Remediating sulfadimethoxine-contaminated aquaculture wastewater using ZVI-activated persulfate in a flow-through system

Antibiotic resistant bacteria can result from the improper discharge of water from the aquaculture farming industry. This calls for the development of a low-cost remediation technology. Our research verified the use of ZVI-activated persulfate (PS) to treat antibiotic-contaminated aquaculture discharge water in a flow-through system. We selected sulfadimethoxine (SDM) as a representative antibiotic residual and tested SDM degradation under varying parameters: activator dose, initial pH, chloride concentration, activator dose and timing. The results demonstrated that increasing the ZVI dosage significantly decreased SDM degradation due to the scavenging effects for the persulfate radical (SO₄⁻). SDM decomposition occurred when SO₄⁻ attacked the aniline moiety via electron transfer prior to undergoing hydrogen abstraction/addition on the sulfonamide. A high pH produced the fastest degradation with reaction rates following the order pH 11 > > pH 9 > pH 3 > pH 5. A high Cl⁻ concentration (>100 mM) enhanced SDM degradation because of the production of chlorine radicals. The experiment results from ZVI sequential addition indicated that only a small continual input of ZVI was sufficient to generate SO₄⁻ to react with SDM. We used a flow-through concept for the real discharge water that was spiked with SDM prior to treating with PS/ZVI. The results showed that our system was able to remove approximately 68% of SDM from filtered and 74% from unfiltered discharge water. These results provide proof-of-concept that our PS/ZVI system could potentially be developed to remediate antibiotic-contaminated aquaculture wastewater.     

Prevention of fungal infestation of rainbow trout (Oncorhynchus mykiss) eggs using UV irradiation of the hatching water

Fungal infestation by water mold Saprolegnia spp. causes great losses in aquaculture and fish egg hatching. To find a safe and effective alternative for the fungal prevention, we studied continuous disinfection of the inlet water by UV irradiation and ozonation combined with low concentration hydrogen peroxide (H₂O₂) treatments in a rainbow trout (Oncorhynchus mykiss) egg hatching system. High dose of UV irradiation (400 mWs/cm²) of the inlet water decreased the mortality of rainbow trout eggs from the 77.3% to 14.3% in a 28 day trial. UV irradiation did not modify water quality parameters, while combination of UV irradiation and H₂O₂ caused up to fivefold increase in the formate levels, and combination of O₃ and H₂O₂ caused even ten-fold increase in the acetate and formate levels. UV suppressed the gradual increase of the heterotrophic bacterial counts on the fish eggs. Based on the molecular profiling high dose of UV reduced the growth of some of the dominating bacterial groups and combination of UV and H₂O₂ had a distinctive effect on the overall bacterial community structure on the fish eggs.     

Pharmacodynamics of amoxicillin against field isolates of Streptococcus parauberis from olive flounder (Paralichthys olivaceus)

Olive flounder is the most important species for the Northeast Asian fish farming industry. However, this species is substantially affected by multiple infectious agents, including Streptococcus parauberis. Evaluation of antibiotics before their application is critical to treat infections and prevent drug resistance. Therefore, in this study, the pharmacodynamics of amoxicillin (AMX) and other antimicrobials against the planktonic‐ and biofilm‐forming bacteria were assessed. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and Time–kill curve assay were analysed using micro‐dilution method. The minimum biofilm eradicating concentration (MBEC) was determined using the Calgary Biofilm device. The effects of temperature, pH, hardness and salinity were detected for both planktonic‐ and biofilm‐forming bacteria. The MIC of AMX ranged from 0.015 to 2 μg/ml, whereas that of cephalexin (CEP), enrofloxacin (ENR) and oxytetracycline (OTC) ranged from 0.125 to 256, 0.125 to >512 and 0.25 to >512 μg/ml respectively. No bacteria were resistant against AMX, while the percentage of resistance to CEP, OTC and ENR were 68.7%, 52.6% and 11.1% respectively. The IC₅₀ of AMX, CEP, ENR and OTC was 0.03, 0.091, 0.015 and 0.213 μg/ml respectively. The MBEC of amoxicillin against S. parauberis ranged from 0.5 to 16 μg/ml. Higher rates of bacterial growth were obtained at 30°C, pH = 8 and salinity of 7.5–10 ppt. The hardness of the media suppressed the bacterial growth. In conclusion, AMX was found to be effective against both the planktonic and the biofilm forms of the prominent fish pathogen, S. parauberis.     

