# Instrumental insemination



## JSL (Sep 22, 2007)

HVH,

I inseminate a few queens...

It is time to get your equipment down off of your shelf and start using it! Far too many people spend a lot of money on II equipment and then use it as a book end.

For CO2, a small 2.5 or 5 pound bottle will last you many years, provided you do not forget to close the valve. A simple regulator, approximately $75, and a small twist valve will give you the best control. The CO2 does not need to be medical grade, go to a welding supply company...

I clean every item with off the shelf rubbing alcohol and rinse with distilled water. The glass tips are soaked in a 5% Clorox solution when not in use.

I am not familiar with PBS, but I assume you are referring to saline. A simple NaCl saline solution will work fine for most applications. I like to use a little more complicated saline if shipping or storing honey bee semen. For a simple saline solution use 100ml distilled water and 1g NaCl. It would be best to sterilize the saline.

To collect the highest proportion of mature drones, a queen excluder leaned in front of the hive during drone flight activity works great. The assumption is if the drones are leaving the hive then chances are they are mature enough to mate. Once collected, give the drones a little honey and store them in a queenless bank for a day or two.

The most challenging part of II tends to be semen collection. Most have a difficult time, at first, keeping mucus out of the syringe. With a little practice, it gets a lot easier!

I have some pics on my website latshawapiaries.com that show the II process.

Hope this helps!


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## HVH (Feb 20, 2008)

JSL said:


> HVH,
> 
> I inseminate a few queens...
> 
> It is time to get your equipment down off of your shelf and start using it! Far too many people spend a lot of money on II equipment and then use it as a book end.


 I do feel guilty.



> For CO2, a small 2.5 or 5 pound bottle will last you many years, provided you do not forget to close the valve. A simple regulator, approximately $75, and a small twist valve will give you the best control. The CO2 does not need to be medical grade, go to a welding supply company...


 I have two CO2 tanks and a good regulator but have read a bubble per second is about right. I assume then that some people use a conical flask or similar with stopper and two holes. The CO2 going through tubing and into sterile liquid to count bubbles. The return hole in the stopper would redirect the CO2 to the queen holder. I have not seen this set up but can easily visualize it. Do you use something downstream of you regulator for fine bubble adjustments? Is this your twist valve?



> I clean every item with off the shelf rubbing alcohol and rinse with distilled water. The glass tips are soaked in a 5% Clorox solution when not in use.


 Sounds good. Do you draw out your own glass syringes?



> I am not familiar with PBS, but I assume you are referring to saline. A simple NaCl saline solution will work fine for most applications. I like to use a little more complicated saline if shipping or storing honey bee semen. For a simple saline solution use 100ml distilled water and 1g NaCl. It would be best to sterilize the saline.


So it doesn't need to be buffered (PBS = phosphate buffered saline)? 



> To collect the highest proportion of mature drones, a queen excluder leaned in front of the hive during drone flight activity works great. The assumption is if the drones are leaving the hive then chances are they are mature enough to mate. Once collected, give the drones a little honey and store them in a queenless bank for a day or two.


I tried this one day and the number of drones flying back was impressive but I didn't have nearly as many drones plastered to the excluder as Sue Cobey shows in her video.



> The most challenging part of II tends to be semen collection. Most have a difficult time, at first, keeping mucus out of the syringe. With a little practice, it gets a lot easier!


 What was the most important discovery that helped you avoid the mucus? 

I have some pics on my website latshawapiaries.com that show the II process.

Hope this helps![/QUOTE]

Crap - it seems like I have no excuses.

Thanks a lot for your time.

Chris


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## JSL (Sep 22, 2007)

Chris,

Yes, a small twist valve of sorts will give you that fine control of the CO2. I call them twist valves, some call them needle valves, thumb valves, just something simple that runs $12 or so. A bubble per second is usually a good rule of thumb, but gauge the proper flow by the queen's response. She should go to sleep fairly quickly and become motionless. If she is moving just turn up the CO2 a little. I do not run the CO2 through a wash flask, but you certainly may. For some it helps to see a constant CO2 flow. The queen will tell you if there is no CO2 flowing.

Yes, I make glass tips. It always helps to have a few extras.

