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Effect of dietary supplementation of brewers y

Aquacult Int (2011) 19:489–496
DOI 10.1007/s10499-010-9364-1

Effect of dietary supplementation of brewers yeast
and nucleotide singularly on growth, survival
and vibriosis resistance on juveniles of the gastropod
spotted babylon (Babylonia areolata)
Nilnaj Chaitanawisuti • Chatchaleeya Choeychom
Somkiat Piyatiratitivorakul



Received: 16 November 2009 / Accepted: 2 August 2010 / Published online: 18 August 2010
Ó Springer Science+Business Media B.V. 2010

Abstract This study was undertaken to evaluate the use of brewers yeast and nucleotides
as a growth promoter and to provide vibriosis resistance for the juveniles of gastropod
spotted babylon (Babylonia areolata). Juvenile spotted babylon (0.3 g initial weight) were
randomly distributed at a density of 50 snails in 45-L aquaria and fed a basic diet (40%
crude protein) containing two incremental levels of 1 and 2% brewers yeast and nucleotides singularly for 4 months. After the feeding trial, snails from each treatment were
challenged by pathogenic bacteria Vibrio alginolyticus given by intramuscular injection

and kept under observation for 5 days to record clinical signs and daily mortality rates.
Results indicated that the snails fed with diets supplemented with brewers yeast or
nucleotides exhibited significantly greater growth than those fed the basic diet (P \ 0.05)
and significantly better food conversion ratios compared to snails fed the basic diet
(P \ 0.05). These results indicated that dietary supplementation of brewers yeast or
nucleotides, at least at the tested dosages, enhanced spotted babylon growth. Supplementing the diet with 1% brewers yeast promises to provide appropriate resistance to
V. alginolyticus.
Keywords Babylonia areolata Á Brewers yeast Á Nucleotides Á Growth performance Á
Disease resistance

Introduction
Spotted babylon Babylonia areolata are popular marine gastropods cultured in Thailand
and a potentially important aquaculture species because of their rapid growth, efficient
N. Chaitanawisuti (&)
Aquatic Resources Research Institute, Chulalongkorn University, Phya Thai Road,
Bangkok 10330, Thailand
e-mail: nilnajc1@hotmail.com
C. Choeychom Á S. Piyatiratitivorakul
Department of Marine Science, Faculty of Science, Chulalongkorn University, Phya Thai Road,
Bangkok 10330, Thailand

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food conversion and high market value. Large-scale rearing of B. areolata in Thailand is
technically feasible using flow-through systems in concrete/canvas ponds. Disadvantages
of these systems that must be solved during growth of the spotted babylon were as follows:
these systems generally require large quantities of water; production systems must be
located near the sea; the stock is vulnerable to external water supply and quality problems;
and their growth rate was significantly influenced by water flow (Chaitanawisuti et al.
2002). Trash fish is used as food at all commercial farms producing spotted babylon in
Thailand. The main problems of this food source are its shortage and discontinuous supply
and the freshness and variable nutritional values of trash fish. This situation has led to
research on the development of cost-effective artificial feeds for farmed spotted babylon
(Chaitanawisuti and Kritsanapuntu 1999; Chaitanawisuti et al. 2005). Proper nutrition has
long been recognized as a critical factor in promoting normal growth and sustaining the
health of mollusks. Prepared diets not only provide the essential nutrients that are required
for normal physiological functioning but also may serve as the medium by which fish
receive other components that may affect their health. Although the concept of functional
feeds is novel to the aquaculture industry, it represents an emerging new paradigm to
develop diets that extend beyond satisfying basic nutritional requirements of the cultured
organisms.
Prebiotics are defined as ‘‘a nondigestible food ingredient which beneficially affects the
host by selectively stimulating growth of and/or activating the metabolism of one or a
limited number of health-promoting bacteria in the intestinal tract, thus improving the
host’s intestinal balance’’ (Li and Gatlin (2004). Moreover, single-cell proteins (SCP)
including microalgae, bacteria and yeast are frequently used as food additives for fish
because they contain nutrients, proteins, vitamins, pigments nucleotides, b-glucans.
Among SCP, some yeast such as Candida sp. and Saccharomyces cerevisiae are used as
probiotics (Scholz et al. 1999; Li and Gatlin 2003). Brewers yeast (Saccharomyces cerevisiae) contains various immunostimulating compounds such as b-glucans, nucleic acids
and chitin as well as mannan oligosaccharides, and it has been observed to be capable of
enhancing immune responses as well as growth of various fish and shellfish species (Li and
Gatlin 2004). In brewers yeast, nucleic acid nitrogen is present mostly in the form of RNA
and represents about 20–25% of the nitrogen (Oliva-Teles and Goncalves 2001; LaraFlores et al. 2003; Li and Gatlin 2003; Li et al. 2005). Dietary nucleotides have been shown
to have several benefits including rapid intestinal repair, improved mucosal gut flora and
mucosal surfaces and elongation of the intestinal tract in aquatic animals (Li and Gatlin
2006; Li et al. 2007). Nucleotides also have been shown to enhance the immune system
and disease resistance of various animals (Murthy et al. 2009). However, the potential
benefit of the dietary supplementation has not been evaluated for the Babylonia areolata.
Therefore, this study was conducted to determine the effects of brewers yeast and
nucleotide supplementation on growth, survival and vibriosis resistance of juvenile spotted
babylon (B. areolata).

