Fish & Shellsh Immunology 56 (2016) 263e271

Contents lists available at ScienceDirect

Fish & Shellsh Immunology

journal homepage: www.elsevier.com/locate/fsi

Full length article

Effects of dietary live and heat-inactive bakers yeast on growth, gut

health, and disease resistance of Nile tilapia under high rearing
density
Chao Ran a, Lu Huang a, Jun Hu a, Philippe Tacon b, Suxu He a, Zhimin Li a, Yibing Wang a,
Zhi Liu a, Li Xu a, Yalin Yang a, Zhigang Zhou a, *
a
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, Peoples
Republic of China
b
Soci

et

e Industrielle Lesaffre, Phileo Lesaffre Animal Care, Marcq-en-Baroeul, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 5 April 2016
Received in revised form
19 May 2016
Accepted 4 July 2016
Available online 5 July 2016

In this study, the effects of bakers yeast as probiotics was evaluated in Nile tilapia reared at high density.
Juvenile tilapia were distributed to tanks at high density (436 sh/m3) and fed with basal diet (CK) or
diets supplemented with live (LY) or heat-inactivated yeast (HIY). Another group of sh reared at low
density (218 sh/m3) and fed with basal diet was also included (LowCK). After 8 weeks of feeding,
growth, feed utilization, gut microvilli morphology, digestive enzymes, and expressions of hsp70 and
inammation-related cytokines in the intestine were assessed. Intestinal microbiota was investigated
using 16S rRNA gene pyrosequencing. Fish were challenged with Aeromonas hydrophila to evaluate
disease resistance. High rearing density signicantly decreased the growth, feed utilization, microvilli
length, and disease resistance of sh (CK versus LowCK). Moreover, the intestinal hsp70 expression was
increased in sh reared at high density, supporting a stress condition. Compared to CK group, supplementation of live yeast signicantly increased gut microvilli length and trypsin activity, decreased intestinal hsp70 expression, and enhanced resistance of sh against A. hydrophila (reected by reduced
intestinal alkaline phosphatase activity 24 h post infection). The gut microbiota was not markedly
inuenced by either rearing density or yeast supplementation. Heat-inactivated yeast (HIY) didnt
display the benecial effects observed in LY except an increase in gut trypsin activity, suggesting the
importance of yeast viability and thus secretory metabolites of yeast. In conclusion, live bakers yeast
may alleviate the negative effects induced by crowding stress, and has the potential to be used as probiotics for tilapia reared at high density.
2016 Elsevier Ltd. All rights reserved.

Keywords:
Yeast
Nile tilapia
High density
Microbiota

1. Introduction
Tilapia is one of the most extensively cultured species in aquaculture with great economic importance, and the production of
tilapia has increased intensely to meet the growing global demand
for shery products [1]. China is the biggest country in tilapia
production, accounting for about 55% of the total production in the
world. In aquaculture systems, high stocking densities have been
used to increase productivity. However, intensive production systems of sh can cause different types of chronic stress, which can

* Corresponding author.
E-mail address: zhouzhigang03@caas.cn (Z. Zhou).
http://dx.doi.org/10.1016/j.fsi.2016.07.001
1050-4648/ 2016 Elsevier Ltd. All rights reserved.

affect the physiological homeostasis, growth rate, reproductive

performance, and immune system to produce sh that are more
susceptible to diseases [2].
Probiotics have received much attention as novel dietary supplements in aquaculture [3e5]. Probiotics have been used in
aquaculture to promote growth, modulate the immune system,
inhibit pathogen, and increase stress tolerance [6e9]. Moreover,
studies have demonstrated an improvement in tolerance to
crowding stress by dietary probiotics in gilhead seabream [10] and
Senegalese sole [11]. Bakers yeast, Saccharomyces cerevisiae, has
been used as probiotics in various aquatic species. Benecial effects
of bakers yeast as probiotics include growth promotion,
enhancement of innate immune response, as well as protection
against pathogens [12e17]. In our previous study, dietary

264

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

supplementation of bakers yeast, Saccharomyces cerevisiae,

improved the growth performance and gut microvilli morphology
of Nile tilapia reared in normal stocking density. Also, it reduced
hsp70 expression, down-regulated intestinal inammation status,
and protected the host against infection by Aeromonas hydrophila
[18]. Moreover, in the same study, comparison of live and heatinactivated yeast (devoid of secretory metabolites as secretion
was suggested to rely on viability) showed that secretory metabolites of yeast didnt play major roles in the growth promotion and
disease protection effects of yeast, while they were the major
contributor toward the improved gut morphology, relieved stress
status, and reduced intestinal inammation of Nile tilapia [18].
Nevertheless, the potential role of bakers yeast in crowded stress
situations has rarely been evaluated in sh, neither has the
contribution of yeast secretory metabolites in sh at crowded
conditions been investigated.
In this study, we investigated the benecial effects of bakers
yeast on tilapia reared in high density condition. Live bakers yeast
was prepared and supplemented to basal diet. A group supplemented with heat-inactivated yeast was also incorporated to
investigate the contribution of secretory metabolites of yeast in sh
under high density condition. The effect of live and heat-inactivated
bakers yeast on growth performance, gut morphology, digestive
enzyme activity, immune response, intestinal microbiota, as well as
disease resistance of Nile tilapia reared under high density was
evaluated.

2. Materials and methods

2.1. Experimental diets
The formulation and chemical composition of the experimental
diets (%) are presented in Table 1. The live bakers yeast was obtained as a commercial preparation Actisaf (Lesaffre, France).

