We sincerely thank the support from Chongqing Medical University Talent Project (No. R4014 to DSP and R4020 to PPJ), National Natural Science Foundation of China (NSFC, No.32200386 to PPJ), Chongqing Postdoctoral Innovation Mentor Studio (X7928 DSP), and Program of China-Sri Lanka Joint Center for Water Technology Research and Demonstration by Chinese Academy of Sciences (CAS)/China-Sri Lanka Joint Center for Education and Research by CAS.

The fish experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Chongqing and the Institutional Animal Care and Use Committee of Chongqing Medical University, China, as well as the standards for experimental animals issued by the State Bureau of Quality and Technical Supervision (Approval ID: GB14922-2001 to GBT14927-2001). Zebrafish (Danio rerio, wild type, AB strain) were sourced from the Institute of Hydrobiology, Chinese Academy of Sciences, and maintained in the laboratory following previously reported procedures36.
1. Adult zebrafish maintenance and embryo collection
NOTE: Drawing upon modifications from previous studies and reports16,37,38,39, the maintenance of adult zebrafish, larvae rearing, and practices for germ-free (GF) models in our laboratory were carried out following the steps outlined below. The ultra-pure flow system for fish culture was commercially obtained (see Table of Materials). The water, maintained with a balanced pH, salt, and alkali, undergoes filtration during cycling to ensure clean conditions.
<ol>
	<li>Maintain adult fish with the following standard procedure in the auto cycle system (see Table of Materials) with a photoperiod of the dark: light = 10 h: 14 h at 28 &#176;C &#177; 0.5&#160;&#176;C and fed with fresh incubated Artemia sp. twice daily.</li>
	<li>Put the sexually mature adult zebrafish (male: female = 2:1) into tanks with fresh and clean water the night before.</li>
	<li>Let the fish spawn the next morning (approximately 8:30 a.m.) under regular light stimulation in the laboratory. After 1-2 h, transfer the fish to another tank.</li>
	<li>Collect embryos into water from the auto-cycling system as the rearing medium in a culture dish using a disposable Pasteur pipette.</li>
</ol>
2. Generation of GF zebrafish from embryos to larvae
NOTE: The procedure for generating germ-free (GF) fish is outlined in Figure 1, while the basic developmental indexes of conventional (CR) and GF models are presented in Supplementary Figure 1. Please follow the steps outlined below in a benchtop biological safety cabinet or laminar flow hood using a sterile aseptic technique in a clean room.
<ol>
	<li>Wash the embryos using the fish culture water and remove the feces impurities and dead or unfertilized eggs.</li>
	<li>Transfer the embryos at 2 hpf to sterilized plates (up to 480 embryos per sterile dish with a 10 cm diameter) filled with antibiotic germ-free zebrafish medium (AB-GZM, see Table of Materials) solution and culture at 28.5 &#176;C for 6-8 h. 
	NOTE: AB-GZM is used to treat the embryos for several hours to eliminate the surface microorganisms, and GZM without antibiotics is used to culture GF fish in subsequence. Usually, the perfected culture time is 6 h).</li>
	<li>Exclude eggs with white spots or a whitish appearance, and select those that are normally developed, displaying transparent and intact envelopes for further processing.</li>
	<li>Rinse the embryos using AB-GZM three times within 10 min.</li>
	<li>Soak the embryos in 0.2 g/L povidone-iodine solution (PVP-I) for 1 min (see Table of Materials). 
	NOTE: Higher concentration and longer than 2 min will influence the death and hatching rate of embryos.</li>
	<li>Wash the embryos using germ-free zebrafish medium (GZM) three times within 10 min.</li>
	<li>Add embryos into sodium hypochlorite (NaClO) working solution, tightly cover the 50 mL microcentrifuge tubes, and bleach for 15 min. During this period, suspend the embryos in bleach solution by gently shaking them.</li>
	<li>Wash the embryos using GZM three times within 10 min.</li>
	<li>Transfer the embryos to sterile 6-well plates with 5 embryos and 10 mL of GZM per well. Culture them in an ultra-clean platform or incubator with a photoperiod of dark: light = 10 h: 14 h at 28 &#176;C &#177; 0.5 &#176;C.The culture density will influence the sterile rate.</li>
	<li>Renew the culture medium daily by removing the spent GZM, collecting it as samples for sterile status detection, and replenishing each well with the same volume of sterile GZM.</li>
	<li>Record the basic developmental indexes of GF embryos/larvae, including the survival rate, hatching rate, heartbeats, body length and weight, etc19.</li>
	<li>Transfer the GF fish to suitable containers (dishes, cell culture flasks, or glass bottles with covers) with expanded volume, providing sterile food throughout the growth process.</li>
</ol>
3. Maintenance of GF zebrafish from larvae to juvenile
<ol>
	<li>Renew the GZM without feeding before 7 dpf and daily detect the sterile states (see step 5).</li>
	<li>Feed zebrafish larvae from 8 dpf to juvenile stages with sterile egg yolk (10 &#181;L prepared stock solution per well) once daily before changing GZM.</li>
	<li>Feed GF juveniles from 14 dpf with sterilized egg yolk mixed with GF Artemia sp. once daily (1-3 Artemia/larvae, see step 4), gradually increasing food mass with the growth and development of GF fish.</li>
	<li>Provide egg yolk until all fish can consume Artemia nauplii. 
	NOTE: GF Artemia should undergo sterility testing before use. Assess the number of Artemia nauplii left, observe pursuit and capturing progress, and examine the fish body with consumed food to indicate feeding success.</li>