Materials and methods
Experimental diets
Four experimental diets were prepared by adding different supplements to the basic diet.
The basic diet consisted of a partially autolyzed brewers yeast produced by Thai Beverage
Public Company Limited, Bangkok, Thailand, and the commercial nucleotide product

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[(NuProÒ), Alltech Biotechnology Corporation Company, Bangkok, Thailand]. NuProÒ is
a functional protein from yeast and contains highly concentrated levels of essential and
functional nutrients, which are important in the diets of young animals. It is rich in
nucleotides, inositol, and amino acids and peptides. Two incremental levels (1 and 2% of
brewers yeast or NuProÒ) were added to the basic diet, and fishmeal, tuna oil and cellulose
were adjusted to provide isonitrogenous (40% crude protein) and isolipidic diets (Table 1).
The diets were prepared by weighing the dry ingredients and mixing them thoroughly in a
mixer. Lipid sources were added drop by drop while the mixture was further blended to
ensure homogeneity. Approximately 200 ml of hot water was then added for each kg of
this mixture. The diets were extruded and dried with an electric fan at room temperature for
24 h. When feeding was undertaken, the diets were made into small rounded pieces
(1.5 cm diameter) to facilitate sucking by the snails. All experimental diets were then
stored in plastic bags at -20°C until use. They were analyzed in duplicate for the proximate compositions according to the standard methods of AOAC (1990).
Husbandry conditions
Juvenile spotted babylon (Babylonia areolata) were obtained from a commercial private
hatchery in Prachuabkirikhan province, Southern Thailand, and maintained in an indoor

Table 1 Composition and proximate composition of the experimental diets
Ingredients (% dry weight) Basic diet 1% brewers yeast 2% brewers yeast 1% NuProÒ 2% NuProÒ
Fishmeal

40.0

40.0

40.0

40.0

40.0

Soybean meal

19.0

18.0

17.0

18.0

17.0

Shrimp meal

3.0

3.0

3.0

3.0

3.0

Wheat flour

17.0

17.0

17.0

17.0

17.0

Wheat gluten

7.0

7.0

7.0

7.0

7.0

Tuna oil

7.0

7.0

7.0

7.0

7.0

Vitamin mixa

2.0

2.0

2.0

2.0

2.0

Mineral mixb

3.0

3.0

3.0

3.0

3.0

b-cellulose

2.0

2.0

2.0

2.0

2.0

Nucleotide (NuProÒ)c



1.0

2.0





Brewers yeastd







1.0

2.0
40.38

Proximate composition (% dry matter)
Protein

40.34

40.31

40.19

40.26

Lipid

9.24

9.18

9.25

9.22

9.27

Ash

13.63

13.71

13.58

13.74

13.67

Fiber
Moisture

4.75

4.78

4.69

4.79

4.70

11.23

11.17

11.32

11.29

11.27

Gross energy (kcal g-1)
a
Vitamins (% kg-1 diet): vitamin A 107 IU, vitamin D 106 IU, vitamin E 0.01%, vitamin K 0.001%,
vitamin B1 0.0005%, vitamin B6 0.01%, Methionin 0.016%
b
Minerals (% kg-1 diet): dicalcium phosphate 14.7%, phosphorus 14.7%, manganese oxide 1.0%, copper
sulfate 0.36%, iron sulfate 0.20%, potassium iodide 0.10%, cobalt sulfate 0.10%, selenium oxide 0.006%
c

