemed to improve the feed conversion ratio (P = 0.09); whereas, other
Keywords:
productive performance parameters were not significantly affected by the supplementations. Using 1.0%
Gut health
Microbial community
AY in the diet significantly decreased the blood urea nitrogen and neutrophil/lymphocyte ratio (N/L ratio)
Piglets but increased the eosinophil count (P < 0.05). Adding AY to the diet did not influence caecal microbial
Productive performance diversity; using 1.0% AY in the diet decreased the abundances of the phylum Actinobacteria, the class
Yeast extract Coriobacteriia and the family Coriobacteriaceae (P < 0.05). At the genus level, an AY inclusion level of
1.0% reduced the abundances of Collinsella, Clostridium and Catenibacterium and increased that of
Marvinbryantia (P < 0.05). Furthermore, the abundance of butyrate-producing bacteria seemed to be
increased by AY supplementation (P = 0.06). Pearson’s correlation coefficient (r) analysis revealed that
AY intake was negatively associated with the abundance of pathogens of the genera Dorea (r =
0.84;
P = 0.03) and Catenibacterium (r =
0.80; P = 0.04). This indicates that AY intake potentially reduces
the population of some pathogenic bacteria at family level. Thus, using an appropriate AY inclusion level
(1.0%) seemed to improve the feed use of postweaning pigs and clearly improved their small intestinal
morphology, blood profiles and caecal microbiota.
Ó 2022 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open
access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Implications Introduction

Weaning piglets are always challenged with low feed intake, The transition period of weaning pigs is a stressful process that
low defensive immune systems and gut dysfunction. Using yeast results in poor performance, poor intestinal functions and high
products in the diet may improve the gut health and growth per- susceptibility to infections (Kiros et al., 2019). Various antibiotics
formance of pigs via the improvement of gut microbiota and mor- have been widely used to eliminate these problems; however, to
phology. Supplementation of autolysed yeast at 1.0% of the diet limit the use of antibiotics, protection against disease in the animal
decreased the concentration of blood urea and the neutrophil/lym- must rely on good feed and management. The yeast Saccharomyces
phocyte ratio, increased the eosinophil number, and decreased the cerevisiae (S. cerevisiae) has been used as an alternative feed addi-
abundance of several bacteria inducing inflammation. Moreover, tive for farm animals. Various yeast products have been used in
the yeast supplementation significantly improved small intestinal animal feed, such as whole yeast, carbohydrates isolated from its
morphology. Therefore, supplementing autolysed yeast at 1.0% of cellular wall (50–55% b-glucans, 35–40% mannan-
the diet potentially improves feed efficiency and gut health of oligosaccharides and 2% chitin) or the isolated nucleotides from
piglets. the cytoplasm of yeast (12–20% of the total nitrogen of yeast)
(Shen et al., 2009).
Recently, Namted et al. (2021) reported that whole-cell yeast
from the autolysis process contain 11.0% b-glucans, 3.0%
mannan-oligosaccharides and 3.9% nucleic acids. The inconsis-
⇑ Corresponding author.
tency of bioactive compounds in yeast products may result in
E-mail address: [email protected] (C. Bunchasak).

https://doi.org/10.1016/j.animal.2022.100660
1751-7311/Ó 2022 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S. Namted, K. Poungpong, W. Loongyai et al. Animal 16 (2022) 100660

different dosages of the recommendation. Autolysed yeast (AY) is Growth performance

