The authors are grateful to the breeders and the society Boy&#233; accouvage (La Boissi&#232;re en G&#226;tine, France) for the supply of fertilized eggs. This study was conducted under the auspices of the research consortium APR-IA &#8220;INTEGRITY&#8217; (2017-2019), which was funded by the R&#233;gion Centre Val de Loire, France.

Animal care procedures were carried out in accordance with the guidelines set by the European Communities Council Directive (86/609/EEC) and by the French regulations on animal experimentation.
1. Isolator preparation
<ol>
	<li>Insert the necessary materials into a rigid isolator under positive pressure: 50 mL tubes, 5, 10 and 25 mL plastic pipettes, irradiated feed, autoclaved water and sterile sealed plastic containers.</li>
	<li>Fill the transfer germicidal trap with a 2% quaternary ammonium solution.</li>
	<li>Sterilize the isolator three times with formaldehyde vapor by using 60 mL of formalin (24% formaldehyde) added to 30 g of potassium permanganate per cubic meter (m3). Move the materials inside the isolator between each sterilization to ensure the sterilization of all contact surfaces.</li>
	<li>Set the isolator temperature to 37 &#176;C for at least 2 days before introducing the eggs. Set the hygrometer to 65-70% of relative humidity (RH) on the day of introduction.</li>
</ol>
2. Egg collection and incubation
<ol>
	<li>Collect the eggs from farms selected on the basis of a good hatching rate of the laying hens (at least 80% at the peak of egg production) and their good sanitary status (i.e., absence of common poultry pathogens and absence of diseases within the flock). Visually select clean and flawless eggs on the treadmill.</li>
	<li>Immediately decontaminate the egg surface by dipping them for 5 min in a 1.5% peracetic acid solution at room temperature.</li>
	<li>Transport the eggs to the experimental facility using a box decontaminated with formaldehyde vapor.</li>
	<li>Store the eggs for 24 hours at 4 &#176;C. Repeat the decontamination process described in step 2.2, and place them in a hatching incubator (Day 0) for 19 days.</li>
</ol>
3. Hatching
<ol>
	<li>On Day 19, verify the fertility, the viability (motility) and the embryo development by candling them using a light egg candler under sterile conditions.&#160;Introduce only live-embryonated eggs into the sterile hatching isolators.</li>
	<li>Decontaminate the selected eggs by spraying 1.5% peracetic acid solution for 30 s or until the entire surface of each egg is covered. Eggs will stay in contact with the spray for 16 min and 30 s during the transfer into isolators.</li>
	<li>Transfer the eggs into the sterile hatching isolators and rinse them with sterile demineralized water before placing them in the hatching space. 
	NOTE: Animals are hatched and reared in sterile isolators. They are fed ad libitum with a commercial diet sterilized by gamma irradiation and watered with autoclaved tap water provided by water dispensers.</li>
</ol>
4. Bacteriological status analysis
<ol>
	<li>One day after hatching, take a fecal sample directly from the rectum of different chicks and pool them in a sterilized glass tube in order to make this sample representative of all animals within a given isolator.</li>
	<li>The fecal samples being more or less liquid, add an equivalent of 1 mL of stool to 9 mL of thioglycolate broth with resazurin. Add the remaining fecal sample to 9 mL of brain heart infusion broth (BHI) and incubate the tubes at 37 &#176;C without shaking for 18 to 48 h. This will allow the growth of a broad range of aerobic, facultative aerobic and nonfastidious anaerobic species. 
	NOTE: Thioglycolate broth with resazurin is intended for the detection of nonfastidious anaerobic bacteria but it also enables the detection of aerobic bacteria. This medium complies with the European, American, and Japanese pharmacopoeias for sterility testing18,19,20.</li>
	<li>Visually observe if any modification in the growth media occurs after 18 hours of incubation. After 48 h, take a drop from the BHI fecal-broth media, place it on a glass slide and observe under a microscope (40X magnification) for the absence or presence of bacteria.</li>
	<li>If there is a suspicion on the presence of bacteria, take a sample from BHI culture and seed it onto BHI agar plates. Incubate at 37 &#176;C for 18 to 48 h.</li>
	<li>If colonies are present, carry out high-throughput techniques such as MALDI-TOF mass spectrometry for precise microbial identification. 
	NOTE: After 72 h, if the bacteriological analyses are negative, the animals are declared germ-free.</li>
</ol>

