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Intestinal neoplasms and preneoplastic lesions are common in zebrafish research facilities. Previous studies have demonstrated that these neoplasms are caused by a transmissible agent, and two candidate agents have been implicated: a Mycoplasma sp. related to Mycoplasma penetrans and the intestinal parasitic nematode, Pseudocapillaria tomentosa, and both agents are common in zebrafish facilities. To elucidate the role of these two agents in the occurrence and severity of neoplasia and other intestinal lesions, we conducted two experimental inoculation studies. Exposed fish were examined at various time points over an 8-month period for intestinal histopathologic changes and the burden of Mycoplasma and nematodes. Fish exposed to Mycoplasma sp. isolated from zebrafish were associated with preneoplastic lesions. Fish exposed to the nematode alone or with the Mycoplasma isolate developed severe lesions and neoplasms. Both inflammation and neoplasm scores were associated with an increase in Mycoplasma burden. These results support the conclusions that P. tomentosa is a strong promoter of intestinal neoplasms in zebrafish and that Mycoplasma alone can also cause intestinal lesions and accelerate cancer development in the context of nematode infection.

In 2017, the zebrafish unit at University of Glasgow experienced a detrimental outbreak of pathogenic bacterium, Mycobacterium haemophilum. The presence of other bacterial species was also confirmed by bacteriology growth in the same unit. The affected individuals composed of a wild-origin parental population sourced from India and their F1 offspring generation. Bacteria were diagnostically confirmed to be present systemically in fish and within the water and biofilm of the recirculating zebrafish system. In the absence of a publicly accessible step-by-step disinfectant protocol for these difficult-to-eliminate pathogens, we devised a successful procedure to eradicate mycobacteria and Aeromonas species after colony removal using Cleanline Chlorine tablets (active ingredient Sodium dichloroisocyanurate) and Virkon Aquatic®. Postdisinfection diagnostics did not detect pathogens in the system or in the new fish inhabiting the system that were tested. Newly established fish colonies have not shown similar clinical signs or disease-induced mortality in the 1-year period following system disinfection and repopulation. We present a historical background of the bacterial outbreak and a disinfection method which can be replicated in other zebrafish facilities—at small or large scales—for reliable mycobacterium removal. This procedure can be implemented as a disinfection protocol before the introduction of a new fish population to a previously contaminated system.

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 zebrafish is a widely used animal model in biomedical research. Despite this, pathogens continue to be common in laboratory zebrafish. It is important to determine and describe the pathophysiology of cryptic infections on zebrafish to elucidate the impacts on experimental endpoints. Body condition is a basic measurement used experimentally and in health monitoring of animals. We exposed three wild-type zebrafish strains: AB, WIK, and 5D to Pseudoloma neurophilia. After 8 weeks postexposure, we individually imaged and processed fish for histology. Morphometric analysis was performed on images and an index of body condition was calculated based on the ratio of length/width from the dorsal aspect. Histological sections from each fish were examined to establish sex, severity of infection, and tissue distribution. We observed a significant decrease in body condition in female fish that was not observed in males. In addition, we observed a negative correlation between the total gonadal area of P. neurophilia exposed females and unexposed controls. These results illustrate the sex-specific impacts of a common chronic pathogen on zebrafish health and a commonly used experimental endpoint, further supporting the establishment of rigorous health monitoring programs in laboratory zebrafish colonies that include screening for chronic infectious diseases.

Small fish species, like zebrafish or medaka, are constantly gaining popularity in basic research and disease modeling as a useful alternative to rodent model organisms. However, the tracking options for fish within a facility are rather limited. Here, we present an aquatic species tracking database, Zebrabase, developed in our zebrafish research and breeding facility that represents a practical and scalable solution and an intuitive platform for scientists, fish managers and caretakers, in both small and large facilities. Zebrabase is a scalable, crossplatform fish tracking database developed especially for research fish facilities but can be easily adapted for a wide variety of aquatic model organisms housed in tanks. It provides sophisticated tracking, reporting, and management functions that help keep the animalrelated records well-organized including a QR code functionality for tank labeling. The implementation of various user roles ensures a functional hierarchy and customized access to specific functions and data. In addition, Zebrabase enables easy personalization of rooms and racks, and its advanced statistics and reporting options make it an excellent tool for creating periodic reports of animal usage and productivity. The communication between the facility and researchers can be streamlined by the requesting capabilities of the database. Finally, Zebrabase also features an interactive breeding history and a smart interface with advanced visualizations and intuitive color coding allowing to speed up the processes.

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

The current body of work on rearing larval/juvenile zebrafish is based on (1) utilization of freshwater and (2) diurnal light/dark cycle, (3) provision of live feed at modest density, and (4) culture in high visibility environment. We challenged these rearing approaches by maintaining zebrafish under constant light for 46-48 days (days postfertilization [dpf]), while securing continuous feeding in high turbidity and saline (1.8-2.1 parts per thousand) environment for the experiment's duration, allowing 24 h feeding/growth of fish from first exogenous feeding to maturation. There was no evidence of negative effects on zebrafish larvae behavior, growth, survival, and life cycle duration at constant illumination when food was continuously available. Zebrafish were stocked at high initial density (100 larvae/L) in a static system and fed high densities of rotifers (Brachionus plicatilis) (200-400/mL) from 6 to 12 dpf. Fish density was then reduced by 50% and two diet treatments, live rotifers or brine shrimp (Artemia) nauplii (10/mL), followed. Fish were reared on these two diets until first maturation. Performance of adult zebrafish fed live rotifer followed by Artemia nauplii diet was the highest recorded in the literature after 42 dpf, 250 ± 29 (males) and 430 ± 5 mg (females). Use of these rearing conditions, during the entire life cycle, until reproduction, resulted in the shortest ever recorded generation time (from egg to egg) of 43-45 dpf and fertilization rate (1 dpf) of 80.3%-94%.

Research into adult zebrafish often requires fish to be of a specific size. Currently, fish must be individually measured to achieve this goal. Here, we design and utilize fish graders to quickly sort fish by width. We characterize graders individually for the length of fish they discriminate between and we also analyze graders in pairs to define the range of lengths for a retained population of fish. We note that a 1 mm increase of fish width increases fish length by 6.2–7.2 mm. We provide the schematics to print a series of eight retention widths, and note that graders of any desired retention width can easily be printed by slightly modifying our design files.

The value of the zebrafish (Danio rerio) as a model organism continues to expand. In developing the model, current feeding practice in zebrafish laboratories includes the use of commercially available diets. In this study, we compared outcomes in growth, body composition, and reproduction among zebrafish fed five highly utilized commercial diets and one formulated chemically defined reference diet. Wild-type zebrafish larvae were raised on live feed until 21 days postfertilization and then fed diets for 16 weeks. All fish received a daily ration of >5% of body weight (adjusted biweekly). Growth varied among diets throughout the feeding trial, and at study termination (week 16), significant differences among diets were observed for terminal weight gain, body condition index, body fat deposition, and reproductive outcomes. In addition, the proportion of viable embryos produced from females fed the formulated reference diet was high relative to the commercial diets. These data suggest that metabolic profiles, most likely reflecting nutrient/energy availability, utilization, and allocation, vary relative to diet in zebrafish. Undefined differences in metabolic profiles could result in erroneous predictions of health outcomes and make comparisons among laboratories more challenging. We recommend that dietary standards should be defined for zebrafish to support their common utility in biomedical research.

Pseudocapillaria tomentosa is an important pathogen in zebrafish facilities. We investigated heat, ultraviolet (UV) light, chlorine, iodine, and dessciation for killing the parasite's eggs. Eggs released with feces larvate in about 5–10 days, and treatments were evaluated by exposing fresh eggs and subsequently comparing larvation to untreated eggs as an indication of survival. Collectively, untreated eggs in all trials showed high levels of survival. Eggs were exposed to elevated temperatures (40°C, 45°C and 50°C) for 1, 8, or 24 h, which resulted in substantial reduction in viability of eggs. UV radiation was effective, with no larvation at 50–300 mWs/cm(2) and

Zebrafish health is a primary research concern because diseases can have unintended impacts on experimental endpoints. Ideally, research would be conducted using disease-free fish or fish with known disease status. Mycobacteriosis is a common bacterial disease in wild and captive fishes, including zebrafish. Despite its prevalence, the dynamics of transmission and potential sources of mycobacterial infections in zebrafish are only partially understood. One suspected natural infection source is surface biofilms on tanks and other system components. This study investigates the role that tank biofilms play in mycobacteriosis in laboratory zebrafish by evaluating the establishment of biofilms from bacteria shed from fish, and conversely, the acquisition of infections in fish from surface biofilms. We found that zebrafish infected with Mycobacterium chelonae shed bacteria through feces, and bacteria are transmitted to tank biofilms from one to 16 weeks postinfection. We also found that zebrafish acquire M. chelonae infections as soon as 2 weeks when introduced to tanks with established M. chelonae biofilms. The results from this study highlight the role that tank biofilms play as both a reservoir and source of mycobacterial infections in zebrafish. Results support the inclusion of biofilm surveillance and prevention as part of a disease control program in zebrafish research facilities.