Alltech Biotechnology Corporation Company, Bangkok, Thailand

d

Thai Beverage Public Company Limited, Bangkok, Thailand

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hatchery at the Research Unit for Completed Commercial Aquaculture of Spotted Babylon,
Chulalongkorn University, Petchaburi province, prior to feeding experiments. Homogenous groups of 1,250 juveniles (initial mean body weight of 0.3 ± 0.1 g per snail) were
distributed randomly into 25 plastic tanks of 50 9 30 9 30 cm at a density of 50 snails per
tank. Each rearing tank was supplied with a flow-through seawater system with a fixed
water flow rate of approximately 150 l/h. The bottom of the rearing tank was covered with
coarse sand (1.0 cm thickness) as substratum. The tanks were provided with a supplemental aerator stone, and water was aerated daily for 20 h to maintain dissolved oxygen
levels at or near saturation. Water temperature and salinity were controlled at 29–31°C and
30 ppt, respectively. A natural photoperiod of 12-h light/ 12-h dark was provided. The
aquaria and sand substrata were cleaned biweekly to remove excess feed and fecal matter
and by scrubbing the sides of the aquarium. No chemical or antibiotic agent was used
throughout the entire experimental period.
Feeding trials
The basic diet was fed to all snails during a 2-week acclimation period. At the beginning of
the feeding trials, each diet was fed to five replicate groups of snails for 4 months. Snails
were hand-fed to apparent visual satiation feeding twice daily (10:00 h and 14:00 h) with
one of the experimental diets. All groups were fed their respective diets at the same fixed
rate of initially 5% of body weight per day. The amount of food was adjusted weekly based
on the amount of food consumed by snails within 0.5 h the previous day to ensure that only
a minimal amount of feed was left. Apparent satiation was determined from observation of
the point at which snails ceased active feeding, moved away from the feeding area and
buried themselves under the sand substratum. Uneaten food was siphoned out daily after
the snails stopped eating to prevent contamination of the water and sand substratum. The
amount of food eaten was recorded daily for the calculation of food intake and food
conversion ratio.
Snail performance
After the feeding trials, snails in each tank were harvested, counted and weighed. Growth
performance was determined, and feed utilization was calculated as follows:
Weight gainðWGÞ ¼ final weightðgÞ À initial weightðgÞ;
Specific growth rateðSGRÞ ¼ 100ðln Wf À ln Wi Þ=T;
Food conversion ratioðFCRÞ ¼ food intakeðgÞ=weight gainðgÞ;
Protein efficiency ratio ðPERÞ ¼ weight gainðgÞ=protein intakeðgÞ;
Survival rateðSRÞ ¼ 100 Â ðfinal snail numberÞ=ðinitial snail numberÞ:
Bacterial challenge
At the end of the experiment, a random sample of snails was taken from each treatment and
distributed into three replicate 50-l aquaria of 20 snails each at the time of challenge. The
water supplied was flow-through dechlorinated seawater at 0.5 l/min maintained at
26 ± 2°C. Aeration was supplied via an airblower and airstones. The snails were challenged with Vibrio alginolyticus at 1 9 106 CFU/ml by intramuscular injection (IM) in the
muscular foot. For the control group, the snails were intramuscularly injected with 0.1 ml

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493

of saline solution. All groups were kept under observation for 5 days to record clinical
asymptomatic signs and daily mortality rates.
Statistical analysis
To determine whether significant differences existed between the different treatments and
the parameters tested, all results were analyzed using one-way analysis of variance
(ANOVA). Duncan’s multiple range tests were used to compare treatment means. Differences were considered significant at P B 0.05.

Results
After the 4-month feeding trial, significant (P \ 0.05) differences in final weight (FW),
weight gain (WG) and specific growth rate (SGR) were observed among snails fed the diets
supplemented with brewers yeast or nucleotides (Table 2). The WG of snails fed with diets
supplemented with 2% brewers yeast (3.70 g/snail), 1% of nucleotides (3.80 g/snail) and
2% of nucleotides (3.60 g/snail) was significantly higher than that of snails fed the basic
diet (3.23 g/snail) and diet supplemented with 1% brewers yeast (3.30 g/snail). The SGR
of the snails fed the experimental diets showed a similar trend to WG. Survival was not
affected by brewers yeast or nucleotide supplementation, and its range was 97.8–99.5%.
Total food intake was identical in all treatments and ranged from 111 to 117 g food/snail
with no significant difference among them. Supplementation with brewers yeast and
nucleotides improved nutrient utilization. Significant (P \ 0.05) differences in FCR and
PER were observed among snails fed any of the diets supplemented with brewers yeast and
nucleotides (Table 2). The FCR of snails fed the diets supplemented with 2% brewers yeast
(1.05), 1% of nucleotides (1.05) and 2% of nucleotides (1.06) was significantly better than
that of snails fed the basic diet (1.22) and diet supplemented with 1% brewers yeast (1.22).
The PER of snails fed any of the experimental diets showed a similar trend to FCR.
After 5 days of intramuscular injection with V. alginolyticus, significant (P \ 0.05)
differences in survival rates were observed among snails fed any of the diets supplemented
Table 2 Growth performance of B. areolata juveniles fed the experimental diets for 4 months
Parameters

