Effect of biofloc technology on growth, digestive enzyme activity, hematology,
and immune response of genetically improved farmed tilapia (Oreochromis
niloticus)
Lina Long, Jing Yang, Yuan Li, Chongwu Guan, Fan Wu
PII:
DOI:
Reference:

S0044-8486(15)30008-9
doi: 10.1016/j.aquaculture.2015.05.017
AQUA 631672

To appear in:

Aquaculture

Received date:
Revised date:
Accepted date:

17 September 2014
11 May 2015
12 May 2015

Please cite this article as: Long, Lina, Yang, Jing, Li, Yuan, Guan, Chongwu, Wu, Fan,
Eect of biooc technology on growth, digestive enzyme activity, hematology, and immune response of genetically improved farmed tilapia (Oreochromis niloticus), Aquaculture
(2015), doi: 10.1016/j.aquaculture.2015.05.017

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Effect of biofloc technology on growth,

digestive enzyme activity, hematology, and

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immune response of genetically improved

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farmed tilapia (Oreochromis niloticus)

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Lina Long a, Jing Yang a,*, Yuan Li b, Chongwu Guan a, Fan Wu a

Key Laboratory of Fishery Equipment and Engineering, Ministry of Agriculture, Fishery Machinery

College of Engineering, Shanghai Ocean University, Shanghai 201306, China

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and Instrument Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200092, China

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* Corresponding author at: Fishery Machinery and Instrument Research Institute,

Chinese Academy of Fishery Sciences, Chifeng Road 63, Shanghai 200092, China.

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Tel.:+86 21 35322363; fax: +86 21 65976741.

E-mail address: yangjing3360@163.com (J.Yang)

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Abstract

An 8-week experiment was conducted to investigate the effects of biofloc

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technology (BFT) on the growth, digestive activity, hematology, and immune

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response of genetically improved farmed tilapia (GIFT) in light-limited and

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zero-water exchange culture tanks. The experiment consisted of one BFT treatment
and a control group with water exchange. Glucose was added to the BFT treatment

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to establish a carbon/nitrogen (C/N) ratio of 15. The stocking density was 3 kg m-3

in each 500-L indoor tank. Nitrite and nitrate concentrations were significantly

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lower in the BFT treatment than in the control (P < 0.05). Fish survival was 100% at

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harvest. BFT significantly increased fish specific growth rate and net yield.
Compared with the control group, the individual fish weight, weight gain and

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protein efficiency ratio of fish in the BFT treatment were 12.54%, 9.46%, and
22.2% higher, respectively, whereas the feed conversion rate was 17.5% lower. The
crude protein, crude lipid, and ash contents of the biofloc were 41.13%, 1.03%, and
6.07%, respectively, while the crude lipid content of BFT fish exhibited an
increasing trend. Significantly higher intestine amylase and liver lipase activities of
fish were found in the BFT treatment. There was no significant difference in
hematology analysis (in terms of white blood cell and red blood cell counts,
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hemoglobin and hematocrit levels), serum total protein content or total superoxide

dismutase activity of fish between the two groups (P > 0.05), but the serum

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glutathione peroxidase and lysozyme activities were considerably higher in the BFT

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treatment than in the control fish (P < 0.05). The results of this study indicate that

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BFT can improve the growth, digestive enzyme activities, and immune response of
GIFT.

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Statement of Relevance

To date, preliminary studies based around the concept of the biooc

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technology (BFT) were focused mainly on shrimp culture, and few studies

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have been conducted on fish in such systems. In addition, BFT have

documented mainly the water quality, growth and production performance of

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tilapia systems. However, little is known about the effect of BFT on the
physiological health of fish particularly concerning digestive enzyme activity,
hematology as well as the immune response, which are very important in
aquaculture. Therefore, the aim of the present study was conducted to
investigate the effects of BFT on the growth, digestive enzyme activity,
hematology, and immune response of genetically improved farmed tilapia
(GIFT) in light-limited and zero-water exchange culture tanks. The
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monitoring indicated that BFT had beneficial effects on the maintenance of

good water quality, improvement of feed utilization, and the growth

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performance of GIFT compared with the control group. The crude lipid

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content of BFT fish exhibited an increasing trend. Furthermore, BFT

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improved the digestive enzyme activity of GIFT. Although there were no

significant differences in the hematological parameters of GIFT, BFT had a

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positive effect on the immune response of cultured tilapia. According to this

study, it is suitable for rearing GIFT in such a light-limited indoor BFT

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system. BFT may be a promising alternative technology for a conventional

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clear water system operated with water exchange in cultivating both

herbivorous and omnivorous sh species including tilapias. In addition, the

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significance of the present study may offer a potential practical approach for
disease prevention and health management in fish aquaculture.

Keywords
Tilapia;

Biofloc

technology;

Growth

performance;

Digestive

activity;

Hematological parameters; Immune response

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1. Introduction

With almost seven billion people on earth, the aquaculture industry is growing

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rapidly. For example, there has been a considerable expansion of tilapia production

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worldwide. The total global production of tilapia and other cichlids was only 107,459

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metric tons MT in the early 1980s; by 2008, however, the production exceeded 2.5
million MT (Food and Agriculture Organization Fisheries and Aquaculture Statistics,

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2010). However, aquaculture expansion is restricted by the usage of land and water

(Widanarni et al., 2012). In addition, environmental problems caused by pollution

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from waste products, especially those from intensive aquaculture systems, are

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becoming increasingly serious (Piedrahita, 2003). Therefore, there is urgent demand

for a relatively new and alternative aquaculture system. Biofloc technology (BFT)

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was recently considered as a more eco-friendly and sustainable technique for use in
zero-water exchange culture systems (Avnimelech, 2007; Azim and Little, 2008; Crab
et al., 2009; De Schryver et al., 2008). Biofloc containing heterotrophic
microorganisms and organic particles are developed with the addition of carbon
sources or the use of a low protein diet to maintain a high carbon/nitrogen (C/N) ratio
in the water (Avnimelech, 1999; Crab et al., 2007; Hargreaves, 2006). Theoretically,
ammonium and other organic nitrogenous waste in such systems will be converted
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into bacterial biomass, which is available as an additional diet for aquatic animals

(Avnimelech, 1999; Avnimelech, 2006; De Schryver et al., 2008). Thus, BFT can

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improve water quality with a reduction in water use, and can also act as an extra food

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source to increase nutrient use efficiency.