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Eur.): Supplement 6.3, Sterility. , (2009).</li><li>Japanese Pharmacopoeia, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Japanese Pharmacopoeia (JP): The 4.06 Sterility Test as it appeared in the partial revision of the JP 15th edition. , (2009).</li><li>United States Pharmacopeia (USP). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> United States Pharmacopeia (USP): Sterility Tests as presented in Pharmacopeial Forum, Interim Revision Announcement No. 6. 34 (6), (2008).</li><li>Harrison, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+germ-free+chicks:+a+comparison+of+the+hatchability+of+eggs+sterilized+externally+by+different+methods.">Production of germ-free chicks: a comparison of the hatchability of eggs sterilized externally by different methods.</a> Laboratory Animals. 3 (1), 51-59 (1969).</li><li>Yokota, H., Furuse, M., Okumura, J., Iwao Task, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+production+and+rearing+of+the+germ-free+chick.">A simple method for production and rearing of the germ-free chick.</a> J-STAGE home Nihon Chikusan Gakkaiho. 55 (8), (1984).</li><li>Ding, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+and+establishment+of+gut+microbiota+in+chickens.">Inheritance and establishment of gut microbiota in chickens.</a> Frontiers in Microbiology. 8, 1967 (2017).</li></ol>

The authors have nothing to disclose.

Several methods to generate germ-free chickens have been previously described7,21,22. Simple methods, such as the one presented here, use different disinfectants to reduce bacterial load in the egg surface and in the isolators. The most commonly used disinfectants are mercuric chloride, quaternary ammonium, iodoform, sodium hypochlorite and chlorine dioxide solutions. The results are often satisfactory. However, despite the accessibility of these methods, few structures can apply the method and raise the animals in large scale, thus making the utilization of germ-free chickens a relatively rare approach, only used to address very specific scientific questions. The methods, materials and equipment described here allow a highly efficient hatching proportion of germ-free broilers, which remain healthy and sterile for at least 3 weeks (the duration of the scientific experiments to which they were produced).
The results show that the adaptation of the protocol to the production of germ-free chicks from eggs collected in commercial poultry farms is successful.&#160;Experience with SPF egg production reveals that hatching efficiency depends primarily on the age of the laying hens and on the quality of collected eggs. Both parameters were taken into consideration when farms were chosen and eggs collected. Using the same method, the average hatching rate of germ-free broilers is much better than that obtained in the last production of SPF laying hens at our facility (79% vs 35%), without affecting animal sterility (87% vs 83%). These differences may be associated to the genetic background of the birds (broilers versus layers) as well as to the quality of the eggshell, which is likely to be more fragile in the eggs of the SPF animals, kept in closed breeding for more than 40 years. Moreover, we also show that transportation of more than 2 h (from the farms to the experimental facility) does not affect hatching efficiency and quality.
Although the sterilization process used for hatching isolators and the protocol for egg decontamination were optimized, around 10% of the isolators were not germ-free.&#160;In order to understand the origin of the contamination, it is very important to carry out routine sterility control of the hatched isolators before egg introduction.
Concerning the birds&#8217; sterility status, we tried to apply methods that allow the growth of aerobic and nonfastidious anaerobic bacteria from fecal samples, thus assuring that we are detecting live, viable bacteria. These methods comply with international pharmacopeias for sterility testing and represent rapid, easy and cost-reduced techniques to be used routinely. However, molecular biology techniques such as 16S rRNA gene sequencing, although not giving information on bacteria viability, can be applied for the confirmation of the presence of noncultivable bacteria. Indeed, a recent study suggested that some bacteria of the maternal oviduct microbiota appear to be transferred to the embryo through the egg white, later constituting most of the embryo gut bacterial population5. Moreover, another study suggested&#160;that part of the microbial colonizers harbored in early embryos were inherited from maternal hens, and the gut microbial abundance and diversity were later influenced by environmental factors and host genetics during development23. However, the results of these studies are based on DNA sequence analysis, where a large number of those bacteria could be dead or not replicable in the egg white (greatly loaded with antimicrobial molecules). Thomas and collaborators16 performed sterility testing through the assessment of cultivable aerobic and facultative aerobic bacteria through fecal dropping on BHI plates, thus highlighting the efficiency of standard bacteriological methods for germ-free sterility control. Moreover, in the protocol proposed, we used growth monitoring in thioglycolate broth with resazurin to be able to detect the growth of nonfastidious anaerobic bacteria.
Already used for the production of germ-free quails and chickens, the protocol is adaptable to the production of germ-free animals from most nesting birds and offers perspectives for the study of microbiota contribution to the physiology of these animals. Besides the use of this model to investigate host-microbiota mutual interactions in the poultry gut, it could also be useful for applied research. For example, it could be used to assess safety and efficacy of probiotics derived from chicken gut commensal microorganisms to improve animal&#8217;s health and robustness.

