Dietary polysaccharide from <Emphasis Type="Italic">Angelica sinensis</Emphasis> enhanced cellular defence responses and disease resistance of grouper <Emphasis Type="Italic">Epinephelus malabaricus</Emphasis>

The purpose of this study was to determine the effect of Angelica sinensis polysaccharide (ASP) supplemented in diet on the innate cellular immune response and disease resistance in grouper, Epinephelus malabaricus. Fish were fed diets containing different doses of ASP (0, 500 and 3,000 mg kg⁻¹ diet) for 12 weeks. After 12 week feeding, the respiratory burst activity, phagocytic activity, and leukocytes proliferation in head kidney were assayed. The functional immunity in terms of cumulative mortality was also assessed by a challenge with live Edwardsiella tarda. Results showed that the respiratory burst activities in ASP-supplemented groups were increased significantly. The respiratory burst index was the highest in fish-fed 3000 mg kg⁻¹ diet and the lowest in control. The phagocytic activities in ASP-supplemented groups were significantly higher than that of control. No significant difference in phagocytic activity was observed between ASP-supplemented groups. ASP stimulated the head kidney leukocytes proliferation significantly, despite the absence of lipopolysaccharide (LPS) or not. The cumulative mortalities of fish fed with 3000 mg ASP kg⁻¹ diet were significantly lower than those fed with 500 mg ASP kg⁻¹ diet and control diet after 96 h of challenge. In conclusion, dietary ASP enhanced some cellular immune parameters and disease resistance against E. tarda in grouper.     

Leukocyte response and phagocytic activity in Nile tilapia experimentally infected with <Emphasis Type="Italic">Enterococcus</Emphasis> sp.

This study evaluated the total and differential leukocyte counting and the phagocytic activity in Nile tilapia Oreochromis niloticus experimentally injected with Enterococcus sp. in the swim bladder. Fish were distributed in four treatments in triplicates of non-injected fish, fish injected with 1 ml of sterile saline solution 0.65%, and fish injected with 1 × 10³ and 1 × 10⁶ colony-forming units (CFU) of Enterococcus diluted in 1 ml sterile saline. Twenty-four hours after injection, the fish were anesthetized and the blood collected for white blood cell (WBC) counts, differential counting of WBC, and phagocytic activity of blood leukocytes. The increased numbers of WBC and lymphocytes were followed by decreased number of monocyte after infection. The percentages of phagocytic activities in the blood were 55.3 and 55.9%, respectively, in tilapia injected with 1 × 10³ and 1 × 10⁶ CFU/ml.     

Effect of dietary protein source and time on alkaline proteolytic activity of Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

A 4 × 3 factorial study was conducted to evaluate the effect of four experimental diets (a control diet and a 0, 50, and 100% fish meal replacement diet) and the period of time Nile tilapia, Oreochromis niloticus, were fed those diets (0, 20, and 40 days) on the alkaline proteolytic activity of the animals’ digestive tract, as well as their potential interaction. Significant differences (at P < 0.05) and a significant interaction were observed among dietary treatments for the alkaline proteolytic activity of tilapia after 40 days of feeding. This study confirmed that, under these experimental conditions, a 50% fish meal replacement formulation elicited the highest alkaline proteolytic activity in the digestive tract of tilapia, which resulted in the highest final weight and specific growth rate (SGR), but further research is needed to establish the relative contribution of the alkaline proteases to the overall proteolytic activity of this omnivorous fish species.     

