# Hygenic test results on breeders



## JBJ

These are just a few of the great results at the 24 hour mark. We arrived at a very hygienic bee by selecting for Varroa tolerance. Dr Royce from OSU came down and showed us how to to the freeze killed brood assay. There were some that did not get 100% in the test so I presume they must have other mechanisms for coping with mites. I have been leaning towards the ones that got 100% for the last few grafts but will continue to monitor the other breeders for mite loads and overall success.


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## JBJ

The one labeled 13.1 actually cleaned both sides of the comb. I think we may have used too much liquid N and froze it all the way through.


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## westernbeekeeper

That amazing! I gotta try that method.


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## julysun

Good work! Another person that did this for years was S. Taber, don't see much mention of his work anymore.


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## canoemaker

Where do you get your nitrogen, and what do you use to store it?


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## JBJ

We used a loaner dewar from a local welding supply shop. I think it was about 10 or 12 liters.


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## robherc

is there any reason why "dry ice" wouldn't work for this purpose? I know it's plenty easier to come by for the average hobbyist/sideliner beek...hopefully it'd work well too?


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## sqkcrk

What were the mite loads like? Does this indicate varroa mite tolerance or resistance? I can see how it would indicate hygenic behavior, but, does hygenic behavior directly lead to mite tolerance/resistance?

What other traits are you selecting for and how do you go about accessing the degree of those traits?


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## Tazcan

How did you get them to lay around that circle? 
I am a nebee so i am just learning about the bees


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## sqkcrk

The OPer can answer from experience, but, you start off w/ a nice patch of capped brood and kill what you can see as an empty circle of brood by applying liquid nitrogen, which freezes the pupae dead. Exactly how this is done, someone else needs to explain. I think a tin can is used somehow, but I haven't done it.


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## irwin harlton

what is the relationship between a cell that has been killed by liquid nitrogen and a cell that has a foundress mite mite in it


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## robherc

@Tazcan
The capped larvae that WERE inside the circles were killed with liquid Nitrogen, as sqkcrk said...then the bees removed the killed larvae within 24 hrs. This is done to test the efficiency with which bees will theoretically remove varroa infested/killed larvae from the comb, thus reducing varroa levels in the hive. (or at least that's my understanding of it)

@irwin harlton
I believe the freeze-killed larvae are supposed to be homologous to mite-infested/killed larvae to the bees... i.e. it's not necessarily "varroa" that the hygiene is sensitive to, so much as "dead larvae" sensitive hygiene...so the same trait should be pretty efficient against EFB too (once again, in my opinion, and according to my understanding of the principles in practice).


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## Tazcan

Thank you for the answer Robherc , i couldn't figure out what was going on from the start of this thread, now i understand.


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## JBJ

The liquid N is poured in and then after it thaws the frame is returned and the clock started.


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## JBJ

sqkcrk said:


> What were the mite loads like? Does this indicate varroa mite tolerance or resistance? I can see how it would indicate hygenic behavior, but, does hygenic behavior directly lead to mite tolerance/resistance?
> 
> What other traits are you selecting for and how do you go about accessing the degree of those traits?


We are looking for hardy mite tolerant bees that are productive. We will be doing some sugar rolls soon to put some metrics on current mite loads. All breeders wintered well and easily made the grade for the almonds. We, of course, are looking for all of the usual economically valuable traits. I like a bee that can pay the bills.
Varroa sensitive hygiene is a well documented trait and does result in lower mite loads. Apparently you can end up with very hygienic bees by selecting from bees that cope with mites well. It is a challenge to keep the selection pressure on but well worth doing. We are taking some steps this year to try to get these traits more "true breeding" throughout the whole population. More isolated mating yards and instrumental insemination will definitely be helpful this year.


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## sqkcrk

Thanks. I can tell you've really thought about this. What are your plans? Queens for yourself or for selling to others?


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## spunky

Thanks for posting and keep up the good work JBJ


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## JBJ

sqkcrk said:


> Thanks. I can tell you've really thought about this. What are your plans? Queens for yourself or for selling to others?


We do both. We usually make between 5 and 7 thousand queens a year and have been working on this project for about 12 years. Its time to start keeping more of them ourselves to grow the operation a bit.


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## Adrian Quiney WI

What is interesting to me is that you said "We arrived at a very hygienic bee by selecting for Varroa tolerance". My understanding is that Marla Spivak and Gary Reuter started the other way, breeding for hygienic bees by using the frozen NO2 test because of the strong correllation of hygienic behavior with bees that remain free of AFB. Yet they make no claim that MH queens are varroa resistant. What percentage of your breeders were that hygienic - if you don't mind sharing.
So, in addition to your bees having hygienic capabilities I suspect they have other positive attributes that make them varroa resistant.


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## cklspencer

I've done the freeze test one many of my breeder hives but I saw a clip in a video the other day where they use a needle and poke the larvea under the cap to kill it and than come back 24 hours later to see if they cleaned it out. What would be the differance between the poke test and the freeze test or do they show the same thing?


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## JBJ

I have heard there are two sets of genes involved, one for uncapping and one for cleaning, and that the piercing of the caps can skew the tests.


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## JBJ

Adrian Quiney WI said:


> What is interesting to me is that you said "We arrived at a very hygienic bee by selecting for Varroa tolerance". My understanding is that Marla Spivak and Gary Reuter started the other way, breeding for hygienic bees by using the frozen NO2 test because of the strong correllation of hygienic behavior with bees that remain free of AFB. Yet they make no claim that MH queens are varroa resistant. What percentage of your breeders were that hygienic - if you don't mind sharing.
> So, in addition to your bees having hygienic capabilities I suspect they have other positive attributes that make them varroa resistant.