	<li>From 28 dpf to early adult stages, feed GF Artemia once daily (3-5 Artemia/larvae) and clean untapped or dead Artemia, and dead fish into the waste GZM as daily samples.</li>
	<li>During GF fish growth, change the 6-well plates after 7 dpf to sterile plates or cell culture flasks from 8 to 21 dpf, and then to sterile bottles after 21 dpf. Increase culture medium and food mass according to the number and weight of GF fish in each container.</li>
	<li>Renew GZM daily and check samples to ensure the success of GF models. 
	NOTE: Maintaining germ-free (GF) fish models until early adulthood and sexual maturity is challenging, with a low average survival rate at 30 dpf, typically less than 10%. This is primarily attributed to weak immunity, delayed development, and impaired intestinal function.</li>
</ol>
4. Preparation of GF Artemia nauplii as the sterile food for chronic GF fish models
NOTE: The cultivation and preparation method of germ-free (GF) Artemia is outlined in Figure 2. Note that all the following steps are conducted in a benchtop biological safety cabinet, using a sterile aseptic technique.
<ol>
	<li>Rinse 0.25 g of Artemia cysts using 2.4 g/L sodium hypochlorite (NaClO, see Table of Materials) for 5 min.</li>
	<li>Filter the rinsed Artemia cysts with a sterilized ultra-dense filter (about 170 meshes in aperture) and wash with sterilized ultrapure water three times, each time for 1-2 min.</li>
	<li>Transfer the washed Artemia cysts into 100 mL of 2.5% sterilized saline water, mix, and package for aseptic hatching according to the steps mentioned below.</li>
	<li>Pack the 50 mL treated Artemia cysts mixture solution into a sterile bottle with a volume of 100 mL, seal it with film, and incubate in a shaking incubator at 150 rpm, 30 &#176;C for 18 h to 24 h with light.</li>
	<li>Extract approximately 30 mL of hatching Artemia nauplii from the middle and lower layers using a disposable Pasteur pipette. 
	NOTE: Prevent floating eggs and empty shells from reaching the upper layer. The indexes of germ-free (GF) Artemia cysts and nauplii incubated for 24 h are presented in Figure 3.</li>
	<li>Filter the Artemia with a sterilizing filter and wash them in a sterilizing beaker with sterilizing ultrapure water three times, about 30 s to 1 min for each time. Finally, add 4 mL of sterilized ultrapure water to prepare the Artemia mixed solution.</li>
	<li>Using a disposable Pasteur pipette, retrieve actively swimming Artemia from the upper layer, wash, and prepare the nauplii solution for feeding and aseptic identification.</li>
</ol>
5. Identification of GF fish models at the whole life stages
NOTE: Throughout the entire life stages of germ-free (GF) fish, key indexes and daily sample detections are crucial to ensure the success of the models. This step summarizes the common classification of samples and the usual methods for identification (Figure 4).
<ol>
	<li>Observe and count the survival status and rate of GF zebrafish larvae daily. In experiments calculating related rates, GF fish are cultured in 24- or 48-well plates with one fish per well.
	<ol>
		<li>Survival rate = (number of live fish at the time of observation / total number of eggs) &#215; 100%.</li>
		<li>Sterility rate = (number of sterile fish/number of surviving fish) &#215; 100%. 
		NOTE: This protocol focuses on the sterile rate of live fish. The number of sterile fish is determined by counting successful GF fish after multiple checks among the live fish. Any dead fish or live fish GF models that have failed due to bacterial presence are eliminated during subsequent GF procedures.</li>
	</ol>
	</li>
	<li>Collect the following samples of GF zebrafish larvae.
	<ol>
		<li>Culture medium of GF zebrafish from each well or bottle container.</li>
		<li>Fish food, such as sterile egg yolk and GF Artemia.</li>
		<li>Waste medium, including fragments of egg membrane after hatching and food residue or feces during the feeding periods from larvae to juvenile fish.</li>
		<li>Dead fish samples to identify the presence of bacteria.</li>
		<li>Fish medium and agents used in embryo treatment as the sterile control.</li>
		<li>Samples from materials, operating platform, and incubator, etc.</li>
	</ol>
	</li>
	<li>Detect samples collected daily using multiple methods.
	<ol>
		<li>First, use the spread plate method and culture plates in an incubator at 30 &#176;C.
		<ol>
			<li>Coat with 20-50 &#181;L of waste medium sample on the Trypticase Soy Agar (TSA) plate (see Table of Materials) under aerobic conditions.</li>
			<li>Coat with 20-50 &#181;L of waste medium sample on the TSA plate under anaerobic conditions.</li>
			<li>Coat with 20-50 &#181;L of waste medium sample on the blood TSA plates (see Table of Materials) under aerobic conditions.</li>
			<li>Coat with 20-50 &#181;L of waste medium sample on the blood TSA plates under anaerobic conditions.</li>
		</ol>
		</li>
		<li>Second, use the liquid culture method.
		<ol>
			<li>Take 200 &#181;L of sample into aerobic tubes with Brain Heart Infusion Broth (BHI) medium (see Table of Materials) and culture in the shaking incubator at 30 &#176;C, 150-180 rpm.</li>
			<li>Add 200 &#181;L of sample into anaerobic tubes with BHI medium and culture in the incubator at 30 &#176;C.</li>
			<li>Add 200 &#181;L of sample into aerobic tubes with Trypticase Soy Broth (TSB) medium, then culture in the shaking incubator at 30 &#176;C, 150-180 rpm.</li>
			<li>Add 200 &#181;L of sample into anaerobic tubes with TSB medium and culture in the incubator at 30 &#176;C.</li>
		</ol>
		</li>
		<li>Third, use molecular techniques-16s polymerase chain reaction (PCR) assay.
		<ol>
			<li>Analyze the wastewater sample with two pairs of primers 27F and 1492R, as well as 63F and 1387R (Table 1). 
