The authors would like to thank Cobb Vantress, Inc. for donation of the eggs and providing technical assistance on egg incubation. The authors would like to thank ChromaDex, Inc. for nicotinamide riboside technical assistance.

All methodologies were approved by the University of Georgia Institutional Animal Care and Use Committee.
1. Egg incubation and treatment administration
<ol>
	<li>Egg procurement and treatment assignment
	<ol>
		<li>Obtain unincubated, fertilized high-yield broiler eggs and transport them to the laboratory.</li>
		<li>Inspect and discard eggs deemed poor quality. 
		NOTE: Eliminate misshapen eggs (round, elongated, slab-sided), cracked, dirty/stained, thin-shelled, and wrinkled. This is important to minimize the risk of rotten eggs.</li>
		<li>Assign individual egg numbers, weigh, and record egg numbers and weights in a spreadsheet software program.</li>
		<li>Utilize the spreadsheet software program to sort eggs by weight.
		<ol>
			<li>Highlight the egg number and egg weight columns.</li>
			<li>Select the Data tab and then Sort&#160;- sort data by egg weight from smallest to largest. 
			NOTE: For the best hatching rate, use eggs weighing between 40 and 70 g.</li>
			<li>Based on the design of the experiment, assign eggs (numerically or alphabetically) an injection treatment and day of euthanasia. Enter treatment number and day of euthanasia in separate columns and randomly assign these factors within each stratum. 
			&#8203;NOTE: For this publication, treatments were randomly assigned within each 8-egg strata.</li>
		</ol>
		</li>
		<li>Generate a PivotTable within the spreadsheet software program to ensure each treatment possesses similar starting egg weights.
		<ol>
			<li>Highlight all data within the spreadsheet to be analyzed.</li>
			<li>Select PivotTable option under Insert tab.</li>
			<li>Select the independent variable (Day of Euthanasia column) within the PivotTable Fields sub-window and drag to Rows field.</li>
			<li>Select the independent variable (Treatment column) within the B sub-window and drag to Rows field, under Day of Euthanasia.</li>
			<li>Select the dependent variable of interest (Egg Weight) and drag it to the Values field.</li>
			<li>Change Value field settings by clicking on the dependent variable and selecting Value Field Settings.
			<ol>
				<li>Change the setting to Average.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tray assignment
	<ol>
		<li>In the spreadsheet software program, assign eggs to a tray (numerically or alphabetically), so treatments are equally represented within a tray.
		<ol>
			<li>Assign the first four eggs with assigned treatments to tray 1. Assign the next four eggs to tray 2 and continue until all eggs are assigned to a tray. 
			&#8203;NOTE: This step will vary depending on the number of incubators and trays used in the experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Ensure all treatments are equally represented on a tray using the PivotTable function.
	<ol>
		<li>Highlight all data within the spreadsheet to be analyzed.</li>
		<li>Select the PivotTable option under the Insert tab.</li>
		<li>Select the independent variable (Tray column) within the PivotTable Fields sub-window and drag to the Rows field.</li>
		<li>Select the dependent variable of interest (Egg Weight) and drag it to the Values field.</li>
		<li>Change Value field settings by clicking on the dependent variable and selecting Value Field Settings.
		<ol>
			<li>Change the setting to Count.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubation
	<ol>
		<li>Place eggs in their appropriate incubation tray and pre-incubate them at 26.6 &#176;C with 40% &#177; 4% relative humidity for 6 h. 
		NOTE: Some incubators have self-monitoring systems that may not be entirely accurate. Use other temperature and humidity monitoring devices to control conditions.</li>
		<li>Increase the incubator temperature to 37 &#176;C with 40% &#177; 4% relative humidity and maintain&#160;these conditions until incubation day 18.
		<ol>
			<li>To ensure proper incubator temperature, measure several eggs&#39; surface temperatures throughout the incubator twice daily with a thermal surface thermometer to ensure surface temperatures are 37 &#176;C.</li>
		</ol>
		</li>
		<li>Rotate eggs hourly to reposition.</li>
		<li>Record egg weights daily to ensure 10%-12.5% egg weight loss during the first 18.5 days of incubation. 
		NOTE: If weight loss is not within the desired range, adjust (increase or decrease) humidity.</li>
	</ol>
	</li>
	<li>Incubation day-10 in ovo injections
	<ol>
		<li>Calculate the amount of nicotinamide riboside needed for each treatment using the formula weight of 290.07 g/mol, with 100 &#181;L of solution injected into each eggs&#39; yolk sac. 
		NOTE: Sterile saline (0.9%) solution will be used as the diluent for all solutions. 