I have heard that also. I have also heard the developers of the VSH and SMR traits ended up with very hygienic bees in the end working from the other direction. Of the ones we have tested, about half were 90 to 100 percent and the other half was all over the place. We plan to monitor mite levels in all but have been grafting off the ones that got 100% lately. The others may still have valuable traits if they test well on the sugar roll later in the week.


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## bend or

Is there a reason to use liquid N2 vs a "freeze spray" like caned air turned upside down? It seems like it would have the same result and be more portable, and using a ring would still be able to control the area affected.


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## Adrian Quiney WI

JBJ, it will be interesting to see how there results match up: Your hygienic bees roll test versus the less hygienic ones.


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## sqkcrk

bend or said:


> Is there a reason to use liquid N2 vs a "freeze spray" like caned air turned upside down? It seems like it would have the same result and be more portable, and using a ring would still be able to control the area affected.


Could be just as effective I guess, having no experience w/ either. All I know is liquid nitrogen is standard operating procedure, tried and true. So, no one sees the need to change. Try it and let us know how well it works. You may be on to an alternative method. Or it could have been tried and rejected for some reason or other.


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## canoemaker

I attended a queen rearing workshop with Dr. Larry Connor this May and asked him about using the canned coolant, like for freezing off warts. He said it would be really expensive, and not very effective. It wouldn't get the brood cold enough for long enough to kill it. He gave an example of how tolerant the brood is by telling about an alternative method to the LN2. If you use wax foundation, you can cut out a 3"x3" plug of brood and put it in the freezer for 24 hours, then place it back in the brood frame. Then you can do your 24 hour hygenic test. He said someone he knew took their brood out of the freezer after 24 hours and it was still alive! They must have soft ice cream! Anyway, the liquid nitrogen must be the gold standard for freezing and killing the brood. If your home freezer is really cold, you can try the plug method.


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## WilliamsHoneyBees

I've seen the pictures around but how is it done. Do you just pour some liquid nitrogen in the pvc and let it sit for X minutes? 

Thanks


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## Adrian Quiney WI

Dan, I'm with you. I would like to see a youtube video, or Beesource pictorial on how it's done and a description of where the supplies came from and the cost.


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## WilliamsHoneyBees

pine_ridge_farms said:


> I've seen the pictures around but how is it done. Do you just pour some liquid nitrogen in the pvc and let it sit for X minutes?
> 
> Thanks


Anyone? Thanks


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## Rusty Hills Farm

I believe this poster is what you are looking for. It's from the Bee Lab and shows you how to test.

HTH

Rusty


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## WilliamsHoneyBees

Rusty Hills Farm said:


> I believe this poster is what you are looking for. It's from the Bee Lab and shows you how to test.
> 
> HTH
> 
> Rusty


Thanks Rusty!


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## RichardsonTX

Is there a list of queen suppliers that use this test to select for hygenic behaviour? 

Is there anyone who can comment on success from using this test?

http://www.sare.org/Learning-Center...ntrolling-Honey-Bee-Diseases-and-Varroa-Mites


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## AR Beekeeper

I once asked Marla Spivak about the pin test for hygienic bees, and she said the colony must be checked within 6 hours of killing the prepupa.

The test for bees that remove varroa that are reproducing in the cells involves placing a known number of mites in cells, and then counting the number that the bees uncap and remove. The two tests do not check for the same behavior. Bees can have one or the other traits, or they may have both traits, they can be hygienic and have Varroa sensitive hygiene.


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## RichardsonTX

AR Beekeeper said:


> I once asked Marla Spivak about the pin test for hygienic bees, and she said the colony must be checked within 6 hours of killing the prepupa.
> 
> The test for bees that remove varroa that are reproducing in the cells involves placing a known number of mites in cells, and then counting the number that the bees uncap and remove. The two tests do not check for the same behavior. Bees can have one or the other traits, or they may have both traits, they can be hygienic and have Varroa sensitive hygiene.


That's what I am also seeing. This behavior is developed to primarily provide resistance to AFB and chalkbrood, along with benefits to alleviate varroa mites. Suppression of Mite Reproduction (SMR) is another trait. I'm interested in learning more about tests for these traits and beekeeper experiences using these tests, outcomes, queen suppliers selecting for these traits, etcetera.


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## Fusion_power

http://www.selectedplants.com/miscan/Ferguson.Hygienic Results_28-05-15.pdf

http://www.selectedplants.com/miscan/Ferguson.Varroa, Nosema and Tracheal Mite Samples_28-05-15.pdf


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## AstroBee

You might find this link interesting: http://www.elgon.es/diary/?p=146


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## kilocharlie

JBJ - The other leg of the task is killing the drones of the less desirable colonies near your mating yards. Of course, re-queen those as soon as possible, but keep killing drones & drone brood until the new queen with "superior" traits is laying a solid pattern.

I like your criteria, "bees that pay the bills"!


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## rick54

Is there a specific brand of can or pipe that you use which is close to 100 cells within the testing diameter? Or do you use any can that you count the number of cells in and use that number in your calculations?


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## Cuttingedgelandinc

rick54 said:


> Is there a specific brand of can or pipe that you use which is close to 100 cells within the testing diameter? Or do you use any can that you count the number of cells in and use that number in your calculations?


We use 3" PVC pipe (approximately 160 cells)


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## John Davis

What AstroBee posted above is the work of John Harbo. There are some other papers of his as well.
His is the protocol for VSH evaluation. Separate from the freeze kill hygienic evaluation. 
Important to understand the difference and how it is used in your choice of breeders for your purposes.


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