Use of vitamin C as an immunostimulant. Effect on growth,nutritional quality,and immune response of <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

Teleost fish lack the enzyme for endogenous synthesis of ascorbic acid (AA), an essential micronutrient for fish. The aim of this study was to examine the effect of higher levels of dietary vitamin C on growth, nutritional quality, and immunomodulation in the Indian major carp, rohu (Labeo rohita). Four groups of L. rohita were fed experimental diets containing either no vitamin C (control) or supplemented with vitamin C at 500 mg kg⁻¹ (Exp-1), 1000 mg kg⁻¹ (Exp-2), or 1500 mg kg⁻¹ (Exp-3) for 60 days. Growth parameters (NWG, ADG, and SGR), serological parameters (TSP, TSA, TSG, and A:G), haematological parameters (TLC, TEC, Hct, MCV, and MCH), and different non-specific immunological parameters (PR, PI, respiratory burst activity, and bactericidal activity) were evaluated during the experimental trial. Fish fed a vitamin C-supplemented diet showed higher specific growth rate (SGR) up to 1000 mg kg⁻¹ compared with control fish. Different haematological and serological parameters along with non-specific immune parameters were influenced by vitamin C supplementation. Among the non-specific immune parameters phagocytic activity (PR and PI) and respiratory burst activity (NBT cells) were significantly (P ≤ 0.05) enhanced by increasing doses of vitamin C supplementation. Higher levels of dietary vitamin C significantly (P ≤ 0.05) enhanced protection against Aeromonas hydrophila (AH1) infection compared with controls. Results from this study help to establish the beneficial effect of vitamin C on growth and immunmodulation in rohu (L. rohita).     

Requirements of vitamin C (L-ascorbyl-2-monophosphate-Mg and L-ascorbyl-2-monophosphate-Na) and its effects on immune responses of grouper, Epinephelus malabaricus

An 8‐week feeding trial was conducted to evaluate two vitamin C derivatives, L‐ascorbyl‐2‐monophosphate‐Mg (C2MP‐Mg) and L‐ascorbyl‐2‐monophosphate‐Na (C2MP‐Na), to satisfy the vitamin C requirement and to test their effects on the immune responses of juvenile grouper, Epinephelus malabaricus. C2MP‐Mg and C2MP‐Na were each supplemented at 20, 50, 80, 150, 250, and 400 mg kg^?1 diet in the basal diet providing of 7, 18, 31, 51, 93, 145 mg ascorbic acid (AA) equivalent of C2MP‐Mg kg^?1 diet and 4, 10, 17, 31, 47, 77 mg ascorbic acid (AA) equivalent of C2MP‐Na kg^?1 diet, respectively. Basal diet without AA supplementation was included as control. Each diet was fed to triplicate groups of grouper (mean initial weight 3.20 ± 0.05 g). Fish fed diets supplemented with either C2MP‐Mg or C2MP‐Na had significantly (P < 0.05) greater weight gain (WG), feed efficiency and survival than those fed the unsupplemented control diet. Liver ascorbate concentrations in fish generally increased as dietary C2MP‐Mg or C2MP‐Na supplementation level increased. Haemolytic complement activity was higher in fish fed diets supplemented with 92 mg AA equivalent of C2MP‐Mg kg^?1 or 10–17 mg AA equivalent of C2MP‐Na kg^?1 than fish fed the unsupplemented control diet. Lysozyme activity was higher in fish fed ≥51 mg AA equivalent of C2MP‐Mg kg^?1 or ≥47 mg AA equivalent of C2MP‐Na kg^?1 than fish fed the unsupplemented control diet. Analysis by broken‐line regression of WG indicated that the adequate dietary vitamin C concentration from each vitamin C derivative in growing grouper is 17.9 mg AA equivalent of 2MP‐Mg kg^?1 and 8.3 mg AA equivalent of C2MP‐Na kg^?1, and it also indicated that C2MP‐Mg is about 46% as effective as C2MP‐Na in meeting the vitamin C requirement of grouper.     