PBS, now I remember... As I shared, a very simple NaCl solution works for most applications, but if you have access to a more complicated saline solution, give it a try, just stay away from those that contain preservatives.

Sue puts drone combs in her colonies, which really helps to produce lots of drones. Also good strong colonies in the spring will have lots of drones!

To avoid mucus, I tell people starting out to only work with drones that have plenty of semen floating on the bed of mucus. The tendency for most is to make several attempts to collect all of the semen. I make one attempt and collect what is possible and then move on to the next drone. There are lots of drones, do not spend much time with one that is not productive. After several attempts to collect the semen, the mucus and the semen start to mix, which only makes the problem worse.

Good luck,
Joe


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## HVH (Feb 20, 2008)

JSL said:


> Chris,
> 
> Yes, a small twist valve of sorts will give you that fine control of the CO2. I call them twist valves, some call them needle valves, thumb valves, just something simple that runs $12 or so. A bubble per second is usually a good rule of thumb, but gauge the proper flow by the queen's response. She should go to sleep fairly quickly and become motionless. If she is moving just turn up the CO2 a little. I do not run the CO2 through a wash flask, but you certainly may. For some it helps to see a constant CO2 flow. The queen will tell you if there is no CO2 flowing.
> 
> ...


Thanks Joe - very helpful.


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## RayMarler (Jun 18, 2008)

The pierco green drone comb frames work very well. Using the drone frames will ensure very large nice looking drones. They are plastic, but the bees draw them out very nicely and quickly when you put them in the hives in spring, the natural time for drone raising and swarming season.


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## HVH (Feb 20, 2008)

Putz said:


> The pierco green drone comb frames work very well. Using the drone frames will ensure very large nice looking drones. They are plastic, but the bees draw them out very nicely and quickly when you put them in the hives in spring, the natural time for drone raising and swarming season.


Thanks. I have a lot to prepare for.
Chris


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## adamf (Jan 28, 2006)

*more II nuggets*

Nice topic!

Keep your workplace and equipment extremely clean when you're doing II.
When in doubt, wipe down. Joe's tips on semen collecting are on target--one
can waste way too much time futzzing around with drones: the importance of
having the correct age drones to produce copious quality semen, cannot be
emphasized enough.

CO2 flow depends on the queen--some queens don't go out right away--and
take more CO2 than others--you'll end up adjusting your CO2 flow--be ready
for that.

A pre-II virgin flight to stimulate defecation is helpful--if the virgin
voids in a flight jar, you'll have an easier time with the II. If she voids
as the CO2 hits her (it happens) you'll need to clean up the whole set-up
which takes time and can possibly lead to contamination. We use gallon pickle
jars as per Steve Taber's book: "Breeding Super Bees".

Adam Finkelstein
www.vpqueenbees.com


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## HVH (Feb 20, 2008)

adamf said:


> Nice topic!
> 
> Keep your workplace and equipment extremely clean when you're doing II.
> When in doubt, wipe down. Joe's tips on semen collecting are on target--one
> ...


Thanks. I will dust off Steve's book and read that section.


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## JBJ (Jan 27, 2005)

Don't feel bad HVH, I am in the same boat, I have been sitting on brand new set up for three years. I still need a stereo scope, but as soon as queen production season kicks in I cant fathom taking more time for something else. Time to delegate and get somebody else on the grafting detail. I will free up some time for learning that tool & craft in 2009 if it is the last thing I do.

Thanks for the tips and encouragement everybody.


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## HVH (Feb 20, 2008)

JBJ said:


> Don't feel bad HVH, I am in the same boat, I have been sitting on brand new set up for three years. I still need a stereo scope, but as soon as queen production season kicks in I cant fathom taking more time for something else. Time to delegate and get somebody else on the grafting detail. I will free up some time for learning that tool & craft in 2009 if it is the last thing I do.
> 
> Thanks for the tips and encouragement everybody.


I here ya. I think I have just come to the point where grafting is easy and reproducible so it is time to take the nest step.


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## magnet-man (Jul 10, 2004)

Talk to Tim Arheit aka tarheit on beesource. He actively does II on his stock.


