Olfactory nerve response of masu salmon (Oncorhynchus masou Brevoort) and rainbow trout (O. mykiss Walbaum) to clove oil and MS-222

Two anaesthetics, clove oil and methane sulphonate (MS-222), were examined for their effects on the olfactory nerve response of masu salmon ( Oncorhynchus masou Brevoort) and rainbow trout ( O. mykiss Walbaum). Exposing both species to clove oil for 3 min at concentrations of 50 and 100 mg L⁻¹, or for 10 min at 50 mg L⁻¹, did not significantly reduce their olfactory response. Directly applying clove oil anaesthesia to the olfactory epithelium significantly reduced olfactory response though after 20 min, olfactory response recovered to 70% and 52% of pre-treatment levels in masu salmon and rainbow trout respectively. Compared with the post-anaesthetic recovery of responses after clove oil (50 mg L⁻¹), buffered MS-222 (100 mg L⁻¹) with NaHCO₃ (100 mg L⁻¹), and unbuffered MS-222 (100 mg L⁻¹) treatment for 3 min, the response after MS-222 treatment declined gradually and significantly, but not after clove oil and MS-222+NaHCO₃ treatments. Clove oil appears to be an effective and relatively safe anaesthetic for salmonids with little long-term impact on their olfactory response, which plays a crucial role in their life history.     

Anaesthetic properties of Propiscin (Etomidaat) and 2-phenoxyethanol in the common carp (Cyprinus carpio L.), neural and behavioural measures

The objective of the study was to evaluate the anaesthetic effects of Propiscin (2% etomidate) and 2-phenoxyethanol on common carp ( Cyprinus carpio L.) by observing neural and behavioural measures. When exposed to the anaesthetic agent, the carp lost equilibrium after approximately 90 s of exposure. They did not breathe and the controls did not respond to tail pinch or prick or 6 V at the skin of the upper jaw. A shift to lower frequencies on the electroencephalogram (EEG) and computing fast Fourier transformation was observed when exposed to water containing Propiscin or 2-phenoxyethanol. Heart rate was reduced after placement in the water containing an anaesthetic agent. It may be concluded from our results that common carp were immobilized and sedated, when exposed to water containing 2 mL L⁻¹ Propiscin and immobilized and anaesthetized in water containing 0.5 mL L⁻¹ 2-phenoxyethanol. In addition, the use of EEG provides for a sound assessment of exposure of carp to these anaesthetics.     

Efficacy of tricaine methanesulphonate and clove oil as anaesthetics for juvenile cobia Rachycentron canadum

Six experiments were designed to determine the optimal anaesthetic dosage of tricaine methanesulphonate (TMS) and clove oil that could be used safely on juvenile cobia Rachycentron canadum of two sizes [G1=4.9±0.8 g; G2=13.9±3.1 g]. We documented the stage of anaesthesia and the acute toxicity as 96 h LC₅₀ (lethal concentration 50% population) at various exposure times of the two anaesthetics. At 10 min induction time, the TMS 96 h LC₅₀ was 93.9 mg L⁻¹ in G1 and 97.0 mg L⁻¹ in G2. Compared with clove oil, the 96 h LC₅₀ was 60.0 mg L⁻¹ in G1 and 69.8 mg L⁻¹ in G2. The difference between the two groups (G1, G2) did not influence anaesthesia safety ( P >0.05). Rachycentron canadum achieved stage 3 anaesthesia more rapidly at a lower clove oil concentration level (40 mg L⁻¹, 10 min) than TMS (60 mg L⁻¹, 10 min), but the recovery period of clove oil, was significantly longer. Clove oil was the most effective in reducing the short-term stress induced by routine biometry (20 mg L⁻¹, 10 min) and also by transporting (1 mg L⁻¹, 8 h). Whereas, for long-term exposure, 40 mg L⁻¹ TMS was found to be safe.     