Assessment of vitamin C different levels in aquaponic system on Nile tilapia (Oreochromis niloticus) and saffron (Crocus sativus): growth performances,hematology, serum biochemistry,and digestive enzyme activity

The aim of this study was to investigate the possibility of improving the growth and physiological indices of plant and fish by adding different levels of ascorbic acid (vitamin C) to water in the aquaponic system using Nile tilapia (Oreochromis niloticus) and saffron plant (Crocus sativus). 240# fish (12.5?±?0.21 gr) and 120# saffron corms (2.8?±?0.12 gr) were randomly assigned to 15 experimental units and underwent treatments of adding 0 (control), 2, 4, 6, and 8 mg L^?1 ascorbic acid to water, every 6 days, for 8 weeks. The fish final weight and subsequently other growth performance indices increased in the treatment of 4 mg L^?1 vitamin C compared to other treatments (P?≤?0.05). Some growth performances of saffron plants such as saffron production, in treatment of 6 mg L^?1, were significantly higher than the control group and reached from 17.34?±?0.27 mg flower^?1 in the control group to 25.4?±?1.61 mg flower^?1 in treatment of 6 mg L^?1. Measuring the serum biochemical parameters of the fish showed that, in the treatment of 8 mg/L ascorbic acid, the cortisol content in the blood reached its maximum (21.49?±?2.42 µg dL^?1). The trypsin activity in proximal intestine and mid-intestine significantly increased in treatments of 2, 4, and 6 mg L^?1, respectively. The current experiment showed that, by adding 4–6 mg/L ascorbic acid to the aquaponic system water (every 6 days), the optimal levels of Nile tilapia and saffron plant growth performances would occur.

     

Bioavailability and interactions with other micronutrients of three dietary iron sources in Atlantic salmon, Salmo salar, smolts

Atlantic salmon, Salmo salar L., with mean initial weight of 60 g were fed a diet based on cod muscle meal supplemented with elemental iron, iron sulphate or haem-bound iron in concentrations of 0, 25, 50, 100, 500 and 1500 mg iron kg^?1 for 8 weeks. No significant differences in growth or mortality were found, except in fish fed 1500 mg haem iron kg^?1, which showed reduced growth. In fish fed diets supplemented with elemental iron below 1500 mg iron kg^?1, blood haemoglobin and hepatic iron concentration decreased compared with fish fed the unsupplemented diet. Fish fed diets supplemented with iron sulphate showed increased blood haemoglobin and hepatic iron concentrations between 25 and 100 mg iron kg^?1. Fish fed diets supplemented with haem-bound iron showed increased hepatic iron at all dietary iron levels, while blood haemoglobin concentration decreased in the group fed 1500 mg haem iron kg^?1. The bioavailability of haem iron relative to sulphate iron was calculated by the slope ratio method to be 239% and 148% using blood haemoglobin and hepatic iron, respectively. Relative bioavailability of elemental iron was zero when dietary supplementation levels were between 25 and 500 mg iron kg^?1, while a small part was utilized when 1500 mg elemental iron kg^?1 was supplemented. Additions of 500 and 1500 mg haem-bound iron kg^?1 resulted in a complete loss of ascorbic acid in these diets. When these groups were discounted, no significant relationship between hepatic iron and hepatic ascorbic acid was found. There was no significant effect of dietary iron on whole-body manganese concentration and only a weak effect on whole-body zinc concentration. No significant correlations between dietary iron and hepatic copper concentration were found in any of the dietary treatments. This study also showed that the level of inorganic iron supplementation may be reduced by inclusion of 20 g blood meal kg^?1 in the diet.     