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Though experiments using the BFT concept have been carried out since the early
1980s (Serfling, 2006), these were focused mainly on shrimp culture. For example,

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many researchers have indicated that BFT can improve the water quality of shrimp

culture systems (De Schryver et al., 2008; Ray et al., 2010; Xu et al., 2012a; Zhao et

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al., 2012), enhance shrimp growth performance through additional natural food and

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stimulated digestive enzyme activities (Xu and Pan, 2012; Xu et al., 2012a; Xu et al.,
2012b), improve the antioxidant status and immune defense of shrimp (Souza et al.,

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2014; Kim et al., 2014; Xu and Pan, 2013), and enhance shrimp biosecurity (Crab et
al., 2010; Zhao et al., 2012). However, few studies have been conducted on fish in
BFT systems. As detritivorous and filter-feeding fish, tilapias are suitable for BFT
systems (Azim and Little, 2008). To date, preliminary studies of tilapia BFT systems
have documented mainly the water quality, growth and production performance
(Avnimelech, 2007; Azim and Little, 2008; Crab et al., 2009; Widanarni et al., 2012),
much is still unknown concerning the effect of BFT on digestive enzyme activity,
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hematology as well as the immune response of tilapia, which are important for tilapia

aquaculture.

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Because of its rapid growth rate, high production, and good disease resistance

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capability, genetically improved farmed tilapia (GIFT) (Oreochromis niloticus) has

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become an important aquaculture species in China. This species also has high market
value in the global fish trade (Dey et al., 2000). Therefore, the aim of the present

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study was conducted to assess the effects of BFT on the growth performance,

digestive enzyme activity, hematology, and immune response of GIFT in a

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light-limited and zero-water exchange culture system. In addition, the water quality

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and nutritional content of the biofloc and fish were investigated, which may provide
insights into exploring the effects of BFT on aquatic animals.

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2. Materials and methods

2.1. Tank facilities and experimental design
The experiment was conducted in indoor fiber-glass tanks (140 cm 70 cm 70
cm), each with a water volume of 500 L. The group with glucose addition was
referred to as the BFT treatment and the control group did not receive glucose. All
groups consisted of triplicate tanks and were assigned randomly. Each tank was filled
with dechlorinated freshwater. During the experimental period, one third of the water
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was exchanged daily in the control tanks. Water was not exchanged in the BFT group,

but evaporation losses were compensated with dechlorinated freshwater. In order to

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well determine the variation of the biofloc and acquire optimal experimental

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conditions, a pre-experiment was conducted with a control group and two BFT

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treatments (C/N ratio of 15 and 20, respectively), with three replicate tanks for each
group. The design and arrangement used here were similar to this trial. Due to the

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more stable condition of the BFT group (C/N ratio of 15), we selected it as the

experimental group after the pre-experiment. Before the fish were stocked, the BFT

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group was inoculated with concentrated biofloc (250 ml per tank) collected from an

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indoor biofloc tank (C/N ratio of 15) during the pre-experiment. The tank water was
aerated and agitated continuously using air-stones connected to an air pump. The

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diameter of the aeration tube was 6 mm, and that of the air-stone was 20 mm. The
laboratory where the trial was conducted had large glass windows and the sunlight
was very strong. Consequently, all tanks were continuously covered with shade cloth
to block out the sunlight. In addition, the shade cloth prevented the fish from jumping
out of the tanks.
2.2. Fish stocking and tank management
Mixed sex Oreochromis niloticus were purchased from Guangdong Aquaculture
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Station (Guangdong, China). After acclimation for 2 weeks, healthy fish (50.43 0.86

g) were selected and randomly stocked into 6 tanks on June 17th. Each tank contained

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30 fish and the biomass at the beginning of the experiment was 3 kg m-3 per tank.

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Pellet feed (Tongyi, Taiwan) containing 46% crude protein was used. The ration was

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split into two equal daily amounts (9:00 and 15:00) for an 8-week period, and the
same amount of feed was applied to each tank. Glucose was added to the BFT group

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to increase the C/N ratio to 15. The daily quantity of glucose added was calculated

according to Avnimelech (1999). At the start of the experiment, feed was added daily

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at a rate of 5% of body weight, and the weight of 20 fish within a tank was measured

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fortnightly to adjust the feeding rate to 3% of total body weight. Feed inputs were
recorded daily for each tank.

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2.3. Assessment of water quality parameters

During the 8-week experimental period, water temperature, dissolved oxygen
(DO) and pH were determined daily at 08:00 using a multiparameter water quality
instrument (YSI plus). Water samples (100 mL) were collected weekly and filtered
through pre-dried and pre-weighed GF/C filter paper under vacuum pressure. Total
ammonia nitrogen (TAN), nitrite (NO2--N) and nitrate (NO3--N) concentrations in the
filtrate were measured according to standard methods (APHA, 1998). Filter paper was
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used for total suspended solid (TSS) determination.

2.4. Biofloc collection and fish sampling

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Concentrated biofloc samples were collected from the BFT group by passing

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tank water through a 10-m mesh nylon bag (Xu et al., 2012a) after the 8-week

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experiment. The samples were dried in an oven at 105 C until constant weight and
then stored in a refrigerator (-20 C) until proximate composition analysis.