produced via cellular degradation activated by the yeast’s own
enzymes. Yang et al. (2016) reported that AY supplements may The BW of each piglet was recorded to calculate the BW gain
stimulate digestion and maintain microbial equilibrium in the (BWG) and the average daily gain (ADG). Feed intake (FI) was
gut of weaning pigs. Upadhaya et al. (2019) and Berto et al. recorded weekly, and the feed conversion ratio (FCR = feed
(2020) found that supplementing AY for approximately 0.2–0.5% intake/gain ratio) was calculated weekly.
of the diet improves growth performance, immunity, and gut
microbiota in weaning pigs. In fattening pigs, Namted et al. Blood sample collection
(2021) reported that adding 0.5% AY in the diet improved the pro-
ductive performance, immune function and meat quality. At the end of the trial, all pigs from two replicates in each treat-
Using yeast as a probiotic modulates the gut microbiome and ment (six pigs per treatment) with a BW close to the group mean
improves the health of piglets (Xu et al., 2018). In addition, Kiros were selected and exsanguinated after withdrawing feed for 6 h.
et al. (2019) reported that supplementing the diet with yeast The blood urea nitrogen (BUN) concentration was analysed using
changes populations of Prevotella spp. in the gut, promoting piglet a commercial test kit (Urea liquicolor No.10505, Human), and
growth. Recently, Berto et al. (2020) found that supplementing AY white blood cell counts (neutrophils, lymphocytes, monocytes,
(0.3–0.5% of the diet) can have an impact on the white blood cell eosinophils and basophils) were determined. Blood films were pre-
profile and improves the growth performance of weaning pigs, pared on triplicate glass slides, air-dried and stained with Giemsa-
although the caecal microbiota was not affected. However, we Wright’s stain; 100 white blood cells were manually counted
hypothesised that using AY (at an inclusion level of 0.5–1.0%) pos- under a microscope (Borges et al., 2004). The blood total
itively impacts the white blood cell profile, gut physiology and gut immunoglobulin G (IgG) was investigated using radial immunodif-
microbiota. The objective of this study was therefore to investigate fusion assay kits (Bethyl Laboratories, Inc., Montgomery, TX).
the effects of AY (containing 30.0% CP, 11.0% b-glucan, 3.0%
mannan-oligosaccharides, 3.9% total nucleic acids and other com- Morphology of the small intestine
pounds) on the health of weaning pigs, focusing on their growth
performance, blood profile, anatomy and morphology of the small Tissue samples from segments of the duodenum, jejunum and
intestine, as well as on the microbiota when fed an antibiotic-free ileum were collected immediately and fixed in 10% neutral buffer
diet. formalin, followed by gently placing them in paraffin. For each
specimen, at least 10 sections with a thickness of 7 lm were pre-
pared and stained with haematoxylin-eosin for histological evalu-
ation (Nunez et al., 1996). Villous height, crypt depth and villous
Material and methods
height/crypt depth ratio were studied using a computer-assisted
image-analysis system (Biowizard, Thaitec, Thailand). Measure-
Animals and management
ments were recorded based on the mean of 10 fields for each spec-
imen, for the villous height from the tip to the villous-crypt
According to Thai Agriculture Standard 6403-2015 (good man-
junction and for the crypt depth from the villous-crypt junction
agement practices for pig farms) and Animal Ethics (licence num-
to the lower limit of the crypt (Saki et al., 2012). According to
ber U1-07385-2561), the pigs were kept in a building with an
Brümmer et al. (2010), goblet cell density was determined as the
evaporative cooling system and a concrete slate floor pen (one pig-
number of goblet cells per 10 000 lm2, with goblet cells found
let per m2). Throughout the study period, the average temperature
on the edge of the villus counted.
in the house was 29.16 °C. Feed and water were provided ad libi-
tum. The pigs were arranged in a completely randomised design.
Gut microbiota
Regarding the experimental diets, the 72 castrated commercial
crossbred piglets (21 days of age and 6.46 ± 0.17 kg BW) were
Sample collection and DNA extraction
divided into three groups, with each group consisting of eight
The caecum contents were stored using a Stool Nucleic Acid
replications of three pigs each. At 21, 38 and 61 days of age, the
Collection and Preservation Tube (Norgen BioTek Corp) and subse-
BW of each pig was determined. At 61 days of age, all pigs from
quently stored at
80 °C. Before DNA extraction, the samples were
two replicates in each treatment (six pigs per treatment) with a
homogenised, weighed into separate aliquots for DNA extraction
BW close to the group mean were selected and exsanguinated after
and extracted using the QIAamp PowerFecal Pro DNA Kit (Qiagen)
withholding feed for 6 h. Pigs were euthanised using CO2 asphyx-
following the manufacturer’s protocol. The purity of the DNA sam-
iation in an atmosphere of less than 2% oxygen (air displaced by
ples was checked using a Nanodrop 2000 spectrophotometer
CO2) at a commercial slaughterhouse (Nikompattana, Rayong,
(Thermo Fisher Scientific).
Thailand).
16S metagenomic library preparation and bioinformatic analysis
Library preparation was carried out according to the standard
Experimental diets Illumina 16S Ribosomal RNA, based on the Illumina MiSeq system
protocol. The V3–V4 region of the microbial 16S rRNA gene was
Without any antimicrobial supplementation, the nutrient den- PCR-amplified using the universal primers 341F: 50 -TCG TCG GCA
sity values of the experimental diets were computed according to GCG TCAGAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC
the recommendations of the National Research Council (National AG-30 and 785R: 50 -GTC TCG TGGGCT CGG AGA TGT GTA TAA
Research Council (NRC), 2012). The diet was divided into two GAG ACA GGA CTA CHV GGG TAT CTA ATC C-30 , following Zhu
phases, namely prestarter (after weaning-14 days or 7–11 kg et al. (2020). The 16S amplicon PCR consisted of 3.5 ll DNA,
BW) and starter (15–40 days after weaning or 11–25 kg BW). 17.5 ll 2X sparQ HiFi PCR Master Mix (Quantabio) and 0.2 uM (fi-
The experimental diets consisted of AY at levels of 0% (control nal concentration) of forward and reverse primers (Zhu et al.,
diet), 1.0% or 3.0% of the diet (Table 1 and Supplementary 2020). The PCR was performed under the following conditions: ini-
Table S1). Feed was offered in pellet form (3 mm diameter) and tial denaturation at 30 cycles of 95 °C for 3 min, 25 cycles of 30 s at
prepared every 3 weeks. 95 °C, annealing 30 s at 55 °C and 30 s at 72 °C and a final extension
2
S. Namted, K. Poungpong, W. Loongyai et al. Animal 16 (2022) 100660

Table 1
Prestarter and starter pig diet composition.

