# My ghetto II station



## beepro

Hi, All!


This is my most ghetto, el cheapo II station I just finish assembling. Tested on the queen and it works!
Can you identify all the cheap main parts of what they are? Let me know if you need any hints.
My next step is to hook up the co2 tubing to complete the entire set up.


El cheapo II parts:


----------



## jcase

beepro said:


> Hi, All!
> 
> 
> This is my most ghetto, el cheapo II station I just finish assembling. Tested on the queen and it works!
> Can you identify all the cheap main parts of what they are? Let me know if you need any hints.
> My next step is to hook up the co2 tubing to complete the entire set up.
> 
> 
> El cheapo II parts:


Check the temp on the light of that microscope, run it for 20minute and see if anything is getting warm. One of mine gets too warm.


----------



## beepro

I'm never seen the warm LED lights before. This one has all cool lights though I don't use
them. I have 2 miner's LED lights that I use. One is a wear on and the 4 phase one is stationary on top of
a can of CA olive shining on the II queen tube. Very sharp focus too. 


Close enough pics:.


----------



## Oldtimer

Nice work Beepro although have to say it is *very* ghetto. With my shaky hands I really need to adjust the sensitive parts by turning a knob rather than pushing them with my fingers. But hey, if it works for you, well done!


----------



## beepro

All the initial trial run are good. Except now that I have to try on a live subject tomorrow.
Keeping my fingers crossed!


----------



## beepro

After 2 week of preparation to gather all the needed II materials, I finally got my 
first II Cordovan queen today. Now that I got my first experience in using this ghetto II station there will be
many more queens to come. Harvesting semen is the hardest part of them all. Next time I will
time the drones maturity better. Smell like a drone too. One of the drones squirted on my face. Luckily I got my eyeglasses on. 
Still I'm not used to the smell not sure why the girls liked it. Not sure if it is normal or not for some white semen to come out on the other end afterward? I should know the laying result within 5 day. Also took out the QE for her to go open mating as it is 17 day passed already . Curious to know what drones are out there now. One more virgin to go next week and 3 more QCs on the way. I'll wear a mask this time!


1st II queen done:


----------



## Oldtimer

Not sure with blurry pic but looks like you got the semen in the wrong place.


----------



## jcase

beepro said:


> After 2 week of preparation to gather all the needed II materials, I finally got my
> first II Cordovan queen today. Now that I got my first experience in using this ghetto II station there will be
> many more queens to come. Harvesting semen is the hardest part of them all. Next time I will
> time the drones maturity better. Smell like a drone too. One of the drones squirted on my face. Luckily I got my eyeglasses on.
> Still I'm not used to the smell not sure why the girls liked it. Not sure if it is normal or not for some white semen to come out on the other end afterward? I should know the laying result within 5 day. Also took out the QE for her to go open mating as it is 17 day passed already . Curious to know what drones are out there now. One more virgin to go next week and 3 more QCs on the way. I'll wear a mask this time!
> 
> 
> 1st II queen done:


You should wait 3 days and inspect her spermatheca, if it is clear then she didnt absorb any, if cloudy then it was a partial success, if really milky then she is sell inseminated.


----------



## beepro

I have 2 more virgins waiting for II today. 
I don't think this one is fully inseminated though. That is why she's allow to go on her
mating flights. Will do a better job next time. On you tube vids I didn't see any semen coming
out after II. This is way harder than I thought.


----------



## beepro

Oldtimer said:


> Not sure with blurry pic but looks like you got the semen in the wrong place.


Nope, not in the wrong place just too much put in I guess. This will not cause an harm to the queen.

This II queen got eggs today! Saw her laying in the cell along with other eggs already laid. I'm very happy. 
She emerged from the cell on 8/11/17. Got II on 9/1/17 which is exactly
21 day within her window of mating opportunity. Today I saw eggs 5 day after her II done. Looks like she's been laying on the last 2 day or so by the eggs deposit pattern. If the math is correct then she can lay on the 3rd day after II. She's a bit small because I haven't been feeding the hive any syrup or honey water. Very interesting for a desperate queen to lay as her hive is shrinking in number. Should know if these are the worker eggs after one week. If not then I still have other II queens waiting to lay.
My first try on the 1st Cordovan queen using el cheapo ghetto II station. And will do II again if she ran out of juice next Spring for an experiment. I will keep her as a breeder after my last expensive Cordovan breeder got squished under my foot on a hive check last time. Now I don't have to buy another breeder queen. You think it will work on the 2nd II try? 
Went out and mark all the remaining drones green for other virgins to II next week. This time I will use the gentamicin since I got it yesterday. For this II queen I only use the saline solution without the gentamicin shortly after the semen harvest. I have more confidence now that even with the white semen squirting out (put in too much?), she can still lay. What is the chance? Lucky shot I guess!

I could not have done it without the many Bee Source members support and encouragement, especially on the many hits and ongoing PMs support in making my first II station a success. OT likes his operation smooth because of the shaky hand. I will try to source local materials next time to see if it can be done on my next improved design. I'll call it El Cheapo II-2 version. I'm sure they are out there only with more time I will find it. If it is not at the dollar store then it will be at the local flea markets. Maybe a 3-D printer can help me with a set too. Quite a learning experience so far. Thanks much, folks!


