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
Wu-Jie Xu, Lu-Qing Pan
PII:
DOI:
Reference:

S0044-8486(14)00054-4
doi: 10.1016/j.aquaculture.2014.02.003
AQUA 631028

To appear in:

Aquaculture

Received date:
Revised date:
Accepted date:

27 October 2013
5 February 2014
6 February 2014

Please cite this article as: Xu, Wu-Jie, Pan, Lu-Qing, 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 biooc-based culture tanks,
Aquaculture (2014), doi: 10.1016/j.aquaculture.2014.02.003

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Evaluation of dietary protein level on selected parameters of immune and

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reared in zero-water exchange biofloc-based culture tanks

antioxidant systems, and growth performance of juvenile Litopenaeus vannamei

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Wu-Jie Xu, Lu-Qing Pan

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Corresponding author

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China, Qingdao 266003, China

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The Key Laboratory of Mariculture, Ministry of Education, Ocean University of

Tel.: +86-532-82032963;

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Fax: +86-532-82032963;

E-mail: panlq@ouc.edu.cn;
Address:

Laboratory of Environmental Physiology of Aquatic Animal, Fisheries

College, Ocean University of China, Yushan Road 5, Qingdao 266003, China.

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AB STRACT

The biofloc technology was proposed as a sustainable solution to culture shrimp with

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low protein feeds even in intensive systems, which can effectively control water

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quality under negligible water exchange and sustain healthy culture of shrimp. This
study was conducted to evaluate the effects of four dietary protein levels (20%, 25%,

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30% and 35%) on selected parameters of immune and antioxidant systems, and
growth performance of Litopenaeus vannamei juveniles reared in zero-water

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exchange biofloc-based intensive culture tanks for a period of 7 weeks. Good water

quality was maintained with the promotion and development of biofloc through

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sucrose addition during the feeding experiment. At the end of the experiment, the total

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hemocyte count in the hemolymph, phagocytic activity of the hemocyte, and

antibacterial activity and bacteriolytic activity in the plasma of shrimp showed no

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significant differences (P > 0.05) between the four treatments with four dietary
protein levels. The shrimp in the treatment with 20% dietary protein level had the
lowest total antioxidant capacity (T-AOC) in both the plasma and the hepatopancreas,
and the lowest reduced glutathione/oxidized glutathione (GSH/GSSG) ratio in the
plasma. No significant differences were found in the antioxidant status (in terms of TAOC, superoxide dismutase activity, GSH level and GSH/GSSG ratio in the plasma
and the hepatopancreas) of shrimp fed with 25%, 30% and 35% dietary protein levels.
Furthermore, except for the suboptimal growth performance of shrimp in the
treatment with 20% dietary protein level, the growth (in terms of final weight, weight

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gain and specific growth rate) and feed conversion rate (FCR) of shrimp in treatments
with 25%, 30% and 35% dietary protein levels showed no significant differences (P >

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0.05). Mean survival rates were above 85%, with no significant differences (P > 0.05)

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between the four treatments with four dietary protein levels. The results of this study
demonstrated that, when juveniles of L. vannamei were reared in zero-water exchange

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biofloc-based intensive culture tanks, dietary protein level can be reduced from 35%
to 25% without affecting survival, growth, FCR, and physiological status of immune

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response and antioxidant capability, indicating that the promoted biofloc could

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contribute to the protein nutrition and physiological health of cultured shrimp.

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Keywords: Litopenaeus vannamei; Biofloc technology; Dietary protein; Growth

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performance; Immune condition; Antioxidant status

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1. Introduction

The application of biofloc technology (BFT) in shrimp aquaculture has gained

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great attention recently because it offers a practical solution to effectively control

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water quality under negligible water exchange and improve shrimp growth
performance, thus achieving efficient and healthy culture of shrimp (Avnimelech,

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2012; Crab et al., 2012; De Schryver et al., 2008; Stokstad, 2010; Xu and Pan, 2013).
In heterotrophic biofloc-based shrimp culture systems, the driving force is dense

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populations of active heterotrophic bacteria which can be promoted by increasing the

C/N ratio of feed input and assimilate the waste nitrogen from culture water resulting

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in the production of new microbial biomass (cellular proteins) (Avnimelech, 2006;

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Crab et al., 2007; Ebeling et al., 2006). As the microbial communities develop,
bioflocs are formed from heterogeneous aggregates of microorganisms and organic

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particles (De Schryver et al., 2008; Hargreaves, 2006). As a supplemental food source
available for cultured shrimp, the biofloc can be consumed and provide a significant
fraction of protein demand (Ballester et al., 2010; Burford et al., 2004; Crab et al.,
2010; Wasielesky et al., 2006; Xu et al. 2012). Some studies suggested that using low
protein feeds in biofloc-based culture systems could also achieve good survival and
growth performance of cultured shrimp (Ballester et al., 2010; Megahed, 2010;
Wasielesky et al., 2006; Moss, 2002). With biofloc supplementing the inadequate
portion of protein intake, the proper use of low protein feed can reduce dietary
fishmeal inclusion, thus reducing the cost of feed and improving the efficency of

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production.