Light–dark cycles mimicking natural settings in a zebrafish facility are crucial for maintaining fish with an entrained circadian clock making them an ideal vertebrate model to study such rhythms. However, failure to provide optimal conditions to include complete darkness can lead to a disturbed circadian pacemaker affecting physiology and behavior in zebrafish. To meet building code requirements, the aquatics facility in use was outfitted with EXIT signs emitting a constant light. To determine if light radiating from the EXIT sign has an effect on zebrafish embryo production, 100 fish (1:1 m/f ratio) were split and housed at 10 fish/L. Half were housed directly in front of the EXIT sign, whereas the other half (control) were housed under a true 14-h light–10-h dark cycle. Reproductive success was evaluated by recording fecundity and viability from 10 weekly matings under two light colors: red (640 nm) and green (560 nm). On average the control group spawned twice as many embryos compared to those housed in front of a red EXIT sign, whereas green EXIT sign showed no difference. This suggests the importance of providing a complete dark environment within the night cycle and a recommendation toward dim green EXIT signs to avoid a decline in reproductive performance.

The use of early-stage zebrafish for biomedical research spans early organogenesis to free-swimming larva. A key benefit of this model organism is that repeated assessments spanning several days can be performed of individual larvae within a single experiment, often in conjunction with administered drugs. However, the initiation of feeding, typically at 5 days postfertilization (dpf), can make serial assessments challenging. Therefore, delayed feeding would increase the utility of the model. To ask whether feeding could be delayed without adversely affecting larval growth and development up to 39 dpf, we systematically raised zebrafish and introduced feeding at 5 dpf or delayed initial feeding up to 9 dpf. We assessed survival into the juvenile stage (39 dpf) and anterior-posterior length at this age as proxies for growth and development. Delaying feeding initiation up to 8 dpf did not decrease baseline survival of greater than 90%; survival decreased to 66% only when delayed to 9 dpf. Larval length was no different under any of these conditions. Our findings define 9 dpf as the critical age before which larval zebrafish must be fed when raising to 39 dpf. The option to delay feeding to 8 dpf will broaden experimental applications for the zebrafish larval model.

Cryopreservation is a common method used to preserve the sperm of various animal species, and it is widely used with zebrafish (Danio rerio). As with other animals, there is a possibility of paternal pathogen transmission through sperm. We evaluated the ability of five common and important pathogens of zebrafish to survive cryopreservation as used with zebrafish sperm and freezing without cryopreservant. We evaluated Mycobacterium chelonae, Mycobacterium marinum, and Edwardsiella ictaluri, each originally isolated from zebrafish, eggs of Pseuodocapillaria tomentosa, and spores of Pseudoloma neurophilia. Each mycobacterial isolate showed relatively minimal reduction in survival after freezing and thawing, particularly when subjected to cryopreservation. E. ictaluri also showed survival after cryopreservation, but exhibited a several log reduction after freezing at -80°C without cryopreservant. With P. neurophilia, two separate experiments conducted 3 years apart yielded very similar results, showing some, but reduced, survival of spores by using three different viability assays: SYTOX stain, Fungi-Fluor stain, and presence of a spore vacuole. Eggs of P. tomentosa showed no survival based on larvation of eggs when subjected to either freezing method. Given that four of the five pathogens exhibited survival after cryopreservation, we recommend that sperm samples or donor male zebrafish fish be tested for pathogens when sperm are to be stored by using cryopreservation.

The zebrafish (Danio rerio) has become a powerful model organism for studying developmental processes and genetic diseases. However, there remain several problems in previous rearing methods. In this study, we demonstrate a novel method for rearing zebrafish larvae by using a new first food, freshwater rotifers (Brachionus calyciflorus). Feeding experiments indicated that freshwater rotifers are suitable as the first food for newly hatched larval fish. In addition, we revisited and improved a feeding schedule from 5 to 40 days postfertilization. Our feeding method using freshwater rotifers accelerated larval growth. At 49 dpf, one pair out of 10 pairs successfully produced six fertilized eggs. At 56, 63, and 71 dpf, 6 out of the 10 pairs constantly produced normal embryos. Our method will improve the husbandry of the zebrafish.

