Like mammals, muscle fiber number, a major determinant of ultimate muscle mass is established during poultry embryogenesis. This method allows researchers to study poultry in ovo myogenesis and manipulation of muscle fiber hyperplasia. This technique requires little investment in equipment and is an easy and relatively low-cost method of studying myogenesis manipulation.
The researchers new to this technique may struggle in properly incubating the eggs. Demonstrating this technique will be myself and Morgan Gravely, both Masters of Science graduate students in our lab. Before starting the experiment, inspect and discard the eggs of poor quality.
Assign and record individual egg numbers to the selected eggs and then weigh the eggs. Next, place eggs in their appropriate incubation tray, and pre incubate them at 26.6 degrees Celsius with 40%relative humidity for six hours. After incubation, increase and maintain the incubator temperature at 37 degrees Celsius with 40%relative humidity until incubation day 18.
Measure the surface temperature of several eggs throughout the incubator twice daily with a thermal surface thermometer to ensure the surface temperature of the eggs is 37 degrees Celsius. Keep repositioning the eggs by rotating every hour. To ensure 10%to 12.5%egg weight loss, record the egg weights daily during the first 18.5 days of incubation.
On day 10 of the incubation, prepare the nicotinamide riboside solution needed for each 100 microliter in ovo injections treatment as described in the manuscript and maintain the solutions at 37 degrees Celsius in a water bath. After removing the eggs one tray at a time from the incubator, and covering with a warm towel, candle each egg to locate the yolk sac. Clean the area of injection with 70%ethanol before inserting a sterile 20 gauge 2.54 centimeter hypodermic needle one centimeter deep into the egg shell to inject the assigned dose of nicotinamide riboside treatment with 100 microliters of sterile saline into the yolk sac.
Immediately covered the injection site with a small piece of absolute waterproof tape to avoid excessive moisture loss. Once all the eggs have received their treatment, place the tray back into the incubator. On incubation day 18, remove the eggs from the trays to place them in the assigned hatching boxes according to their treatments, then place the hatching boxes into the incubator with the humidity increase to 60%until all the eggs hatch or until the 23rd day of the incubation.
Before the sample collection, determine the crown to rump length of the euthanized chicks by laying the chick on its side with head tucked down and legs under the body and measuring the length from the top of the head to the tail, then measure the head width from one ear hole to the other ear hole in the head length from the rear of the beak to the back of the cranium. To assess the head circumference, wrap a non-elastic string around the skull from one ear hole to the other, and then obtain the measurement of the string with a metric ruler. In the same way, determine the chest circumference by wrapping a string around the chest under where the wings contact the body before placing the string on a metric ruler to acquire the measurement.
Spray the breasts of the chick with 70%ethanol, and with fingers, pull the feathers and skin to reveal the pectoralis major muscles followed by taking measurements with digital calipers. Measure the chest width across the chest where the wings contact the body and the chest length from the bottom of the clavicle to the top of the fat pad. To extract the right pectoralis major muscle, use surgical scissors or scalpel and forceps to cut along the keel bone before releasing the muscle from the body wall.
After removing the pectoralis major muscle, lay the muscle flat on a Popsicle stick to collect the measurements using digital calipers. Find out the muscle length from the cranial to the caudal part of the muscle and the muscle width at the widest portion of the cranial part of the muscle. For determining the muscle thickness, pick up the breast with forceps and measure at the thickest portion of the cranial section of the muscle.
When done with the measurements, the right and left pectoralis major muscles can be stored for further analyses at minus 80 degrees Celsius for up to a year. The embryos were injected with four nicotinamide riboside doses or dose to study the effect of in ovo feeding. It was observed that there were no dose effects on the body weight of day 18 embryos and hatched chicks.
Similarly, no dose effects were detected on the pectoralis major muscle measurements for all day 18 embryos. For hatched chicks, there were no dose effects on the pectorals major muscle length and width measurements. However, dose did affect the muscle weight and depth.
The chicks developed from the embryos that were not injected with nicotinamide riboside had pectoralis major muscles that weighed less than the chicks from the embryos injected with 501, 000 millimolar nicotinamide riboside. The chicks from embryos injected with zero and 250 millimolar nicotinamide riboside had less pectoralis major depth than the chicks from embryos injected with 501, 000 millimolar nicotinamide riboside. The key to execute a robust experiment is selecting defect free and uniform eggs.
This will ensure that all effects are treatment catalyzed. With the harvest of pectoralis major muscle, histology can be used to measure muscle fiber morphometrics, and total RNA and protein can be extracted for gene and protein expression analysis. We are currently studying the effects of this technology on the growth performance in fresh meat quality of treated birds.