Probiotic bacteria <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> influences the blood profile in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> (Walbaum)

This paper reports the effect of feeding probiotic diets on blood profiles in rainbow trout. Two experiments were performed: in the first, fish of average weight 75 g were offered either a commercial feed or the same incorporated with 10⁹ CFU g⁻¹ of lactic acid bacteria Lactobacillus rhamnosus for 30 days; in the second study performed for a similar duration, fish of average weight 126 g were offered formulated diets that either contained the same bacteria in heat-killed or freeze-dried form (nearly 10¹¹ CFU g⁻¹), or the basal diet without the bacteria. Blood samples were collected at different times after commencement of probiotic feeding to determine the total cholesterol, triglyceride contents, the plasma alkaline phosphatase activity, plasma protein and hematocrit value. The plasma cholesterol significantly increased upon probiotic feeding in the first experiment. A significant elevation (P < 0.05) of plasma cholesterol and triglyceride and alkaline phosphatase activity level was found in the freeze-dried probiotic fed groups at 20 and 30 days postfeeding. This was concomitant with the increased plasma protein and hematocrit values in FD group at 20 and 30 days. Likewise, the heat-killed probiotic fed group registered significantly high values of triglycerides, alkaline phosphatase activity, and plasma protein compared to the control diet fed groups after 20 days of feeding. Thus, alterations in the blood profiles could serve as supplementary information when examining the benefits of probiotics for fish.     

Effects of a probiotic bacterial <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> dietary supplement on the crowding stress response of juvenile Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

To test the effects of probiotic bacteria against crowding stress, juvenile Nile tilapia Oreochromis niloticus were fed commercial feed supplemented with 1 × 10¹⁰ cfu/g pellets of Lactobacillus rhamnosus for 3 weeks. The fish were confined and subjected to crowding for 7 and 14 days after which their physiological condition was investigated. The administration of probiotic prophylaxis improved growth performance even in the stressed fish. After 7 days of stress exposure, fish receiving probiotic-supplemented feed showed proactive behavior and coping responses to the stressor, as evidenced by elevated plasma glucose levels and osmolality, stabilized plasma electrolytes, and a higher RNA:DNA ratio. Fish stressed for 7 days and fed normal commercial diet showed impairment of plasma electrolytes; after 14 days, the plasma osmolality, electrolytes, glucose, and RNA:DNA ratio decreased, indicating physiological maladaptation to the stressor. The growth rate was also found to be reduced, suggesting that energy demand exceeded the energy available from metabolism and dietary uptake. This latter effect was not observed in fish fed the probiotic-supplemented diet. The results suggest that probiotic bacteria prophylaxis may increase energy availability for metabolic support of the crowding stress response and improve the stress coping capacity of fish.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Immune response of largemouth bass, <Emphasis Type="Italic">Micropterus salmoides</Emphasis>, to whole cells of different <Emphasis Type="Italic">Nocardia seriolae</Emphasis> strains

Largemouth bass Micropterus salmoides were immunized with four different N. seriolae strains—two α-glucosidase-positive (961113, KU040801) and two α-glucosidase-negative (94260, OTTS) strains—along with Freund’s incomplete adjuvant. After primary immunization (week 0), a booster was administered at weeks 4 and 8. Nonspecific immune responses to multiple immunizations with the different N. seriolae strains were determined based on serum lysozyme activity and nitroblue tetrazolium (NBT)-positive cells in peripheral blood. The serum lysozyme activity and NBT-positive cells in peripheral blood were not significantly increased even after the two booster immunizations. Specific antibody responses against N. seriolae cells were investigated by enzyme-linked immunosorbent assay. At 4 weeks after immunization, all groups immunized with N. seriolae antigens showed significant increases in their specific antibody levels. The sera from fish immunized with different N. seriolae strains exhibited reactivity with N. seriolae sonicated antigens of 28, 30, 36 and 84 kDa by western blot analysis. After two boosters, fish were challenged with live N. seriolae to assess the vaccine’s efficacy; however, multiple injections of the N. seriolae strains did not reduce mortality, irrespective of the bacterin.     

Immunostimulatory effects of fermented vegetable product on the non-specific immunity of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

The stimulatory effect of fermented vegetable product (FVP) upon the phagocytic and superoxide generation of leukocytes was studied in the Japanese flounder Paralichthys olivaceus. The phagocytic activity of casein-induced, intraperitoneal leukocytes was investigated and quantified, that is the activity significantly increased (P<0.05 or <0.01) by the addition of FVP beyond 3 mg/kg body weight. Further analysis investigated the effect of FVP on superoxide generation in leukocytes. Established in vitro cytochrome c reduction assay was used to measure superoxide generation; reduced levels of FVP in assay samples had a profound effect on superoxide generation. FVP was also incorporated in commercial diets and fed to Japanese flounder for 4 weeks. The phagocytic activities and superoxide generation of peritoneal induced leukocytes were significantly higher (P<0.05, <0.01) in fish fed the FVP supplemented diet than fish fed the control diet. FVP feeding in fish had a significantly higher (P<0.05) activity of lysozyme than in the control fish.     