The lack of standardized diet for laboratory animals can have profound effects on animal health and lead to less reproducible research outcomes. Live diets are commonly used in zebrafish culture and, although they are a more natural feed than flake or pellet food, are also a potential source of pathogens and toxic compounds. Heavy metals are a group of such compounds, which can accumulate in fish leading to developmental abnormalities, reduced growth, and increased rates of mortality. Two to three weeks after feeding adult zebrafish a new lot of nonhatching decapsulated brine shrimp cysts (Decaps), embryos at the University of Minnesota Zebrafish Core Facility (ZCF) and the University of Utah Centralized Zebrafish Animal Resource (CZAR) began to exhibit an orange color in the yolk, and larval health began to decline. The concentration of chromium in the Decaps (69.6 mg/kg) was more than 30 times that of other zebrafish diets tested (up to 2.1 mg/kg) and is thought to be the cause of the observed symptoms. Within 3 weeks of removing the Decaps from the feeding regimen, the orange coloration in the yolks began to diminish, the morphological abnormalities began to subside, and larval survival rates began to increase. Thus, implementation of standardized zebrafish diets and regular feed-quality testing may help to prevent the introduction of contaminants to zebrafish research facilities.

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

Zebrafish are a powerful model organism to study disease. Like other animal models, Danio rerio colonies are at risk of pathogenic infection. Microsporidia, a group of intracellular fungus-like parasites, are one potential threat. Microsporidian spores germinate and spread causing pathological changes in the central nervous system, skeletal muscle, and other anatomic sites. Infection can impair breeding, cause other morbidities, and ultimately be lethal. Previously, detecting microsporidia in zebrafish has required sacrificing animals for histopathologic analysis or microscopic examination of fresh tissues. Here, we show that fish with microsporidial infection often have autofluorescent nodules, and we demonstrate infectious spread from nodule-bearing fish to healthy D. rerio. Histologic analyses revealed that fluorescent nodules are granulomatous lesions composed of spores, degenerating muscle, and inflammatory cells. Additional histologic staining verified that microsporidia were present, specifically, Pseudoloma neurophilia. Polymerase chain reaction (PCR)-based testing confirmed the presence of P. neurophilia. Further PCR testing excluded infection by another common zebrafish microsporidial parasite, Pleistophora hyphessobryconis. Collectively, these studies show that P. neurophilia can induce skeletal muscle granulomas in D. rerio, a previously unknown finding. Moreover, since granulomas autofluoresce, microscopic screening for P. neurophilia infection is feasible in live fish, avoiding the need to sacrifice fish for surveillance for this pathogen.

In the underwater environment, the properties of light (intensity and spectrum) change rapidly with depth and water quality. In this article, we have described how and to what extent lighting conditions can influence the development, growth, and survival of zebrafish. Fertilized eggs and the corresponding larvae were exposed to different visible light wavelengths (violet, blue, green, yellow, red, and white) in a 12-h light–12-h dark (LD) cycle until 30 days posthatching (dph), when the expression of morphometric parameters and growth (igf1a, igf2a)- and stress-related (crh and pomca) genes were examined. Another group of larvae was raised under constant darkness (DD) until 5 or 10 dph, after which they were transferred to a LD of white light. A third group remained under DD to investigate the effects of light deprivation upon zebrafish development. The results revealed that the hatching rate was highest under blue and violet light, while total length at 30 dph was greatest under blue, white, and violet light. Red light led to reduced feeding activity and poor survival (100% mortality). Larvae raised under constant white light (LL) showed a higher proportion of malformations, as did larvae raised under LD violet light. The expression of growth and stress factors was upregulated in the violet (igf1a, igf2a, pomca, and chr) and blue (igf2a) groups, which is consistent with the higher growth recorded and the higher proportion of malformations detected under the violet light. All larvae kept under DD died before 18 dph, but the survival rates improved in larvae transferred to LD at 5 dph and at 10 dph. In summary, these findings revealed that lighting conditions are crucial factors influencing zebrafish larval development and growth.

Mycobacterium chelonae is widespread in aquatic environments and can cause mycobacteriosis with low virulence in zebrafish. The risk of infection in zebrafish is exacerbated in closed-recirculating aquatic systems where rapidly growing mycobacteria can live on biofilms, as well as in zebrafish tissues. We have discovered a method of identifying and visualizing M. chelonae infections in living zebrafish using endogenous autofluorescence. Infected larvae are easily identified and can be excluded from experimental results. Because infection may reduce fertility in zebrafish, the visualization of active infection in contaminated eggs of transparent casper females simplifies screening. Transparent fish are also particularly useful as sentinels that can be examined periodically for the presence of autofluorescence, which can then be tested directly for M. chelonae.