Administration of gulonolactone does not evoke ascorbic acid synthesis in teleost fish

Evidence is presented that for common carp (Cyprinus carpio L.), intraperitoneal injection of L-gulonolactone, a precursor of ascorbic acid synthesis in the D-glucuronic acid pathway, does not result in an increased concentration of ascorbate in tissue. Control fish injected with an equimolar amount of ascorbic acid have shown a significant increase in ascorbic acid concentration in the kidney, hepatopancreas, plasma and spleen. The ascorbate status in the carp body,i.e., the ascorbate nutritional history, produced significant differences in ascorbate withdrawal from circulation and probably in the catabolic rate. Acute fasting decreased ascorbate uptake into tissues as compared to fish fed a diet lacking ascorbate. Intraperitoneally injected ascorbate affects common carp being fed a diet containing 295 mg of total ascorbic acid kg^–1 by causing tissue to become saturated with vitamin C, similar to the tissues in the group undergoing acute fasting. There was no gulonolactone oxidase activity in the hepatopancreas of the common carp. These results suggest that the metabolic rate induced by feeding is the primary factor regulating ascorbate requirement.     

Effects of cyclic and regular feeding of a Quillaja saponin supplemented diet on growth and metabolism of common carp (Cyprinus carpio L.)

Fifteen common carp weighing 19.2±2.9 g were reared individually in chambers of a respirometer system which allowed feeding, and continuous measurement of oxygen consumption. The fish were divided into three groups of 5 each, and were given control feed (C group), control feed supplemented with 150 mg kg⁻¹ of Quillaja saponins (QS) (S150 group), and control and the saponin supplemented diet during alternate weeks (S150b group), for 56 days. Supplementation with QS at a level of 150 mg kg⁻¹ caused a significant (P<0.05) increase in metabolic growth rate, food conversion efficiency (FCE), protein productive value (PPV) and apparent lipid conversion (ALC) of carp, even as the metabolic rate was lower than the C group. The efficiency of energy utilisation indicated by parameters such as energy expenditure (EE), energy retention (ER) and amount of oxygen consumed per unit body mass gain (OPM) were also significantly (P<0.05) better in the S150 group. The performance of the S150b group was intermediate between the C and S150 group in this experiment. Possible reasons for the effects of dietary QS are discussed. It was concluded that there has to be continuous dietary supply of QS at the level of 150 mg kg⁻¹, for maximum positive effects of these substances on growth of common carp. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of dietary vitamin C on growth and parr-smolt transformation in Atlantic salmon, Salmo salar L.

Atlantic salmon (Salmo salar) parr were fed fish meal based pelleted diets supplemented with graded levels of ascorbate-2-monophosphate (AP), equivalent to 0, 20, 60 and 1000 mg ascorbic acid (AA) kg”¹ throughout smoltification on a continuous light regime from February to June. No differences were observed in growth rate and body length distribution between the dietary regimes. The condition factor and the hepatosomatic index were somewhat elevated in fish fed no vitamin C throughout the smoltification, which can reflect changes in lipid metabolism in fish with suboptimal vitamin C nutrition. Sea water challenge tests (exposure to sea water with salinity of 34 gL^?1 and ambient temperature for 24 h) performed monthly did not reveal differences attributed to the vitamin C status, as measured by mortality, serum chloride and cortisol concentrations, haematological parameters and liver and head kidney ascorbate concentrations after 24 h. Elevated serum cortisol concentrations most probably reflected stress in the challenge tests, and some lower concentrations in fish fed high vitamin C levels may indicate a certain stress-ameliorating effect. The present results do not, however, support the anticipation of increased requirement of dietary vitamin C above the minimum requirement during smoltification in Atlantic salmon.     