Ingredient (%, DM) Prestarter pig Starter pig

Control Autolysed yeast (%) Control Autolysed yeast (%)
1.0 3.0 1.0 3.0
Broken rice 55.35 55.45 55.60 59.60 59.68 59.83
Rice bran oil 1.56 1.53 1.49 1.27 1.25 1.20
Soybean meal 16.92 16.49 15.64 24.25 23.83 23.01
(48%CP)
Full-fat soybean meal 10.00 10.00 10.00 5.00 5.00 5.00
Fish meal (63.5%CP) 3.00 3.00 3.00 - - -
Rice bran solvent meal 3.17 2.71 1.84 2.86 2.40 1.52
Whey powder, sweet 6.00 6.00 6.00 3.00 3.00 3.00
L-Lysine hydrochloride 0.57 0.52 0.41 0.48 0.42 0.32
DL-Methionine 0.22 0.22 0.21 0.16 0.16 0.15
L-Threonine 0.22 0.21 0.19 0.18 0.17 0.14
Monodicalcium-phosphate 0.88 0.88 0.87 0.95 0.95 0.94
Limestone 0.95 0.83 0.60 1.01 0.90 0.67
Salt 0.26 0.26 0.29 0.35 0.34 0.37
Vitamin and Mineral Premix1 0.25 0.25 0.25 0.25 0.25 0.25
Sodium bicarbonate 0.40 0.40 0.40 0.40 0.40 0.35
Antioxidant and toxin binder 0.25 0.25 0.25 0.25 0.25 0.25
Autolysed yeast2 0.00 1.00 3.00 0.00 1.00 3.00
Total 100.00 100.00 100.00 100.00 100.00 100.00
Nutrient compositions (%, DM)
Metabolisable energy 3 400 3 400 3 400 3 350 3 350 3 350
(kcal/kg)
Protein 19.59 19.58 19.57 19.07 19.07 19.07
(20.99)3 (20.51) (20.94) (20.76) (19.24) (20.17)
Fat 5.00 5.00 5.00 3.50 3.50 3.50
(5.51) (5.17) (5.21) (4.93) (4.79) (4.71)
Fibre 2.57 2.48 2.32 2.71 2.62 2.62
Lysine 1.53 1.53 1.53 1.40 1.40 1.40
Methionine 0.56 0.56 0.56 0.47 0.47 0.47
Methionine + Cysteine 0.87 0.87 0.87 0.79 0.79 0.79
Threonine 0.95 0.95 0.95 0.87 0.87 0.87
Tryptophan 0.27 0.27 0.27 0.27 0.28 0.28
Calcium 0.80 0.80 0.80 0.70 0.70 0.70
Total Phosphorus 0.63 0.63 0.63 0.57 0.57 0.57
Dietary electrolyte balance (mEq) 242.34 245.76 244.59 244.74 247.17 246.07
1
Provided per kilogram of complete diet: Vitamin and mineral premix and amino acid contents were met the recommendation of NRC (2012).
2
The compositions of autolysed yeast: Supplementary Table S1.
3
Values in parentheses are proximate analyses of diet.

step for 5 min at 72 °C, with a holding temperature of 4 °C (Zhu levels of the experimental groups were defined at the phylum,
et al., 2020). All assays were performed in duplicate on a T100TM family and genus levels. Rarefaction analysis was used to evaluate
Thermal Cycler (Bio-Red Laboratories, Inc.), and the 16S V3–V4 the rationality and efficiency of the sequencing depth based on
amplicon was purified using AMPure beads (Beckman Coulter). alpha diversity indicators (Observed OUTs, Shannon’s, Faith’s
The PCR mix consisted of 5 ll DNA, 25 ll 2X Phusion Hot Start II diversity and Pielou’s Evenness). b diversity analysis was used to
High-Fidelity PCR Master Mix (Thermo ScientificTM), 5 ll Nextera XT estimate differences between samples in species complexity,
index Primer 1, 5 ll Nextera XT index Primer 2 and 10 ll PCR- including the Principal Coordinate Analysis (PCoA) with the
grade water. The PCR was performed under the following condi- Unweighted UniFrac Pair-group method (Wang et al., 2021).
tions: eight cycles (95 °C for 3 min, 95 °C for 30 s, 55 °C for 30 s,
72 °C for 30 s), and 72 °C for 5 min, with a 4 °C holding tempera- Statistical analysis
ture. The PCR products were purified using AMPure beads, quanti-
fied based on Qubit Fluorometric quantitation and pooled in All data were subjected to ANOVA, using the GLM procedure of
equivalent concentrations. The pooled samples were sequenced the SAS software (SAS, 1998). The least-square mean of each treat-
using an Illumina MiSeq instrument with a V3 reagent kit ment was compared using the least significant difference test. The
(2  250 cycles). means of growth performance, BUN and haematological profiles, as
In terms of analysis of bioinformatics, Quantitative Insights Into well as the morphology and anatomy of the small intestine and gut
Microbial Ecology 2 was used. Briefly, the qiime dada2 denoise- microbiota, were compared based on Tukey’s studentised range
paired function in Quantitative Insights Into Microbial Ecology 2 test. Data normality was verified, and no outliers were detected.
was used to combine the forward and reverse reads contained in The statistical model was as follows:
the FASTQ files of the samples. Paired-ends FASTQ files were qual-
Yij ¼ l þ Ai þ eij ;
ity, filtered, sequence trimmed and dereplicated with DADA2
(Bolyen et al., 2019). In addition, curve sequences were rarefied where Yij is the observed response, Ai is the effect of AY supplemen-
at 25 000 sequences per sample. The sequences were grouped into tation in the diet, eij is the experimental error; eij  NID (0, d2).
operational taxonomic units (OTUs) above 97% similarity against Statistical significance (P < 0.05) and tendency (0.05  P < 0.10)
the Silva reference database, version 132, with 99% for the region were used. Microbiota alpha diversity analysis was determined
V3–V4. After normalisation of the reads, the relative abundance based on OTUs as well as Shannon’s, Faith’s diversity and Pielou’s
3
S. Namted, K. Poungpong, W. Loongyai et al. Animal 16 (2022) 100660