Large breeding tubercle (BT) clusters, spike-like keratinized epidermal structures, have been identified along the middorsal surface of wild-type (AB, Ekkwill, Wik, Tuebingen) and long-fin mutant male zebrafish pectoral fin rays.1–4 The androgen-dependent growth of BT clusters occurs only in males and appears around the time of sexual maturation.1 Once present, these structures are maintained through the continuous renewal of the most superficial keratinized layer for the entire life of the fish, playing an essential role in stimulating egg release during spawning.2 The constant presence of these easily visualized structures only in males allows for the quick distinction of sex. Current guidelines available for distinguishing males from females rely on differences in the color, shape, and behavior of these fish. Generally, males appear pinkish-gold, possess a streamlined body, and chase females before mating. Females appear bluish-white and possess a protruding egg-filled abdomen.5–7 These guidelines have been proven subjective. For example, not all females possess an obvious distended abdomen, and there are reports of similar coloring in males and females.8 Although females are distinguishable from males by the presence of genital papilla,9 these ventrally located structures are small and difficult to observe in nonanesthetized fish. Overall, the ambiguities of previously set guidelines make the determination of zebrafish sex difficult, particularly for new students and staff members. In this study, we report the use of sexually dimorphic pectoral fin BTs as a means to facilitate in the identification of male and female zebrafish. The determination of sex using BTs requires that zebrafish are placed in a small fish tank that sits on a light-colored background. Examination of the dorsal surface of each spread pectoral fin will reveal the presence or absence of BTs. BTs are clearly visible from above as brown lines along the central fin rays of males (Fig. 1A). Females lack BTs and thus their pectoral fins are translucent (Fig. 1B). Together, these characteristics allow for the immediate distinction of zebrafish sex (Fig. 1C). BTs are easily visible in adult (AB, Ekkwill, Wik, Tuebingen, and long-fin mutant) zebrafish without any microscopic aids. However, a stereomicroscope can be used to identify small BTs on young, lightly anesthetized, fish as small as 1.3 cm or 2.5 months.1 A small bowl of 0.17 mg/mL of tricaine (ethyl-aminobenzoate) can be placed directly under the stereomicroscope.7 Fish immersed in tricaine solution are immobilized in seconds, spreading their pectoral fins laterally, enabling an immediate unobstructed view of the fin rays. Under the stereomicroscope, the spike-like BT structures are easily identifiable along the central fin rays1 (Fig. 1D–G). Fish exposed to tricaine will eventually flip upside down; however, dorsally located BTs are still visible from the ventral surface of pectoral fins due to inter-ray tissue transparency. Given the ease at which BTs are observed under the stereomicroscope, the determination of sex should take a maximum of 30 seconds. FIG. 1. Distinguishing male and female zebrafish based on large clusters of breeding tubercles (BTs). (A) Male zebrafish possess BT clusters, observed as brown lines, along the central pectoral fin rays (blue arrowheads). (B) Female pectoral fins are translucent, ...

A workshop to address husbandry and animal welfare was held during the 9th European Zebrafish Meeting in Oslo, Norway, from June 28 to July 2, 2015. The husbandry workshop took place on Monday, June 29, and was well attended by ∼100 audience members. It highlighted problems arising from the diversity of current husbandry practices and included presentations on recent efforts to find common husbandry and animal welfare standards from a variety of international contributors, from Norway, Portugal, the United Kingdom, as well as the United States and Japan. Presentations included zebrafish and medaka as representatives of aquatic species used in biomedical research and addressed a diverse range of topics such as proposed European guidelines for zebrafish husbandry, general fish facility health and husbandry standards, cryopreservation, publication standards, and feeding strategies. The workshop highlighted the desire to develop common husbandry standards for the aquatic research community across the world.

The goal of this study was to evaluate plasma membrane integrity and motility for zebrafish sperm quality assessment along the cryopreservation pathway-from sample collection through refrigerated storage, cryoprotectant equilibration, freezing, thawing, and fertilization. The objectives were to: (1) evaluate the effects of osmolality, extender, and refrigerated storage on sperm plasma membrane integrity and motility, and (2) compare cryopreservation of sperm from farm-raised and well-characterized research populations by evaluating motility and membrane integrity of fresh, post-equilibration (before freezing) and post-thaw sperm, and post-thaw fertility. Osmolality, extender, and storage time each influenced sperm motility and membrane integrity. Isotonic osmolality showed the best protection for motility and membrane integrity compared to hypotonic and hypertonic osmolalities. Of the four tested extenders, Hanks' balanced salt solution (HBSS) and Ca(2+)-free HBSS showed the best protection compared with NaCl and glucose, and sperm retained motility and membrane integrity for 24 h of refrigerated storage. Sperm cryopreservation of zebrafish from a farm population (n = 20) and an AB research line (n = 20) showed significant differences in post-thaw fertility (32% ± 18% vs. 73% ± 21%). No differences were found in post-thaw motility, although the farm-raised zebrafish possessed a larger body size, testis weight, and higher fresh motility. Correlation analysis of pooled data did not identify correlations among motility, flow cytometry analysis of membrane integrity and recognizable cells, and post-thaw sperm fertility (p ≥ 0.202). More research is needed to standardize the fertilization conditions especially sperm-to-egg ratio to avoid possible overabundance of sperm to obscure the differences.