Effect of ascorbic acid supplementation on zootechnical performance,haematological parameters and sperm quality of lebranche mullet Mugil liza

The feed provided to breeding fish is one of the main factors influencing the quality of fish gametes. This study was carried out to evaluate the influence of ascorbic acid on growth, haematological parameters and sperm quality of Lebranche mullet males (Mugil liza). Six diets with different levels of ascorbic acid (0; 53; 107; 216; 482 and 708 mg/kg) were tested in triplicate for 75 days. During spermiation (first gonadal maturation), 144 individuals (205.7 ± 11.5 g and 25.7 ± 0.4 cm) were randomly distributed in 18 experimental tanks. Growth parameters were evaluated at the beginning and end of the experiment. Fish blood was collected to analyse glucose, total protein and erythrocyte count (EC) (n = 9). Fish (n = 12) from each treatment were euthanized to determine hepatosomatic index (HSI) and gonadosomatic index (GSI). Semen was collected to evaluate spermatic density, cell membrane integrity and sperm motility. No difference (p > .05) was found on growth parameters, GSI, HSI and total protein. However, EC was lower in fish fed without ascorbic acid (the control group). Ascorbic acid supplementation provided positive effects on sperm characteristics. Fish from treatments with 53 and 107 mg/kg presented the best results for motility time (133.30 ± 4.25 and 135.00 ± 2.77 respectively). Treatments with 107, 216 and 708 mg/kg provided the best results for motility rate (92.0 ± 2.9%, 93.0 ± 5.8% and 93.0 ± 5.8% respectively). Supplementation with 107 and 216 mg/kg provided the best results for plasma membrane integrity (70.12 ± 9.10% and 76.3 ± 3.1% respectively). Lower spermatic density was observed in fish without ascorbic acid supplementation, although no difference was found in sperm density among the treatments with ascorbic acid (p < .05). Considering these results, supplementation of dietary ascorbic acid between 107 and 216 mg/kg optimizes the spermatic quality in males of lebranche mullet.     

Fatty acid,fat‐soluble vitamin and sterol contents in testis and semen,testis‐somatic index and spermatologic values of broodstock rainbow trout (Oncorhynchus mykiss) fed with different levels of omega‐3 fatty acids under regular stripping conditions

In this study, the testis and semen fatty acid, fat‐soluble vitamin and sterol levels, testis‐somatic index and spermatological values were investigated in the broodstock rainbow trout fed with the different levels of omega‐3 polyunsaturated fatty acid (PUFA) under the regular stripping conditions. For this purpose, a control and three test diets were prepared. The control, D1, D2 and D3 diets were contained the omega‐3 PUFA at 3.14%, 7.84%, 13.63% and 20.54% as a percentage of the total fatty acids respectively. The fish in the experimental groups were fed with these diets for 60 days. The testis‐somatic index (3.81–4.50), semen volume (9.30–18.5 ml) and pH (8.05–8.99), initial sperm motility (68.1%–93.7%), duration of 50% sperm motility (141.3–258.9 s), total duration of sperm motility (268.7–489.6 s) and sperm density (9.95–17.5 cells × 10⁹ ml^?1) values of the fish fed the control diet were significantly decrease (p < 0.05) depending on decline of the omega‐3 PUFA, fat‐soluble vitamin and sterol contents in the testis and semen. The omega‐3 PUFA (3.40%–14.76% and 3.96%–16.31%), vitamin A (33.9–61.2 ml/mg and 9.12–26.7 ml/mg), D₂ (0.10–0.19 ml/mg and 0.04–0.10 ml/mg), D₃ (0.75–1.30 ml/mg and 0.06–0.24 ml/mg), alpha‐tocopherol (30.1–60.4 ml/mg and 8.93–25.3 ml/mg), K₁ (0.30–0.94 ml/mg and 0.03–0.08 ml/mg) and K₂ (2.0–3.19 ml/mg and 0.25–0.34 ml/mg) vitamins, cholesterol (1,201–1,588 ml/mg and 330–596 ml/mg), ergosterol (3.12–6.40 ml/mg and 2.10–4.08 ml/mg), stigmasterol (5.57–10.4 ml/mg and 10.2–12.6 ml/mg) and beta‐sitosterol (0.20–0.60 ml/mg and 0.14–0.41 ml/mg) contents in the testis and semen of the fish were significantly affected (p < 0.05) by the dietary omega‐3 PUFA supplementation. The highest values for these parameters were provided from the fish fed the D2 and D3 diets (p > 0.05), followed by the fish fed the D1 diet (p < 0.05). These results showed that the dietary omega‐3 PUFA at 13.63% of the total fatty acids could increase the omega‐3 PUFA, fat‐soluble vitamin and sterol contents in the testis and semen, the testis‐somatic index and spermatologic values of the broodstock rainbow trout under the regular stripping conditions.     

