作者感谢育种者和Boyé accouvage协会（法国La Boissière en Gâtine）提供受精卵。这项研究是在研究联盟APR-IA"INTEGRITY"（2017-2019）的主持下进行的，该联盟由法国卢瓦尔河谷地区中心资助。

动物护理程序是根据欧洲共同体理事会指令（86/609/EEC）和法国动物实验条例制定的准则进行的。
1. 隔离器准备
<ol><li>在正压下将必要的材料插入刚性隔离器:50 mL 管、5、10 和 25 mL 塑料移液器、辐照进料、高压灭菌水和无菌密封塑料容器。</li>
	<li>用2%季铵盐溶液填充转移杀菌阱。</li>
	<li>使用60 mL福尔马林（24%甲醛）加入30g高锰酸钾（m 3），用甲醛蒸气灭菌隔离器三次。在每次灭菌之间移动隔离器内的材料，以确保所有接触面的灭菌。</li>
	<li>在引入卵子之前，将隔离器温度设置为37°C至少2天。在引入当天将湿度计设置为相对湿度（RH）的65-70%。</li>
</ol>2. 种蛋收集和孵化
<ol><li>根据蛋鸡的良好孵化率（在产蛋高峰期至少80%）及其良好的卫生状况（即没有常见的家禽病原体和羊群中没有疾病）从选择的农场收集鸡蛋。在跑步机上直观地选择干净无瑕的鸡蛋。</li>
	<li>通过在室温下将其浸入1.5%过氧乙酸溶液中浸渍5分钟，立即对卵表面进行去污。</li>
	<li>使用用甲醛蒸气净化的盒子将鸡蛋运输到实验设施。</li>
	<li>将鸡蛋在4°C下储存24小时。 重复步骤2.2中描述的去污过程，并将其置于孵化箱（第0天）中19天。</li>
</ol>3. 孵化
<ol><li>在第19天，通过在无菌条件下使用轻鸡蛋蜡烛将它们倒入来验证生育能力，活力（运动性）和胚胎发育。仅将活胚胎卵引入无菌孵化隔离器中。</li>
	<li>通过喷洒1.5%过氧乙酸溶液30秒或直到每个鸡蛋的整个表面被覆盖来对选定的鸡蛋进行净化。在转移到隔离器的过程中，卵子将与喷雾保持16分钟和30秒的接触。</li>
	<li>将卵转移到无菌孵化隔离器中，并用无菌软化水冲洗，然后将其放入孵化空间。 注意:动物在无菌隔离器中孵化和饲养。他们 随意 喂食通过伽马辐照消毒的商业饮食，并用饮水机提供的高压灭菌的自来水浇水。</li>
</ol>4. 细菌学状态分析
<ol><li>孵化后一天，直接从不同雏鸡的直肠中取出粪便样本，并将其池化在灭菌玻璃管中，以使该样本代表给定隔离器内的所有动物。</li>
	<li>将粪便样品或多或少地是液体，用利沙林向9mL硫代甘氨酸盐肉汤中加入相当于1mL的粪便。将剩余的粪便样品加入9mL脑心输液（BHI）中，并将管子在37°C下孵育18至48小时而不摇动。这将允许广泛的有氧，兼性有氧和非快速厌氧物种的生长。 注意:含有刃天青的硫代乙酸盐肉汤用于检测非快速厌氧细菌，但它也可以检测好氧细菌。该培养基符合欧洲，美国和日本药典的无菌测试18，19，20。</li>
	<li>目视观察孵育18小时后生长培养基是否有任何修饰。48小时后，从BHI粪汤培养基中滴出一滴，将其放在载玻片上，并在显微镜下观察（40X放大倍率）是否没有或存在细菌。</li>
	<li>如果怀疑细菌的存在，请从BHI培养物中取样并将其播种到BHI琼脂平板上。在37°C下孵育18至48小时。</li>
	<li>如果存在菌落，则进行高通量技术，例如MALDI-TOF质谱法，以进行精确的微生物鉴定。 注意:72小时后，如果细菌学分析为阴性，则宣布动物无菌。</li>
</ol>