1st II laying queen:


----------



## jcase

beepro said:


> Nope, not in the wrong place just too much put in I guess. This will not cause an harm to the queen.
> 
> This II queen got eggs today! Saw her laying in the cell along with other eggs already laid. I'm very happy.
> She emerged from the cell on 8/11/17. Got II on 9/1/17 which is exactly
> 21 day within her window of mating opportunity. Today I saw eggs 5 day after her II done. Looks like she's been laying on the last 2 day or so by the eggs deposit pattern. If the math is correct then she can lay on the 3rd day after II. She's a bit small because I haven't been feeding the hive any syrup or honey water. Very interesting for a desperate queen to lay as her hive is shrinking in number. Should know if these are the worker eggs after one week. If not then I still have other II queens waiting to lay.
> My first try on the 1st Cordovan queen using el cheapo ghetto II station. And will do II again if she ran out of juice next Spring for an experiment. I will keep her as a breeder after my last expensive Cordovan breeder got squished under my foot on a hive check last time. Now I don't have to buy another breeder queen. You think it will work on the 2nd II try?
> Went out and mark all the remaining drones green for other virgins to II next week. This time I will use the gentamicin since I got it yesterday. For this II queen I only use the saline solution without the gentamicin shortly after the semen harvest. I have more confidence now that even with the white semen squirting out (put in too much?), she can still lay. What is the chance? Lucky shot I guess!
> 
> I could not have done it without the many Bee Source members support and encouragement, especially on the many hits and ongoing PMs support in making my first II station a success. OT likes his operation smooth because of the shaky hand. I will try to source local materials next time to see if it can be done on my next improved design. I'll call it El Cheapo II-2 version. I'm sure they are out there only with more time I will find it. If it is not at the dollar store then it will be at the local flea markets. Maybe a 3-D printer can help me with a set too. Quite a learning experience so far. Thanks much, folks!
> 
> 
> 1st II laying queen:


Congrats, no II after she starts laying won't work.

Watch out for queen cells, they often make them with ii queens, her pheromones sometimes take a minute to kick in fully


----------



## beepro

Dups!


----------



## beepro

Dups!


----------



## beepro

Thanks, I will keep that in mind and will be looking for the QCs. More practice for me. So far a very nice solid laying pattern.
Now that I have an initial success, I'm trying it on the 2nd queen the second time today.
She's 2 week old and more yellow than the first sister queen. Another queen is waiting for II that emerged on 9/1.
Keeping it going with more virgins to practice on. Each time improving the process with better skills and learning experience. Getting better at semen extraction now. All drones on CO2 going asleep. Hurry up to get through that learning curve, eh. 



Another II queen today:


----------



## jcase

beepro said:


> Thanks, I will keep that in mind and will be looking for the QCs. More practice for me. So far a very nice solid laying pattern.
> Now that I have an initial success, I'm trying it on the 2nd queen the second time today.
> She's 2 week old and more yellow than the first sister queen. Another queen is waiting for II that emerged on 9/1.
> Keeping it going with more virgins to practice on. Each time improving the process with better skills and learning experience. Getting better at semen extraction now. All drones on CO2 going asleep. Hurry up to get through that learning curve, eh.
> 
> 
> 
> Another II queen today:


Can you post pictures of your syringe setup and your tip messurements


----------



## beepro

My 7.1 MP digi cam cannot pick up the capillary tip pic because it is too small. So cannot give you a measurement of it. I don't even have a caliper device to use. I just follow the you tube vids to make the tips as close as I can. To ensure a more successful II, I am doing a 2nd time on the same queen.
For the syringe setup I have a mounting bracket that is movable at 360 degree. The entire syringe is tug inside a
white foam with a channel in the middle. I use wax to sealed the cap. tube inside the syringe for an air tight fit. The
entire cap. tube is fit inside the syringe through a clear plastic syringe cap all filled with wax inside so the syringe tip cannot move at all. All the connection is air tight and very secure otherwise it cannot suck up the solution and semen. Still ghetto but works for me so far.


Setup and the syringe connection:


----------



## AstroBee

Congrats on making this work. One thing you might consider is getting or making better tips. This one in your photo is WAY shorter and more blunt than those typically used. I just compared my tips (from Cobey and Schley) to your picture and there appears to be a big difference. Again, the ones I use have a very long "narrow" section of nearly constant diameter. Perhaps this explains the excess semen after your procedure?


----------



## jcase

AstroBee said:


> Congrats on making this work. One thing you might consider is getting or making better tips. This one in your photo is WAY shorter and more blunt than those typically used. I just compared my tips (from Cobey and Schley) to your picture and there appears to be a big difference. Again, the ones I use have a very long "narrow" section of nearly constant diameter. Perhaps this explains the excess semen after your procedure?


Which do you prefer, the cobey or schley? The ones I got from Sue are more consistant than the ones from I got with my schley unit. The schley ones are not perfectly straight.

On that note, I finally have my tip puller mock up working, waiting for machinist to build the real one for me now. Ended up using 24g nichrome wire, you can get it working with a much lower end power supply that the gauge most people are recommending. My glass sandpaper just arrived yesterday, so I'll probably give it a go at polishing and finishing tips sometime this month.


