Successful Rearing of Nutritionally Supplemented Rotifers (Brachionus plicatilis) at Reduced Salinity for Zebrafish (Danio rerio) Polyculture.

Rotifers, Brachionus plicatilis, are a valuable first exogenous feed for zebrafish because they can provide continuous nutrition for growing zebrafish larvae when used in a rotifer-zebrafish polyculture. Typically cultured at high salinities (>10 ppt), B. plicatilis are temporarily immobilized when moved to lower salinities (5 ppt) used for polycultures, decreasing their accessibility and attractiveness to the larvae. The nutritional value of rotifers varies based on their diet, typically live algae, which has limited nutritional value and may pose biosecurity risks. After confirming that rotifers consume and can reproduce when fed an irradiated, processed larval fish diet (PD), they were reared at 5 or 15 ppt, and fed various combinations of an algae mix and/or PD. Population densities and percentages of egg-bearing rotifers were quantified daily until the population density plateaued, and then their nutritional value was assessed. Results indicated that rotifers thrived at both salinities. Those fed PD were successfully maintained at >500 rotifers per mL and contained a greater ω-6/ω-3 fatty acid ratio. Our findings indicate that enriching rotifers with PD raised at 5 ppt can potentially eliminate rotifer immobilization in polyculture, while providing a nutritious, attractive diet for zebrafish larvae and decreasing biosecurity risks.

Evaluation of Media Conductivity and a Combination of Iodine and Sodium Hypochlorite Surface Disinfection on Zebrafish (Danio rerio) Embryo Viability and Morphology.

Embryo surface disinfection in either an iodine or sodium hypochlorite (NaOCl) solution is commonly performed on imported zebrafish embryos to decrease pathogen introduction into a facility. The impact of the consecutive use of these disinfectants and the conductivity of the culture media on embryo survival and development post-disinfection have not been evaluated. Iodine (12.5-25 ppm) is effective at eliminating several Mycobacterium species, whereas NaOCl (50-100 ppm) reduces the number of viable Pseudoloma neurophilia spores. Casper and T5D (tropical 5D wild type) embryos reared in media of differing conductivities (0-10, 100-200, 750-950, and 1500-2000 µS) with and without exposure to NaOCl 100 ppm at 6 h post-fertilization were evaluated for survival, hatching success, and morphological defects at 5 days post-fertilization. Additionally, the consecutive use of iodine (12.5 ppm for 2 min) followed by NaOCl (75 or 100 ppm for 10 min), as well as the inverse, was evaluated. Embryo survival was not impacted by embryo rearing media alone; however, survival significantly decreased when embryos were disinfected with 100 ppm NaOCl in media with a conductivity >750-950 µS. Iodine (12 ppm) and NaOCl (75 ppm) used sequentially resulted in >50% survival, whereas the use of 100 ppm NaOCl resulted in high levels of embryo mortality.

Impact of Pronase, Sodium Thiosulfate, and Methylene Blue Combinations on Development and Survival of Sodium Hypochlorite Surface-Disinfected Zebrafish (Danio rerio) Embryos.

Embryo surface disinfection is utilized in aquaculture to decrease the risk of pathogen introduction into established colonies. Zebrafish embryos are commonly disinfected with unbuffered sodium hypochlorite at 25-50 ppm for 10 min with or without concurrent treatment with chemicals, including pronase (Pron), sodium thiosulfate, and/or methylene blue; however, the impact of these chemicals on embryo survival and development has not been evaluated. In this study, AB and casper embryos were exposed to disinfection protocols that used Pron, sodium thiosulfate, and/or methylene blue (given alone, in various combinations, or all three combined) with 50 and 100 ppm sodium hypochlorite performed 6 and 24 h postfertilization (HPF). All groups were evaluated for survival, hatching, and malformations at 5 days postfertilization. Maximal survival (69%-97%) and hatching rates (66%-94%) were generally observed with sodium hypochlorite disinfection followed by exposure to both Pron and sodium thiosulfate and maintenance in standard embryo medium without methylene blue. Methylene blue had variable effects on survival and hatching. Higher survival and hatching rates were seen in AB embryos disinfected at 6 HPF and casper embryos disinfected at 24 HPF. Susceptibility to sodium hypochlorite toxicity differed by strain, emphasizing the need to test disinfection protocols on small embryo cohorts.

The Influence of Behavioral, Social, and Environmental Factors on Reproducibility and Replicability in Aquatic Animal Models.

The publication of reproducible, replicable, and translatable data in studies utilizing animal models is a scientific, practical, and ethical necessity. This requires careful planning and execution of experiments and accurate reporting of results. Recognition that numerous developmental, environmental, and test-related factors can affect experimental outcomes is essential for a quality study design. Factors commonly considered when designing studies utilizing aquatic animal species include strain, sex, or age of the animal; water quality; temperature; and acoustic and light conditions. However, in the aquatic environment, it is equally important to consider normal species behavior, group dynamics, stocking density, and environmental complexity, including tank design and structural enrichment. Here, we will outline normal species and social behavior of 2 commonly used aquatic species: zebrafish (Danio rerio) and Xenopus (X. laevis and X. tropicalis). We also provide examples as to how these behaviors and the complexity of the tank environment can influence research results and provide general recommendations to assist with improvement of reproducibility and replicability, particularly as it pertains to behavior and environmental complexity, when utilizing these popular aquatic models.

Transmission of Pseudoloma neurophilia in Laboratory Zebrafish (Danio rerio) When Using Mass Spawning Chambers and Recommendations for Chamber Disinfection.

Pseudoloma neurophilia, a microsporidium that primarily infects neural tissues, is a common pathogen in laboratory zebrafish. The risk of parasite transmission with different spawning apparatuses and the effectiveness of disinfection are unknown. In this study, we spawned uninfected zebrafish with P. neurophilia-infected zebrafish in either 50 L mass spawning chambers (MSCs) or 1 L standard breeding tanks (BTs). Fish were spawned once or thrice, with and without chamber disinfection between uses, to evaluate risk of vertical and horizontal transmission. Six disinfection protocols were tested to determine which effectively eliminated residual spores. We demonstrated that three consecutive uses of an MSC significantly increased the risk of transmission to other fish when compared to the use of BTs or only one spawning event in an MSC (both p < 0.0001). Vertical transmission was not detected with any method. Disinfection with ∼100 ppm bleach soak (pH ∼7.0), 75 ppm Wescodyne® soak, and 175 ppm Wescodyne Plus spray was 100% effective in eliminating spores from the MSCs. Disinfection of MSCs before spawning did not decrease P. neurophilia transmission when infected fish remained present in the breeding population. Researchers should avoid using endemically infected fish in MSCs to minimize transmission of pathogens within their colonies.

Recommendations for Health Monitoring and Reporting for Zebrafish Research Facilities.

The presence of subclinical infection or clinical disease in laboratory zebrafish may have a significant impact on research results, animal health and welfare, and transfer of animals between institutions. As use of zebrafish as a model of disease increases, a harmonized method for monitoring and reporting the health status of animals will facilitate the transfer of animals, allow institutions to exclude diseases that may negatively impact their research programs, and improve animal health and welfare. All zebrafish facilities should implement a health monitoring program. In this study, we review important aspects of a health monitoring program, including choice of agents, samples for testing, available testing methodologies, housing and husbandry, cost, test subjects, and a harmonized method for reporting results. Facilities may use these recommendations to implement their own health monitoring program.

Developmental and behavioral effects of toe clipping on neonatal and preweanling mice with and without vapocoolant anesthesia.

Toe clipping is used to identify and genotype preweanling mice, but the procedure generates concerns relevant to pain and distress. The few pertinent studies available evaluated mice between postnatal days (PND) 3 and 7, advocate the use of toe clipping in mice PND 7 or younger, and identify handling as the most distressing aspect of the procedure. Because both toe and tail clipping may be necessary in older mice to obtain sufficient DNA for genotyping, we surmised that performing these procedures concurrently to minimize handling would be beneficial. We also examined reflex development until PND 21 and adult behavior at 8 to 10 wk of age in mice toe clipped at PND 7 or 17 and the benefits of using topical vapocoolant anesthesia. C57BL/6J pups at PND 7 and 17 were assigned to 1 of 4 groups: 1) clipping of digit 3 of contralateral fore- and hindpaws; 2) toe clipping after topical vapocoolant anesthesia; 3) unclipped, unsprayed controls; and, 4) unclipped and vapocoolant-sprayed. Compared with unanesthetized pups, those sprayed with vapocoolant vocalized and struggled more when handled and had more bleeding, erythema, and swelling, which persisted for as long as 12 h after toe clipping. Reflex development, anxiety, locomotion, and motor coordination were not different among groups or with regard to the age of toe clipping. No tissue reaction was noted microscopically in paws collected at 10 wk of age. We conclude that the use of vapocoolant cannot be recommended due to its harmful effects and that toe clipping at PND 7 or 17 does not significantly affect the long-term welfare of mice.

Efficacy and safety of 5 anesthetics in adult zebrafish (Danio rerio).

Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other anesthetics used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) with MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss of equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced short-term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the upper half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered regardless of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful for short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must be maintained at approximately 10 °C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for surgical procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic agents due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthe- sia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures.

Evaluation of 5 cleaning and disinfection methods for nets used to collect zebrafish (Danio rerio).

Few standardized methods of cleaning and disinfecting equipment in zebrafish facilities have been published, even though the effectiveness of these procedures is vital to preventing the transmission of pathogenic organisms. Four chemical disinfectants and rinsing with municipal tap water were evaluated for their ability to disinfect nets used to capture zebrafish. The disinfectants included benzalkonium chloride+methylene blue, sodium hypochlorite, chlorine dioxide, and potassium peroxymonosulfate+sodium chloride for a soak time of 5 or 30 min. Disinfection effectiveness was evaluated by using an ATP-based system that measured the reduction in absolute number and percentage of relative light units. In addition, nets were cultured aerobically on blood and MacConkey agar plates to determine the number of bacteria remaining after disinfection procedures. Soaking nets in sodium hypochlorite for 30 min and in potassium peroxymonosulfate+sodium chloride for 5 or 30 min were effective means of disinfection, according to at least 90% reduction in the number of relative light units and no bacterial growth after cleaning. These results will aid facility managers, veterinarians and investigators in selecting net cleaning and disinfection protocols.

