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Dietary administration of chitooligosaccharides to enhance growth, innate immune response and disease resistance of Trachinotus ovatus
Fish & Shellfish Immunology 32 (2012) 909e913
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Dietary administration of chitooligosaccharides to enhance growth, innate immune response and disease resistance of Trachinotus ovatus Shimei Lin a, b, *, Shuhong Mao a, Yong Guan a, Xin Lin a, Li Luo a, b a b
College of Animal Science and Technology, Southwest University, Chongqing 400716, PR China Key Laboratory of Freshwater Fish Resources and Reproductive Development (Ministry of Education), Southwest University, Chongqing 400716, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2011 Received in revised form 9 February 2012 Accepted 12 February 2012 Available online 18 February 2012
The present study was conducted to investigate the effects of dietary chitooligosaccharides (COS) supplementation on the innate immune response and protection against Vibrio harveyi infection in Trachinotus ovatus. A basal diet was supplemented with 0.0 (control), 2.0, 4.0 and 6.0 g COS kg1 to formulate four experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 1.0 2.0 m), and each cage was stocked with 80 fish (initial average weight 10.8 0.05 g). After 8 weeks of feeding trial, Both the final weight and specific growth rate (SGR) significantly increased with increasing dietary COS levels up to 4.0 g kg1, whereas there were no significant differences for COS levels from 4.0 to 6.0 g kg1. A decreased feed conversion ratio (FCR) was observed with increasing dietary COS levels. The total leukocyte counts (WBC), differential leukocyte counts, respiratory burst activity, lysozyme and superoxide dismutase (SOD) activity were significantly increased with the increased levels of dietary COS (P < 0.05), and reached a maximum at level of 4.0 g kg1 COS. There were no significant differences in those immunological parameters between 4.0 and 6.0 g kg1 COS. Moreover, the dietary COS supplementation groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with V. harveyi. These results indicated that dietary intake containing COS could enhance the immune responses of fish and improve its resistance to infection by V. harveyi. Especially supplementation with 4.0 g kg1 COS to the fish for 56 days showed considerable improvement in the growth, survival and immune response of the fish. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Chitooligosaccharides Growth Non-specific immunity Trachinotus ovatus
1. Introduction Chitosan has been widely used in many fields as a source of potential bioactive material during past few decades [1e3]. However, chitosan has several drawbacks to be utilized in biological applications, including poor solubility under physiological conditions. Chitosan’s biofunctionalities are highly related to its molecular weight and degree of acetylation. Therefore, a new interest has recently been emerged on partially hydrolyzed chitosan. Chitooligosaccharide (COS), which is one type of the oligosaccharides, is produced from chitin or chitosan by chemical or enzymatic decomposition methods [4]. The COS has a higher activity and more physiological functions than chitosan due to its lower molecular weight or its ready solubility in water. Moreover, the chitosan is found abundantly in natural sources, it is produced commercially on a large scale in different countries [5]. For these reasons, the biological activities of COS are of increasing interest in recent
research. In fact, some studies regarding COS have already been conducted in either pigs [6], broilers [4] or fishes [7,8], and beneficial effects on growth performance, immunity, or blood profiles have been reported. The ovate pompano Trachinotus ovatus (Linnaeus) is one of the most commercially cultured marine fish species in the South China. Owing to the intensive culture, bacterial infectious diseases often occurred and resulted in serious losses. However, little information is available on its basal immune response and the increased immunity and protection against pathogen infection which might be stimulated by immunostimulants. Therefore, the purpose of the present study was to examine the effects of dietary chitooligosaccharides (COS) on immunity and disease resistance of this fish.
2. Materials and methods 2.1. Experimental diets
* Corresponding author. College of Animal Science and Technology, Southwest University, Chongqing 400716, PR China. Tel./fax: þ86 23 68251196. E-mail address: [email protected] (S. Lin). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2012.02.019
The COS used in the current study was provided by Bioengineering Institute, Ocean University of China (Qingdao, China) and
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S. Lin et al. / Fish & Shellfish Immunology 32 (2012) 909e913
produced by microbial fermentation of the shells of crustaceans by Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus acidophillus. The average molecular weight of COS was lower than 5000 Da. The COS premix contained 8.6% crude protein, 14.5% crude fat, 22.6% crude ash, 10.7% crude fiber, 9.0% moisture, 3.0% chitooligosaccharide. The practical basal diet was formulated to contain approximately 40% crude protein and 9% crude lipid. Four experimental diets were formulated so as to contain different concentrations of COS at 0.0 (control), 2.0, 4.0 or 6.0 g kg1. All the ingredients were passed through a grinder, and passed through a paste extruder (1 mm diameter), and stored at 20 C until required. 2.2. Experimental animals T. ovatus were obtained from a commercial fish farm located in Zhanjiang, Guangdong, China, transported to sea cages and acclimatized for one weeks. At the start of the experiment, the fish were fasted for 24 h and weighed after being anesthetized with 3aminobenzoic acid ethyl ester (MS 222; 100 mg mL1). Young T. ovatus (average weight 10.8 0.05 g) were randomly selected and distributed into 12 sea cages (1.5 1.0 2.0 m) for the growth trial (80 fish/cage). Fish were hand-fed to apparent satiation twice (08:00 and 17:00) daily. The feeding trial lasted for 8 weeks. During the experimental period, the temperature ranged from 26.5 to 29.5 C, the salinity from 26 to 29& and dissolved oxygen content was approximately 7.6 mg l1.