Characterization of the testicular semen of the African catfish, Clarias gariepinus (Burchell, 1822), and its short-term storage

Semen of the African catfish, Clarias gariepinus (Burchell, 1822), was investigated with respect to its cellular composition, sperm cell density, maturation grade, motility and fertility. Storage conditions were tested, whereby sperm viability was assessed by measurement of the motility after activation and by fertility tests. Testicular semen differed in its composition, i.e. the sperm density and numbers of spermatids, according to the maturity grade of the testis. Two semen types could be distinguished: semen type I was characterized by high sperm densities and low numbers of spermatids and semen type II had lower sperm densities and higher numbers of spermatids. Two semen types did not differ in motility and fertility (when adjusted for differences in sperm density). During storage, the sperm viability was influenced by the sodium concentration of the storage medium, temperature, membrane stabilizers as bovine serum albumen (BSA) or hen egg yolk, antibiotics and oxygen. Semen viability was maintained best when it was diluted at a ratio of 1:5 in storage solution (150 mmol L^?1 NaCl, 2.5 mmol L^?1 KCl, 1 mmol L^?1 CaCl₂, 1 mmol L^?1 MgSO₄, 20 mmol L^?1 Tris (pH 8.5) and 0.5% BSA or 0.5% hen egg yolk) and stored at 4 °C. Oxygen gassing and addition of antibiotics (1 mg mL^?1 gentamycine sulphate) to the storage solution affected the two semen types in different ways. Antibiotics had no effect on type I semen, but had a positive effect on type II semen. Oxygen gassing had a positive effect on type I semen but a negative effect on type II semen.     

Determination of spermatological properties of male Liza abu (Heckel, 1843) in Atatürk Dam Lake, Şanlıurfa

The aim of this study was to determine the spermatological characteristics in male L. abu during the spawning season. Semen was collected weekly by abdominal massage from 26 males in March. In collected semen, volume, motility, duration of motility, concentration and pH were determined. In the L. abu sperm, volume (μl), motility (%), duration of motility (s), concentration (×10⁹/ml), and pH values were found 45.76 ± 3.55, 54.25 ± 2.93, 330.15 ± 37.92, 4.27 ± 0.40 and 7.87 ± 0.05, respectively. A correlation was found between semen volume and semen pH. Semen volume and the duration of sperm motility were higher in the 2nd and 3rd sampling dates than in the 1st and 4th sampling dates (P < 0.05; P < 0.01, respectively). Neither sperm motility nor sperm concentration was affected by sampling dates. Major changes in semen pH were observed in the 4th sampling date (P < 0.001). The Pearson correlation test presented significant relationships with the duration of motility, semen volume, and motility. Semen pH values were significantly correlated with the sperm concentration and semen volume. Sperm concentration was inversely correlated with semen volume. Sperm motility and duration significantly correlated with total weight. Total length significantly correlated with the duration of motility and total weight. In conclusion, these characteristics represent a valuable baseline dataset for establishing a semen quality standard and provide background information that may be useful for assisted breeding programs in this species.     