		&#8203;Calculation: 50 eggs &#215; 100 &#181;L = 5,000 &#181;L (5 mL) of solution needed. Round up to 6 mL to ensure enough solution is available for injection (Figure 1).
		<ol>
			<li>Once solutions are made, place them in a 37 &#176;C water bath to keep them at the temperature of the eggs.</li>
		</ol>
		</li>
		<li>Remove eggs from the incubator one tray at a time and cover with a warm towel.</li>
		<li>Candle egg to locate the yolk sac and clean the area of injection with 70% ethanol.</li>
		<li>Insert a sterile 20 G, 2.54 cm hypodermic needle ~1 cm into the eggshell and inject the assigned dose into the yolk sac. Inject the eggs from the 0 mM nicotinamide riboside treatment with 100 &#181;L of sterile saline (0.9%).</li>
		<li>Immediately, cover the injection site with a small piece of absolute waterproof tape to avoid excessive moisture loss.</li>
		<li>Once all eggs have received their treatment, place the tray back into the incubator.</li>
		<li>On incubation day 18, remove eggs from trays and place them in hatching boxes according to their treatments.</li>
		<li>Place hatching boxes into the incubator and increase humidity to 60 &#177; 2% until all eggs hatch or until day 23 of incubation. 
		NOTE: If eggs do not contain an embryo at candling, discard the egg. This will prevent the occurence of rotten eggs.&#160;</li>
	</ol>
	</li>
</ol>
2. Euthanasia and pectoralis major muscle sample collection
<ol>
	<li>Chick euthanasia
	<ol>
		<li>On incubation day 18, remove the embryonic eggs from the incubator and place them at room temperature for 1 h to cease metabolism. Remove the embryos from the eggs, weigh without the yolk sac, and then decapitate. 12 h post-hatching, euthanize the chicks by exposure to CO2 for 10 min, weigh, quickly collect crown-to-rump length measurement, and then decapitate. 
		NOTE: The fact that the bird no longer has their head ensures euthanasia.</li>
		<li>Consider the following measurements (steps 2.1.2.1-2.1.2.4) using digital calipers for embryos and chicks.
		<ol>
			<li>For determining crown-to-rump length, lay the chick on its side with its head tucked down and legs under its body. Measure from the top of the head to the tail.</li>
			<li>To measure the head width, measure from one ear hole to the other ear hole.</li>
			<li>For determining head length, measure from the rear of the beak to the back of the cranium.</li>
			<li>Take a non-elastic string and wrap it around the skull from one ear hole to the other to measure the head circumference. Place string on a metric ruler to obtain a measurement.</li>
		</ol>
		</li>
		<li>Collect chest circumference by wrapping a string around the chest, under where the wings contact the body and placing the string on a metric ruler to obtain the measurement.</li>
		<li>Spray breasts with 70% ethanol, and using fingers, pull the feathers and skin to reveal the pectoralis major muscles and take the measurements (steps 2.1.4.1-2.1.4.2) with digital calipers.
		<ol>
			<li>For determining the chest width, measure across the chest where the wings contact the body.</li>
			<li>For determining the chest length, measure from the bottom of the clavicle to the top of the fat pad.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Extraction of pectoralis major muscle, measurement, and collection
	<ol>
		<li>Using surgical scissors or scalpel and forceps, remove the right pectoralis major muscle by cutting along the keel bone and releasing the muscle from the body wall. 
		NOTE: Be sure not to collect the pectoralis minor muscle by visually identifying that the muscle remains on the rib cage.</li>
		<li>After removing the pectoralis major muscle, lay the muscle flat on a popsicle stick, and collect the following measurements (steps 2.2.2.1-2.2.2.3) using digital calipers.
		<ol>
			<li>For determining the muscle length, measure from the cranial to the caudal portion of the muscle.</li>
			<li>For determining the muscle width, measure at the widest portion of the cranial part of the muscle.</li>
			<li>For determining the muscle thickness, pick up the breast with forceps and measure at the thickest portion of the cranial part of the muscle.</li>
		</ol>
		</li>
		<li>If desired, store this muscle and the left pectoralis major muscle for further analyses (such as histology, protein and gene expression, etc.) at -80 &#176;C for up to a year.</li>
	</ol>
	</li>
</ol>
3. Statistics
<ol>
	<li>Analyze the data as a completely randomized design with egg/chick as the experimental unit. 
	NOTE: Nicotinamide riboside dose (DOS) served as the fixed effect. All data were analyzed with a statistical analysis software program (see Table of Materials), and pairwise comparisons between least-square means of treatments were computed. Differences were considered significant at P &#60; 0.05.</li>
</ol>