Differential leukocyte counts (neutrophil, lymphocyte, monocyte) were determined using blood smears under a light microscope. 2.5.2. Respiratory burst activity The respiratory burst activity of the phagocytes was carried out by nitroblue tetrazolium (NBT, Shanghai Reagent Corp., China) following the method of Secombes [9] subsequently modified by Ai et al. [12]. The absorbance at 630 nm was measured with a Model Multiskan spectrum (Thermo, USA) using KOH/DMSO alone as a blank. Respiratory burst was expressed as NBT-reduction in 100 ml of cell suspension. 2.5.3. Phagocytic activity Phagocytic activity (PA) for five fish in each tank was determined by a method of Lin et al. [10]. PA ¼ number of phagocytic cells with engulfed yeast O number of peritoneal cells 100 2.5.4. Lysozyme activity Lysozyme level in serum of five fish in each tank was determined by turbidimetric assay according to the method described by Ellis [13]. Briefly, test serum (0.1 ml) was added to 1.9 ml of a suspension of Micrococcus lysodeikticus (Sigma) (0.2 mg ml1) in a 0.05 M sodium phosphate buffer (pH 6.2). The reaction was carried out at 25 C and absorbance was measured at 530 nm after 0.5 and 4.5 min in a spectrophotometer. One unit of lysozyme activity was defined as the amount of sample causing a reduction in absorbance of 0.001 min1.
2.3. Sample collection After being fasted for 24 h, the fish were anesthetized with MS 222 (Sigma, USA). Blood was collected by the caudal venipuncture with a 27 gauge needle and 1 ml syringe, and pooled from a random sample of five fish per tank. The blood samples were collected into both nonheparinized and K3EDTA vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) in order to obtain the serum and whole blood, respectively. After collection, whole blood from nonheparinized tubes was allowed to clot at room temperature for 4 h. Following centrifugation (3000 g, 10 min, 4 C), the serum was removed and frozen at 80 C until use. Head-kidney macrophages from five fish in each tank were isolated as described by Secombes [9] with some modifications [10]. 2.4. Survival and growth performance At the termination of the experiment, the fish were fasted for 24 h before harvest. The fish were moderately anaesthetized (3aminobenzoic acid ethyl ester, MS 222; 100 mg mL1), and then each group was counted and weighed. Based on recording the weight of each fish and counting the number of fish, specific growth rate (SGR), feed conversion ratio (FCR) and survival were calculated using the following equations: SGR ¼ 100 [in final weight in initial weight] O total duration of the experiment FCR ¼ feed given (dry weight) O weight gain (wet gain) Survival (%) ¼ (final number of kois O initial number of kois) 100.
2.5.5. Serum superoxide dismutase Superoxide dismutase (SOD) activity was measured by its ability to inhibit superoxide anion generated by xanthine and xanthine oxidase reaction system according to Wang and Chen [14] using an SOD detection kit (Nanjing Jiancheng Bioengineering Institute, China). The optical density was measured at 550 nm. One unit of SOD was defined as the amount required for inhibiting the rate of xanthine reduction by 50% in 1 ml reaction system. Specific activity was expressed as SOD unit per ml serum. 2.6. Challenge test The Vibrio harveyi strain was originally isolated from infected T. ovatus. The seven day LD50 was determined by intraperitoneal injection of 48 fish with graded doses of V. harveyi (106, 107, 108, 109 and 1010 cfu/fish) at 24 C, and the result showed that the LD50 on day 7 was 108 cfu/fish. Challenge tests were conducted in triplicate with 12 fish per replicate. Each fish was injected intraperitoneally with 0.3 ml PBS containing 2.4 108 live V. harveyi from a 24 h culture in 2216E medium at 25 C. The fish were then kept in separate 100 l glass aquaria (12 fish each). A total of 144 fish (36 4) were used for the study. No diet was given to the animals during the test. The fish were observed for the presence of disease manifested. Mortality of fish in each tank was observed over 14 days, and the average of the triplicate tanks was used to express cumulative mortality and relative percent survival (RPS) values were calculated as follows: RPS ¼ 100 [(treatment mortality O control mortality) 100]
2.5. Immunological assays
2.7. Statistical analysis
2.5.1. Total leukocyte count (THC) Number of total leukocyte count (WBC), was calculated using a Neubauer chamber, according to Ranzani-Paiva et al. [11]. The leukocytes were counted manually in all 25 squares (¼0.1 mm3).