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At the end of the experiment, fish were starved for 24 h before sampling. Then,

the fish were harvested by draining the tanks and were anesthetized using tricaine

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methanesulfonate (MS-222) at 120 mg/L for weighing and counting purposes. For

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hematological assays, blood samples of three fish cultured in each tank were collected
from the heart using heparinized (100 IU/mL) tuberculin syringes. In addition,

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nonheparinized tuberculin syringes were used to collect the blood of another three fish
per tank. After clotting for one night and centrifuging at 4000 rpm for 10 min at 4 C,
serum samples were collected and stored at -20 C for immune analyses. Three fish
per tank were randomly collected for sampling digestive tissues. The liver and
intestine of each fish were excised and the adipose tissue carefully cleaned. Then
samples were rinsed and homogenized with chilled normal saline solution (1:3 w/v)
using an electric blender operating at 8000 rpm for 30 s each time. The homogenate
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was centrifuged at 10,000 g, 4 C for 30 min to eliminate tissue debris and lipids.

The supernatant of the enzyme extract was gently pipetted into 1.5-mL Eppendorf

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tubes and frozen at -20 C until enzymatic assay. Each sample preparation step was

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conducted on ice to maintain a low temperature. Three additional fish were randomly

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sampled from each tank, and each fish was minced, pooled, and frozen at -20 C for
whole-body proximate composition analysis.

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2.5. Fish digestive enzyme activity, hematological and immune parameters

By diluting (1:10,000) the whole blood in a phosphate buffer saline (PBS)

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solution, total red blood cell (RBC) and white blood cell (WBC) counts of each

Hemoglobin

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sample were determined three times using a hemocytometer (Lim et al., 2009).
(Hb)

was

determined

by

measuring

the

formation

of

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cyanomethemoglobin (Van Kampen and Zijlstra, 1961). Haematocrit (Ht) was

determined by centrifuging the whole blood using heparinised microhaematocrit tubes
at 5000 g for 5 min (Azim and Little, 2008).
The fish immune and digestive enzyme activities were determined using
commercial assay kits (Nanjing Jiancheng Institute, Nanjing, China) according to the
instructions of the manufacturer.
The total superoxide dismutase (SOD) activity in the serum was measured
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according to the method of Beauchamp and Fridovich (1971). One unit of SOD

activity was determined by the amount of superoxide dismutase used to inhibit the

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reduction of nitroblue tetrazolium at 50%. The level of glutathione peroxidase (GPX)

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activity was measured using the method of Floh and Gnzler (1984). The amount of

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GPX needed to oxidize 1 mol of NADPH per min was defined as one unit. The
activity of serum lysozyme (LSZ) was determined following the turbidimetric method

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(Boman et al., 1974; Hultmark et al., 1980), which involved the lysis of

lysozyme-sensitive Gram-positive bacteria by LSZ. The serum total protein

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concentration of fish, which was determined using the biuret protein assay kit, was

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calculated using bovine serum albumin as the protein standard and expressed as mg
mL1 of fish serum.

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The amylase activity was measured following the method of Robyt and Whelan
(1968) using soluble starch as the substrate. One unit of amylase activity was defined
to hydrolyze 10 mg of starch in 30 min at 37 C. The lipase activity was determined
according to the method of Winkler and Stuckman (1979). One unit of lipase activity
was expressed as 1 mol of p-nitrophenyl palmitate (pNPP) released from the
substrate in 1 min at 37 C. The trypsin activity was assayed using
Na-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as the substrate (Erlanger et al.,
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1961). One unit of trypsin activity was calculated using the amount of trypsin required

to release 1 mol of nitroanilide from substrate in 1 min at 37 C. The soluble protein

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concentration was measured in diluted homogenates following the Bradford method

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(Bradford, 1976) using bovine serum albumin (BSA) as the standard. The trypsin and

activity is expressed as U mg-1 of protein.

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2.6. Proximate composition analysis

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lipase activities are both presented as specific activity U g-1 of protein, and amylase

The fish sampled for proximate composition analysis were later dried in an oven

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at 105 C until constant weight, then ground and analyzed for crude protein, crude

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lipid, and ash contents using the standard methods (GB/T6432-1994, GB/T6433-2006
and GB/T6438-2007, respectively). The crude protein content was determined by

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measuring nitrogen using the Kjeldahl method and multiplying this value by 6.25; the
crude lipid content was measured by ether extraction using a Soxhlet extractor, and
the ash content was assayed by oven incineration at 550 C. The biofloc samples were
analyzed using methods similar to those described above for fish.
2.7. Calculations and statistics
At the end of the experiment, the observed body weight and food intake data
were calculated using the following growth indices:
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Specific growth rate (SGR, % day1) = 100 [Ln(final body weight) Ln(initial body

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Survival rate (%) = 100 (final fish count/initial fish count),

weight)](days of experiment),

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Weight gain (%) = 100 (final body weight initial body weight)initial body

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weight,

Feed conversion rate (FCR) = total dry weight of feed supplytotal fish wet biomass

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increase,

Protein efficiency ratio (PER) = total fish wet biomass increase/total dry weight of

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feed protein consumed

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Data obtained from the experiment were analyzed using SPSS 18.0 software
(SPSS, Chicago, USA) for Windows. One-way ANOVA was performed on the

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experimental parameters including growth parameters, proximate composition of the

biofloc and fish, digestive enzyme activity, hematology and immune indicators after
conducting a homogeneity of variance test. Differences were considered significant at
P<0.05. When significant differences were found, Duncan's multiple range test was
used to identify differences between the experimental groups.