<ol>
	<li>Biressi, S., Molinaro, M., Cossu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+heterogeneity+during+vertebrate+skeletal+muscle+development.">Cellular heterogeneity during vertebrate skeletal muscle development.</a> Developmental Biology. 308 (2), 281-293 (2007).</li><li>Chal, J., Pourquie, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+muscle:+Skeletal+myogenesis+in+vivo+and+in+vitro.">Making muscle: Skeletal myogenesis in vivo and in vitro.</a> Development. 144 (12), 2104-2122 (2017).</li><li>Sharma, J., Burmester, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+of+Marek's+disease+at+hatching+in+chickens+vaccinated+as+embryos+with+the+Turkey+herpesvirus.">Resistance of Marek's disease at hatching in chickens vaccinated as embryos with the Turkey herpesvirus.</a> Avian Diseases. 26 (1), 134-149 (1982).</li><li>Al-Murrani, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+injecting+amino+acids+into+the+egg+on+embryonic+and+subsequent+growth+in+the+domestic+fowl.">Effect of injecting amino acids into the egg on embryonic and subsequent growth in the domestic fowl.</a> British Poultry Science. 23 (2), 171-174 (1982).</li><li>Ohta, Y., Kidd, M. T., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+growth+and+amino+acid+concentration+profiles+of+broiler+breeder+eggs,+embryos,+and+chicks+after+in+ovo+administration+of+amino+acids.">Embryo growth and amino acid concentration profiles of broiler breeder eggs, embryos, and chicks after in ovo administration of amino acids.</a> Poultry Science. 80 (10), 1430-1436 (2001).</li><li>Zhao, M. M., 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=In+ovo+feeding+of+creatie+pyruvate+increases+hatching+weight,+growth+performance,+and+muscle+growth+but+has+no+effect+on+meat+quality+in+broiler+chickens.">In ovo feeding of creatie pyruvate increases hatching weight, growth performance, and muscle growth but has no effect on meat quality in broiler chickens.</a> Livestock Science. 206, 59-64 (2017).</li><li>Bieganowski, P., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discoveries+of+nicotinamide+riboside+as+a+nutrient+and+conserved+NRK+genes+establish+a+Preiss+Handler+independent+route+to+NAD1+in+fungi+and+humans.">Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss Handler independent route to NAD1 in fungi and humans.</a> Cell. 117 (4), 495-502 (2004).</li><li>Chi, Y., Sauve, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+riboside,+a+trace+nutrient+in+foods,+is+a+Vitamin+B3+with+effects+on+energy+metabolism+and+neuroprotection.">Nicotinamide riboside, a trace nutrient in foods, is a Vitamin B3 with effects on energy metabolism and neuroprotection.</a> Current Opinion in Clinical Nutrition and Metabolic Care. 16 (6), 657-661 (2013).</li><li>Gonzalez, J. M., Jackson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+feeding+of+nicotinamide+riboside+affects+pectoralis+major+muscle+development.">In ovo feeding of nicotinamide riboside affects pectoralis major muscle development.</a> Translational Animal Science. 4 (3), 1-7 (2020).</li><li>Xu, X., Jackson, A. R., Gonzalez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+in+ovo+nicotinamide+riboside+dose+on+broiler+myogenesis.">The effects of in ovo nicotinamide riboside dose on broiler myogenesis.</a> Poultry Science. 100 (3), 100926 (2021).</li></ol>

The authors have no conflict of interests to declare.