All datas were expressed as means SE (standard error of the means). Differences between the means were tested by Tukey’s multiple range tests, using SPSS 11.0 statistical analysis software. P values <0.05 were considered significant.
S. Lin et al. / Fish & Shellfish Immunology 32 (2012) 909e913
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Table 1 Growth response and survival of T. ovatus fed dietary chitooligosaccharides (COS) at the end of 8th weeks of feeding trial (means S.E.M).a Parameters
Diet no. (COS supplementation level g kg1) Diet 1 (0.0)
Initial weight (g) Final weight (g) SGR (% day1) FCR Survival (%)
10.9 76.2 3.47 1.38 97.3
0.06 0.3c 0.04c 0.06a 1.3
Diet 2 (2.0) 10.9 80.7 3.58 1.27 99.3
0.09 1.1 b 0.06b 0.03b 0.7
Diet 3 (4.0) 10.7 85.3 3.71 1.15 99.3
0.07 0.4a 0.05a 0.09c 0.7
Diet 4 (6.0) 10.7 84.5 3.69 1.18 98.7
0.12 0.8a 0.03a 0.04c 0.7
a Means S.E.M having the same letter in the same row are not significantly different at P < 0.05.
3. Results 3.1. Growth After the 56 day feeding period, the final mean weight and SGR of fish improved as dietary COS increased from 0.0 to 4.0 g kg1 (P < 0.05), without any further improvement at the highest COS content (6.0 g kg1). The poorest growth was observed in the fish fed the diet containing 0.0 g kg1 COS (control group). Feed conversion ratio (FCR) was improved by increasing the dietary COS content, maximum and significantly (P < 0.05) higher FCR was achieved with the 0.0 g kg1 COS diet followed by the 2.0 g kg1 COS diet. However, FCR was not significantly affected by the dietary COS content from 4.0 to 6.0 g kg1 COS (Table 1). Similarly, no significant difference (P > 0.05) was observed in survival.
3.2. Immune response Fish fed diets with 4.0 and 6.0 g kg1 COS had the highest total leukocytes (WBC), neutrophil and monocyte counts, followed by fish fed the diets with 2.0 g kg1 COS, and lowest in fish fed the diets with 0.0 g kg1 COS (control group). The lymphocyte counts decreased with increasing dietary level of COS from 0.0 to 4.0 g kg1 COS (P < 0.05) (Table 2). The respiratory burst activity and phagocytic capacity of headkidney macrophages obtained with diets containing 4.0 and 6.0 g kg1 COS were similar but significantly (P < 0.05) higher than those obtained with diets containing 0.0 or 2.0 g kg1 COS. Fish supplemented with 0.0 g kg1 COS (control group) showed the lowest the respiratory burst activity and phagocytic capacity in comparison with the other groups (Figs. 1 and 2, n ¼ 15). Over the experimental period, the fish fed supplemented with COS exhibited significant increase in lysozyme activity or SOD activity of serum (P < 0.05) compared with the control, and the 4.0 and 6.0 g kg1 COS groups showed the maximum increase (P < 0.05) compared with all other groups. While no significant
Fig. 1. Respiratory burst activity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
differences in lysozyme activity or SOD activity were observed between 4.0 and 6.0 g kg1 COS (Figs. 3 and 4, n ¼ 15). 3.3. Challenge test The challenge test (n ¼ 36 for each dietary treatment) showed that long-time oral administration of the COS enhanced the protection against bacterial infection (Table 3). The average total mortality rate in fish fed diets with graded levels of COS was significantly lower than the control. RPS was lowest in the control group (P < 0.05) and increased with increasing dietary level of COS from 2.0 to 4.0 g kg1 COS. However, no significant differences in average mortality and RPS were detected between 4.0 and 6.0 g kg1 COS (P > 0.05). 4. Discussion The current study demonstrated the benefit of dietary supplementation with chitooligosaccharides (COS) on the growth of T. ovatus. Influences of dietary chitosan supplementation on growth have been evaluated with several aquacultured species with varied results. According to Gopalakannan and Arul [15], dietary chitosan supplementation enhanced the growth of Cyprinus carpio. Furthermore, a chitosan-coated diet was also shown to enhance the
Table 2 The effects of chitooligosaccharides (COS) on differential leukocyte counts of T. ovatus at the end of 8th weeks of feeding trial (means S.E.M).a Parameters
Diet no. (COS supplementation level g kg1) Diet 1 (0.0)
Diet 2 (2.0)
Diet 3 (4.0)
Diet 4 (6.0)
Total leukocyte count 1.19 0.36c 1.26 0.29b 1.38 0.52a 1.37 0.74a ( 104 mm3) Neutrophils (%) 4.6 0.4c 6.7 0.3b 8.2 0.6a 8.4 0.5a Lymphocytes (%) 92.5 1.3a 89.1 0.9b 83.7 1.1c 84.6 0.7c Monocytes (%) 2.6 0.5c 3.9 0.3b 6.6 0.7a 6.3 0.8a a
Means S.E.M having the same letter in the same row are not significantly different at P < 0.05.