			NOTE: The PCR master mix was prepared according to Table 2 using a commercially available DNA polymerase kit (see Table of Materials), and the PCR machine was set up with the program specified in Table 3.</li>
			<li>After the PCR program is completed, examine the products by 1% agarose gel electrophoresis40 at target bands of 1465 bp (using primers 27F and 1492R) or 1324 bp (using primers 63F and 1387R) to infer the sterility of samples.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Record and observe sterility tests, including plate and medium culture from 24 h to 3 days and 7 days, during which no colony on plates and no turbidity in liquid indicate the successful construction of GF models. Observe and record the plate and medium at 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h.</li>
</ol>
6. Identification of GF Artemia samples before feeding on GF fish
NOTE: To detect the GF Artemia samples, which are indispensable as live food before feeding on GF fish (Figure 4), follow the steps below.
<ol>
	<li>Collect samples of GF Artemia models.
	<ol>
		<li>Untreated Artemia cysts.</li>
		<li>Treated GF Artemia cysts.</li>
		<li>Hatched GF Artemia nauplii.</li>
		<li>Sterilized ultrapure water as a control.</li>
	</ol>
	</li>
	<li>Detect GF Artemia with the following methods.
	<ol>
		<li>Use the spread plate method to detect the samples by coating them on TSA plates and blood TSA plates under aerobic and anaerobic conditions. Incubate them at 30 &#176;C.</li>
		<li>Use a liquid medium to detect the samples in aerobic and anaerobic TSB and BHI tubes. Culture in a shaker at 150-180 rpm, 30 &#176;C.</li>
		<li>Use PCR assay to detect the presence of bacteria in the samples collected using 16s ribosomal RNA target genes primers 27F and 1492R, 63F and 1387R (Table 1). The volume and program are shown in Table 2 and Table 3.</li>
	</ol>
	</li>
	<li>Record the results: Detect the PCR product by 1% agarose gel electrophoresis40 for target bands measuring 1465 bp (using primers 27F and 1492R) or 1324 bp (using primers 63F and 1387R). The results of plates and liquid medium can ensure the sterile status of GF Artemia before being used as GF fish food.</li>
</ol>
7. Isolation and identification of intestinal bacterial strains from zebrafish
NOTE: To explore intestinal functions in vitro and in vivo, it is necessary to isolate bacterial strains from zebrafish, which also provide the species source for potential probiotics screened by infection using GF animals (Figure 5). In this protocol, we describe the traditional dissection method to obtain intestinal contents, while the samples can also be collected directly from live anesthetized fish (data not shown).
<ol>
	<li>Choose healthy adult zebrafish and wash them with sterile water. Perform the following steps in the clean bench.</li>
	<li>Anesthetize the fish with 100 mg/L MS-222 for 5-10 min.</li>
	<li>Use 75% alcohol to treat the fish&#39;s body surface.</li>
	<li>Dissect the fish intestines and extrude the intestinal contents with TSB or BHI medium.</li>
	<li>Incubate the intestinal supernatant by applying TSA, blood plate, TSB, and BHI medium in aerobic and anaerobic conditions.</li>
	<li>Translate the bacteria to gain the signal strain and record the characteristics of different colonies.</li>
	<li>Store the bacteria within 30% glycerol at -80 &#176;C.</li>
	<li>Then, identify the intestinal bacteria by genomic DNA extraction and PCR sequencing. 
	NOTE: The main steps of bacterial genomic DNA extraction include liquid centrifugation, RNase A and Protease K dissociation, ethyl alcohol extraction, rinsing, and dissolution using commercially available kits following the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Analyze the 16S sequences using the National Center for Biotechnology Information (NCBI) and the Basic Local Alignment Search Tool (BLAST) with the Bacteria and Archaea database40,41(see Table of Materials).</li>
	<li>Pick the best-matched bacteria and characters to identify the isolated intestinal strains at the genus level.</li>
	<li>Detect the metabolic functions of bacterial strains using commercially available kits (see Table of Materials).</li>
</ol>
8. Flu-label, infection, and colonization of bacteria in GF zebrafish intestines
NOTE: Before infecting GF zebrafish, the isolated bacteria were modified with dyes and counted, making microbial colonization and migration visible continuously and in vivo (Figure 6).
<ol>
	<li>Choose potential beneficial bacteria or dominant strains with high abundance in the host to infect the GF fish models. 
	NOTE: The bacteria used in this study were Aeromonas sp. and Vibrio sp. (see Table of Materials), selected from the 8 genera that were isolated and identified from adult wild-type zebrafish in the laboratory42.</li>
	<li>Label the fresh bacteria with CM-Dil dyes (see Table of Materials) and expose them to GF zebrafish at 5 dpf with a concentration of 106-108 Colony-Forming Units (CFUs)/mL.</li>
	<li>Observe the fluorescence under the fluorescent microscope with 3&#215; magnification to indicate the colonization and distribution of bacteria in the gut of GF zebrafish larvae from 6 dpf and 14 dpf.</li>
	<li>Manually count the number of bacteria in GF fish by plate culture at each time point.</li>
	<li>Detect the colonies and sequences to ensure the infection was successful with the target bacterial strains.</li>
	<li>Collect samples of GF fish with bacterial infection for subsequent assays, including neuro-behavior, developmental indexes, histopathological analysis, and hormone measurements, etc43,44,45. 
	NOTE: The bacteria used in the infection experiment should be newly recovered and freshly cultured by the liquid medium.</li>
</ol>