Anaesthetic efficacy and physiological responses to clove oil-anaesthetized kelp grouper Epinephelus bruneus

The efficacy of clove oil as an anaesthetic and at producing a physiological response (plasma cortisol and glucose) was evaluated in the kelp grouper, Epinephelus bruneus . To acquire complete anaesthesia in less than 3 min and recovery in <10 min, three doses of clove oil were tested at 18, 22 and 26 °C. Although higher anaesthetic doses resulted in shorter induction times and longer recovery times, and a lower temperature resulted in longer anaesthesia induction and slower recovery, we found the optimal dose and administering temperature of clove oil to be 250–300 mg L⁻¹ at water temperature of 18 °C, 150–200 mg L⁻¹ at water temperature of 22 °C and 50–100 mg L⁻¹ at water temperature of 26 °C respectively. Following the administration of 150 mg L⁻¹ of clove oil at 22 °C, the plasma cortisol level was highest (4.24 ± 1.571 μg dL⁻¹) after 12 h and the plasma glucose was highest (92.7 ± 9.61 mg dL⁻¹) after 2 h. These results should be useful to the aquaculture industry, where anaesthesia is necessary for a range of activities.     

Use of salt to control ichthyophthiriosis and prevent saprolegniosis in silver perch, Bidyanus bidyanus

The ciliate protozoan, Ichthyophthirius multifiliis , and the fungus, Saprolegnia parasitica , cause the diseases ichthyophthiriosis and saprolegniosis respectively. Both diseases are difficult to control and can cause high mortalities of freshwater fish, including the Australian silver perch, Bidyanus bidyanus (Mitchell). The efficacy of salt (NaCl) in controlling and preventing these diseases in silver perch was evaluated in aquaria and tanks. Low pH levels were also evaluated as a control for ichthyophthiriosis. Concentrations of 2 or 3 g L⁻¹ salt controlled infestations of I. multifiliis , and fish were free of both theronts and trophonts by day 8 at temperatures of 17.3–21.3 °C and by day 6 at 19.2–23.5 °C. Fish treated with 1 g L⁻¹ salt remained infested and all fish in a control treatment (0 g L⁻¹ salt) died. Although the mean survival rates of infested fish at pH levels of 5 or 6 were only 13.9% and 7.6%, respectively, there were no theronts or trophonts on surviving fish after 12 days. Silver perch harvested from a pond and treated with 2 or 3 g L⁻¹ salt did not become infected with S. parasitica and survival was 100%, whereas 16.6% of untreated (0 g L⁻¹ salt) fish became infected and survival was only 66.7%. A concentration of 2 g L⁻¹ NaCl is recommended for the control of ichthyophthiriosis and the prevention of saprolegniosis in silver perch held in tanks, aquaria and re-circulating aquaculture systems.     

The ability of clove oil and MS-222 to minimize handling stress in rainbow trout (Oncorhynchus mykiss Walbaum)

The effects of 60‐mg L^?1 clove oil and 60‐mg L^?1 tricaine methanesulphonate (MS‐222) on the blood chemistry of rainbow trout were compared after exposure to handling stress via caudal puncture blood sampling. Fish sampled by caudal puncture and subsequently exposed to anaesthetics showed a typical handling stress response over a 48‐h period. There were no significant differences between the responses of fish exposed to equal concentrations of clove oil and MS‐222, with the following exceptions: the blood glucose at full anaesthesia, and lactate at full recovery increased significantly in the clove oil‐exposed fish. In a subsequent experiment, the stress response observed in fish sampled by caudal puncture and exposed to clove oil and MS‐222 was compared with a non‐anaesthetized control group. The increases in plasma cortisol levels were significantly lower at recovery in fish treated with either anaesthetic compared with the control fish. Fish exposed to MS‐222 had significantly higher cortisol levels at 1 h. These findings show that few differences exist between the anaesthetic effects of clove oil and MS‐222 on the physiological response of fish to stress. However, clove oil is more effective at reducing the short‐term stress response induced by handling and blood sampling, and is recommended as an alternative fish anaesthetic.     

Comparative efficacy of MS-222 and benzocaine as anaesthetics under simulated transport conditions of a tropical ornamental fish Puntius filamentosus (Valenciennes)

There is a growing commercial interest in the fish, Puntius filamentosus , in the ornamental fish trade in India and elsewhere. The trade is, however, hampered by severe mortalities during transport of the fish owing to insufficient data available on the use of anaesthetics. To resolve this problem, we evaluated the efficacy of two anaesthetics, MS-222 and benzocaine, in sedating P. filamentosus in simulated transportation experiments and used stress response parameters such as cortisol and blood glucose levels to perform assessments. We observed that MS-222 at 40 mg L⁻¹ and benzocaine at 20 mg L⁻¹ were sufficient to induce sedation for 48 h. Above these concentrations, both the anaesthetics adversely affected the fish and resulted in mortalities. Both anaesthetics significantly lowered the blood cortisol and glucose levels compared with the unsedated controls. Importantly, the anaesthetics treatment significantly lowered the post-transport mortality in the fish. The results of the study show that MS-222 and benzocaine could be used as sedatives to alleviate transport-related stress in P. filamentosus to improve their post-transport survival and hence reduce economic loss.     

Potential benefit of clove oil sedation on animal welfare during salmon smolt, Salmo salar L. transport and transfer to sea

The purpose of this study was to determine the effect of clove oil (4.0 mg L⁻¹) sedation, compared with non-sedation, on the primary (plasma cortisol), secondary (osmoregulation) and tertiary (mortality) stress responses in Atlantic salmon smolts during transport and transfer to sea. Clove oil sedation during on- and off-loading sufficiently reduced the primary stress response to lower mortality (2.1%) during transfer to sea compared with unsedated fish, which experienced a mortality rate above 12.2%. The unsedated fish experienced an acute mortality that stabilized only 6 days after the transport. None of the secondary stress responses measured in this experiment could contribute towards explaining this phenomenon, with the possible exception of plasma magnesium (Mg²⁺). Plasma Mg²⁺ differed between the groups; while plasma Mg²⁺ in the clove oil sedated group returned to pre-stress levels 72 h after transport, the unsedated group showed no such recovery even 1 week after transport, which may indicate a disturbance in the hydromineral balance, and provides a plausible explanation for the delayed mortality in this group. Eugenol-based anaesthetics appear to be promising as a stress-reducing sedative for Atlantic salmon smolts, and, if used properly, this chemical could improve animal welfare and survivability during and after common aquaculture-related incidents.     

Effects of a peroxide-based commercial product on bacterial load of larval rearing water and on larval survival of two species of Sparidae under intensive culture: preliminary study

Larvae of two Mediterranean Sparidae species, Sparus aurata and Dentex dentex , were used to test the efficacy of a peroxide-based product (Ox-Aquaculture^©) on the reduction in bacterial load in larval rearing water and its effects on larval survival. Eleven-day-old S. aurata larvae and 15-day-old D. dentex larvae were exposed to different concentrations of Ox-Aquaculture^© (50, 100 and 200 mg L⁻¹, and 20 and 50 mg L⁻¹ respectively) for 1 h. Results indicated that 50 and 20 mg L⁻¹ were the most effective concentrations for the reduction in bacterial load (at least one order of magnitude) after 1 h treatment, without affecting larval survival and/or vitality in 11 dph S. aurata and 15 dph D. dentex larvae respectively. Ox-Aquaculture^© concentrations of 200 and 50 mg L⁻¹ during 1 h affected negatively final survival rate of the larvae of S. aurata and D. dentex respectively.     

Evaluation of lipid spray beads for the delivery of water-soluble materials to a marine suspension-feeder, the Manila clam Tapes philippinarum (Deshayes 1853)

We describe the development and evaluation of a new microparticle for delivering low-molecular weight, water-soluble materials to suspension feeders. Spray beads successfully incorporated materials dissolved in an aqueous phase or as dry particulate, within a triacylglyceride bead composed of tripalmitin, 600 mg g⁻¹ tripalmitin/400 mg g⁻¹ triolein, or 600 mg g⁻¹ tripalmitin/400 mg g⁻¹ fish oil.
Riboflavin was successfully incorporated (up to 44 mg g⁻¹ lipid) and retained (up to 98% over 24 h in seawater) as dry particles in all three mixtures of lipid. Aqueous oxytetracycline hydrochloride or polymeric dye were incorporated (45.6 mg g⁻¹ lipid and 18.1 mg g⁻¹ lipid, respectively) and retained best (99% and 94%, respectively) in spray beads composed of tripalmitin. The addition of triolein or fish oil to the lipid bead reduced incorporation and retention efficiencies for aqueous core materials by up to 75%.
Manila clam seed readily ingested and digested lipid microparticles, spray beads and lipid-walled microcapsules. Microparticles composed of tripalmitin were excreted with their payloads intact. Intact microparticles composed of 600 mg g⁻¹ tripalmitin/400 mg g⁻¹ fish oil were largely absent in faecal strands suggesting successful release and delivery of microparticle contents to clams.
Spray beads composed of tripalmitin softened with 400 mg g⁻¹ fish oil represent an effective microparticle type for delivering low-molecular weight, water-soluble materials to aquatic suspension feeders.     