Structural Changes,Volatile Compounds and Antioxidant Activities of Maillard Reaction Products Derived from Scallop (Patinopecten yessoensis) Female Gonad Hydrolysates

In order to improve the utilization of scallop byproducts, scallop female gonad hydrolysates (SFGHs) were prepared using alcalase and ribose at a mass ratio of 1:2 and pH 7.0 (95°C, up to 12 h) to develop heterogeneous Maillard-type antioxidant and flavor compounds. The formation of Schiff’s base as well as modification of the amide bands in Maillard reaction products (MRPs) were investigated using ultraviolet–visible, fluorescence, and Fourier transform infrared spectroscopy. The significant decrease of amino acid residues explained the strong Maillard reactivity of SFGHs. MRPs with enhanced ABTS⁺ radical scavenging capacity over SFGHs promoted the survival of HepG2 cells treated with hydrogen peroxide. Moreover, 19 new volatile compounds were produced by the reaction of SFGHs with ribose. These results suggest that SFGHs–ribose MRPs can be potentially used as antioxidants with flavor properties.

Abbreviations: SFGs: scallop female gonads; SFGHs: scallop female gonad hydrolysates; ABTS: 2?2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); BHT: butylated hydroxytoluene; Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; MRPs: Maillard reaction product; H₂O₂: hydrogen peroxide; HS-SPME/GC/MS: Headspace solid phase micro-extraction/gas chromatography/mass spectrometry.     

Oxidative damage repair by glutamine in fish enterocytes

Fish intestine is very sensitive to oxidative damage. Repair of damaged enterocytes may be involved to restore normal function of fish intestine. However, studies of fish enterocyte repair are scarce. The present study aimed to investigate the potential repair role of glutamine after a H₂O₂ challenge. In this study, fish enterocytes were post-treated with graded levels of glutamine (0, 4, 8, 12 and 20 mM of glutamine) after expose to 100 μM H₂O₂. The basal control cells were kept in the glutamine-free minimum essential medium only. Results showed that the H₂O₂-induced decreases in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide optical density, alkaline phosphatase and Na⁺, K⁺-ATPase activities were completely restored by subsequent glutamine treatments. In addition, cellular injury (lactate dehydrogenase), lipid peroxidation (malondialdehyde) and protein oxidation (protein carbonyls) caused by H₂O₂ were reversed by subsequent glutamine treatments. Furthermore, the H₂O₂-induced decreases in glutathione contents, glutathione reductase, superoxide dismutase and glutathione peroxidase activities were completely restored by subsequent glutamine treatments. In summary, the present study indicated that glutamine improved the repair activity in fish enterocytes after challenge with H₂O₂.     

Antibacterial activity of oxytetracycline on microbial ecology of Nile tilapia (Oreochromis niloticus) gastrointestinal tract under laboratory condition