Physiological functions such as immune and antioxidant systems are essential for

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shrimp in maintaining their health and guaranteeing satisfactory growth performance,

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especially under the condition of environmental stresses (e.g. pollutions and

pathogens) (Bachre, 2000; Castex et al., 2010), and thereby ensure healthy culture of

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shrimp. Otherwise, the induced decrease in immune and antioxidant defense functions
of cultured shrimp could easily lead to the outbreak of diseases and cause a great

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economic loss (Bachre, 2000). The supply of feed nutrition, mainly protein, is

required for the normal physiological metabolism and growth of cultured shrimp

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(Kureshy and Davis, 2002). This implies that the shrimp should not only be fed with

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adequate dietary protein for superior growth but also be maintained in a healthy
nutritional and physiological state. The ingested proteins are not only involved in the

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synthesis of hormones and enzymes but are some components of the immune and
antioxidant systems. Previous studies have shown that very low protein feeds can
weaken the physiological functions of cultured shrimp, such as protein metabolism
and immune response (Goimier et al., 2006; Pascual et al., 2004). These results are
demonstrated by the experiments conducted in clear water or recirculating systems.
There may be some change in biofloc-based shrimp culture systems because of the
presence of biofloc. Our recent study have shown that biofloc could enhance immune
cellular response and antioxidant status of cultured shrimp probably because of being
rich in natural microbes and bioactive compounds (Ju et al., 2008a; Xu and Pan,

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2013). This result was found after juviniles L. vannamei were fed a formulated 35%
crude protein diet in biofloc-based culture tanks for a period of 30 days; however, it is

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not clear if feeding appropriate low protein feed (< 35% ) with the contribution of

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biofloc could sustain the health status of cultured shrimp in such systems.
The white shrimp L. vannamei (Boone) is a commercially important shrimp

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species and has become the main farmed species in many parts of the world. Over the
past decade, production of L. vannamei in biofloc-based intensive systems under

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negligible or zero water exchange has achieved sustainable stage (Avnimelech, 2012;

Haslun et al. 2012; Taw, 2010). However, the information concering optimal dietary

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protein level for the well performance of cultured shrimp is still limited in such

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systems, especially through the evaluation of both growth performance and

physiological health status with the potential contribution of biofloc. Therefore, this

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study was conducted to evaluate the effects of different dietary protein levels on some
selected parameters of immune and antioxidant systems, and growth performance of L.
vannamei juveniles in zero-water exchange biofloc-based culture tanks.
2. Materials and methods
2.1. Diets preparation and formulation
Four practical diets containing grade levels of 20%, 25%, 30% and 35% crude
protein (CP) were formulated (Table 1). A mixture of Peruvian fish meal, squid
visceral meal, defatted soybean meal and wheat meal was used to obtain the desired

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protein levels. Soybean meal and wheat flour were utilized as the main carbon source,
and lipid sources were soybean lecithin oil and fish oil. Other dietary ingredients were

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added to fulfill the nutritional requirements of L. vannamei (Hu et al., 2008) and were

Table 1

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kept in the same levels in all diets.

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The diets (Table 1) were prepared by mixing the dry ingredients in a mixer

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followed by supplementation of soybean lecithin oil and fish oil. Then an appropriate
amount of hot water was added, followed by further mixing. The wet mash was

compressed through a 1.5-mm die using a meat grinder. The resulting pellets were

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dried in a parallel flux forced air drier at 60 C until the moisture content was reduced

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to about 10%. After drying, strings were broken and pellets were sieved in a 2-mm
sieve and stored in plastic bags at 20 C until use.