Requirements of vitamin C (l-ascorbyl-2-sulphate and l-ascorbyl-2-polyphosphate) and its effects on non-specific immune responses of grouper, Epinephelus malabaricus

An 8‐week feeding trial was conducted to determine two vitamin C derivatives, l ‐ascorbyl‐2‐sulphate (C2S) and l ‐ascorbyl‐2‐polyphosphate (C2PP), to satisfy vitamin C requirement and to test their effects on the non‐specific humoral immune responses of juvenile grouper, Epinephelus malabaricus. C2S and C2PP were each supplemented at 20, 50, 80, 150, 250 and 400 mg kg^?1 diet in the semi‐purified basal diet providing of 7, 16, 28, 55, 86, 142 mg ascorbic acid (AA) equivalent of C2S kg^?1 diet and 4, 9, 15, 31, 49, 75 mg AA equivalent of C2PP kg^?1 diet, respectively. Basal diet without AA supplemented was included as a control. Each diet was fed to triplicate groups of grouper (mean initial weight: 6.69 ± 0.07 g). Fish fed diets with ≥28 mg AA equivalent of C2S or ≥4 mg AA equivalent of C2PP kg^?1 had significantly (P < 0.05) greater weight gain (WG) than fish fed the unsupplemented control diet. Liver AA concentrations were higher in fish fed diets with ≥16 mg AA equivalent of C2S or ≥9 mg AA equivalent of C2PP kg^?1 than fish fed the control diet. Alternative pathway of complement activation (ACP) was higher in fish fed diets with ≥55 mg AA equivalent of C2S or ≥15 mg AA equivalent of C2PP kg^?1 than fish fed the control diet. Lysozyme activity was higher in fish fed ≥86 mg AA equivalent of C2S or ≥15 mg AA equivalent of C2PP kg^?1 than fish fed the control diet. Analysis by broken‐line regression of WG indicated that the adequate dietary vitamin C concentration from each vitamin C derivative in growing grouper is 46.2 mg AA equivalent of C2S kg^?1 diet and 17.8 mg AA equivalent of C2PP kg^?1 diet, and it also indicated that C2S is approximately 39% as effective as C2PP in meeting the vitamin C requirement for grouper. The data suggest that both C2S and C2PP supplementation support non‐specific immune responses of grouper.     

Effects of dietary vitamin C on survival, growth, and immunity of large yellow croaker, Pseudosciaena crocea

An 8 weeks growth experiment was conducted to determine the effects of dietary vitamin C on the survival, growth, tissue ascorbic acid contents and immunity of large yellow croaker (Pseudosciaena crocea) with initial weight of 17.82 ± 0.68 g. Seven practical diets were formulated to contain 0.1, 12.2, 23.8, 47.6, 89.7, 188.5 and 489.0 mg ascorbic acid equivalent kg^− 1 diet, supplied as l-ascorbyl-2-polyphosphate (LAPP). Each diet was fed to triplicate groups of fish in seawater floating cages (1.5 × 1.5 × 2.0 m), and each cage was stocked with 120 fish. Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 8 weeks. The water temperature fluctuated from 19.5 to 25.5 °C, the salinity from 25 to 28‰ and dissolved oxygen content was approximately 7 mg l^− 1 during the experimental period. Results showed that the specific growth rate (SGR) (from 1.80 to 1.96% d^− 1) had an increasing trend with the increase of dietary vitamin C, but no significant difference was observed among dietary treatments. No gross deficiency signs were observed in any of the experimental fish. Survival rate, however, significantly increased with increasing dietary vitamin C (P < 0.05). The vitamin C contents in liver and muscle correlated positively with the vitamin C in diets. The vitamin C requirement was estimated to be 28.2 mg kg^− 1 based on survival, and 87.0 mg kg^− 1 on liver content of vitamin C. The activities of serum lysozyme and alternative complement pathway (ACP), phagocytosis percentage (PP) and respiratory burst activity of head kidney significantly increased with increasing dietary vitamin C. The challenge experiment with Vibrio harveyi showed that fish fed the diets with supplementation of vitamin C had significantly lower cumulative mortality compared to the control group (66.7%), and the cumulative mortality (16.7%) in fish with 489.0 mg kg^− 1 ascorbic acid was significantly lower than that (41.7%) in fish with 23.8 mg kg^− 1 ascorbic acid. These results suggested that vitamin C significantly influenced the immune response and disease resistance of large yellow croaker.     

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.     