The antibacterial effect of oxytetracycline (OTC) on bacterial load, pathogen and possible development of antibiotic resistance in intestinal flora of apparently healthy Nile tilapia (Oreochromis niloticus) was investigated under laboratory condition. OTC, a broad spectrum approved antibiotic widely used in aquaculture, was fed to Nile tilapia through medicated diet at a rate of 2 g/ kg of feed for consecutive 7 days in treatment 1 (T₁) and 30 days in treatment 2 (T₂). Changes in physico‐chemical parameters of water were also recorded where pH and dissolved oxygen influenced bacterial load and coliform count respectively. At the start of the experiment, initial bacterial load in fish intestine was 1.61 ± 2.25 × 10¹⁰ cfu/g, which after OTC treatment decreased significantly (p < .05) to 3.06 ± 2.08 × 10⁸ cfu/g on day 7 and 3.45 ± 4.46 × 10⁷ cfu/g on day 30 when compared with the control group. Among the 10 bacterial genera identified in the intestine, 4 potential public health concern bacteria viz., Salmonella, Escherichia, Enterobacter and Staphylococcaus were predominant. There was a sharp increase in bacterial load on day 18 in T₂ that may be attributed to the development of antibiotic resistance in intestinal bacteria. Antibiotic susceptibility test for isolates against seven antibiotics: amoxycillin, ciprofloxacin, co‐trimoxazole, erythromycin, gentamicin, nitrofurantoin and tetracycline using disc diffusion method revealed significantly increased resistance of Gram‐negative rods (p < .05) that possibly caused increased frequency of OTC‐resistant microorganisms. In conclusion, short‐ and long‐term exposure to OTC treatment affected the distribution of bacterial genera including pathogens in the Nile tilapia gastrointestinal tract and concomitantly influenced their antimicrobial resistance.     

Effect of pH on Structural Changes in Perch Hemoglobin that Can Alter Redox Stability and Heme Affinity

pH can be manipulated to alter the oxidative stability of fish-based foods during storage. X-ray diffraction was used to investigate the ability of reduced pH to cause structural changes in fish hemoglobins that lead to enhanced oxidative degradation. Decreasing pH from 8.0 to 6.3 and 5.7 created a large channel for solvent entry into the heme crevice of perch hemoglobin beta chains. The proton-induced opening of this channel occurred between site CD3 and the heme-6-propionate. Solvent entry into the heme crevice can enhance metHb formation and hemin loss, processes that accelerate lipid oxidation. Reduced pH also decreased the distance between Ile at E11 in one of the alpha chains and the ligand above the heme iron atom. This sterically displaces O₂ and protonated O₂ which increases metHb formation. These studies demonstrate that pH reduction causes structural changes in perch hemoglobin which increase oxidative degradation of the heme pigment.     

Characterization of a novel marine origin aerobic nitrifying–denitrifying bacterium isolated from shrimp culture ponds

A novel marine origin Bacillus subtilis strain H1 isolated from a shrimp culture pond effectively removed NH₄⁺‐N, ‐N and ‐N, with a maximum ammonium, nitrite and nitrate removal rate of 2.35 mg NH₄⁺‐N L^?1 hr^?1 per OD, 9.64 mg ‐N L^?1 hr^?1 per OD and 0.75 mg ‐N L^?1 hr^?1 respectively. The gas chromatography–isotope ratio mass spectrometry results indicated that N₂O was emitted when ¹⁵NH₄Cl, Na¹⁵NO₂ or Na¹⁵NO₃ was used. Additionally, N₂ was also produced when Na¹⁵NO₂ was used. Single‐factor experiments suggested that the optimal conditions for NH₄⁺‐N and ‐N removal were glucose as a carbon source, C/N 15, initial pH 7.5, 30 g/L NaCl, 28°C and a shaking speed of 160 rpm. Orthogonal tests showed that the optimal conditions for NH₄⁺‐N removal were C/N 15, pH 9, 10 g/L NaCl and shaking speed 160 rpm when ammonium chloride was used as the substrate. The optimal conditions for ‐N removal were C/N 10, pH 6, 10 g/L NaCl and a shaking speed of 160 rpm when sodium nitrite was used as the substrate. In summary, B. subtilis strain H1 had highly efficient aerobic nitrifying–denitrifying ability and high adaptability, suggesting that it is potentially valuable to marine aquaculture.     

