D. Murrell wrote:
> It appears that some of the advantages of instrumental insemination
> in a breeding program could be achieved or even accelerated by hand
> fertilizing. Mr. Haberl, could you share the details of this procedure
> with the list for those of use not having access to the German journals.
Personally, I do not have any experience in hand fertilisation of
previously unfertilised eggs, but I came across these articles some
time ago. A great potential for bee breeding lies in this technique
and it makes me wonder why it has received so little attention so far,
especially, as it was in principle performed already in 1919 by Barrett.
I will use the term 'artificial fertilisation' (AF) for
fertilisation of previously unfertilised eggs.
Unfortunately, many people, at least in Germany, do not use the
adequate terminology with regard to insemination. What is called
'artificial insemination' should better be called 'artificial mating'
(AM) in bees IMHO.
The following procedure was published by Frank Neumann in the
'Deutsches Bienenjournal', issue March 1995):
1. Preparation of drones/semen:
- Let the queen lay unfertilised eggs in drone combs about
40 days (24 days development + ca. 16 days for sexual maturity)
before AF. These drones should hatch under controlled conditions
to be sure about their origin. If e.g. marked with colour paint
on their thorax, they can be allowed to freely fly because
only a few are needed (one?).
- Here comes the most difficult part:
Fill a sterile syringe (volume about 1 ml) about half with
sterile sperm dilution buffer (same as used as a stop solution in AM).
Attach a sterile glass capillary (about same as with AM) to the
syringe
and make the dilution buffer fill the capillary, then draw back to
make a small volume of air enter the capillary. This air bubble
is used to separate the semen from the dilution buffer (this large
portion in the 'back' of the syringe).
The semen of one selected drone (about 1 ul) is then collected in the
glass capillary (same procedure as with AM). Thereafter, draw about
eight to ten times the volume of the semen (i.e. 10 ul) of semen
dilution
buffer into the syringe and mix the semen and this small volume of
buffer
through repeated draw and push cycles on a sterile glass plate.
The prepared, diluted semen can be used several hours if stored at
room
temperature and in the dark.
2. Preparation of unfertilised eggs:
- In the morning, cage the queen on one side of a empty fully
drawn drone comb. It will take some time till she begins
egg laying. In the afternoon transfer the queen to the other
side of the same comb. Now, she will continue egg laying after a few
minutes. These eggs can now be used for AF.
(My comment: In another article I read that if unfertilised
eggs will enter development spontaneously after about 4 hours.
So in practice, one could wait about 2-3 hours before taking
the drone comb to ensure that enough eggs have been laid).
- The syringe with the diluted sperm is pushed so that the
diluted sperm forms half a droplet at the end of the glass capillary.
A egg to be fertilised must now be covered with diluted sperm
at it's upper 25 % (the free end of the egg not being attached to the
cell)
for a second. That's it! To prevent the sperm from drying, the droplet
is drawn
back into the syringe each time after AF. Be sure to mark the
respective cells (using e.g. an overhead transparency) on the comb.
- The comb with AF-eggs is then transferred to a previously dequeened
colony.
After 3 days the larvae can be grafted as usual. The raised queens
can be used for AM too, of course.
If queens are reared from AF-eggs and mated uncontrolled, you may
profit from heterosis effects (in workers) in each generation, but
at the same time be able to keep 'your' race/breeding line/etc. 'pure'.
But one can think of many more applications. Compared to AM, time
schedules are reduced significantly.
Except from the syringe and the glass capillary, you do not need any
special equipment. For sterilisation you can use a high pressure
cooking pot (about 120 degrees Celsius, 20 min).
--
Michael Haberl
Hessische Landesanstalt fuer Tierzucht, Abt. Bienenzucht
Erlenstr. 9, 35274 Kirchhain, Germany, 51n 9w
Tel: ++49-6422-9406-12
Fax: ++49-6422-9406-33
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