There has been a dramatic increase in scientific and popular interest concerning the contribution of the intestinal microbiota to animal health. The microbiota, consisting of bacteria, viruses, fungi, and archaea that inhabit different niches in the animal&#8217;s intestine, is implicated directly or indirectly in the regulation of inflammatory, infectious and metabolic diseases affecting not only mammalian species but also livestock, such as poultry1. Several animal models were developed to better study the contribution of the gut microbiota to health and disease. For example, germ-free and gnotobiotic animals allow the study of the complete absence of microbes or of a known microbiota, respectively, on the physiopathology of infections2,3. However, generating and maintaining these animals requires specialized techniques and facilities, and the costs, labor, and skills required to maintain them limit their access to many researchers. Indeed, germ-free animals must be monitored regularly for possible contamination using a combination of bacteria cultivation methods, microscopy, serology, gross morphology, and sequencing-based detection techniques. Similar procedures also apply to other species, such as livestock, where animals are generally bigger and require larger facilities for their breeding and maintenance, which may hamper, to some extent, research on the microbiota.
Poultry, more specifically chickens, are the cornerstone of livestock production worldwide, with a flock population that may exceed 40 billion birds per year. It is the most important source of animal protein in the world (http://www.fao.org/poultry-production-products/en/). Moreover, there are no cultural or religious taboos associated to the rearing or consumption of chickens. The poultry gut microbiota is importantly involved in animal&#8217;s growth, feed conversion ratio, immunity, pathogen resistance, among many other nutritional, physiological or pathological processes4. The generation of germ-free chickens is therefore indispensable to underscore the dialogue between the microbiota and its host4. Even if microbial communities inhabit the chicken oviduct5, the content of an egg freshly laid by a healthy hen is mostly free of microorganisms, the eggshell and membranes possessing mechanical barriers to avoid microorganism invasion4. In addition, chicks are easily raised in the absence of their relatives, which, unlike mammals, allows the production of germ-free animals without parental rearing under sterile conditions.
The experimental facility &#8220;Infectiology of Farm, Model and Wild Animals&#8221; (PFIE, UE-1277, Centre INRAE Val de Loire, Nouzilly, France, https://doi-org.remotexs.ntu.edu.sg/10.15454/1.5572352821559333e12) is part of the French National Infrastructure Network EMERG&#8217;IN (https://www.emergin.fr/). PFIE has mastered the production of germ-free chickens to perform various experimental studies for more than 40 years7,8,9,10,11. These animals were produced from specific pathogen-free (SPF) eggs from a white leghorn laying line raised in closed breeding since the 1970&#8217;s. Mainly used for microbiological studies7,8,9, germ-free birds are experiencing a resurgence of interest with questions such as the contribution of the gut microbiota to behavior13, nutrient utilization14, immune development15 and endocrine activity. Even if some studies have been published using germ-free broiler lines16, these studies remain underrepresented compared to studies using experimental layer lines. The evolution of scientific questions towards the crosstalk between the microbiota and its host in poultry health and welfare has led us to adapt our historical protocol to produce germ-free broilers of the Ross PM3 line, the world&#8217;s most utilized broiler chicken line.

<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

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines.&#160;The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry.&#160;Eggs were quickly collected after laying at a broiler breeder farm.&#160;They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator.&#160;The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Six runs of germ-free chicks&#8217; generation were conducted with Ross PM3 eggs coming from two different French farms (Table 1).&#160;A total of 853 eggs were collected and after two decontamination steps and 19 days of incubation, 86.40% were viable. 490 of these viable eggs underwent a third decontamination step and were introduced into various hatching isolators, for an average hatching rate of 79.80%. This represents a hatching rate of 68.94% compared to the initial number of eggs collected.
However, hatching results vary substantially according to the series carried out: from 41.67% to 88.16% of viable chicks compared to the number of eggs collected. These variations were also observed among the different hatches during the same experiment. Since not all isolators have been used for all runs, it is difficult to exclude an isolator-dependent effect. However, this batch effect was directly correlated with the age of the laying hens (Figure 1), where eggs coming from older hens were less viable.
The six runs were carried out using four different hatching isolators.&#160;After bacteriological control, the animals from 14 of the 16 isolators were confirmed to be germ-free.&#160;This corresponds to a success rate of 87.5%.&#160;The two remaining isolators were contaminated by a single environmental and nonpathogenic bacteria.
All animals used for the scientific experiments remained germ-free until the end of the studies, for at least three weeks after hatching.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61148/61148fig1v2.jpg" /> 
Figure 1: Effect of the hen&#8217;s age on hatching rate&#160;The present figure highlights the hatching rates observed based on the age of the laying hen flock, expressed in weeks of laying. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61148/61148fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:font-size="5px" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>Collected eggs</td>
			<td>D19 viable eggs</td>
			<td>D19 viable eggs/collected eggs</td>
			<td>Eggs tranfered into isolator 1</td>
			<td>Eggs tranfered into isolator 2</td>
			<td>Eggs tranfered into isolator 3</td>
			<td>Eggs tranfered into isolator 4</td>
			<td>total of transfered eggs</td>
			<td>viable chicks hatched into isolator 1</td>
			<td>viable chicks hatched into isolator 2</td>
			<td>viable chicks hatched into isolator 3</td>
			<td>viable chicks hatched into isolator 4</td>
			<td>total of viable hatched chicks</td>
			<td>viable hatched/transfered eggs isolator1</td>
			<td>viable hatched/transfered eggs isolator2</td>
			<td>viable hatched/transfered eggs isolator3</td>
			<td>viable hatched/transfered eggs isolator4</td>
			<td c="">viable hatched/transfered eggs global</td>
		</tr>
		<tr>
			<td>Run 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>Run 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>Run 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>Run 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>Run 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>Run 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>Total</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>
Table 1: Technical results of the different hatching experiments.&#160;The present table highlights the results of the 6 series of germ-free hatching carried out: number of eggs collected, viability of the embryo at 19 days and viable hatched chicks in the various isolators.

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

Here we describe a method for the generation of germ-free chicks from eggs of a commercial broiler line, the Ross PM3. This method can be adapted for the generation of germ-free animals from other poultry species.

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

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.0K Views.  INRAE, PFIE, UE-1277. Here we describe a method for the generation of germ-free chicks from eggs of a commercial broiler line, the Ross PM3. This method can be adapted for the generation of germ-free animals from other poultry species.

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

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.

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

Developmental Biology

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.

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 ...

Environment

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.