Table 1
Formulations and chemical compositions of the experimental diets (%).
Ingredients
a

Fish meal
Corn gluten meal
Soybean meal
Cottonseed meal
Rapeseed meal
Wheat our
Soybean oil
Zeolite powder
CMCb
Vitamin C phosphate
Mineral Premix
Vitamin Premixc
Lysine sulphated
Calcium methionine hydroxy
Ca(H2PO4)2
Live yeast
Heat-inactivated yeast
Crude protein
Crude lipid
Crude ash
Crude ber
a

2.2. Fish and rearing conditions

All experimental and animal care procedures were approved by
the Feed Research Institute of Chinese Academy of Agricultural
Sciences Animal Care Committee, under the auspices of the China
Council for Animal Care (Assurance # 2012 ZZGCC01). MS-222 was
used as the anaesthetic. Nile tilapia, Oreochromis niloticus (L.), ngerlings were obtained from a local aquaculture farm in Beijing,
China. The sh were acclimatized in plastic aquaria
(50  30  38 cm) in a recirculation system (at 0.5 L/min) for at
least 2 weeks before the feeding trial. After the acclimatization, sh
with similar size (9.8 0.04 g) were randomly chosen and
distributed into 12 tanks at a density of 24 sh per tank (436 sh/
m3). Fish were fed with basal diet (CK) or diets supplemented with
live (LY) or heat-inactivated yeast (HIY), with each diet randomly
assigned to four tanks. Another 4 tanks were loaded with sh at 12
sh per tank (218 sh/m3) and fed with basal diet (LowCK). The sh
were handfed to apparent satiation three times daily (8:00, 11:30
and 17:30). The rearing conditions were same as described by
Huang et al. [19]. The feeding trial was conducted for 8 weeks.
2.3. Growth performance and sampling

CK/LowCK

LY

HIY

5.00
4.00
28.00
10.00
20.00
24.00
4.40
1.48
0.20
0.050
0.20
0.20
0.19
0.28
2.00
0
0
32.36
6.83
5.50
6.84

5.00
4.00
28.00
10.00
20.00
24.00
4.40
1.38
0.20
0.050
0.20
0.20
0.19
0.28
2.00
0.10
0
32.36
6.83
5.50
6.84

5.00
4.00
28.00
10.00
20.00
24.00
4.40
1.38
0.20
0.050
0.20
0.20
0.19
0.28
2.00
0
0.10
32.36
6.83
5.50
6.84

Domestic sh meal. Tianshen Corporation, Tangshan, China.

Carboxymethylcellulose sodium, as feed adhesive.
c
Vitamin premix (g/kg): thiamine, 0.438; riboavin, 0.632; pyridoxine$HCl,
0.908; d-pantothenic acid, 1.724; nicotinic acid, 4.583; biotin, 0.211; folic acid,
0.549; vitamin B-12, 0.001; inositol, 21.053; menadione sodium bisulte, 0.889;
retinyl acetate, 0.677; cholecalciferol, 0.116; dl-a-tocopherol-acetate, 12.632.
d
Mineral premix (g/kg): CoCl2$6H2O, 0.074; CuSO4$5H2O, 2.5; FeSO4$7H2O, 73.2;
NaCl, 40.0; MgSO4$7H2O, 284.0; MnSO4$H2O, 6.50; KI, 0.68; Na2SeO3, 0.10;
ZnSO4$7H2O, 131.93; Cellulose, 501.09.
b

Heat-inactivated yeast was prepared by incubating live yeast at

80
C for 30 min. The inactiveness was conrmed by culturing on
YPD agar, and there were no yeast colony growing on the plates.
The basal diet was supplemented with live yeast or its heatinactivated counterpart. The procedures of feed preparation have
been described by Huang et al. [19]. Briey, the dietary ingredients
were blended with water (100 ml water per 1 kg diet) to form a
paste which was then passed through a meat grinder equipped
with a 3-mm die to obtain uniform pellets. The pelleted diets were
air-dried till the moisture was lower than 10%. The levels of
S. cerevisiae in diets supplemented with live yeast were veried by
pellet homogenization, serial dilution and spreading of the dilutions on YPD plates. All the diets were stored in plastic bags at
4
C until use. The viability of the supplemented live yeast during
storage was conrmed by spread plate method described above.

After 8 weeks feeding, all the sh were batch weighed and

counted after 24 h starvation, and then feeding schedules
continued at least 5 days before sampling. Four sh were randomly
sampled from each tank and anaesthetised with MS-222 (50.0 mg/
l). The intestine and head kidney were sampled. For three of the
sampled sh, a piece of the midgut (~1 cm) was cut and gently
agitated three times in PBS (pH 7.2) for 1 min to remove the digesta
for intestinal mucosal morphology investigation; the rest of midgut
was washed the same way and used for RT-PCR analysis. The
hindgut was used for analysis of autochthonous and allochthonous
microbiota [18]. The whole intestine from another sh was
immediately frozen in liquid nitrogen and stored at 80
C for
digestive enzyme activity assay [20]. Survival rate % (SR), Weight
gain (WG), Feed conversion ratio (FCR) were calculated as described
previously [18].
2.4. Digestive enzyme activity in intestine
The whole gut of sh were homogenized in ice-cold PBS and
centrifuged at 30,000 g for 30 min at 4
C [20]. The supernatant was
collected. The digestive enzyme activity (amylase, lipase, trypsin)
was tested by kits following the manufacturers specications
(Nanjing Jiancheng Bioengineering Institute, China; Kit C016-1,
A054, A080-2 for amylase, lipase, and trypsin, respectively). The
enzymatic activity was expressed as U/mg protein. The concentration of total protein was measured using a BCA Protein Assay Kit