Cryopreservation of sperm from Xiphophorus fishes has produced live young in three species: X. hellerii, X. couchianus, and X. maculatus. In this study, the goal was to establish protocols for sperm cryopreservation and artificial insemination to produce live young in X. variatus, and to identify needs for repository development. The objectives were to: 1) collect basic biological characteristics of males; 2) cryopreserve sperm from X. variatus, 3) harvest live young from cryopreserved sperm, and 4) discuss the requirements for establishment of sperm repositories. The 35 males used in this study had a body weight of 0.298±0.096 g (mean±SD), body length of 2.5±0.2 cm, and testis weight of 6.4±3.4 mg. The sperm production per gram of testis was 2.33±1.32×10(9) cells. After freezing, the post-thaw motility decreased significantly to 37%±17% (ranging from 5% to 70%) (p=0.000) from 57%±14% (40%-80%) of fresh sperm (N=20). Artificial insemination of post-thaw sperm produced confirmed offspring from females of X. hellerii and X. variatus. This research, taken together with previous studies, provides a foundation for development of strategies for sperm repositories of Xiphophorus fishes. This includes: 1) the need for breeding strategies for regeneration of target populations, 2) identification of minimum fertilization capacity of frozen samples, 3) identification of fish numbers necessary for sampling and their genetic relationships, 4) selection of packaging containers for labeling and biosecurity, 5) assurance of quality control and standardization of procedures, 6) information systems that can manage the data associated with cryopreserved samples, including the genetic data, 7) biological data of sampled fish, 8) inventory data associated with frozen samples, and 9) data linking germplasm samples with other related materials such as body tissues or cells saved for DNA and RNA analyses.

Despite the growing popularity of the zebrafish model system, the optimal husbandry conditions for this animal are not well defined. The aim of this study was to examine the effect of stocking density on reproductive performance in zebrafish. In this study, undertaken by eight different zebrafish facilities, clutches of at least 200 wild-type zebrafish embryos from a single pairwise mating were produced at each participating institution and subsequently reared according to "in-house protocols" until they were 14 weeks old. Fish were then randomly assigned into treatment groups with balanced sex ratios and densities of 3, 6, or 12 fish/L. After a 1-month acclimation period, fish were spawned in pair crosses every 2 weeks for 3 months, for a total of six spawning dates. The number of viable and nonviable embryos produced in each clutch were counted at 1 day postfertilization. Although there was a great deal of variability in clutch size and percent spawning success among laboratories, there were no significant differences in average clutch size, spawning success, or percent viable among the treatment densities. These data suggest that using stocking densities as high as 12 fish/L does not have a negative impact on performance, when measured by reproductive performance.

One caveat to current loss-of-function approaches in zebrafish is that they typically disrupt gene function from the beginning of development. This can be problematic when attempting to study later developmental events. In vivo electroporation is a method that has been shown to be effective at incorporating reagents into the developing nervous system at multiple later developmental stages. The temporal and spatial characteristics of in vivo electroporation that have been previously demonstrated suggest that this could be a powerful approach for time-resolved loss-of-function analysis. Here, in an attempt to demonstrate the efficacy of this approach for analysis of a specific developmental timeframe--that of initial development of the zebrafish visual system-we have done a systematic characterization of the efficiency of in vivo electroporation in zebrafish across multiple developmental stages, from 24 to 96 h postfertilization. We show that electroporation is efficient at delivering expression plasmids to large numbers of neurons at multiple developmental steps, including 24, 48, or 96 h postfertilization. Expression from electroporated plasmids is maximal within 24 h, and significant and useful expression is seen within 6 h. Electroporation can be used to deliver two separate expression plasmids (green fluorescent protein and mCherry), resulting in coexpression in 97% of cells. Most importantly, electroporation can be used to incorporate siRNA reagents, resulting in 84% knockdown of a target protein (green fluorescent protein). In conclusion, in vivo electroporation is an effective method for delivering both DNA-based expression plasmids and RNA interference-based loss-of-function reagents, and exhibits the appropriate characteristics to be useful as a time-resolved genetic approach to investigate the molecular mechanisms of visual system development.

