Name: 作者聚焦：建立无菌斑马鱼用于微生物群-宿主关系研究
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

我们衷心感谢重庆医科大学人才项目（项目编号：R4014，项目编号：R4020）、国家自然科学基金项目（国家自然科学基金项目，编号：32200386）、重庆市博士后创新导师工作室（X7928 DSP）、中国科学院中斯水技术研究示范联合中心项目/中科院中斯联合教育研究中心等项目的支持。

鱼类实验是按照重庆市动物护理和使用专业委员会和重庆医科大学动物护理和使用机构的指导原则以及国家质量技术监督局颁布的实验动物标准（批准号：GB14922-2001-GBT14927-2001）进行的。斑马鱼（Danio rerio，野生型，AB品系）来源于中国科学院水生生物研究所，并按照先前报道的程序在实验室中保存36。
1.成年斑马鱼的维护和胚胎采集
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优化 GF 斑马鱼模型：生成、维护和微生物研究

GF鱼类和GF食品制备协议中的关键步骤 在GF鱼模型的生成过程中，涉及几个关键步骤，包括无菌材料的制备、胚胎的灭菌、GZM的每日更新、各种样品的收集以及使用多种方法对每个样品进行无菌检查。在这些步骤中，胚胎的初始处理对于GF模型的成功是基础和决定性的。控制药剂、药剂浓度和处理时间对于实现最佳灭菌率和孵化成功率至关重要。在将GF鱼胚胎处理到幼虫时，需要特别...