Diets
Basic diet

Initial weight (g)
Final weight (g)
Total feed intake (g)
Weight gain (g/snail)
Specific growth rate
(% day-1)
Survival (%)
Food conversion ratio
Protein efficiency ratio

1% brewers
yeast

0.35 ± 0.06a

0.33 ± 0.05a

b

b

3.57 ± 0.25

a

116.8 ± 6.20

b

3.23 ± 0.31

a

1% NuProÒ

2% brewers
yeast

3.60 ± 0.10

a

116.9 ± 5.90

ab

3.30 ± 0.10

ab

4.00 ± 0.36

a

115.4 ± 7.70

ab

3.70 ± 0.36

0.31 ± 0.02a
a

4.10 ± 0.20

3.93 ± 0.25ab

a

111.2 ± 8.20a

a

3.60 ± 0.30a

b

117.2 ± 4.70
3.80 ± 0.20

1.99 ± 0.01

2.16 ± 0.08

2.18 ± 0.04

2.14 ± 0.04b

98.90 ± 0.51a

98.9 ± 0.55a

99.5 ± 0.60a

97.8 ± 0.46a

97.8 ± 0.72a

a

a

b

b

1.06 ± 0.07b

b

2.36 ± 0.09b

a

2.06 ± 0.21

1.22 ± 0.07

a

2.06 ± 0.11

b

0.30 ± 0.0a

1.93 ± 0.05

1.22 ± 0.08

ab

0.30 ± 0.01a

2% NuProÒ

1.05 ± 0.06

b

2.39 ± 0.25

1.05 ± 0.06
2.38 ± 0.17

Values are means ± SD. Means having the same superscript letters in the same row are significantly
different at P \ 0.05

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with brewers yeast and nucleotides (Table 3). Survival of snails fed diets containing
brewers yeast or nucleotides was significantly (P \ 0.01) higher than snails fed the basic
diet after the same period of 4 months. Snails fed diets supplemented with 1% brewers
yeast showed the highest survival rate (80.0%), followed by those of snails fed diets
supplemented with 1% nucleotides (70.0%), 2% brewers yeast (65.0%) and 2% nucleotides
(60.0%), respectively, and snails fed the basal diet showed the lowest survival rate
(30.0%). The snails showed typical symptoms of V. alginolyticus infection including
protruding of the proboscis without retraction, pinkish color at the top of proboscis at the
beginning of infection, extreme reddish color and no movement (Fig. 1).

Discussion
In the present study, snails fed diets supplemented with brewers yeast or nucleotides
(NuProÒ) exhibited improvement in growth and food utilization compared to those fed the
basic diet. These results agree with those obtained with Nile tilapia (Abdel-Tawwab et al.
2008; Lara-Flores et al. 2003), white shrimp (Li et al. 2007; Murthy et al. 2009), hybrid
striped bass (Li and Gatlin 2004) and sea bass (Oliva-Teles and Goncalves 2001). This
study is in accordance with the study of Lara-Flores et al. (2003) in which the 40% protein
diet supplemented with yeast produced the best growth performance and food efficiency of
Nile tilapia Oreochromis niloticus, suggesting that yeast is an appropriate growth-stimulating additive in tilapia cultivation. The improved snail growth and feed utilization may
possibly be due to improved nutrient digestibility. In this regard, Lara-Flores et al. (2003)
found that the addition of live yeast improved diet and protein digestibility, which may
explain the better growth and food efficiency seen with yeast supplements. Abdel-Tawwab
et al. (2008) also reported growth-promoting influences of bakers’ yeast, Saccharomyces
cerevisiae, on Nile tilapia fry, Oreochromis niloticus. Optimal growth, food utilization and
protein turnover were obtained with 0.5–1.0 g yeast per kg diet. Li and Gatlin (2003)
demonstrated that brewers yeast positively influenced growth performance and food efficiency of hybrid striped bass, Morone chrysops 9 M. saxatilis, as well as resistance to
Streptococcus iniae infection and that brewers yeast can be administered for relatively long
periods without causing immunosuppression. Li et al. (2007) also reported that dietary
supplementation of nucleotides at two levels of crude protein diet (25 and 35%) significantly enhanced the final weight of Penaeus vannamei. However, additional information is
needed in regard to age/size-related responses of the animals as well as the appropriate
dose and timing of administration to obtain optimum growth of selected animals. In the
present study, the snail survival 1 day after IM injection with V. alginolyticus showed that
Table 3 Survival rate of B. areolata juveniles fed a 40% protein diet with different supplementation of
brewers yeast and nucleotides in aquarium for 4 months and challenged by V. alginolyticus for 1 day
Diets

