Hand fertilisation of bee eggs.

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Hivemaker. 

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Had a link passed on to me about this subject by Norton,have since looked into it a bit more, and believe it could be very useful in queen breeding,depending on success rate,has anyone else tried this.

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 takes 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 were 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 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).
 

drstitson 

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what sort of success rates were quoted in the reference(s) you two have read???

"Here comes the most difficult part"

easier if you have a 0-20u micropipette handy and a box of sterile tips.
 
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Norton 

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As far as I know it has not been performed successfully yet. Could make a really good and useful project for a university undergraduate. Might even be useful for AMM fanatics!
 

Hivemaker. 

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There is a thread about it on beesource,and comment from M Bush Below..

I discussed this with Roger Hoopingarner at HAS this last summer. He said he'd done it many times but only with mixed success. he says it's difficult to get them fertilized. Perhaps if one were to catch it right AS the queen layed the egg and have the semen ready...

This is the other link...http://beenatural.wordpress.com/queen-rearing/hand-fertilization/

Also Read Steve Taber's "Breeding Super Bees". He has an extensive writeup about ways to manipulate drone eggs including how to artificially fertilize them.
 
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Poly Hive 

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Ye old trap, acronyms.

Af=?

Am=?

If it's worth the bother of trying surely it's worth the bother of spelling it out.

Mark the cells using an over head transparency? Needs expounding as I for one, (aye village ***** I know) need an explanation.

PH
 

Hivemaker. 

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Ye old trap, acronyms.
If it's worth the bother of trying surely it's worth the bother of spelling it out.
PH
Here is another ..........II=? or Kiss=?

So you know which cells/eggs you have fertilised maybe,athough i could think of easier ways to do that.
 
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Poly Hive 

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I think most of us know what KISS means Hivemaker and I am asking an honest question so if you wear the Mod badge you need to think that way.

In case you forgot... Keep it simple silly....

PH
 

Hivemaker. 

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I am asking an honest question so if you wear the Mod badge you need to think that way.
Okay i will explain your question,it is explained in the link.
Below is a quote from the link which explains the acronyms.

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.
 
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drstitson 

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what needs thinking about is what is done to the semen during normal storage in HM - various media used for II are just physiologic buffer solutions for diluting and keeping viable prior to insemination ie thus restoring the semen to their expected normal pathway/fate. whatever HM's reproductive tract does to sperm (potentiation or removal of inhibition) needs to be reproduced too for success - unless we are to head down the ICSI route.

Most people here are aware of the normal physiologic change that semen undergoes naturally before one even takes into account potential physiologic contributions from where it is deposited!!!!
 

DanBee 

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How do the nurse bees take to worker in drone cells?
 

Hivemaker. 

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Okay from what i have read Dan,the comb is placed into a Q- colony,only until it emerges,then the larvae is grafted into a queen cup.
 

fatshark 

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Nice one Hivemaker ... all sorts of possibilities here for using pre-screened sperm and eggs which are known to have low or negligible levels of virus in them.

What does the "mixed success" reported by Roger Hoopingarner mean? 10% or something more manageable?

Cheers
 

Poly Hive 

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"Most people here are aware of the normal physiologic change that semen undergoes naturally before one even takes into account potential physiologic contributions from where it is deposited!!!!"

I read the English and ain't a clue as to what you mean.

Can we keep to basics please as this thread had a great deal of promise if we try not to assume we are all on a superior level of knowledge.

Thanks

KISS>>>>> Keep it simple silly

PH
 

DanBee 

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Okay from what i have read Dan,the comb is placed into a Q- colony,only until it emerges,then the larvae is grafted into a queen cup.
Oh yes, I understood that was what was done when fertilising drone eggs for queen raising purposes, but just wondered what happened if you left fertilised eggs where they were laid...
 

alanf 

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A point that comes to mind is that if fertilisation was easy, it would make Instrument Insemination pointless. Specifying both parents of the next queen is a big advance and time saving on mating a virgin queen with a dozen drones.

It would be much more widely practiced if it worked reliably. Am I missing something?
 

Hivemaker. 

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Am I missing something?
Yes Alan,this is simply to fertilise an infertile drone egg with one sperm,and then use the larvae to raise a queen from,and not to inseminate or otherwise mate a virgin queen.The queen produced from the larvae still has to then be mated,either naturally or by II. Hope that helps to explain.
 
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drstitson 

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"How do the nurse bees take to worker in drone cells?"

presumably not a problem - so long as eggs don't smell like diploid drones i'd imagine they are left alone (at least for long enough to graft).

remember the relatively few eggs that you do fertilise will be removed within 24 hrs of hatching (with aid of your trusty OHP transparancy "map" overlay PH!).

Edit: Just check out the literature on cell size and varroa control. Here is an example:

" Martynov, Michailov and Tuenin have conducted long series of observations
in Russia upon a similar matter and have shown scientifically that
worker bees reared in drone cells are larger than their worker cell sisters.
The most outstanding of these three works is that of Michailov. He
chanced upon a comb in one of his colonies that in the upper half, con-
tained normal worker brood and, in the lower half, contained worker
brood in drone cells covered with level cappings. Michailov took ad-
vantage of this phenomenon and preserved the bees for further treat-
ment. He then measured the length and width of the right fore wing,
the length of the tongue and the width of the abdominal tergite seg-
ments. He showed that the bees reared in the worker cells were smaller
than their sisters which were reared in the drone cells. It is of interest
to record at this time that the tongue length showed an increase of over 4 per cent.
"
 
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Storm™ 

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I am sorry for adding to this as clearly I am in no way qualified however two points raised are:

What is the mixed success rate
What would happen if they were just left in the comb. (presumably this means would they draw a queen cell off from it because they sense it is different.)

I dont know the answer to the first question however it would make sense to duplicate the conditions of the hive as closely as possible. Now I am guessing that this is done in a lit area in comb but not actually inside a full working hive. If I am wrong on that sorry from now on. But it would have to be dark. As in the hive. The temp range would have to be similar. And also and I would say most importantly there will probably be a humidity threshold where optimal fertilisation occurs. I dont know what that is but the kit is literally a few quid and less than a tenner. Orchid suppliers have these. If it is essential that you have the best I used to use a thing called a wet globe bulb thermometer which does everything from temp to humidity.

Finally , the egg is laid in a cell which you monitor using your marking system. Then you graft, and its drawn out presumably, as long as it was fertilised. So this is a substitution game with it all hinging on the fertilisation. I dont know (as I said because I know nothing) but can you not help this trickery, by transferring the egg to a used queen cell from which a queen has already emerged, or to use a partially built cup, thus replicating the circumstances and look of the aim you are trying to achieve. I dont know it was just a thought.

Edit - scratch the second bit, i've just seen HM's post saying just that.
2nd Edit - Just read the whole thread now on beesource, and of course any queen you rear will have to be mated, artificially by a drone of your choice (or naturally (not the aim I assume)). So you control both ends of the process and therefore a greater retention of the traits you are trying to breed into the queens. Instead of just the back end of the process?
 
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