Potential of Chinese chive oil as a natural antimicrobial for controlling <Emphasis Type="Italic">Flavobacterium columnare</Emphasis> infection in Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

Chinese chive Allium tuberosum oil was studied for its diallyl sulfide content and its antimicrobial activity against Flavobacterium columnare in fish both in vitro and. The oil was found to have a very low concentration of diallyl monosulfide relative to the other diallyl sulfides (diallyl disulfide, diallyl trisulfide, and diallyl tetrasulfide) identified. In the in vitro study, the Chinese chive oil had a bacteriocidal effect on all tested strains of F. columnare, with varied minimal inhibitory concentrations. The median lethal dose (LD₅₀) of FC4 for Nile tilapia Oreochromis niloticus was determined to be 3.72 × 10³ CFU/fish. In the in vivo trial, no mortality was observed in fish fed fish diets supplemented with 800 mg/kg Chinese chive oil and 100 mg/kg of oxytetracycline hydrochloride 5 days prior to infection with F. columnare strain 4 at a LD₅₀. These results indicate that Chinese chive oil has the potential to replace antibiotics for controlling fish disease caused by F. columnare.     

Evaluation of dried seaweed <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis> as an ingredient in diets for teleost fish <Emphasis Type="Italic">Siganus canaliculatus</Emphasis>

In order to evaluate the feasibility of dried seaweed Gracilaria lemaneiformis (Bory) as a dietary ingredient for the rabbitfish Siganus canaliculatus (Park), juvenile fish (average weight 15.64 ± 0.15 g) were fed with two isonitrogenous (32% crude protein) and isolipid (8% lipid) diets for 8 weeks, with one diet incorporating 33% of dried G. lemaneiformis (DGL), which accounted for 6% protein and 17% carbohydrate (DGL-diet), and the other containing no DGL (control diet). After 8 weeks, the growth performance and feed utilization efficiency of fish fed with the DGL-diet were inferior to fish fed with the control diet, although some non-specific immunity parameters were better in fish fed with the DGL-diet compared with those of fish fed control diet. Nutritional composition (whole body composition, amino acid, and fatty acid composition in dorsal muscle) except methionine and tyrosine in muscle showed no differences between the two groups. Furthermore, the survival rate of fish and apparent digestibility coefficient of diets were the same between the two groups. These results indicated that incorporation of DGL in diet of S. canaliculatus is feasible, and further studies are recommended to optimize the level of DGL in diet of S. canaliculatus to improve growth performance.     

The immune modulatory effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) essential oil on <Emphasis Type="Italic">Tilapia zillii</Emphasis> following intraperitoneal infection with <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

The current study aimed to evaluate the possible effect of Origanum essential oil on innate immune parameters as well as the hematological profiles of Tilapia zillii following challenge with Vibrio anguillarum. Fifty-four of Tilapia zillii weighing 180?±?10.2 g were randomly distributed into three identical closed recirculating seawater systems. The study included three groups (G1, G2, and G3) repeated in triplicates. Fish of the first two groups were fed on a basal diet without herbs, whereas fish of the last group were fed on a basal diet supplemented with Origanum essential oil at concentration 1 g kg^?1 for 15 days. Subsequently, fish of G2 and G3 subjected to a peritoneal inflammation by intraperitoneally injecting V. anguillarum (5.5?×?10⁵ CFU mL^?1), whereas fish of G1 injected with saline and served as control. Fish of all groups were then sampled at 4, 12, and 24 h post injection. No mortalities were observed in both basal and Origanum fed groups. However, some specimens of fish fed basal diet showed dorsal fin erosions, eroded mouth, and detached skin. Although the kinetics of RBCs, WBCs, Hb, and differential leukocyte values remained unchanged in fish fed different diets at the beginning of the trial, significant increases in those values were observed in fish fed Origanum essential oil at 12 and 24 h post injection. Similarly, an augmentation of plasma proteases, antiproteases, and lysozyme activities were recorded in fish fed Origanum essential oil at the same particular sampling points. A significant enhancement in plasma bactericidal capacity was only recorded in fish fed Origanum essential oil at 12 and 24 h post challenge compared to those fed basal diet. In conclusion, Origanum essential oil had a pronounced influence on the innate immunity and increased the fish resistance to V. anguillarum. These data gave insight into the potential use of Origanum in prophylactic strategies against threatening pathogens.     