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

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

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.

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.

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.

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.

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

Biology

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.

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

Studying Development of Axenic Delia antiqua and Ensuring Bacteria-Free Growth

Rearing Axenic Delia antiqua with Half-Fermented Sterile Diets

Axenic insects are obtained from sterile artificial rearing systems using sterile media. These insects, characterized by their small size, short growth cycle, and low feed requirements, are ideal for studying the relationship between microorganisms and hosts. The gut microbiota significantly influences the physiological characteristics of insect hosts, and introducing specific strains into axenic insects provides a method for verifying gut microbial functions. Delia antiqua, a threatening pest in the order Diptera, family Anthomyiidae, and genus Delia, primarily feeds on onions, garlic, leeks, and other vegetables of the family Liliaceae. Its larvae feed on the bulbs, causing rotting, wilting, and even death of entire plants. By rearing axenic larvae, follow-up studies can be conducted to observe the effects of intestinal microflora on the growth and development of D. antiqua. Unlike the method involving antibiotic elimination of associated microbes, this article presents a low-cost and high-efficiency approach to raising axenic D. antiqua. After surface sterilization of D. antiqua eggs, half-fermented sterile diets were used to raise larvae, and the axenic state of D. antiqua was verified through culture-dependent and culture-independent assays. In conclusion, the combination of insect egg sterilization and the preparation of sterile diets for larval culture has enabled the development of an efficient and simple method for obtaining axenic D. antiqua. This method provides a powerful approach to studying insect-microflora interactions.

Author Spotlight: Axenic Rearing of Delia antiqua to Detect the Impact of Intestinal Microbes on Its Growth and Development 

A simple procedure for rearing axenic Delia antiqua with half-fermented sterile diets is described. Only one Wolbachia strain was detected in each instar of axenic D. antiqua using PCR.

Axenic insects are obtained from sterile artificial rearing systems using sterile media. These insects, characterized by their small size, short growth ...

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

Cultivation Strategies for Uncultured Microbial Diversity in the Chicken GI Tract

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, digestion, nutrient absorption, immune system maturation, and prevention of pathogen intrusion. For optimal animal health and productivity, it is imperative to characterize these microorganisms and comprehend their role. While the GIT of poultry holds a reservoir of microorganisms with potential probiotic applications, most of the diversity remains unexplored. To enhance our understanding of uncultured microbial diversity, concerted efforts are required to bring these microorganisms into culture. Isolation and cultivation of GIT-colonizing microorganisms yield reproducible material, including cells, DNA, and metabolites, offering new insights into metabolic processes in the environment. Without cultivation, the role of these organisms in their natural settings remains unclear and limited to a descriptive level. Our objective is to implement cultivation strategies aimed at improving the isolation of a diverse range of anaerobic microbes from the chicken&#39;s GIT, leveraging multidisciplinary knowledge from animal physiology, animal nutrition, metagenomics, feed biochemistry, and modern cultivation strategies. Additionally, we aim to implement the use of proper practices for sampling, transportation, and media preparation, which are known to influence isolation success. Appropriate methodologies should ensure a consistent oxygen-free environment, optimal atmospheric conditions, appropriate host incubation temperature, and provision for specific nutritional requirements in alignment with their distinctive needs. By following these methodologies, cultivation will not only yield reproducible results for isolation but will also facilitate isolation procedures, thus fostering a comprehensive understanding of the intricate microbial ecosystem within the chicken&#39;s GIT.

Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry

The protocol presents two methodologies to improve the isolation of anaerobic intestinal bacteria. The first focuses on the isolation of a diverse range of bacteria using different culture media. The second focuses on the cultivation steps of a specific microbial group, possibly assimilating myo-inositol, to fully comprehend its ecological significance.

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...

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

Summary

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Chapters in this video

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

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

Isolation of Preadipocytes from Broiler Chick Embryos

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

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

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

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

Rearing Axenic Delia antiqua with Half-Fermented Sterile Diets

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken