Abstract
The effects of dietary supplementation with Clostridium butyricum on growth performance and humoral immune response in Miichthys miiuy were evaluated. One hundred and fifty Miichthys miiuy weighing approximately 200~260 g were divided into five groups and reared in 15 tanks with closed circuiting culture system. The animals were fed 5 diets: basal diet only (control) or supplemented of the basal diet with C. butyricum at doses of 103 (CB1), 105 (CB2), 107 (CB3) or 109 (CB4) CFU/g. Compared with the control, the serum phenoloxidase activity was significantly increased by the supplementation (P<0.05), acid phosphatases activity was increased significantly (P<0.05) at the doses of 109 CFU/g. Serum lysozyme activity peaked at dose of 107 CFU/g and in the skin mucus at dose of 109 CFU/g. Immunoglobulin M level in the serum and skin mucus was increased except at dose of 103 CFU/g (P<0.05). The growth at the dose of 109 CFU/g was higher than that of the control (P<0.05). It is concluded that supplementation of C. butyricum can mediate the humoral immune responses and improve the growth performance in Miichthys miiuy.
Keywords: Clostridium butyricum, Growth performance, Humoral immune response, Miichthys miiuy
INTRODUCTION
It is well known that antibiotics are used as effective agents to control diseases and as growth promoters in domestic animals. However, the continuous use of antimicrobials increases the bacterial resistance. Therefore, it is essential to find effective and non-resistance replacements. Probiotics are viable cell preparations that have beneficial effects on health of the host by improving its intestinal balance (Fuller, 1989) via producing nutrients, enhancing immune responses and improving the water quality in aquaculture (Gatesoupe, 1999; Verschuere et al., 2000). In recent years, probiotics have been increasingly used in the biological control to prevent diseases in aquaculture (Verschuere et al., 2000), which will make aquaculture products more acceptable to consumers.
Clostridium butyricum has been used as probiotics for many years in Eastern Asia countries, such as Japan, Korea and China (Ito et al., 1997; Kamiya et al., 1997) and is a butyricum-acid producing, spore-forming, gram-positive and oblige anaerobe rod bacterium, found in soil and intestines of healthy animals and humans, and is used clinically to prevent disturbances of microflora in the human intestine and to treat diarrhea. C. butyricum also has cellular immunostimulatory effects, such as stimulation of production of macrophage and NK (natural killer) cells (Young et al., 1987). Sakai et al.(1995) demonstrated that enhancement of resistance to Vibrosis in rainbow trout by oral administration of C. butyricum bacteria was mediated by leucocyte activation, including phagocytosis and increased superoxide anion production. We hypothesize that C. butyricum enhances humoral immune response in marine fish. This hypothesis was examined by measuring growth performance and humoral immune response in Miichthys miiuy that originally grow in the North Pacific west coast in response to dietary supplementation with C. butyricum.
MATERIALS AND METHODS
Bacterial strain and preparation of C. butyricum
The bacterial strain C. butyricum was isolated from healthy chicken intestine, and identified by biochemistry and molecular experiments in the School of Science, Zhejiang University, China.
C. butyricum was fermented in a 70-liter-fermentation tank (GUJS7A-70, Dongfang Bioengineering Co., China). After 48 h fermentation, C. butyricum was collected, centrifuged and freezer-dried. The freezer-dried powder was then enumerated by plate counting on MRS agar (Sigma Chemical Co., USA). The viability of the freezer-dried bacteria was 1.86×1011 CFU/g. They were stored at −20 °C until further use. In the supplementation, dried bacteria were added into the basal diet (see below), mixed thoroughly and pelleted into 5 mm diameter (Mingo Tech-Bank Co., Ltd.). Subsequently, the pellets were dried immediately in oven at 50~60 °C, and then stored at −20 °C. Plate counting method was used to detect the number of C. butyricum in dried pellets. The viability of C. butyricum was approximately 90% in the four dried experimental diets.
