Name: in ovo feeding of commercial broiler eggs: an accurate and reproducible method to affect muscle development and growth
Uploaded: 2021-09-20T15:00:00.000+00:00
Duration: 6 min 38 s
Description: 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

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 current study, did not increase pectoralis major muscle morphometrics beyond the 250 mM dose10. These two studies utilized a commercial yield broiler line; therefore, this study was conducted to demonstrate the effects of in ovo feeding of high yield broiler embryos with nicotinamide riboside.
Through these studies, several critical steps have been identified located in this protocol that determines success. This process is critically important for those unfamiliar with selecting eggs for incubation to reduce bacteria spread and not biasing hatched-chick results. First, it is critically important not to choose dirty or misshapen eggs because they possess bacteria that could hamper the other eggs. These bacteria will spread rapidly through the incubator and cause rotten egg incidence to increase drastically; thus, affecting the number of embryos and chicks available for sampling.
As for assigning eggs to experimental treatments, researchers must utilize spreadsheet software methods described above to ensure all treatment starting egg weights are equal. Completing this step will be demonstrated in the embryo and hatched-chick whole-body morphometric data. This will ensure that all experimental treatment muscle differences are due to treatment application. There were no nicotinamide riboside effects on all body morphometric measures in the Gonzalez and Jackson9 and Xu et al.10 studies. Because of these consistent findings, only embryo and hatched-chick body weights were measured in the current research to establish a lack of a nicotinamide riboside effect on whole-body morphometrics; however, methodologies for collecting whole body morphometrics are presented in this publication for those wishing to collect those data. There were no nicotinamide riboside dose effects on embryo or chick body weights in the current study, continuing the trend reported previously.
Because this methodology strictly affects secondary myogenesis, future research teams may be tempted to inject embryos at an earlier time point. In the authors&#39; experience, early injection, from incubation days 0 to 5, drastically reduces the hatchability of eggs by up to 70 to 80%. An early injection is a significant limitation of the technique. It could serve as a future area of research, but in the authors&#39; experience, early injection is detrimental to hatchability which severely reduces the value of this technology.
When measuring morphometrics of the pectoralis major muscle, researchers must ponder two crucial considerations. First, the authors advise a single, well-trained researcher to remove all muscles utilized for morphometric analysis. Because the pectoralis major muscle is so small, much-unwanted variation or bias could be introduced into data by collecting&#160;other muscles outside the muscle of interest. Utilizing a single researcher will ensure the same muscle will be collected according to consistent landmarks used to identify the muscle. Second, when placing muscles on the wood surface for measurement, care must be taken in laying all muscles in a natural position. This is especially true for the length measurement, as it can be manipulated by stretching the muscle when laying it down on the measurement surface. No nicotinamide riboside effects were seen in the current study for pectoralis major muscle morphometrics at incubation day 18. Xu et al.10 reported no pectoralis major muscle weight and length differences at incubation day 19; thus, indicating nicotinamide riboside&#39;s effect on whole muscle morphometrics may not manifest until after incubation day 19 in these two genetic broiler lines.
Compared to previously published studies, one of the major modifications in the current study was the use of commercially available capsule-form nicotinamide riboside. In the previous studies9, 10, pure nicotinamide riboside was secured from a manufacturer. With assistance from the manufacturer, the research group was informed that the commercial product utilized in the current study also had cellulose ingredients mixed into the product, reducing the calculated concentration of nicotinamide ribose by 34%. Therefore, in the present study, pectoralis major muscle weight from hatched chicks injected with 500 and 1,000 mM nicotinamide riboside was greater than chicks from embryos injected with 0 mM nicotinamide riboside by 15 and 10%, respectively. This weight increased mainly due to these treatments&#39; pectoralis major muscle depth increasing by 17 and 7%, respectively. This response was less than half of the previous responses. Xu et al.10 reported nicotinamide riboside supplementation, 250 to 1,000 mM concentrations, increased pectoralis major muscle weight by 35% due to increased muscle length, width, and depth. While reduced response could be primarily due to supplementing less nicotinamide riboside than calculated, it is also unknown if cellulose material hindered myogenesis. Therefore, the authors recommended all future research utilize pure nicotinamide riboside and not commercially available products.
Regardless of the present results, following the methodologies outlined in this publication will ensure robust execution of in ovo feeding studies. Future researchers can utilize the above methods to test other compounds that may positively affect broiler chicken in ovo muscle development and growth.

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><tbody><tr><td>Air-Tite&amp;trade; 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&amp;trade; Isotemp&amp;trade; General Purpose Deluxe Water Baths</td><td>Fisher Scientific</td><td>FSGPD02&amp;nbsp;</td><td>https://www.fishersci.com/shop/products/isotemp-general-purpose-water-baths/p-6448020</td></tr><tr><td>Fisherbrand&amp;trade; 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&lt;br/&gt; &amp;amp;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; Figure 4). Chicks from embryos not injected with nicotinamide riboside had pectoralis major muscles that weighed less than chick from embryos injected with 500 and 1,000 mM nicotinamide riboside (P &#60; 0.03), but these treatments did not differ (P = 0.41) from each other. Chicks from embryos injected with 250 mM nicotinamide riboside did not vary in pectoralis major weight compared to the other treatments (P &#62; 0.06). Chicks from embryos injected with 0 and 250 mM nicotinamide riboside had less pectoralis major depth than chicks from embryos injected with 500 and 1,000 mM nicotinamide riboside (P &#60; 0.05), but these treatments did not differ (P = 0.95). Chick from embryos injected with 500 and 1,000 mM nicotinamide riboside did not vary (P = 0.73) in pectoralis major depth.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63006/63006fig01.jpg" /> 
Figure 1: Nicotinamide riboside dose general calculation and examples of the three doses utilized in the current experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63006/63006fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63006/63006fig02.jpg" /> 
Figure 2: Effect of in ovo feeding of four doses of nicotinamide riboside on (A) day-18 embryo and (B) hatch chick body weights. Embryos were injected into the yolk sac with four nicotinamide riboside doses at day-10 of incubation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63006/63006fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63006/63006fig03.jpg" /> 
Figure 3: Effect of in ovo feeding of four doses of nicotinamide riboside on day-18 embryo pectoralis major muscle. (A)&#160;Weight. (B)&#160;Length. (C)&#160;Width.&#160;(D)&#160;Depth. Embryos were injected into the yolk sac with one of four nicotinamide riboside doses at day 10 of incubation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63006/63006fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63006/63006fig04.jpg" /> 
Figure 4: Effect of in ovo feeding of four doses of nicotinamide riboside on&#160;hatch chick body pectoralis major muscle. (A)&#160;Weight. (B)&#160;Length. (C)&#160;Width. (D)&#160;Depth. Embryos were injected into the yolk sac with one of four nicotinamide riboside doses at day 10 of incubation. a,b indicates the statistical difference from each other within a sub-figure (P &#60; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63006/63006fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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 ...

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Biology

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Neuroscience

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

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. ...

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.

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, ...

Immunology and Infection

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

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

Summary

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Chapters in this video

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

Windowing Chicken Eggs for Developmental Studies

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

In Ovo Electroporations of HH Stage 10 Chicken Embryos

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

In Ovo Intravascular Injection in Chicken Embryos

Isolation of Preadipocytes from Broiler Chick Embryos

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

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake