Randall W. Oplinger

The Safety and Effectiveness of Various Hydrogen Peroxide and Iodine Treatment Regimens for Rainbow Trout Egg Disinfection

Abstract Four tests were conducted to evaluate iodine and hydrogen peroxide for the disinfection of rainbow trout Oncorhynchus mykiss eggs at higher doses for shorter durations than previously studied. In the first test, eyed eggs were exposed to (1) 2,000 mg iodine/L for 10 min, (2) 100 mg iodine/L for 15 min, (3) 30 g hydrogen peroxide/L for 1 min, (4) 6 g hydrogen peroxide/L for 5 min, or (5) no treatment. Iodine (2,000 mg/L) or hydrogen peroxide (30 g/L) significantly reduced bacterial loads on eggs but did not significantly affect egg survival or fry deformity rates. Hydrogen peroxide at 30 g/L for 1 min was generally better for bacterial control than the other treatments, but the 2,000-mg/L iodine treatment also was effective. A second test assessed the effect of hydrogen peroxide on pH at various levels of water hardness. The pH of hydrogen peroxide solutions dropped as total hardness levels decreased, but buffering with at least 1.32 g NaHCO3/L returned pH to approximately neutral levels. In the t...

Toxicity of Common Aquaculture Disinfectants to New Zealand Mud Snails and Mud Snail Toxicants to Rainbow Trout Eggs

Abstract The New Zealand mud snail (NZMS) Potamopyrgus antipodarum is an invasive species that threatens North American fish populations. Establishment of NZMS in fish hatcheries is a concern because fish stocking practices could expedite the spread of the species. We evaluated the potential use of chemicals to remove snails that are inadvertently collected during egg take operations involving wild broodstock from the field. We exposed green eggs of rainbow trout Oncorhynchus mykiss for 5 min to several chemicals that have been proven lethal to NZMS: 1,940-ppm Hyamine 1622; Clorox Commercial Solutions 409 Cleaner, Degreaser, and Disinfectant (full strength; hereafter, Clorox Commercial 409); Pine-Sol (50% dilution); household ammonia (full strength); and a 504-ppm copper solution (as copper sulfate). All of the eggs exposed to Hyamine 1622, Clorox Commercial 409, and ammonia died within 24 h. Among eggs treated with Pine-Sol and copper sulfate, hatch and cripple rates did not differ from the control (wate...

Control of Flavobacterium psychrophilum: Tests of Erythromycin, Streptomycin, Osmotic and Thermal Shocks, and Rapid pH Change

Flavobacterium psychrophilum, the etiological agent that causes bacterial coldwater disease, has been implicated in significant reductions in the numbers of salmonids reared at fish hatcheries. In this study, we performed a series of in vitro experiments to test the effectiveness of rapid temperature, pH, and osmotic pressure changes in killing three strains of the bacterium. We also evaluated the effectiveness of antibiotics (erythromycin, streptomycin, and a penicillin-streptomycin mixture) against F. psychrophilum. The bacterium tolerated temperatures of 40-50°C for up to 30 min (when acclimated to 15°C). The bacterium can survive lower temperatures for >60 min. Although temperatures ≥55°C appeared to kill F. psychrophilum on contact, we found that eyed eggs of Rainbow Trout Oncorhynchus mykiss were not able to survive short (<60-s) exposures at these temperatures. We found that rapid changes in pH (15-min exposures to pH 3.0, 4.0, 5.0, 7.0, 9.0, 10.0, and 11.0) and osmotic pressure (15-min exposures to 0, 6, 8, 10, 12% sodium chloride) were not effective at killing the bacterium. Erythromycin concentrations up to 2,000 mg/L for 15 min were also ineffective. However, streptomycin concentrations ≥5,000 mg/L killed the bacterium during a 15-min exposure. The combination of penicillin and streptomycin was also effective, killing the bacterium at doses as low as 2.5 × 10(6) IU penicillin + 2,500 mg/L streptomycin. Our trials demonstrate that elevated temperatures and the combination of penicillin and streptomycin can kill F. psychrophilum under in vitro conditions. Erythromycin and rapid changes in pH and osmotic pressure are not effective at killing the bacterium.