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## KQ6AR (May 13, 2008)

Hi,
I use co2 & a bubble counter in my calcium reactor, for raising coral. Here's a link to where I bought mine.
http://www.marinedepot.com/ps_searchItem.aspx?IdCategory=&SearchText=bubble+counter&parsed=1


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## JSL (Sep 22, 2007)

Hi,

I always like to keep the II setup as simple as possible. The queens will show you when you have the CO2 flow rate properly adjusted. My preference is to have the rate high enough so that the queens are anesthetized fairly quickly and remain completely motionless during the insemination procedure. As Adam shared, the queens will vary with regard to how quickly they respond to the CO2.

Another point to consider is they size and shape of the queen holder used. Most European queen holders are tapered at one end and work very well with a CO2 flow rate of 1-3 bubbles per second. For the instrument I designed, I use an untapered queen holder, so a little higher flow rate of 3-4 bubbles per second usually works best. Again, keep it simple and watch the queens.


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## tarheit (Mar 26, 2003)

There is no need to get overly complicated with the Co2 setup. I use a 5lb cylinder and regulator from a local welding supply outfit and a valve and some tubing from Lowes. Mainly because it was readily available and relatively cheap (compaired to some of the rest of the equipment). You could use the small Co2 cylindars used for pellet guns, the larger ones sold as bike tire inflators, etc. It's not critical. (Don't use fix a flat, queens tend not to lay well once sealed) A 5lb tank, regulator, valve, etc. could run $200, but refills are cheap.

I simply turn it on, stick the end in a cup of water and adjust until I get a steady bubble. Pretty much like JSL said, the queen will let you know quickly if it's enough. I do notice some queens do seem to go to sleep more easily than others. I probably use a higher rate now that I am comfortable and pretty quick with the proceedure.

The most important part though is to remember to turn it off. It's easy after spending a couple hours inseminating queens to clean everything up and forget to turn off the CO2 resulting in an empty tank 2 days later when you remember it.

-Tim


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## HVH (Feb 20, 2008)

Thanks everyone. You make it sound easy enough to give it a whirl.


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## KQ6AR (May 13, 2008)

You're beer making supply store, should also carry CO2 & accessories.


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## Pooh (Mar 8, 2007)

If anyone is tired of that equipment sitting around I am looking for a used II setup


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## Aspera (Aug 1, 2005)

HVH said:


> I do feel guilty.
> 
> 
> So it doesn't need to be buffered (PBS = phosphate buffered saline)?
> ...


PBS can mean either Physiologic or Phosphate buffered saline. The formula is usually something like 0.9% solution of salt buffered to a pH of 7.4 with phosphates. I'm not sure that this pH is appropriate for bees or drone sperm. You can probably buy pre-made sterile isotonic saline or lactated Ringer's for about $6 a liter.


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## magnet-man (Jul 10, 2004)

I don't do II that much and never did get around to buying a co2 system. I have a $2 rig for CO2 using an empty 2 liter coke bottle with some tubing from Home Depot. A quick trip to the grocery store for a $5 piece of dry ice and you are in business. I control the flow of gas with a plastic I.V. flow clamp I picked up during a trip to the minor emergency clinic. *Yes,yes I know the bottle can blow up so I use a very small piece of dry ice.*

If you are really desperate you can use baking soda, marble or limestone with vinegar to make CO2. just make sure you run it through a bubblier. 

We do have two paint ball guns and should really get around to making an adapter to use that CO2 source. Lets see make CO2 adapter or Ultra-Breeze beekeeping suits. Hum, I think I will make beekeeping suits.  I can't get $200 plus for my queens like Sue can.

Don't use the little CO2 cartridges for filling up bicycle flats. I saw some that said they were charged using CO2 from a volcanic source. That doesn't sound to pure to me, buy why the heck would they use that as a source and how? :scratch:


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## magnet-man (Jul 10, 2004)

JSL said:


> I inseminate a few queens...
> 
> The most challenging part of II tends to be semen collection. Most have a difficult time, at first, keeping mucus out of the syringe. With a little practice, it gets a lot easier!


Joe, I think you do more than just a few queens. 

The most challenging part is learning how to pop drones. It took most of us half of the first day of Sue's course before we managed to pop drones successfully. That is everybody but Tim. Tim practiced before he attended the course.