Lack of arterial PO<Subscript>2</Subscript> downregulation in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) during long-term normoxia and hyperoxia

Regulation of arterial partial pressure of O₂ (P_aO₂) in Atlantic salmon (Salmo salar) was investigated during resting conditions in normoxic and hyperoxic water. Dorsal aorta cannulated adult Atlantic salmon (1.2–1.6 kg, n = 8) were exposed to 2 week sequential periods of normoxia [16.7 ± 1.1 kPa (mean ± SD)] and hyperoxia (34.1 ± 4.9 kPa) in individual tanks containing seawater (33.7 ± 0.2 ppt) at stable temperature conditions (8.7 ± 0.7°C) and a light regime of L:D = 12:12. Tank design and sampling procedures were optimized to provide suitable shelter and current for the fish, and to allow repeated, undisturbed sampling of blood from free-swimming fish. Fish were sampled regularly through the experimental period. P_wO₂, P_aO₂, blood ion composition (Na⁺, K⁺, Cl⁻), acid–base status (pH, PCO₂, HCO₃ ⁻), haematocrit and glucose were measured. The most frequently observed P_aO₂ values were in the range of 60–80% of P_wO₂, both during normoxia and hyperoxia, and P_aO₂ values were significantly lower during normoxia than during hyperoxia. Blood pH, PCO₂ and HCO₃ ⁻ were significantly elevated during hyperoxia, while, Na⁺, Cl⁻ and Hct were significantly lower. K⁺ and glucose showed no significant differences. This study demonstrates a lack P_aO₂ regulation in Atlantic salmon to low partial pressures, in contrast to previous reports for many aquatic gill breathing animals. Both during normoxia and hyperoxia, P_aO₂ reflects P_wO₂, and alterations in external PO₂ consequently result in proportional arterial PO₂ changes. Physiological adaptation to hyperoxia, as illustrated by changes in several blood parameters, does not include down-regulation of P_aO₂ in Atlantic salmon. The lack of P_aO₂ regulation may make Atlantic salmon vulnerable to the oxidative stress caused by increased free radical formation in hyperoxic conditions.     

Hydrogen peroxide oxygenation and disinfection capacity in recirculating aquaculture systems

Hydrogen peroxide (H₂O₂) treatment is an alternative for disinfection in aquaculture, which may be advantageous as it dissociates and disinfects while increasing water oxygen concentration. Yet, accurate dosing remains undeveloped in Recirculating Aquaculture Systems (RAS). Dosage requirements can depend on organic burden, stocking density, feeding frequency, salinity, temperature and biofilter performance. The present case study investigated the dual effect of H₂O₂ application for oxygen enrichment and disinfection when continuously applied to a RAS rearing European seabass. H₂O₂ addition equivalent to 2.4 and 15.8 H₂O₂ mg L⁻¹ were applied for 4 h per day in three 5-days experiments. H₂O₂ was injected at the inlet of protein skimmer and/or the rearing tanks in or without combination with traditional disinfection methods. Water microbial load and oxygen saturation were determined, along with stress markers glucose and cortisol in blood plasma of fish. Doses of 15.8 mg L⁻¹ H₂O₂ steadily increased oxygen levels in holding tank water from ∼50 % to over 100 % saturation while reducing microbial load (from 604.4 CFU ml⁻¹ in the rearing tanks before dosing to 159.8 CFU ml⁻¹ after application), achieving suitable conditions for commercial fish densities in RAS. The doses used had negligible impact on biofilter performance and did not affect the fish in terms of stress. Overall results indicate H₂O₂ is effective for disinfection and oxygenation of RAS systems when applied at appropriate dosage and we recommend the protein skimmer as the safest position in order to protect the bacterial community of the biofilters and the reared fish.     

Effects of foam fractionation and chemical disinfection on the removal of different microalgae cultures

