I think there is no clear manual for the sites of avian In-Ovo. However, this will depend on the objective of the study and according to that you should study the suitable embryonic stage and the needed needle length in order to reach the target site. For example; in In-Ovo feeding experiments you need to reach the amniotic fluid of the embryo and the time which it starts ingest orally, this meet the day 18 of the incubation also in this time the embryo is almost complete you you need needle size = 21-25 gauge inserted from the broad end passing the air sac....... and so on with different objective...

Also you may have a look to the attached materials

https://www.researchgate.net/publication/263698546_Standardization_of_injection_site_needle_length_embryonic_age_and_concentration_of_amino_acids_for_in_ovo_injection_in_broiler_breeder_eggs

Article Standardization of injection site, needle length, embryonic ...

Thanks alot Dr. Yahya, your effort and effective answer are appreciated :), but if i need to inject some probiotic in yolk sac at the day 18 of incubation what is the most suitable method for this?

from my point of view it will be little difficult, unless you do X ray to determine the position (see the attached pic)  of yolk sac , but this will be time and money consuming. In the attached paper the author said that he inject in yolk sac with needle 21 g but he did not explain how did he spot the yolk sac.

Also, I would like to take your attention that in the E18 the embryo starts ingest orally the amniotic fluid, so any injection in the amniotic fluid will go directly to the digestive tract

Just to note. Is it safe enough to inject the drugs into the YS or  amnion? It may cause hemorrhages  if you got into the vessels. It would be much safer and practicable to inject into the air sack. The injected solution must diffuse through the inner shell membrane. Anyway, it should be examined.      

For the embryos incubated for 6 days the best mode of injection is to make a hole on the upper side of egg (center) and use the needle 4-5 cm long. The amount of injected fluid (suspension) could be .2-.5 ml.

So Dr. Hrvoje, what about the mode of injection for the yolk sac of embryo incubated for 17 days?

Sorry no idea! But, I would try to inject an egg placed horizontally into into mid part of the lower edge.  The needle should be inserted to the center of this space.  

Thank you Dr. Hrvoje, and i'm looking forward to share our opinions and ideas with each other constantly.

The egg injection companies (Embrex and others) have established methods for days e17, e18, and e19. Since the machines for commercial hatcheries are now owned by vaccine companies, methods have been optimized for vaccine delivery at transfer from the incubator to the hatcher (e18). It means that delivery of nutritional enrichment and probiotics may be tough to have accepted at the commercial broiler industry level. This is unfortunate, because there is lots of opportunity to encourage early growth of the gut and healthy growth of the chick. Peter Ferket and Zahava Uni have been leaders in this field who have faced some of these challenges. 

If you plan to do a lot of egg injection work and want to mimic commercial egg injection conditions, it is possible to get a single egg-injecting bench-top version. Our research hatchery at the University of Alberta has one. The benefits for us were that we could quite quickly inject a number of eggs under identical injection conditions. 

Please Dr. Robert, can you explain to me more about the single egg-injecting bench-top version; manual instructions, video about its work and the way to achieve it. Because in my PhD work i'll inject a lot of eggs in the yolk sac or amniotic fluid, if applicable, at the day 18 of incubation with different probiotic strains.

About four years ago, i asked for the help in determining the appropriate method for manually injecting the yolk sac or amniotic fluid of avian embryos. Now, it is my pleasure to introduce you my innovative manual method for injection in the yolk sac or amniotic fluid in the late stage of avian embryogenesis.

Article Assessment of In Ovo Administration of Bifidobacterium bifid...

I hope it will help you in your upcoming relevant investigations.

Best regards

Dr. Abd El-Moneim Eid Abd El-Moneim

We currently developed a method to deliver the injection materials into the amniotic cavity. On day e18 the eggs were individually exposed to the light emitted from the candling box window in a way that their lateral bulge abutted the edge of lighting window with the blunt end held upward. Then the egg was turned around its longitudinal axis until the nearest point of the embryonic shadow to the air sac appeared, and then the point was marked on the eggshell using a carbon marker. We finally concluded that, 13-mm tip needles (25-ga) should be used for the injection 5 mm upper than carbon mark point. Syringe body was parallel to the long axis of the egg during the injection.

For more details please refer to following paper:

Effects of in ovo injection of chrysin, quercetin and ascorbic acid on hatchability, somatic attributes, hepatic oxidative status and early post-hatch performance of broiler chicks.

DOI: 10.1111/jpn.12760

https://www.youtube.com/watch?v=HnBrjLprzi4