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Hello everyone!
We have obtained several fresh fertilized eggs from а private farm with my wife. Instead of eating them we decided to check if it is possible to hatch a single egg without using an incubator. We have set up the tungsten lamp at such a height above the rice-filled dish with an egg, at such height that ensured that the temperature at rice surface was around 41C which is recommended for the first 12 hours of hatching. We used rice because it is cheaper than sand, and the sand is not widely available in the city, so instead of digging kids sandbox we just used available cheap rice. We have put an egg into the rice and have constantly rotated it, every 2 hours. The arrow marks the rotation direction. After 19 hours of incubating the egg we have opened the shell and isolated the whole embryonic disc. We have fixed it in neutral buffered formalin. After 48 hour fixation I'm going to image it under the microscope and then embed in paraffin and make sections and stain them. This embryo seems to be the late gastrula at the stage 4-5 according to Hamburger and Hamilton chicken development stages. Thus , the eggs can be hatched without an incubator.
Very good solution. I have hatched chicken, duck, and peacock eggs in an old 10 gallon aquarium with a 60 watt bulb and thermometer with pretty good results.
I look forward to seeing images of your sections.
CE
Hi Naphtalene,
I have never done this (it has been on my "to do" list) so I will be interested in seeing how it turns out. Have you read up on the micro technique for this project? I only ask as you seem to be missing some steps that I have seen in my research.
Best of luck.
Peter.
Thank you for your replies!
I will update the topic as I will get more photos of the project.
Peter, I'm not sure I understand your question: what is that you call the missing step in micro technique? Are you regarding to egg hatching or to further embryo processing?
We have incubated the other two eggs for 48 and 72 hours, when the growth was interrupted and the embryos harvested. They seem to develop normally. Though the rice works well as the thermostating media , the lamp height requires the constant attention because it tend to overheat at prolonged use. We had to use three thermometers and we rotated the eggs, even once at night to prevent the accident temperature changes.
We used the Bouin's fixative as it works better than formalin for embryos, making them more tough and better preserving morphology, so we changed from NBF to Bouins solution.
After the fixation. we have preembedded the embryos in agar. The embryos are very fragile, and agar greatly eases their handling during processing and paraffin embedding.
The reflected light view of 48 hour embryo -13-14 Hamburger-Hamilton stage (13 of 46) :
Transmitted and a high mag of tail portion, showing somite mesoderm (square segments)
72 hour embryo looks more compact because it is in classical embryo U-like pose, 19 HH stage (19 of 46):
The agar preembedded embryos after 24 hour fixation, before the paraffin processing.
Hi Naphtalene,
I was meaning the handling and processing of the embryo after removing it from the egg. As I said I have never don this procedure only read a very detailed description in P. Gray's "The Microtomist's Formulary and Guide". I realize this is an old publication so techniques have probably moved on since, I was just wondering if you are using recent techniques.
Peter.
Peter, I would greatly appreciate if you would post some details on the wmbryo isolation procedure. This part is the most problematic, and actually we have lost 3 out of 6 embryos during isolation. I would describe the procedure if you are interersted.
Briefly, the embryos consist of zona pellucida in the center, containing the embryo and surrounding zona opaca containing the supporting cells. This round structure lies on the surfece of the yolk. We usually cut the yolk surrounding the zona opaca and then insert the spatula under the round embryo with zona opaca. Then we lift the spatula while pulling the embryo with the forceps along the spatula. The embryos is then washed from the yolk with phosphate-buffered saline and fixed with either 4% neutral buffered formaldehyde or Bouins fixative.
Hi Naphtalene,
The description in Peter Gray's book is over fie pages, I would suggest you borrow this through your library or if you prefer I will scan the relevant pages and PM them to you.
Peter.
Thank you much Peter for this reference! I have found the "The Microtomist's Formulary and Guide" you indicated, this is the best resource on the topic so I have seen so far. Previiously I have only seen the SpringerProtocols procedure using the wet paper, and it was described in quite few detail so we had to find our own solutions for tasks like lifting the embryo from the yolk.
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