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Proximate composition analysis of crude protein, crude lipid and ash content of
the experimental diets were performed by the standard methods of AOAC (1995).
Protein was determined by measuring nitrogen using the Kjeldahl method and
multiplying by 6.25; lipid by ether extraction using Soxhlet; ash by combustion at
550 C. Gross energy of the experimental diets was determined by an adiabatic bomb
calorimeter (PARR1281, PARR, Moline, Illinois, USA).
2.2. Shrimp and experimental setup
Healthy juveniles of L. vannameiweighing 6.5 0.4 g, were obtained from

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Laoshan Aquaculture Station in Qingdao, China. Shrimp were acclimated in an indoor
fiberglass tank (6 m2, 5 tons) containing aerated clear seawater (salinity 32 0.4 g L1,

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pH 8.2 0.3) at 26 0.4 C for 7 days prior to the experiment. The water used for the

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experiment was pumped from the sea near Qingdao and sand-filtered. During the
acclimation period, half of the water was renewed daily and shrimp were fed three

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times per day with the commercial feed (35% CP) applied in the shrimp farm.
The feeding experiment was carried out in indoor fiberglass tanks (72 cm 56 cm

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40 cm) with a water volume of 125 L each. Prior to stocking shrimp, each tank was

inoculated with 62.5 mL concentrated biofloc (equivalent to 0.5 mL L1 of tank water

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volume) collected from an indoor biofloc-based shrimp culture pond. The inoculant

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was characterized by Bacillus sp. and proteobacterium as predominant bacteria (Zhao

et al., 2012), and harvested by passing culture water through a 10-m mesh size nylon

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bag filter (Burford et al., 2004). All tanks were aerated and mixed continuously using
air-stones connected to an air pump. No water was exchanged during the experimental
period, and dechlorinated freshwater was added to compensate for evaporation losses.
The photoperiod was maintained on a 12:12 h light-dark cycle (artificial luminosity of
~600 lux).
Acclimatized shrimp in the intermolt phase were selected and weighed to obtain
their initial body weights (wet weight), and then randomly stocked into 16 tanks.
Intermolt phase of shrimp was determined following the criteria of Robertson et al.
(1987) in this study. Each tank contained 28 shrimp (equivalent to a shrimp density of

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224 m3 water volume), and four replicate tanks were randomly assigned to each of

the four diets containing CP levels of 20%, 25%, 30% and 35%.

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2.3. Feeding management and water quality monitoring

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The experimental diets were fed to shrimp for a period of 7 weeks. Feeding was
done by hand to apparent satiation 3 times per day at 06:00, 14:00 and 22:00 hours.

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The daily feeding rate was slowly reduced from approximately 5% of total body

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weight to 3% by the end of the feeding experiment, and it was adjusted daily based on
the observation of slight excess diets on feeding trays after 1 h of feeding. Diet inputs

in all tanks were recorded daily. Locally purchased sucrose (~95% purity) contained

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38% (w/w) carbon was used as a carbohydrate to manipulate an optimum C/N of the

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feed inputs (diet and sucrose) to approximately 15 for promoting the development of
biofloc in the experimental tanks (Hargreaves, 2006; Xu and Pan, 2012). The pre-

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weighed sucrose was added daily concomitantly with the 14:00 h feed, calculating to
be 20%, 30%, 40% and 52% of the daily diets inputs for 20%, 25%, 30% and 35% CP
diets, respectively.
During the 7-week experimental period, water temperature, salinity, dissolved
oxygen (DO) and pH were monitored daily at 08:00 ~ 10:00 h using a hand-held
multi-meter (YSI-6600V2, Yellow Springs Instruments Inc., Ohio, USA). Whenever
the pH of the water in any tank dropped below 7.2, NaHCO3 solution (8%, w/v) was
added into the tank water to raise the pH slowly. Water samples (100 mL) were
collected weekly at 14:00 h from each tank for total ammonia nitrogen (TAN), nitrite

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nitrogen (NO2-N) and total suspended solids (TSS) analyses following the Standard
methods for the examination of water and wastewater (APHA, 1998). Biofloc

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volume (BFV) was determined on site using Imhoff cones weekly, registering the

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volume taken in by the flocs in 1000 mL of the tank water after 30 min sedimentation
(Xu et al., 2012).

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2.4. Sampling procedures and performance calculations

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At the end of the experiment, 8 shrimp per tank were randomly sampled. Only
shrimp in the intermolt phase were used. This was to minimize internal variations, as

changes in physiological functions are generally observed during the molting phase in

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crustaceans (Bonilla-Gmez et al., 2012). Hemolymph (250 L) was withdrawn from

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the ventral sinus at the base of the first abdominal segment of each shrimp into a 1 mL
sterile syringe (25 gauge needle) containing an equal volume of anticoagulant solution

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(30 mM trisodium citrate, 0.34 M sodium chloride, and 10 mM EDTA-Na2, at a pH of