----------



## FlowerPlanter

:thumbsup:


----------



## beepro

Thanks for the feedback. I've since change the tip to a narrower and longer tube.
Without this change the semen cannot go in because the previous tip is too blunt.
Sorry about the blurry vids. Something to time the co2 with. Might not be interesting to
you but to the new beekeeper like me it is.
https://youtu.be/QifBBNon6hs
https://youtu.be/FyfA_rgCU_o
https://youtu.be/WZ5bTN2_sow
https://youtu.be/nEpSd4ytRzI


----------



## jcase

beepro said:


> Thanks for the feedback. I've since change the tip to a narrower and longer tube.
> Without this change the semen cannot go in because the previous tip is too blunt.
> Sorry about the blurry vids. Something to time the co2 with. Might not be interesting to
> you but to the new beekeeper like me it is.
> https://youtu.be/QifBBNon6hs
> https://youtu.be/FyfA_rgCU_o
> https://youtu.be/WZ5bTN2_sow
> https://youtu.be/nEpSd4ytRzI


frame rate on that usb microscope is horrid. Really should pick up an amscope (~$180) if you plan to do this more than a couple times


----------



## Oldtimer

The videos show her whole abdomen outside of the housing, the end of the housing should be a little smaller and keep most of her abdomen inside it, because her breathing tubes are in the abdomen and you want her breathing the Co2 inside the housing.

The bubble rate for the Co2 is too high, although I wondered if that was intentional because of the issue with the queens abdomen not being in the housing.

There's a few issues, but as far as a ghetto inseminator built for almost nothing, you have done well and I'm sure will gradually mess with and improve it.


----------



## JRG13

My only question is how do you know II was successful, you said you were going to let her go on a mating flight or she wasn't restricted from doing so after II.


----------



## beepro

Good observation, OT. Yes, it is intentional on high CO2 otherwise she's breathing too much air. That does not
affect her waking up after the II procedure. The you tube vid said it does not affect her if OD on the CO2. Put her in with the nurse bees to take care of her right away. I took some short cuts and did not use any attendant bees through out the entire short process. The CO2 is cheap here at $5 dollars for a 5 lbs. cylinder that I cannot even use it that much with only a few II queens. The sales clerk said I have the option to upgrade to 10 lbs. too. Very easy to source it here. 
Learning more facts on you tube vids. An II queen can lay up to 3 year under normal condition. A well taken breeder can lay up to 4 year inside a nuc hive. Already I'm improving the process and doing a little II experiment on the virgin queens. I did 2 consecutive days of II then skip a day. And resume II on the 4th day. If left alone she will be laying on the 5th day. Do you know what I'm trying to accomplish here? By then this queen should be full of semen to last her a life time. If they run out of juice then I will put them inside a booming hive on drone frames over a QE to collect the Cordovan drones. Doesn't matter to me either way. But first to do another II to see if she can lay the normal workers. This I have not try and very curious as j.case said it cannot be done. I can keep on experimenting on the II for as long as there are drones available through the winter. They will make queens when you make them queen less even in Dec. I have 2 normal golden color and one orange/peach color queen to experiment on.


----------



## jcase

beepro said:


> Good observation, OT. Yes, it is intentional on high CO2 otherwise she's breathing too much air. That does not
> affect her waking up after the II procedure. The you tube vid said it does not affect her if OD on the CO2. Put her in with the nurse bees to take care of her right away. I took some short cuts and did not use any attendant bees through out the entire short process. The CO2 is cheap here at $5 dollars for a 5 lbs. cylinder that I cannot even use it that much with only a few II queens. The sales clerk said I have the option to upgrade to 10 lbs. too. Very easy to source it here.
> Learning more facts on you tube vids. An II queen can lay up to 3 year under normal condition. A well taken breeder can lay up to 4 year inside a nuc hive. Already I'm improving the process and doing a little II experiment on the virgin queens. I did 2 consecutive days of II then skip a day. And resume II on the 4th day. If left alone she will be laying on the 5th day. Do you know what I'm trying to accomplish here? By then this queen should be full of semen to last her a life time. If they run out of juice then I will put them inside a booming hive on drone frames over a QE to collect the Cordovan drones. Doesn't matter to me either way. But first to do another II to see if she can lay the normal workers. This I have not try and very curious as j.case said it cannot be done. I can keep on experimenting on the II for as long as there are drones available through the winter. They will make queens when you make them queen less even in Dec. I have 2 normal golden color and one orange/peach color queen to experiment on.


CO2 OD will kill her for sure. I've put down animals with co2 before (mice).

I dont think you need to insemiante her multiple times. I certainly wouldnt inseminate her in a period of time greater than 48hours.


----------



## beepro

Thanks, because her abdomen is rather long extending out, there will be a mixture of oxygen along
with the CO2. Maybe this is why she cannot be on OD yet? I can see that after the 2nd II her abdomen is
2x larger than normal on the 2nd day. I know I'm pushing it a bit with this experiment. This is the only way to find out what is the max and the min of it. Other than the bigger abdomen all queens are are still alive and accepted by the bees.