3. Results and discussion

3.1. Water quality
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Daily changes in the water quality parameters are shown in Table 1. There were

no significant differences in temperature (29.8-23.0 C) or pH (7.98-6.48) between the

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two groups (P > 0.05). DO was maintained at a level greater than 6 mg L1, and

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differed significantly between the two groups (P < 0.05). This likely resulted from the

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higher respiration rates caused by the bacteria and other microorganisms in the BFT
group. Similar results were reported in other studies (Emerenciano et al., 2012; Kim et

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al., 2014). However, the DO level in the BFT treatment was well within the

acceptable range for the survival and growth of fish.

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The fluctuation of TSS over time is presented in Fig. 1. The TSS levels in the

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BFT treatment increased gradually throughout the experimental period. The average
TSS concentration was 24.61 mg L-1 in the control group and 484.48 mg L-1 in the

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BFT treatment. As demonstrated by Ray et al. (2010), the TSS concentration in the
BFT tanks should be well controlled because it is closely related to water quality and
anti-clogging of fish gills. In our study, we attempted to maintain the TSS level below
400 mg L-1. Although we removed some water from the light-limited BFT tanks and
replaced it after removing most of the floc using buckets, the TSS levels reached
almost 1000 mg L-1 in the last two weeks. This uncontrollable situation was also
reported by Azim and Little (2008) for light-limited BFT systems. However, no
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apparent negative influence on fish survival or growth was observed in the BFT group

throughout the experiment. Avnimelech (2012) demonstrated that in the case of fish

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ponds, TSS concentrations can reach levels of up to 1000 mg L-1, and the level should

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be limited to approximately 400 mg L-1, though data that indicate this level are lacking.

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Moreover, in tilapia culture, Avnimelech (2007) reported values ranging from 460 to
643 mg L-1, while Azim and Little (2008) presented average levels of 597 and 560 mg

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L-1. Consequently, the control of the TSS level as well as the optimum value warrants

further investigation.

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The changing tendency of TAN concentrations was fairly consistent over time

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between the two groups (Fig. 2A). The TAN concentrations in the biofloc group
remained stable from week 1 to the end of the experiment. During the 8-week

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experimental period, no water was discharged from the BFT treatment. By adding a
carbon source (glucose) to stimulate the growth of heterotrophic bacteria, TAN can be
used and stored by the formation of biofloc microbes (Asaduzzaman et al., 2008;
Avnimelech, 1999; Ebeling et al., 2006; Emerenciano et al., 2012), which may explain
why the TAN concentration did not exhibit significant differences between the clean
water control group and the zero-water exchange BFT treatment.
The NO2--N concentration in the two groups was less than 0.8 mg L-1 throughout
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the culture period, except for the highest level (1.62 mg L-1), which was observed in

the control group in week 7 (Fig. 2B). The NO3--N concentration in the control group

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fluctuated throughout the culture period (Fig. 2C) and a similar trend was observed in

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the BFT treatment. The highest NO3--N level in the BFT treatment was 5.45 mg L-1,

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observed in week 3, which then gradually decreased over time. The NO3--N
concentration was significantly higher in the control (P < 0.05), and showed a

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tendency to accumulate in the first 5 weeks of the culture period to levels of 11.64 mg

L-1. However, this level decreased and then increased again in week 8. The

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accumulation of NO2--N and NO3--N in the first few weeks may have been caused by

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nitrification processes, which are common in BFT systems (Azim and Little, 2008;
Widanarni et al., 2012; Xu et al., 2012a; Zhao et al., 2012). However, the reduction in

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NO2--N and NO3--N from the third week to the end of the experiment likely occurred
due to immobilization by heterotrophic bacteria, which inhibited the nitrification
process. In addition, denitrification may have occurred during the experiment (Azim
and Little, 2008; Luo et al., 2013).
3.2. Fish growth and yield parameters
The fish growth and yield parameters of both groups are shown in Table 2.
Weight gain and individual fish weight in the BFT treatment was 9.46% and 12.54%
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higher, respectively, than in the control group. Furthermore, BFT significantly

increased the SGR of fish in the BFT group at harvest (P < 0.05). The survival rate of

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fish in the BFT treatment and control tanks was 100%. Compared with the control

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group, the FCR of the BFT group was 17.5% lower, while the PER was 22.2% higher.

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The net yield of the BFT group (6.22 kg m3) was significantly higher than that of the
control (5.88 kg m3) (P < 0.05). Luo et al. (2014) reported similar results for fish

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growth and feed utilization parameters. Biofloc contributes significantly to the growth

of fish (Avnimelech, 2007; Azim and Little, 2008), and this finding is consistent with

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the present results. Burford et al. (2004) found that over 29% of the daily food

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consumed by L. vannamei could be biofloc. In addition, biooc has been

demonstrated to be an effective potential food source for tilapia using a stable

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nitrogen isotope labelling technique (Avnimelech, 2007). Avnimelech et al. (1994)

also estimated that tilapia in BFT ponds was fed a ration 20% less than conventional
amounts, while feed utilization was higher. Similar results were observed in the
present experiment, i.e., the FCR in the BFT treatment was lower than that in the
control.
In the present study, no substantial differences were observed in the fish whole
body crude protein or ash contents between the two groups (P > 0.05, Table 3).
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However, the lipid content of the fish whole body in the BFT treatment was

significantly higher than that in the control group (P < 0.05). According to Xu and Pan

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(2012), biofloc can influence the whole body composition of cultured shrimp, and the

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lipid content in the whole shrimp body was increased considerably. Izquierdo et al.

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(2006) also found that the whole body lipid content of shrimp growing in mesocosms
systems with biofloc exhibited an increasing trend. This can be attributed to the

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essential amino acids, fatty acids (PUFA and HUFA) and other nutritional elements

provided by the biofloc in the BFT treatment (Izquierdo et al., 2006; Ju et al., 2008b).

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In contrast, Luo et al. (2014) reported that there was no significant difference in the

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crude lipid content of fish back muscles between the BFT and RAS treatments. This
might be related to the different parts of the fish analyzed for crude lipid content in

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the experiments, i.e., Luo et al. (2014) used the back muscle of tilapia whereas the
whole body was used in the current trial.
3.3. Nutritional content of biofloc
The proximate composition of the biofloc collected from the tanks of the BFT
treatment is presented in Table 3. The crude protein content of the biofloc in the BFT
treatment was 41.13%. Some authors suggest that 25-30% crude protein in diets is
appropriate for the growth of tilapia (Chou and Shiau, 1996; Jauncey, 2000), which
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indicates that the crude protein level of biofloc in our study was suitable.

In contrast, the crude lipid and ash contents were somewhat low: 1.03% and

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6.07%, respectively. A very low crude lipid content of the biofloc was also detected by

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Luo et al. (2014). Although the crude lipid content of biofloc in the BFT treatment

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was lower than the level (2-5%) found in other studies (Azim and Little, 2008; Azim
et al., 2008; Crab et al., 2010), it was higher than the value (0.47%) reported by

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Emerenciano et al. (2012). However, the content was not sufficient according to the

dietary lipid requirement of 5-12% for tilapia (Lim et al., 2009), suggesting that

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commercial feed will be still needed for these fish. Although the ash content of biofloc

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was lower than that reported by other studies (Azim and Little, 2008; Crab et al., 2010;
Xu and Pan, 2012; Xu et al., 2012a; Xu et al., 2012b), there are some authors who

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suggest that the ash content of fish diets should be less than 13% (Craig and Helfrich,
2009; Tacon, 1988).
3.4. Digestive enzyme activity of fish
The activities of trypsin, amylase and lipase in the intestine and liver of fish at
harvest are shown in Fig. 3. No significant differences were found in the liver trypsin
and amylase activities of fish between the two groups (P > 0.05). However, the lipase
activity in the liver of fish cultured in the BFT treatment was significantly higher than
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that in the control group (P < 0.05). There were no significant differences in the

trypsin and lipase activities in the fish intestines between the BFT treatment and the

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control group (P > 0.05), whereas the intestine amylase activity of fish cultured in the

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BFT tanks was significantly higher than that in the control tanks (P < 0.05).

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It is possible that biofloc plays a role in stimulating the activities of digestive

enzymes (Moss et al., 2001; Xu et al., 2012a; Xu et al., 2012b). In addition, Xu and

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Pan (2012) found that biofloc displayed relatively high amylase activities, which may

account for the increased intestinal amylase activity of fish growing in the BFT

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treatment in this study. Furthermore, enhanced liver lipase may facilitate the digestion

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and utilization of lipid, which could have increased the crude lipid content of tilapia in
the BFT group. It is evident that the enzyme activities were tissues-specific, i.e.,

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highest in the liver and lowest in the intestine. The BFT treatment had a stimulatory
effect on the digestive enzyme activities, which may have contributed to the enhanced
growth performance of the tilapia, and the influence of BFT on enzyme activity varied
between the liver and intestine.
3.5. Hematology and immune response of fish
Fish hematological parameters reflect the health status of fish (Harikrishnan et al.,
2011). In this study, biofloc had no apparent effect on RBC, WBC, Hb, or Ht (Table 4),
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indicating that the biofloc system in our study had no negative effect on the physical

conditions of the tilapia. Similarly, Azim and Little (2008) reported that blood Ht of

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Nile tilapia did not vary statistically between the biofloc treatment and the control

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group. Furthermore, the total hemocyte count of shrimp did not vary significantly

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between biofloc-treated and control groups (Souza et al., 2014; Xu and Pan, 2014). In
contrast, Xu and Pan (2013) found that the total hemocyte count in shrimp was

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significantly higher in biofloc treatments than in the control group. It seems that

different experimental conditions and culture species may account for this

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phenomenon.

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In the present study, the total protein content in fish serum did not differ
significantly between the two groups (P > 0.05, Table 4). Although the serum total

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SOD activity of fish cultured in the BFT treatment was higher than that in the control
group, these values were not statistically different. However, BFT significantly
increased serum GPX and LSZ activities of fish (P < 0.05). On the contrary, Luo et al.
(2014) demonstrated that the serum total SOD activity in BFT fish was significantly
higher than in the fish cultured in the RAS, whereas the serum LSZ activity showed
no substantial difference. These results may be due to the relatively short duration of
our culture period and differences in stocking density compared with their study.
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In the immune defense of fish, LSZ is an important factor. The main ability of

this enzyme is antibacterial activity against Gram-positive and Gram-negative bacteria

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(Saurabh and Sahoo, 2008). Results indicate that biofloc can improve the immune

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response of tilapia to a certain extent. Biofloc is not only a source of additional

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nutrition, such as proteins, lipids, minerals and vitamins (Izquierdo et al., 2006; Ju et
al., 2008b; Moss et al., 2006; Xu et al., 2012a), but also provides abundant natural

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microbes and bioactive compounds such as carotenoids and fat-soluble vitamins (Ju et

al., 2008a), and other immunostimulatory compounds (Crab et al., 2012) that may

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stimulate the immune response of cultured fish. In addition, we found that BFT

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played a positive role in feed utilization and the digestive enzyme activities of fish
(Xu and Pan, 2012; Xu et al., 2012a), which may have enhanced the assimilation of

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dietary bioactive substances from the feed and then exerted an immune-stimulating
effect on the fish cultured in the BFT system. Further investigation is required to
determine the mechanisms of biofloc on the fish immune system.