Anaesthetic efficacy of clove oil,benzocaine, 2‐phenoxyethanol and tricaine methanesulfonate in juvenile marbled spinefoot (Siganus rivulatus)

Anaesthetics are used in aquaculture and fisheries to facilitate routine procedures, such as capture, handling, transportation, tagging, grading and measurements that can often cause injury or induce physiological stress. Two experiments were performed to assess the efficacies of four anaesthetic agents, clove oil, benzocaine, 2‐phenoxyethanol and MS‐222 on juvenile marbled spinefoot rabbitfish (Siganus rivulatus). In the first experiment we tested the lowest effective doses that produced induction and recovery times in 3 min or less and 5 min or less respectively. Dosages were 70 mg L^?1 for clove oil, 60–70 mg L^?1 for benzocaine, 400 μL L^?1 for 2‐phenoxyethanol and 100–125 mg L^?1 for MS‐222. In the second experiment, we determined optimal concentrations of the four anaesthetics if they were to be used to transport rabbitfish fry. Anaesthetic concentrations suitable for handling and transport were: 10–15 mg L^?1 of MS‐222, 5–10 mg L^?1 of benzocaine, 5 mg L^?1 of clove oil and 50–100 μL L^?1 of 2‐phenoxyethanol. All anaesthetic agents are acceptable for use on S. rivulatus, however, 2‐phenoxyethanol, MS‐222 and clove oil appear to be more suitable than benzocaine. Further studies need to be conducted on effects of high and low doses of anaesthetic agents on physiology of marbled spinefoot.     

Effects of water temperature and body weight on anaesthetic efficiency in marbled rabbitfish (Siganus rivulatus)

The effects of four anaesthetic agents, tricaine methanesulphonate (MS‐222) (112.5 mg L^?1), 2‐phenoxyethanol (400 μL L^?1), clove oil (70 mg L^?1) and benzocaine (65 mg L^?1) on juvenile marbled spinefoot (Siganus rivulatus) of three mean body weights (7.3 g, 19.1 g, 55.5 g) and at three temperatures (20, 25, 30°C) were evaluated. In addition, the relationship between body lipid content and efficacy of the four anaesthetic agents was evaluated in juvenile S. rivulatus. Times necessary for induction and recovery were recorded. Significant effects of temperature on induction and recovery times were observed. Induction and recovery times decreased with increasing water temperature. No uniform relationship between body weight of juvenile marbled spinefoot and anaesthetic efficacy was observed. Body fat content was positively correlated with induction time only when MS‐222 was used but did not affect induction times of fish exposed to 2‐phenoxyethanol, clove oil or benzocaine. Recovery times were generally longer for all fish containing more body fat. Results of the study show that anaesthetic efficiency increases with increasing water temperature but is not strongly affected by body weight for juvenile marbled spinefoot. In addition, body fat in fish affected the efficacy of the various anaesthetic agents tested in this study, generally slowing down recovery.     

Effect of combined photoperiod, water calcium concentration and pH on survival, growth, and moulting of juvenile crayfish (Procambarus clarkii ) cultured under laboratory conditions

The red swamp crayfish, Procambarus clarkii (Girard), is one of the most commonly farmed freshwater species in inland China due to its high market value and consumer demand. The aim of this study was to determine the optimum combinations of photoperiod, water calcium concentration and pH for juvenile survival, growth and moulting. In our orthogonal experiment, the three environmental factors were varied at three levels (photoperiod: 16L:8D, 12L:12D and 8L:16D; calcium concentration: 45.5, 65.5 and 85.5 mg L⁻¹; and pH: 6.8, 7.8 and 8.8). Range analysis showed that the maximum survival of juvenile crayfish occurred at photoperiods of 16L:8D or 8L:16D, water calcium concentration of 45.5 mg L⁻¹ and pH of 7.8; maximum weight gain at photoperiod 16L:8D, water calcium concentration 65.5 mg L⁻¹ and pH 7.8; maximum length increase at photoperiod 16L:8D, water calcium concentration 65.5 mg L⁻¹ and pH 7.8; and the highest moult frequency at photoperiod 12L:12D, water calcium concentration 65.5 mg L⁻¹ and pH 7.8. Analysis of variance indicated that photoperiod, water calcium concentration and pH significantly influenced only the weight gain of juvenile crayfish ( P <0.05). Taking growth into consideration, we suggest that a photoperiod of 16L:8D, calcium concentration of 65.5 mg L⁻¹ and pH 7.8 might be optimal conditions for rearing juvenile P. clarkii .     

Acute LD50 and kidney histopathology following injection of erythromycin (Erythro-200) and its carrier in spring chinook salmon, Oncorhynchus tshawytscha (Walbaum)

We evaluated the toxicity of commercial injectable erythromycin (Erythro-200) and the injectable drug carrier alone (PEG-400, ethyl alcohol and ethyl acetate) in sub-adult spring chinook salmon, Oncorhynchus tshawytscha (Walbaum). Both compounds were toxic to salmon when administered in high dosages. The 50% lethal dose (LD₅₀) of the erythromycin within its carrier (Erythro-200) at 12 °C was estimated at 429 mg erythromycin kg^–1 or 2.145 mL kg^–1 when measured by volume. The LD₅₀ of the drug carrier alone was 2.95 mL kg^–1 by volume or equivalent to 3.21 g kg^–1 of the carrier. We evaluated and scored the histopathology in kidney sections removed from fish 96 h after injection with Erythro-200, the drug carrier alone, or sterile phosphate buffered saline (PBS) that served as a reference control. Kidneys from fish injected with saline had lowest average pathology scores; those injected with the carrier had moderate scores; and the pathology score was highest in tissues from erythromycin-injected fish. In tests at 12 °C, vacuoles were more prominent in proximal and distal tubules of fish injected with higher dosages of 403 and 545 mg erythromycin kg^–1 than in tissues from fish injected with 299 mg kg^–1.     

The synthetic antioxidant, ethoxyquin, adversely affects immunity in tilapia (Oreochromis niloticus)

Ethoxyquin (EQ) has been used as an antioxidant in livestock, aquaculture and pet foods. Animal food safety law has established the upper limit of EQ in animal feed at 150 mg kg⁻¹. However, the risk of EQ at the approved level for aquaculture feed (150 mg kg⁻¹) to fish health is unknown. Here, we examine the effect of EQ on the immunity of tilapia ( Oreochromis niloticus ). EQ concentration in the blood reached 0.16 mg L⁻¹ in fish fed EQ at the approved level. This level of EQ inhibited phagocytic activity of leucocytes in vitro and antibacterial activity of whole blood in vivo . Furthermore, pyknosis in the liver was observed throughout the duration of feeding. However, after 30 days of experimental challenge with feed containing 150 mg kg⁻¹ of EQ, no significant difference was observed in mortality. Although EQ at the approved level in feed causes immunosuppression in fish, the severity of immunosuppression does not lead to a lowering of disease resistance for short feeding periods.     

The efficacy of MS‐222 as anaesthetic agent in four freshwater aquarium fish species

The efficacy of anaesthetic tricaine methanesulfonate (MS‐222) was evaluated in four freshwater aquarium fish species, Zebrafish (Danio rerio), Guppy (Poecilia reticulata), Discu (Symphysodon discus) and Green swordtail (Xiphophorus helleri). The correct dose of anaesthetic should induce the plane 4 of anaesthesia in less than 180 s, recovery in less than 300 s and must survive when exposed during 30 min to anaesthetic. Fishes were exposed to six concentrations of anaesthetic (75, 100, 125, 150, 200 and 250 mg L^?1) and the time of fish reaching plane 4 of anaesthesia, post exposure recovery, and the percentage of survival when fish were subject to 30 min in the anaesthetic were recorded. The optimal doses varied according to the species: D. rerio – 75, 100 and 125 mg L^?1, P. reticulata – 125, 150 and 200 mg L^?1, S. discus – 75 and 100 mg L^?1 and X. helleri – 125 and 150 mg L^?1. The induction time generally decreased significantly with increasing concentration of MS‐222 for all of the species evaluated. The recovery time had a tendency to increase with the increase of the MS‐222 concentration for D. rerio, P. reticulata and S. discus. On the other hand, X. helleri recovery time decreased with the increase of MS‐222 concentration. MS‐222 proved to be effective in anaesthesia for all the freshwater ornamental species studied. The main results clearly show that the optimal dose to anesthetize is fish species dependent and it is completely wrong to extrapolate optimal anaesthetic concentrations between different species.     

