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Effect of biofloc technology on growth, digestive enzyme activity, hematology,and immune response of genetically improved farmed tilapia (Oreochromisniloticus)

Lina Long, Jing Yang, Yuan Li, Chongwu Guan, Fan Wu

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

To appear in: Aquaculture

Received date: 17 September 2014Revised date: 11 May 2015Accepted date: 12 May 2015

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

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Effect of biofloc technology on growth,

digestive enzyme activity, hematology, and

immune response of genetically improved

farmed tilapia (Oreochromis niloticus)

Lina Long a, Jing Yang

a,*, Yuan Li

b, Chongwu Guan

a, Fan Wu

a

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

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

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

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

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

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

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

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

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

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

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

harvest. BFT significantly increased fish specific growth rate and net yield.

Compared with the control group, the individual fish weight, weight gain and

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

glutathione peroxidase and lysozyme activities were considerably higher in the BFT

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

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

GIFT.

Statement of Relevance

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

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

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

documented mainly the water quality, growth and production performance of

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

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

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

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

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

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

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

clear water system operated with water exchange in cultivating both

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

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

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

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

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,

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

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

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

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

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

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

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

source to increase nutrient use efficiency.

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,

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

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

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.,

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.

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

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

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

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

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

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

and nutritional content of the biofloc and fish were investigated, which may provide

insights into exploring the effects of BFT on aquatic animals.

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

well determine the variation of the biofloc and acquire optimal experimental

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

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

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

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

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

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

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

per tank.

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

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

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

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

fortnightly to adjust the feeding rate to 3% of total body weight. Feed inputs were

recorded daily for each tank.

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

Concentrated biofloc samples were collected from the BFT group by passing

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

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.

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

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

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,

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

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

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

sampled from each tank, and each fish was minced, pooled, and frozen at -20 °C for

whole-body proximate composition analysis.

2.5. Fish digestive enzyme activity, hematological and immune parameters

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

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

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

Hemoglobin (Hb) was determined by measuring the formation of

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

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

activity was measured using the method of Flohé and Günzler (1984). The amount of

GPX needed to oxidize 1 μmol of NADPH per min was defined as one unit. The

activity of serum lysozyme (LSZ) was determined following the turbidimetric method

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

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

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

calculated using bovine serum albumin as the protein standard and expressed as mg

mL−1

of fish serum.

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 μmol 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 μmol of nitroanilide from substrate in 1 min at 37 °C. The soluble protein

concentration was measured in diluted homogenates following the Bradford method

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

lipase activities are both presented as specific activity U g-1

of protein, and amylase

activity is expressed as U mg-1

of protein.

2.6. Proximate composition analysis

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

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

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

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, % day−1

) = 100 × [Ln(final body weight) − Ln(initial body

weight)]∕(days of experiment),

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

Weight gain (%) = 100 × (final body weight − initial body weight)∕initial body

weight,

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

increase,

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

feed protein consumed

Data obtained from the experiment were analyzed using SPSS 18.0 software

(SPSS, Chicago, USA) for Windows. One-way ANOVA was performed on the

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

two groups (P > 0.05). DO was maintained at a level greater than 6 mg L−1

, and

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

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

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

acceptable range for the survival and growth of fish.

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

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

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

ponds, TSS concentrations can reach levels of up to 1000 mg L-1

, and the level should

be limited to approximately 400 mg L-1

, though data that indicate this level are lacking.

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

L-1

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

further investigation.

The changing tendency of TAN concentrations was fairly consistent over time

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

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

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

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

-1,

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

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

accumulation of NO2--N and NO3

--N in the first few weeks may have been caused by

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

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

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

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

The net yield of the BFT group (6.22 kg m−3

) was significantly higher than that of the

control (5.88 kg m−3

) (P < 0.05). Luo et al. (2014) reported similar results for fish

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

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

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

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

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

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

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

(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

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.,

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

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

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

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

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

different experimental conditions and culture species may account for this

phenomenon.

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

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

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

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

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

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

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

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

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

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

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

which consequently enhanced the feed utilization and growth performance. Although

there were no significant differences in the hematological parameters of GIFT

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

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

manipulation of the microbial communities in the biofloc. This may allow better

understanding of the pathways and mechanisms of BFT on the growth, digestive

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 People’s Republic

of China for their support towards this study.

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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 ±

0.11a

27.11 ±

0.04a

Dissolved oxygen

(mg L−1

)

7.70 ±

0.10a

6.90 ± 0.06b

pH 7.55 ±

0.03a

7.57 ± 0.05a

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

(g)

50.25 ±

0.78a

50.61 ± 0.91a

Final individual weight (g) 146.66 ±

0.85b

160.54 ±

3.06a

Initial number (fish

tank−1

)

Weight gain (%)

30a

191.93 ±

2.81b

30a

216 ± 0.83a

Specific growth rate

(SGR,%day−1

)

1.92 ± 0.02b 2.04 ± 0.01

a

Survival rate (%)

Feed conversion rate

(FCR)

100a

0.97 ± 0.01a

100a

0.83 ± 0.03b

Protein efficiency ratio

(PER)

2.20 ± 0.03b 2.59 ± 0.09

a

Net yield (kg m−3

) 5.88 ± 0.09b 6.31 ± 0.08

a

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 ±

0.88

53.65 ±

0.25a

51.87 ± 0.99a

Crude lipid 1.03 ±

0.03

14.30 ±

2.20b

18.30 ± 0.62a

Ash 6.07 ±

0.12 21.30 ± 0.4

a 18.80 ± 0.35

a

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 cell（×107/ul） 8.86 ± 2.36

a 7.54 ± 0.94

a

Red blood cell（×109/ul） 1.46 ± 0.19

a 1.33 ± 0.09

a

Hemoglobin（g/dL） 5.18 ±

0.367a 5.02 ± 0.21

a

Haematocrit（%）

26.83 ± 1.2a

23.86 ±

2.57a

Total superoxide

dismutase（U/ml）

120.75 ±

6.27a

133.85 ±

14.24a

Glutathione peroxidase

（U/ml）

1137.51 ±

35.36b

1335.00 ±

32.87a

Lysozyme（U/ml） 2409.09 ±

63.64b

2684.85 ±

39.74a

Total protein（mg/ml） 40.03 ±

5.05a

36.73 ±

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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Highlights

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

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

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

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