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

(Novagen, Darmstadt, Germany) following the manufacturers

specications described by Qin et al. [21].
2.5. Intestinal mucosal morphology
The intestine sample were processed for scanning electron microscopy (SEM) and transmission electron microscope (TEM)
analysis according to the methods described by Liu et al. [22]. TEM
images (magnication  30,000) were analyzed to measure the
microvilli length. SEM images (magnication  20,000) were used
to measure the microvilli density. The details for data calculation
and processing were described previously [18].
2.6. Expression of hsp70 and immune-related cytokine genes
Total RNA of intestine and kidney was extracted from each of the
three sh sampled from each tank as described above using a
TRIzon Reagent RNA kit (Promega, Germany). The integrity of total
RNA was veried by visualization on a 1.2% agarose gel. RNA was
dissolved in 50 ml RNase-free water and stored at 70
C until use.
cDNA was synthesized for quantitative reverse transcription PCR
(RT-qPCR) using the ReverTra Ace-a-RT-PCR kit (TRAN, Beijing,
China) according to the manufacturers instructions. The qPCR
primers were designed using Primer 5.0 software based on the
available cytokine sequences in GenBank (S1 Table). Additional
dissociation curve analysis was performed and a single melting
curve was observed in all cases. The qPCR was performed with the
SYBR Green SuperReal PreMix Plus (TIANGEN, Beijing, China) in
StepOne Software V2.2 real-time PCR Detection system (ABI,
Beijing, China). The total volume of the PCR reactions was 20 ml and
consisted of 10 ml SYBR Green SuperReal PreMix Plus (2), 1 ml
primer of each, 2 ml cDNA, and 6 ml RNase-free H2O. The cycling
conditions were as follows: 95
C for 10 min and then 40 cycles of
95
C for 20 s, 58
C for 20 s, and 72
C for 20 s. All qPCRs were
performed at least two times. Data analysis was conducted using
the 2DDCT method [23] and the b-actin gene was chosen as the
internal standard.
2.7. Gut alkaline phosphatase activity 24 h after challenge with
Aeromonas hydrophila NJ-1
After the feeding trial, four replicate tanks of each dietary
treatment were infused with pathogenic strain A. hydrophila NJ-1 at
a concentration of 108 CFU/ml. After 24 h, 2 sh were randomly
chosen from each tank and anaesthetised with MS-222 (50.0 mg/l).
The whole intestine was sampled and gently agitated three times in
PBS (pH 7.2) for 1 min to remove the digesta. The alkaline phosphatase activity was determined according to Qin et al. [21].
2.8. Gut autochthonous and allochthonous bacterial communities
The gut wall and content of the hindgut were separated for
analysis of autochthonous and allochthonous microbiota, as
described by Ran et al. [18]. In brief, the DNA was extracted from
samples according to Liu et al. [24]. Bacterial 16S rRNA genes were
amplied by PCR from DNA samples using a range of V3eV4
oligonucleotide primers specic for bacteria. We amplied a standard 450 bp V3eV4 region using the modied primers 349F and
806R. All PCR products were checked for size and specicity by
electrophoresis on 1.2% w/v agarose, gel puried and adjusted to
10 ng/ml using molecular grade water and pooled equally for subsequent sequencing. V3eV4 amplicons were sequenced using pairend method by Illumina Hiseq2500. The sequence reads have been
submitted to European Nucleotide Archive (ERA) (accession number PRJEB12551).

265

The raw data was processed and trimmed by FastQC and NGS
toolkits. Sequences were analyzed in UCLUST v1.2.22 to determine
Operational Taxonomic Units (OTU), which was dened as
comprising sequences with less than 3% difference by the furthestneighbor method. Taxon annotation was conducted using one
representative sequence from an OTU by RDP classier referring to
GreenGenes database. The relative abundance of OTUs was calculated, and microbial composition in different taxonomic levels was
analyzed for each group. Heatmaps for the top 10 abundant genera
were made for both autochthonous and allochthonous microbiotas.
2.9. Statistical analysis
Data are expressed as mean values SE. Data were analyzed by
one way analysis of variance (ANOVA) with Duncans post hoc test.
Normality was checked by KeS test and QeQ plot. Variance homogeneity of the data was examined with Levenes test. All the
analyses were conducted by SPSS 17.0 (SPSS, Inc.), and differences
with P < 0.05 were considered signicant.
3. Results
3.1. Effect of rearing density and yeast on production
Effects of the dietary treatments on the production of juvenile
Nile tilapia after 8 weeks of feeding are displayed in Table 2. The
growth and feed utilization were decreased in sh reared at high
density (CK) compared to those at normal density (LowCK)
(P < 0.05). Under the high density condition, no signicant improvements were observed in the growth performance and feed
utilization of tilapia fed with diets supplemented with the live or
heat-inactivated yeast compared with the control group (CK).
3.2. Effect of rearing density and yeast on gut morphology
No signicant difference was observed in the midgut microvilli
density of sh reared at different densities or dietary groups
(Table 3). The microvilli length was decreased in sh reared at high
density compared to LowCK (P < 0.05). Supplementation of LY
increased microvilli length of sh reared at high density (compared
to CK) (P < 0.05) (Fig. 1 & Table 3). The microvilli length of sh in LY
group was similar to that of LowCK sh. HIY supplementation
didnt improve the microvilli length of sh compared to the CK
group (Fig. 1 & Table 3).
3.3. Gut digestive enzyme activity
No signicant difference was observed for the amylase and
lipase activity in all treatments (P > 0.05). However, the trypsin
activity in LY and HIY groups were higher than that in CK and
LowCK (P < 0.05) (Fig. 2).
Table 2
Effects of the experimental treatments on weight gain, feed conversion ratio, and
survival rates of tilapias at the end of the feeding period.a
Treatments

LowCK

CK

LY

HIY

FBW (g)
WG (%)
FCR
SR (%)

32.7 2.8b
226 17b
1.71 0.06b
94.4 4.8

28.2 1.5a
191 16a
1.85 0.14a
96.9 3.9

27.8 1.5a
185 16a
1.89 0.10a
94.8 5.2

28.5 1.3a
192 14a
1.81 0.11a
95.8 5.9

a
Values are means SEMs (n 4). Means sharing a common letter were not
signicantly different (P > 0.05). IBW, initial body weight; FBW, nal body weight;
WG, weight gain; FCR, feed conversion ratio; SR, survival rate; LowCK, low stocking
density control; CK, high stocking density control; LY, live yeast group at high
density; HIY, heat-inactivated yeast group at high density.

