Why not dissect out the vas deferens from one side of an animal, and
stitch her back up for a second (later) usage? Once the vas deferens
(slim, zig-zag tube) is in a few drops of solution one can snip it into
segments (like "sausage links"), grip one end of a segment with
watchmaker's forcepts, and grip the link--somewhat tightly, but not enough
to completely crimp it-- so that by pulling on the first forcepts the
sperm inside are extruded like compact "sausage filling". With a pipette
the "filling" can be easily transferred to a test tube and teased apart.
That gives a VERY concentrated sperm solution.
Normally, for artificial insemination, the vas deferens is simply
"minced". With the above protocol a very "pure" sperm preparation is
obtained.
Good luck. Please keep the uronet informed of your progress. Many of us
are interested in making axolotl transgenics!
cheers, gmm
George M. Malacinski, Ph.D.
Professor of Biology
Department of Biology
Indiana University
Bloomington, IN 47405
Fax (812) 855-6705
---------- Forwarded message ----------
Date: Mon, 10 Feb 1997 09:49:13 -0500 (EST)
From: MCLEANM at vax.cs.hscsyr.edu
To: nobody at net.bio.net
Newgroups: bionet.organisms.urodeles
Subject: Axolotl spermatophores
I am trying to design an axolotl transgenic by using a restriction enzyme
mediated insertion of DNA into sperm nuclei. Now the protocol that is
used in Xenopus requires sacrificing the frog and though periodically we do
sacrifice 1 or 2 for tissue, I may need more sperm than what that would give.
So, being the males lay the nice spermatophore cones I want to try to isolate
the nuclei from there. The only problem I am having is getting rid of the
extra gel coat of the cones from the sperm itself. After spinning the tips of
the cones down and several washes etc I can't get rid of the gel. I have not
yet tried a 2.5% cysteine solution that is used to chemically dejelly Xenopus
eggs but will be trying that soon.
I was wondering if you knew of any method to separate the sperm from the jelly
cones they are laid in? I need to remove this before I can demembranate the
sperm to isolate the nuclei.
Thank you for your time and suggestions.
Sincerely,
Matt McLean
MCLEANM at VAX.CS.HSCSYR.EDU