The recent development of recirculation aquaculture systems (RAS) potentially allows more sustainable and controlled rearing conditions. However, control of suspended microparticles and microalgae in RAS is challenging, as uncontrolled blooms of toxic algae or heterotrophic dinoflagellates can have catastrophic impacts on the production of fish. In this study, we tested the potential of protein skimmers to remove microalgae. In 100 L batch tests, protein skimmers were tested separately and in combination with ozone (O₃), ultraviolet (UV) or hydrogen peroxide (H₂O₂). Three different and distinct microalgae cultures were tested with densities from 30,000 to 120,000 algae cells/ml in a triplicated experimental setup. Trial 1 included twelve 24‐hr replicated tests where protein skimmers with air or with two levels of ozone (low and high O₃ exposure) were compared. The protein skimmer with air alone had a limited effect on the removal of microalgae compared to the untreated control algae tanks. When ozone and protein skimmers were combined, a clear additive effect was found, and all added microalgae were removed. Low O₃ dosage and protein skimmers completely removed the algae cultures within 12 hr, while more than 95% of the algae were removed within 7 hr when a higher ozone dose was used. The second trial compared the removal capacity of protein skimmers in combination with UV, H₂O₂ and O₃. These experiments showed no to limited additive effect of UV combined with protein skimming, but significantly increased removal efficiency (270% and 1,300%, respectively) was found when H₂O₂ and ozone were combined with a protein skimmer. The study showed an algae species‐specific response to a protein skimmer with ozonation and provided information on the transition from reactivity and consumption to accumulation of ozone‐produced oxidants.     

The effect of temperature on the resting and post-exercise metabolic rates and aerobic metabolic scope in shortnose sturgeon <Emphasis Type="Italic">Acipenser brevirostrum</Emphasis>

The effects of acclimation temperature (15, 20, 25 °C) on routine oxygen consumption and post-exercise maximal oxygen consumption rates (MO₂) were measured in juvenile shortnose sturgeon (Acipenser brevirostrum LeSueur, 1818). The routine MO₂ of shortnose sturgeon increased significantly from 126.75 mg O₂ h^?1 kg^?1 at 15 °C to 253.13 mg O₂ h^?1 kg^?1 at 25 °C. The temperature coefficient (Q ₁₀) values of the routine metabolic rates ranged between 1.61 and 2.46, with the largest Q ₁₀ values occurring between 15 and 20 °C. The average post-exercise MO₂ of all temperature groups increased to a peak value immediately following the exercise, with levels increasing about 2-fold among all temperature groups. The Q ₁₀ values for post-exercise MO₂ ranged from 1.21 to 2.12, with the highest difference occurring between 15 and 20 °C. Post-exercise MO₂ values of shortnose sturgeon in different temperature groups all decreased exponentially and statistically returned to pre-exercise (resting) levels by 30 min at 15 and 20 °C and by 60 min at 25 °C. The aerobic metabolic scope (post-exercise maximal MO₂-routine MO₂) increased to a maximum value ～156 mg O₂ h^?1 kg^?1 at intermediate experimental temperatures (i.e., 20 °C) and then decreased as the temperature increased to 25 °C. However, this trend was not significant. The results suggest that juvenile shortnose sturgeon show flexibility in their ability to adapt to various temperature environments and in their responses to exhaustive exercise.     

The conversion of plasma HCO 3 − to CO2 by rainbow trout red blood cells in vitro: adrenergic inhibition and the influence of oxygenation status

This study employed a recently developed radioisotopic assay (Wood and Perry 1991) to examine the inhibition, induced by catecholamines, of the conversion of plasma HCO ₃ ^– to CO₂ in acidotic trout blood, and the influence of oxygenation status on the response. Blood was incubated in vitro at P_{CO 2}= 2 torr, and 10^–6 M noradrenaline was employed as the adrenergic stimulus. In particular we investigated whether the inhibition of plasma HCO ₃ ^– conversion could be explained by a limited supply of H⁺s for the intracellular HCO ₃ ^– dehydration reaction because of competition by the adrenergically activated Na /H⁺ exchanger. Hypoxia (P_{O 2}= 15 torr) was employed as a tool to intensify this competition. Hypoxia raised RBC pHi, pHe, and plasma total CO₂ concentration (C_{CO 2}) by the Haldane effect, and increased the magnitude of Na⁺/H⁺ activation, expressed as the change in the transmembrane pH gradient (pHe-pHi). However hypoxia did not alter the inhibition of the conversion of plasma HCO ₃ ^– to CO₂ caused by noradrenaline. Hypoxia itself stimulated the RBC-mediated conversion of plasma HCO ₃ ^– to CO₂ by about 20% in the presence or absence of noradrenaline. The conversion rate was strongly correlated with pHe, pHe-pHi, and plasma C_{CO 2} in these experiments, but not with pHi. We conclude that adrenergically mediated inhibition in the conversion of plasma HCO ₃ ^– to CO₂ by trout RBCs is not due to competitive limitation on intracellular H⁺s, but rather to changes in the electrochemical gradient for HCO ₃ ^– entry and/or to CO₂ recycling from plasma to RBC. The deoxygenated condition helps to promote CO₂ excretion at the level of the RBC.     

