Aquacult Int (2014) 22:1823–1835 DOI 10.1007/s10499-014-9785-3
The improvement of growth, digestive enzyme activity and disease resistance of white shrimp by the dietary citric acid Xionggao Su • Xiaoqin Li • Xiangjun Leng • Chonggui Tan Bo Liu • Xianqi Chai • Ting Guo
•
Received: 20 October 2013 / Accepted: 16 April 2014 / Published online: 6 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract A 45-day feeding trial was conducted to evaluate the effects of dietary citric acid on growth, digestive enzyme and disease resistance of white shrimp, Litopenaeus vannamei. Shrimp with initial body weight of 5.57 ± 0.21 g were fed with basal diet supplemented with 0.0 g kg-1 (control), 1.0, 2.0, 3.0, 4.0 and 5.0 g kg-1 citric acid. Results showed that weight gain was increased by 15.9 % and feed conversion ratio was decreased by 0.17 by 2.0 g kg-1 dietary citric acid compared with control group (P \ 0.05). Intestinal protease activity of shrimp fed 2.0 g kg-1 citric acid was significantly higher (P \ 0.05) than that of control group. No significant difference was found in intestinal amylase activity among treatments (P [ 0.05). The activities of serum phenoloxidase, superoxide dismutase and lysozyme in 2.0 and 3.0 g kg-1 citric acid group were significantly higher, and accumulative mortalities of the two groups on the fourth day after injection of Vibrio alginolyticus were significantly lower than those of control group (P \ 0.05). Results above demonstrated that dietary citric acid could improve growth performance, immunity and resistance against V. alginolyticus. The supplementation level of citric acid in diet was suggested to be 2.0–3.0 g kg-1 for white shrimp. Keywords
Shrimp Citric acid Growth Immunity Disease resistance
X. Su X. Li X. Leng (&) C. Tan B. Liu X. Chai T. Guo College of Fisheries and Life Science, Shanghai Ocean University, 999 Huchenghuan Road, Lingang New City 201306, Shanghai, People’s Republic of China e-mail:
[email protected] X. Li X. Leng Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai 201306, People’s Republic of China X. Li X. Leng Shanghai Engineering Research Center of Aquaculture, Shanghai, People’s Republic of China X. Li X. Leng Shanghai University Knowledge Service Platform, Shanghai Ocean University Aquatic Animal Breeding Center (ZF1206), Shanghai, People’s Republic of China
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Introduction White shrimp, Litopenaeus vannamei, is an important shrimp cultured in the world. With the expansion of farming scale and the increase in stocking density, risks of shrimp infected by various diseases are rising simultaneously (Xie and Yu 2007; Qi et al. 2009). To prevent these diseases, antibiotics and chemical drugs are widely used. But the use of antibiotics and chemical drugs leads to the transfer of resistance to bacterial species for animals and humans and serious pollution for the environment. Thus, a greater interest has arisen in seeking alternatives to antibiotic and chemical substances that could inhibit pathogens, improve the immunity and act as growth promoters in aquaculture (Lim et al. 2010). Citric acid (CA) is an organic acid widely applied in food and pharmaceuticals industry. As an acidifier in diet, CA can enhance digestive function and alleviate stress, which has been reported in land animals such as pig (Partanen and Mroz 1999; Øverland et al. 2000, 2008; Partanen et al. 2002) and broiler chick (Agustı´n et al. 2003; Gauthier 2005), but relatively few studies were reported in aquatic animals. The addition of 1 % CA to a lowfish-meal diet without inorganic P supplementation enhanced the growth and P retention of rainbow trout and decreased P load to the environment (Adria´n et al. 2012). Study by Khajepour and Hosseini (2012a) indicated that Beluga, Huso huso, has a limited ability to utilize soybean meal as a protein source, whereas CA can improve growth and nutrient utilization in Beluga. The improvement in growth and nutrients digestibilities by dietary CA was also reported in tilapia, Oreochromis niloticus 9 O. aureus (Pan et al. 2004), allogynogenetic crucian carp, Carassius auratus gibelio (Leng et al. 2006), rainbow trout, Oncorhynchus mykiss (Sugiura et al. 2001; Pandey and Satoh 2008) and red sea bream, Pagrus major (Sarker et al. 2005). However, studies on shrimp fed with organic acids or their salts are limited. Bruno et al. (2013) evaluated the potential of several organic acid salts to be used as additives for marine shrimp and concluded that the use of salts of organic acids could improve marine shrimp nutrition and health and that the salt propionate has the greatest potential for use as a diet supplement for L. vannamei. Based on the above results in land animals and fishes, we consider CA as a potential growth promoter and immune stimulant in shrimp culture. So the present study was conducted to evaluate effects of different levels of dietary CA on the growth, digestive enzyme activity, immune parameters and resistance against V. alginolyticus of white shrimp to develop environmentfriendly feed for shrimp.