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作者没有什么可透露的。

以前已经描述了几种产生无菌鸡的方法7，21，22。简单的方法，例如这里介绍的方法，使用不同的消毒剂来减少卵子表面和隔离器中的细菌负荷。最常用的消毒剂是氯化汞、季铵盐、碘仿、次氯酸钠和二氧化氯溶液。结果往往令人满意。然而，尽管这些方法易于获得，但很少有结构可以应用该方法并大规模饲养动物，因此使得利用无菌鸡成为一种相对罕见的方法，仅用于解决非常具体的科学问题。这里描述的方法，材料和设备允许无菌肉鸡的高效孵化比例，这些肉鸡保持健康和无菌至少3周（生产它们的科学实验的持续时间）。
结果表明，将该方案改编为从商业家禽养殖场收集的鸡蛋中生产无菌雏鸡是成功的。SPF产蛋经验表明，孵化效率主要取决于蛋鸡的年龄和收集的蛋的质量。在选择农场和收集种蛋时，这两个参数都被考虑在内。使用相同的方法，无菌肉鸡的平均孵化率远远优于我们工厂上次生产SPF蛋鸡（79%对35%），而不影响动物不育（87%对83%）。这些差异可能与鸟类的遗传背景（肉鸡与蛋鸡）以及蛋壳的质量有关，蛋壳在SPF动物的蛋中可能更脆弱，在封闭繁殖中保持了40多年。此外，我们还表明，超过2小时的运输（从养殖场到实验设施）不会影响孵化效率和质量。
虽然用于孵化隔离器的灭菌过程和鸡蛋去污方案得到了优化，但大约10%的隔离器不是无菌的。为了了解污染的来源，在引入卵子之前对孵化的隔离器进行常规的无菌控制非常重要。
关于鸟类的不育状态，我们试图应用允许粪便样品中需氧和非快速厌氧细菌生长的方法，从而确保我们检测到活的活细菌。这些方法符合国际无菌测试药典，代表了常规使用的快速，简单和降低成本的技术。然而，分子生物学技术，如16S rRNA基因测序，虽然没有提供细菌活力的信息，但可以应用于确认不可培养细菌的存在。事实上，最近的一项研究表明，母体输卵管微生物群的一些细菌似乎通过蛋清转移到胚胎上，后来构成了大部分胚胎肠道细菌种群5。此外，另一项研究表明，早期胚胎中携带的部分微生物定殖者是从母鸡遗传的，肠道微生物的丰度和多样性后来受到发育过程中环境因素和宿主遗传学的影响23。然而，这些研究的结果是基于DNA序列分析，其中大量这些细菌可能在蛋清中死亡或不可复制（大量含有抗菌分子）。Thomas和16 名合作者通过粪便滴落在BHI平板上评估可培养的需氧和兼性需氧细菌，进行了无菌测试，从而突出了标准细菌学方法在无菌无菌控制方面的效率。此外，在提出的方案中，我们使用乙酰草素在硫代乙酸盐肉汤中的生长监测，以便能够检测非快速厌氧菌的生长。
该方案已经用于生产无菌鹌鹑和鸡，可适应大多数筑巢鸟类的无菌动物的生产，并为研究微生物群对这些动物的生理学贡献提供了视角。除了使用该模型来研究家禽肠道中的宿主 - 微生物群相互相互作用外，它还可用于应用研究。例如，它可用于评估来自鸡肠道共生微生物的益生菌的安全性和有效性，以改善动物的健康和健壮性。