Fig. 2. Phagocytic capacity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
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S. Lin et al. / Fish & Shellfish Immunology 32 (2012) 909e913 Table 3 Total average mortality (%) 14 days after challenge with V. harveyi in T. ovatus fed with the basal diet and diets containing graded levels of chitooligosaccharides (COS) (means S.E.M).a Diet no. (COS supplementation level g kg1)
Average mortality (%)
Diet Diet Diet Diet
70.8 53.2 38.1 36.5
1 2 3 4
(0.0) (2.0) (4.0) (6.0)
1.2a 1.7b 1.3c 0.9c
RPS (%)
0c 25b 46a 48a
a Data are the means S.E.M from 36 fish. Values in any one column not followed by the same superscripts are significantly different at P < 0.05.
Fig. 3. Lysozyme activity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
growth of the olive flounder, Paralichthys olivaceus [16]. Similar result was observed in fish fed with COS in this study. However, Kono et al. [17] reported that dietary chitosan exerted no significant influence on the growth of the red sea bream, Japanese eel, or yellowtail. Recently, Luo et al. [8] reported that dietary COS did not improve growth performance of rainbow trout. On the contrary, Shiau and Yu [18] observed depressed growth in tilapia fed with chitosan. To date, there is no exact explanation on how COS works to enhance growth rate. In addition, growth enhancing effect of immunostimulants is dependent on dosage, molecular weight, duration of feeding, environmental temperature, the route of administration and species [19]. The results of the present study demonstrated that the growth-promoting effect of COS was related to doses of polysaccharides, and supplementation of COS at 4.0 g kg1 was optimal for the growth of T. ovatus. Leukocytes are the key components of immune system in fish [20]. The leukocyte numbers or the proportion of different cell types is affected by factors such as sex, growth, life-stage, nutritional status, stressors, and bacteria infection [21]. The different types of leukocytes may possess different the pattern recognition receptors (PRRs) which may bring about different immunological responses [22]. In fish, glucan receptors have been reported to exist on neutrophils [20,23] and macrophages [24]. This study showed that the COS can alter the leukocyte counts and their proportion in fish. COS was a positive regulator of resting neutrophils [25], and resulted in higher survival rate of the fish in this study. The use of natural immunostimulants is a promising area because they are biodegradable, biocompatible and safe both for
the environment and human health [26]. Consequently, chitosan has been tested as immunostimulants in aquaculture [27e29]. As the derivatives of chitosan, chitooligosaccharides (COS) have better biocompatibility and solubility [30]. The present study also demonstrated that the dietary application of COS had beneficial effects on the some non-specific immune functions of fish. Similar findings have also been reported in other animals including pigs [6], broilers [4] or fishs [7,8]. Neutrophil of T. ovatus is the main cell type for production of lysozme. As shown in this study the significant increase in neutrophil count was just observed at level of 2.0 g kg1, 4.0 g kg1 or 6.0 g kg1 COS that is in parallel with increase in serum lysozyme activity, respectively. The mechanism by which COS enhance innate immune responses is still not elucidated to date. In the present study, those immunological parameters in fish fed high dose of COS (6.0 g kg1) remained relatively unchanged compared with 4.0 g kg1. The similar phenomenon was also seen in rainbow trout [8]. However, some researches reported that feeding the high level of immunostimulants resulted in immunosuppression or feedback regulation of fish, and further decreased immune responses [11,31]. Moreover, the biofunctionalities of COS are highly related to its molecular weight. The beneficial effects of low molecular COS (MW < 5.0 kDa) had been proved in preventing negative mineral balance [32], whereas the molecular weight of COS is critical for microorganism inhibition and required higher than 10 kDa [33]. In addition, the effect of COS administration on animal immunity greatly depended on their degree of polymerization (DP) [34]. Wei et al. [35] found that COS with the DP equal to or greater than 6 had greater bioactivities. These differences may be due to the type of immunostimulants used and experimental species, which resulted in different availability of immunostimulants. Numerous studies have shown that oral administration of immunostimulants can effectively improve resistance of fishes to pathogenic bacteria or virus [8,15,19,36]. The present study showed that the oral administration of COS significantly reduced cumulative mortality of T. ovatus after injecting with V. harveyi. The increase in resistance against V. harveyi in T. ovatus fed with COS can be possibly explained on the basis of increased non-specific immune response. In conclusion, the present study indicated that dietary COS supplementation could enhance the performance and the immune response of T. ovatus. The results suggested that oral administration of COS at 4.0 g kg1 was better than lower doses. However, further research needs to be conducted to ascertain the mechanisms of COS with reduced length of administration period and doses.
Acknowledgments Fig. 4. Superoxide dismutase(SOD) activity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
The authors would like to thank Zhang L and Wu YG for taking care of the fish. Special thanks to Yao CF for helping with the chemical analysis.