266

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

Table 3
Effects of rearing density and yeast on intestinal microvilli length and density of Nile
tilapia.a

Length (mm)
Density (count/mm2)

LowCK

CK

LY

HIY

1.06 0.01a
110 5.3a

0.94 0.02b
112 7.1a

1.10 0.01a
120 5.7a

0.95 0.02b
109 3.7a

a
Values are means SEMs (n 4). Means sharing a common letter were not
signicantly different (P > 0.05). LowCK, low stocking density control; CK, high
stocking density control; LY, live yeast group at high density; HIY, heat-inactivated
yeast group at high density.

3.4. Expression of hsp70 in intestine and head kidney

The intestinal hsp70 expression was lower in the low stocking
condition (LowCK) compared with high stocking control (CK)

(P < 0.05) (Fig. 3). Live yeast decreased the intestinal expression of
hsp70, to a level similar to that in LowCK. However, the inactive
yeast had no signicant effect on hsp70 expression (P > 0.05),
although the value was numerically lower (Fig. 3). In head kidney,
the hsp70 expression was lower in the live yeast group compared to
the high density control (CK) (P < 0.05), while no signicant difference was observed between the inactive yeast group and CK (S1
Fig).
3.5. Expression of immune-related cytokine genes in intestine and
head kidney
No difference was observed in the expression of intestinal
cytokine genes among different groups (P > 0.05) (Fig. 4). In head
kidney, there was no signicant difference in the expression of tnf-a
and tgf-b among groups (P > 0.05). Nevertheless, the expression of

Fig. 1. Electron microscope images of the gut microvilli. (A) The TEM images for microvilli length; (B) The SEM images of the microvilli density. LowCK, low stocking density control;
CK, high stocking density control; LY, live yeast group at high density; HIY, heat-inactivated yeast group at high density.

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

267

Fig. 2. Effects of rearing density and yeast on intestinal digestive enzymes activity of tilapias after 8 weeks feeding. (A) amylase, (B) lipase, (C) trypsin. Values are means SEMs.
Means sharing a common superscript letter were not signicantly different (P > 0.05). LowCK, low stocking density control; CK, high stocking density control; LY, live yeast group at
high density; HIY, heat-inactivated yeast group at high density.

il-1b in LY was higher compared to the other three groups (CK, HIY,
LowCK) (P < 0.05) (S2 Fig).
3.6. Resistance against A. hydrophila
The gut alkaline phosphatase activity was positively correlated

Fig. 3. Effects of rearing density and yeast on hsp70 gene expression in intestine of
tilapias after 8 weeks feeding. Values are means SEMs. Means sharing a common
superscript letter were not signicantly different (P > 0.05). LowCK, low stocking
density control; CK, high stocking density control; LY, live yeast group at high density;
HIY, heat-inactivated yeast group at high density.

with mortality of tilapia after challenge by A. hydrophila [25]. Protection of the sh against pathogen can be reected by lower gut
alkaline phosphatase activity after challenge [25]. The alkaline
phosphatase activity of sh reared at high density (CK) was higher
than LowCK sh after challenge by A. hydrophila NJ-1 (P < 0.05),
pointing to a lower resistance against A. hydrophila infection
(Fig. 5). Compared to the CK group, LY reduced the gut alkaline

Fig. 5. Effects of rearing density and yeast on gut alkaline phosphatase activity of tilapias 24 h post challenge by Aeromonas hydrophila NJ-1. Values are means SEMs.
Means sharing a common superscript letter were not signicantly different (P > 0.05).
LowCK, low stocking density control; CK, high stocking density control; LY, live yeast
group at high density; HIY, heat-inactivated yeast group at high density.

Fig. 4. Effects of rearing density and yeast on gut immune-related cytokine expression of tilapias after 8 weeks feeding. (A) il-1b, (B) tnf-a, (C) tgf-b. Values are means SEMs. Means
sharing a common superscript letter were not signicantly different (P > 0.05). LowCK, low stocking density control; CK, high stocking density control; LY, live yeast group at high
density; HIY, heat-inactivated yeast group at high density.

268

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

phosphatase activity of sh after challenge (P < 0.05), indicating a

protection effect, while there was no signicant difference in HIY
compared to CK (P > 0.05) (Fig. 5).
3.7. Gut autochthonous and allochthonous bacterial communities
For the autochthonous bacterial communities, the observed
OTUs ranged from 1055 to 1193 for the four treatments. Fusobacteria and Proteobacteria were the dominant phyla in all the microbiotas. In the genus level, Cetobacterium was the dominant group
for each treatment, representing 34%e54% abundance of the total
microbiota in different samples. PCoA of weighted UniFrac distances demonstrated that the microbiotas from different groups
were closely clustered (S3 Fig), indicating that rearing density and
yeast supplementation didnt markedly inuence the autochthonous microbiota. Hierarchical clustering of the microbiotas based
on the top 10 abundant genera showed that the microbiotas were
well separated by density, i.e., LowCK versus LY/HIY/CK (Fig. 6).
Moreover, relative to CK, the alteration of microbiota by LY was
more profound than HIY.
The observed OTUs in the allochthonous microbiotas were between 5117 and 5321 for the four treatments. Fusobacteria, Proteobacteria and Actinobacteria were the dominant phyla in all the
microbiotas. Cetobacterium was the dominant genus in all the

Fig. 6. Heatmap showing the relative abundance of the top 10 genera of the microbiota. The gure describes autochthonous microbiota (A) and autochthonous microbiota (B) of Nile tilapia after 8 weeks of feeding. The microbial proles of the 4 groups
were clustered by complete linkage method. LowCK, low stocking density control; CK,
high stocking density control; LY, live yeast group at high density; HIY, heat-inactivated
yeast group at high density.