关于肠道微生物群对动物健康的贡献的科学和大众兴趣急剧增加。微生物群由栖息在动物肠道不同生态位的细菌，病毒，真菌和古菌组成，直接或间接地与炎症，传染病和代谢性疾病的调节有关，这些疾病不仅影响哺乳动物物种，还影响牲畜，如家禽1。开发了几种动物模型，以更好地研究肠道微生物群对健康和疾病的贡献。例如，无菌和无菌动物允许分别在感染的物理病理学上研究完全没有微生物或已知微生物群2，3。然而，产生和维护这些动物需要专门的技术和设施，而维护它们所需的成本，劳动力和技能限制了它们与许多研究人员的联系。事实上，必须使用细菌培养方法、显微镜检查、血清学、大体形态学和基于测序的检测技术的组合，定期监测无菌动物是否存在可能的污染。类似的程序也适用于其他物种，如牲畜，其中动物通常体型较大，需要更大的设施进行繁殖和维护，这可能在一定程度上阻碍对微生物群的研究。
家禽，更具体地说是鸡，是全球畜牧业生产的基石，每年的家禽数量可能超过400亿只。它是世界上最重要的动物蛋白来源（http://www.fao.org/poultry-production-products/en/）。此外，没有与养鸡或食用鸡有关的文化或宗教禁忌。家禽肠道微生物群参与动物的生长、饲料转化率、免疫力、病原体抗性等诸多营养、生理或病理过程4。因此，无菌鸡的产生对于强调微生物群与其宿主之间的对话是必不可少的4。即使微生物群落栖息在鸡的输卵管5中，健康母鸡新鲜产下的蛋的含量也大多不含微生物，蛋壳和膜具有机械屏障，以避免微生物入侵4。此外，雏鸡在没有亲戚的情况下很容易饲养，与哺乳动物不同，这允许在无菌条件下没有父母抚养的情况下生产无菌动物。
实验设施"农场，模型和野生动物的感染学"（PFIE，UE-1277，法国努齐利卢瓦尔河谷中心，https://doi.org/10.15454/1.5572352821559333e12）是法国国家基础设施网络EMERG'IN（https://www.emergin.fr/）的一部分。PFIE已经掌握了无菌鸡的生产，进行了40多年的各种实验研究7，8，9，10，11。这些动物是由自20世纪70年代以来在封闭育种中饲养的白色腿角产蛋中产生的特定无病原体（SPF）卵产生的。主要用于微生物研究7，8，9，无菌鸟类正在经历兴趣的复苏，例如肠道微生物群对行为13，营养利用14，免疫发育15和内分泌活性的贡献等问题。即使一些研究已经使用无菌肉鸡系16发表，与使用实验层系的研究相比，这些研究仍然代表性不足。在家禽健康和福利中，微生物群与其宿主之间的串扰的科学问题演变促使我们调整我们的历史协议，以生产Ross PM3系列的无菌肉鸡，这是世界上使用最多的肉鸡系列。

<table><tbody><tr><td>2 mL sterile plastic pipettes</td><td>Starsted</td><td>86.1252.001</td><td></td></tr><tr><td>50 mL tubes</td><td>Falcon</td><td></td><td></td></tr><tr><td>BHI agar plates</td><td>Thermo fisher diagnostic</td><td>PO1198A</td><td></td></tr><tr><td>Brain Heart Infusion broth</td><td>Thermo fisher diagnostic</td><td>CM1135</td><td></td></tr><tr><td>Glass tubes with 9 mL BHI broth</td><td></td><td></td><td>home made and sterilized by autoclaving</td></tr><tr><td>Glass tubes with 9 mL thioglycolate broth with resazurin</td><td></td><td></td><td>home made and sterilized by autoclaving</td></tr><tr><td>Hatching incubator</td><td>Fieme</td><td>MG 576</td><td></td></tr><tr><td>Incubator</td><td>Memmert</td><td></td><td>for bacteriological culture, 37 &amp;deg;C</td></tr><tr><td>Irradiated feed</td><td>Safe</td><td>U8983G10R</td><td>40 kG irradiated</td></tr><tr><td>Isolators</td><td></td><td></td><td>home made. 1 m&lt;sup&gt;3&lt;/sup&gt; rigid isolator under positive pressure</td></tr><tr><td>Microbiological safety cabinet</td><td>thermon electron corporation</td><td></td><td>model: Hera Safe</td></tr><tr><td>Microscope Visiscope series 300</td><td>VWR</td><td></td><td></td></tr><tr><td>Pipette aid</td><td>Drummond</td><td></td><td></td></tr><tr><td>Plastic pipettes</td><td></td><td></td><td></td></tr><tr><td>Sterile sealed boxes</td><td>Tuperware</td><td></td><td>diameter</td></tr><tr><td>Sterilized glass tube</td><td></td><td></td><td>&quot;sovirel&quot;</td></tr><tr><td>Thioglycolate Broth with Resazurin</td><td>Merck</td><td>90404-500G</td><td></td></tr><tr><td>Water bath</td><td>Fisher scientific</td><td></td><td>model: polystat 36, used to incubate 10 min at 100 &amp;deg;C the glass tubes with 9 mL thioglycolate broth with resazurin in order to regenerate the medium</td></tr></tbody></table>

production of germ-free fast-growing broilers from a commercial line for microbiota studies