The need to develop standardized diets to support zebrafish (Danio rerio) research is supported by the knowledge that specific dietary ingredients, nutrients, or antinutritional factors in diets have been shown to affect development and growth of adult D. rerio and their offspring. In this study, there were seven dietary treatments consisting of five commercially available diets and two laboratory-prepared diets, three replicates per treatment. Fish were fed ad libitum twice daily for 9 weeks. At 9 weeks, both weight and length were recorded to determine condition indices. D. rerio fed one of the laboratory-prepared diets had significantly higher weights than individuals fed any of the other diets and exhibited significantly higher lengths than those fed five of the six remaining diets. Although there were significant differences in general growth demographics (length/weight) after the 9-week feeding trial, no significant differences in overall health of D. rerio were observed for the different dietary treatments as determined by statistical analysis of condition factor indices (K = [weight x 100]/length(3)). The success achieved with the laboratory-prepared diets represents the foundation for establishing an open-formulation nutritional standard to ensure that the D. rerio model for research does not generate confounding research results caused by nutritional vagaries.

Captive zebrafish (Danio rerio) exhibit a limited repertoire of mating behaviors, likely due to the somewhat unnatural environment of aquaria. Observations in their natural habitat led us to believe that a depth gradient within the mating setup would positively affect fish mating. By tilting the tank to produce a depth gradient, we observed novel behaviors along with a preference for oviposition in the shallow area. Although we did not see an increase in the likelihood of a pair of fish to mate, we did see an increase in the embryo output in both adults and juveniles. In the adults, tilting led to a significant increase in embryo production (436 +/- 35 tilted vs. 362 +/- 34 untilted; p < 0.05). A similar effect was seen in juvenile fish as they progressed through sexual maturity. These results suggest that tilting of mating cages in the laboratory setting will lead to demonstrable improvements in embryo production for zebrafish researchers, and highlights the possibility of other manipulations to increase fecundity.

Previously established rearing protocols for zebrafish begin feeding with marine rotifers (Brachionus plicatilis), followed by Artemia nauplii until the fish reach subadult stage, the developmental time point at which they can be most easily transitioned onto a processed diet. However, the inclusion of Artemia is less than ideal, given its fluctuating availability and high costs. We tested whether or not we could replace Artemia with rotifers during our normal rearing sequence and still meet published performance standards for (i) weaning fish onto a processed diet by 25 days postfertilization (dpf) and (ii) successful breeding by 60 dpf. Here, we present the results of trials where wild-type and casper zebrafish were fed exclusively with rotifers (R) or rotifers followed by Artemia (RA) for the first 25 dpf after which point all fish were transitioned to a processed diet (Gemma Micro 300). We measured growth and survival at days 25 and 60, and tested for reproductive capability at 60 dpf. While growth performance was significantly better in the RA groups, we were still able to meet goals for both weaning and generation time in the R groups without compromising survival or sex ratios.

Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

Commercially available aquatic housing systems provide excellent and relatively trouble-free hardware for rearing and housing juvenile as well as adult zebrafish. However, the cost of such systems is quite high and potentially prohibitive for smaller educational and research institutions. The need for tank space prompted us to experiment with various additions to our existing Aquaneering system. We also noted that high water exchange rates typical in commercial systems are suboptimal for quick growth of juvenile fish. We devised a housing system we call “SideRack,” which contains 20 large tanks with air supply and slow water circulation. It enables cost-effective expansion of existing fish facility, with a key additional benefit of increased growth and maturation rates of juvenile fish.

A 5-week study was conducted to evaluate commercially available Artemia, Ziegler zebrafish diet, and Calamac diet fed in five different feeding regimes on the growth and reproductive development of 7-month-old zebrafish. Zebrafish were fed to satiation three times daily during the normal work week and twice daily during the weekend and holidays. Zebrafish in dietary groups CCC (Calamac three times daily) and CCA (Calamac twice daily, Artemia once daily) had a significantly (p

The importance of nutritional components on growth and body composition outcomes has been demonstrated in multiple model organisms. Although zebrafish (Danio rerio) have an established role in research laboratories for its utility in understanding developmental biology and genetics, the influence of diet composition on basic growth outcomes is less well demonstrated. In the current study, four protein sources were tested in isolation using isonitrogenous diets or combined using a defined lab diet. Fish (n≈60/group) were group housed (n≤10 fish/1.8 L tank) and fed ad libitum three times daily for 12 weeks. Fish were assessed for effects on length, body weight, and body composition (lean and fat mass). Individuals fed wheat gluten protein were significantly shorter in length, with significantly lower body weight and lean mass in both male and female fish, although percent body fat was high compared with other diets. Casein-fed fish similarly had significantly reduced body length, body weight, and lean and fat mass in both male and female fish, with a low percent body fat compared with other diets (leanest). Fish protein hydrolysate-fed fish had significantly lower lean mass and a high percent body fat, whereas soy protein isolate diet performed similarly to a mixed-protein control diet for all measured outcomes. These results suggest that the protein source, with accompanying amino acid ratios or additional protein source differences, has a significant impact on growth and body composition outcomes in zebrafish when fed in a semipurified, defined diet background.