Trichodina xenopodus, a ciliated protozoan, in a laboratory-maintained Xenopus laevis.

A postmortem evaluation of a domestically bred, adult, female Xenopus laevis revealed the presence of a urinary bladder protozoan consistent with Trichodina xenopodus. T. xenopodus is considered an incidental finding, as its presence in the urinary bladder in frogs has not been correlated with disease or with urinary bladder epithelial lesions. Trichodina spp. are ciliated protozoa known to colonize many species of amphibians and fish. These protozoa frequently inhabit the skin and gills, but may also be present in the urinary bladder of infected animals. Their presence on the skin and gills in low numbers is not related to disease; however, large numbers may indicate poor water quality and overcrowding.

Mycobacteriosis in zebrafish colonies.

Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward.

An evaluation of preparation methods and storage conditions of tribromoethanol.

This reports the in vitro portion of a study designed to establish guidelines for the preparation, storage, and use of tribromoethanol (TBE). We evaluated: 1) the purity of TBE powder from three suppliers; 2) nine methods of preparation of a 25-mg/ml (working) solution for formation of particulates and breakdown products; 3) formation of particulates and breakdown products and pH change in 1-g/ml (stock) solutions and working solutions stored under four conditions (25 degrees C and 5 degrees C in light and in dark); and 4) stock and working solutions of TBE that caused lethal effects in mice. These objectives were met by using nuclear magnetic resonance spectroscopy, gas chromatography-mass spectroscopy, particle-size and turbidity analyses, and pH strips. TBE powder from three suppliers varied in purity. No significant differences in breakdown product formation, particle size, or turbidity were noted between the nine preparation methods evaluated. Stock solutions and the working solution stored at 5 degrees C in the dark maintained a pH of 6.5 to 7.0, whereas the pH dropped for all other working solutions. A low level of dibromoacetaldehyde (DBA), a potential breakdown product reported to cause toxic effects, was detectable in all newly prepared solutions. Regardless of the storage condition or pH, DBA concentration did not increase measurably in any of the solutions after 8 weeks. The stock and working solutions that demonstrated lethal effects in mice had a pH of 6.5 and did not differ notably from newly prepared, non-lethal solutions, when evaluated for DBA. A decrease in pH could not be correlated to an increase in DBA or potential lethality, as suggested in the literature. The toxicity associated with the lethal TBE in this study appears to be a result of a chemical reaction or breakdown product that has not yet been reported.

Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice.

This study, performed in conjunction with an in vitro evaluation of tribromoethanol (TBE), consisted of three trials with three objectives. The first objective was to compare anesthetic efficacy and short-term pathologic findings of TBE, ketamine-xylazine (K-X), and sodium pentobarbital (NaP). The second objective was to evaluate how changes of TBE that occur during the perceived most favorable and least favorable storage conditions (8 weeks at 5 degrees C in the dark [5D] and 25 degrees C with exposure to light [25L], respectively) affect anesthetic efficacy and short-term pathology when compared to newly prepared TBE. The third objective was to perform a 6-week clinical assessment of animals that received newly prepared TBE. All animals that received TBE (400 mg/kg) and 14 of 15 that received K-X (K, 120 mg/kg; X, 16 mg/kg) were anesthetized, as defined by loss of pedal reflex. In comparison, only 8 of 15 animals administered NaP (60 mg/kg) were anesthetized. Anesthetic duration for animals that received K-X was 31.7 min, which was significantly (P = 0.0085) longer than animals that received TBE (18.5 min). Recovery times for TBE and K-X were not significantly different (26.5 and 27.5 min, respectively). Pathologic lesions associated with TBE administration were significantly (P = 0.001) greater than those associated with K-X. NaP was not associated with any pathologic lesions. The pH of newly prepared and 5D TBE was 6.5 to 7.0, whereas that for 25L TBE was 3.0. Anesthetic induction, duration, recovery times, and pathologic lesions were not significantly different, regardless of the pH or storage condition of the solution. It was noted, however, that the average anesthetic duration for animals administered newly prepared TBE in the second trial was longer (37.7 min) than the first trial that used newly prepared TBE. For the third trial (long-term clinical assessment), the average anesthetic duration for TBE was 46.5 min, significantly (P < 0.025) longer when compared to the first trial that used newly prepared TBE. During the third trial, 10 animals were found dead or moribund. All animals that were found moribund were necropsied and found to exhibit a marked ileus. Because of the variability in anesthetic effectiveness, pathology, and morbidity and mortality associated with the use of TBE, we do not recommend the use of this anesthetic agent in ICR mice.