4. Conclusions
The major objective of this study was to evaluate the effects of BFT on the
growth, digestive enzyme activity, hematology, and immune response of GIFT in
light-limited and zero-water exchange culture tanks. BFT had beneficial effects on the
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maintenance of good water quality, improvement of feed utilization, and the growth

performance of GIFT compared with the control group. The crude lipid content of

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BFT fish exhibited an increasing trend, which suggests that BFT can influence the fish

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body composition. Furthermore, BFT improved the digestive enzyme activity of fish,

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which consequently enhanced the feed utilization and growth performance. Although
there were no significant differences in the hematological parameters of GIFT

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between the two groups, BFT produced a stimulatory effect on the immune response

of GIFT in some way, which may enhance the growth of these fish. Further research

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should focus on an optimal method to manage the biofloc concentration and

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manipulation of the microbial communities in the biofloc. This may allow better
understanding of the pathways and mechanisms of BFT on the growth, digestive

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enzyme activity, hematology, and immune response of aquatic animals.

Acknowledgments
This work was supported by the National Science and Technology Support
Program (2012BAD25B03). The authors thank the staff at the Key laboratory of
Fishery Equipment and Engineering, Ministry of Agriculture of the Peoples Republic
of China for their support towards this study.
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References:
Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Huque, S., Salam, M.A., Azim, M.E., 2008. C/N

ratio control and substrate addition for periphyton development jointly enhance freshwater prawn

IP

Macrobrachium rosenbergii production in ponds. Aquaculture 280, 117-123.

Avnimelech, Y., 1999. Carbon nitrogen ratio as a control element in aquaculture systems. Aquaculture

SC
R

176, 227-235.

Avnimelech, Y., 2006. Bio-filters: the need for an new comprehensive approach. Aquaculture
Engineering 34, 172-178.

Avnimelech, Y., 2007. Feeding with microbial flocs by tilapia in minimal discharge bioflocs

NU

technology ponds. Aquaculture 264, 140-147.

Avnimelech, Y., 2012. Biofloc technology-A Practical Guide Book, 2nd ed. The World Aquaculture

MA

Society, Baton Rouge, LA, USA 73-91.

Avnimelech, Y., Kochva, M., Diab, S., 1994. Development of controlled intensive aquaculture systems
with a limited water exchange and adjusted C to N ratio. Israeli Journal of Aquaculture-Bamidgeh

46, 119-131.

TE

Azim, M.E., Little, D.C., 2008. The biofloc technology (BFT) in indoor tanks: water quality, biofloc
composition, and growth and welfare of Nile tilapia (Oreochromis niloticus). Aquaculture 283,
29-35.

CE
P

Azim, M.E., Little, D.C., Bron, J.E., 2008. Microbial protein production in activated suspension tanks
manipulating C:N ratio in feed and the implications for fish culture. Bioresource Technology 99,
3590-3599.

AC

Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to
acrylamide gels. Analytical Biochemistry 44, 276-287.
Boman, H.G., Nilsson-faye, I., Paul, K., Rasmuson, T., 1974. Insect immunity I. Characteristics of an
inducible cell-free antibacterial reaction in hemolymph of Samia Cynthia pupae. Infection and
Immunity 10, 136-145.
Bradford, M.A., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.
Burford, M.A., Thompson, P.J., McIntosh, R.P., Bauman, R.H., Pearson, D.C., 2004. The contribution
of flocculated material to shrimp (Litopenaeus vannamei) nutrition in a high-intensity,
zero-exchange system. Aquaculture 232, 525-537.
Chou, B., Shiau, S.Y., 1996. Optimal dietary lipid level for growth of juvenile hybrid tilapia,
Oreochromis niloticusOreochromis aureus. Aquaculture 143, 185-195.
Crab, R., Avnimelech, Y., Defoirdt, T., Bossier, P., Verstraete, W., 2007. Nitrogen removal techniques
in aquaculture for a sustainable production. Aquaculture 270, 1-14.
25

ACCEPTED MANUSCRIPT
Crab, R., Chielens, B., Wille, M., Bossier, P., Verstraete, W., 2010. The effect of different carbon
sources on the nutritional value of bioflocs, a feed for Macrobrachium rosenbergii postlarvae.
Aquaculture Engineering 41, 559-567.

Crab, R., Defoirdt, T., Bossier, P., Verstraete, W., 2012. Biofloc technology in aquaculture: beneficial

IP

effects and future challenges. Aquaculture 356-357, 351-356.

Crab, R., Kochva, M., Verstraete, W., Avnimelech, Y., 2009. Bio-flocs technology application in

SC
R

over-wintering of tilapia. Aquaculture Engineering 40, 105-112.

Craig, S., Helfrich, L.A., 2009. Understanding fish nutrition, feeds, and feeding. Virginia: College of
Agriculture and Life Sciences, Virginia Polytechnic Institute and State University.
De Schryver, P., Crab, R., Defoirdt, T., Boon, N., Verstraete, W., 2008. The basics of bio-flocs

NU

technology: the added value for aquaculture. Aquaculture 277, 125-137.

Dey, M.M., Paraguas, F.J., Bimbao, G.B., Regaspi, P.B., 2000. Socioeconomics of disseminating

MA

genetically improved Nile tilapia in Asia: an introduction. Aquaculture Economic and

Management 4, 5-11.

Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the stoichiometry of

photoautotrophic, autotrophic, and heterotrophic removal of ammonia-nitrogen in aquaculture

TE

systems. Aquaculture 257, 346-358.

Emerenciano, M., Ballester, E.L.C., Cavalli, R.O., Wasielesky, W., 2012. Biofloc technology
application as a food source in a limited water exchange nursery system for pink shrimp

CE
P

Farfantepenaeus brasiliensis (Latreille, 1817). Aquaculture Engineering 43, 447-457.