Diet preparation and experiment design
The components of a basal diet presented in Table 1 should provide adequate nutrient supply to Miichthys miiuy. White fishmeal, soybean meal and protein powder of blood corpuscles were used for sources of protein; fish oil and alpha-starch for non-protein energy. Five treatments consisted of the basal diet only (control) and four doses of supplementation with C. butyricum respectively at 103 (CB1), 105 (CB2), 107 (CB3) and 109 (CB4) CFU/g in freezer-dried form.
Table 1.
Formulation and proximate composition of the basal diet (on dry matter basis)
| Composition (%) | |
| Ingredients | |
| White fish meal | 66.5 |
| Soybean meal | 4.75 |
| Alpha-cornstarch | 19 |
| Protein powder of blood powder | 1.9 |
| Vitamin pre-mixture* | 0.675 |
| Mineral pre-mixture* | 2 |
| Cellulose | 0.175 |
| Fish oil |
5 |
| Proximate | |
| Moisture | 10.88 |
| Crude protein | 48.45 |
| Ash | 9.86 |
| Crude fat | 5.14 |
Vitamin and mineral pre-mixtures were provided by the Mingo Tech-Bank Co., Ltd. China
Total energy is 15.59 kJ/kg
Experimental animals and rearing facilities
Miichthys miiuy weighing approximately 200~260 g were purchased from net-cage culture in Xihu Port, Zhejiang Province, China and transferred to the experimental laboratory (Institute of Mingo Tech-Bank Co., Ltd.). Upon arrival, after being bathed in penicillin solution, they were assigned to 600-liter-tanks and acclimatized to a circulating rearing system for three weeks, during which the fish were fed the basal diet. Subsequently, 150 healthy, energetic and no-disease fish were put into 15 tanks and divided into 5 groups. Each group was repeated in triplicates. The supplementation lasted for 8 weeks. The fish were fed twice daily at 07:30 a.m. and 17:30 p.m., with the feed offered being about 1.5% of the fish biomass.
The closed circulating rearing system consisted of microorganism-filtration, heating, cooling, protein-separating and ultraviolet sterilization devices. Each tank was a part of the whole system with a common reservoir of water at 29‰~32‰ salinity. The data on pH, DO (dissolved oxygen), temperature, conductance and ORP (oxidation-reduction potential) was recorded and saved in a computer. Water was circulated 2 times per hour through a separate biofilter to remove impurities and reduce ammonia concentration. Photoperiod was 12 h light (08:00 a.m.~20:00 p.m.) and 12 h dark. Water temperature 25~30 °C was maintained. Total ammonia and nitrite concentrations were maintained at 0.01 mg/L and 0.01 mg/L, respectively.
Sample collection and assays
1. Sampling
At the end of the research, blood was drawn from the caudal vein of the individual fish after anaesthetization (Fengyuan Co., Ltd., China). The blood samples were collected in heparinized tube and plasma was harvested by centrifuging at 1500×g for 5 min at 4 °C. The whole blood was collected in a syringe, allowed to clot for an hour in microtubes at room temperature, followed by five hours at 4 °C and then serum was harvested by centrifuging at 1500×g for 5 min at 4 °C. Both plasma and serum samples were preserved at −20 °C prior to analysis. The plasma samples were used for immunoglobulin M (IgM) analysis and the serum samples for determining the activities of lysozyme, phenoloxidase and acid phosphatase.
The skin mucus sample was collected by scalpels. The mucus was mixed with four volumes of 8.5% salty solution and centrifuged at 1500×g for 5 min at 4 °C. The supernatant was collected for lysozyme and IgM assays.