evenness indices. b diversity ordination (unweighted UniFrac dis- increase (P = 0.07). Using AY at 1.0% of the diet significantly
tance matrix) was calculated after rarefaction correction. Pearson’s enhanced the percentage of eosinophils compared to the control
correlation coefficient (r) was used to investigate correlations diet (P = 0.02). The total IgG was not significantly affected by diet-
between gut microbiota and all parameters. Statistical significance ary treatments.
was based on P < 0.05. Compared to the control diet, using AY at inclusion levels 1.0%
and 3.0% reduced the concentration of BUN by 26.32% and
13.53%, respectively. Based on a previous study, BUN is the end
Results and discussion
product of amino acid catabolism in mammals, increasing the
BUN concentration and reducing protein deposition and the feed
Growth performance
efficiency ratio (Wei, 2001). Based on the tendency to improve
FCR, the suppression of BUN by using AY (1.0% of the diet) may
The effects of dietary AY on the growth performance of weaning
indicate increased protein use and/or decreased metabolic stress
pigs are presented in Table 2. During the prestarter period (14 days
from protein degradation. Although supplementation of AY at
after weaning) and the starter period (15–40 days after weaning),
3.0% of the diet also significantly reduced the stress index (N/L
BW, BWG, ADG, FI and FCR were not significantly affected by the
ratio), BUN was not drastically decreased. This indicates that the
inclusion of AY in the diets. For the overall period, the FCR tended
supplemental AY at 3.0% in the diet is not an appropriate level
to be improved by supplementing AY in the diets (P = 0.09);
for maximal nitrogen utilisation.
whereas, other parameters were not significantly affected by AY
Yeast extracts contain immune-stimulating factors, most likely
supplementation.
as a function of b-glucans and a-mannans contained in the yeast
Weaning pigs must cope with several problems, such as diar-
cell wall. In a previous study, yeast b-glucans reduced proinflam-
rhoea, impaired growth performance and low feed intake.
matory cytokine levels while elevating the anti-inflammatory
Although Hahn et al. (2006) showed that supplementing the diet
cytokines in piglets challenged with Escherichia coli (Li et al.,
with yeast products positively affected the growth rate of weaned
2006). Hyun et al. (2005) reported that a high N/L ratio reflected
piglets, van der Peet-Schwering et al. (2007) did not find any
a weakened immune system (poor health) of an animal. A decrease
growth rate improvement in weaned piglets or young pig fed yeast
in neutrophils and the N/L ratio as well as an increase in eosino-
products. In the present study, there were no significant differ-
phils were observed following AY supplementation in our study.
ences in AGD, whereas FCR seemed to improve. Under the good
Elsewhere, the use of yeast products in diets decreased the level
feeding management conditions in the present study, no diarrhoea
of neutrophils in the plasma of piglets (van der Peet-Schwering
was observed, and the growth rate seemed to achieve the maximal
et al., 2007). Since the N/L ratio in the serum is directly related
genetic potential (ADG = 640–660 g/days). Therefore, the predom-
to both inflammation and stress in animals, a low N/L ratio caused
inant advantage in using dietary AY under these conditions may be
by AY supplementations (1.0% and 3.0% AY groups) indicates the
related to gut health or the immunological function rather than
benefit of AY regarding anti-stress and anti-inflammation.
any improvement in the growth rate.
Normally, at 28 days of age, concentrations in the range of 0.4–
3.2 mg/ml are used for IgG in pigs (Markowska-Daniel and
Blood profiles Pomorska-Mól, 2010). However, in the present study, the total
IgG in the control group was 5.13 mg/ml and declined from 3.53
The effects of AY on BUN, WBC and total IgG values in the blood to 3.82 mg/ml following AY supplementation. The normalisation
of piglets are presented in Table 3. Using AY at 1.0% of the diet sig- of the total IgG concentration by AY supplementation may be
nificantly decreased BUN compared to the control group. The neu- related to the potential decrease in the neutrophil count or the
trophil and neutrophil/lymphocyte ratio (N/L ratio) values were N/L ratio in the blood due to AY inclusion.
significantly reduced by AY supplementation (1.0% and 3.0% of In this study, the percentage of eosinophils was increased by
the diet; P < 0.05); whereas, the lymphocyte count tended to using AY at 1.0% in the diet, and this cell count was also negatively

Table 2
Effects of dietary autolysed yeast on growth performance of weaning pigs.

