Name: В центре внимания автора: Создание стерильных рыбок данио-рерио для исследований отношений между микробиотой и хозяином
Uploaded: 2024-04-12T13:50:00
Description: Watch this Scientific Journal Video about Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies at JoVE.com

Мы искренне благодарим за поддержку Проект талантов Чунцинского медицинского университета (No R4014 для DSP и R4020 для PPJ), Национальный фонд естественных наук Китая (NSFC, No 32200386 для PPJ), Студию наставников по инновациям в постдокторантуре Чунцина (X7928 DSP) и Программу Китайско-Шри-Ланкийского совместного центра исследований и демонстрации водных технологий Китайской академии наук (CAS) / Китайско-шри-ланкийского совместного центра образования и исследований CAS.

Эксперименты с рыбами проводились в соответствии с руководящими принципами Комитета по уходу за животными и их использованию г. Чунцин и Институционального комитета по уходу за животными и их использованию Чунцинского медицинского университета, Китай, а также стандартами для экспери...

Оптимизация моделей рыбок данио-рерио GF: генерация, обслуживание и микробиологические исследования

Важнейшие этапы в рамках протоколов приготовления рыбы и пищи для ГФ Во время создания моделей рыб GF было задействовано несколько важных этапов, включая подготовку стерильных материалов, стерилизацию эмбрионов, ежедневное обновление GZM, сбор различных образцов и стерильное...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AB-GZM</td>
			<td>Amphotericin:Solarbio;&#160; kanamycin:Solarbio; Ampicillin:Solarbio.</td>
			<td>Amphotericin:CAS:1397-89-3; 
			kanamycin:CAS: 25380-94-0; Ampicillin:CAS: 69-52-313.</td>
			<td>49.6 mL GZM, 50 &#181;L amphotericin stock solution (250 &#181;g/mL), 25 &#181;L kanamycin stock solution (10 mg/mL), and 250 &#181;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&#160;</td>
			<td>11112</td>
			<td>To culture fish</td>
		</tr>
		<tr>
			<td>Aeronomas</td>
			<td>NCBI database</td>
			<td>No.MK178499</td>
			<td>2019-JPP-ESN</td>
		</tr>
		<tr>
			<td>Anaerobic TSA plates</td>
			<td>tryptone:Oxoid ; 
			soy peptone:Solarbio ;NaCl:Biosharp; 
			agar powder:BioFroxx.</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112; 
			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>&#160;BioMerieux SA, France</td>
			<td>No.1005915090</td>
			<td>Ref 20100 Kits to detect bacterial metabolism</td>
		</tr>
		<tr>
			<td>Artemia (Brine shrimp)</td>
			<td>Shangjia Aquarium Co., Ltd.</td>
			<td>Aquamaster brand</td>
			<td>Artemia cysts, and brine shrimp eggs&#160;</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&#160;&#160;</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/ 
			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&#160;</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&#160;</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 ; 
			soy peptone:Solarbio ;NaCl:Biosharp; 
			agar powder:BioFroxx.</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			NaCl:BS112; 
			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 ; 
			soy peptone:Solarbio ;NaCl:Biosharp;</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			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 ; 
			soy peptone:Solarbio ;NaCl:Biosharp;</td>
			<td>tryptone:LP0042B; 
			soy peptone:Cat#S9500; 
			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>Vibrio</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

Рыбки данио служат ценными моделями для исследований роста, иммунитета и микробиоты кишечника из-за их геномного сходства с млекопитающими, прозрачных эмбрионов, развивающихся в относительно чистой среде хориона, и чрезвычайно быстрого развития личинок по сравнению с моделями грызунов. Стерильные (GF) данио-рерио (Danio rerio) имеют решающее значение для оценки токсичности загрязняющих веществ и создания моделей заболеваний, подобных человеческим, связанных с микробными функциями. По сравнению с моделями, выращенными традиционным способом (CR) (рыбы в общем хозяйстве), рыбки данио-рерио GF позволяют более точно манипулировать микробиотой хозяина, помогая определить причинно-следственную связь между микроорганизмами и хозяевами. Следовательно, они играют решающую роль в продвижении нашего понимания этих отношений. Тем не менее, модели рыбок данио-рерио GF обычно создаются и исследуются на ранних стадиях жизни (от эмбрионов до личинок) из-за ограничений иммунной функции и усвоения питательных веществ. Это исследование оптимизирует создание, поддержание и идентификацию ранних моделей рыбок данио рерио без кормления и с длительным кормлением с использованием корма GF (например, Artemia sp., артемия, артемия). На протяжении всего процесса выполнялся ежедневный отбор проб и бактериологическое исследование, которые были идентифицированы с помощью нескольких обнаружений, включая секвенирование планшетов и 16S рРНК. Для обеспечения качества и количества сгенерированных моделей были зарегистрированы показатели асептической скорости, выживаемости и развития рыбок данио-рерио. Важно отметить, что это исследование предоставляет подробную информацию о бактериальной изоляции и методах заражения рыб GF, что позволяет эффективно создавать модели рыб GF от личинок до молодых стадий с пищевой поддержкой GF. Применяя эти процедуры в биомедицинских исследованиях, ученые могут лучше понять взаимосвязь между бактериальными функциями кишечника и здоровьем хозяина.

