Our survival rate to 14 days has probably varied as widely as 30 to 80%, depending on the age of the flock, the conditions under which the birds live, the quality of their feed, the season of the year, and the duration of egg storage prior to their incubation.  Unless you maintain your own flock you have little control over these things.  What you can control, however, is your egg source.  If survival rate is a problem, it may be well worth your while to compare eggs from different sources in your area.

Heartbeat is greatly influenced by ambient temperature.  I'm going to guess, based on your report, that you are maintaining your embryos at room temperature throughout your observation period.  If you want to maintain anything close to the initial rate you will need to keep the eggs at a constant temperature (38o C or so) throughout.  Depending on your mode and frequency of observation, you will need either to place the eggs in a constant-temperature chamber with a transparent top or return the eggs to their initial incubator between observations. 

If the heart is beating at all, most of the embryos are probably still viable.  I have often controlled the timing of developmental stages by removing eggs to room temperature for hours at a time, even overnight, before returning them to the incubator.  You have to expect, though, that viability among a population of embryos will decline in direct proportion to the length of time at lower temperature.

The eggs were kept in a 38 degrees Celsius  clear top incubator. 

If your eggs never left this incubator even for observation I would suggest either that your incubator is not functioning as you think it is or that there is something wrong with your method of ex ovo culture.  Does your incubator provide air flow sufficient to keep all eggs at the same temperature?  Does it provide an inflow of fresh air sufficient to supply the embryos with adequate oxygen?  What is its source of heat? 

My students (undergraduates) and I, over a period of nearly 50 years, have made thousands of ex ovo cultures with both chick and quail eggs and kept many of them alive until they were almost fully developed.  If you provide me with a full description of each step you perform in making these cultures I may be able to provide additional help.  For example:  How do you treat or handle the eggs before placing them in culture?  Into what kind of vessel do you empty the egg's contents?  How old are your embryos when you place them in culture?  Do your embryos exhibit any sign of vascular damage, if only a broken capillary, after being placed in culture?  What is your angiogenic stimulus?  How do you place it on the CAM?  ... and any other details you can think to provide.

Good answers from Mr.Glenn.

I suggest you to consider a possible degree of drying up that ex ovo culture depending on time and exposure to air flow conditions.

I'm not sure about the airflow of our incubator. We have an Innova 4000 incubator shaker so the air flow isn't too well regulated. We have thermometers placed throughout the incubator and they're reading a steady 38 degrees Celsius.

Our basic protocol so far is set the eggs on their side in the incubator with a pencil to mark the top of the egg for future reference. We give them about 72 hours to incubate. Because of the time of the eggs, I'm usually teaching whenever the undergrads come in to crack the eggs so I have yet to observe the process myself. Otherwise, we discard any unfertilized eggs or eggs with broken yolks. The vasculature have been fine and we have a heartbeat when initially opened. Our first batch of eggs were cooked because we believe they were too close to the heat source. The second batch we placed the embryos in a pentagonal weigh boat (all we had at the time) and placed them in a circular ziploc container. I'm sure you can guess where this one went wrong (haha), but because of the shape of the weigh boat, we couldn't completely cover the eggs and they dried overnight. We also had one egg that was placed in a petri dish again but was elevated using an egg carton. This one showed a little more promise since it didn't dry at all or get cooked but the embryo died from damage to the yolk. However, Before the ones in the weigh boats dried out, The heart beats still slowed down to a beat per 2 seconds after about 3 or 4 hours.

We currently have another 7 eggs incubating and will be cracked on the 16th. We plan on using a Cell culture incubator instead (Therm Scientific CO2 Water Jacket incubator to be exact set to 5% CO2) to see how that works.

We have yet to actually conduct any experiments because of our issues with culturing the eggs. 

First, don't use CO2 with chick eggs!  CO2 is used only for cell and tissue culture, where, in balance with bicarbonate ion, which the embryos need for metabolic purposes, it forms a weak buffer.  The embryos will be producing CO2 on their own, which should be dissipated with a small fan; some incubators have one already built in. 

I think your problem lies primarily in the way your current incubator is heated.  Use of your cell culture incubator should solve that problem.  If it doesn't, go to a different method of shell-less culture, which you might want to try anyway.

The version I've used requires one 100 mm plastic Petri dish (I prefer those with a depth of 25 mm), one 8-12 oz. Styrofoam cup, and 1 rubber band for each egg, and a supply of Handi-Wrap.  A tissue culture dish can be used in place of a Petri dish, but the former costs more without providing any additional benefit.  The volume of the cup is less important than the diameter of its open end; make sure that the bottom of the Petri dish fits over the open end.  None of the components need be sterilized.  Assemble this apparatus (see below) using sterile technique, and the lysozyme in the egg albumin will take care of the rest.  The procedure for making these cultures is described below.  It is best done after the eggs have been incubated for 2.5 to 2.75 days; embryos of this age will produce cultures of the highest viability.