Use of penicillin and streptomycin to reduce spread of bacterial coldwater disease I: antibiotics in sperm extenders.

Bacterial coldwater disease caused by Flavobacterium psychrophilum has led to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid. Methods for disinfecting ovarian fluid and unfertilized eggs are available, but methods for disinfecting sperm have not been described. In this study we determined whether sperm extenders containing a mixture of penicillin and streptomycin can be used to eliminate F. psychrophilum. In vitro trials demonstrated that when Rainbow Trout Oncorhynchus mykiss sperm is mixed with an extender, a 15-min exposure to 0.197 mg penicillin plus 0.313 mg/mL streptomycin is effective at killing the bacteria and has no effect on sperm motility. Small-scale trials showed that egg fertilization rates were not reduced when sperm held in an extender solution containing the same antibiotic mixture for 15 min was used to fertilize eggs. Production-scale trials, however, showed a roughly 18% decrease in egg fertilization rate when sperm stored in an antibiotic containing extender was used. To determine why a reduction in fertilization capacity was observed, a small-scale experiment testing the fertilization of eggs with larger quantities of sperm was performed and showed that increasing the volume of sperm used did not increase fertilization rates. Our results demonstrate that extenders containing penicillin and streptomycin can be used to disinfect sperm, especially when small quantities of eggs are fertilized, but factors negatively affecting egg fertilization and survival on a production scale still need further investigation.

Egg Disinfection to Improve Conservation Aquaculture of Leatherside Chub

Abstract Leatherside chub Lepidomeda copei eggs were obtained as part of separate spawning and life history experiments. This paper summarizes several egg disinfection tests conducted between 2007 and 2010 to develop protocols for controlling fungus and improving hatching success. Several disinfection strategies that have worked for other species did not result in improved hatch rates, namely, formalin (1,000–2,000 mg/L for 15 min), ultraviolet light (10,200 mW/cm2), or sodium sulfite (1.5% for 5 min). However, petri dish incubation after treatment with copper sulfate (CuSO4) or hydrogen peroxide (H2O2) provided high hatch rates. Eggs treated with 40 or 60 mg/L CuSO4 for 2 min had significantly higher hatch rates than untreated controls. Daily or repeated (2-d) treatments using H2O2 or CuSO4 did not significantly improve survival to hatch relative to treating only once, but fungal growth was better controlled in the daily H2O2 treatments. The highest percentage of hatching (100%) was observed in petri dis...

Culture of Tubifex tubifex: Effect of Feed Type, Ration, Temperature, and Density on Juvenile Recruitment, Production, and Adult Survival

Abstract The oligochaete worm Tubifex tubifex is widely cultured as a fish food. Stocking of certain T. tubifex strains has been shown to help prevent the occurrence or reduce the severity of whirling disease caused by Myxobolus cerebralis. Optimal culture conditions (e.g., temperature, density, and feed type) are not known for T. tubifex. We conducted four experiments to improve our knowledge of culture methods for T. tubifex. First, we evaluated the survival, growth, and recruitment of T. tubifex that were fed one of three different diets. We found that the performance of worms fed cow manure was poor and that growth and recruitment were best when they were fed either a commercial fish-flake food (Tetramin) or a commercial sinking fish feed containing spirulina Spirulina spp. In the second experiment, we evaluated the performance of worms fed Tetramin at rations of 0, 2.5, 5.0, and 10.0% of body mass/d and found the greatest growth and recruitment at the 5.0% and 10.0% rations. In the third experiment, ...