S. Lin et al. / Fish & Shellfish Immunology 32 (2012) 909e913
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Dietary administration of chitooligosaccharides to enhance growth, innate immune response and disease resistance of Trachinotus ovatus Shimei Lin a, b, *, Shuhong Mao a, Yong Guan a, Xin Lin a, Li Luo a, b a b
College of Animal Science and Technology, Southwest University, Chongqing 400716, PR China Key Laboratory of Freshwater Fish Resources and Reproductive Development (Ministry of Education), Southwest University, Chongqing 400716, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 October 2011 Received in revised form 9 February 2012 Accepted 12 February 2012 Available online 18 February 2012
The present study was conducted to investigate the effects of dietary chitooligosaccharides (COS) supplementation on the innate immune response and protection against Vibrio harveyi infection in Trachinotus ovatus. A basal diet was supplemented with 0.0 (control), 2.0, 4.0 and 6.0 g COS kg1 to formulate four experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 1.0 2.0 m), and each cage was stocked with 80 fish (initial average weight 10.8 0.05 g). After 8 weeks of feeding trial, Both the final weight and specific growth rate (SGR) significantly increased with increasing dietary COS levels up to 4.0 g kg1, whereas there were no significant differences for COS levels from 4.0 to 6.0 g kg1. A decreased feed conversion ratio (FCR) was observed with increasing dietary COS levels. The total leukocyte counts (WBC), differential leukocyte counts, respiratory burst activity, lysozyme and superoxide dismutase (SOD) activity were significantly increased with the increased levels of dietary COS (P < 0.05), and reached a maximum at level of 4.0 g kg1 COS. There were no significant differences in those immunological parameters between 4.0 and 6.0 g kg1 COS. Moreover, the dietary COS supplementation groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with V. harveyi. These results indicated that dietary intake containing COS could enhance the immune responses of fish and improve its resistance to infection by V. harveyi. Especially supplementation with 4.0 g kg1 COS to the fish for 56 days showed considerable improvement in the growth, survival and immune response of the fish. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Chitooligosaccharides Growth Non-specific immunity Trachinotus ovatus
1. Introduction Chitosan has been widely used in many fields as a source of potential bioactive material during past few decades [1e3]. However, chitosan has several drawbacks to be utilized in biological applications, including poor solubility under physiological conditions. Chitosan’s biofunctionalities are highly related to its molecular weight and degree of acetylation. Therefore, a new interest has recently been emerged on partially hydrolyzed chitosan. Chitooligosaccharide (COS), which is one type of the oligosaccharides, is produced from chitin or chitosan by chemical or enzymatic decomposition methods [4]. The COS has a higher activity and more physiological functions than chitosan due to its lower molecular weight or its ready solubility in water. Moreover, the chitosan is found abundantly in natural sources, it is produced commercially on a large scale in different countries [5]. For these reasons, the biological activities of COS are of increasing interest in recent
research. In fact, some studies regarding COS have already been conducted in either pigs [6], broilers [4] or fishes [7,8], and beneficial effects on growth performance, immunity, or blood profiles have been reported. The ovate pompano Trachinotus ovatus (Linnaeus) is one of the most commercially cultured marine fish species in the South China. Owing to the intensive culture, bacterial infectious diseases often occurred and resulted in serious losses. However, little information is available on its basal immune response and the increased immunity and protection against pathogen infection which might be stimulated by immunostimulants. Therefore, the purpose of the present study was to examine the effects of dietary chitooligosaccharides (COS) on immunity and disease resistance of this fish.
2. Materials and methods 2.1. Experimental diets
* Corresponding author. College of Animal Science and Technology, Southwest University, Chongqing 400716, PR China. Tel./fax: þ86 23 68251196. E-mail address: [email protected] (S. Lin). 1050-4648/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2012.02.019
The COS used in the current study was provided by Bioengineering Institute, Ocean University of China (Qingdao, China) and
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produced by microbial fermentation of the shells of crustaceans by Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus acidophillus. The average molecular weight of COS was lower than 5000 Da. The COS premix contained 8.6% crude protein, 14.5% crude fat, 22.6% crude ash, 10.7% crude fiber, 9.0% moisture, 3.0% chitooligosaccharide. The practical basal diet was formulated to contain approximately 40% crude protein and 9% crude lipid. Four experimental diets were formulated so as to contain different concentrations of COS at 0.0 (control), 2.0, 4.0 or 6.0 g kg1. All the ingredients were passed through a grinder, and passed through a paste extruder (1 mm diameter), and stored at 20 C until required. 2.2. Experimental animals T. ovatus were obtained from a commercial fish farm located in Zhanjiang, Guangdong, China, transported to sea cages and acclimatized for one weeks. At the start of the experiment, the fish were fasted for 24 h and weighed after being anesthetized with 3aminobenzoic acid ethyl ester (MS 222; 100 mg mL1). Young T. ovatus (average weight 10.8 0.05 g) were randomly selected and distributed into 12 sea cages (1.5 1.0 2.0 m) for the growth trial (80 fish/cage). Fish were hand-fed to apparent satiation twice (08:00 and 17:00) daily. The feeding trial lasted for 8 weeks. During the experimental period, the temperature ranged from 26.5 to 29.5 C, the salinity from 26 to 29& and dissolved oxygen content was approximately 7.6 mg l1.