肠道微生物群对宿主生理学和免疫能力的贡献的研究通过无菌动物模型的可用性得到促进，这被认为是黄金标准。筑巢鸟类是生产无菌动物的理想模型，因为不需要在无菌条件下饲养亲戚。无菌鸡主要由无特异性病原体（SPF）实验品系产生，这些试验品系在商业鸡系中代表性较差。这里提出的方法允许从快速生长的肉鸡生产线Ross PM3生产无菌鸡，这是家禽业常用的。在肉鸡饲养场产卵后，迅速收集了鸡蛋。它们经历了严格的去污过程，从收集到引入无菌蛋孵化隔离器。雏鸡已经孵化并保存在这些无菌隔离器中，以控制其不育所必需的时间。该方案最初是为实验性SPF白叉角线开发的，不仅适用于Ross PM3肉鸡系，也适用于鹌鹑。因此，它代表了对其他家禽物种和具有经济，生物或生态相关性的筑巢鸟类的强大且易于适应的程序。

使用来自两个不同法国农场的Ross PM3卵进行了六次无菌雏鸡的生成（表1）。共收集了853枚卵，经过两个去污步骤和19天的孵化，86.40%是可行的。这些活蛋中有490个经过了第三个去污步骤，并被引入各种孵化隔离器中，平均孵化率为79.80%。这表示与收集的初始种蛋数量相比，孵化率为68.94%。
然而，孵化结果根据所进行的系列而有很大差异:与收集的种蛋数量相比，存活雏鸡的41.67%至88.16%。在同一实验中，在不同的舱口中也观察到了这些变化。由于并非所有隔离器都用于所有运行，因此很难排除隔离器相关效应。然而，这种批次效应与蛋鸡的年龄直接相关（图1），其中来自年长母鸡的鸡蛋不太可行。
六次运行是使用四种不同的孵化隔离器进行的。经过细菌学控制，16个隔离器中的14个动物被确认为无菌。这相当于87.5%的成功率。剩下的两个隔离器被一种环境和非致病性细菌污染。
所有用于科学实验的动物在孵化后至少三周内保持无菌状态，直到研究结束。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61148/61148fig1v2.jpg"> 图1:母鸡年龄对孵化率的影响 本图突出显示了根据蛋鸡群的年龄观察到的孵化率，以产蛋周数表示。 <a href="https://www.jove.com/files/ftp_upload/61148/61148fig1v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:font-size="5px" fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>收集的鸡蛋</td>
			<td>D19 活卵</td>
			<td>D19 活卵/收集的卵子</td>
			<td>将卵子转入隔离器 1</td>
			<td>将卵子转入隔离器 2</td>
			<td>将卵子转入隔离器 3</td>
			<td>将卵子转入隔离器 4</td>
			<td>转移的卵子总数</td>
			<td>孵化成隔离器1的活雏鸡</td>
			<td>孵化成隔离器2的活雏鸡</td>
			<td>孵化成隔离器3的活雏鸡</td>
			<td>孵化成隔离器的活雏鸡 4</td>
			<td>存活的孵化雏鸡总数</td>
			<td>活孵化/转移的种蛋分离器1</td>
			<td>活体孵化/转移的种蛋隔离器2</td>
			<td>活孵化/转移的种蛋隔离器3</td>
			<td>活孵化/转移的种蛋隔离器4</td>
			<td c="">全球可行的孵化/转移种蛋</td>
		</tr>
<tr>
<td>运行 1</td>
			<td>101</td>
			<td>93</td>
			<td>92.08%</td>
			<td>23</td>
			<td>24</td>
			<td>-</td>
			<td>-</td>
			<td>47</td>
			<td>22</td>
			<td>23</td>
			<td>-</td>
			<td>-</td>
			<td>45</td>
			<td>95.65%</td>
			<td>95.83%</td>
			<td></td>
			<td></td>
			<td>95.74%</td>
		</tr>
<tr>
<td>运行 2</td>
			<td>130</td>
			<td>117</td>
			<td>90.00%</td>
			<td>25</td>
			<td>25</td>
			<td>25</td>
			<td></td>
			<td>75</td>
			<td>20</td>
			<td>24</td>
			<td>18</td>
			<td>-</td>
			<td>62</td>
			<td>80.00%</td>
			<td>96.00%</td>
			<td>72.00%</td>
			<td></td>
			<td>82.67%</td>
		</tr>
<tr>
<td>运行 3</td>
			<td>132</td>
			<td>97</td>
			<td>73.48%</td>
			<td>26</td>
			<td>-</td>
			<td>26</td>
			<td>45</td>
			<td>97</td>
			<td>14</td>
			<td>-</td>
			<td>13</td>
			<td>28*</td>
			<td>27</td>
			<td>53.85%</td>
			<td></td>
			<td>50.00%</td>
			<td>62.22%</td>
			<td>56.70%</td>
		</tr>
<tr>
<td>运行 4</td>
			<td>130</td>
			<td>116</td>
			<td>89.23%</td>
			<td>36</td>
			<td>35</td>
			<td>-</td>
			<td>-</td>
			<td>71</td>
			<td>33</td>
			<td>34</td>
			<td>-</td>
			<td>-</td>
			<td>67</td>
			<td>91.67%</td>
			<td>97.14%</td>
			<td></td>
			<td></td>
			<td>94.37%</td>
		</tr>
<tr>
<td>运行 5</td>
			<td>180</td>
			<td>148</td>
			<td>82.22%</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>120</td>
			<td>26*</td>
			<td>25</td>
			<td>17</td>
			<td>24</td>
			<td>66</td>
			<td>86.67%</td>
			<td>83.33%</td>
			<td>56.67%</td>
			<td>80.00%</td>
			<td>76.67%</td>
		</tr>
<tr>
<td>运行 6</td>
			<td>180</td>
			<td>166</td>
			<td>92.22%</td>
			<td>-</td>
			<td>40</td>
			<td>40</td>
			<td>-</td>
			<td>80</td>
			<td>-</td>
			<td>35</td>
			<td>35</td>
			<td>-</td>
			<td>70</td>
			<td>86.67%</td>
			<td>87.50%</td>
			<td>87.50%</td>
			<td></td>
			<td>87.50%</td>
		</tr>
<tr>
<td>总</td>
			<td>853</td>
			<td>737</td>
			<td>86.40%</td>
			<td>140</td>
			<td>154</td>
			<td>121</td>
			<td>75</td>
			<td>490</td>
			<td>89</td>
			<td>141</td>
			<td>83</td>
			<td>24</td>
			<td>337</td>
			<td>82.14%</td>
			<td>91.56%</td>
			<td>68.60%</td>
			<td>69.33%</td>
			<td>79.80%</td>
		</tr>
</tbody></table>表1:不同孵化实验的技术结果。 本表突出显示了进行6系列无菌孵化的结果:收集的种蛋数量，19天胚胎的存活率以及各种隔离器中存活的孵化雏鸡。