Materials and methods Experimental design and diets Based on the study conducted by our laboratory (Leng et al. 2006), six diets were formulated by adding 0.0 (control), 1.0, 2.0, 3.0, 4.0 and 5.0 g kg-1 CA in basal diet, respectively. The CA is an analytical reagent (AR) with the purity of 99.5 % and produced by Shanghai Shenbo Chemical Co., Ltd., Shanghai, China. After the addition of CA, a corresponding reduction in the amount of flour was conducted to maintain the balance of the formula. Ingredients were powdered, screened by 60-mesh sieve, gradually mixed and granulated into sinking pellet with a diameter of 1.0 mm by pellet machine (produced by Xinchang Chenshi Mechanical Factory, Zhejiang province, China, KL series). The pellet was post-cooked in a sealed oven at 95 °C for 25 min, air-dried naturally and stored at
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4 °C until use. The diet pH was measured according to the method described by Radecki et al. (1988), and the pH of the six diets was 5.95, 5.83, 5.69, 5.56, 5.48 and 5.41, respectively. The composition and proximate analyses of basal diet are shown in Table 1. Experimental shrimp and culture condition The experimental shrimp were obtained from Qingpu Aquaculture Farm in Shanghai, China, and acclimatized for 1 week. During the acclimation, shrimp were fed with commercial diet (42 % crude protein) as they consumed in the aquaculture farm. One thousand and two hundred shrimp (initial body weight 5.57 ± 0.21 g) were distributed to 24 cages (2.5 m 9 1.25 m 9 1.0 m) with 50 shrimp per cage. The cages were randomly hung in six indoor concrete pools (5.0 m 9 3.0 m 9 1.2 m) with four cages per tank. The mesh size of the bottom of the net was so small (20-mesh sieve) to avoid the diet dropping from the cage. The culture water came from the same source, dechlorinated tap water (aerated before using). The shrimp were fed to apparent satiation three times a day at 06:00, 12:00 and 18:00, with a feeding rate of 5 % of the body weight. Two hours after feeding, the uneaten feed was removed and weighed after air-drying to calculate the exact feed intake. Feces were siphoned daily, and 15 % of the water was exchanged every 3 days. During the feeding trial, water temperature was maintained at 25–30 °C, with dissolved oxygen (DO) 5.1–6.7 mg L-1, salinity 0.5–1.0 %, pH 7.1–8.0 and NH4?– N \ 0.1 mg L-1. The feeding trial was carried out at Nanhui Aquaculture Farm, Shanghai, and lasted for 45 days. Measurement indicators and methods At the end of the feeding trial, the shrimp were deprived of feed for 24 h, then weighed and counted to calculate growth performance as follows: Weight gainðWG; %Þ ¼ 100 ðfinal weight initial weightÞ=ðinitial weightÞ Feed conversion ratioðFCRÞ ¼ ðfeed consumedÞ=ðweight gainÞ Survival rateð%Þ ¼ final number of shrimp=initial number of shrimp 100 Muscles of four shrimp were sampled from each cage (16 shrimp from each treatment) and stored at -20 °C for composition analysis. Moisture, ash, crude protein, crude fat, calcium and phosphorus content were determined following standard methods (AOAC 1999). Moisture was determined by oven-drying at 105 °C until a constant weight was achieved. Ash content was estimated by combusting the samples in a muffle furnace at 550 °C. The crude protein was determined by the Kjeldahl method (Foss Tecator AB, Kjeltec 2200, Switzerland), and the crude fat was measured by the ether extraction method (Foss Tecator AB, Soxtec Avanti 2050, Switzerland).Samples for Ca and P determination were digested in a solution of nitric acid and perchloric acid (2:1) and then measured by atomic absorption