Optimizing GF Zebrafish Models: Generation, Maintenance, and Microbial Studies

The protocol of GF Artemia has been granted as a patent by the China National Intellectual Property Administration, which will relieve the restricted usage of the method after gaining the permission of the authors. The authors declare that they have no other competing or financial interests.

Critical steps within the protocols of GF fish and GF food preparation 
During the generation of GF fish models, several critical steps were involved, including the preparation of sterile materials, sterilization of embryos, daily renewal of GZM, collection of various samples, and the sterile examination of each sample using multiple methods. Among these steps, the initial treatment of embryos is fundamental and decisive for the success of GF models. Controlling agents, their concentrations, and treatment times are crucial for achieving optimal sterilization rates and hatching success. Careful attention is necessary when handling GF fish embryos to larvae, involving daily medium changes and ensuring that materials and platforms are thoroughly sterilized using ultraviolet light or autoclaving when removing plates from the incubator. Monitoring temperature conditions and operating time during winter is essential to prevent larval death caused by lower temperatures in clean benches.
Sterile egg yolk and GF Artemia should undergo detection before being fed to fish after 7 dpf to ensure their sterile state for long-term GF models. During the feeding period, cleaning food residues is important as part of the samples collected for detection when renewing the waste medium. The container, volume of GZM, and food mass should be adjusted along with the growth and development stages to prevent GF fish loss due to limited surroundings and water quality issues. Regular monitoring and adjustments in these parameters contribute to the successful maintenance of GF fish models.
Modifications and significance of these protocols 
The simplified three-step process for generating GF zebrafish embryos, involving optimized agents, concentrations, and exposure times of AB-GZM, PVP-I, and NaClO solutions, ensures a high sterile rate and healthy development of models. Additionally, the sterile detection methods for daily samples have been perfected to include plates and liquid medium cultures under aerobic and anaerobic conditions, along with molecular techniques using common bacterial primers. Further experiences in testing for bacterial and fungal contaminants aim to optimize isolator contaminants and sterile testing during GF animal techniques48.
The specific protocol for GF Artemia nauplii as live food for fish models has been investigated in our study, and a patent was granted to PPJ and DSP by the China National Intellectual Property Administration (No. ZL202210482684.8). Long-term GF fish models are consistently constrained by inadequate and unsuitable nutrition, exacerbating weaknesses in gut functions related to digestion and absorption. As a credible food source for medaka and zebrafish models at different life stages, GF Artemia sp. should be easy to prepare with a simple procedure, short incubation time, and real-time sterile detection for daily use. In this method, normal Artemia cysts are rinsed in a sufficient concentration of NaClO to be aseptic and then incubated in a film-coated bottle, as well as on a plate and in a liquid medium for detection. This supports freshly hatching nauplii for GF fish feeding. The sterile rate and hatching rate of Artemia, as well as the body length, weight, and activity of, are excellent, indicating available applications in practice.
The significance of these modifications in GF fish and GF food protocols benefits researchers in generating models at embryos, larvae, juvenile stages, and even early adult and adult stages in marine and freshwater mode&#181;species. Long-term GF animals will provide permission as in vivo models in research on drug screening and microbial functions during host growth and development, especially in the maturity of immunity and related intestinal cell lines. For example, zebrafish larvae only possess innate immunity in the first week, then develop the adaptive immunity system during 7-21 dpf, along with thymus maturity31,49. Moreover, GF fish models can provide the possibility to analyze bacteria-bacteria and microbiota-host interactions with live observation of dynamic colonization and migration processes in organisms50,51.
Limitations of the method 
Culturing GF fish from juveniles to early adults (28 dpf-2.5 mpf) and sexually mature adults (&#62;2.5 mpf) remains challenging due to increased mortality and the risk of bacterial contamination during model maintenance. These limitations primarily arise from the absence of suitable autoculture systems for fish living in a GF environment. These systems should include clean air, water, and a food system to effectively maintain the animals and their environments, ensuring complete isolation from external microorganisms. Unlike other GF animal models such as mice and chickens52, obtaining GF zebrafish through anatomical operations from the CR parents is challenging due to their aquatic habits and natural fertilization style.
Treating adult animals as completely devoid of intestinal microbiota is also challenging. While antibiotic treatment may induce a state similar to specific pathogen-free (SPF) animals, characterized by a reduced microbiota detected in fecal samples, achieving true GF adult fish models remains difficult. The challenges involved in establishing and maintaining GF models, especially during the transition from juvenile to long-term states, are significant. The importance of GF offspring, such as GF F1 or F2 eggs from adult GF fish, in microbial research is acknowledged. However, overcoming challenges in establishing and maintaining long-term adult GF fish models requires substantial time.
It&#39;s worth noting that gnotobiotic zebrafish with specific bacterial colonization has been developed, showing chronic survival based on GF models. Nevertheless, it remains uncertain whether these models can serve as viable alternatives to GF models in bacterial infection and biomedical research.
Future applications of GF fish models 
In this study, we have refined the protocol for generating GF zebrafish models, progressing from embryos to juveniles. This advancement holds significant applications in developmental biology, immunology, human disease research, and organogenesis. Through mass isolation and identification, the study explores the functions, mechanisms, and gut-derived metabolites of single bacterial strains. The investigation involves infecting GF zebrafish models after the vent opens stage47,53. Additionally, labeled bacteria with fluorescence facilitate the observation of colonization and distribution in live GF larvae, free from other microbial influences54. Similar GF fish models, such as marine or freshwater medaka, can be created using comparable methods with various agents or medium changes.
To probe human disease mechanisms, the study employs FMT methods, transferring samples from patients or donors to healthy animals or GF models55. Unlike conventionally reared (CR) animals, GF models offer robust evidence of microbial functions, impacts, or responses in the host, particularly when combined with bacteriophages to regulate target bacteria in the microbiota46.
In summary, GF zebrafish serve as powerful models for assessing bacterial safety, drug effectiveness, pollutant toxicity, or compound interference, owing to their sensitive and pure intestinal environment. Future exploration of GF fish applications should encompass various fields, with a need to create adult GF fish models for a deeper understanding of microbial functions across different life stages.