Erlanger, B., Kokowsky, N., Cohen, W., 1961. The preparation and properties of two new chromogenic
substrates of trypsin. Archives of Biochemistry and Biophysis 95, 271-278.

AC

Floh, L., Gnzler, W.A., 1984. Assay of glutathione peroxidase. Methods in Enzymology105,
115-121.

Hargreaves, J.A., 2006. Photosynthetic suspended-growth systems in aquaculture. Aquaculture

Engineering 34, 344-363.
Harikrishnan, R., Kim, M.C., Kim, J.S., Balasundaram, C., Heo, M.S., 2011. Protective effect of herbal
and probiotics enriched diet on haematological and immunity status of Oplegnathus fasciatus
(Temminck & Schlegel) against Edwardsiella tarda. Fish & Shellfish Immunology 30, 886-893.
Hultmark, D., Steiner, H., Rasmuson, T., Boman, H.G., 1980. Insect immunity. Purification and
properties of three inducible bactericidal proteins from hemolymph of immunized pupae of
Hyalophoracecropia. European Journal of Biochemistry 106, 7-16.
Izquierdo, M., Forster, I., Divakaran, S., Conquest, L., Decamp, O., 2006. Effect of green and clear
water and lipid source on survival, growth and biochemical composition of pacific white shrimp
Litopenaeus vannamei. Aquaculture Nutrition 12, 192-202.
Jauncey, K., 2000. Nutritional requirements. Tilapias: Biology and Exploitation. Springer 327-375.
26

ACCEPTED MANUSCRIPT
Ju, Z.Y., Forster, I., Conquest, L., Dominy, W., 2008a. Enhanced growth effects on shrimp (Litopenaeus
vannamei) from inclusion of whole shrimp floc or floc fractions to a formulated diet. Aquaculture
Nutrition 14, 533-543.

Ju, Z.Y., Forster, I., Conquest, L., Dominy, W., Kuo, W.C., Horgen, F.D., 2008b. Determination of

IP

microbial community structures of shrimp floc cultures by biomarkers and analysis of floc amino
acid profiles. Aquaculture Research 39, 118-133.

SC
R

Kim, S., Pang, Z., Seo, H., Cho, Y., Samocha, T., Jang, I., 2014. Effect of bioflocs on growth and
immune activity of Pacific white shrimp. Aquaculture Engineering 45, 362-371.
Lim, C., Yildirim-Aksoy, M., Li, M.H., Welker, T.L., Klesius, P.H., 2009. Influence of dietary levels of
lipid and vitamin E on growth and resistance of Nile tilapia to Streptococcus iniae challenge.

NU

Aquaculture 298, 76-82.

Luo, G.Z., Avnimelech, Y., Pan, Y.F., Tan, H.X., 2013. Inorganic nitrogen dynamics in sequencing batch

MA

reactors using bioflocs technology to treat aquaculture sludge. Aquaculture Engineering 52, 73-79.
Luo, G.Z., Gao, Q., Wang, C.H., Liu, W.C., Sun, D.C., Li, L., Tan, H.X., 2014. Growth, digestive
activity, welfare, and partial cost-effectiveness of genetically improved farmed tilapia

(Oreochromis niloticus) cultured in a recirculating aquaculture system and an indoor biooc

TE

system. Aquaculture 422-423, 1-7.

Moss, S.M., Divakaran, S., Kim, B.G., 2001. Stimulating effects of pond water on digestive enzyme

125-131.

CE
P

activity in the Pacic white shrimp, Litopenaeus vannamei (Boone). Aquaculture Research 32,

Moss, S.M., Forster, I.P., Tacon, A.G.J., 2006. Sparing effect of pond water on vitamins in shrimp diets.
Aquaculture 258, 388-395.

AC

Piedrahita, R.H., 2003. Reducing the potential environmental impact of tank aquaculture effluents
through intensification and recirculation. Aquaculture 226, 35-44.
Ray, J.A., Lewis, B.L., Browdy, C.L., Leffler, J.W., 2010. Suspended solids removal to improve shrimp
(Litopenaeus vannamei) production and an evaluation of a plant-based feed in minimal-exchange,
superintensive culture systems. Aquaculture 299, 89-98.
Robyt, J.F., Whelan, W.J., 1968. The b-amylases. In:Starch and Its Derivates. London, pp. 477-497.
Saurabh, S., Sahoo, P.K., 2008. Lysozyme: an important defence molecule of fish innate immune
system. Aquaculture Research 39, 223-239.
Serfling, S.A., 2006. Microbiol flocs: Natural treatment method supports freshwater, marine species in
recirculating systems. Global Aquaculture Advocate 9, 34-36.
Souza, D.M.D., Suita, S.M., Romano, L.A., Jr, W.W., Ballester, E.L.C., 2014. Use of molasses as a
carbon source during the nursery rearing of Farfantepenaeus brasiliensis (Latreille, 1817) in a
Biofloc technology system. Aquaculture Engineering 45, 270-277.
Tacon, A.G.J., Maciocci, G., 1988. The nutrition and feeding of farmed fish and shrimp-A Training
27

ACCEPTED MANUSCRIPT
Manual.3. Feeding Methods. FAO Field Document. Projet GCP/RLA/075/ITA, Field
Document, 7.
Van Kampen, E.J., Zijlstra, N.C., 1961. Determination of haemoglobin. Clinica Chimica Acta 5,

719-720.

IP

Widanarni, Ekasari, J., Maryam, S., 2012. Evaluation of biofloc technology application on water
quality and production performance of Red Tilapia Oreochromis sp. cultured at different stocking

SC
R

densities. Hayati Journal of Biosciences 19, 73-80.