Items Control Autolysed yeast (%) SEM P-value

1.0 3.0
Initial weight (kg) 6.48 6.46 6.44 0.03 0.91
Prestarter pigs
BW (kg) 12.15 12.23 11.83 0.16 0.60
BWG (kg) 5.67 5.77 5.39 0.15 0.61
ADG (kg/day) 28.91 29.44 27.51 0.80 0.61
FI1 (kg/day) 0.47 0.46 0.47 0.01 0.51
FCR 1.16 1.16 1.14 0.02 0.91
Starter pigs
BW (kg) 32.28 33.03 32.15 0.31 0.47
BWG (kg) 20.14 20.79 20.32 0.26 0.59
ADG (kg/day) 0.77 0.80 0.78 0.01 0.59
FI (kg/day) 1.18 1.15 1.15 0.02 0.74
FCR 1.53 1.45 1.47 0.02 0.16
Overall
BWG (kg) 25.81 26.56 25.71 0.32 0.50
ADG (kg/day) 0.64 0.66 0.64 0.01 0.42
FI (kg/day) 0.93 0.91 0.90 0.01 0.61
FCR 1.45 1.37 1.40 0.01 0.09

Abbreviations: BWG = BW gain; ADG = average daily gain; FI = feed intake; FCR = feed conversion ratio.
1
as fed basis.

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S. Namted, K. Poungpong, W. Loongyai et al. Animal 16 (2022) 100660

Table 3
Effects of dietary autolysed yeast on blood urea nitrogen and haematological profiles of weaning pigs.

Item Control Autolysed yeast (%) SEM P-value

1.0 3.0
BUN (mg/dl) 13.60a 10.02b 11.76ab 0.56 0.02
Neutrophils (%) 43.60a 34.60b 34.00b 1.73 0.02
Lymphocytes (%) 49.60 56.20 60.00 1.95 0.07
N/L ratio 0.89a 0.63b 0.58b 0.05 0.03
Monocytes (%) 6.82 4.68 5.02 0.51 0.19
Eosinophils (%) 1.18a 3.98b 2.12ab 0.45 0.02
Basophils (%) 0.30 0.40 0.38 0.02 0.25
Total IgG (mg/ml) 5.13 3.82 3.53 0.37 0.18

Abbreviations: BUN = blood urea nitrogen; N/L ratio = neutrophil/lymphocyte ratio; IgG = immunoglobulin G.
a-b
Values within a row with different superscripts differ significantly at P < 0.05.

correlated with the BUN level (r =
0.86, P = 0.03). Similarly, Wang intestine are shown in Table 4. Supplementing AY at 1.0% signifi-
et al. (2017) reported that at 28 and 42 days of age, pigs fed yeast cantly increased villous height and villus height/crypt depth ratio
had cell walls with a higher eosinophil level compared to the con- in the segment of the duodenum (P < 0.05), and the ratio tended
trol group. The Interleukin 5 is one of the key cytokines involved in to increase in the segment of the jejunum (P = 0.07). In contrast,
the development of eosinophils (Kouro and Takatsu, 2009), and these parameters decreased in the ileum (P < 0.05). The density
adding AY to the diet increased the level of blood eosinophils, of goblet cells in the jejunum tended to increase the following sup-
which may be related to the production of Interleukin 5, which is plementation with AY at 1.0% of the diet (P = 0.09) compared to the
a key anti-inflammatory factor in the body. However, a high level control group. The inclusion of AY (1.0% and 3.0%) significantly
of AY supplementation (3.0% of diet) did not significantly affect increased the length and surface area of the duodenal segment
the number of eosinophils, although this white blood cell was (P < 0.05).
slightly elevated. Therefore, the reduction in neutrophils and the In the present study, villus height and villus height/crypt depth
N/L ratio and/or an increase in eosinophils following the use of ratio in the duodenum segment increased following AY supple-
1.0% AY in the diet indicate its anti-inflammatory properties. mentation. Considering the FCR, a higher villous height/crypt
depth ratio indicates greater digestive processing following AY
supplementation. In addition, the villus height in the jejunum
Small intestine morphology and size
was positively correlated with AY intake (r = 0.87, P = 0.03). Simi-
larly, Shen et al. (2009) reported that dietary yeast increased villus
The effects of AY supplementation on villous height, crypt
height and the villus/crypt ratio in the jejunum of weaned piglets;
depth, villous height/crypt depth ratio and surface area of the small

Table 4
Effects of dietary autolysed yeast on small intestinal morphology and anatomy of weaning pigs.