2. Growth performance
At beginning and end of the feeding trial, the animals were fasted for one day and their weights were determined. Growth performance was expressed as the total weight gain (TWG), daily weight gain (DWG), feed utilization efficiency (FUE), feed conversion ratio (FCR), relative weight gain (RWG) and specific growth rate (SGR). The calculation formulas were as follows:
| TWG=Wt−Wi, |
| DWG=(Wt−Wi)/t, |
| RWG=(Wt−Wi)×100%/Wi, |
| SGR=(lnWt−lnWi)/t×100%, |
| FUE=TWG×100%/feed consumption, |
| FCR=total feed consumption/TWG, |
where Wt and W i are the final and initial mean weight (g), respectively, and t is the feeding trial period (d).
3. Assay
Lysozyme activity in the serum and skin mucus was measured with spectrophotometry based on lysis of freezer-dried particles of Micrococcus lysodeikitcus (Sigma Chemical Co., USA) (Ellis, 1990). One unit of enzyme activity was defined as the amount of enzyme causing a decrease in absorbance of 0.001 min−1·ml−1 serum or skin mucus. Phenoloxidase (PO) activity was measured according to Ashida and Säderhäll (1984) with some modifications. A 0.5 ml sample serum, 0.5 ml phosphate buffer solution (0.1 mol/L, pH=6.0), and 0.5 ml L-DOPA (L-dihydroxy-phenylalanine) (0.01 mol/L) were added into a 1.5-ml centrifuge tube. The mixture was kept at 25 °C in water bath for 10 min, and then the OD value was measured at 2-min intervals for 10 min at 490 nm. One unit of enzyme activity was defined as an increase in absorbance of 0.001 min−1·ml−1 serum. Acid phosphatase (ACP) activity in serum was measured with spectrophotometry, wherein sodium nitropheny-phosphate was used as substrate (Pipe, 1990). One unit of ACP activity was defined as the amount of enzyme in 100 ml serum necessary to produce 1 mg nitrophenol for 30 min at 37 °C. Levels of total IgM in serum and mucus of body surface were analyzed by enzyme-linked immunosorbent assays (ELISA) according to Bowden et al.(2003). The optical density (OD) was measured at 560 nm. The OD-value was directly related to the amount of total IgM.
Statistical analysis
Rank-sum test was used to determine the significant variation in growth (P≤0.05). One-way analysis of variance (ANOVA) was used to determine the significant variation between the treatments in the humoral immune responses (SYSTAT 10.0, SPSS, USA). The least significant difference (LSD) was used for multiple comparisons, and all tests used a significance level of P≤0.05.
RESULTS
Growth performance
Table 2 and Table 3 of the results show that C. butyricum supplemented to the diet could increase the TWD, RWG, FUE and SGR, decrease the FCR at dose of 109 CFU/g (P<0.05); and that, the growth showed increasing tendency with C. butyricum dose.
Table 2.
Effect of Clostridium butyricum on RWG, FUE and SGR in Miichthys miiuy
| RWG (%) | FUE (%) | SGR (%) | |
| Control | 18.0±0.34b | 28.00±2.10b | 0.31±0.01b |
| CB1 | 26.9±2.2ab | 37.80±1.60ab | 0.43±0.03ab |
| CB2 | 29.1±8.1ab | 40.87±10.6ab | 0.45±0.10ab |
| CB3 | 36.3±1.3ab | 49.20±16.4ab | 0.55±0.17ab |
| CB4 | 39.2±7.1a | 54.20±12.2a | 0.58±0.09a |
Values with different superscript letters are significantly different (P<0.05); Values are means±SD (n=3)
Table 3.
