Hand Fertilization

A drone egg can be fertilized to produce a female larva. That larva could be grafted to raise a queen. This process could enhance queen breeding and selection.

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Below is a copy of some correspondence between Michael Haberl and I concerning hand fertilization:

>It appears that some of the advantages of instrumental insemination in a breeding program are 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....

Michael Haberl replied:

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 bufferthrough 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).

--Michael Haberl

The conversation continues with questions from Ernie Huber:

> ...could you please explain what the "stop solution" is?

In artificial insemination (AM in 'my' sense) the sperm from several drones is USUALLY NOT diluted with sperm dilution buffer. However, one uses sperm dilution buffer to handle such a small volume of sperm. The largest portion of a syringe is filled with sperm dilution buffer and just a little air bubble separates the sperm from the buffer. The buffer's function is only to replace the air in the syringe because as fluid it is not compressible to the degree air is and hence enables accurate small volume transfers. I do not know where the word 'stop' originates from, perhaps because the buffer sets the limit what has to be injected into the queen.

Obviously, there's a chance that sperm comes into contact with the fluid used as 'stop solution'. So it is recommended to use the same fluid that is used in real sperm dilution. There are several different recipes for such buffers. One of them is e.g. Hyes-Buffer (0.9 percent NaCl, 0.02 percent CaCl2, 0.02 percent KCl, 0.01 percent NaHCO3, in distilled water at pH 8.5).

> Also, could you please say what the 1919 reference to Barrett appeared in?

My citation refers to the publication of Frank Neumann in the Deutsches Bienenjournal 3/98, but with in this article there's no detailed reference list.

> Is the Neumann article in Deutsches Bienenjournal in English?

No, sorry, it is in German.

> Apparently it is possible to control the genetics of both selected queens And selected drone donors through this process, and if so, does that make it unnecessary to do artificial insemination to accomplish the same thing? Why would anyone prefer to do artificial insemination if this technique is available?

Yes, you could control the male side of reproduction by artificial fertilisation as you do with the aid of artificial insemination (AM) (but why the queen side!?).

I see the difference between the both mainly in the number of fertilisation events you control. An artificially inseminated queen will produce thousands of workers and queens that carry the genes of the drones you used for insemination. With artificial fertilisation you only produce a few individuals of the desired genetic constitution. However, what you need for breeding are only a few individuals (queens) to produce males (sperm) and eggs!

A problem might be, however, that you need a performance test for your breeder colonies to select the colonies for further breeding. But most commercially interesting traits (honey yield, etc.) result from workers AND the queen. With artificial fertilisation used as I mentioned, the workers influence the result of the performance test but carry only half the genes (those from her mother) you select for. In short: you can not test what you want to.

> Sorry to ask so many questions but it really is interesting.

Thanks for asking!!! I am curious myself why this technique has not become more popular so far. Or is someone on this list already practising AF?

--Michael Haberl