Item Control Autolysed yeast (%) SEM P-value

1.0 3.0
Villus height (lm)
Duodenum 322.57b 444.12a 358.21b 18.93 0.01
Jejunum 309.27 342.50 367.20 16.53 0.38
Ileum 328.20a 252.32b 209.81b 18.17 0.01
Crypt depth (lm)
Duodenum 144.54 139.65 150.42 7.29 0.85
Jejunum 149.09 145.76 153.39 4.97 0.84
Ileum 134.25 140.17 143.69 6.71 0.86
Villus height to crypt depth ratio
Duodenum 2.32b 3.19a 2.44b 0.16 0.03
Jejunum 2.09 2.37 2.43 0.12 0.07
Ileum 2.45a 1.81b 1.54b 0.13 0.01
2
Average goblet cell density (goblet cells per 10 000 lm villus area)
Duodenum 13.56 17.20 14.12 0.73 0.51
Jejunum 14.59 23.22 12.32 2.12 0.09
Ileum 21.44 21.48 19.90 1.77 0.93
Anatomy of small intestinal
Width (cm)
Duodenum 4.08 4.78 5.50 0.32 0.21
Jejunum 3.90 3.96 4.82 0.24 0.23
Ileum 4.50 4.80 5.10 0.22 0.58
Length (cm)
Duodenum 31.34b 46.78a 57.72a 4.07 0.01
Jejunum 1 656.60 1 458.80 1 560.60 50.43 0.29
Ileum 42.48ab 30.06ab 52.50a 3.59 0.02
Small intestinal 1 730.42 1 535.64 1 670.82 62.52 0.32
Surface area (cm2)
b
Duodenum 134.85 215.85a 326.08a 29.41 0.01
Jejunum 6 512.60 5 778.04 7 678.14 535.63 0.37
Ileum 192.66ab 141.44b 262.30a 18.26 0.01
a-b
Values within a row with different superscripts differ significantly at P < 0.05.

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S. Namted, K. Poungpong, W. Loongyai et al. Animal 16 (2022) 100660

however, van der Peet-Schwering et al. (2007) did not find any Gut microbiota play an important role in immune development
effect of yeast cell walls on the jejunal morphology in piglets. and nutrient use and maintain good health in animals. More than
The goblet cell density in the jejunum segment tended to 90% of the bacteria in the hind gut of neonatal piglets belong to
increase following feeding AY at 1.0% (an increase of approxi- the phyla Firmicutes, Bacteroidetes and Actinobacteria (Kiros
mately 59.15% compared to the control group). Sauerwein et al. et al., 2019). In the present study, Firmicutes, Bacteroidetes and
(2007) found that dietary mannan-oligosaccharides in the yeast Actinobacteria were predominant, accounting for 84.98%, 12.95%
cell wall increased the proliferation of goblet cells on the surface and 0.87% of the total sequences (98.80%), respectively. The other
of the villus membrane and subsequently improved the immune phyla contained only 1.20% of the total sequences. Only the abun-
system. This leads us to infer that AY supplementation may also dance of the phylum Actinobacteria (genus Collinsella) was signifi-
improve gut absorptive and defensive aspects by stimulating the cantly decreased by using AY at an inclusion level of 1.0%. A high
proliferation of goblet cells. abundance of the phylum Actinobacteria (genus Collinsella) has
Notably, in the anatomical study, the length in the duodenum been reported from feeding a high-fat, low-fibre diet; whereas,
segment was positively correlated with the AY intake (r = 0.93, the activities of Collinsella seemed to increase gut leakage and
P < 0.03); using AY in the diet increased the surface area of the duo- reduce tight junction proteins, which cause metabolic endotox-
denum segment. Intestinal length has been used as an indicator of emia (Gomez-Arango et al., 2018). Therefore, the reduction of this
the digestion and absorption capacity of the small intestine (Masri bacterial genus by using AY in the diet of weaning pigs may
et al., 2015). A high surface area of the small intestine with optimal improve gut functions via the development of gut morphology
enterocyte maturity is important to young pigs to achieve maxi- and an increase in goblet cells in the jejunum.
mum digestive and absorptive potential (Masri et al., 2015). There- Although the phylum Firmicutes, which is the dominant bacte-
fore, not only the morphology but also anatomical aspects may be ria in the gut, was not significantly influenced by AY supplementa-
related to the activities of microorganisms in the gut following AY tion, the abundances of the genera Clostridium and Catenibacterium
supplementation. in this phylum clearly decreased; whereas, the abundance of the
genus Marvinbryantia increased with the addition of AY at 1.0%.
Gut microbiota Generally, the primary virulence factors implicated in Clostridium-
associated disease are toxin A and toxin B. Toxin A has mild cyto-
Gut microbiota toxic activity, causing fluid accumulation in the intestine; whereas,
There were no significant differences (P > 0.05) between the AY toxin B is a potent cytotoxin (Yaeger et al., 2007). Hence, using AY
dietary supplementation and control group in the number of (1.0–3.0%) in the feed may also reduce factors inducing inflamma-
observed operating OTUs and a diversity indices (Supplementary tion by a reduction of the Clostridium population.
Fig. S1). Also, there was no significant difference in the dispersion On the other hand, the abundance of the genus Marvinbryantia
of microbial communities of caecum contents between treatment decreased at the AY inclusion level of 1.0%. In previous studies,
groups (P > 0.05). Marvinbryantia had anti-proinflammatory effects (Wang et al.,
Neither a-diversity nor b-diversity were significantly different 2018) and helped in mucosa regeneration (Nava and
with AY supplementation (Supplementary Fig. S2), indicating that Stappenbeck, 2011). Since the mucus layer lies between bacteria
AY did not have a significant effect on the diversity of microbiota in and host epithelial and immune cells, including AY in the diet
weaning pigs. may improve gut health by increasing the population of Marvin-
bryantia and, consequently, increasing the mucin production. This
Microbial community composition is supported by the tendency of goblet cells to increase in the jeju-
The effects of using AY in the diet on the microbiota in the cae- num when the piglets were fed AY at an inclusion level of 1.0%
cum are presented in Table 5. Supplementing AY at 1.0% of the diet (Table 5). However, a high level of AY supplementation (3.0% of
significantly decreased the abundance of the phylum Actinobacte- diet) did not affect the abundance of Marvinbryantia, in order to
ria (P = 0.04). Within this phylum, using AY (1.0% and 3.0% of the achieve the beneficial gut microbiota profile, this indicates that
diet) significantly decreased the abundance of Collinsella spp. the appropriate level of AY in diet must be carefully concerned.
(P = 0.01). In the phylum Firmicutes, the genera Clostridium The effects of AY supplementation on the population of short-
(P < 0.01) and Catenibacterium (P = 0.02) were significantly chain fatty acid-producing bacteria in piglets are shown in Table 6.
reduced; whereas, using AY at 1.0% of the diet significantly Supplementing AY at 1.0% of the diet tended to increase butyrate-
increased the population of the genus Marvinbryantia (P = 0.04) producing bacteria (P = 0.06), whereas, lactate-producing and
compared to the control group. propionate-producing bacteria were not significantly affected.