7.55 with osmolality adjusted to 780 mOsm kg1 using 0.115 M glucose) (Liu and
Chen, 2004). The anticoagulant-hemolymph of 8 shrimp were pooled and gently
mixed in a sterile Eppenddorf tube immediately. Then one part was used to determine
hemocyte count and phagocytic activity immediately; the remainder was centrifuged
at 800 g for 10 min at 4 C, and the supernatant fluid was dispensed into 2.0 mL
Eppenddorf tubes as plasma samples and stored at 80 C for analysis of other
parameters.
After hemolymph sampling, the shrimp were weighed individually. Then the

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hepatopancreas of the shrimp were excised, washed in cold normal saline (0.8%, w/v),
blotted dry, and flash frozen with liquid nitrogen in a mortar. The frozen

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hepatopancreas of 8 shrimp from each tank were pooled, ground, weighted, dispensed

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into 2.0 mL Eppenddorf tubes and stored at 80 C. Prior to analysis, samples were
defrosted at 4 C, and then were homogenized in 9 volumes w/v of chilled buffer (50

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mM Tris-HCl, 1 mM EDTA-Na2 and 0.25 mM sucrose at pH 8.2) with the help of an

electric homogenizer operating at 800 rpm. The crude homogenates were centrifuged

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at 3, 000 g for 10 min at 4 C and then the supernatants were collected for

immediate analysis of antioxidant parameters.

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After sample collection and preparation, the remaining shrimp in the tanks were

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harvested after draining off water: live shrimp were counted and final body weight
(wet weight) of each individual was weighed. Survival rate, weight gain, specific

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growth rate and feed conversion rate were calculated using the following equations:
Survival rate (%) = 100 (final shrimp count initial shrimp count), Weight gain (%)
= 100 (final body weight initial body weight) initial body weight, Specific growth
rate (% day1) = 100 [Ln(final body weight) Ln(initial body weight)]
experimental duration (days), Feed conversion rate = total dry weight of feed offered
total shrimp wet weight gained. In the present study, the feed conversion rate is
referred to as apparent efficiency and is of more practical than biological
significance, because actual consumption of the diets could not be monitored in the
biofoc-based tanks, nor could the impact of cannibalism and consumption of biofloc

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be directly assessed (Tacon et al., 2002).

2.5. Determination of immune parameters

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To measure total hemocyte count (THC) in hemolymph, a drop of anticoagulant-

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hemolymph was placed on the hemocytometer; and hemocyte were counted by

observing under Olympus light microscope and expressed as cells mL1 hemolymph.

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The immune response was determined by phagocytic activity of hemocyte, and

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antibacterial activity and bacteriolytic activity in plasma. Phagocytic activity of

hemocyte was measured using Vibrio alginolyticus by the method of Yue et al. (2010).

Phagocytic activity, defined as phagocytic percent was calculated as: Phagocytic

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percent (%) = (number of phagocytic hemocytes 200 hemocytes) 100%.

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Antibacterial activity and bacteriolytic activity in plasma were measured using Vibrio
harveyi and Micrococus lysoleikticus (Sigma), respectively, following the method of

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Jiang et al. (2005). Antibacterial activity and bacteriolytic activity, defined as Ua and
UL respectively, were calculated as follows:

Ua = (0 ) ,

UL = (A0 A) A;
Where A0 and A represent the initial and terminal optical density of the reaction
measured at 570 nm, respectively.
2.6. Determination of antioxidant parameters
The antioxidant status was determined by total antioxidant capacity (T-AOC),

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superoxide dismutase (SOD) activity and glutathione levels (both reduced (GSH) and
oxidized (GSSG) forms). T-AOC was determined using commercial kits (Nanjing

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Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's

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instructions. T-AOC of a sample is a quantitative measurement of the state of balance

of various antioxidant components using the ferric reducing/antioxidant power assay

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under specified reaction conditions. One unit of T-AOC was defined as the total
amount of sample antioxidants increasing the optical density of reaction system by

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0.01 per minute. SOD activity was assayed following the method of Marklund and
Marklund (1974) which is based on the auto-oxidation of pyrogallol. One unit of SOD

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activity was defined as the amount of enzyme inhibiting the autooxidation of

pyrogallol by 50% per minute. Total amounts of GSH and GSSG were determined

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spectrophotometrically using 5,5-Dithio-bis(2-nitrobenzoic acid) (DTNB) according

to the method of Anderson (1985) using commercial GSH (Sigma) as a standard.