Zebrafish (Danio rerio) has recently emerged as a new animal model in neuroendocrinology and behavior (e.g., stress physiology and ecotoxicology studies). In these areas, the concentrations of steroid hormones in the blood are often used to study the endocrinological status of individuals. However, due to the small body size of zebrafish, blood sampling is difficult to perform and the amount of plasma obtained per sample for assaying hormones is very small (ca. 1–5 μL), and therefore most studies have been using whole-body hormone concentrations, which implies sacrificing the individuals and hampers sequential sampling of the same individual. Here a noninvasive method to assay steroid hormones from zebrafish holding-water, based on the fact that steroids are released into the fish holding-water through the gills by passive diffusion, is validated. Cortisol and the androgen 11-ketotestosterone (KT) were measured in water samples and compared to plasma levels in the same individuals. Cortisol released to holding-water correlates positively with plasma concentrations, but there was a lack of correlation between KT water and circulating levels. However, KT levels showed a highly significant sex difference that can be used to noninvasively sex individuals. An ACTH challenge test demonstrated that an induced increase in circulating cortisol concentration can be reliably detected in holding-water levels, hence attesting the responsiveness of holding-water levels to fluctuations in circulating levels.

One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

Research involving model organisms necessitates recording and archiving many types of animal maintenance and use data. We developed a comprehensive inventory system using FileMaker Pro® to incorporate, record, and archive data on zebrafish stocks, tank organization, husbandry, and fish usage. Our relational database is constructed of tables containing detailed information on fish identity, parents of origin, tank location, mutant phenotypes, caretakers, natural mating and in vitro fertilization experiments, and fish mortality. In addition to its basic annotation and reporting capabilities, the database allows barcode scan entry of several actions, for example, moving a tank of fish, mating or performing in vitro fertilization with specific fish, and recording dead fish. All data are input in real time using either barcode scanning or manual entry. The database provides several types of preformatted reports, as well as printed labels for tank location and stock identification. In summary, we have created a versatile, multipurpose inventory system that can be personalized and enhanced for any zebrafish facility and can be further adapted to organize data and archival information for other model systems or applications.

The goal of this study was to establish an efficient method for determination of sperm concentration requiring only 1-2 microL of sample by use of microspectrophotometry. The objectives were (1) determination of wavelengths with absorbance profiles appropriate for analysis of sperm suspensions from zebrafish Danio rerio, green swordtail Xiphophorus helleri, and medaka Oryzias latipes collected by crushing of dissected testis or by stripping of live males; (2) generation of standard curves and equations between sperm sample absorbance and sperm concentration estimated by hemocytometer counts; (3) accuracy verification of equations for estimating concentration by microspectrophotometry; and (4) analysis of the precision in generating equations and estimation of sperm concentration. Within the visible wavelengths (380-750 nm) there was no single maximal absorbance peak. For zebrafish, a linear correlation was established with an effective absorbance range of 0.034-0.936 for crushed samples, and 0.028-0.961 for stripped samples at 400 nm. For Xiphophorus, the effective absorbance range was 0.014-1.154 for crushed samples, and the effective range was 0.038-1.082 for stripped samples. For medaka, the effective range was 0.041-0.896 for crushed samples. The accuracy of these equations was verified by comparison of sample concentrations counted with hemocytometer and calculated with equations, and no significant differences (p = 0.447) were observed. Measurement of serially diluted aliquots from pooled samples verified the precision of techniques used. Overall, this confirmed that microspectrophotometric estimation of sperm concentration is accurate, efficient, and sample-saving for use with small-bodied fishes.

The growth of the zebrafish as a model organism has so far greatly outpaced regulatory oversight governing its care and use in research. However, the same levels of regulation that characterize the use of traditional mammalian model animals will likely be extended to zebrafish, especially as use of the animal continues to increase. The challenge for both the zebrafish research community and regulatory agents is to ensure that emerging guidelines are sensible and serve to promote quality science and the highest standards of animal care. This brief review summarizes a panel discussion that was convened on this subject at the World Aquaculture Society's Aquaculture America 2009 Conference in Seattle, Washington. The current regulatory environment in the United States is discussed, and a set of generalized recommendations for developing new guidelines is presented.

We have constructed a standalone, modular, low-cost water recirculation zebrafish facility in our laboratory. This is easy to maintain and will take only approximately $500 and a half a day to build an 80-tank module. The system described here will be useful to even investigators who are new to zebrafish aquaculture.