Effect of Potassium Permanganate Treatments on New Zealand Mud Snail Behavior and Survival and Rainbow Trout Growth and Condition

Abstract The New Zealand mud snail (NZMS) Potamopyrgus antipodarum, a nonnative species recently introduced into North America, has been shown to negatively influence ecosystem function. Methods to control or eliminate NZMS are needed. Twenty-four-hour treatments with 2 mg of potassium permanganate (KMnO4) per liter of water were evaluated against NZMS held for 24 h in artificial stream sections that contained refuge areas. The snails did not seek refuge when placed into an area that contained KMnO4 and also had neutral preference when provided the opportunity to select between artificial stream sections with and without KMnO4. In a separate series of experiments, NZMS and rainbow trout Oncorhynchus mykiss were placed into hatchery raceway sections that were treated with a 2-mg/L KMnO4 solution for 2 h/d over a period of 14 consecutive days. Mortality of NZMS and rainbow trout was not elevated in these raceways relative to nontreated control raceways. There was evidence, however, that the presence of the ...

Effect of Sodium Chloride, Tricaine Methanesulfonate, and Light on New Zealand Mud Snail Behavior, Survival of Snails Defecated from Rainbow Trout, and Effects of Epsom Salt on Snail Elimination Rate

Abstract The New Zealand mud snail (NZMS) Potamopyrgus antipodarum is an invasive species that threatens fish populations in North America. Establishment of NZMS in fish hatcheries is particularly problematic because NZMS could be inadvertently spread through fish stocking. Herein, we present the results of tests conducted to improve our understanding of (1) the risk of stocking fish from NZMS-infested hatcheries and (2) methods of NZMS control. We determined the proportion of NZMS that can survive passage through the digestive tracts of rainbow trout Oncorhynchus mykiss and whether the purgative Epsom salt (MgSO4) could increase excretion rates. We tested NZMS phototropism and whether light preference is influenced by food availability. Finally, we determined whether NZMS would contract into their shells when exposed to sodium chloride (NaCl) and whether the anesthetic tricaine methanesulfonate (MS-222) would induce NZMS muscle relaxation. Only 4.5% of snails survived passage through the digestive tracts...

Use of Penicillin and Streptomycin to Reduce Spread of Bacterial Coldwater Disease II: Efficacy of Using Antibiotics in Diluents and During Water Hardening

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, has lead to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid and can enter the egg during fertilization. The addition of antibiotics to diluents and water-hardening solutions could prevent the spread of the disease. In separate trials, a mixture of 0.197 mg/mL penicillin plus 0.313 mg/mL streptomycin was added to both a 0.5% sodium chloride fertilization diluent and hatchery well water during hardening. Tests showed that the addition of the antibiotics to the diluent and during up to 60 min of water hardening had no effect on the eye-up, hatch and deformity rates of Rainbow Trout Oncorhynchus mykiss eggs compared with the nonantibiotic-treated controls. Also, significant reductions in the prevalence of F. psychrophilum on the surface and inside eggs were observed when compared with controls. These results indicate that the addition of penicillin and streptomycin to diluents and during water hardening can prevent the vertical transmission of bacterial coldwater disease.

Effect of Single or Double Exposures to Hydrogen Peroxide or Iodine on Salmonid Egg Survival and Bacterial Growth

Abstract Four disinfection tests were conducted on the eggs of cutthroat trout Oncorhynchus clarkii, rainbow trout O. mykiss, and brown trout Salmo trutta. In each egg test, the effects of treating twice were determined, which simulated treatment at both the brood source and the receiving hatchery. Cutthroat trout eggs were treated at 1 h and again at 3 or 6 h after fertilization. Survival to the eyed stage was greater for eggs treated with 100 mg/L iodine for 10 min (86.9%) than with 1.5% hydrogen peroxide (H2O2) for 2 min (82.4%); the timing of the second disinfection had no effect on egg survival. Bacterial growth was significantly greater among H2O2-treated eggs. For eyed rainbow trout eggs, 2,000 mg/L iodine for 10 min was toxic (<11% hatch), but eggs treated either once or twice with hydrogen peroxide (1% H2O2 for 2 min) survived to hatch, as did the controls. Bacterial growth was significantly greater on eggs treated with H2O2 than control eggs treated with 100 mg/L iodine for 10 min. In a subseque...