Effect of Clostridium butyricum on the TWG, DWG and FCR in Miichthys miiuy
| Wi (g) | Wt (g) | TWG (g) | DWG (g) | FCR | |
| Control | 258±17 | 306.5±18 | 48.5±3.5b | 0.86±0.06b | 3.40±0.25b |
| CB1 | 238±12 | 301.5±20 | 63.5±2.7ab | 1.13±0.05ab | 2.64±0.11ab |
| CB2 | 236±11 | 304.7±16 | 68.7±17.8ab | 1.20±0.30ab | 2.93±0.93ab |
| CB3 | 228±14 | 310.1±14 | 82.1±11.0ab | 1.47±0.49ab | 2.18±0.68ab |
| CB4 | 231±12 | 322.3±13 | 97.3±20.0a | 1.62±0.36a | 1.89±0.39a |
Values with different superscript letters are significantly different (P<0.05); Values are means±SD (n=3)
Lysozyme activity of the serum and skin mucus in Miichthys miiuy
The lysozyme activity of the serum and the skin mucus in Miichthys miiuy are given in Table 4 showing similar increasing tendency with supplementation of the bacteria, which significantly increased at dose of 107 and 109 CFU/g C. butyricum in comparison with the control (P<0.05). Although the lysozyme activity in serum of CB1 and CB2 treatments was higher than those in the mucus, they reached almost the same level in CB3 and CB4.
Table 4.
Effect of Clostridium butyricum on lysozyme activity of the serum and skin mucus in Miichthys miiuy (U/ml)
| Control | CB1 | CB2 | CB3 | CB4 | |
| Serum | 333±35b | 475±28b | 526±33b | 813±45a | 782±36a |
| Skin mucus | 140±38b | 156±46b | 317±29b | 716±35a | 786±35a |
Values with different superscript letters are significantly different (P<0.05); Values are means±SD (n=3)
Activity of phenoloxidase and acid phosphatase in serum of Miichthys miiuy
The activities of acid phosphatase and phenoloxidase in serum of Miichthys miiuy are given in Table 5. Acid phosphatase activity of serum in Miichthys miiuy was significantly higher than control at dose of 109 CFU/g (P<0.05) showed significant increase by dietary supplementation of the bacteria from 103 CFU/g to 109 CFU/g compared with control for phenoloxidase activity (P<0.05).
Table 5.
Effect of Clostridium butyricum on activity of phenoloxidase and acid phosphatase in serum of Miichthys miiuy (U/ml)
| Control | CB1 | CB2 | CB3 | CB4 | |
| Acid phosphatase | 6.90±1.4b | 6.6±0.4b | 7.1±1.2b | 7.4±0.4b | 8.9±0.8a |
| Phenoloxidase | 3.90±0.4d | 13.6±1.2c | 18.9±0.7b | 25.9±1.8a | 26.6±0.7a |
Values with different superscript letters are significantly different (P<0.05); Values are means±SD (n=3)
Total IgM level of serum and the skin mucus in Miichthys miiuy
The total IgM level of serum and skin mucus in Miichthys miiuy is given in Table 6. Total IgM level of serum peaked at (0.65±0.1) mg/ml for CB3 treatment and was significantly different from the control (P<0.05), while there were no significant differences among CB2, CB3 and CB4 treatment. Although there was similar variation tendency of the serum and skin mucus IgM level, obviously the total IgM level in serum was always higher than that in skin mucus.
Table 6.
Effect of Clostridium butyricum on total IgM levels in serum and skin mucus in Miichthys miiuy (mg/ml)
| Control | CB1 | CB2 | CB3 | CB4 | |
| Serum | 0.16±0.06c | 0.29±0.03bc | 0.62±0.05a | 0.65±0.10a | 0.42±0.07b |
| Skin mucus | 0.11±0.01c | 0.20±0.04b | 0.20±0.05ab | 0.26±0.04a | 0.17±0.02c |
Values with different superscript letters are significantly different (P<0.05); Values are means±SD (n=3)
DISCUSSION
Probiotics used as the alternative of antibiotics to promote fishes and shrimp growth was shown in (Wang and Xu, 2006; Ziaei-Nejad et al., 2006; Lara-Flores et al., 2003). In the present experiment, C. butyricum could decrease the FCR (feed conversion ratio) and improve Miichthys miiuy growth. The results were consistent with the results of previous researches. Effects of C. butyricum to promote the growth have been demonstrated early in chicken (Han et al., 1984). A compound additive containing protease, lipase, lactic acid and C. butyricum was proved to increase the protein and fat absorption, decrease feed conversion ratio and reduce disease in fish (Yang, 1998). Therefore, C. butyricum used singly as the additive or mixed with other additives could improve animal growth.