treatment groups, representing 37%e41% of the total microbiota in

abundance. Similarly, the allochthonous microbiotas from different
groups were closely clustered on the PCoA plot of weight UniFrac
distances, pointing to minor changes in the microbiota by rearing
density and yeast supplementation. Hierarchical clustering also
showed well-separated microbiotas by rearing density, and more
profound alteration of the microbiota by LY compared to HIY under
the high density condition (Fig. 6).
4. Discussion
Results about the effects of high stocking densities on sh production have been divergent, probably depending on the sh species investigated. While adverse effects of high stocking densities
on growth have been reported in halibut (Paralichthys californicus)
[26], turbot (Scophthalmus maximus) [27], and Dover sole (Solea
solea) [28], a null effect of high densities on production was
observed in Senegalese sole (Solea senegalensis) [29], wedge sole
(Dicologlossa cuneata) [30], winter ounder (Pseudopleuronectes
americanus) [31]. In this study, the growth performance of tilapia
was signicantly reduced at the high density condition, which is in
agreement with previous results observed in this species reared at
high stocking density [32,33]. The crowding stress caused by high
density was reected by increased expression of hsp70 in the intestine and kidney. The stress may enhance energy reserves consumption and reallocation of metabolic energy, which interferes
negatively with growth of sh [34]. In our previous study, dietary
supplementation of live bakers yeast improved the growth and
feed utilization of tilapia reared under normal stocking density;
marginal improvement in growth performance was registered in
the heat-inactivated yeast group [18]. Moreover, growth promotion
effect has been reported in various sh species reared at normal
density, upon dietary supplementation of either live [12,14e17] or
inactive yeast [35,36]. However, no improvements in the growth
performance of sh at the high stocking density were observed
upon yeast supplementation in this study. This might be due to that
the growth promotion effect of yeast was counteracted by the energy mobilization induced by crowding stress.
It has been reported that the intestinal villus height and diameter were signicantly decreased in Senegalese sole under high
stocking density [11], indicating that the crowding stress can impair
the digestive function of sh intestine. In this study, the microvillus
length and density were used as the morphology parameters
reecting intestinal health. The microvilli length was signicantly
lower in sh reared at high stocking density compared to those at
low density, which was in agreement with the previous result in
Senegalese sole [11], indicating that high stocking density can
compromise intestinal health by impairing the microscopic
morphology. Live yeast signicantly improved the microvilli length
of tilapia under high stocking density while heat-inactivated yeast
didnt. This result was consistent with our previous one that live
yeast improved the microvilli morphology of tilapia at normal
stocking density while heat-inactivated yeast was devoid of this
activity [18], indicating that the benecial effect of yeast on
microvillus are not impacted by the crowding stress.
The inuence of crowding stress on the digestive enzymes activities of sh was rarely reported. The results in this study indicated that there was no difference in the amylase, lipase and trypsin
activity of tilapia reared at high or normal stocking densities.
Nevertheless, the trypsin activity of sh at high density was
improved by live and heat-inactivated yeast. Previous studies reported that dietary S. cerevisiae enhanced alkaline phosphatase and
disaccharidase activities in Nile tilapia fry [37] and amylase activity
in juveniles [38]. The activation of different digestive enzymes
compared to our study might be due to differences in diet and

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

rearing conditions. Costas et al. reported that crowding stress

increased amino acids requirements in Senegalese sole, which was
attributed to higher demand for energy production to cope with the
stressful conditions, higher rate of protein synthesis, or synthesis of
other important metabolites related to stress response [39]. In this
regard, the enhanced trypsin activity by yeast could increase protein digestibility and putatively free amino acids level in the
plasma, which may improve the tolerance of sh to crowding
stress.
Heat shock proteins (HSPs) are highly conserved molecular
chaperones. They protect the cells by preventing protein aggregation, facilitating folding of newly and damaged protein, and targeting misfolded proteins to specic degradative pathways. In
particular, HSP70 have been used as biomarkers of stress in aquatic
animals [40]. Generally, hsp70 expression decreases in sh given
probiotics/prebiotics and coincides with increased welfare of sh
[41e43]. The expression of hsp70 was increased after a crowding
stress in the liver and blood of sea bass [44] and Atlantic cod [45],
respectively. Consistent with these results, the hsp70 expression
was signicantly up-regulated in the intestine of tilapia under
crowding condition, indicating a cellular stress process in the
digestive tract of sh. Live yeast supplementation signicantly
decreased the intestinal hsp70 expression of sh under crowding
condition. The hsp70 expression level in live yeast group was
comparable to that in sh reared at normal stocking density, suggesting that live yeast supplementation improved the crowding
stress tolerance of tilapia and alleviated the cellular stress response
in the intestine of sh. Heat-inactivated yeast didnt decrease hsp70
expression of sh reared at high stocking density, which is
consistent with previous results observed in sh reared at normal
stocking density [18], conrming the importance of yeast viability
in the benecial effect regarding hsp70 expression.
Crowding stress has been previously associated with an immune
depression effect in sh, inuencing the resistance against infection in some cases [11,27,46]. However, the intestinal expression of
il-1b and tnfa was not signicantly affected by crowding stress in
this study. Both of the cytokines are the biomarker immune regulators and activate lymphocytes, macrophages, and natural killer
cells, which result in subsequent enhancement of immune
response [47]. In this regard, specimens under the crowding condition didnt show immune depression. However, the intestinal
expression of cytokines is local immune response, which doesnt
completely reect the systemic immune status. The disease resistance of tilapia reared at high stocking density was signicantly
lowered compared to the LowCK group, suggesting an overall
compromised immune status of sh under crowding condition.
This suggests that immune parameters other than those correlated
with the intestinal cytokines might have been impaired by
crowding stress. Live yeast supplementation signicantly improved
disease resistance of sh under crowding condition. In contrast,
heat-inactivated yeast failed to have such a benecial effect. In
previous study, both live and dead yeast signicantly improved
disease resistance of tilapia reared at low density [18]. The
discrepancy of results in the two studies suggested an interaction
between the inuence of yeast viability and stocking density
regarding the effect of yeast on disease resistance of sh.
Our result showed that high rearing density didnt markedly
change the intestinal microbiotas (both autochthonous and
allochthonous) of tilapias. Consistent with our result, the variations
in rearing density resulted in only minor changes in intestinal
microbiota composition of rainbow trout despite signicant effects
on sh health [48]. The alteration of microbiota by live yeast was
more profound than dead yeast, implying the involvement of
secreted metabolites in alteration of the microbiota. The functional
implication of the changed microbiota in LY group is unknown.