Winkler, U.K., Stuckman, M., 1979. Glycogen, hyaluronate, and some other polysaccharides greatly
enhance the formation of exolipase by Serratia marcescens. The Journal of Bacteriology138,
663-670.

NU

Xu, W.J., Pan, L.Q., 2012. Effects of bioflocs on growth performance, digestive enzyme activity and
body composition of juvenile Litopenaeus vannamei in zero-water exchange tanks manipulating

MA

C/N ratio in feed. Aquaculture 356-357, 147-152.

Xu, W.J., Pan, L.Q., 2013. Enhancement of immune response and antioxidant status of Litopenaeus
vannamei juvenile in biofloc-based culture tanks manipulating high C/N ratio of feed input.

Aquaculture 412-413, 117-124.

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Xu, W.J., Pan, L.Q., 2014. Evaluation of dietary protein level on selected parameters of immune and
antioxidant systems, and growth performance of juvenile Litopenaeus vannamei reared in
zero-water exchange biofloc-based culture tanks. Aquaculture 426-427, 181-188.

CE
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Xu, W.J., Pan, L.Q., Sun, X., Huang, J., 2012a. Effects of bioflocs on water quality, and survival,
growth and digestive enzyme activities of Litopenaeus vannamei (Boone) in zero-water exchange
culture tanks. Aquaculture Research 44, 1093-1102.

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Xu, W.J., Pan, L.Q., Zhao, D., Huang, J., 2012b. Preliminary investigation into the contribution of
bioflocs on protein nutrition of Litopenaeus vannamei fed with different dietary protein levels in
zero-water exchange culture tanks. Aquaculture 350-353, 147-153.
Zhao, P., Huang, J., Wang, X., Song, X., Yang, C., Zhang, X., Wang, G., 2012. The application of
bioflocs technology in high-intensive, zero exchange farming systems of Marsupenaeus japonicus.
Aquaculture 354-355, 97-106.

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Figure. 1. Changes of total suspended solids (TSS) in the control group

and biofloc technology (BFT) treatment during the experimental period.
Values are means S.D. of three replicate tanks per sampling time in
each group.

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Figure. 2. Changes of dissolved inorganic nitrogen concentrations in the

control group and biofloc technology (BFT) treatment during the
experimental period. Values are means S.D. of three replicate tanks per
sampling time in each group.
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Fig. 3. Specific activities of trypsin, amylase and lipase in liver and

intestine of tilapia raised in the control group and biofloc technology
(BFT) treatment at the end of 8-week experiment. Each value represents
mean S.E. (n=9). Values within the same tissue with different
superscript letters are significantly different at P<0.05.

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Table 1 Comparison of water quality parameters in the control group

and biofloc technology (BFT) treatment during the 8-week
experimental period.
Parameter
Control
BFT
Temperature (C)
27.06
27.11

a
a
0.11
0.04
Dissolved oxygen
7.70
6.90 0.06b
(mg L1)
0.10a
pH
7.55
7.57 0.05a
0.03a
Each value represents mean S.D. (n=168). Values in the same row with
different superscript letters are significantly different at P<0.05.

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Table 2 Growth performance and feed utilization of tilapia growing

in the control group and biofloc technology (BFT) treatment at the
end of 8-week feeding experiment.
Parameter
Control
BFT
Initial individual weight
50.25
50.61 0.91a
(g)
0.78a
Final individual weight (g) 146.66
160.54

b
a
0.85
3.06
Initial number (fish
30a
30a
tank1)
191.93
216 0.83a
Weight gain (%)
2.81b
Specific
growth
rate 1.92 0.02b 2.04 0.01a
(SGR,%day1)
Survival rate (%)
100a
100a
Feed conversion rate
0.97 0.01a 0.83 0.03b
(FCR)
Protein efficiency ratio
2.20 0.03b 2.59 0.09a
(PER)
Net yield (kg m3)
5.88 0.09b 6.31 0.08a
Each value represents mean S.E. (n=9). Means in the same row with
different superscripts are significantly different at P<0.05.

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Table 3 Proximate composition (% dry matter) of the biofloc

collected from biofloc technology (BFT) treatment, and the whole
body of tilapia cultured in the control group and BFT treatment at
the end of 8-week feeding experiment.
Composition(%DM) Biofloc
Whole body of tilapia
Control
BFT
Crude protein
41.13
53.65
51.87 0.99a
a
0.88
0.25
Crude lipid
1.03
14.30
18.30 0.62a
b
0.03
2.20
Ash
6.07
21.30 0.4a 18.80 0.35a
0.12
Each value represents mean S.E. (n=9). Means in the same row with
different superscripts are significantly different at P<0.05.

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Table 4 Hematology and immune response of tilapia reared in the

control group and biofloc technology (BFT) treatment at the end of
8-week feeding experiment.
Parameter
Control
BFT
White blood cell107/ul 8.86 2.36a 7.54 0.94a
Red blood cell109/ul 1.46 0.19a 1.33 0.09a
5.18
Hemoglobing/dL
0.367a
5.02 0.21a
23.86
Haematocrit%
26.83 1.2a 2.57a
Total superoxide
120.75
133.85
a
6.27
14.24a
dismutaseU/ml
Glutathione peroxidase
1137.51
1335.00
b
35.36
32.87a
U/ml
2409.09
2684.85
LysozymeU/ml
b
63.64
39.74a
40.03
36.73
Total proteinmg/ml
a
5.05
3.24a
Each value represents mean S.E. (n=9). Means in the same row with
different superscripts are significantly different at P<0.05.

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1. BFT improved the crude lipid content of cultured tilapia.

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2. BFT could enhance the digestive enzyme activity of cultured tilapia.

3. BFT had a positive effect on immune response of cultured tilapia.

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4. BFT showed no apparent effect on hematology analysis of cultured tilapia.

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