Correlation between C-reactive protein level,immunology, and hematology of a <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> infected with <Emphasis Type="Italic">Lactococcus garvieae</Emphasis>

The aim of this experiment was to evaluate the symptoms, variation, and correlation of hematological factors and some parameters of the innate immune response of the infected rainbow trout (Oncorhynchus mykiss) fish with Lactococcus garvieae bacteria. A total of 270 fish were divided into three groups including infected fish with a high and low concentration of L. garvieae and a control group without infection. The blood and tissue (brain, head, kidney, and spleen) samples were collected from each group (n?=?6 fish) at 0, 3, 14, and 21 days after treatment. The most observed symptoms during infection were lethargy, exophthalmia, and ascites. The mortality rates for the high and low dose-infected fish were defined as 60 and 25%, respectively. The infected groups had significantly (p?<?0.05) higher activity of serum lysozyme, myeloperoxidase, classical and alternative pathways of complement, serum bactericidal effects, and the specific antibody titer. Also, a significant (p?<?0.05) lower hematocrit and hemoglobin levels and higher white blood cell numbers were observed in the infected groups. A significant correlation was observed between the CRP levels and some of the hematological and immunological indices as bactericidal effects, classical complement pathways, lysozyme activity myeloperoxidase activity, white blood cell numbers, and hematocrit levels. The clinical symptoms, immune responses, and hematological indices variation among L. garvieae-infected fish are dependent on the duration and bacterial dose of the infection.     

Epigallocatechin-3-gallate (EGCG) affects the antioxidant and immune defense of the rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Epigallocatechin-3-gallate (EGCG), a very potent antioxidant derived from green tea, was compared with vitamin E in terms of its effects on antioxidant defense and immune response of rainbow trout, by means of a feeding trial of eight weeks. Two of the experimental diets were supplemented with EGCG at either 20 or 100 mg kg⁻¹ diet (which contained only 30% of the intended levels) and the third was provided with 100 mg kg⁻¹ vitamin E but not EGCG. The control diet was not supplemented with the test components. Observation of tissue levels indicated that the high amount of EGCG helped to increase the availability of the lipid-soluble antioxidant vitamin E. The lower levels of lipid hydroperoxide in the liver of fish fed the higher amount of EGCG suggested that it was an effective antioxidant. Considering the immune indices, EGCG and vitamin E at 100 mg (actual amounts 31.9 and 94.1 mg kg⁻¹ diet, respectively) had identical capabilities in improving phagocytic activity and controlling hydrogen peroxide production by leucocytes. However, EGCG could possibly be more effective at enhancing serum lysozyme activity and the alternative complement activity. This work revealed the potential of EGCG as an antioxidant and an immunostimulant for rainbow trout, at least at the inclusion level of 32 mg kg⁻¹ diet.     

