Hand fertilisation of bee eggs.

[alanf]

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.

Not questioning experience. I'm trying to work out the practicalities that have led to II being widely written about but very little about AF. I guess it depends on what the aim of your breeding is.

If your intention is only to maintain a blood line you can do it solely through artificial fertilization. You control the parents of each queen, that does the job without having to get the queen on the bench again. Mating those queens randomly isn't a problem for the genetic purity because the offspring will be non breeding workers.

If you want the genetics to be be expressed productively thoughout all your colonies you have to control the worker genetics through II or other restrictions on mating. But that won't have any influence on the next generation if you use AF. Producing queens for sale, I can see why controlled mating is an advantage. II does use a dozen or more drones but if these are from the same hive is there any real advantage using a single drone with AF?

Random mating may be riskier than II in experienced hands if the concern is losing a queen. It's unlikely to be higher risk than mating at an isolated site. Is the main reason II is more prominent is that once II is mastered for productive queens, AF doesn't add much practical advantage?

 

[mbc]

"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.


PH

I was about to spell out what I understood from this but remembered your last pm to me PH and refrained

 

[Hivemaker.]

I guess it depends on what the aim of your breeding is.

Absolutely correct,and this would be useful.

 

[drstitson]

couple of interesting papers (well they are for those of us with proteomics interests) - the first offering the following conclusion:

"Our large-scale identification of proteins within the spermathecal
fluid of honeybee queens offers an intriguing insight
into the details of female sperm storage. Our data indicate
that females provide stored sperm with a complex mixture of
proteins that form a metabolically connected network. They
also suggest that some essential physiological requirements
of sperm have effectively been 'outsourced' and are now provided
by the female. In this respect, sperm storage could be
regarded as a specialized from of endosymbiosis between
males and females, post-copulation but pre-fertilization."

spermatheca proteome: http://www.springerlink.com/content/l04332j228034182/fulltext.pdf

drone semen proteome:
http://ddr.nal.usda.gov/bitstream/10113/9132/1/IND43847942.pdf

Finally - this abstract below states:
"We provide evidence that the cellular machinery of sperm is plastic and differs between sperm within the ejaculate and within the female’s storage organ. Future work will be required to test whether these changes are a consequence of active adaptation or sperm senescence and whether they alter sperm performance in different chemical environments or impact on the cost of sperm storage by the female. However, these changes can be expected to influence sperm performance and therefore determine sperm viability or sperm competitiveness for storage or egg fertilization."

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.2011.05029.x/full

 

[ebee]

What's the reason for diluting the semen with buffer solution? Is it just to make it go farther or easier to use. Do AI practitioners dilute drone semen?

 

[drstitson]

presume it needs diluting a bit in II to allow adequate mixing of the multiple samples.

 

[Hivemaker.]

presume it needs diluting a bit in II to allow adequate mixing of the multiple samples.

For normal semen collection and insemination no dilution is needed,but for homogenising semen from say 300 drones, then dilution can be used, and then the semen is put into a centrifuge,the dilution fluid is then removed,but centrifuging is not a good idea,breaks the very long tails off many of the sperm.
Tris buffer can is used if the semen is to be stored for longer periods.

 

[susbees]

What's the reason for diluting the semen with buffer solution? Is it just to make it go farther or easier to use. Do AI practitioners dilute drone semen?

The tubes are very fine and easily blocked if any mucus gets admitted into the tube following the semen harvest. A little buffer is then sucked up to buffer the sample from the air and pushed partly out again before the next harvest. I'm quite new to II and can say without a doubt that this is the hardest bit!

So yes and no is the answer.

 

[susbees]

And what HM said...whilst I was composing

 

[Hivemaker.]

These are some of types of fluid used.

Simple Saline Solution- 100 ml.
100 ml. distilled water
1.0 g. sodium chloride
0.25 g. dihydrostreptomycin

Tris-Buffer Diluent- 100 ml.

100 ml. distilled water
1.11 g. sodium chloride
0.10 g. glucose
0.01 g. L - lysine
0.01 g. L - arginine
0.01 g. L - glutamic acid
0.329 g. Trizma HCL
0.329 g. Trizma base
0.25 g dihydrostreptomycin

Note:
You can also use this antibiotic instead of dihydrostreptomycin

Gentomycin sulfate, cell culture tested ( 250 micrograms per mill. )



Gentamycin 0.462g in 10ml sterile distilled water

Add the antibiotic solution to 1 Liter buffer just before use.

The antibiotic in only viable for about 5 antibiotic days in solution


Water Source
The water source used to make up solutions is important. Use double distilled water. When mixing or storing semen it is best to use HPLC (high pressure liquid chromotography) water.


Ph
The pH should be adjusted to 8.7-8.9. If the pH is too high use dilute HCL to adjust. If the pH is too low use NaOH to adjust.

To Sterilize
Bacteriological filtering is recommended. Use filter with a pore size of 0.2 um. Solutions can also be sterilized by heating to 250 F for 30 minutes. Note that heating will denature the antibiotic, add this after the solution has cooled.