Watch this Scientific Journal Video about Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies at JoVE.com

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

在这里，我们描述了一种从商业肉鸡系的鸡蛋Ross PM3中生成无菌雏鸡的方法。这种方法可以适用于从其他家禽物种中产生无菌动物。

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, ...

production-germ-free-fast-growing-broilers-from-commercial-line-for

Research

JoVE Journal

Immunology and Infection

3.0K 次观看.  INRAE, PFIE, UE-1277. 在这里，我们描述了一种从商业肉鸡系的鸡蛋Ross PM3中生成无菌雏鸡的方法。这种方法可以适用于从其他家禽物种中产生无菌动物。

视频: Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

发育生物学

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

环境科学

Isolation of Preadipocytes from Broiler Chick Embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

从肉鸡雏鸡胚胎中分离出前脂肪细胞

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

优化 GF 斑马鱼模型:生成、维护和微生物研究

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.

作者聚焦:建立无菌斑马鱼用于微生物群-宿主关系研究

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.

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

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

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations. 
Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.

This protocol describes the best practices for germ-free mouse transfer to and housing in experimental single-cage isolators (isocages) while maintaining sterile conditions. Methods for fecal transplant into germ-free mice and the collection of viable bacteria from these gut "humanized" mice for further applications are discussed.

用于人粪便移植到无菌小鼠体内的生物排斥 IsoPositive 笼实验的制备和维持

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

生物学

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

摘要

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此视频中的章节

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

饲养果蝇

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

从肉鸡雏鸡胚胎中分离出前脂肪细胞

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

用于人粪便移植到无菌小鼠体内的生物排斥 IsoPositive 笼实验的制备和维持

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

摘要

探索更多视频

此视频中的章节

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

饲养果蝇

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

从肉鸡雏鸡胚胎中分离出前脂肪细胞

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

用于人粪便移植到无菌小鼠体内的生物排斥 IsoPositive 笼实验的制备和维持

建立和维护侏罗育美国蟑螂 （佩里普拉内塔美洲）

用组织培养幼苗制备和饲养轴性昆虫用于叶甲虫的宿主 - 肠道微生物群相互作用研究

建立和维护侏罗育美国蟑螂（佩里普拉内塔美洲）