Differential leukocyte counts (neutrophil, lymphocyte, monocyte) were determined using blood smears under a light microscope. 2.5.2. Respiratory burst activity The respiratory burst activity of the phagocytes was carried out by nitroblue tetrazolium (NBT, Shanghai Reagent Corp., China) following the method of Secombes [9] subsequently modified by Ai et al. [12]. The absorbance at 630 nm was measured with a Model Multiskan spectrum (Thermo, USA) using KOH/DMSO alone as a blank. Respiratory burst was expressed as NBT-reduction in 100 ml of cell suspension. 2.5.3. Phagocytic activity Phagocytic activity (PA) for five fish in each tank was determined by a method of Lin et al. [10]. PA ¼ number of phagocytic cells with engulfed yeast O number of peritoneal cells 100 2.5.4. Lysozyme activity Lysozyme level in serum of five fish in each tank was determined by turbidimetric assay according to the method described by Ellis [13]. Briefly, test serum (0.1 ml) was added to 1.9 ml of a suspension of Micrococcus lysodeikticus (Sigma) (0.2 mg ml1) in a 0.05 M sodium phosphate buffer (pH 6.2). The reaction was carried out at 25 C and absorbance was measured at 530 nm after 0.5 and 4.5 min in a spectrophotometer. One unit of lysozyme activity was defined as the amount of sample causing a reduction in absorbance of 0.001 min1.
2.3. Sample collection After being fasted for 24 h, the fish were anesthetized with MS 222 (Sigma, USA). Blood was collected by the caudal venipuncture with a 27 gauge needle and 1 ml syringe, and pooled from a random sample of five fish per tank. The blood samples were collected into both nonheparinized and K3EDTA vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) in order to obtain the serum and whole blood, respectively. After collection, whole blood from nonheparinized tubes was allowed to clot at room temperature for 4 h. Following centrifugation (3000 g, 10 min, 4 C), the serum was removed and frozen at 80 C until use. Head-kidney macrophages from five fish in each tank were isolated as described by Secombes [9] with some modifications [10]. 2.4. Survival and growth performance At the termination of the experiment, the fish were fasted for 24 h before harvest. The fish were moderately anaesthetized (3aminobenzoic acid ethyl ester, MS 222; 100 mg mL1), and then each group was counted and weighed. Based on recording the weight of each fish and counting the number of fish, specific growth rate (SGR), feed conversion ratio (FCR) and survival were calculated using the following equations: SGR ¼ 100 [in final weight in initial weight] O total duration of the experiment FCR ¼ feed given (dry weight) O weight gain (wet gain) Survival (%) ¼ (final number of kois O initial number of kois) 100.
2.5.5. Serum superoxide dismutase Superoxide dismutase (SOD) activity was measured by its ability to inhibit superoxide anion generated by xanthine and xanthine oxidase reaction system according to Wang and Chen [14] using an SOD detection kit (Nanjing Jiancheng Bioengineering Institute, China). The optical density was measured at 550 nm. One unit of SOD was defined as the amount required for inhibiting the rate of xanthine reduction by 50% in 1 ml reaction system. Specific activity was expressed as SOD unit per ml serum. 2.6. Challenge test The Vibrio harveyi strain was originally isolated from infected T. ovatus. The seven day LD50 was determined by intraperitoneal injection of 48 fish with graded doses of V. harveyi (106, 107, 108, 109 and 1010 cfu/fish) at 24 C, and the result showed that the LD50 on day 7 was 108 cfu/fish. Challenge tests were conducted in triplicate with 12 fish per replicate. Each fish was injected intraperitoneally with 0.3 ml PBS containing 2.4 108 live V. harveyi from a 24 h culture in 2216E medium at 25 C. The fish were then kept in separate 100 l glass aquaria (12 fish each). A total of 144 fish (36 4) were used for the study. No diet was given to the animals during the test. The fish were observed for the presence of disease manifested. Mortality of fish in each tank was observed over 14 days, and the average of the triplicate tanks was used to express cumulative mortality and relative percent survival (RPS) values were calculated as follows: RPS ¼ 100 [(treatment mortality O control mortality) 100]
2.5. Immunological assays
2.7. Statistical analysis
2.5.1. Total leukocyte count (THC) Number of total leukocyte count (WBC), was calculated using a Neubauer chamber, according to Ranzani-Paiva et al. [11]. The leukocytes were counted manually in all 25 squares (¼0.1 mm3).
All datas were expressed as means SE (standard error of the means). Differences between the means were tested by Tukey’s multiple range tests, using SPSS 11.0 statistical analysis software. P values <0.05 were considered significant.