Find a cafeteria tray or something similar, and line the top with two layers of aluminum foil.  Place the top of the Petri dish on the foil, place the cup on the dish, and fill the cup with tap water to about 0.75 inch from the top.  (This will vary depending on the size of your eggs and the diameter of the cups.)  Place a sheet of plastic wrap over the cup, push it down so that it makes a pouch with sufficient volume to contain the contents of one egg, and hold it in place with a rubber band.  The bottom of the pouch should touch the water so that the latter will serve to cushion the egg when it is dropped into the pouch.  (The water also will help to maintain temperature when cultures are removed from the incubator for observation and prevent the cultures from being top-heavy, thereby minimizing the likelihood of cultures being tipped over.)  Now crack an egg, pour its contents into the pouch, and place the Petri dish bottom (cover) over the top of the cup, and remove the excess plastic wrap scissors.  In the completed assembly, the top of the egg should be no more than 0.25 to 0.5 inch below the cover.  (The Petri dish top is placed at the bottom of the culture vessel so that its larger diameter will help to catch the inevitable dripping of albumin.)  It's best to replace both the bottom of the culture assembly (i.e., the Petri dish top) and the aluminum foil when they get soiled; otherwise they will become contaminated with bacteria and fungi, and you'll find yourself having to scrub out your incubator.  In the 

The top (the Petri dish bottom) of this assembly will become clouded because of the high humidity.  You'll need to remove it to make your observations.

I hope this goes well for you.  Please let me know. 

Thanks Glenn Sherer,

I've found the method you described before and considered it, however, my adviser was skeptical of the protocol. Maybe if our next batch fails to develop she might change her mind. 

Our protocol this time was to use a square weigh boat to hold the egg, a petri dish to cover the egg, and a ziploc container filled with water and a kimwipe. We place the weigh boat containing the egg into the ziploc container and close it, making sure to leave 2 corners open to allow air flow. We only had 3 eggs left (still practicing cracking the eggs, my climbing hands applying too much pressure...). 1 of the 3 died from being cracked too hard and the other one died because the egg was rotated just prior to cracking. Our final egg is surviving so far, I'll attach a picture. Although, it's still kind of difficult to determine immediately if it's alive or dead.

Our plan is to see how well this one develops and continue on from there.

I suggest you read this article:' Avian Area Vasculosa and CAM as Rapid In Vivo Pro-angiogenic and Antiangiogenic Models,

Andrew N Makanya · Beata Styp-Rekowska · Ivanka Dimova · Valentin Djonov 

Methods in molecular biology (Clifton, N.J.) 01/2015; 1214:185-96. DOI:10.1007/978-1-4939-1462-3_11 · 1.29 Impact Factor

You may want to use plastic cups rather than petri dishes.

First I'd like to express my thanks to Andrew Makanya for his citation.  I was unaware of this article and have not yet seen it, but I agree, judging from the title alone, that it is something you should read.  In fact, I would suggest that both you and your advisor need to read more.  Her skepticism over the technique I described alone suggests this as does your conciliation to it.  That method and its many variants have been used successfully for tens of thousands of cultures by my own and many other laboratories here and abroad.  You seem to be trying to reinvent a wheel that has already been turning for many decades.  I would also suggest that you make many more cultures at any one time, since you will inevitably lose some fraction of them with each passing day.

The embryo in your photograph is dead.  Its white (rather than pink) body color indicates that it is not circulating blood; its emaciated yolk vasculature suggests this as well.  I suggest you search the literature for a good color picture of a living embryo at this stage so you know what to look for.

Once again, I wish you the best of luck.

 Hello Glenn Sherer, I hope all has been well.

We've had a few weeks to try different protocols. We've had some success with culturing the embryos to about day 10. Since the last time I gave an update, We are still using the method of placing the eggs in a weigh boat, placing the weighboat in a ziploc container filled with water, then closing the container with 2 corners still open to allow air circulation. 

Again, these eggs made it about 5 days post cracking, we then applied pipette rings of 1mm thickness, and 8mm diameter. We also used a whatman filter. We applied cells to the eggs and allowed them to incubate for several more days. However, we've been having a fungal problem with our cells and after 3 days, found a fungus growing on where the cells were applied, killing our embryos. We plan on using the cup method next but I wanted to know what percentage of your eggs survive to about 14 days?

Thank you.

Our survival rate to 14 days has probably varied as widely as 30 to 80%, depending on the age of the flock, the conditions under which the birds live, the quality of their feed, the season of the year, and the duration of egg storage prior to their incubation.  Unless you maintain your own flock you have little control over these things.  What you can control, however, is your egg source.  If survival rate is a problem, it may be well worth your while to compare eggs from different sources in your area.

That seems pretty similar to what we've been experiencing. Are there any sterilization techniques you follow? A lot of protocols I see tend to spray and dry their containers with ethanol and even spray the eggs when they receive them. This doesn't seem like a good idea to me as other articles say that ethanol decreases survival rates.