The microbiota (i.e., Archaea, Bacteria, Eukarya, and viruses) play crucial roles in maintaining host health and contributing to the development of various diseases by influencing physiological and pathological processes through symbiotic interactions within the intestinal barrier, epithelial surface, and mucin functions in individuals1,2,3. The composition of the microbiota across different life stages, from infancy to juvenility, adulthood, and aging, as well as its presence in various locations such as nares, oral, skin, and gut sites, is dynamically shaped by diverse habitats and environments4. The intestinal microbiota in organisms is involved in nutrient absorption, immune response, pathogen invasion, metabolic regulation, etc5,6. Studies on patients have demonstrated that disruptions in gut microbiota are related to human obesity, sleep disorders, depression, inflammatory bowel disease (IBD), neurodegenerative diseases (Parkinson&#39;s, Alzheimer&#39;s), aging, and various cancers7,8,9. Furthermore, interactive pathways between gut microbiota and hosts involve inflammatory factors, neurotransmitters, metabolites, intestinal barrier, and oxidative stress, as observed in previous research using mice and fish models10,11.
Recently, multiple bacteria-related approaches or therapies, including potential probiotics and fecal microbiota transplantation (FMT), have been explored for these disorders in clinical and animal models. These explorations are based on discoveries related to the microbiota-gut-brain/liver/kidney axis, microbiota-derived products, and altered receptor activity12,13. However, the development, various functions, and mechanisms of the microbiota-host system are still incompletely understood and identified due to the complexity of the microbial community and the challenge of generating powerful human-like disease models.
To address these issues, germ-free (GF) animal models were urgently proposed in the mid-19th century and primarily developed during the 20th century. Subsequent refinements, including antibiotic-treated and gnotobiotic models, along with advancements in microbial detection and observation technologies, further perfected these models14,15,16. GF animals, created by erasing their own background and avoiding environmental microbes, offer an excellent strategy for exploring the interactions between microorganisms and their hosts17. Through the application of animal models and refined protocols, researchers have successfully replicated similar microbial compositions found in patients in GF mice and fish. Additionally, other GF animal models, such as dogs, chickens, and pigs, provide diverse options as research subjects18,19,20,21. This approach has enabled investigations into the potential therapeutic effects of commensal microbiomes on various diseases, including cancer immunotherapy in humans16,18. GF models offer more accurate insights into the characteristics and mechanisms of specific bacterial colonization, migration, multiplication, and interaction within hosts. This provides crucial novel insights into the occurrence and development of microbiota-related diseases22,23. The history of establishing and applying GF zebrafish in microbial research has evolved from the reports of Rawls et al. in 2004 and Bates et al. in 2006 to Melancon et al.&#39;s protocol in 201716,24,25. However, the feasibility of adult or breeding GF models is still a prolonged process, accompanied by variable longevity, success rates, and health challenges.
Among various animal models, zebrafish (Danio rerio) stands out as a critical tool for both basic and biomedical research due to its advantageous similarity to human organs and genomics, short developmental cycle, high fecundity, and transparent embryos19,26. Zebrafish, serving as reliable human disease models, offer a visual representation of physiological and pathological processes in vivo, providing insights into the attractive features of host-microbe interactions. Notably, zebrafish exhibit distinct cell lineages, allowing imaging of intestinal physiology, microbial dynamics, gonads and reproductive development, maturation of the host immune system, behavior, and metabolism27. Zebrafish embryos develop within protective chorions until hatching, becoming larvae at 3 days post-fertilization (dpf). They actively hunt for food at 5 dpf and reach sexual maturity around 3 months post-fertilization (mpf)28. The first successful germ-free (GF) zebrafish, reported by Rawls et al.24, showed that larvae fed with autoclaved feed after yolk absorption exhibited tissue necrosis from 8 dpf and total death at 20 dpf. This indicated the effects of diet or the importance of considering exogenous nutrient supply in experiments involving long-term (&#62;7 dpf) GF fish29. Subsequent studies improved the generation protocol of GF fish, employing sterile food and methods perfected in different fish models16.
However, most research on GF zebrafish models has focused on early life stages, involving bacterial infection at 5 dpf for 24 h to 48 h, with samples collected before 7 dpf at the conclusion of the experiments25,30,31. It&#39;s widely acknowledged that the microbiota in organisms, including humans and zebrafish, is colonized at the beginning of life and shaped during growth and development. The composition remains stable at adult stages, with the roles of microbiota in the host being crucial throughout life, especially in aging, neurodegenerative, metabolic-related obesity, and intestinal disease aspects3. Thus, perspectives from GF animals with longer survival can provide insights into the mechanisms of microbial roles in host organ development and functions, considering the immature immune and reproductive systems of fish larvae in early life. While bacterial strains in zebrafish intestines have been isolated and identified in previous studies, offering the potential for infecting GF animal models to select probiotics or research bacterial functions in the host19,25, the generation and application of GF fish models have primarily been restricted to early life stages. This limitation, attributed to the complex production process, high maintenance costs, and associated issues with food and immunity, hinders research efforts aimed at investigating the developmental and chronic effects of microbiota in the host.
The survival rate, behavior, growth, maturation, and overall health of fish, especially in germ-free (GF) models, are significantly influenced by feeding practices, encompassing nutrition intake and absorption during the mouth-open period from early larvae to juveniles32,33. However, one of the challenges in GF fish husbandry is the scarcity of suitable sterile diets, limiting the effectiveness of nutritional support for sustaining the growth and survival of larvae. Resolving this issue is crucial to restoring the life of GF fish, considering their developmental defense mechanisms and weak digestion abilities due to the absence of an intestinal microbiome. In terms of food, live brine shrimp (Artemia sp.) emerges as the most suitable diet for mouth-open larvae to juvenile fish. It has been observed that fish fed with live brine shrimp exhibit higher growth and survival rates compared to those fed with cooked egg yolk or other natural and synthetic baits34. While early life models of GF fish can survive with yolk support and GF larvae models can be maintained with sterile feeding, generating long-term models from larvae to juveniles and reaching sexual maturity remains challenging. Additionally, flake or powder food is limited by unequal nutritional composition and can impact water quality. In contrast, live Artemia has advantages such as survival in both salt and freshwater, small size suitable for larvae to adults, ease of batching, and higher hatching quality35. Building upon previous methods16,24,30, we have simplified the complex treatment process and addressed the diet challenge by establishing easily incubated GF live Artemia sp. as sterile food for longer durations than early-life GF fish.
This study presents an optimized protocol covering (1) generation, (2) maintenance, (3) identification of sterile rate, and (4) maintenance and feeding to ensure the growth of germ-free (GF) zebrafish from embryos to larvae and juvenile stages. The results offer preliminary evidence on the hatching, survival, growth, and sterility of GF zebrafish, along with essential indices for GF Artemia sp. as sterile food. The detailed steps in model generation and preparation of sterile live foods provide crucial technical support for constructing and applying long-term GF fish models, as well as GF Artemia sp. in microbiota-host interaction research. The protocol addresses bacterial isolation, identification, and infection on GF fish models, outlining methods for bacterial fluorescence labeling and observing their colonization in fish intestines under a microscope. GF fish, gnotobiotic fish with bacterial infection, or transferred human microbiota models will undergo various detections to elucidate their functions and effects on host immunity, digestion, behavior, transcriptomic regulation, and metabolic aspects. In the long term, this protocol can be extended to different wild-type fish species, such as marine medaka, and potentially to other selected transgenic zebrafish lines correlated to specific tissues or diseases.