Table 5
Effects of dietary autolysed yeast on gut microbiota of weaning pigs at the phylum level.

Phylum Genus Control Autolysed yeast (%) SEM P-Value

1.0 3.0
Firmicutes 87.18 84.63 83.13 2.69 0.84
Clostridium 1.92a 0.26b 0.41b 0.25 <0.01
Dorea 0.99 0.61 0.57 0.08 0.07
Catenibacterium 0.84a 0.38b 0.37b 0.05 0.02
Marvinbryantia 0.13b 0.43a 0.07b 0.06 0.04
Bacteroidetes 10.92 13.18 14.77 2.53 0.84
Actinobacteria 1.16a 0.61b 0.83ab 0.09 0.04
Collinsella 0.78a 0.31b 0.40b 0.07 0.01
Euryarchaeota 0.42 0.30 0.25 0.04 0.33
Proteobacteria 0.19 0.94 0.72 0.18 0.21
Spirochaetes 0.03 0.14 0.12 0.03 0.54
Epsilonbacteraeota 0.01 0.04 0.07 0.02 0.67
a-b
Values within a row with different superscripts differ significantly at P < 0.05.

6
S. Namted, K. Poungpong, W. Loongyai et al. Animal 16 (2022) 100660

Table 6
Effects of dietary autolysed yeast on short-chain fatty acid-producing bacteria in caecum of weaning pigs.

Item Control Autolysed yeast (%) SEM P-value

1.0 3.0
Lactate-producing bacteria1 55.84 53.70 53.89 1.84 0.97
Propionate-producing bacteria2 1.24 1.10 0.90 0.09 0.18
Butyrate-producing bacteria3 16.32 22.93 17.83 1.24 0.06
1
Lactate-producing bacteria = Family Enterococcaceae + Lactobacillaceae + Leuconostocaceae + Streptococcaceae (Salvetti et al., 2013).
2
Propionate-producing bacteria = Genus Bacteroidaceae + Ruminococcus + Acidaminococcus + Christensenellaceae (Shimiz et al., 2018).
3
Butyrate-producing bacteria = Family Bifidobacteriales + Ruminococcaceae + Lachnospiraceae (Louis and Flint, 2017).

