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

Summary

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.

Abstract

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 muscle development. When injected into a developing embryo'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.

Introduction

Since 1960, the United States' 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's main objective is to produce muscle for conversion to meat, their efforts have drastically changed the bird'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 growth^4,5,6. 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'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.

Protocol

All methodologies were approved by the University of Georgia Institutional Animal Care and Use Committee.

1. Egg incubation and treatment administration

1. Egg procurement and treatment assignment
   1. Obtain unincubated, fertilized high-yield broiler eggs and transport them to the laboratory.
   2. 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.
   3. Assign individual egg numbers, weigh, and record egg numbers and weights in a spreadsheet software program.
   4. Utilize the spreadsheet software program to sort eggs by weight.
      1. Highlight the egg number and egg weight columns.
      2. Select the Data tab and then Sort - sort data by egg weight from smallest to largest.
         NOTE: For the best hatching rate, use eggs weighing between 40 and 70 g.
      3. 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.
         ​NOTE: For this publication, treatments were randomly assigned within each 8-egg strata.
   5. Generate a PivotTable within the spreadsheet software program to ensure each treatment possesses similar starting egg weights.
      1. Highlight all data within the spreadsheet to be analyzed.
      2. Select PivotTable option under Insert tab.
      3. Select the independent variable (Day of Euthanasia column) within the PivotTable Fields sub-window and drag to Rows field.
      4. Select the independent variable (Treatment column) within the B sub-window and drag to Rows field, under Day of Euthanasia.
      5. Select the dependent variable of interest (Egg Weight) and drag it to the Values field.
      6. Change Value field settings by clicking on the dependent variable and selecting Value Field Settings.
         1. Change the setting to Average.
2. Tray assignment
   1. In the spreadsheet software program, assign eggs to a tray (numerically or alphabetically), so treatments are equally represented within a tray.
      1. 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.
         ​NOTE: This step will vary depending on the number of incubators and trays used in the experiment.
3. Ensure all treatments are equally represented on a tray using the PivotTable function.
   1. Highlight all data within the spreadsheet to be analyzed.
   2. Select the PivotTable option under the Insert tab.
   3. Select the independent variable (Tray column) within the PivotTable Fields sub-window and drag to the Rows field.
   4. Select the dependent variable of interest (Egg Weight) and drag it to the Values field.
   5. Change Value field settings by clicking on the dependent variable and selecting Value Field Settings.
      1. Change the setting to Count.
4. Incubation
   1. Place eggs in their appropriate incubation tray and pre-incubate them at 26.6 °C with 40% ± 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.
   2. Increase the incubator temperature to 37 °C with 40% ± 4% relative humidity and maintain these conditions until incubation day 18.
      1. To ensure proper incubator temperature, measure several eggs' surface temperatures throughout the incubator twice daily with a thermal surface thermometer to ensure surface temperatures are 37 °C.
   3. Rotate eggs hourly to reposition.
   4. 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.
5. Incubation day-10 in ovo injections
   1. Calculate the amount of nicotinamide riboside needed for each treatment using the formula weight of 290.07 g/mol, with 100 µL of solution injected into each eggs' yolk sac.
      NOTE: Sterile saline (0.9%) solution will be used as the diluent for all solutions.
      ​Calculation: 50 eggs × 100 µL = 5,000 µL (5 mL) of solution needed. Round up to 6 mL to ensure enough solution is available for injection (Figure 1).
      1. Once solutions are made, place them in a 37 °C water bath to keep them at the temperature of the eggs.
   2. Remove eggs from the incubator one tray at a time and cover with a warm towel.
   3. Candle egg to locate the yolk sac and clean the area of injection with 70% ethanol.
   4. 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 µL of sterile saline (0.9%).
   5. Immediately, cover the injection site with a small piece of absolute waterproof tape to avoid excessive moisture loss.
   6. Once all eggs have received their treatment, place the tray back into the incubator.
   7. On incubation day 18, remove eggs from trays and place them in hatching boxes according to their treatments.
   8. Place hatching boxes into the incubator and increase humidity to 60 ± 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. 

2. Euthanasia and pectoralis major muscle sample collection

1. Chick euthanasia
   1. 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.
   2. Consider the following measurements (steps 2.1.2.1-2.1.2.4) using digital calipers for embryos and chicks.
      1. 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.
      2. To measure the head width, measure from one ear hole to the other ear hole.
      3. For determining head length, measure from the rear of the beak to the back of the cranium.
      4. 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.
   3. 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.
   4. 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.
      1. For determining the chest width, measure across the chest where the wings contact the body.
      2. For determining the chest length, measure from the bottom of the clavicle to the top of the fat pad.
2. Extraction of pectoralis major muscle, measurement, and collection
   1. 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.
   2. 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.
      1. For determining the muscle length, measure from the cranial to the caudal portion of the muscle.
      2. For determining the muscle width, measure at the widest portion of the cranial part of the muscle.
      3. For determining the muscle thickness, pick up the breast with forceps and measure at the thickest portion of the cranial part of the muscle.
   3. If desired, store this muscle and the left pectoralis major muscle for further analyses (such as histology, protein and gene expression, etc.) at -80 °C for up to a year.

3. Statistics

1. 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 < 0.05.

Results

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

Discussion

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 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 ...

Materials

List of materials used in this article

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