微生物群（即古细菌、细菌、真核菌和病毒）在维持宿主健康方面发挥着至关重要的作用，并通过在肠道屏障、上皮表面和个体粘蛋白功能内的共生相互作用影响生理和病理过程，从而促进各种疾病的发展 1,2,3.从婴儿期到少年期、成年期和衰老等不同生命阶段的微生物群组成，以及其在鼻孔、口腔、皮肤和肠道部位等不同部位的存在，是由不同的栖息地和环境动态塑造的4.生物体中的肠道菌群参与营养吸收、免疫反应、病原体入侵、代谢调节等 5,6.对患者的研究表明，肠道菌群的破坏与人类肥胖、睡眠障碍、抑郁症、炎症性肠病 （IBD）、神经退行性疾病（帕金森氏症、阿尔茨海默氏症）、衰老和各种癌症有关 7,8,9。此外，肠道微生物群和宿主之间的相互作用途径涉及炎症因子、神经递质、代谢物、肠道屏障和氧化应激，正如之前使用小鼠和鱼类模型的研究中观察到的那样10,11。
最近，在临床和动物模型中，已经探索了多种与细菌相关的方法或疗法，包括潜在的益生菌和粪便微生物群移植 （FMT），用于治疗这些疾病。这些探索基于与微生物群-肠-脑/肝/肾轴、微生物群衍生产物和受体活性改变相关的发现12,13。然而，由于微生物群落的复杂性和生成强大的类人疾病模型的挑战，微生物群-宿主系统的发育、各种功能和机制仍未完全理解和识别。
为了解决这些问题，无菌（GF）动物模型在19世纪中叶被紧急提出，并在20世纪主要发展起来。随后的改进，包括抗生素处理和gnotobiotic模型，以及微生物检测和观察技术的进步，进一步完善了这些模型14,15,16。GF动物通过消除自身背景和避免环境微生物而产生，为探索微生物与其宿主之间的相互作用提供了一种极好的策略17。通过应用动物模型和精细的方案，研究人员成功地复制了在GF小鼠和鱼类患者中发现的相似微生物组成。此外，其他GF动物模型，如狗、鸡和猪，作为研究对象提供了多样化的选择18,19,20,21。这种方法使人们能够研究共生微生物组对各种疾病的潜在治疗效果，包括人类癌症免疫疗法16,18。GF模型为特定细菌在宿主内定植、迁移、繁殖和相互作用的特征和机制提供了更准确的见解。这为微生物群相关疾病的发生和发展提供了重要的新见解22,23。GF斑马鱼在微生物研究中的建立和应用的历史从2004年Rawls等人的报告和2006年Bates等人的报告发展到2017年Melancon等人的方案16,24,25。然而，成年或繁殖GF模型的可行性仍然是一个漫长的过程，伴随着可变的寿命、成功率和健康挑战。
在各种动物模型中，斑马鱼（Danio rerio）因其与人体器官和基因组学的有利相似性、发育周期短、繁殖力高和胚胎透明等优点，成为基础和生物医学研究的重要工具19,26。斑马鱼作为可靠的人类疾病模型，提供了体内生理和病理过程的视觉表示，提供了对宿主-微生物相互作用的吸引人特征的见解。值得注意的是，斑马鱼表现出独特的细胞谱系，可以对肠道生理学、微生物动力学、性腺和生殖发育、宿主免疫系统的成熟、行为和新陈代谢进行成像27。斑马鱼胚胎在保护性绒毛膜内发育，直到孵化，在受精后 3 天 （dpf） 成为幼虫。它们在 5 dpf 时积极寻找食物，并在受精后 （mpf） 3 个月左右达到性成熟 28。Rawls 等人 24 报道的首次成功的无菌 （GF） 斑马鱼表明，在卵黄吸收后用高压灭菌饲料喂养的幼虫表现出 8 dpf 的组织坏死和 20 dpf 时的总死亡。这表明了饮食的影响或在涉及长期（>7 dpf）GF鱼的实验中考虑外源性营养供应的重要性29。随后的研究改进了GF鱼的生成方案，采用了在不同鱼类模型中完善的无菌食品和方法16。
然而，对 GF 斑马鱼模型的大多数研究都集中在早期生命阶段，涉及细菌感染在 5 dpf 下持续 24 小时至 48 小时，在实验结束时在 7 dpf 之前收集样本 25,30,31。人们普遍认为，包括人类和斑马鱼在内的生物体中的微生物群在生命开始时就被定植，并在生长和发育过程中形成。该成分在成虫阶段保持稳定，宿主体内微生物群的作用在整个生命过程中都至关重要，尤其是在衰老、神经退行性、代谢相关肥胖和肠道疾病方面3.因此，考虑到鱼类幼虫在早期生命中的免疫和生殖系统不成熟，来自存活时间较长的GF动物的观点可以深入了解微生物在宿主器官发育和功能中的作用机制。虽然斑马鱼肠道中的细菌菌株在以前的研究中已被分离和鉴定，为感染 GF 动物模型以选择益生菌或研究宿主中的细菌功能提供了潜力19,25，但 GF 鱼模型的生成和应用主要局限于早期生命阶段。这种局限性归因于复杂的生产过程、高昂的维护成本以及与食物和免疫力相关的问题，阻碍了旨在调查宿主中微生物群的发育和慢性影响的研究工作。
鱼类的存活率、行为、生长、成熟和整体健康状况，尤其是在无菌 （GF） 模型中，受到摄食方式的显着影响，包括从早期幼虫到幼鱼张口期间的营养摄入和吸收32,33。然而，GF鱼类养殖业面临的挑战之一是缺乏合适的不育日粮，这限制了维持幼鱼生长和存活的营养支持的有效性。考虑到GF鱼的发育防御机制和由于缺乏肠道微生物组而导致的消化能力薄弱，解决这个问题对于恢复GF鱼的生命至关重要。在食物方面，活盐水虾（卤虫属）是张口幼鱼到幼鱼最合适的食物。据观察，与用煮熟的蛋黄或其他天然和合成饵料喂养的鱼相比，用活盐水喂养的鱼表现出更高的生长率和成活率34。虽然GF鱼的早期生活模型可以在卵黄支持下生存，而GF幼虫模型可以通过无菌喂养来维持，但从幼虫到幼鱼并达到性成熟的长期模型仍然具有挑战性。此外，片状或粉状食物受到营养成分不均的限制，并可能影响水质。相比之下，活卤虫具有在盐水和淡水中都能存活、体型小、适合幼虫到成虫、易于配料和更高的孵化质量等优点35.在先前方法16、24、30 的基础上，我们简化了复杂的处理过程，并通过建立易于孵育的 GF 活卤虫作为无菌食品，其持续时间比早期 GF 鱼更长，从而解决了饮食挑战。
本研究提出了一个优化的方案，涵盖 （1） 代、（2） 维持、（3） 确定不育率以及 （4） 维持和饲喂，以确保无菌 （GF） 斑马鱼从胚胎生长到幼虫和幼年阶段。研究结果为GF斑马鱼的孵化、存活、生长和不育提供了初步证据，并为GF 卤虫 属作为不育食品提供了重要指标。无菌活食模型生成和制备的详细步骤为构建和应用长期GF鱼模型以及 GF卤虫 属在微生物群-宿主相互作用研究中提供了重要的技术支持。该协议解决了GF鱼模型上的细菌分离，鉴定和感染，概述了细菌荧光标记的方法，并在显微镜下观察它们在鱼肠道中的定植。GF鱼、细菌感染的gnotobiotic鱼或转移的人类微生物群模型将进行各种检测，以阐明其功能和对宿主免疫、消化、行为、转录组调控和代谢方面的影响。从长远来看，该协议可以扩展到不同的野生型鱼类，例如海洋青鳉鱼，并可能扩展到与特定组织或疾病相关的其他选定的转基因斑马鱼品系。