In general, intestinal microbiota produce short-chain fatty of Clostridium, although the lymphocyte count was negatively
acids, which positively influence the metabolism and physiology related to the abundance of this genus (r =
0.50; P = 0.04).
of the host. In 2020, van den Abbeele et al. reported that yeast cell Modulation of gut microbiota can improve the health and feed
walls increased the production of health-related acetate, propi- use of pigs via microbiota-generated metabolites (Richards et al.,
onate and butyrate in canines. High amounts of butyrate inside 2005). Using AY at 1.0% of the diet significantly increased the abun-
the caecum can benefit gut health, increase intestinal cell prolifer- dance of Marvinbryantia, and this genus was negatively related to
ation, provide more energy and promote the growth performance feed intake and the FCR. Zhu et al. (2020) found that the relative
of animals (Brown et al., 2018). In the present study, supplement- abundance of Marvinbryantia was positively correlated with the
ing AY at 1.0% of the diet seemed to increase the abundance of short-chain fatty acid concentration in the faeces of piglets at
butyrigenic bacteria (P = 0.06), which have been associated with 42 days of age, indicating that Marvinbryantia (in the phylum Fir-
the improvement of the intestinal morphology and an increased micutes) may use AY as substrate for growth to improve feed use
duodenal surface area. and, consequently, reduce feed intake. Accordingly, supplementing
Pearson’s correlations between the abundance of gut micro- AY at 3.0% did not increase the abundance of Marvinbryantia, while
biota (at the genus level) and yeast intake, growth performance the FCR was slightly increased when compared to 1.0% AY group.
or white blood cell count are shown in Table 7. The abundance of The relative abundance levels of the genera Catenibacterium and
the genus Dorea was negatively correlated with AY intake Clostridium were significantly decreased by AY supplementation;
(r =
0.84, P = 0.03), feed intake (r =
0.84, P = 0.03) and lympho- whereas, there was a positive correlation between these bacterial
cyte count (r =
0.64, P < 0.01) and positively related to the neu- populations and the neutrophil count. Reductions in the neutrophil
trophil count (r = 0.57, P = 0.03) and the N/L ratio (r = 0.61; count and N/L ratio, following AY supplementation, could have
P < 0.01). The Catenibacterium count was negatively correlated with been associated with the suppression of Catenibacterium and/or
the AY intake (r =
0.80; P < 0.04), but there was a positive corre- Clostridium in the gut. Shin et al. (2016) found that Catenibacterium
lation with the neutrophil count (r = 0.52; P = 0.04). Feed intake was more abundant in the high-fat diet of Koreans. Our recent
(r =
0.94; P < 0.01) and FCR (r =
0.80; P = 0.05) were negatively study showed that AY supplementation reduced fat deposition in
related with the abundance of Marvinbryantia. The abundance of fattening pigs (Namted et al., 2021). Thus, it seems that AY may
Collinsella paralleled an increase in the BUN (r = 0.72; P < 0.01), have suppressed the growth of Catenibacterium via some mecha-
neutrophil count (r = 0.86; P < 0.01) and N/L ratio (r = 0.88; nisms related to fat metabolism in the body.
P < 0.01); whereas, it was negatively related to lymphocyte Neutrophils are the principal cells that respond to infection by
(r =
0.79; P < 0.01) and eosinophil (r =
0.6; P < 0.01) levels. Clostridium difficile, with neutrophilic inflammation being the indi-
Finally, the neutrophil count (r = 0.60; P < 0.01) and N/L ratio cator of Clostridium difficile-related disease (Jose and Madan, 2016).
(r = 0.59; P = 0.02) increased with an increase in the abundance The present study identified a positive correlation between the
abundance of the genus Clostridium and neutrophils or the N/L
ratio, whereas supplementing AY in the diet significantly
decreased the concentrations of neutrophils and the N/L ratio in
Table 7 the blood, indicating that AY supplementation may have poten-
Correlations between relative abundance levels of microbial taxa with growth tially reduced the toxin produced by a reduction of the abundance
performance of weaning pigs at family and genus levels.
of the genus Clostridium.
Item Genus r1 P-value Using AY at 1.0% in the diet reduced the abundances of the phy-
Autolysed yeast intake Dorea
0.84 0.03 lum Actinobacteria and the genus Collinsella. This genus belongs to
Catenibacterium
0.80 0.04 the family Coriobacteriaceae, which are considered pathobionts,
Feed intake Dorea
0.84 0.03 with Collinsella being the dominant taxon (Chow et al., 2011).
Marvinbryantia
0.94 0.01
There was a positive correlation between the abundance of Collin-
Feed conversion ratio Marvinbryantia
0.80 0.05
Blood urea nitrogen Collinsella 0.72 <0.01
sella and BUN, neutrophil count and N/L ratio in the blood. Menni
Neutrophil Collinsella 0.86 <0.01 et al. (2021) found links between inflammatory cells and the genus
Catenibacterium 0.52 0.04 Collinsella in the gut. Samra et al. (2021) found that Collinsella was
Clostridium 0.60 <0.01 negatively related with total IgE or percent eosinophil in children
Dorea 0.57 0.03
with allergies. Supplementing AY at 1.0% of the diet significantly
Lymphocyte Collinsella
0.79 <0.01
Dorea
0.64 <0.01 increased the number of eosinophils; whereas, these white blood
Clostridium
0.50 0.04 cells were negatively correlated with the abundance of Collinsella.
Neutrophil/Lymphocyte ratio Collinsella 0.88 <0.01 Thus, using AY at 1.0% of diet may reduce proinflammatory cyto-
Clostridium 0.59 0.02
kine levels by increasing the eosinophil level in the blood via sup-
Dorea 0.61 <0.01
Eosinophils Collinsella
0.60 <0.01
pressing the abundance of Collinsella. Unfortunately, a high level of
AY supplementation (3.0% of diet) may reduce this potential, while
1
Correlation coefficient between genus levels and parameters based on repli- the exact mechanism is unclear.
cation (n = 6).

7
S. Namted, K. Poungpong, W. Loongyai et al. Animal 16 (2022) 100660

Conclusions Financial support statement

Using an antibiotic-free diet and good animal husbandry, sup- This research was supported by the Graduate Program Scholar-
plementation with AY in the diet seemed to improve feed use ship from The Graduate School (2/2563, 6217100151), Kasetsart
rather than the growth rate. Adding AY at 1.0% of the diet reduced University.
blood inflammation and stress indicators while developing gut
morphology and the anatomy. Finally, supplementing AY modu-
lated gut microbiota; in particular, it suppressed the abundance
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