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.

В центре внимания автора: Создание стерильных рыбок данио-рерио для исследований отношений между микробиотой и хозяином

Создание, поддержание и идентификация стерильных моделей данио-рерио от личинок до ювенильных стадий

The scope of this research is generation of novel, germ-free animal models, which will be helpful and powerful tools in investigation of various microbiome functions. In this field, the early germ-free fish models have been used recently, but the long-term models are still limited due to various culture conditions and the staff developmental defense. The current GF fish models are normally early life ones.Thus, the technologies including the sterile scale, the sample detection methods, and providing sterile food are very important challenges to overcome. The germ-free zebrafish model is crucial for studying host-microbiota interaction and their impacts on development and the immunity and disease. This model allows researchers to precisely manipulate the single gut bacteria and observe its effect on the transparent zebrafish embryos.It provides valuable insights into the roles of gut microbes inheriting the disease. In the future, our research will mainly focused on establishing a germ-free zebrafish model for entire life cycle to better understanding the function of the gut microbiome.

Модели GF данио рерио могут быть эффективно получены за счет использования обильной икры, отнерестящейся парами рыбок данио, с протоколом, оптимизированным на основе предыдущих моделей рыб GF35. Один 6-луночный планшет может культивировать примерно 30-48 эмбрионов/личинок, что ...

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

В этом протоколе изложены основные шаги по получению стерильных эмбрионов рыб (GF) и поддержанию их от личинок до ювенильной стадии, включая отбор проб и определение их стерильного статуса. Использование моделей GF с инфекцией важно для понимания роли микробов в здоровье хозяина.

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

Целью этого исследования является создание новых, стерильных моделей животных, которые будут полезными и мощными инструментами в исследовании различных функций микробиома. В этой области недавно были использованы ранние модели стерильных рыб, но долгосрочные модели все еще ограничены из-за различных условий культивирования и защиты развития персонала. Современные модели рыб GF обычно являются моделями раннего возраста.Таким образом, технологии, включая стерильные весы, методы обнаружения образцов и обеспечение стерильной пищей, являются очень важными проблемами, которые необходимо преодолеть. Модель стерильных рыбок данио имеет решающее значение для изучения взаимодействия хозяина и микробиоты и их влияния на развитие, иммунитет и болезни. Эта модель позволяет исследователям точно манипулировать одной кишечной бактерией и наблюдать ее влияние на прозрачные эмбрионы данио-рерио.Он дает ценную информацию о роли кишечных микробов, наследующих болезнь. В будущем наши исследования будут в основном сосредоточены на создании модели стерильных рыбок данио для всего жизненного цикла, чтобы лучше понять функцию кишечного микробиома.

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Research

JoVE Journal

Developmental Biology

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.

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 of Germ-Free Zebrafish Models from Embryos

Создание стерильных моделей данио-рерио из эмбрионов

GF Artemia nauplii Preparation for Chronic GF Zebrafish Nutrition

To begin, obtain 0.25 grams of Artemia cysts and rinse them with 2.4 grams per liter sodium hypochlorite for five minutes. Filter the rinsed cysts with a sterilized ultra dense filter and wash with sterilized ultrapure water for one to two minutes. After three washes, transfer them to 100 milliliters of 2.5%sterile saline and mix well.Pack 50 milliliters of treated Artemia cyst mix into a sterile 100-milliliter bottle. Using a film, seal the bottle thoroughly and place it in a shaking incubator with light. Next, using a Pasteur pipette, extract 30 milliliters of hatching Artemia nauplii from the middle and lower layers.After filtering, wash the Artemia three times with sterile ultrapure water for about one minute, and add four milliliters of sterile ultrapure water. Finally, using a disposable Pasteur pipette, retrieve actively swimming Artemia from the upper layer, wash, and prepare the solution for feeding. Microscopic observation confirmed efficient hatching of Artemia cysts after the treatment.