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GSH/GSSG ratio, a valuable biomarker of antioxidant status, was systematically

calculated. The total protein content of hepatopancreas samples were measured in
diluted homogenates by the Bradford method (1976) using bovine serum albumin
(Sigma) as a standard. The results of antioxidant parameters were expressed as U
mL1 plasma in hemolymph and U mg1 protein in hepatopancreas.
2.7. Statistical analysis
All statistics were performed using IBM SPSS Statistics 20.0 software for
windows (IBM Corp., NY, USA). Data were analyzed using one-way ANOVA after

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checking tests of normality and homogeneity of variance. Arcsine transformation was
used for survival rate, weight gain and phagocytic percent. Significant differences

3. Results

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3.1. Biofloc development and water quality

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was used to identify the differences between treatments.

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were considered at P < 0.05. When significant differences were found, Tukeys test

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The biofloc development in terms of BFV and TSS over time is shown in Fig. 1.
Both BFV and TSS levels increased gradually throughout the experiment period and

the changing tendency of them over time were basically consistent. The treatments

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with higher dietary protein level had slightly higher BFV and TSS levels, and at the

Figure 1

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respectively.

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end of the experiment both of them were below 26 mL L1 and 430 mg L1,

The results of main water quality parameters monitored are shown in Table 2.
Except for the occasional regulation of pH, all water quality parameters remained
within recommended levels for shrimp culture throughout the experimental period.
Table 2
3.2. Immune condition of shrimp
The THC in the hemolymph, phagocytic activity (expressed as phagocytic percent)
of the hemocyte, and antibacterial activity and bacteriolytic activity in the plasma of

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shrimp showed no significant differences (P > 0.05) between the four treatments with

four dietary protein levels (Fig. 2A, B, C, D).

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Figure 2

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3.3. Antioxidant status of shrimp

The shrimp in the treatment with 20% CP level had the lowest T-AOC level in

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both the plasma and the hepatopancreas among the four treatments with four dietary

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protein levels, and there were no significant differences (P > 0.05) between the
treatments with 25%, 30% and 35% CP levels (Fig. 3A and E). The SOD activity and

the GSH level in both the plasma and the hepatopancreas of shrimp showed no

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significant differences (P > 0.05) between the four treatments with four dietary

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protein levels (Fig. 3B,F, C and G). The shrimp in the treatment with 20% CP level
had the lowest GSH/GSSG ratio in the plasma among the four treatments with four

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dietary protein levels, and there were no significant differences (P > 0.05) between
the treatments with 25%, 30% and 35% CP levels (Fig. 3D). The ratio of GSH/GSSG
in the hepatopancreas of shrimp showed no significant differences (P > 0.05) between
the four treatments with four dietary protein levels (Fig. 3H).
Figure 3
3.4. Survival, growth and FCR of shrimp
Mean survival rates were above 85%, with no significant differences (P > 0.05)
between the four treatments with four dietary protein levels (Table 3). The growth of

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shrimp in terms of final weight, weight gain (expressed as a percent of initial body
weight) and specific growth rate showed a tendency of increase as dietary protein

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level increases from 20% to 30%, and there were no significant differences (P > 0.05)

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between the treatments with 25%, 30% and 35% CP levels (Table 3). There was a
decrease of FCR with the increase of dietary protein level from 20% to 35%, and no

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significant differences (P > 0.05) were found between the treatments with 25%, 30%
and 35% protein levels (Table 3).

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Table 3

4. Discussion

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The immune and antioxidant systems are two major physiological mechanisms to

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maintain health of aquatic animals, ensuring their survival and resistance to possible
environmental stresses (Bachre, 2000; Castex et al., 2010; Liu and Chen, 2004). Like

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other crustaceans, shrimp lack a specific, adaptive immune system and rely entirely
on their innate immune mechanisms that include both cellular responses (such as
phagocytosis) and humoral responses (such as releasing antimicrobial peptide and
lysozyme to kill bacteria) for defense against pathogens (Bachre et al., 2004;
Vazquez et al., 2009). As the undertaker of the cellular immune and the supplier of the
humoral immune, the circulating hemocytes play a central role in the immune system
(Bachre et al., 2004). On the other hand, shrimp also possess an integrated
antioxidant system, including enzymatic (such as SOD) and non-enzymatic
antioxidants (such as GSH) to maintain normal oxidant status and especially to cope

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with natural or induced stressors (Castex et al., 2009, 2010; Parrilla-Taylor and
Zenteno-Savn, 2011). Generally, the immune responses and antioxidant capabilities

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of cultured shrimp under certain condition can reflect their health status. Since the

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biofloc not only can provide supplemental nutrition, like protein, lipid, mineral and
vitamin for the growth of cultured shrimp (Izquierdo et al., 2006; Ju et al., 2008b;

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Moss et al., 2006; Xu et al., 2012), but also is a source of abundant natural microbes
and bioactive compounds that could exert a positive effect on the physiological health

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of the shrimp in biofloc-based culture systems (Ju et al., 2008a; Xu and Pan, 2013),
there is a great potential in reducing crude protein level of feeds while maximizing the

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contribution of biofloc to the growth and health of the shrimp.