spectrophotometer (GBC Scientific Equipment Pty Ltd., GBC 932 plus, Dandenong, Victoria, Australia) and the spectrophotometer (UNICO (Shanghai) Instruments Co., Ltd., WFZ UV-480 2H, Shanghai, China), respectively. Nine shrimp from each replication were dissected on ice, and the gut (washed in distilled water) and hepatopancreas were extracted and preserved at -80 °C. For the measurement, tissues from three shrimp were pooled as one sample and then were thawed at 4 °C, and ice-cold distilled water with a volume (ml) ten times the weight (g) of the tissues was added. The tissues were then homogenized at 4 °C and centrifuged for 10 min
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Table 1 The composition and proximate analyses of basal diet Ingredients (g kg-1) Fish meala
230.0
Soybean mealb
200.0
Peanut mealc
100.0
Squid visceral meald Meat and bone meale Wheat flourf
50.0 50.0 265.5
Brewers dried yeastg
30.0
Ca (H2PO4)2H2O
20.0
Fish oil
20.0
Soybean lecithin
15.0
Vitamin premixh
2.5
Mineral premixi
5.0
Choline chloride
6.0
Ecdysterone
1.0
KCl Total
5.0 1,000.0
Proximate analyses Moisture Crude proteinj Crude fatj
88.1
Crude ashj
116.4
Gross energy (kJ/g dry matter)k a
98.8 429.1
18.3
Fish meal (Peruvian), crude protein 64.5 % and crude lipid 7.8 %
b
Soybean meal (defatted), crude protein 43.5 % and crude lipid 1.8 %
c
Peanut meal (defatted), crude protein 46.1 % and crude lipid 1.7 %
d
Squid visceral meal, crude protein 54.8 % and crude lipid 15.3 %
e
Meat and bone meal, crude protein 51.2 % and crude lipid 9.5 %
f
Wheat flour, crude protein 13.5 % and crude lipid 1.9 %
g
Brewers dried yeast, crude protein 50.6 % and crude lipid 0.7 %
h
One kilogram of vitamin premix contained: vitamin A 4 000 000 IU, vitamin D 2 000 000 IU, tocopherol acetate 30 g, menadione 10 g, thiamine 5.0 g, riboflavin 15 g, Vitamin C phosphate 140 g (35 % available VC), nicotinic acid 40 g, Ca pantothenate 25 g, pyridoxine 8 g, vitamin B12 0.02 g, folic acid 2.5 g, biotin 0.08 g, inositol 150 g i One kilogram of mineral premix contained: Ca(H2PO4)2 600 g, KCr(SO4)2 0.55 g, CuCO3 0.30 g, FeC6H5O7 10 g, MgO 30 g, MnSO4 3.5 g, C6H5K3O7H2O 220 g, KI 0.02 g, K2SO4 52 g, NaCl 74 g, Na2SeO3 0.02 g, ZnCO3 3.0 g j
Valued in dry weight (%)
k
Calculated value (kJ/g Dry matter), GE = 23.4 9 CP (%) ? 39.2 9 EE (%) ? 17.2 9 CARB (%) (Cho et al. 1982). Carbohydrate was determined as the total minus the sum of moisture, protein, ash and lipid
(5,505 g), and the supernatant was collected for measurement of digestive enzyme activities and soluble protein. Digestive enzyme activities were expressed as a relative unit per milligram of soluble protein (U mg-1). Protease was measured according to the method of Divakaran and Ostrowski (1990). For the protease activity of intestines and
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hepatopancreas, one unit was defined as 1 lg of tyrosine released per minute by 1 mg of tissue protein hydrolyzing casein at pH values of 7.2 and 9.8, respectively. Amylase was determined with the iodine–starch colorimetric method by kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). One unit of amylase activity was defined as 10 mg of starch hydrolyzed in the reaction with 1 mg tissue protein acting on the substrate for 30 min at 37 °C. After the feeding trial, shrimp were not fed for 24 h, and nine shrimp were randomly selected from each cage to be bled from the pericardial cavity. Blood from three shrimp was pooled as one sample and was centrifuged at 4 °C for 10 min (5,505 g), and then the supernatant was collected for measuring non-specific immune parameters. The assay of phenoloxidase (PO) was