<table><tbody><tr><td>AB-GZM</td><td>Amphotericin:Solarbio;&amp;nbsp; kanamycin:Solarbio; Ampicillin:Solarbio.</td><td>Amphotericin:CAS:1397-89-3;&lt;br/&gt; kanamycin:CAS: 25380-94-0; Ampicillin:CAS: 69-52-313.</td><td>49.6 mL GZM, 50 &amp;micro;L amphotericin stock solution (250 &amp;micro;g/mL), 25 &amp;micro;L kanamycin stock solution (10 mg/mL), and 250 &amp;micro;L ampicillin stock solution (20 mg/mL).</td></tr><tr><td>1.5 mL, 15 mL, 50 mL EP tubes</td><td>biosharp</td><td>BS-15-M</td><td>To collect samples, and hold agents</td></tr><tr><td>2.4 g/L NaClO</td><td>XILONG SCIENTIFIC Co., Ltd.</td><td>CAS: 7681-52-9</td><td>Diluted with 8% sodium hypochlorite aqueous solution.</td></tr><tr><td>6-well plates, 24-, 48- well plates</td><td>LABSELECT&amp;nbsp;</td><td>11112</td><td>To culture fish</td></tr><tr><td>&lt;em&gt;Aeronomas&lt;/em&gt;</td><td>NCBI database</td><td>No.MK178499</td><td>2019-JPP-ESN</td></tr><tr><td>Anaerobic TSA plates</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;&lt;br/&gt; agar powder:BioFroxx.</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;&lt;br/&gt; agar powder:9002-18-0.</td><td>The TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, and 6 g agar powder under the anaerobic system.</td></tr><tr><td>Anaerobic work station</td><td>GENE SCIENCE</td><td>E200G</td><td>Bacterial isolation, sterile testing</td></tr><tr><td>Analysis</td><td>GraphPad Prism 5</td><td>v6.07</td><td>To analysis the data</td></tr><tr><td>API 20 E kits</td><td>&amp;nbsp;BioMerieux SA, France</td><td>No.1005915090</td><td>Ref 20100 Kits to detect bacterial metabolism</td></tr><tr><td>&lt;em&gt;Artemia &lt;/em&gt;(Brine shrimp)</td><td>Shangjia Aquarium Co., Ltd.</td><td>Aquamaster brand</td><td>Artemia cysts, and brine shrimp eggs&amp;nbsp;</td></tr><tr><td>Auto cycle system for fish culture</td><td>Ningbo Hairui Technology Co., Ltd</td><td>No Cat</td><td>Maintain the fish</td></tr><tr><td>Autoclave</td><td>Zeal Way</td><td>G154DWS</td><td>Prepare the materials</td></tr><tr><td>BHI Aerobic</td><td>Coolaber</td><td>Cat#PM0640</td><td>BHI medium was prepared, wherein 100 mL medium included 3.7 g BHI powder.</td></tr><tr><td>BHI Anaerobic</td><td>Coolaber</td><td>Cat#PM0640</td><td>BHI medium was prepared and divided into anaerobic tubes under the anaerobic system.</td></tr><tr><td>Biochemical incubator</td><td>LongYue Co., Ltd</td><td>SPX</td><td>For fish and plates</td></tr><tr><td>Biosafety cabinet</td><td>Haier</td><td>HR40-IIA2</td><td>Sterile treatment and testing</td></tr><tr><td>Bleaching agent of 0.02 g/L NaClO</td><td>XILONG SCIENTIFIC Co., Ltd.</td><td>CAS: 7681-52-9</td><td>Working solution with sodium hypochlorite (NaClO) concentration: Diluted with 8% sodium hypochlorite aqueous solution or 166.6 uL 6% sodium hypochlorite with 500 mL distilled water.</td></tr><tr><td>Blood plates</td><td>sheep blood:Solarbio</td><td>Cat. NO. TX0030</td><td>Sterile-defibrinated sheep blood was added into TSA to prepare 5% blood plates.</td></tr><tr><td>Cell culture flask</td><td>Corning</td><td>430639</td><td>To culture fish</td></tr><tr><td>CM-Dil dyes</td><td>Molecular Probes</td><td>Cat#C7000&amp;nbsp;&amp;nbsp;</td><td>To label the bacteria</td></tr><tr><td>Constant temperature shaking incubator</td><td>Peiving Co., Ltd</td><td>HZQ-X100</td><td>Bacterial culture</td></tr><tr><td>Database</td><td>NCBI</td><td>Bacteria and Archaea database</td><td>Link: Archaea FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Archaea/&lt;br/&gt; Bacteria FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Bacteria/</td></tr><tr><td>Disposable Pasteur pipette</td><td>biosharp</td><td>bs-xh-03l</td><td>Used to change water, and transfer eggs</td></tr><tr><td>Disposable petri dish</td><td>biosharp</td><td>BS-90-D</td><td>To culture fish</td></tr><tr><td>DNA kits</td><td>Solaribio</td><td>Cat#D1600</td><td>Bacterial genomic DNA extraction kits&amp;nbsp;</td></tr><tr><td>Electric pipette</td><td>SCILOGEX</td><td>Levo me</td><td>Change water</td></tr><tr><td>Exiguobacterium</td><td>NCBI database</td><td>No.MK178504</td><td>2019-JPP-ESN</td></tr><tr><td>GZM</td><td>Sea salt:LANDEBAO Co., Ltd.</td><td>No Cat</td><td>Composed of 1 L of water and 1.5 mL of sea salt solution (40 g/L), autoclaved. The content of sea salt in the GZM solution was 60 mg/L.</td></tr><tr><td>Laboratory pure water system</td><td>Hitech Co., Ltd</td><td>Prima-S15</td><td>Prepare the agents</td></tr><tr><td>Microscope</td><td>Nikon</td><td>SMZ18</td><td>With fluorescent light to observe fish larvae</td></tr><tr><td>PCR kits</td><td>TIANGEN</td><td>Cat#ET101</td><td>Taq DNA Polymerase kit</td></tr><tr><td>Pipette</td><td>LABSELECT&amp;nbsp;</td><td>sp-013-10</td><td>Change water</td></tr><tr><td>Povidone iodine (PVP-I)</td><td>Aladdin</td><td>Lot#H1217005</td><td>Aqueous solution povidone iodine 0.4 g/L pure water.</td></tr><tr><td>Timing converter</td><td>PinYi Co., Ltd</td><td>AL-06</td><td>To regulate the light</td></tr><tr><td>TSA plates</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;&lt;br/&gt; agar powder:BioFroxx.</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;&lt;br/&gt; agar powder:9002-18-0.</td><td>TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, 6 g agar powder.</td></tr><tr><td>TSB Aerobic</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;</td><td>TSB medium was prepared, wherein 400 mL medium included 6 g tryptone, 2 g soy peptone, and 2 g NaCl.</td></tr><tr><td>TSB Anaerobic</td><td>tryptone:Oxoid ;&lt;br/&gt; soy peptone:Solarbio ;NaCl:Biosharp;</td><td>tryptone:LP0042B;&lt;br/&gt; soy peptone:Cat#S9500;&lt;br/&gt; NaCl:BS112;</td><td>TSB medium was prepared and divided into the anaerobic tubes under the anaerobic system.</td></tr><tr><td>Ultra-clean workbench</td><td>Airtech</td><td>SW-CJ-2FD</td><td>Sterile treatment and testing</td></tr><tr><td>Ultra-pure flow system for fish culture</td><td>Marine Biological Equipment company</td><td>No Cat</td><td>Produce water for fish</td></tr><tr><td>&lt;em&gt;Vibrio&lt;/em&gt;</td><td>NCBI database</td><td>No.MK178501</td><td>2019-JPP-ESN</td></tr></tbody></table>

generation, maintenance, and identification of germ-free zebrafish models from larvae to juvenile stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