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Previous studies have shown that the dietary protein level could affect immune
condition of shrimp (Goimier et al., 2006; Pascual et al., 2004) in the experimental

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condition. For example, Pascual et al. (2004) studied the immunological response of L.
vannamei juveniles to dietary protein in clear water system, and found that 15% of
dietary protein level could induce a severe reduction in immune capacities of the
shrimp. In the present study, the THC and immune responses of shrimp showed no
significant differences between the treatments with dietary protein levels from 20% to
35%, indicating that the immune state of the shrimp was not affected by decreasing
the dietary protein level in the presence of biofloc. This result is not contradictory to
the results of the previous studies, because their experiment systems worked in clear
water without natural productivity. While in biofloc-based culture system, the

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developed biofloc could provide an important source of food protein for cultured
shrimp (Ballester et al., 2010; Decamp, et al., 2002; Hari et al., 2004), and thereby

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supplemented the relatively inadequate portion of dietary protein intake in the present

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experiment. Moreover, the biofloc could exert a positive effect on the immune system
of cultured shrimp probably because of being rich in natural microbial bioactive

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compounds such as polysaccharides and carotenoids (Ju et al., 2008a; Xu and Pan,
2013). Therefore, the dietary protein level could be reduced from 35% to 20% without

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affecting the immune condition of the shrimp in this study.

On the other hand, the T-AOC in both the plasma and the hepatopancreas showed

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a decreasing trend as dietary protein level decreased in the present study. Also the

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shrimp in the treatment with 20% protein level had the lowest GSH/GSSG ratio in the
plasma among the four treatments. These results indicate that the antioxidant

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capability and redox homeostasis of cultured shrimp could be weakened when they
were fed with very low protein feed (20%). However, no significant differences were
found in the antioxidant status (in terms of T-AOC, SOD activity, GSH level and
GSH/GSSG ratio) of shrimp fed with 25%, 30% and 35% CP levels, indicating that
feeding 25% protein feeds could provide adequate protein nutrition for the shrimp to
maintain a normal antioxidant function with the supplement of biofloc. It should be
noted that biofloc contain an appropriate amount of antioxidants such as carotenoids
and fat-soluble vitamins (Ju et al., 2008a), which could contribute to sustaining
sufficient antioxidant status of the shrimp (Xu and Pan, 2013). For example,

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carotenoids have been reported to improve animal immune systems, increase stress
tolerance and perform an antioxidant function (Babin et al., 2010; Linan-Cabello et al.,

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2002).

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Dietary protein requirements for juvenile L. vannamei have been widely studied with
values most reported more than 30% under experimental conditions (Smith et al. 1984;

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Kureshy & Davis, 2002; Xia et al., 2010). As dietary protein is the most important
factor affecting growth performance of shrimp (Kureshy & Davis, 2002), most shrimp

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farmers prefer to use high protein feeds, especially in intensive culture systems. In

China, for example, shrimp feeds are typically formulated to contain 38% ~ 42% of

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crude protein. In most of the cases, however, those formula feeds do not consider the

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contribution of natural production in practical culture systems, such as biofloc-based

system, and the use of high protein feeds could be unnecessary and wasteful.

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Reduction of dietary protein level without affecting growth performance of cultured

shrimp in the presence of biofloc has been reported by several authors (Ballester et al.,
2010; Decamp, et al., 2002; Hari et al., 2004). For example, Ballester et al. (2010)
demonstrated that the dietary protein level of F. paulensis can be reduced up to 10%
(from 45% to 35%) without impairing shrimp growth performance when the shrimp
were nursed in a suspended bioflocs system. In the present study, the development of
biofloc not only maintained the water quality suitable for shrimp culture under zerowater exchange but also could supplement a portion of protein nutrition, thereby
resulting high survival and similar growth performance (growth and FCR) of the

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shrimp fed with 25%, 30% and 35% dietary protein levels.

In aquaculture, there is growing awareness that the high quality of feeds should not

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only ensure superior growth, but also return prime health (Kiron, 2012). In this

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context, as the main and costliest nutrient component of shrimp feeds, the dietary
protein level should be optimized to not only meet growth demand but also maintain

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health status of cultured shrimp while maximizing the use of natural food source
under practical conditions. The present study demonstrated that, when juveniles of L.

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vannamei were reared at a stocking density of 224 shrimp m3 in zero-water exchange

biofloc-based culture tanks, dietary protein level can be reduced from 35% to 25%

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without affecting survival, growth, FCR, and physiological status of immune response

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and antioxidant capability, indicating potential contribution of biofloc in the protein

nutrition and physiological health of cultured shrimp. It can be deduced that the

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promoted biofloc could not only supplement a portion of food protein for low protein
feeds to satisfy nutrition demand but also contribute to maintaining physiological
functions, thereby sustaining efficient and healthy culture of shrimp. Additionally,
according to the results of this study, the antioxidant parameters (e.g. T-AOC,
GSH/GSSG) of shrimp may be more sensitive than immune parameters in response to
the insufficiency of dietary protein, and they could be chosen as physiological health
indicators for the evaluation of feed protein nutrition.
Acknowledgements
This work was supported by the Special Fund for Agro-scientific Research in the

ACCEPTED MANUSCRIPT
Public Interest from the Ministry of Agriculture of China (Grant No. 201103034). We
thank the staff at the Laboratory of Environmental Physiology of Aquatic Animal for

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their assistance in conducting the experiment. A special thank to Dr. Yong-Jun Chen

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for revising the manuscript.

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Fig. 1

20%

25%

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24.0

16.0

400

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300

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P

200

MA

500

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0.0

100

35%

32.0

8.0

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TSS (mg L1)

30%

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BFV (mL L 1)

40.0

Sampling time (week)

ACCEPTED MANUSCRIPT
Fig. 2

1.15
0.90

0.40
0.15

38.0

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34.0

36.0

a
a

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32.0
30.0
28.0

24.0
20%

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26.0
25%

30%

15.0

25%

30%

12.5

10.0
7.5
5.0

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0.65

a
a

1.40

17.5

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Antibacterial activity (units mL 1 )

Bacteriolytic activity (units mL 1 )

1.65

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Phagocytic percent (%)

1.90

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Total hemocyte count (107 mL 1)

35%

2.5
0.0

1.4
1.2

1.0

0.8
0.6
0.4
0.2

0.0
20%

35%

ACCEPTED MANUSCRIPT
Fig. 3
Plasma

14.50
14.00
13.50

13.00
12.50

25.00

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SOD (U mL 1)

30.00

20.00
15.00

SOD (U mg 1 protein)

a
a

0.77

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0.73

5.00

4.90

a
a

4.80
4.70
4.60

0.81
0.78

0.72

0.75
0.72

a
a

0.69
0.66

0.63
0.60

3.00

2.70
ab

3.15
a

3.00

ab

2.55
a

GSH/GSSG

2.85
GSH/GSSG

5.10

GSH (mol mg 1 protein)

0.78

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GSH (mol mL 1)

0.79

2.40

7.50

4.40

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5.00

8.00

ab

4.50

10.00

0.74

8.50

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35.00

0.75

ab

9.00

6.50

B 40.00

0.76

9.50

7.00

12.00

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10.00

b
b

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15.50
15.00

T-AOC (U mL 1)

Hepatopancreas
E

T-AOC (U mg 1 protein)

2.85
2.70

20%

25%

2.55

2.25

2.40

2.10

2.25
2.10

1.95
20%

25%

30%

35%

30%

35%

ACCEPTED MANUSCRIPT
Figure captions
Fig. 1 Changes of bioflocs volume (BFV) and total suspended solids (TSS) in the four

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treatments with four dietary protein levels throughout the 7-week feeding experiment

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for L. vannamei. Values are means (S.D.) of four replicate tanks per sampling time
in each treatment.

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Fig. 2 Total haemocytes count (A), phagocytosis percent (B), antibacterial activity (C)
and bacteriolytic activity (D) of L. vannamei in the four treatments with four dietary

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protein levels at the end of 7-week feeding experiment. Each bar represents the mean
value from four replicate tanks with the standard error. Means ( S.E.) with different

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letters are significantly different (P < 0.05).

Fig. 3 Antioxidant activities in the plasma and the hepatopancreas of L.vannamei in

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the four treatments with four dietary protein levels at the end of 7-week feeding
experiment. Each bar represents the mean value from four replicate tanks with the

0.05).