performed with L-dopa as the substrate, by the method of Ashida and Soderhall (1984). Three milliliter phosphate buffer (0.1 mol L-1, pH 6.4), 100 lL serum and 100 lL L-dopa were mixed at room temperature. Using a wavelength of 490 nm, the luminosity value was recorded every 2 min. A unit (U) of activity was equal to 0.001 increment of the luminosity density per minute under the experimental conditions. Superoxide dismutase (SOD) was measured by xanthine oxidase method. One SOD activity unit (U) was defined as the corresponding SOD amount when the SOD inhibition rate in the serum per mL reached 50 %. Lysozyme (LYZ) was measured by the turbidimetric method improved by Hultmark and Rasmusnt (1980). One unit of LYZ activity was defined as the amount of sample resulting in a decrease in absorbance of 0.001/min. Both were measured with kits produced by Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Shrimp from control group, 1.0, 2.0 and 3.0 g kg-1 CA groups were chosen for disease resistance trial according to the growth performance (shown in Table 2) after growth trial. Four replications were conducted in each group. Ten shrimp from each replication were injected intramuscularly with 30 lL (4 9 106 cell mL-1) Vibrio alginolyticus, which was obtained from the Alge Lab of Shanghai Ocean University. The injection dose of V. alginolyticus was determined in our previous study (Tan et al. 2014). Then these injected shrimps were kept in aquariums of 50 L with continuous aeration and circulation without feeding. The number of dead shrimp was recorded every 24 h for 4 days to calculate accumulative mortality. Accumulative mortality ¼ number of dead shrimps=number of total shrimps Statistical analysis Results are presented as mean ± SD (standard deviation of means). SPSS (version 17.0) programs were used for the statistical analyses. One-way analysis of variance (one-way ANOVA) and Tukey multiple comparisons were used to determine whether significant difference existed between the treatments. All tests used a significance level of P \ 0.05. The relationship between dietary CA level and WG was analyzed by linear regression with SPSS programs (version 17.0).
Results As shown in Table 2, WG of shrimp was increased by 15.9 % and FCR decreased by 0.17 (P \ 0.05) by the addition of 2.0 g kg-1 CA in diet compared with the control group. In 1.0 and 3.0 g kg-1 CA groups, WG was improved and FCR decreased, but the significant level (P [ 0.05) was not reached. Survival rates of shrimp fed 0.1–0.4 % CA were all significantly higher than those of control group (P \ 0.05). The relationship of additional
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94.5 ± 3.4
97.6 ± 1.1
b
1.30 ± 0.04
Means in the same row with different superscripts indicate significant (P \ 0.05)
Survival rate (%)
a
1.36 ± 0.08
Feed conversion ratio
ab
191.3 ± 12.9ab
178.2 ± 6.9a
Weight gain (%)
b
5.6 ± 0.3 16.4 ± 0.5ab
5.6 ± 0.2
15.9 ± 0.5a
1.0 g kg-1
Initial body weight (g)
0 g kg-1
CA supplements
Final body weight (g)
Index
Table 2 Effects of citric acid on growth performance of white shrimp (n = 4)
98.5 ± 1.9
bc
1.19 ± 0.04
a
206.7 ± 24.9b
17.3 ± 0.8c
5.6 ± 0.3
2.0 g kg-1
99.0 ± 2.0
c
1.25 ± 0.05
ab
193.0 ± 8.9ab
16.8 ± 0.7bc
5.7 ± 0.2
3.0 g kg-1
99.0 ± 1.2
c
1.31 ± 0.08
b
186.2 ± 17.6ab
16.2 ± 0.4a
5.6 ± 0.2
4.0 g kg-1
95.5 ± 2.5ab
1.34 ± 0.05b
182.2 ± 9.5a
16.1 ± 0.5a
5.6 ± 0.1
5.0 g kg-1