Lysozyme is one of the major components in the immune defense system in both invertebrates and vertebrates and can split peptidoglycan in bacterial cell walls especially of the gram-positive species and cause lysis of the cells (Chipman and Sharon, 1969). It exists in the mucus, lymphoid tissue, plasma and other body fluids of most fish (Grinde et al., 1988; Grinde, 1989; Yousif et al., 1994) and plays an important role in defense against pathogens invasion. Lysozyme concentrations in fish have been reported to be increased after injection of a bacterial product (Chen et al., 1996) and in response to bacterial infection (Møyner et al., 1993). Panigrahi et al.(2004) demonstrated that the Lactobacillus rhamnosus JCM 1136 could modulate the lysozyme activity in fish. In his study, both LAB (Lactobacillus rhamnosus) fed groups showed elevated level of lysozyme activity, but the group receiving the higher density was observed to have significantly higher lysozyme activity compared with that of the control, indicating activation of the immune system. The present study is the first report that lysozyme activity in marine fish was increased by dietary supplementation with C. butyricum and showed increasing tendency with supplemented dose. The mechanism that C. butyricum could increase lysozyme activity in Miichthys miiuy is not clear.
Phenoloxidase (PO) is known to be a defense enzyme, and serves as a non-self recognition system in the host defense reactions, and is usually an indicator of the immune response in shrimp and crab. The present study showed that phenoloxidase is detectable in fish serum and enhanced by C. butyricum supplementation. Phenoloxidase usually exists as an inactive precursor (proPO) (Smith and Säderhäll, 1991; Asokan et al., 1997), which can readily be activated to phenoloxidase (PO) by an endogenous activating system or exogenous elicitors such as trypsin, lipopolysaccharide and zymosan (β-1,3-glucan) (Asada et al., 1993; Sugumaran and Nellaiappan, 1991). Therefore, it could be assumed that C. butyricum might trigger proPO activation via a limited proteolytic cleavage of the proenzyme as exogenous elicitor.
Acid phosphatase, an enzyme localized within lysosomes, is important for intracellular digestion of phagocytized antigens, has been used as a marker of macrophage activation in mammalian models and invertebrates. In the present study, it was also observed that an increase in acid phosphatase activity occurred in Miichthys miiuy when they were fed with C. butyricum at the dose of 109 CFU/g (P<0.05), which indicated that C. butyricum could stimulate immune response.
Immunoglobulins are well recognized to provide protection in animals and human beings against various diseases. In teleost fish, two types of immunoglobulin (belonging to the IgM and IgD subpopulations) have been characterized (Watts et al., 2001), with IgM being the main immunoglobulin which resembles mammalian IgM in both structure and physiological characteristics. IgM is present in blood and other body fluids, and plays a role as an immune effector molecule (Ross et al., 1998). Some studies have shown the effects of probiotics on immunoglobulin secretion. Perdigon et al.(1990) reported an increase of IgA in the intestine via an oral administration of lactic acid bacteria which protects mice against Salmonella typhmirium. However the LAB feeding resulted in a higher total Ig level in rainbow trout without significant differences in comparison with the control (Panigrahi et al., 2004). In this study, dietary supplementation with C. butyricum at higher dose treatment (P<0.05) caused secretion of IgM in the blood sera and the skin mucus in comparison with the control. Stimulatants, such as vitamin A, chitin, yeast cells or levamisole supplemented to the diet resulted in the increase of IgM level (Cuesta et al., 2004). Therefore, it was assumed that C. butyricum might be an effective immune stimulatant in Miichthys miiuy.
Footnotes
Project supported by the Bureau of Science and Technology of Zhejiang Province (No. 2004201), China and the Youth Fund of Ningbo City (No. 2004A620008), China
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