Sperm motility and fertilizing ability in the Persian sturgeon Acipenser persicus

The motility and fertilizing ability of the Persian sturgeon, Acipenser persicus, spermatozoa were investigated. Optimum ionic content (Na⁺, K⁺, Ca²⁺ and Mg²⁺) and pH of activation solution as well as the optimum dilution rate were determined. The results show optimum motility characteristics of spermatozoa in buffered solutions containing 25, 0.2, 3 and 10 mM L^?1 Na⁺, K⁺, Ca²⁺ and Mg²⁺, respectively, at dilution rate 1:50 and pH 8.0. To test the fertilizing ability of sperm, two buffered saline solutions were used as activation solution of sperm motility. The present study indicated (1) spermatozoa motility is one of key factors that influence on fertilizing ability of sperm, (2) a high fertilizing ability of sperm is obtained after dilution in saline solutions rather than in freshwater and (3) a maximum fertilization rate occurs in buffered saline solution containing 0.2 mM L^?1 K⁺. There is also a good correlation between biochemical characteristics of seminal plasma and fertilizing ability of sperm.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Quantifying the dietary biotin requirement of the catfish,Clarias batrachus

Asian catfish, Clarias batrachus, were fed semi-purified basaldiets containing 0, 0.1, 0.5, 1, 3 and 5 mg biotin kg^–1diet for 60 days. Fish fed the control diet (no biotin) showed(P < 0.05) higher mortality, lower weight gain, specificgrowth rate (SGR), feed efficiency ratio (FER) and protein efficiencyratio (PER) than in fish fed diets supplemented with biotin. The highestweight gain, SGR, FER and PER were noticed in fish fed 1 mg biotinkg^–1, followed by 0.5, 5, 3 and 0.1 mg biotinkg^–1, except for PER (followed by 0.5, 5, 0.1 and 3 mgbiotin kg^–1). Quadratic analysis showed that the optimumdietary biotin requirements for maximal weight gain, PER and PER were2.49, 2.54 and 2.52 mg kg^–1, respectively. Liver biotinconcentrations were influenced by levels of biotin in the diet.Concentration of liver biotin increased as level of dietarysupplementation increased and no biotin was detected in the liver of thecontrol fish. Liver pyruvate carboxylase and acetyl CoA carboxylaseactivities were higher in fish fed biotin-supplemented diets than incontrols. Biotin concentrations, pyruvate carboxylase and acetyl CoAcarboxylase activities in liver associated with normal growth rangedfrom 10.59 to 10.66 g g^–1, 147.97 to 148.18 units mgprotein^–1 and 12.76 to 12.78 units mg protein^–1, respectively. Biotin deficiency symptoms such as anorexia, darkskin colour and convulsions were observed in fish fed the control diet.The optimum dietary biotin requirement for maximal growth of C.batrachus is about 2.49 mg kg^–1 diet.     

Effects of dietary vitamin C on growth,gonad development and antioxidant ability of on‐growing gibel carp (Carassius auratus gibelio var. CAS III)

An 88‐day experiment was conducted in a flowing system to evaluate the effects of dietary vitamin C on growth, body composition, antioxidant and gonad development of on‐growing gibel carp. Triplicate tanks of gibel carp (77.2 ± 0.1 g) were randomly fed with one of seven experimental diets containing l ‐ascorbic acid of 0, 101.1, 188.5, 313.1, 444.1, 582.1 and 747.0 mg/kg, respectively. The results showed that specific growth rate (SGR) and feed efficiency (FE) of fish were not affected by dietary l ‐ascorbic acid. Dietary l ‐ascorbic acid of 444.1 mg/kg diet led to low levels (p < .05) of gonadosomatic index (GSI) and hypothalamic gonadotropin‐releasing hormone (GnRH) in on‐growing fish, as well as the early ovarian stages (Developing stages) compared with fish (Maturing stages) from the other groups. Dietary l ‐ascorbic acid supplementation increased (p < .05) the dorsal muscle collagen content, but did not affect the protein or lipid content of dorsal muscle in gibel carp. Plasma total antioxidant capacity (T‐AOC) and superoxide dismutase (SOD) activity increased (p < .05) and then remained unchanged with the increase in dietary l ‐ascorbic acid levels. Dietary l ‐ascorbic acid of 101.1 mg/kg diet improved (p < .05) plasma lysozyme activity of the fish. Broken‐line regression indicated that dietary l ‐ascorbic acid requirement of 77 g gibel carp was 223.3 or 225.0 mg/kg diet based on plasma T‐AOC or SOD activity, which was a little higher than that based on plasma l ‐ascorbic acid concentration (193.2 mg/kg).     