Or Amo lens plus,ocupure,steri-neb,SafeCell,boar sperm diluter,Boar semen is fairly dilute, and SafeCell has a specific osmolarity formulation to closely match that of boar semen. Honey bee semen has a higher osmolarity. To make SafeCell for bees mix 540ml distilled water with 100ml SafeCell concentrate.
__________________

 

[DorsetB]

I read the Beesource thread a couple of weeks back - one of the things I dind't quite understand was this comment by M. Bush, (underlining is mine):

"How about this. You put some drone comb in the hive you want pure queens from. You wait for the queen to lay eggs in it. You squeeze off a few drones to collect semen. You add a bit of diluted honey to the semen to activate it. you apply this to the newly laid eggs in the drone comb. "

How does dilute honey activate sperm? Curious as to how this works.

 

[Hivemaker.]

I think perhaps MB was just being a little flippant perhaps,but just getting the point over about this in his kind of way.

 

[newportbuzz]

fasinating thread.
lots of potential it can almost half the work needed to produce a viable breeding line.
can i raise a question,
is the sperm altered in any significant way during its stay in the queens spermethea and is it (wrong wording i am sure) put to sleep/sedated in anyway and then woken/revived ?

there could be some possible steps missed in the journey of sperm to fertelised egg? As in a sense raw sperm never actually contacts an egg in nature but stored sperm does????

 

[DorsetB]

I emailed MB a couple of weeks back, and asked him about this technique as I might try it out next year, I also asked if it was preferable to use distilled or reverse osmosis water, which I have plenty of, and what the dilution ratio should be. Here is his answer:


"I have discussed the method with three people who have actually done it.
The results varied somewhat between the three, only one was
enthusiastic about it, but that is the gist of it.
Yes, it's probably a good idea to start with sterile distilled water. The main thing is that the diluted honey activates the sperm and allows it to get in. No one gave particulars on that, but I would assume from the conversations, 1:1
or thinner. I have never had an interest in it other than academic, so
I haven't had time to try it.

I would like to just for curiosity, but I don't want to control breeding. I've seen what it's done to other domestic animals. Sometimes you end up perpetuating genetics that can't even reproduce by themselves as you have bypassed the "tests" for reproduction, in this case strength, endurance, sense of direction, and maybe even the instincts of mating themselves etc. when you intervene.

One of the downsides is rather than having a queen who is now
inseminated with all the genes you wanted, as in II, you have an unmated
queen who, albeit, has the genes from both sides that you wanted, but is
still unamated."

I can see his point about perpetuating genetics which would not naturally be selected for reproduction.

 

[drstitson]

Critical Flaw in this Technique

Whilst reading Snelgrove in the "little library" this morning I came across something rather critical, confirmed by a few quick checks.

Like all hymenoptera eggs, those of honey bees are fertilised through the broad ANTERIOR micropylar end - which has pores for sperm to enter. This is the end which, obviously, emerges from the queen first and hence is the attachment point to the cell base. So the core instruction in the methodology is wrong:

"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! "

If it were me - i'd cover the BASE of the egg with diluted sperm AND ensure i was doing it ASAP after laying.

 

[drstitson]

"How does dilute honey activate sperm?"

Honey contains catalase, other antioxidants and amino acids and is weakly acid. This is similar to spermathecal gland secretion.

However, not sure about the fact that honey is very hypertonic.

Might do a small lab study this season to see how honey affects sperm (drone, i might add).

have also done some further reading re fertilisation:

it seems that mechanical stress to the egg during passage through the oviduct (or a capillary tube) may "activate" the meiotic division ready for receipt of male pro-nucleus after fertilisation. This obviously happens shortly before fertilisation and so any manual addition of semen to "drone" eggs would need to be done very soon (mins) after laying to be successful.

 

[Finman]

.
Some organs are very sensitive to pH. You cannot do what ever..
I do not bee blood but human blood pH is very stable, 7,5.

Mothers milk and cow milk is 6,5.

Honey pH IS ABOUT 4. If you put one cell sperm into pH 4, it is one question, how it stand it.

 

[Hivemaker.]

Saline for II is usually set to around Ph 8.7 to 8.9

 

[RoofTops]

So we have a method which as far as I can see from this thread no one has actually succeeded in getting to work?* For the reasons drstitson suggests perhaps?

*There is a reference to "mixed success" which might mean it worked occasionally - or perhaps the queen inadvertently fertilised the eggs herself?

 

[drstitson]

So Barrett had positive result in 1919 (perhaps due to his saliva); not much else since.

"Saline for II is usually set to around Ph 8.7 to 8.9"

HM BUT insemination and fertilisation are two completely different processes.

making sure sperm can get to spermatheca successfully then survive 2+ years is different to the requirements for actively penetrating a passing oocyte at some point down the line.

Highly likely that both the egg and sperm need potentiation for the process to go as planned. Giving the egg a kickstart squeeze and activating small number of stored sperm sounds reasonable to me.