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level of WG (Y) with CA (X) presented a two-slope broken line model, and the two regression equations were Y = 14.25X ? 177.8 (R2 = 0.997) and Y = -8.03X ? 220.1 (R2 = 0.930). At the cross point of the two lines, maximum WG was achieved at the additional level of 1.90 g kg-1. Treatments showed no significant difference in the contents of muscle moisture, ash, crude protein, crude fat, calcium and phosphorus (P [ 0.05) (Table 3). Shrimp fed 2.0 g kg-1 CA showed significantly higher intestinal protease activity than the control group (P \ 0.05), and hepatopancreas protease activity tended to be increased by the addition of 2.0 and 3.0 g kg-1 CA (P [ 0.05), when compared with the control group. No significant difference was found in intestinal amylase activities among all treatments (P [ 0.05) (Table 4). The activities of phenoloxidase (PO) in 1.0–3.0 g kg-1 CA groups, superoxide dismutase (SOD) in 2.0–5 g kg-1 CA groups and lysozyme (LSZ) in 2.0 and 3.0 g kg-1 CA group were all significantly higher than those of the control group (P \ 0.05) (Table 5). At the first day after injection of V. alginolyticus, the mortality of shrimp fed 2.0 g kg-1 CA group was lower than that of control group (P \ 0.05). At the second day, there was no significant difference in mortality among all groups (P [ 0.05). At the third day, the mortality of shrimp fed 3.0 g kg-1 CA was lower than that of control group (P \ 0.05). At the fourth day, the mortality of shrimp fed 2.0 and 3.0 g kg-1 CA was 60.0 and 56.7 %, respectively, both were significantly lower than that of the control group (80.0 %) (P \ 0.05) (Fig. 1).
Discussion The promoting effect of CA on growth of terrestrial animals has been widely reported in pigs (Partanen and Mroz 1999; Øverland et al. 2000, 2008; Partanen et al. 2002) and poultry (Agustı´n et al. 2003; Gauthier 2005). Studies with aquatic animals showed that adding 2.0 and 3.0 g kg-1 CA in diet increase the WG of tilapia, Oreochromis niloticus 9 O. aureus (Pan et al. 2004) and allogynogenetic crucian carp, Carassius auratus gibelio (Leng et al. 2006) by 15.3 and 10.3 %, while decreasing FCR by 15.2 and 7.2 %, respectively. Dietary 30.0 g kg-1 CA improved the WG of red sea bream, Pagrus major by 19.1 % (Sarker et al. 2005). In the diet with fish meal partly replaced by soybean meal, the WG of beluga sturgeon, Huso huso, was improved by 11.1 % and FCR decreased by 7.6 % by the addition of 30 g kg-1 CA (Khajepour and Hosseini 2012a). In the present study, 2.0 g kg-1 CA in diet increased WG and decreased FCR of the shrimp (P \ 0.05). Studies above indicated that proper level of dietary CA can improve the growth performance of aquatic animals. It is noteworthy that the additional level of CA in diet of aquatic animals mentioned above is remarkably different, for example, the suitable level of CA was 2.0 g kg-1 for Carassius auratus gibelio (Leng et al. 2006), 3.0 g kg-1 for Oreochromis niloticus 9 O. aureus (Pan et al. 2004), 30.0 g kg-1 for Pagrus major (Sarker et al. 2005) and Huso huso (Khajepour and Hosseini 2012a). In the present study, the proper level of CA was 2.0–3.0 g kg-1 for white shrimp. Higher dietary CA levels may disturb the acid–alkaline balance in the digestive tract and then lead to the damage for the body and the growth. The difference in dietary CA levels for different aquatic animals may be attributed to factors such as the feeding habits of aquatic animals, the existence and the development of stomach, growth stage and the feed composition. Organic acids have the function of stimulating the secretion of gastric acid and digestive enzymes to improve protein digestion, or chelating cations to promote the absorption of Ca,
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Table 3 Effects of citric acid on muscle composition of white shrimp (% fresh tissue, n = 16) Index