Effects of clove oil and MS-222 on blood hormone profiles in rainbow trout Oncorhynchus mykiss, Walbaum

Clove oil has been demonstrated to be an effective, inexpensive anaesthetic and euthanizing agent for a number of fish species, including rainbow trout, used in aquaculture and fisheries research. However, the potential for clove oil to cause perturbations in important plasma hormone concentrations has not been investigated. The effect of anaesthesia and euthanasia in trout with eugenol (the active ingredient in clove oil) on plasma cortisol, glucose, growth hormone (GH) and two thyroid hormones [tri‐iodothyronine (T₃) and thyroxine (T₄)] was compared with tricaine methanesulfonate (MS‐222) anaesthesia, and stunning by cranial concussion in two experiments. Effects on blood chemistry were different when comparing the particular anaesthetic method being used. Stunning fish significantly increased plasma cortisol and glucose levels (both P<0.05), while euthanizing fish using either clove oil or MS‐222 had no effect on these hormone levels. In contrast, the levels of GH, T₃ and T₄ hormones were unaffected regardless of whether fish were euthanized by stunning, MS‐222 or clove oil. Variation in effects between hormones were observed using clove oil eugenol. In fish sampled 10 min after anaesthetizing with 150 mg L^?1 of eugenol, cortisol levels were significantly decreased (P<0.03), while there were no differences in either glucose or GH levels. Tri‐iodothyronine and T₄ also showed significantly elevated levels (P<0.05) after 10‐min exposure to eugenol. These results highlight the importance of investigating the potential effects of any new anaesthetic or euthanizing compounds on blood plasma parameters, prior to using them in a research setting, or when comparing results to other studies which have utilized alternative anaesthetic compounds.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Using Efinol®L during transportation of marbled hatchetfish, Carnegiella strigata (Günther)

The objective of this experiment was to test the efficacy of a probiotic (Efinol^®L) during transportation of marbled hatchetfish, Carnegiella strigata . Wild specimens were captured from a small stream and transported for 24 h in plastic fish boxes with a probiotic (10 mg L⁻¹) and probiotic-free water. The boxes were sampled at 3, 12 and 24 h of transport. At the end of the experiment, the survival rate was close to 100% in both treatments. Dissolved oxygen diminished with time in both treatments, but the probiotic group had significantly higher levels. Conductivity, pH and ammonia increased significantly during the transport, demonstrating higher levels in the probiotic-free group. Fish from both treatments presented very high net Na⁺ and K⁺ effluxes after 3 h of transport. At 24 h, net K⁺ effluxes in fish of the probiotic treatment reached values close to zero and a significantly lower Na⁺ efflux was observed. Cortisol levels in both treatments at 3 and 12 h were significantly higher than that in control samples. Higher body cortisol levels were observed in the probiotic-free group than that in the probiotic group at 3 and 12 h. The results demonstrate that addition of a probiotic during fish transport improves water quality and leads to fish presenting a lower stress response intensity.     

Effect of plant active compounds on immune response and disease resistance in Cirrhina mrigala infected with fungal fish pathogen, Aphanomyces invadans

The antibacterial activity of individual and mixed medicinal plant compounds, azadirachtin (Az), camphor (Ca) and curcumin (Cu), was tested at 100, 200, 300, 400, 500, 600 and 700 ppm (mg L⁻¹) against fungal fish pathogen, Aphanomyces invadans, in vitro . At the lower concentrations between 100 and 300 ppm, the mixture of the tri-herbal (Az+Ca+Cu) compound yielded a higher ( P >0.05) zone of inhibition (ZI) of 7 mm than the positive control; the maximum ZI values (8–15 mm) were realized between 400 and 700 ppm ( P <0.05). At the lowest minimum inhibitory concentration (MIC) the tri-herbal compound (100 ppm) yielded 13 colony-forming units; hence, this dose can be effectively used at the lowest concentration of 100 mg L⁻¹ to ward off the growth of A. invadans in vitro . In Cirrhina mrigala , intramuscular administration (100 μL) of the selected doses of 100, 400 and 700 ppm (mg L⁻¹) significantly enhanced ( P <0.05) the serum lysozyme activity (Ly), production of reactive oxygen species and reactive nitrogen species (RNS or NO) by peripheral blood leucocytes on the 10th, 20th and 30th day. A priori administration of the compound in the fish (100 ppm on 30th day) decreased the percentage mortality when challenged with the pathogen while in the untreated group the mortality increased ( P <0.05). This study indicates that intramuscular administration of the tri-herbal compound Az+Ca+Cu at a concentration of 100 ppm could augment the immune response in C. mrigala against A. invadans .