Immunological effects of glucan and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> GG,a probiotic bacterium,on Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> intestine with oral <Emphasis Type="Italic">Aeromonas</Emphasis> challenges

The present study histologically examined the effects of glucan-containing and Lactobacillus rhamnosus GG (LGG)-containing diets on intestinal damages inflicted on Nile tilapia by Aeromonas challenges. Tilapia were fed control, glucan, and LGG diets for 2 weeks and were subsequently challenged with Aeromonas. The intestines were then histologically examined at 1, 7, 14, and 21 days post-infection. Mortality following the challenge was lower for the fish fed the glucan and LGG diets. The intestines of these groups also showed increased inflammatory cell infiltration and reduced intestinal damage from Aeromonas. Moreover, inflammatory cell infiltration occurred more rapidly in the glucan-fed than in the LGG-fed fish following the challenge. Before the challenge, the dominant mucous cell was the acid type in all the tests. After the challenge, the main mucus cell type in the proximal intestine of the glucan-fed fish shifted to AB-PAS double-staining cells, while in the LGG-fed fish, it remained the acid type throughout the test period, and the number of double-staining cells was smaller than in the control fish after the challenge. Thus, the different mucous cell and inflammatory cell responses show that glucan and LGG might have different immunostimulative effects, although they both reduced the intestinal damage following Aeromonas challenges.     

Non-specific immune potentiating activity of fucoidan from a tropical brown algae (Phaeophyceae), <Emphasis Type="Italic">Sargassum cristaefolium</Emphasis> in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

Fucoidan mainly produced by brown algae is a sulfated polysaccharide with fucose as a main molecule backbone. The objective of this research was to evaluate the non-specific immune potentiating activity of fucoidan from a tropical brown alga, Sargassum cristaefolium indigenous to the Gunungkidul coast, Yogyakarta, Indonesia. Fucoidan was extracted by acidic extraction, separated from alginate by CaCl₂ precipitation and precipitated with ethanol. To know the component of fucoidan, acid hydrolysis was conducted and analyzed by using silica gel thin layer chromatography. In these analyses, a commercial fucoidan from Fucus vesiculosus was used as a standard fucoidan. Fucoidan isolated from S. cristaefolium was composed by monosaccharide components of fucose and galactose. The result revealed that injection of fucoidan increased phagocytic activity, total plasma protein, leukocrit and leukocyte count significantly (p < 0.05). The immunomodulating activity of fucoidan from S. cristaefolium was comparable to that of fucoidan standard from F. vesiculosus. However, administration of fucoidan did not affect the phagocytic index and leukocyte differentiation. Injection of fucoidan from S. cristaefolium effectively increased innate immunity parameters of tilapia at doses of 0.4–0.6 mg/kg fish suggested that this fucoidan was potential use for immunostimulant to control fish diseases.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.     

Alterations in blood electrolytes of a freshwater catfish <Emphasis Type="Italic">Heteropneustes fossilis</Emphasis> in response to treatment with a botanical pesticide, <Emphasis Type="Italic">Nerium indicum</Emphasis> leaf extract

The present study aimed at investigating the effects of Nerium indicum leaf extract on the blood electrolytes of Heteropneustes fossilis for short- and long term. Fish were subjected to Nerium indicum leaf extract for short term (11.27 mg/L i.e. 0.8 of 96 h LC₅₀) and long term (2.81 mg/L i.e. 0.2 of 96 h LC₅₀). Fish were killed on each time intervals from control and experimental (Nerium indicum) groups after 24, 48, 72, and 96 h in short-term exposure and after 7, 14, 21, and 28 days in long-term experiment. Blood samples were analyzed for calcium and inorganic phosphate levels. Acute exposure of Nerium indicum leaf extract caused a progressive decrease in the serum calcium levels after 48 h in fish H. fossilis, which persists till the close of the experiment (96 h). The serum inorganic phosphate levels remain unaffected till 48 h in the Nerium indicum leaf extract–exposed fish. After 72 and 96 h, the levels exhibit a decrease. Chronic Nerium indicum leaf extract treatment provoked a decrease in serum calcium levels at day 14. This decrease continues till 28 days. The serum phosphate level of the Nerium indicum leaf extract–treated fish decreases on day 14 and 21. However, on day 28, the levels become close to the normal values. We conclude that Nerium indicum leaf extract exposure alters the blood electrolytes of the fish, thus causing physiological disturbances which might affect seriously the normal vital functions, growth rate, reproduction, and their survival in nature.     