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standard error. Means ( S.E.) with different letters are significantly different (P <

ACCEPTED MANUSCRIPT
Table 1

Ingredient and proximate composition of the experimental diets (% dry weight basis)

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containing four crude protein levels for Litopenaeus vannamei juvenile

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Dietary protein level (% dry weight basis)

Item

20

25

30

35

15

20

25

10

15

20

58.5

50.0

41.5

33.0

1.9

1.4

0.85

0.35

0.2

0.2

0.2

0.2

0.1

0.1

0.1

0.1

Choline choride

0.3

0..3

0.3

0.3

Sodium alginate

Fillerf

12

10

8.05

6.05

Total

100

100

100

100

20.1

25.2

30.2

35.0

8.0

8.1

8.0

8.0

Ash

12.2

13.5

14.0

15.9

Gross energy (kJ g1)

15.8

15.9

16.3

16.5

Peruvian fish meala

10
5

Squid visceral mealc

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Soybean mealb

Wheat meal

Mineral premixd

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Vitamin Ce

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Vitamin premixd

Soybean lecithin
Fish oil

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Ingredients

Proximate composition
Crude protein
Crude lipid

Peruvian fish meal: crude protein 69.2% (dry weight basis).

ACCEPTED MANUSCRIPT
Soybean meal (defatted): crude protein 48.0% (dry weight basis).

Squid visceral meal: crude protein 44.0% (dry weight basis).

Kindly provided by Qingdao Master Bio-Tech CoLtd (Qingdao, Shandong,China).

Ascorbyl phosphate (Stay C-35%, Roche).

Cellulose.

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CE
P

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MA

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ACCEPTED MANUSCRIPT

DOa (mg L1)

pH
TANb (mg L1)
NO2-N (mg L1)

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DO: dissolved oxygen.

TAN: total ammonia nitrogen.

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25.1 1.1
(23.8, 26.7)
32.0 0.7
(31.4, 32.8)
7.5 1.3
(6.4, 8.7)
7.85 0.17
(7.41, 8.07)
0.06 0.08
(0.00, 0.19)
0.32 0.25
(0.03, 0.90)

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30%

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Salinity (g L1)

25%
25.3 1.2
(23.9, 26.8)
32.2 0.8
(31.4, 33.0)
7.2 1.6
(6.3, 8.8)
7.76 0.19
(7.37, 8.08)
0.12 0.13
(0.00, 0.48)
0.38 0.24
(0.02, 0.91)

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Temperature (C)

20%

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Parameters

Table 2
The overall means S.D. and range values (minimum, maximum) of main water
quality parameters in the treatments with four dietary protein levels during the 7-week
experimental period
Dietary protein levels

25.0 1.0
(23.7, 26.6)
31.8 0.8
(31.3, 32.7)
7.3 1.8
(6.0, 8.5)
7.62 0.26
(7.18, 8.05)
0.11 0.13
(0.00, 0.43)
0.41 0.24
(0.04, 0.82)

35%
25.5 1.3
(24.0, 26.9)
32.1 1.0
(31.4, 33.2)
7.1 2.1
(5.7, 8.3)
7.60 0.28
(7.13, 8.06)
0.10 0.10
(0.00, 0.35)
0.44 0.26
(0.01, 0.88)

ACCEPTED MANUSCRIPT
Table 3
Growth, survival and FCR of L. vannamei in the treatments with four dietary protein

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levels at the end of the 7-week feeding experiment

parameters

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Dietary protein levels

25%

30%

35%

Initial weight (g)

6.68 0.05a

6.68 0.04a

6.69 0.05a

6.68 0.06a

Final weight (g)

12.95 0.37a

13.46 0.69ab

14.08 0.54b

14.05 0.48b

Weight gain (%)

93.9 5.6a

101.5 8.8ab

110.6 7.5b

110.3 5.0b

SGRa (% day1)

1.35 0.07a

1.43 0.11ab

1.52 0.09b

1.52 0.06b

Survival rate (%)

92.0 3.3a

90.0 6.9a

85.0 7.6a

86.0 5.2a

FCRb

2.40 0.07a

2.19 0.09ab

2.06 0.08b

1.95 0.10b

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20%

SGR: specific growth rate.

FCR: feed conversion rate.

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Each value represents mean S.D. (n = 4). Values in the same row with different
superscripts are significantly different (P < 0.05).

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Research Highlights
We evaluated dietary protein level on performance of L. vannamei in biofloc

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tanks.

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The dietary protein level (20%~35%) didnt affect the immune condition of
shrimp.

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The dietary protein level (25%~35%) didnt affect the antioxidant status of shrimp.

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Reducing protein level from 35% to 25% didnt affect growth and FCR of shrimp.

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The biofloc may contribute to protein nutrition and physiological health of shrimp.