<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

斑马鱼因其与哺乳动物的基因组相似性、在相对干净的绒毛膜环境中发育的透明胚胎以及与啮齿动物模型相比幼虫发育极快，因此可以作为生长、免疫和肠道微生物群研究的宝贵模型。无菌（GF）斑马鱼（Danio rerio）对于评估污染物毒性和建立与微生物功能相关的类人疾病模型至关重要。与传统饲养（CR）模型（共同饲养的鱼类）相比，GF斑马鱼可以更准确地操纵宿主微生物群，有助于确定微生物与宿主之间的因果关系。因此，它们在促进我们对这些关系的理解方面发挥着关键作用。然而，由于免疫功能和营养吸收的限制，GF斑马鱼模型通常是在生命早期（从胚胎到幼虫）生成和研究的。本研究优化了早期GF斑马鱼模型的生成、维持和鉴定，无需摄食和长期使用GF食物（如 卤虫 、盐水虾）摄食。在整个过程中，每天进行采样和培养，并通过多次检测进行鉴定，包括平板和 16S rRNA 测序。记录GF斑马鱼的无菌率、存活率和发育指标，保证所生成模型的质量和数量。重要的是，这项研究提供了关于GF鱼的细菌分离和感染技术的详细信息，使得在GF食物支持下能够有效地创建从幼虫到幼鱼阶段的GF鱼模型。通过在生物医学研究中应用这些程序，科学家们可以更好地了解肠道细菌功能与宿主健康之间的关系。

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.

作者聚焦：建立无菌斑马鱼用于微生物群-宿主关系研究

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

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.

GF斑马鱼模型可以通过利用成对的斑马鱼产卵来有效地生产，并根据以前的GF鱼模型35优化了协议。单个 6 孔板可培养约 30-48 个胚胎/幼虫，可进行充足的数据收集和统计分析。无菌处理后，将GF胚胎在干净的培养箱中培养至48-72小时孵化为幼虫，并每天检测收集的样品以改变GZM，这对于保持无菌状态至关重要（图1）。7 dpf后，应为幼虫至幼虫阶段准备蛋黄和?...

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

该协议概述了获得无菌（GF）鱼胚胎并将其从幼虫维持到幼虫阶段的主要步骤，包括取样和检测其不育状态。使用GF模型处理感染对于理解微生物在宿主健康中的作用非常重要。

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

这项研究的范围是生成新颖的、无菌的动物模型，这将是研究各种微生物组功能的有用和强大的工具。在该领域，早期的无菌鱼模型是最近才得到应用的，但由于养殖条件和工作人员发育防御的不同，长期模型仍然有限。目前的GF鱼模型通常是早期生命的。因此，包括无菌秤、样品检测方法和提供无菌食品在内的技术是需要克服的非常重要的挑战。无菌斑马鱼模型对于研究宿主-微生物群相互作用及其对发育、免疫和疾病的影响至关重要。该模型使研究人员能够精确地操纵单个肠道细菌，并观察其对透明斑马鱼胚胎的影响。它为遗传疾病的肠道微生物的作用提供了宝贵的见解。未来，我们的研究将主要集中在建立斑马鱼全生命周期的无菌模型上，以更好地了解肠道微生物组的功能。

generation-maintenance-identification-germ-free-zebrafish-models-from

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.

科研

发育生物学

Generation of Germ-Free Zebrafish Models from Embryos

从胚胎中生成无菌斑马鱼模型

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.