269

Putatively, this alteration in microbiota could be either contributing

factor or a paralleling result of the benecial effects of live yeast in
tilapia. Liu et al. reported that changes in intestinal hsp70 expression were coincident with the scale of autochthonous microbiota
alteration in tilapia [49]. In this study, the difference in hsp70
expression relative to CK was more signicant in LY compared to
HIY, which was in accordance with a more profound alteration of
microbiota by LY versus HIY (relative to CK), supporting the correlation between hsp70 expression and the alteration of intestinal
autochthonous microbiota. We observed that Cetobacterium somerae was the predominant in the gut microbiota of tilapia, constituting around 30%e50% of the microbiome in all treatments. It has
been reported that C. somerae is an indigenous bacterium in the
intestinal tract of freshwater sh including goldsh, common carp,
tilapia and ayu [50]. This bacterium produced vitamin B12 at high
efciency, and its abundance in the intestine involved with the
requirement of sh for vitamin B12 [51]. The abundance of
C. somerae in autochthonous microbiota was markedly reduced in
LY compared to other groups, which might have contributed much
to the larger dissimilarity (UniFrac distance) of LY microbiota to the
microbiotas of other treatments (S3 Fig). In view of its high abundance, the function of C. somerae in the intestine of tilapia deserves
further investigation.
The advantage of live yeast compared to dead yeast reects the
contribution of secretory metabolites, as the secretion of metabolites by yeast was suggested to rely on yeast viability [52]. The inuence of yeast viability, i.e., the contribution of secretory factors,
to the probiotic effects has been thoroughly investigated in Nile
tilapia reared under normal density [18]. In this study, live yeast
showed advantage in improving the microvillus morphology and
alleviating the intestinal stress (reduced hsp70 expression), which
was in agreement with previous study. This conrmed the contribution of secretory metabolites to those benecial effects, and
indicated that such contribution was not impacted by crowding
stress. Nevertheless, the effect of yeast viability on disease resistance of sh was enlarged under high stocking density, as only live
yeast improved sh disease resistance. This suggested that the
contribution of yeast secreted components to disease resistance of
sh might be inuenced by stocking density, with a larger contribution in sh under crowding conditions. This advantage of live
yeast might be of particular relevance in the aquaculture eld,
where a chronic crowding stress was prevalent. On the other hand,
the complex secretory metabolites of live yeast may contain some
components that exert negative effects on growth, which may
counteract the positive contributions expected from the observed
benecial effects of live yeast, such as improved microvilli length,
decreased hsp70 expression, and higher trypsin activity. The
increased pro-inammatory factor il-1b expression in head kidney
in LY group might be such an effect negatively affecting growth.
Moreover, the effect of intestinal microbiota induced by live yeast
supplementation on growth of tilapia deserves further investigation. Future research with new technologies in metabonomics,
microbial engineering, and gnotobiotic sh will help to answer
these questions.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (31272672), Key Project of Chinese National
Programs for Fundamental Research and Development (973 Program) (2015CB150605), the National Science and Technology Support Program Project of China (2012BAD25B02), and Beijing
earmarked fund for Modern Agro-industry Technology Research
System (SCGWZJ 20151104-4).We would like to thank Yang Deng
for the sh work.

270

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.fsi.2016.07.001.
References
[1] FAO, World Aquaculture Production by Inland and Marine Waters, 2015.
[2] G.S. Telli, M.J.T. Ranzani-Paiva, D. de Carla Dias, F.R. Sussel, C.M. Ishikawa,
L. Tachibana, Dietary administration of Bacillus subtilis on hematology and
non-specic immunity of Nile tilapia Oreochromis niloticus raised at different
stocking densities, Fish Shellsh Immunol. 39 (2) (2014) 305e311.
[3] A. Irianto, B. Austin, Probiotics in aquaculture, J. Fish Dis. 25 (11) (2002)
633e642.
[4] A. Newaj-Fyzul, A. Al-Harbi, B. Austin, Review: developments in the use of
probiotics for disease control in aquaculture, Aquaculture 431 (2014) 1e11.
[5] D. Merrield, E. Ring, Aquaculture Nutrition: Gut Health, Probiotics and
Prebiotics, Wiley-Blackwell, New Jersey, 2014.
[6] L. Verschuere, G. Rombaut, P. Sorgeloos, W. Verstraete, Probiotic bacteria as
biological control agents in aquaculture, Microbiol. Mol. Biol. Rev. 64 (4)
(2000) 655e671.

zar, I. De Blas, I. Ruiz-Zarzuela, D. Cunningham, D. Vendrell,
[7] J.L. Balca
J.L. Mzquiz, The role of probiotics in aquaculture, Vet. Microbiol. 114 (3)
(2006) 173e186.
[8] A. Kesarcodi-Watson, H. Kaspar, M.J. Lategan, L. Gibson, Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes, Aquaculture 274 (1) (2008) 1e14.
[9] Y.-B. Wang, Z.-Q. Tian, J.-T. Yao, W.-F. Li, Effect of probiotics, Enteroccus faecium, on tilapia (Oreochromis niloticus) growth performance and immune
response, Aquaculture 277 (3) (2008) 203e207.

n-Rubio, I. Garca[10] J. Varela, I. Ruiz-Jarabo, L. Vargas-Chacoff, S. Arijo, J. Leo
Mill

an, et al., Dietary administration of probiotic Pdp11 promotes growth and
improves stress tolerance to high stocking density in gilthead seabream
Sparus auratus, Aquaculture 309 (1) (2010) 265e271.