Effects of adrenaline on ionic equilibria in red blood cells of rainbow trout (Salmo gairdneri)

Effects of adrenaline on the equilibrium distributions of Na⁺ , K⁺ , H⁺ , Cl^– , and H₂O across the cell membrane of rainbow trout (Salmo gairdneri) erythrocytes were determinedin vitro, as a function of P CO₂ (1.76–7.77 torr). CO₂-carrying capacity of the blood was also examined. Plasma catecholamine concentrations inunanaesthetized, unrestrained trout were 3.1 nM adrenaline and 1.2 nM noradrenaline. Elevation of the plasma adrenaline concentrationin vitro to 4.6 × 10³ nM resulted in net gains of Na⁺ , Cl^– and H₂O by red cells, a net loss of H⁺ from red cells, and a pronounced red cell swelling. Adrenaline also reduced the CO₂-carrying capacity of trout bloodin vitro. The magnitudes of these effects increased with P_CO2 and, thus, were sensitive to blood HCO₃ ^– concentrations. The distribution of K⁺ between red cells and plasma was unaffected by adrenaline. Adrenergic-mediated ion movements and red cell swelling were sensitive to both propranolol and SITS. These results are consistent with the symport NaCl uptake model for adrenergic-mediated swelling of Baroinet al. (1984). The adrenergic response of fish erythrocytes may function to ameliorate the effects of blood acidoses on O₂-carrying capacity by maintaining red cell pH in the face of a decrease in plasma pH.     

Assessment of microbial activity in water based on hydrogen peroxide decomposition rates

This study proposes a new and simple assay that allows rapid assessment of microbial activity in water samples. The assay consists of standardized hydrogen peroxide (H₂O₂) addition to a water sample and subsequent spectrophotometric determination of H₂O₂ reduction over time. The H₂O₂ decomposition rate constant reflects the level of enzymatic activity from planktonic and particle-associated bacteria as well as algae and protozoans. The proof of concept was verified on water samples from recirculating aquaculture systems (RAS), showing that the vast majority of H₂O₂ decomposition was related to microbial activity. Only 3% of the total H₂O₂ decomposition was related to abiotic processes when 0.20 μm sterile filtered RAS water was compared with unfiltered RAS water. Planktonic bacteria (size range 0.20–1.6 μm) accounted for 16% of H₂O₂ decomposition, while bacterial aggregates, particle-associated bacteria and microbiota above 1.6 μm were responsible for the remaining 81%. H₂O₂ decomposition rate constants were positively correlated to BOD₅ (r = 0.893; p < 0.001; n = 18) and to the number of 1–30 μm micro particles (r = 0.909; p < 0.001; n = 72) in RAS water, substantiating the biologically mediated decomposition processes in the water phase. The H₂O₂ decomposition assay thus represents a new alternative to existing methods that allows rapid (1–2 h) and simple quantification of microbial activity in fresh- and saltwater samples from aquaculture systems. Potential applications of the assay are discussed.     

Basal H2-receptor stimulated and pH-dependent acid secretion from an isolated stomach mucosa preparation of the cod,Gadus morhua,studied using a modified pH-static titration method

Gastric acid secretion from isolated cod stomach mucosa was measured using a pH-static titration method. A basal acid secretion rate (BASR) of 6.0±0.6 nEqH⁺min^–1cm^–1 was measured when using 0.9% NaCl as luminal solution. There was a dose-dependent increase in response to histamine between 0.12 and 0.20 M (EC₅₀=0.15 M), above which gastric acid secretion plateaued at 13.5±1.8 nEqH⁺min^–1cm^–1. Ranitidine, a H₂-receptor antagonist, completely blocked the stimulatory effect of histamine and reduced the BASR. The H₁-receptor antagonist, clemastine, did not inhibit the response to histamine. Acid secretion rates decreased significantly when the pH of the luminal side of the mucosa was lowered from pH 5.75 to pH 4.50, indicating that a negative feedback mechanism was operating. Histological staining showed that oxynticopeptic cells were uniformly distributed throughout the cardiac stomach.It is concluded that the acid secretion in the isolated stomach mucosa of cod can be measuredin vitro with a pH-static titration method. The method was used to demonstrate that the BASR is downregulated by a decrease in pH. Furthermore, we conclude that the histamine receptor in the cod stomach mucosa resembles the mammalian H₂-receptor and that histamine is secreted under basal conditions.     

Natural dietary additive yellow loess as potential antibiotic replacer in Japanese eel,Anguilla japonica: Effects on growth,immune responses,serological characteristics and disease resistance against Edwardsiella tarda

The present experiment was conducted to evaluate the efficacy of an additive derived from the nature as an alternative of dietary antibiotic in Japanese eel, Anguilla japonica. Six experimental diets were formulated to contain no antibiotics or additive (yellow loess/YL) (control/CON), three graded levels of yellow loess at 5 (YL₅), 10 (YL₁₀) and 20 g/kg (YL₂₀), oxytetracycline at 5 (OTC) and amoxicillin at 10 g/kg amoxicillin (AMX) of diet. Weight gain (WG) and specific growth rate (SGR) from fish fed CON or YL₅ diets were significantly lower than those of fish fed YL₂₀ or OTC diets. Among non‐specific enzyme, lysozyme activity of fish fed YL₂₀, OTC or AMX was detected to be significantly higher than that from fish fed CON or YL₅ diets, whereas superoxide dismutase (SOD) activity of the fish fed CON was significantly lower than that for fish fed other experimental diets. Challenge test with bacteria, Edwardsiella tarda, showed improved disease resistance among the fish fed different levels of natural additive without any statistical difference from those fed antibiotics (OTC and/or AMX) supplemented diets. Therefore, these results demonstrated the potential of natural feed additive, yellow loess to replace oxytetracycline and/or amoxicillin in Japanese eel, A. japonica.     

Effects of pH on contractility of rainbow trout (Oncorhynchus mykiss) intestinal muscle in vitro

Extreme acid-base imbalances in salmonids have been shown to impact on gastrointestinal motility. Changes in pH are known to affect cardiac and skeletal muscle in fish and mammalian intestinal smooth muscle preparations. This study was conducted to determine if the contractility of rainbow trout intestinal muscle was sensitive to pH fluctuations and to begin the investigation into the mechanisms by which pH affected contractility. Isolated duplicate or triplicate segments of rainbow trout intestine were suspended in organ baths containing modified Krebs-Henseleit solution. The pH was adjusted by varying the concentration of CO₂ aerating the solution and with HCl or lactic acid. Contractility was determined as response to the administration of 5-hydroxytryptamine (5-HT, serotonin), KCl, and transmural stimulation within the pH range of 6.3 to 8.5. Optimum pH for proximal segments was 7.85 while the range was much wider for distal segments. Decreasing the pH with CO₂ resulted in a greater inhibition of smooth muscle contractility than when the pH was decreased in 100% O₂ with either HCl or lactic acid, particularly when the tissues are stimulated electrically. This effect was ameliorated as the fish attained sexual maturity, although the mechanisms involved were not clear. The Cl^-/HCO ₃ ^- exchanger appeared to be involved in recovery from acidotic stress, particularly at pH levels below 6.5. The role played by the Na⁺/H⁺ exchanger is still unclear. Although contractility was inhibited in the presence of the amiloride analog EIPA (5-(N-ethyl-N-isopropyl)amiloride), whether the inhibition was due to blockage of ion transport or to cytosolic effects is yet to be determined.