S. Lin et al. / Fish & Shellfish Immunology 32 (2012) 909e913
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Table 1 Growth response and survival of T. ovatus fed dietary chitooligosaccharides (COS) at the end of 8th weeks of feeding trial (means S.E.M).a Parameters
Diet no. (COS supplementation level g kg1) Diet 1 (0.0)
Initial weight (g) Final weight (g) SGR (% day1) FCR Survival (%)
10.9 76.2 3.47 1.38 97.3
0.06 0.3c 0.04c 0.06a 1.3
Diet 2 (2.0) 10.9 80.7 3.58 1.27 99.3
0.09 1.1 b 0.06b 0.03b 0.7
Diet 3 (4.0) 10.7 85.3 3.71 1.15 99.3
0.07 0.4a 0.05a 0.09c 0.7
Diet 4 (6.0) 10.7 84.5 3.69 1.18 98.7
0.12 0.8a 0.03a 0.04c 0.7
a Means S.E.M having the same letter in the same row are not significantly different at P < 0.05.
3. Results 3.1. Growth After the 56 day feeding period, the final mean weight and SGR of fish improved as dietary COS increased from 0.0 to 4.0 g kg1 (P < 0.05), without any further improvement at the highest COS content (6.0 g kg1). The poorest growth was observed in the fish fed the diet containing 0.0 g kg1 COS (control group). Feed conversion ratio (FCR) was improved by increasing the dietary COS content, maximum and significantly (P < 0.05) higher FCR was achieved with the 0.0 g kg1 COS diet followed by the 2.0 g kg1 COS diet. However, FCR was not significantly affected by the dietary COS content from 4.0 to 6.0 g kg1 COS (Table 1). Similarly, no significant difference (P > 0.05) was observed in survival.
3.2. Immune response Fish fed diets with 4.0 and 6.0 g kg1 COS had the highest total leukocytes (WBC), neutrophil and monocyte counts, followed by fish fed the diets with 2.0 g kg1 COS, and lowest in fish fed the diets with 0.0 g kg1 COS (control group). The lymphocyte counts decreased with increasing dietary level of COS from 0.0 to 4.0 g kg1 COS (P < 0.05) (Table 2). The respiratory burst activity and phagocytic capacity of headkidney macrophages obtained with diets containing 4.0 and 6.0 g kg1 COS were similar but significantly (P < 0.05) higher than those obtained with diets containing 0.0 or 2.0 g kg1 COS. Fish supplemented with 0.0 g kg1 COS (control group) showed the lowest the respiratory burst activity and phagocytic capacity in comparison with the other groups (Figs. 1 and 2, n ¼ 15). Over the experimental period, the fish fed supplemented with COS exhibited significant increase in lysozyme activity or SOD activity of serum (P < 0.05) compared with the control, and the 4.0 and 6.0 g kg1 COS groups showed the maximum increase (P < 0.05) compared with all other groups. While no significant
Fig. 1. Respiratory burst activity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
differences in lysozyme activity or SOD activity were observed between 4.0 and 6.0 g kg1 COS (Figs. 3 and 4, n ¼ 15). 3.3. Challenge test The challenge test (n ¼ 36 for each dietary treatment) showed that long-time oral administration of the COS enhanced the protection against bacterial infection (Table 3). The average total mortality rate in fish fed diets with graded levels of COS was significantly lower than the control. RPS was lowest in the control group (P < 0.05) and increased with increasing dietary level of COS from 2.0 to 4.0 g kg1 COS. However, no significant differences in average mortality and RPS were detected between 4.0 and 6.0 g kg1 COS (P > 0.05). 4. Discussion The current study demonstrated the benefit of dietary supplementation with chitooligosaccharides (COS) on the growth of T. ovatus. Influences of dietary chitosan supplementation on growth have been evaluated with several aquacultured species with varied results. According to Gopalakannan and Arul [15], dietary chitosan supplementation enhanced the growth of Cyprinus carpio. Furthermore, a chitosan-coated diet was also shown to enhance the
Table 2 The effects of chitooligosaccharides (COS) on differential leukocyte counts of T. ovatus at the end of 8th weeks of feeding trial (means S.E.M).a Parameters
Diet no. (COS supplementation level g kg1) Diet 1 (0.0)
Diet 2 (2.0)
Diet 3 (4.0)
Diet 4 (6.0)
Total leukocyte count 1.19 0.36c 1.26 0.29b 1.38 0.52a 1.37 0.74a ( 104 mm3) Neutrophils (%) 4.6 0.4c 6.7 0.3b 8.2 0.6a 8.4 0.5a Lymphocytes (%) 92.5 1.3a 89.1 0.9b 83.7 1.1c 84.6 0.7c Monocytes (%) 2.6 0.5c 3.9 0.3b 6.6 0.7a 6.3 0.8a a
Means S.E.M having the same letter in the same row are not significantly different at P < 0.05.