J. Jurado, H. Cordero, et
[11] S. Tapia-Paniagua, S. Vidal, C. Lobo, M. Prieto-Alamo,
al., The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their
resistance to disease, Fish Shellsh Immunol. 41 (2) (2014) 209e221.

ndez, W. Lo

pez-Madrid,
[12] M. Lara-Flores, M.A. Olvera-Novoa, B.Z.E. Guzm

an-Me
Use of the bacteria Streptococcus faecium and Lactobacillus acidophilus, and
the yeast Saccharomyces cerevisiae as growth promoters in Nile tilapia
(Oreochromis niloticus), Aquaculture 216 (1) (2003) 193e201.
~ o, A. Cuesta, A. Rodrguez, M.A. Esteban, J. Meseguer, Oral adminis[13] J. Ortun
tration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream (Sparus aurata L.), Vet. Immunol.
Immunopathol. 85 (1) (2002) 41e50.
[14] P. Li, D.M. Gatlin, Evaluation of brewers yeast (Saccharomyces cerevisiae) as a
feed supplement for hybrid striped bass (Morone chrysops  M. saxatilis),
Aquaculture 219 (1) (2003) 681e692.
[15] M. Abdel-Tawwab, A.M. Abdel-Rahman, N.E. Ismael, Evaluation of commercial
live bakers yeast, Saccharomyces cerevisiae as a growth and immunity promoter for fry Nile tilapia, Oreochromis niloticus (L.) challenged in situ with
Aeromonas hydrophila, Aquaculture 280 (1) (2008) 185e189.
[16] M. Abdel-Tawwab, M.A. Mousa, M.A. Mohammed, Use of live bakers yeast,
Saccharomyces cerevisiae, in practical diet to enhance the growth performance
of Galilee tilapia, Sarotherodon galilaeus (L.), and its resistance to environmental copper toxicity, J. World Aquacult. Soc. 41 (s2) (2010) 214e223.
[17] C.-H. Chiu, C.-H. Cheng, W.-R. Gua, Y.-K. Guu, W. Cheng, Dietary administration of the probiotic, Saccharomyces cerevisiae P13, enhanced the growth,
innate immune responses, and disease resistance of the grouper, Epinephelus
coioides, Fish Shellsh Immunol. 29 (6) (2010) 1053e1059.
[18] C. Ran, L. Huang, Z. Liu, L. Xu, Y. Yang, P. Tacon, et al., A comparison of the
benecial effects of live and heat-inactivated bakers yeast on Nile Tilapia:
suggestions on the role and function of the secretory metabolites released
from the yeast, PloS One 10 (12) (2015) e0145448.
[19] L. Huang, C. Ran, S. He, P. Ren, J. Hu, X. Zhao, et al., Effects of dietary
Saccharomyces cerevisiae culture or live cells with Bacillus amyloliquefaciens
spores on growth performance, gut mucosal morphology, hsp70 gene
expression, and disease resistance of juvenile common carp (Cyprinus carpio),
Aquaculture 438 (2015) 33e38.

rez-Jime

nez, F. Coutinho, P. Pousa
~o-Ferreira, T.M. Branda
~o,
[20] C. Castro, A. Pe
A. Oliva-Teles, H. Peres, Digestive enzymes of meagre (Argyrosomus regius)
and white seabream (Diplodus sargus): effects of dietary brewers spent yeast
supplementation, Aquaculture 416e417 (2013) 322e327.
[21] C. Qin, L. Xu, Y. Yang, S. He, Y. Dai, H. Zhao, et al., Comparison of fecundity and
offspring immunity in zebrash fed Lactobacillus rhamnosus CICC 6141 and
Lactobacillus casei BL23, Reproduction 147 (1) (2014) 53e64.
[22] W. Liu, Y. Yang, J. Zhang, D.M. Gatlin, E. Ring, Z. Zhou, Effects of dietary
microencapsulated sodium butyrate on growth, intestinal mucosal
morphology, immune response and adhesive bacteria in juvenile common
carp (Cyprinus carpio) pre-fed with or without oxidised oil, Brit. J. Nutr. 112
(01) (2014) 15e29.
[23] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]
[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