CA supplements 0 g kg-1
1.0 g kg-1
2.0 g kg-1
3.0 g kg-1
4.0 g kg-1
5.0 g kg-1 75.82 ± 0.50
Moisture
76.27 ± 0.35
75.72 ± 0.27
75.28 ± 0.67
75.93 ± 0.65
75.82 ± 0.50
Crude ash
1.50 ± 0.02
1.53 ± 0.03
1.55 ± 0.02
1.49 ± 0.03
1.51 ± 0.01
1.51 ± 0.02
Crude protein
19.58 ± 0.05
20.00 ± 0.10
20.45 ± 0.09
19.84 ± 0.06
19.90 ± 0.05
19.93 ± 0.08
Crude fat
1.29 ± 0.12
1.22 ± 0.05
1.20 ± 0.12
1.19 ± 0.13
1.17 ± 0.11
1.18 ± 0.10
Ca
0.28 ± 0.03
0.29 ± .04
0.30 ± 0.03
0.29 ± 0.06
0.28 ± 0.05
0.29 ± 0.04
P
0.58 ± 0.03
0.59 ± 0.02
0.61 ± 0.02
0.59 ± 0.02
0.59 ± 0.04
0.58 ± 0.04
Means in the same row with different superscripts indicate significant (P \ 0.05)
P and other minerals, which has been extensively reported in land animals (Mroz et al. 1997; Øverland et al. 2000; Boling et al. 2001). So the promotion of CA for aquatic animal growth may be attributed to the improvement in nutrients digestion and utilization. Adding 10.0 g kg-1 CA in diet increased pepsin activity of Oreochromis niloticus 9 O. aureus by 29.6 % (Li et al. 2005). Adding 30.0 g kg-1 CA in diet decreased the N and P excretion rate of Pagrus major by 7.5 and 43.5 %, respectively (Sarker et al. 2005), and increased the protein and phosphorus digestibility of Huso huso (Khajepour and Hosseini 2012a). The addition of 20.0 or 30.0 g kg-1 CA to the diet of Beluga increased the bioavailability of Ca and P, thereby increasing muscle and scute mineralization (Khajepour and Hosseini 2012b). Fe content of rainbow trout was also increased by the addition of 4.0 g kg-1*16.0 g kg-1 CA in diet (Vielma et al. 1999). Our previous study in Carassius auratus gibelio showed that 2.0 g kg-1 CA can improve the digestibilities of dry matter and phosphorus by 5.6 % and 12.0 %, respectively (Leng et al. 2006). In the present trial, shrimp fed 2.0 g kg-1 CA showed significantly higher intestinal protease activity than control group (P \ 0.05), which maybe come from the stimulation of the relatively low diet pH, but more dietary CA would negatively affect the activities of enzymes. Due to the difficulty in fecal collection for shrimp, the determination of nutrient digestibility was not conducted in the present trial, which requires further study in digestibility to provide more direct proofs to explain the mechanism of CA for shrimp. In addition, organic acids inhibited the harmful microorganisms to improve microecosystem (Øverland et al. 2008; Partanen and Mroz 1999) and reduced the intestinal pH value to enhance the reproduction of beneficial bacteria (Knarreborg et al. 2002; Partanen and Mroz 1999). It has been proved that CA can inhibit the adhesion of Escherichia coli with disturbing DNA synthesis in nucleus (Gedek et al. 1993) and prevent bacteria breeding and kill the bacteria by penetrating cell wall (Lambert and Stratford 1999). In a recent study by Bruno et al. (2013), the addition of 2 % sodium propionate decreased the vibrio species concentration in the intestinal tract of white shrimp. In the present study, the diet pH was decreased by the addition of CA, which may affect the pH in the digestive tract, but more studies on micro-ecosystem in the digestive tract are needed to illustrate the possible mechanism of CA for shrimp in the future. With respect to immune enhancement and disease resistance, studies indicated that dietary CA improves the resistance against thermal stress and bacterial infection of chicken (Gauthier 2002; Ma 2002) and promotes the antibody titers and immune protein content of pigs (Li et al. 2009). As for the crustacean, CA was considered having similar functions
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ab
0.21 ± 0.03s
33.2 ± 5.9
Means in the same row with different superscripts indicate significant (P \ 0.05)
0.21 ± 0.01
Intestinal amylase
ab
32.0 ± 3.1
a
108.3 ± 16.9ab
110.2 ± 9.4ab
a
1.0 g kg-1
0 g kg-1
CA supplements
Intestinal protease
Hepatopancreas protease
Index
a
0.15 ± 0.02
42.4 ± 2.1
b
125.6 ± 10.9b
2.0 g kg-1
Table 4 Effects of citric acid on digestive enzyme activity of white shrimp (U mg-1 prot) (n = 12)
a
0.15 ± 0.07
40.8 ± 6.2
ab
120.5 ± 6.7ab
3.0 g kg-1
b
0.28 ± 0.03
39.0 ± 3.6
ab
104.3 ± 12.2ab
4.0 g kg-1
0.21 ± 0.06ab
40.9 ± 5.9ab
99.2 ± 6.9a
5.0 g kg-1
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58.53 ± 6.58
63.10 ± 7.75
ab
61.35 ± 7.16
a
49.48 ± 9.03
ab
1.47 ± 0.12b
1.17 ± 0.07a
a
1.0 g kg-1
0 g kg-1
CA supplements
81.42 ± 6.44
PO phenoloxidase, SOD superoxide dismutase, LYZ lysozyme
b
64.57 ± 1.52
b
2.17 ± 0.07d
2.0 g kg-1
Means in the same row with different superscripts indicate significant (P \ 0.05)
LYZ
SOD
PO
Index
Table 5 Effects of citric acid on immune parameters of white shrimp (U ml-1) (n = 12)
103.81 ± 9.25
c
66.67 ± 2.27
b
1.69 ± 0.08c
3.0 g kg-1
51.15 ± 2.10
a
66.34 ± 1.38
b
1.21 ± 0.19a
4.0 g kg-1
55.47 ± 4.51a
68.12 ± 8.97b
1.17 ± 0.07a
5.0 g kg-1
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Fig. 1 Accumulative mortality of shrimp after attacked with V. alginolyticus
with b-N-acetyl glucosaminidase (NAGase). NAGase plays an important role in molting, chitin digestion, and resistance to viruses and parasites (Koo et al. 2008; Zhao et al. 2007), thus influencing the immune function of crustacean (Xie et al. 2010). The prophenoloxidase (proPO) system, superoxide dismutase (SOD) and lysozyme (LYZ) are important immune response factors for shrimp. ProPO activation system is an important defense system to identify foreign materials in shrimp (Washington and Dankert 1997), reflecting the health status and immune sensitivity of the body. SOD plays an important role in scavenging superoxide free radicals and preventing the biomolecular damage (Holmblad and Soderhall 1999). Lysozyme can hydrolyze the acetyl polysaccharide in the viscosity polypeptide of bacterial cell wall to kill bacteria. In this experiment, 2.0 g kg-1 and 3.0 g kg-1 CA significantly increased activities of serum PO, SOD and LYZ (P \ 0.05) and decreased shrimp mortality at the fourth day after injection with V. alginolyticus (P \ 0.05). These results indicated that CA can enhance immunity of white shrimp and enhance the resistance against pathogens. The reasons are not clear, but may be attributed to one or more of the following interdependent factors. First, CA may act as an immune-stimulating cofactor that helps in activating proPO system. Second, CA owns antioxidation function, which plays an important role in body protection. Finally, as the intermediate product of tricarboxylic acid cycle, CA forms energy more quickly than glucose and can be used in the emergency synthesis of ATP under stress to improve the body’s non-specific immunity and stress resistance ability. Acknowledgments This study was supported by Shanghai Municipal Agricultural Commission (No. 2009-6-6), Shanghai aquatic fishery key project (No. Y1101).
References Adria´n JH, Shuichi S, Viswanath K (2012) Supplementation of citric acid and amino acid chelated trace elements in low-fish meal diet for rainbow trout affect growth and phosphorus utilization. J World Aquacul Soc 43(5):688–696 Agustı´n B, Agustı´n V, Ignacio A, Carmen C, Manuel P, Carmen B (2003) The effect of citric acid and microbial phytase on mineral utilization in broiler chicks. Anim Feed Sci Technol 110:201–219
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