The effect of vitamin C on stress withstanding capability in the juvenile soft-shelled turtle (Pelodiscus sinensis)

The study was conducted to determine the effect of dietary supplementation of vitamin C on anti‐acid stress ability in juvenile soft‐shelled turtle. The soft‐shelled turtles were fed vitamin C supplemented diets at levels of 0, 250, 500, 2500, 5000 and 10 000 mg kg^?1 for 4 weeks. The results showed that the phagocytic rate of blood cell in the group fed vitamin C deficient diets, the serum bacteriolytic activity in the two groups fed vitamin C supplemented diet at 0 and 250 mg kg^?1 and the serum bactericidal activity in all groups after acid stress significantly decreased compared with those of before stress (P < 0.1). The phagocytic rate of blood cells in the two groups fed vitamin C supplemented diet at 2500 and 5000 mg kg^?1 were significantly higher than those of the four groups fed at 0, 250, 500 and 10 000 mg kg^?1 (P < 0.01). The serum bacteriolytic activity in the groups fed vitamin C supplemented diet at 500, 2500, 5000 and 10 000 mg kg^?1 were significantly higher than that of the group fed vitamin C deficient diets (P < 0.05), and the two groups fed vitamin C at 2500 and 5000 mg kg^?1 were significantly higher than those of the groups fed at 250 and 10 000 mg kg^?1 (P < 0.05). The serum bactericidal activities in the four groups fed vitamin C supplemented diet at 500, 2500, 5000 and 10 000 mg kg^?1 were significantly higher than those of the two groups fed at 0 and 250 mg kg^?1 diet (P < 0.01), and the two groups fed vitamin C at 2500 and 5000 mg kg^?1 were significantly higher than those of the two groups fed at 500 and 10 000 mg kg^?1 (P < 0.1). These results suggest that supplementation of vitamin C higher than 250 mg kg^?1 is necessary to reduce the adverse effects of acid stress.     

The effect of synthetic and natural pigments on the colour of the cichlids (<Emphasis Type="Italic">Cichlasoma severum</Emphasis> sp., Heckel 1840)

In this study, we have investigated the effects of Porphyridium cruentum (Rodophyta) as a natural pigment source and astaxanthin and β-carotene as synthetic pigment sources on the skin colour of cichlid fish (Cichlasoma severum sp., Heckel 1840), which are generally light orange with white patches and becomes shiny orange in the reproductive phase. The fish were fed diets containing 50 mg kg⁻¹ astaxanthin and β-carotene, and P. cruentum powder. The amount of both natural and synthetic pigment sources given as feed was 50 mg kg⁻¹, and the experiment was continued for 50 days. Total carotenoid content of the fish was determined spectrophotometrically at the end of the experiment. As a result, while a visible change of colour in the skin of the fish fed on the feed containing astaxanthin was observed with 0.34 ± 0.2 mg g⁻¹ of pigment accumulation, a relatively small change of colour was observed in the skin of other fish that were fed on the feed containing P. cruentum and β-carotene with 0.22 ± 0.2 mg g⁻¹ and 0.26 ± 0.1 mg g⁻¹ of pigment accumulations, respectively. Therefore, it was determined that these pigment sources have an effect on the colour of cichlid fish.