# Instrumental insemination, CO2 time



## Juhani Lunden (Oct 3, 2013)

Just curious how long CO2 gasification times you folks are using. My system has been over the last few years 3-4 min one day before insemination ( and second gas comes with insemination)

Is there need to keep the queen in sleep during the insemination for at least couple minutes? Sometimes insemination itself doesn´t last that long.


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## AstroBee (Jan 3, 2003)

I usually do 5 minutes the day prior. During the procedure, I only run the CO2 only during the insemination. No further CO2 is used unless I find one not laying, and then I'll give them a 3rd treatment.


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## Juhani Lunden (Oct 3, 2013)

How long do you wait for the start of laying?


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## AstroBee (Jan 3, 2003)

I usually see laying in less than 10 days. If I find one not laying for more than 14 days, I'll give a 3rd treatment. 

There is an enormous amount of literature on CO2 times out there. Some say as long as 10 minutes, others 1 minute. I'm not sure you're going to see much difference between 1 to 5 minutes, but perhaps a real expert will chime in. My feeling is that this is a "fine tuning" detail, and the real issues are: cell builder, pre- and post-insemination conditions, and of course the insemination procedure (excluding the CO2).

Here are a few references I found on the CO2 times:


http://naldc.nal.usda.gov/download/CAT87209055/PDF

On page 12 (PDF page 16) it says 10 minutes. 

Here’s another reference (by Sue) suggesting 10 minutes is too long I believe It references higher death rates in their third year.

https://hal.archives-ouvertes.fr/hal-00892280/document

This reference says 3 minutes:

https://www.researchgate.net/public..._predisposition_period_1_Onset_of_oviposition

Looks like another says 3 minutes:

https://www.researchgate.net/public...anaesthetised_honey_bee_Apis_mellifera_queens


http://www.degruyter.com/dg/viewart...2fv10289-012-0016-8$002fv10289-012-0016-8.xml

This also says 3 minutes:

http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.708.346&rep=rep1&type=pdf


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## Juhani Lunden (Oct 3, 2013)

Thanks AstroBee! 

I did the first inseminations on the 17th, so I´ll wait couple days and give them then a short CO2 "treatment" if there is no laying. I have never before done a third treatment.


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## Juhani Lunden (Oct 3, 2013)

Interesting would be to know how they in these studies counted the length of CO2 treatment and narcosis. Was it the actual time the queen was kept in plastic bag (or similar) or was it the time she did not move a leg?


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## rfgreenwell (Feb 14, 2010)

Is there an advantage to doing a 5 minute CO2 treatment the day prior? I have not done that before. I have been running CO2 during insemination, and then glue on my ID tag, cage her, and make sure she comes around on the heating pad before I return the cages to the bank. For spring and summer queens I have given a second 5 minute CO2 24 hours after insemination. For fall I havent given a second treatment but would if she did not begin laying. This was how I learned it from Joe Latshaw in 2013. Has there been more recent information leading to a "prior" CO2 treatment or just a different protocal?


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## Brandy (Dec 3, 2005)

rfgreenwell, did he explain why the different treatments for spring, summer, then fall??? Also you mentioned returning the virgin to a bank, are they not going into nucs after insemination?


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## rfgreenwell (Feb 14, 2010)

Joe did have an explanation for why it wasn't as needed for fall queens, but it is foggy in my mind now what the reasoning was, and I don't have it in my notes to refer back to. I usually inseminate anywhere from 8-24 in a sitting to make it worth all the set-up prep and followup work, and bank them in mini cages for observation and until I am ready to place them into nucs to prove their patterns. This got me in real trouble my first time out, because I ended up with a rogue queen in the bank. I am much more careful with the bank after that frustrating loss of my hard earned queens. I had been using a cloake board starter/finisher, and turned it into a bank. In swapping in capped brood frames at some point I failed to check back and kill cells, and ended up with a rogue. I guess what Astro was saying about treating the day prior and then the treatment during insemination acting as the followup, an inseminated queen could be caged and immediately introduced to a queenless nuc without the likelihood of making mating flights. Seems like Joe thought they were less likely to try and make mating flights in September, but I can't remember now. I actually always have done a CO2 treatment a day or two after insemination since I have been doing it myself, and before that when Adam Finklestein did II for me, he recommended I followup with two CO2 treatments after taking the queens home.


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## rfgreenwell (Feb 14, 2010)

Well, all that and I failed to tell you that they go into the bank to await the second CO2 treatment prior to being placed into nucs. I was wondering if the reason Astro was treating prior was to eliminated the banking after ii. The virgins are caged and held in the finisher for a week or 10 days prior to insemination, so if prior treating does the trick, it streamlines things a bit.


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## Juhani Lunden (Oct 3, 2013)

rfgreenwell said:


> I was wondering if the reason Astro was treating prior was to eliminated the banking after ii.


Yep, that is the reason at least for me.
When the queens are 8 days old I gage them, 1 day before insemination, give them 3-4 minutes CO2 treatment and return them into the hive in cages. Each one in her own mating hive. These mating hives have 6 frames, about 8x6 inches. The cages have two 5mm holes, so the nursing bees can come to them.
At the day of insemination I just do the semen collection and then I have all queens ready in cages. After insemination I return them, marked and wing clipped, among the bees without cage. 