The GF zebrafish models can be efficiently produced by utilizing the abundant eggs spawned by pairs of zebrafish, with the protocol optimized based on previous GF fish models35. A single 6-well plate can culture approximately 30-48 embryos/larvae, allowing for ample data collection and statistical analysis. After sterile treatment, the GF embryos are cultured in a clean incubator till hatching to larvae at 48-72 h, and changed GZM daily with the detection of collected samples, which is crucial to maintaining the germ-free state (Figure 1). After 7 dpf, the food of egg yolk and live Artemia should be prepared for larvae to juvenile stages. During the growth and development of fish, the volume, container, and density of GZM should be regulated or changed in time to avoid death and failure of sterile status. If the detection shows no bacterial pollution, the successful GF models can be maintained as chronic ones from juvenile to early adulthood, but the survival rate is much lower in practice. Finally, the sterile rate, survival rate, development index, behavior, histopathological analysis, and transcription profile of GF and CR fish models can be measured to explore the influence of microbiota and drug screen fields. In this study, the key developmental indexes were compared (Supplementary Figure 1), and the hatching rate of GF fish was 45.7% lower than CR fish, with 60% at 72 hpf. The heart rates of GF larvae at 7 dpf were significantly lower at 18.2 beats/10 s compared to CR fish with 22.6 beats/10 s, but there was no difference between the two groups at 14 dpf with rates of 23.5-23.8 beats/10 s. The survival rate of GF fish at 7 dpf was the same as for CR fish at 86.7%, but decreased to 60% at 14 dpf compared to 80% for CR fish. However, the survival rates of both GF and CR fish decreased to 25%-10% after the mouth-open period when they acquired feeding abilities or habits. The failure to maintain the sterile status of fish was one of the reasons for the lower survival rate. Therefore, feeding and nutrient maintenance from juvenile to early adulthood stages are critical challenges in fish culture.
Another key factor that affects the growth and development of fish is feeding. In this study, we explore multiple methods to obtain GF Artemia as live food for long-term GF zebrafish larvae to juveniles and adults (process shown in Figure 2). Due to the daily use of food for GF fish, the optimized protocol for GF Artemia should be easy, time-saving, and economical in practice. After comparing the active movement and death/immobility of nauplii observed by the microscope, characteristics of nauplii, unhatched eggs, and shell, the method is perfected. After daily incubation, the indexes of fresh GF Artemia obtained from bottles, including the hatching, survival and deformity rate, and the length and weight of nauplii, were observed and measured under the microscope (Figure 3). After treatment, the hatching rate of Artemia cysts was high, with an average of 88.15%, the survival of swimming nauplii was 77.83%, and the malformation rate was 1.87%. The body length of nauplii was 546.70 &#181;m, and the body weight index was 3.80 mg/100, which are suitable for fish catching and feeding from the larvae month-open period to juvenile and adult stages.
The detection of GF fish and GF Artemia is also important for model maintenance. According to sample collection and multiple test methods, the results can provide the success of treated zebrafish embryos and Artemia cysts in the cultured plate and the liquid medium (Figure 4). The various samples collected daily should be detected as sterile in all test methods, which can then indicate the GF models at the time of collecting. The GF Artemia should be detected before feeding on GF fish, or the plate and medium incubator results should refer to the sterile state of fresh live nauplii. That is, cultivate GF Artemia from cysts on TSA plates or TSB or BHI liquid medium glass in shake incubation, which could be used for generating GF Artemia and sterile verification at the same time. The GF Artemia protocol can accommodate a limited number of larvae for feeding. During the feeding process, it was evident that the GF Artemia swam within the GZM and then was captured and consumed by the GF fish larvae.
The applications of GF fish models involve various fields in biology and medicine, allowing the investigation of microbial functions, growth and development, disease mechanisms, and probiotic or drug screening, among others46. In this study, for example, two major intestinal bacteria, Aeromonas sp. and Vibrio sp., were isolated from zebrafish. The colony characteristics and identification were performed, and multiple metabolic functions were measured in vitro using commercially available kits (Figure 5). These bacteria were isolated and identified with 16S sequencing, and the blast results were presented in a previous report42. The identified bacterial strains enable scientific research on the impact of single or combined microbiota on host health by infecting the GF fish models. The transparency of fish larvae allows the observation of fluorescence-labeled bacterial cells using CM-Dil dyes, revealing colonization and distribution in the intestines of GF fish (shown in Figure 6). After bacterial infection at 5 dpf, GF zebrafish larvae can be imaged with a fluorescence microscope and continuously observed from 6 to 14 dpf, providing insights into microbial functions combined with developmental indexes, histopathological changes, and molecular alterations47.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66512/66512fig01.jpg" /> 
Figure 1: Protocol of GF zebrafish from embryos to larvae and juveniles. Key steps in the generation of GF zebrafish include: (1) Collection of embryos from wild-type zebrafish and placement in AB-GZM. (2) Rinse treatment with PVP-I and NaClO agents to ensure complete sterility. (3) Culture of GF embryos/larvae in 6-well plates with GZM, renewing the medium daily. (4) Regulation of culture conditions for optimal growth and development. (5) Sampling at various developmental stages, followed by sterility testing using plate and liquid medium methods. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66512/66512fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66512/66512fig02.jpg" /> 
Figure 2: Protocol of GF Artemia preparation as live food for GF fish. Key steps include: (1) Rinse treatment of Artemia cysts for complete sterility. (2) Preparation of GF cysts in the medium. (3) Incubation in a salt solution for 24 h to promote hatching. (4) Filtration of failed hatching and empty eggs to collect active nauplii. (5) Washing collected nauplii to remove debris, ensuring high-quality and contaminant-free live food. (6) Sterility testing of the GF Artemia before feeding to GF fish larvae. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66512/66512fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66512/66512fig03.jpg" /> 
Figure 3: Indexes of GF Artemia cysts and nauplii incubated for 24 h. (A) Untreated Artemia cysts with impurity film (scale bar: 500 &#181;m). (B) Treated Artemia cysts with a slightly convex surface after water absorption and a cleaner appearance (scale bar: 500 &#181;m). (C) Microscopic examination of fresh incubated live GF Artemia nauplii (scale bar: 2000 &#181;m) and (D) magnification with a scale bar of 1000 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66512/66512fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66512/66512fig04.jpg" /> 
Figure 4: Sterile detection of GF fish at each life stage and GF Artemia. (A) Sterile tests of CR and GF fish samples using TSB and BHI liquid medium, TSA, and blood plates under aerobic and anaerobic conditions. (B) Sterile tests of untreated Artemia cysts, GF Artemia cysts, and nauplii using TSB and BHI liquid medium, TSA, and blood plates under aerobic and anaerobic conditions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66512/66512fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66512/66512fig05.jpg" /> 
Figure 5: Isolation and identification of intestinal bacteria in zebrafish. (A) Bacterial strains, characteristics of colonies, 16S sequencing maps (examples: Aeromonas sp. and Vibrio sp.). (B) Genus and NCBI accessions. (C) Metabolic functions of bacteria isolated from zebrafish gut. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66512/66512fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66512/66512fig06.jpg" /> 
Figure 6: Infection and colonization of gut bacteria strains in GF zebrafish. (A) Recovery of isolated and identified bacterial strains for infection experiments. (B) Labeling of bacteria with fluorescent dyes and infection of GF zebrafish larvae at 5 dpf. Magnification: 1x. (C) Observation of the distribution and colonization of bacteria (Vibrio sp.) in gut tissues in vivo. Magnification: 3x. (D) Counting of CFUs of bacteria (Aeromonas sp. and Vibrio sp.) in each fish larva by daily sampling. Data presents mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66512/66512fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primers</td>
			<td>Sequences(5&#8217;-3&#8217;)</td>
			<td>Product length</td>
		</tr>
		<tr>
			<td>27F</td>
			<td>AGAGTTTGATCMTGGCTCAG</td>
			<td rowspan="2">1465 bp</td>
		</tr>
		<tr>
			<td>1492R</td>
			<td>ACGGYTACCTTGTTACGACTT</td>
		</tr>
		<tr>
			<td>63F</td>
			<td>CAGGCCTAACACATGCAAGTC</td>
			<td rowspan="2">1324 bp</td>
		</tr>
		<tr>
			<td>1387R</td>
			<td>GGGCGGWGTGTACAAGGC</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Primers used for bacterial detection.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Components</td>
			<td>Units</td>
			<td>Volume (&#181;L)</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>2.5 U/&#181;L</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>10&#215; Buffer(Mg2+)</td>
			<td>---</td>
			<td>2.1</td>
		</tr>
		<tr>
			<td>dNTP Mixture</td>
			<td>2.5 mM</td>
			<td>1.6</td>
		</tr>
		<tr>
			<td>Primer 27F</td>
			<td>10 &#181;M</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Primer 1492R</td>
			<td>10 &#181;M</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DNA/RNA free H2O</td>
			<td>---</td>
			<td>13.2</td>
		</tr>
		<tr>
			<td>Template DNA</td>
			<td>---</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>---</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;Final concentration and volume of components per reaction.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature</td>
			<td>Time</td>
			<td>Cycles</td>
		</tr>
		<tr>
			<td>Initial denaturation</td>
			<td>95 &#176;C</td>
			<td>4 min</td>
			<td>--</td>
		</tr>
		<tr>
			<td>Denaturation&#160;</td>
			<td>94 &#176;C</td>
			<td>1 min</td>
			<td rowspan="3">35</td>
		</tr>
		<tr>
			<td>Annealing</td>
			<td>55 &#176;C</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Extension</td>
			<td>72 &#176;C</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Final extension</td>
			<td>72 &#176;C</td>
			<td>10 min</td>
			<td>--</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &#176;C</td>
			<td>&#8734;</td>
			<td>--</td>
		</tr>
	</tbody>
</table>
Table 3: PCR program for amplification of the 16S rRNA gene.
Supplementary Figure 1: The critical developmental indexes of GF and CR zebrafish models. (A) The hatching rate of CR and GF zebrafish at 72 hpf, and (B) heart beats/10 s of CR and GF fish at 7 dpf and 14 dpf. The survival rates (%) of CR and GF fish decreased from 7 days and 14 days with 86%, 60%-80% to 10%-25% at 30 dpf. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66512/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies at JoVE.com

Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

1.0K Views.  Chongqing Medical University. This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

Video: Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Genetics

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Biology

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Cancer Research

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

An Introduction to the Zebrafish: Danio rerio

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their high degree of genetic conservation with humans and their simple, inexpensive maintenance. Additionally, gene expression can be easily manipulated in zebrafish embryos, and their transparency allows for observation of developmental processes.
This overview video first introduces basic zebrafish biology, including their phylogeny, life cycle, and natural environment, before presenting the features that make them so useful in the lab. A brief history of zebrafish research is also provided through a review of major discoveries made in fish, ranging from the early establishment of methods for efficient genetic screening to the discovery of novel therapeutics for human diseases such as cancer. Finally, some of the many avenues of experimentation performed in zebrafish are discussed, including immunological and developmental studies.

Danio rerio Zebrafish as a Model Organism

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their ...

Education

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Basic Biology

Biology II: Mouse, Zebrafish, and Chick

Zebrafish Maintenance and Husbandry

The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to their small size, large numbers of zebrafish can be housed in the laboratory at low cost. Although zebrafish are relatively easy to maintain, special consideration must be given to both diet and water quality to in order to optimize fish health and reproductive success.
This video will provide an overview of zebrafish husbandry and maintenance in the lab. After a brief review of the natural zebrafish habitat, techniques essential to recreating this environment in the lab will be discussed, including key elements of fish facility water recirculation systems and the preparation of brine shrimp as part of the zebrafish diet. Additionally, the presentation will include information on how specific zebrafish strains are tracked in a laboratory setting, with specific reference to the collection of tail fin samples for DNA extraction and genotyping. Finally, experimental modifications of the zebrafish environment will be discussed as a means to further our understanding of these fish, and in turn, ourselves.

Maintenance and Husbandry of Danio rerio Zebrafish

The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to ...

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

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Chapters in this video

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Generating Chimeric Zebrafish Embryos by Transplantation

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

An Introduction to the Zebrafish: Danio rerio

Zebrafish Maintenance and Husbandry