----------



## AstroBee

jcase said:


> Which do you prefer, the cobey or schley? The ones I got from Sue are more consistant than the ones from I got with my schley unit. The schley ones are not perfectly straight.
> 
> On that note, I finally have my tip puller mock up working, waiting for machinist to build the real one for me now. Ended up using 24g nichrome wire, you can get it working with a much lower end power supply that the gauge most people are recommending. My glass sandpaper just arrived yesterday, so I'll probably give it a go at polishing and finishing tips sometime this month.


I don't have a strong preference either way. The ones from Sue were purchased when I took her class and seem just fine. I bought these in addition to the ones that came with the Schley unit. The other Schley tips I have came with the "Harbo-like" syringe that he sells on his website. These tips are much shorter than those that come with the Schley unit that Sue resells.


----------



## beepro

Thanks, as long as they work then it doesn't matter that much. I use a similar one from the
you tube vid that I made. Mine is not perfect either with a slightly narrower tip and longer body. I know when to
stop without ending up on the other side of her abdomen though. It is a very delicate process indeed!


One from you tube:


----------



## beepro

I tried some extra CO2 on the drones waiting for II. They all got frozen given too much of it.
Without the outside air mixed in they will turn to popsicle inside a small plastic bag. You are correct. Excessive CO2 will
freeze the queen while she's waiting to be II or is already inside the II tube. Some of these you tube vids. have misleading
information too. So nothing beats the real hands on experience on the entire II process. I have to better regulate the
amount given to the queen next time. Don't want a frozen queen afterward! Thanks to OT for his tips and keen observation.
Hoping that after this last time of II she can wake up and make it through. Three time is just too much for her. I will never do it again as 2 time is definitely enough. Learning my lesson along the way. I have another one going through the CO2 1st time tonight. Will be II-ing on her tomorrow night. Then I will have 3 breeder queens until next Spring time to resume this process. I intend to do more than a few of them when queen rearing time starts again. 
The first II queen is getting fat and laying well. When she is laying, the big fat young nurse bees are all over her to the point of suffocation. These are some of the strongest most prolific queens I've ever seen this late in the season. The extra high protein homemade patty subs not only carry them through our summer dearth but also added some of the fattest QCs ever made. Resulted in some of the hardiest Cordovan queens ever. I'm glad that this II station got done in time otherwise these queens would be wasted on the local carnis drones. Now I have a gadget against these local carnis drones out there. Is this the only way to control the bees genetics in absent of an isolated station? I've also swap the hive position with the most booming hive inside the metal tent today. This will boost up the hive population a bit to better take care of her and the broods. I should know whether or not this is a successful II when I see the cap worker broods in a week. I'm improving on the method and gaining new skills set along the way. All is going well so far!



1st big fat queen is laying:


----------



## beepro

This little queen experiment is a total failure. The 2nd II queen died sometime last night. Got thrown out
of the hive today. Now I know what not to do next time. Either 1 or 2 time II and that's it. Whoever want to
repeat this failed experiment can do so. I for one will not do it again. Maybe the day to laying is too close or there was
too much CO2 administered this time. Either way I'm still trying to refine my II process with better improvement the
next time. Got a narrower pull cap. tube to use this time. The 3rd queen is waiting for II later today.


----------



## jcase

beepro said:


> This little queen experiment is a total failure. The 2nd II queen died sometime last night. Got thrown out
> of the hive today. Now I know what not to do next time. Either 1 or 2 time II and that's it. Whoever want to
> repeat this failed experiment can do so. I for one will not do it again. Maybe the day to laying is too close or there was
> too much CO2 administered this time. Either way I'm still trying to refine my II process with better improvement the
> next time. Got a narrower pull cap. tube to use this time. The 3rd queen is waiting for II later today.


You are wrong, it isn't a failure, its called practice and experimentation. You are doing fine. Your queens are only going to store 1ul or so of semen, so a single insemination of 8-12ul is fine. I'd double check your syringe tip. How are you polishing the glass? What kind of end are you putting on the tip? What is your buffer solution makeup?


----------



## beepro

I have gentamicine and saline solution to use 50/50. The tip is small enough to go inside the queen. I have very fine sandpaper and use flame torch to refine the tip down. It is not the tip or solution issue. It is more of my angle to deliver the semen. I'm going to try the 90 degree angle to see if it is better than the 45 degree I've been using. This will be the last queen that I can practice on until next time again. It is a good learning and practice lesson either way. You mean for her entire laying life she can only use 1 ul? That is hard to believe.


----------



## jcase

beepro said:


> I have gentamicine and saline solution to use 50/50. The tip is small enough to go inside the queen. I have very fine sandpaper and use flame torch to refine the tip down. It is not the tip or solution issue. It is more of my angle to deliver the semen. I'm going to try the 90 degree angle to see if it is better than the 45 degree I've been using. This will be the last queen that I can practice on until next time again. It is a good learning and practice lesson either way. You mean for her entire laying life she can only use 1 ul? That is hard to believe.


50% gentamicin / saline solution, that cant be right, that would be VERY Expensive, and would kill 100% of the semen. What is the specific gentamicin solution you are using?

Yes she will only store about 1ul in her spermatheca, for her entire life.


----------



## beepro

I meant a mixture of 50/50 gentamicin/saline solution. Keep in mind I'm only using it for one queen at a time. So a
few drops are enough to fill up the small syringe. All I know is the package said gentamicine for dogs eye wash. They are not that expensive compare to the Tigent antibiotic I bought earlier but cannot use. Going to donate this bottle to the local vet.
So I bought 4 small 30 ml bottles and the 100 pack saline disposable single use solution. If the gentamicin does not work then I can just skip it. Using the saline solution is fine too.


----------



## jcase

Right but a 50% strength solution of saline would be crazy strong. You want something like 100ml of 0.9 saline to 0.25g of gentamicin. Saying 50/50 doesnt mean anything, as we dont know what your initial gentamicin solition was. You very well could be using a too strong solution, depending on what that starting solution was.



beepro said:


> I meant a mixture of 50/50 gentamicin/saline solution. Keep in mind I'm only using it for one queen at a time. So a
> few drops are enough to fill up the small syringe. All I know is the package said gentamicine for dogs eye wash. They are not that expensive compare to the Tigent antibiotic I bought earlier but cannot use. Going to donate this bottle to the local vet.
> So I bought 4 small 30 ml bottles and the 100 pack saline disposable single use solution. If the gentamicin does not work then I can just skip it. Using the saline solution is fine too.


----------



## beepro

I have never thought of it that way. Maybe 1 drop of gentamicin into the .9 saline solution bottle should be fine then. The package did not say what is the strength of the solution. I also found out that on the 1st queen I did not use the 
antibiotic to II her. She's still laying now. No sign of any supersedure cell so far. Now I'm learning more.


----------



## jcase

beepro said:


> I have never thought of it that way. Maybe 1 drop of gentamicin into the .9 saline solution bottle should be fine then. The package did not say what is the strength of the solution. I also found out that on the 1st queen I did not use the
> antiobiotic to II her. She's still laying now. No sign of any supersedure cell so far. Now I'm learning more.


If you cant get the dosage right, you are better off with none. Antibiotics need to be near spot on, too much is bad, too little is worthless or actually could be bad.


----------



## JRG13

beepro said:


> This little queen experiment is a total failure. The 2nd II queen died sometime last night. Got thrown out
> of the hive today. Now I know what not to do next time. Either 1 or 2 time II and that's it. Whoever want to
> repeat this failed experiment can do so. I for one will not do it again. Maybe the day to laying is too close or there was
> too much CO2 administered this time. Either way I'm still trying to refine my II process with better improvement the
> next time. Got a narrower pull cap. tube to use this time. The 3rd queen is waiting for II later today.


How many times did you II this one?


----------



## beepro

Will leave off the gentamicin until I can get the dosage right. The 1st II queen without antibiotic is still laying. The
2nd one died last night after the 3rd time. The 3rd virgin will be II tonight without the antibiotic depending on how it goes.


----------



## teplov

beepro said:


> I'm going to try the 90 degree angle to see if it is better than the 45 degree I've been using. .


Angle of the capillary relative to the holder for the queen.


----------



## AstroBee

beepro said:


> Now I know what not to do next time. Either 1 or 2 time II and that's it. Whoever want to
> repeat this failed experiment can do so. I for one will not do it again.



Not quite sure what you're saying here. 

For the CO2, test the flow rate by placing the tube in a glass of water, that way you can observe the flow rate. Without looking back at my notes, a bubble per second is roughly about right. I've never been to precise on this rate, but I've never chilled a queen either. If you're chilling queens, then I suspect that your flow rate is way too high.

Don't expect perfection right out of the box, particularly using non-standard equipment. It may take lots of queens before you become proficient with the procedure. At Sue's class, they provided a LOT of practice queens with ideal lab conditions too, and still I had many failures.


----------



## jcase

AstroBee said:


> Not quite sure what you're saying here.
> 
> For the CO2, test the flow rate by placing the tube in a glass of water, that way you can observe the flow rate. Without looking back at my notes, a bubble per second is roughly about right. I've never been to precise on this rate, but I've never chilled a queen either. If you're chilling queens, then I suspect that your flow rate is way too high.
> 
> Don't expect perfection right out of the box, particularly using non-standard equipment. It may take lots of queens before you become proficient with the procedure. At Sue's class, they provided a LOT of practice queens with ideal lab conditions too, and still I had many failures.


1 to 2 bubble second is correct, i think i run a little higher than most at 2p/s, but I want her out fast as I want to get in and out fast.

Same thing at Sue's class, I did rougly 20-30. Only the last one did we wait any period of time to check spermatheca, rest we only waited a day or so to check them. Last one came out great.


----------



## AstroBee

beepro said:


> Will leave off the gentamicin until I can get the dosage right. The 1st II queen without antibiotic is still laying. The
> 2nd one died last night after the 3rd time. The 3rd virgin will be II tonight without the antibiotic depending on how it goes.


You may want to consider leaving the antibiotic out permanently. I used it initially, but dropped it after the first year and saw no difference in the results. I achieved multi-year survival with excellent laying up until the end. Keep your conditions as sterile as possible and liberal use of saline. Clean the tips and syringe very well after use.


----------



## beepro

Thanks all, for all the good encouragement. I have so many pics to document the entire II process including a complete revamping of my station set up. Every thing got elevated to see the queen better. This will make the II much more smoother when lowering the syringe. Ergonomic is very important considering I can sit there for hours on the entire process. Leaning and refining my process down. Cannot do it free style without suspending the syringe because of my shaky hand when nervous approaching the II site. Imagine jabbing extra holes on the queen when not needed. 
Since combining the 2 nuc hives together, the 1st queen is laying strong. Seems like every time I inspected the hive she is there laying right in front of me. The genetics is so stable that they are the most calm and gentle bees around. Definitely showing very good sign of the allogrooming activity in and outside of the hive. With the 4 expensive bought queens all dead only with II will continue the Cordovan lineage. Any mixing with the local carnis drones the genetics will be diluted. 
Did the 2nd round of II on the second queen tonight. After the first round last night, she's noticeably more plump today. Found her bury under a pile of bees trying to protect her. At 11 day post emergence, they already recognized her as the queen. This time I only use 14 drones instead of 30 to extract 20 ul from them and have almost 9 ul left over after the II. This queen only use 12 ul or so which is more than enough to satisfy her. I'm still using the 90 degree angle to do the II. Maybe one day I will experiment on the 45 degree when getting more proficient at it with improved II equipment too. Found out that an II virgin will not likely to leave for her mating flights when she's all filled up with more than the recommended 10 ul. There is no time for her to explore her surrounding with a QE on and the consecutive days of II on her at the right time. That is why neither of the 2 queens go on their mating flights afterward even though the QE was off after the 1st II. Because this experiment is such a success, I decided to use 20 ul from now on because not all will go in as seen from the pics. There is no harm to the queen as the extra will just leak out. At least some will go in to fill up the spermatheca. Also spent an hour or so to make 2 cap. tubes after I broke the first one on accident yesterday while cleaning it. These are very fine pointed and fragile glass tubes. The one I made only 2 out of 17 tube was a successful pull with a very fine point at the end and enough to take in 30 ul at one time. With 2 tubes of 400 total I still have plenty to work on. Seems like a longer tube is easier to work with. This is the end of my II session unless the new queen will lay me some drone eggs later on. Put in 2 drone comb frame in there hoping that she will lay some. If she does then my II experiment will continue and with some improvement too. 




Are these queen pics look familiar to you?:


----------



## beepro

More pics! Intro. the queen back after the II:


----------



## AstroBee

beepro said:


> Did the 2nd round of II on the second queen tonight. After the first round last night, she's noticeably more plump today.


Are you inseminating twice on two separate days? If so, you should probably drop back to a single insemination. I perform a single insemination, but with two CO2 doses on consecutive days. During the first CO2 treatment, she gets a wind clipped while under (you should seriously consider doing this too), and the following day she is inseminated with 10 - 12 ul and if all goes well gets a numbered disc (you should definitely at least mark your II queens). Clipping is to prevent her from flying and mating. The whole point in doing II is controlling the crosses, and you insure this by preventing the flights. Sue Cobey told us that a single insemination is just as good as two separate inseminations. In fact, one may ultimately prove more efficient since there will be less possibility of failure because of the fewer times she's handled. 

Also, the vertical technique seems to hyper-extend her abdomen (at least from your pictures). You can do that with the 45 deg setup too, but probably less likely to happen. The motion you're trying to achieve is not all axial extension, but some axial and some transverse. Regardless, VERY little extension (in either direction) is needed. It is mostly a stabilization (with some small extension) of the abdomen that you're trying to achieve.


----------



## beepro

Yes, the first day on CO2 then follow by 2 consecutive day of II on the same queen. The reason being my aim is a bit
off thinking that there isn't much done on the first round. The first queen was just a lucky shot now that she's laying. And not clipping her wing to allow her on a mating flight is to test out what drones are out there at this time. Also to see if she will go on her flights and if successful, what are the workers look like at each hatch. Are they a mixture of the Cordovan and carnis? Or they are the LIFO or FIFO of workers since someone claimed that they are a mixture of both. I think they are the LIFO of workers though which will be the carnis. But so far it seems that I've over saturated the queen not enabling her to go on her mating flights. That is o.k. since the Cordovan genetics have been preserve as well. Either way it is fine with me. If she lay the carnis then it will be a production queen the next Spring. If not then will pick her as the breeder queen. 
What is more interesting is that approaching our mild winter time my hives can still make the virgin queens. If I can somehow make the queen lay the drones to overwinter then I can still continue with this process. Some small nuc hives will overwinter their drones as I have seen before. This may turn out to be a year round II adventure. There are so many variable I have not test yet. Once the perfect II angle has been achieved and bypassing the valve fold is not an issue anymore, I will go back to a single dose with 20 ul. Almost one month into this process and I'm still learning and experimenting. So far I know what cap. tube tip size to make. This cannot be done by asking question alone as it is a hands on operation. Knowledge will only take you so far that you still have to do it in order to learn.



1st queen is still plump:


----------



## beepro

Just a quick hive check tonight. Saw the recent II queen hiding among the bee cluster again. There are still many mature
Cordovan drones left. What a waste. She's acting like a mated queen already looking into the empty cells like a real mated queen does. So maybe that my II did work the first time. But her abdomen is still too small to tell. Like the first II queen her abdomen will not swell up until she is laying for a few days. Then she will be more plump when others are feeding her more RJ. While this queen is roaming on the empty cells I 
did a combine with a weaker nuc hive that is queen less now. This should give them an instant population boost if they
liked this queen. I will know for sure in another 5 day if this II is a successful one by 9/18. With II I'm able to cut down her
laying time by almost 2 week instead of the normal on month time frame. Not that bad either. So you think I did it right this time?



Still a virgin queen?:


----------



## beepro

Hi, All!


So I did a hive inspection today. Found some cap worker broods from the first II queen. It is a good sign! Have
confidence that these are the Cordovan workers since the queen never take her mating flights even though the QE is
off after the 1st day. I inspected the nuc hive every night for the entire duration of the II process. This queen is the light yellow Cordovan color that I'm looking to propagate. Also found her roaming on the drone frame right in the middle of the brood nest that I put in last week. Upon inspection into the drone cells I found some eggs scattered through out the frame. Don't expect perfection the first time around. And saw the queen laid in one of the drone cells too. It is my first time seeing a queen tucking her head all the way down into a drone cell inspecting it before depositing her egg. So I took out my cam and snap a few pics.
Since we have the drone eggs, if the weather holds then in mid-October I can do another round of the II process with more graft virgin queens either from the first queen or from the 3rd II queen if she lays. We also have the half-carnis/half Italian queen mated with the local carnis drones to experiment on. This time I will try the 45 degree on the II station to see if there can be better insemination improvement. Pushing the queen rearing season to the max, do you think it is possible to make II queens all the way to December?


----------



## beepro

A quick update:


I'm very happy today! Finally on the 5th day of waiting, seems like forever, I saw an egg laid by the 3rd II queen this afternoon. So I gave them a pint jar of honey water. Last time I re-II the 2nd queen on the 4th days causing her death. This time around I learn that being patience enough is everything. I did check the nuc hive a few times but cannot find any eggs in the last few days. So like the 1st queen I have to wait 5 days in order to see some eggs after the II process. During this time it is better to wait and check for sign of any mating flights she might be doing. Of the 5 days that I look after this queen she did not go to her mating flights retaining all the II value of the Cordovan genetics. So even though the II process doesn't go as smooth as I like it to be with 20 ul of semen used and some leaked out, it is still a successful one on both occasion. Might have been the 3rd time if the 2nd queen did not died. This means that now I have 2 breeder queens for next year if everything holds through this winter. Interesting how a virgin behaved after the first II done.
The II station have some improvement now with an adjustable know to control the lowering of the II syringe at 90 degree still. There are still many ways to improve on this station which I will adjust accordingly.


----------



## Oldtimer

It's actually pretty amazing the transformation a queens body goes through after being inseminated. For her mating flight she is trimmed down to a lean, fit, flying machine. Once she is inseminated there is a massive transformation in her abdomen with oviaries plumping up and getting ready for the job of turning food into around in season, one thousand and five hundred eggs daily. That her body can make this transformation in just 5 days is a remarkable thing in itself.


----------



## beepro

JRG13 said:


> My only question is how do you know II was successful, you said you were going to let her go on a mating flight or she wasn't restricted from doing so after II.


Today is the bees emergence day on SC (small cells) from the 1st II breeder queen. Finally I have waited for that long now.
Yep, all the newly emerged worker bees are the Cordovan color, alright.
So when I over II the queen she was too fat to make her next mating flight. No
choice but to stay in until after the 3-5 days before laying. In order for a queen 
to take her first mating flight, she has to explored her outside hive surrounding first like a mini orientation flight. These queens never got that chance to explore theirs when confined inside a QE from emergence until II time. Anyways, using that much II will bogged down the queen making her unwillingly to take her mating flight anymore. The tiny bits of spermatheca stored sperm after the II, when successful, is enough to make her full and stay in to lay. I'm sure she knows when it is full just like a pregnant woman. This was a test to see if after II the queen will fly to mate or not. Now I know after this little bee experiment. It is that easy!

Saw lots of cap drones today too. Enough on a frame for the next test of II queens, same color. Need to get the
timing right for making the QCs this time.



Same yellow emerged workers:


----------



## JRG13

Looks ok, but I hope you realize you're kind of inbreeding them very rapidly with your limited donor pool of queens and drones?


----------



## beepro

For now it is. I'm starting to source compatible bees for the II process. Can also open mate the daughters with the
local carnis and cross back to the Cordovan drones again via II. Reading said that after the 2nd generation the
bees will revert back to the Cordovan color. As long as I have a Cordovan breeder everything
should be fine. After a few generation, the Cordovan genetics will be back to normal again. My experience is 4 generation to revert back. Not sure about the Cordovan until I have try them out. So it will be II to maintain the Cordovan line and a mix for the production queens with open mating. I'm sure 100 II Cordovan breeders will maintain the genetics here somewhat. I'll be spreading the little nuc hives through out the area to ensure the bees are there.


----------



## beepro

Hi, All!

Update: II a DL Cordovan queen today.


In many aspect of beekeeping many will tell me that it cannot be done. Or it might
not work using my way just because they tend to follow the norm. I like to experiment a little after
being more proficient at beekeeping. So today I re-II a DL (drone layer) Cordovan queen for the
first time. So they said that it cannot be done either.

After a little improvement made on my II station, I figured that it was time to give it a little trial run. Now the II
station has smoother syringe adjustment lever and better II hooks. I was a bit afraid that the queen might die when she's a laying queen already even though they are the drones. Maybe this was the reason that other beekeepers said it cannot be done? I know for a fact that when I was keeping tropical fish when a different color male mated with the laying female, the result is a different color offspring from the most recent male. So why can't a DL queen be II again assuming that her spermatheca has not been full of semen yet? If this queen is still alive after 48 hours then
this will be a successful re-II done. Of course, she has to lay the normal worker eggs in order to count. If not then I will devise a better method of re-II another DL queen in the future. This way a good laying queen is not wasted-- squish! I can see that her abdomen is a lot larger (ballooned) right after the re-II process. If it cannot be done, why not? 



Cordovan queen re-II:


----------



## beekuk

beepro said:


> If it cannot be done, why not?


No reason, as it can be done.


----------



## beepro

So can I re-do the II the 2nd time after the 48 hours?
She's not laying now since her abdomen is till fat up. Maybe she needed more time to
prepare the semen to migrate to the spermatheca?


----------



## beepro

beekuk said:


> No reason, as it can be done.


Oh well, this re-II of the DL Cordovan queen had failed miserably. Yesterday supposed
to be the 48 hours time frame but I did not see the queen in there. Check again today and
she's not in there either. So I did a combine with another laying queen, a Fat Bee Man's queen.
Let's see how an east coast bee can survive on our dry and hot summer west coast condition.

Not sure why the queen died. Might be that while doing the shut down laying period her 
body chemistry changed again therefore the workers cannot recognized her and started the
balling process. Anyways, next time I will caged the queen after the re-II to see it past
the 48 hours. The good news is that there isn't a mite inside that I can find. With a 
low mite level like that now I'm sure the coming Spring build up will not be affected. Let's
see if this tf queen will hold too.


----------



## beepro

beekuk said:


> No reason, as it can be done.


Oh well, this re-II of the DL Cordovan queen had failed miserably. Yesterday supposed
to be the 48 hours time frame but I did not see the queen in there. Check again today and
she's not in there either. So I did a combine with another laying queen, a Fat Bee Man's queen.
Let's see how an east coast bee can survive on our dry and hot summer west coast condition.

Not sure why the queen died. Might be that while doing the shut down laying period her 
body chemistry changed again therefore the workers cannot recognized her and started the
balling process. Anyways, next time I will caged the queen after the re-II to see it past
the 48 hours. The good news is that there isn't a mite inside that I can find. With a 
low mite level like that now I'm sure the coming Spring build up will not be affected. Let's
see if this tf queen will hold too.


----------



## jcase

beepro said:


> Oh well, this re-II of the DL Cordovan queen had failed miserably. Yesterday supposed
> to be the 48 hours time frame but I did not see the queen in there. Check again today and
> she's not in there either. So I did a combine with another laying queen, a Fat Bee Man's queen.
> Let's see how an east coast bee can survive on our dry and hot summer west coast condition.
> 
> Not sure why the queen died. Might be that while doing the shut down laying period her
> body chemistry changed again therefore the workers cannot recognized her and started the
> balling process. Anyways, next time I will caged the queen after the re-II to see it past
> the 48 hours. The good news is that there isn't a mite inside that I can find. With a
> low mite level like that now I'm sure the coming Spring build up will not be affected. Let's
> see if this tf queen will hold too.


Died probably due to stress. Virgins are much more able to handle stress. Non virgins can't be reinseminated after the initial mating period. Too much stress, and I doubt her body is even able to asorb more semen at that point.

The workers also probably notice something "wrong" and dispatch her anyhow.


----------



## beepro

Quite possibly that the changed in hormones will confused the workers. There was one day that
she did not lay. Either find a better way to not stress her out and then isolate her afterward or do it
right the first time.


----------



## beekuk

beepro said:


> So can I re-do the II the 2nd time after the 48 hours?
> She's not laying now since her abdomen is till fat up. Maybe she needed more time to
> prepare the semen to migrate to the spermatheca?


 The drone laying queen would need to be caged long enough for her abdomen to of shrunk back to the size of a virgin queen and be free of any eggs.


----------



## beepro

Oh well, this queen already laid the worker eggs and then the drone eggs afterward. I say she is not a
free eggs laying queen anymore. Maybe this was my mistake for not letting her abdomen to shrink down first. Or
it could be that she can never be eggs free anymore. Anyways, let this one to be the future re-II reference then.


----------



## beepro

Oh well, this queen already laid the worker eggs and then the drone eggs afterward. I say she is not a
free eggs laying queen anymore. Maybe this was my mistake for not letting her abdomen to shrink down first. Or
it could be that she can never be eggs free anymore. Anyways, let this one be the future re-II reference then.


----------



## beekuk

Keep at it with your II projects Beepro, practice makes perfect as they say.


----------