Influence of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> on testicular enzyme in fresh water ornamental fish <Emphasis Type="Italic">Poecilia latipinna</Emphasis>

The influence of Tribulus terrestris on the activities of testicular enzyme in Poecilia latipinna was assessed in lieu of reproductive manipulation. Different concentrations of (100, 150, 200, 250, and 300 mg) Tribulus terrestris extract and of a control were tested for testicular activity of enzymes in Poecilia latipinna for 2 months. The testis and liver were homogenized separately in 0.1 mol/l potassium phosphate buffer (0.1 mol/l, pH 7.2). The crude homogenate was centrifuged, and supernatant obtained was used as an enzyme extract for determination of activities. The activities of testicular functional enzyme ALP, ACP, SDH, LDH, and G6PDH levels were changed to different extent in treated groups compared with that of the control. The total body weight and testis weight were increased with the Tribulus terrestris-treated fish (Poecilia latipinna). These results suggest that Tribulus terrestris induced the testicular enzyme activity that may aid in the male reproductive functions. It is discernible from the present study that Tribulus terrestris has the inducing effect on reproductive system of Poecilia latipinna.     

Adverse and beneficial effects of long-term high-concentration ascorbic acid supplementation in rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

The present study examined the effects of high-concentration ascorbic acid (AsA) supplementation over a long period on growth, plasma components, nonspecific immune responses, and thermal tolerance in rainbow trout Oncorhynchus mykiss. Four commercial diets supplemented with 0, 100, 1000, and 5000 mg AsA per kg of diet (designated AsA0, AsA100, AsA1000, and AsA5000, respectively) were fed to rainbow trout (initial weight 1.85 g) for 100 days. AsA contents in liver increased with increasing dietary AsA levels. Feeding period, growth performance, and plasma components did not differ significantly between the AsA groups. On the other hand, the phagocytic assay [nitro-blue tetrazolium (NBT) assay] was significantly higher in fish fed AsA1000 and AsA5000 than those fed a diet without AsA (AsA0) at the end of feeding trial. Superoxide dismutase (SOD) activity in the intestines of fish fed AsA5000 was also significantly higher than that of fish fed AsA0. In the thermal tolerance test, fish fed AsA5000 only showed significantly lower cumulative mortality compared with the AsA0 group. In conclusion, high-concentration AsA supplementation such as AsA5000 over a long period does not induce any adverse effects and can enhance disease and stress resistance in rainbow trout.     

Oral and parenteral vaccines against <Emphasis Type="Italic">Flavobacterium columnare</Emphasis>: evaluation of humoral immune response by ELISA and in vivo efficiency in Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

Flavobacterium columnare is a bacterial pathogen for many freshwater fish species. It is responsible for outbreaks in fish farms worldwide, causing high mortality rates. Fish vaccination is a potential approach for prevention and control of disease, with oral vaccines suitable for fish because of their easier application, low cost and minimum stress to fish. Alginate microparticles have been widely used as controlled release systems, including for fish vaccination. The aim of this study was to evaluate the capacity of oral and parenteral vaccines against F. columnare to induce a humoral response, as well as the in vivo efficiency in Nile tilapia fingerlings. The fingerlings were immunized with bacterin by intraperitoneal (i.p.), intramuscular (i.m.), oral and immersion routes, as well as orally with alginate microparticles containing formalin-killed bacteria. A sandwich ELISA was developed to detect specific antibodies against F. columnare. The animals were challenged with pathogenic strain BZ-1 to determine the relative percentage of survival. A significant humoral response was induced by bacterin administered by i.p. and i.m. routes (P < 0.05). However, none of the vaccine preparations were effective in protecting fish against F. columnare infection (P < 0.05). In spite of high antibody levels, there was no relation between immunoglobulin titers and resistance to columnaris for Nile tilapia fingerlings. These data suggest that use of serological analysis as the only method to determine vaccine efficiency against F. columnare infection in Nile tilapia can lead to imprecise results for the usefulness of these products in vivo.