We crossed!
I just read the paper and it sounds very promising. I think the size difference between the W and Z chromosomes in chickens is significant. It sounds like there is a difference in the staining protocol with whole cells versus measuring for apoptosis. Would this be significant if we extracted the nuclei?
Pip and a sick chick.
I don't use PI for measuring apoptosis (I think apototic cells can actually exclude it) so I am not sure about the protocol difference. I think something that I read just indicated incubation with PI for 24 hrs for nuclei. Odd since dead cells fluoresce pretty much as soon as you drop it in there. Have you done any nuclei extraction? I think I saw that you use some sort of glass crushing device, but I am not sure. If I can figure out that part, I would be inclined to try it with cells I have hanging around before embarking on chicken plucking or bleeding. Running things on the Caliber takes no time so if we come up with a protocol I should be able to try it pretty quickly.
Myrrh
My goodness you 2 are so smart. I wouldn't know where to even begin with all this, but I am enjoying reading it all. I hope you 2 can figure out a way to make it work! Good luck!
Thanks so much ladybugs, but its not smarts, just education. I am sure that if it was something you were interested in you too could learn and contribute.
So I had another thought. Do you do any geimsa staining? I am wondering if one could see a difference in nucleus size visually.
Here are some more interesting links about measuring DNA content using Flow:
http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstract&ArtikelNr=132514&Ausgabe=236198&ProduktNr=224037
http://www.icms.qmul.ac.uk/flowcytometry/uses/dnaanalysis/propidiumiodide/
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Cancer_Research/Key_Resources/Flow_Cytometry.html
Hi Granny! We just need to get rid of those pesky roos!
Hmmm, Myrrh--the only extraction I've done was mitochondrial, using a density gradient.
We don't do geimsa staining--I think you would need an excellent standard, a big size difference and/or maybe some sort of algorithmic analysis to make it work. But I would be willing to give it a try. The thing is you want a better result than the 80-90% certainty they claim for vent sexing (I think that is an absolute "best case" result using superb sexers and morphologically consistant birds).
I think Nakamura's paper and its citations would clarify the nuclei extraction protocol. The abstract suggest it could be used in the field--which means it should be pretty straight forward. I can't get the paper on line, but here is the abstract: http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowAbstract&ArtikelNr=132930&Ausgabe=236183&ProduktNr=224037
And here is the abstract for a paper suggesting why it might not work if the difference in size between the chromosomes is not sufficient: http://www.bioone.org/perlserv/?request=get-document&doi=10.1648%2F0273-8570(2002)073%5B0239%3AACOFCA%5D2.0.CO%3B2.
If you can get the Nakamura paper, I would love to see it. I might be able to come up with a work related reason to go the UC Library at Berkeley or Davis and get it there. It would be great to solve this.
This message was edited Jun 17, 2008 6:09 PM
anything for a work related field trip, eh?
i know a relatively reliable way to sex RIRs... crow=roo, egg=hen
;P
Hey tf--Remember the crowing hen! So far no egg laying roos that I know of. Just because I am willing to make the arduous trip from Lodi to Berkeley is no reason to snigger!
lol tf.....I think I could follow that one! lol
I got the paper (I'll send it to you by normal e-mail), but it's no good. The whole paper talks about being able to do it and how great it is, with no methods, materials, anything. He wrote another paper that might have details but it is not available on-line. A friend is going to pick it up at school tomorrow, so hopefully I'll have it by Thursday. Ah the joys of working at a tiny start-up....everyone is either going to school on the side, or planning to soon.
Myrrh
No citations? Maybe the second paper on larks or whatever--Oh, I just found the link isn't working. I'll look for it again.
Okay for some reason everyone is beeing very cagey about this. Here is one citation, but no abstract: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VJ1-3WJDV4C-1H&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=5a8bb6ee23a1e82914ad4969738a3957#bibl1
Now this looks useful--or at least encouraging--and it is a whole paper! http://elibrary.unm.edu/sora/JFO/v064n01/p0018-p0026.pdf
This message was edited Jun 17, 2008 6:31 PM
Ya know, TF, I think even I could sex them that way...LOL. I tried to sex turkeys once... Toms = fans and beard, Hens = no fan, no beard, but then here came, you guessed it, a bearded hen. She actually has a 10 inch beard. It seems that we have a flock of bearded hens around here.
GG
How odd granny! We had a royal palm that we were just sure was a hen, it was our only royal palm, the rest were Black Spanish. It would not fan it's tail out or puff up at any time. It wasn't until it started growing a beard and his face started turning shades of blue that we were pretty sure she was a he. Then our head honcho and breeding BS was killed by a dog and Thomas or Mr. Big Stuff we ended up calling him, stepped right up and took his place. He strutted proudly around all over the place all puffed up. He even started breeding our last BS hen........she is now hiding across the road in the tall grass and sitting on a nest. If she is successful this time, I can't wait to see those babies! If Mr. Big Stuff had not turned out to be a he after all, I would still be sitting here scratching my head about the hen with a beard and face that turned blue. I sure bet a bearded hen is a site!
Well, their heads are blue, but the toms turn red in breeding season, and the hens stay bluish. But, when I checked with DNR, they told me they knew about them and that they were legal in turkey season. But, after DH getting his (a tom) a few years ago, we haven't bothered with them.
GG