To date, the Gonzalez Laboratory at the University of Georgia is the only group to demonstrate positive effects of nicotinamide riboside in ovo feeding on pectoralis major muscle&#160;development and growth. The first study found that in ovo feeding of 250 mM nicotinamide riboside increased muscle weight and dimensions when injected in the yolk sac9. In the follow-up study, injecting increasing nicotinamide riboside dose into the yolk, similar to the doses tested in the ...

Since 1960, the United States&#39; per capita consumption of meat from poultry has risen at an astounding rate, while other primary protein sources have remained stagnant, declined, or minimally increased. The poultry industry invested considerable time and research efforts optimizing nutrition and genetics to produce an efficient bird to keep up with demand. Because the poultry industry&#39;s main objective is to produce muscle for conversion to meat, their efforts have drastically changed the bird&#39;s ultimate muscle mass at harvest.
Like most species, poultry develops muscle in a biphasic manner. Primary myogenesis utilizes mesenchymal stem cells to produce primary muscle fibers, which serve as the scaffold for the second wave of muscle fiber development1. In poultry, primary myogenesis occurs during embryonic days 3 to 8, and secondary myogenesis happens from days 8 to 212. Once developed, primary and secondary muscle fibers serve as the basis for all future muscle growth through cellular hypertrophy. Therefore, scientists and industry spent considerable effort attempting to manipulate primary and secondary myogenesis in all meat-producing species to maximize meat yield.
One technology explored in poultry, called in ovo feeding, involves feeding compounds through injection. In ovo feeding, a technology employed by the poultry industry for almost 40 years, was initially developed for vaccine administration3. The literature documents that in ovo feeding of various compounds and nutrients at different developmental periods and locations within the egg positively affected in ovo muscle development and growth4,5,6. To date, the Gonzalez Laboratory at the University of Georgia is the pioneer in utilizing nicotinamide riboside in ovo feeding to manipulate poultry muscle development.
Nicotinamide riboside, a pyridine-nucleoside analog of vitamin B3, produces NAD+ through the salvage pathway7. Since this pathway utilizes fewer enzymatic steps to produce NAD+, production is the most efficient8. Gonzalez and Jackson9 demonstrated that supplementation of developing broiler embryo yolk sac with nicotinamide riboside increased hatched chick pectoralis major muscle weight and muscle fiber density. This was later confirmed by Xu et al.10, who found that increasing nicotinamide riboside dose increased muscle weight and increased muscle fiber density. These first two studies were conducted in a commercial yield broiler. Because high yield broilers possess a more significant genetic potential for ultimate muscle mass size, the study&#39;s objective was to determine the effects of nicotinamide riboside dose on high yield broiler hatched chick pectoralis major muscle development and growth at hatch.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Air-Tite&#8482; Sterile Hypodermic Needles- 20 G; 1 inch</td>
			<td>Fisher Scientific</td>
			<td>14-817-208</td>
			<td>https://www.fishersci.com/shop/products/sterile-hypodermic-needles-32/p-7182916#?keyword=</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>VWR</td>
			<td>VWR-214B2</td>
			<td>https://us.vwr.com/store/product/20970740/vwr-b2-series-analytical-and-precision-balances</td>
		</tr>
		<tr>
			<td>Complete Dissection Set</td>
			<td>DOCAZON</td>
			<td>DK1001</td>
			<td>https://www.amazon.com/DOCAZON-Complete-Dissection-Set-Dissecting/dp/B07VBHKSW3</td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Isotemp&#8482; General Purpose Deluxe Water Baths</td>
			<td>Fisher Scientific</td>
			<td>FSGPD02&#160;</td>
			<td>https://www.fishersci.com/shop/products/isotemp-general-purpose-water-baths/p-6448020</td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Sterile Syringes for Single Use</td>
			<td>Fisher Scientific</td>
			<td>14-955-464</td>
			<td>https://www.fishersci.com/shop/products/sterile-syringes-single-use-12/p-7114739#?keyword=</td>
		</tr>
		<tr>
			<td>HIGH INTENSITY EGG CANDLER</td>
			<td>Titan Incubators</td>
			<td>N/A</td>
			<td>https://www.titanincubators.com/collections/egg-candlers/products/egg-candler-high-intensity</td>
		</tr>
		<tr>
			<td>Infrared Forehead Thermometer</td>
			<td>HALIDODO</td>
			<td>XZ-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Neiko Tools Digital Caliper</td>
			<td>Neiko Tools</td>
			<td>01408A</td>
			<td>https://www.amazon.com/Neiko-01407A-Electronic-Digital-Stainless/dp/B000NEA0P8?th=1</td>
		</tr>
		<tr>
			<td>Nexcare Absolute Waterproof Tape</td>
			<td>Nexcare Brand</td>
			<td>732</td>
			<td>https://www.nexcare.com/3M/en_US/nexcare/products/catalog/~/Nexcare-Absolute-Waterproof-Tape/?N=4326+3294529207+3294631805 
			&#38;rt=rud</td>
		</tr>
		<tr>
			<td>Pen Size Temperature and Humidity USB Data Logger with Display</td>
			<td>Omega</td>
			<td>OM-HL-SP-TH</td>
			<td>https://www.omega.com/en-us/temperature-measurement/temperature-and-humidity-data-loggers/p/OM-HL-SP-Series</td>
		</tr>
		<tr>
			<td>SAS 9.4 for Windows</td>
			<td>SAS Institute</td>
			<td>N/A</td>
			<td>https://www.sas.com/en_us/home.html</td>
		</tr>
		<tr>
			<td>Sportsman 1502 Incubator</td>
			<td>GQF Manufacturing</td>
			<td>1502</td>
			<td>https://www.gqfmfg.com/item/1502-digital-sportsman/</td>
		</tr>
		<tr>
			<td>Tru Niagen (Nicotinamide riboside)</td>
			<td>ChromaDex, Inc.</td>
			<td>N/A</td>
			<td>https://www.truniagen.com/truniagen-300mg/ - note, contact company for pure product</td>
		</tr>
		<tr>
			<td>Wood Craft Sticks</td>
			<td>Creatology</td>
			<td>M20001547</td>
			<td>https://www.michaels.com/wood-craft-sticks-by-creatology/M20001547.html</td>
		</tr>
	</tbody>
</table>