real-time quantitative PCR and the 2DDCT method, Methods 25 (4) (2001)
402e408.
Y. Liu, Z. Zhou, B. Yao, S. He, L.B. Hlvold, E. Ring, Effect of intraperitoneal
injection of immunostimulatory substances on allochthonous gut microbiota
of Atlantic salmon (Salmo salar L.) determined using denaturing gradient gel
electrophoresis, Aquacult. Res. 39 (6) (2008) 635e646.
Z. Liu, W. Liu, C. Ran, J. Hu, Z. Zhou, Abrupt suspension of probiotics administration may increase host pathogen susceptibility by inducing gut dysbiosis,
Sci. Rep. 6 (2016) 23214.
G.E. Merino, R.H. Piedrahita, D.E. Conklin, The effect of sh stocking density on
the growth of California halibut (Paralichthys californicus) juveniles, Aquaculture 265 (1) (2007) 176e186.
S. Irwin, J. Ohalloran, R. FitzGerald, Stocking density, growth and growth
variation in juvenile turbot, Scophthalmus maximus (Ranesque), Aquaculture
178 (1) (1999) 77e88.
E. Schram, J. Van der Heul, A. Kamstra, M. Verdegem, Stocking densitydependent growth of Dover sole (Solea solea), Aquaculture 252 (2) (2006)
339e347.
E. Salas-Leiton, V. Anguis, B. Martn-Antonio, D. Crespo, J.V. Planas, C. Infante,
et al., Effects of stocking density and feed ration on growth and gene
expression in the Senegalese sole (Solea senegalensis): potential effects on the
immune response, Fish Shellsh Immunol. 28 (2) (2010) 296e302.
M. Herrera, L. Vargas-Chacoff, I. Hachero, I. Ruz-Jarabo, A. Rodiles, J.I. Navas, et
al., Physiological responses of juvenile wedge sole Dicologoglossa cuneata
(Moreau) to high stocking density, Aquacult. Res. 40 (7) (2009) 790e797.
E.A. Fairchild, W.H. Howell, Optimal stocking density for juvenile winter
ounder Pseudopleuronectes americanus, J. World Aquacult. Soc. 32 (3) (2001)
300e308.
M.T. Ridha, Comparative study of growth performance of three strains of Nile
tilapia, Oreochromis niloticus, L. at two stocking densities, Aquacult. Res. 37 (2)
(2006) 172e179.
G.A. Gall, Y. Bakar, Stocking density and tank size in the design of breed
improvement programs for body size of tilapia, Aquaculture 173 (1) (1999)
197e205.
K. de Punder, L. Pruimboom, Stress induces endotoxemia and low-grade
inammation by increasing barrier permeability, Front. Immunol. (2015) 6.
S.H. Hoseinifar, A. Mirvaghe, D.L. Merrield, The effects of dietary inactive
brewers yeast Saccharomyces cerevisiae var. ellipsoideus on growth, physiological responses and gut microbiota of juvenile beluga (Huso huso), Aquaculture 318 (2011) 90e94.
P. Li, D.M. Gatlin, Evaluation of the prebiotic GroBioticR-A and brewers yeast
as dietary supplements for sub-adult hybrid striped bass (Morone chrysops
M. saxatilis) challenged in situ with Mycobacterium marinum, Aquaculture
248 (2005) 197e205.
M. Lara-Flores, L. Olivera-Castillo, M.A. Olvera-Novoa, Effect of the inclusion of
a bacterial mix (Streptococcus faecium and Lactobacillus acidophilus), and the
yeast (Saccharomyces cerevisiae) on growth, feed utilization and intestinal
enzymatic activity of Nile tilapia (Oreochromis niloticus), Int. J. Fish. Aquacult. 2
(4) (2010) 93e101.
M.A. Essa, S. El-Serafy, M.M. El-Ezabi, S.M. Daboor, N.A. Esmael, S.P. Lall, Effect
of different dietary probiotics on growth, feed utilization and digestive enzymes activities of Nile tilapia, Oreochromis niloticus, J. Arab. Aquacult. Soc. 5
(2) (2010) 143e161.
~o, J.M. Mancera, M.T. Dinis, L.E. Concei~
B. Costas, C. Araga
ao, High stocking
density induces crowding stress and affects amino acid metabolism in Senegalese sole Solea senegalensis (Kaup 1858) juveniles, Aquacult. Res. 39 (1)
(2008) 1e9.
A. Rollo, R. Sulpizio, M. Nardi, S. Silvi, C. Orpianesi, M. Caggiano, et al., Live
microbial feed supplement in aquaculture for improvement of stress tolerance, Fish Physiol. Biochem. 32 (2) (2006) 167e177.
M.A. Avella, I. Olivotto, S. Silvi, C. Ribecco, A. Cresci, F. Palermo, A. Polzonetti,
O. Carnevali, Use of Enterococcus faecium to improve common sole (Solea
solea) larviculture, Aquaculture 315 (2011) 384e393.
W. Liu, P. Ren, S. He, L. Xu, Y. Yang, Z. Gu, Z. Zhou, Comparison of adhesive gut
bacteria composition, immunity, and disease resistance in juvenile hybrid
tilapia fed two different Lactobacillus strains, Fish Shellsh Immunol. 35
(2013) 54e62.
P. Yarahmadi, H.K. Miandare, H. Farahmand, A. Mirvaghe, S.H. Hoseinifar,
Dietary fermentable ber upregulated immune related genes expression,
increased innate immune response and resistance of rainbow trout (Oncorhynchus mykiss) against Aeromonas hydrophila, Fish Shellsh Immunol. 41
(2014) 326e331.
R. Gornati, E. Papis, S. Rimoldi, G. Terova, M. Saroglia, G. Bernardini, Rearing
density inuences the expression of stress-related genes in sea bass (Dicentrarchus labrax, L.), Gene 341 (2004) 111e118.
C.M.A. Caipang, M.F. Brinchmann, I. Berg, M. Iversen, R. Eliassen, V. Kiron,
Changes in selected stress and immune-related genes in Atlantic cod, Gadus
morhua, following overcrowding, Aquacult. Res. 39 (14) (2008) 1533e1540.

n
~ ez, et
L. Vargas-Chacoff, D. Martnez, R. Oyarzn, D. Nualart, V. Olavarra, A. Ya
al., Combined effects of high stocking density and Piscirickettsia salmonis
treatment on the immune system, metabolism and osmoregulatory responses
of the Sub-Antarctic Notothenioid sh Eleginops maclovinus, Fish Shellsh
Immunol. 40 (2) (2014) 424e434.
K.M. Selim, R.M. Reda, Improvement of immunity and disease resistance in
the Nile tilapia, Oreochromis niloticus, by dietary supplementation with

C. Ran et al. / Fish & Shellsh Immunology 56 (2016) 263e271

Bacillus amyloliquefaciens, Fish Shellsh Immunol. 44 (2) (2015) 496e503.
[48] S. Wong, T. Waldrop, S. Summerfelt, J. Davidson, F. Barrows, P.B. Kenney, et al.,
Aquacultured rainbow trout (Oncorhynchus mykiss) possess a large core intestinal microbiota that is resistant to variation in diet and rearing density,
Appl. Environ. Microbiol. 79 (16) (2013) 4974e4984.
[49] W. Liu, P. Ren, S. He, L. Xu, Y. Yang, Z. Gu, et al., Comparison of adhesive gut
bacteria composition, immunity, and disease resistance in juvenile hybrid
tilapia fed two different Lactobacillus strains, Fish Shellsh Immunol. 35 (1)
(2013) 54e62.

271

[50] C. Tsuchiya, T. Sakata, H. Sugita, Novel ecological niche of Cetobacterium

somerae, an anaerobic bacterium in the intestinal tracts of freshwater sh,
Lett. Appl. Microbiol. 46 (1) (2008) 43e48.
[51] H. Sugita, J. Takahashi, C. Miyajima, Y. Deguchi, Vitamin B12-producing ability
of the intestinal microora of rainbow trout (Oncorhynchus mykiss), Agric.
Biol. Chem. 55 (3) (1991) 893e894.
[52] F. Gatesoupe, Live yeasts in the gut: natural occurrence, dietary introduction,
and their effects on sh health and development, Aquaculture 267 (1) (2007)
20e30.