Fig. 2. Phagocytic capacity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
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S. Lin et al. / Fish & Shellfish Immunology 32 (2012) 909e913 Table 3 Total average mortality (%) 14 days after challenge with V. harveyi in T. ovatus fed with the basal diet and diets containing graded levels of chitooligosaccharides (COS) (means S.E.M).a Diet no. (COS supplementation level g kg1)
Average mortality (%)
Diet Diet Diet Diet
70.8 53.2 38.1 36.5
1 2 3 4
(0.0) (2.0) (4.0) (6.0)
1.2a 1.7b 1.3c 0.9c
RPS (%)
0c 25b 46a 48a
a Data are the means S.E.M from 36 fish. Values in any one column not followed by the same superscripts are significantly different at P < 0.05.
Fig. 3. Lysozyme activity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
growth of the olive flounder, Paralichthys olivaceus [16]. Similar result was observed in fish fed with COS in this study. However, Kono et al. [17] reported that dietary chitosan exerted no significant influence on the growth of the red sea bream, Japanese eel, or yellowtail. Recently, Luo et al. [8] reported that dietary COS did not improve growth performance of rainbow trout. On the contrary, Shiau and Yu [18] observed depressed growth in tilapia fed with chitosan. To date, there is no exact explanation on how COS works to enhance growth rate. In addition, growth enhancing effect of immunostimulants is dependent on dosage, molecular weight, duration of feeding, environmental temperature, the route of administration and species [19]. The results of the present study demonstrated that the growth-promoting effect of COS was related to doses of polysaccharides, and supplementation of COS at 4.0 g kg1 was optimal for the growth of T. ovatus. Leukocytes are the key components of immune system in fish [20]. The leukocyte numbers or the proportion of different cell types is affected by factors such as sex, growth, life-stage, nutritional status, stressors, and bacteria infection [21]. The different types of leukocytes may possess different the pattern recognition receptors (PRRs) which may bring about different immunological responses [22]. In fish, glucan receptors have been reported to exist on neutrophils [20,23] and macrophages [24]. This study showed that the COS can alter the leukocyte counts and their proportion in fish. COS was a positive regulator of resting neutrophils [25], and resulted in higher survival rate of the fish in this study. The use of natural immunostimulants is a promising area because they are biodegradable, biocompatible and safe both for
the environment and human health [26]. Consequently, chitosan has been tested as immunostimulants in aquaculture [27e29]. As the derivatives of chitosan, chitooligosaccharides (COS) have better biocompatibility and solubility [30]. The present study also demonstrated that the dietary application of COS had beneficial effects on the some non-specific immune functions of fish. Similar findings have also been reported in other animals including pigs [6], broilers [4] or fishs [7,8]. Neutrophil of T. ovatus is the main cell type for production of lysozme. As shown in this study the significant increase in neutrophil count was just observed at level of 2.0 g kg1, 4.0 g kg1 or 6.0 g kg1 COS that is in parallel with increase in serum lysozyme activity, respectively. The mechanism by which COS enhance innate immune responses is still not elucidated to date. In the present study, those immunological parameters in fish fed high dose of COS (6.0 g kg1) remained relatively unchanged compared with 4.0 g kg1. The similar phenomenon was also seen in rainbow trout [8]. However, some researches reported that feeding the high level of immunostimulants resulted in immunosuppression or feedback regulation of fish, and further decreased immune responses [11,31]. Moreover, the biofunctionalities of COS are highly related to its molecular weight. The beneficial effects of low molecular COS (MW < 5.0 kDa) had been proved in preventing negative mineral balance [32], whereas the molecular weight of COS is critical for microorganism inhibition and required higher than 10 kDa [33]. In addition, the effect of COS administration on animal immunity greatly depended on their degree of polymerization (DP) [34]. Wei et al. [35] found that COS with the DP equal to or greater than 6 had greater bioactivities. These differences may be due to the type of immunostimulants used and experimental species, which resulted in different availability of immunostimulants. Numerous studies have shown that oral administration of immunostimulants can effectively improve resistance of fishes to pathogenic bacteria or virus [8,15,19,36]. The present study showed that the oral administration of COS significantly reduced cumulative mortality of T. ovatus after injecting with V. harveyi. The increase in resistance against V. harveyi in T. ovatus fed with COS can be possibly explained on the basis of increased non-specific immune response. In conclusion, the present study indicated that dietary COS supplementation could enhance the performance and the immune response of T. ovatus. The results suggested that oral administration of COS at 4.0 g kg1 was better than lower doses. However, further research needs to be conducted to ascertain the mechanisms of COS with reduced length of administration period and doses.
Acknowledgments Fig. 4. Superoxide dismutase(SOD) activity of T. ovatus fed with graded levels of chitooligosaccharides (COS). Data are expressed as mean (S.E.M). Means in the same column sharing the same superscript letter are not significantly different determined by Tukey’s test (P > 0.05). n ¼ 15.
The authors would like to thank Zhang L and Wu YG for taking care of the fish. Special thanks to Yao CF for helping with the chemical analysis.
S. Lin et al. / Fish & Shellfish Immunology 32 (2012) 909e913
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