in ovo feeding of commercial broiler eggs: an accurate and reproducible method to affect muscle development and growth

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during embryonic and fetal development. This area continues to be an area of focus because muscle fiber number is established during this time and determines the basis for all future growth. In poultry, numerous studies demonstrated in ovo feeding of growth factors, vitamins, or other nutrients improved chick embryonic muscle and intestinal development. Improving in ovo muscle development could benefit the poultry industry by possibly influencing meat yield, growth rate, or myopathy conditions. During the past five years, the Gonzalez Laboratory at the University of Georgia developed a nicotinamide riboside in ovo feeding methodology for broiler-chicken embryos, which altered&#160;muscle development. When injected into a developing embryo&#39;s yolk sac, nicotinamide riboside increased pectoralis major muscle weight and muscle fiber density at hatch. This protocol will demonstrate a methodology to accurately and reproducibly conduct in ovo feeding studies utilizing commercial standard- and high-yielding broiler embryos. These data and methods will allow other research groups to perform in ovo feeding studies with much success and reproducibility.

There were no DOS effects for the bodyweight of day-18 embryos and hatched chicks (P &#62; 0.52; Figure 2). There were no DOS effects for all day-18 embryo pectoralis major muscle measurements (P &#62; 0.24; Figure 3). There were no DOS effects for hatched chick pectoralis major muscle length and width measurements (P &#62; 0.26); however, DOS did affect muscle weight and depth (P &#60; 0.03; <strong class="x...

Watch this Scientific Journal Video about In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth at JoVE.com

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during ...

in-ovo-feeding-commercial-broiler-eggs-an-accurate-reproducible

Research

JoVE Journal

Developmental Biology

2.3K Views.  University of Georgia. A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

Video: In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding.

The Complete and Updated &quot;Rotifer Polyculture Method&quot; for Rearing First Feeding Zebrafish

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science.  One of the most demanding technical aspects of ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.