Survival rate (%)
30e ± 20.92

Basal diet
Ò

70b ± 20.92

Ò

2% NuPro

60d ± 28.50

1% brewers yeast

80a ± 20.92

2% brewers yeast

65c ± 28.50

1% NuPro

Treatment means with different superscripts are significantly different (P \ 0.05)

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495

Fig. 1 Mean body weight of B. areolata juveniles throughout the 4-month feeding trials

snails fed the diets supplemented with 1% brewers yeast had the highest survival rate,
followed by those of snails fed diets supplemented with 1% nucleotides, 2% brewers yeast
and 2% nucleotides, and that of snails fed the basic diet showed the lowest survival rate
(30.0%). These data suggest that the yeast supplementation could increase the non-specific
immune system of spotted babylon resulting in a snail resistance to V. alginolyticus
infection. This result agrees with Abdel-Tawwab et al. (2008) who evaluated commercial
live baker’s yeast S. cerevisiae as a immunity promoter for the fry Nile tilapia challenged
in situ with Aeromonas hydrophila. They found that total fish mortality 10 days after IP
injection with A. hydrophila decreased with the increased yeast level in fish diets and that
the lowest fish mortality was found in fish fed 5.0 g yeast/kg. They also indicated that
bakers’ yeast supplement is promising as an alternative method to antibiotics for disease
prevention in Nile tilapia and that the optimum level of live bakers’ yeast is about 1.0 g per
kg diet, corresponding to this study in that the best result for V. alginolyticus resistance was
found in snails fed with 1% of brewers yeast per kg diet. It is possible that the negative
effect on survival of B. areolata, found for nucleotides in this study, could be due to the
dose administered, especially when taking into account that the brewers yeast diets gave
relative better survival, though not at a significant level. These findings suggest that there is
need for caution when administering nucleotides long term and/or in high dose. Shelby
et al. (2009)observed that protection from disease challenge by pasteurellosis caused by
Photobacterium damselae in gilthead seabream was dependent on duration and dose. For
example, fish fed glucans for 2 weeks had no protection from disease challenge at the
higher inclusion rates of glucan. Scholz et al. (1999) also reported that Atlantic salmon fry,
fed a diet containing baker’s yeast at 2%, displayed better survival than control animals
when challenged with Vibrio anguillarum, while juvenile European seabass showed similar
results when fed a diet containing experimental treated yeast at 1%. Furthermore, brewers
yeast chemically treated to enhance its digestibility and to increase access to the glucan on
the surface of the yeast gave better survival than fresh yeast. However, further research is
needed as there are numerous gaps in existing knowledge about exogenous nucleotides and
brewers yeast application to spotted babylon including various aspects of digestion,

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absorption, metabolism and influences on various physiological responses especially
expression of immunogenes and modulation of immunoglobin production.
In conclusion, this study represents the first attempts to utilize the by-product of brewers
industry, brewers yeast, supplemented in feed as the protein source. The results of this trial
indicate that brewers yeast provided the good results in growth and feed efficiency, with no
negative effects in spotted babylon juvenile performance (growth, survival and disease
resistance). Moreover, the inclusion of up to 2% brewers yeast in the diet improved feed
efficiency and protein utilization. Based on the result of this study, it is concluded that
brewers yeast positively influenced growth performance and feed efficiency of spotted
babylon as well as resistance to V. alginolyticus infection. In addition, brewers yeast can be
administered in the feeds as the alternative, less expensive protein source for this species.
Acknowledgments This study was supported by the National Research Council of Thailand (NRCT), who
provided funding for this research in the fiscal years 2005–2008. We are especially grateful to Associate
Professor Dr. Somkiat Piyatiratitivorakul, Faculty of Science, Chulalongkorn University, and Professor Dr.
Yutaka Natsukari, Faculty of Fisheries, Nagasaki University for their encouragement, suggestions and
critical reading of the manuscript.

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