In this manner I can leave the queen and her hive in peace after insemination. The queens have been in cage just for one day. Caging is harmful. 

I have understood the effect of CO2 is that it makes the queens start laying sooner. Autumn queens do not need it because they start laying in spring.


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## Brandy (Dec 3, 2005)

Juhani, that's closer to what I've been doing since I "think" the most important aspect is being able to return the inseminated queen uncaged into her nuc so she can be attended to similarly to a naturally mated scenario...


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## AstroBee (Jan 3, 2003)

rfgreenwell said:


> I was wondering if the reason Astro was treating prior was to eliminated the banking after ii.


Very simply, because Sue Cobey suggested that's the way we should do it. 

My procedure is very similar to what Juhani sketched out.

I'm not sure I understand why Fall queens should be handled differently. To me, it would be more important to know that she is laying prior to going into Fall/winter. The sooner I know this, the sooner I can make corrections if needed.


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## kilocharlie (Dec 27, 2010)

Juhani - You might ask the Queen herself, Dr. Susan W. Cobey:

www.honeybeeinsemination.com

She, Dr. Joe Latshaw, and Adam Finkelstein are probably some of the best resources on the subject you can find. Another would be Dr. Peter Schley in Germany. Find out what they say, try to set up an experiment to see what works best for your setup.

50% CO2 may be different that 100% CO2, etc.

Good luck!

Astrobee - great post! Thank you for the list


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## adamf (Jan 28, 2006)

Hi folks!

Everyone does AI a little different. Successful results help form a good protocol.

The idea is that with 2 CO2 narcosis events, the artificially inseminated queen will oviposit (lay eggs readily). In nature, she'll do this a day or so after her last mating.

Why the CO2 narcosis works is another very interesting subject. However, one has to be careful not to give too much CO2 as overdoses shorten her life.


Standards are to give her two treatments, one while she's being inseminated and one at a different time but 24 hours apart from the exposure during AI.

Some do 5 min while inseminating, then 5 later. Some say 10 min and AI time exposure. Some pre-treat with CO2 then AI and the queen is all set to be introduced.


We've arrived at this scenario: virgin gets CO2 while being inseminated. 24 or more hours later, she gets another treatment for 7-8 min. That means the timer starts

when she's out and after 7-8 min, she's taken out into fresh air. (Treatment is done in a large plastic bag, usually in groups of 20 or more).


This has worked well for us. As we got more proficient with AI, the first treatment while being inseminated, has shortened considerably. The 2nd treatment takes care of her

though, and she'll begin laying 2-3 days after she is introduced. Remember, egg laying depends on the newly inseminated queen's nutritional intake. Those eggs have to come from 

somewhere you know? A proper nuc, with the right types of bees, does the trick. That's another thread 


Have a good one!

Adam (& Kelly)
http://vpqueenbees.com


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## JRG13 (May 11, 2012)

Does C02 narcosis kind of simulate the queen taking a mating flight in terms of biological metabolites?


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## kilocharlie (Dec 27, 2010)

I've been trying inseminating the VQ, placing her in a mating nuc, letting her mate, then gassing her a second time, and replacing her into the nuc'. Some do go out and mate after I.I. 

Naturally, I get high expectations from these queens, but so far the one that inspired this effort was the best one. I tried it on 3 queens last year, and on 2 this year. All performed well, but none like the first one, who laid up 18 frames of brood 2 years ago.

I have not performed this as a scientific experiment. Just noticed that the one queen had mated after I.I., and she was laying up a storm, so I tried this on the last few queens last year. I started this year and sold off all the bees. Any ideas on an experimental setup are welcome.


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## adamf (Jan 28, 2006)

JRG13 said:


> Does C02 narcosis kind of simulate the queen taking a mating flight in terms of biological metabolites?


That's a good question. I don't think anyone knows. I think there's some trigger in some part of the queen's brain that is tripped during mating and during the CO2 narcosis.

This initiates the metabolism of oviposition (egg-laying). Sometimes, there will be AI queens that are fully extended, throwing off all the "queen-right" pheromonal compounds to

make the colony act queen-right, but for some physiological reason (clogged oviduct/deformed oviduct) she doesn't lay.

Also, after the virgins get a CO2 treatment, workers treat them much more like laying queens. In colonies or if they are caged.


Lot's of really interesting Virgin Queen Honey Bee research could be done sometime!

Adam
http://vpqueenbees.com


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## Juhani Lunden (Oct 3, 2013)

adamf said:


> Remember, egg laying depends on the newly inseminated queen's nutritional intake. Those eggs have to come from
> 
> somewhere you know? A proper nuc, with the right types of bees, does the trick.


Seems to me the best TF bees regulate egg laying. This regulation is done by feeding bees and the queen together? The stock I have at the moment is getting fairly weak in August, after that they build up again for winter.

Maybe this regulation is one of the causes some queens start egg laying very late: they don´t get fed.


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## Sunday Farmer (Nov 13, 2013)

Hey- hoping to learn how to instrumentally inseminate this fall. Where did y'all learn? Has anyone self taught? Or did you go to a class or meet up with another beek?


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