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## HVH (Feb 20, 2008)

Aspera said:


> PBS can mean either Physiologic or Phosphate buffered saline. The formula is usually something like 0.9% solution of salt buffered to a pH of 7.4 with phosphates. I'm not sure that this pH is appropriate for bees or drone sperm. You can probably buy pre-made sterile isotonic saline or lactated Ringer's for about $6 a liter.


Good point about the pH. In humans semen is about pH 8 and vaginal tract about 6. Fortunately for me I no less about bee sex.


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## HVH (Feb 20, 2008)

tarheit said:


> There is no need to get overly complicated with the Co2 setup. I use a 5lb cylinder and regulator from a local welding supply outfit and a valve and some tubing from Lowes. Mainly because it was readily available and relatively cheap (compaired to some of the rest of the equipment). You could use the small Co2 cylindars used for pellet guns, the larger ones sold as bike tire inflators, etc. It's not critical. (Don't use fix a flat, queens tend not to lay well once sealed) A 5lb tank, regulator, valve, etc. could run $200, but refills are cheap.
> 
> I simply turn it on, stick the end in a cup of water and adjust until I get a steady bubble. Pretty much like JSL said, the queen will let you know quickly if it's enough. I do notice some queens do seem to go to sleep more easily than others. I probably use a higher rate now that I am comfortable and pretty quick with the proceedure.
> 
> ...


I got all the toys, and have watched the Sue Cobey video many times. I guess I just need to be willing to fail a few times. Getting past the valve fold looks the hardest to me. Is positioning the queen and getting past the valve fold difficult?


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## magnet-man (Jul 10, 2004)

> Is positioning the queen and getting past the valve fold difficult?


It takes some practice to get the right touch to get past the valve fold. Remember in class how the glass tube did not want to go far if you missed the valve fold? Once past it just slid right in. Since it has been two or three years since you took your course this is my recommendation.

1. Breed a whole butch of queens to practice on. I wouldn't even bother using semen on the first 5 or so queens. Too much time wasted on popping drones for queens that likely won't produce. 
2. Mix up a little blue food coloring and put a fraction of a drop on the queen's business end. Do this for the first few queens you practice on. It will help your aim.

The worst part of II is doing all the prep work. Popping drones and collecting semen. :waiting:


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## HVH (Feb 20, 2008)

magnet-man said:


> It takes some practice to get the right touch to get past the valve fold. Remember in class how the glass tube did not want to go far if you missed the valve fold? Once past it just slid right in. Since it has been two or three years since you took your course this is my recommendation.
> 
> 1. Breed a whole butch of queens to practice on. I wouldn't even bother using semen on the first 5 or so queens. Too much time wasted on popping drones for queens that likely won't produce.
> 2. Mix up a little blue food coloring and put a fraction of a drop on the queen's business end. Do this for the first few queens you practice on. It will help your aim.


I've been married for 27 years so I might need extra practice.


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## JSL (Sep 22, 2007)

All very good suggestions!

As for bypassing the valve fold, I find it has more to do with the position of the sting. With the older style spoon shaped hooks, it was difficult to really pull the sting structure up and out. Now with the forceps and perforated sting hooks it is much easier to manipulate the sting. By lifting the sting structure a bit further, the valve fold is really moved out of the way. Individual queens will vary, but it is often unnecessary to perform the zig-zag motion described in the older literature to bypass the valve fold.

Joe


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## HVH (Feb 20, 2008)

JSL said:


> All very good suggestions!
> 
> As for bypassing the valve fold, I find it has more to do with the position of the sting. With the older style spoon shaped hooks, it was difficult to really pull the sting structure up and out. Now with the forceps and perforated sting hooks it is much easier to manipulate the sting. By lifting the sting structure a bit further, the valve fold is really moved out of the way. Individual queens will vary, but it is often unnecessary to perform the zig-zag motion described in the older literature to bypass the valve fold.
> 
> Joe


Which do you prefer, the forceps or perforated sting hook?


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## JSL (Sep 22, 2007)

I am biased...

I learned on a Mackensen using the old spoon shaped hook, then used a Schley with a perforated sting hook. Now my instrument designs are based on the use of forceps. The work by Laidlaw and Kuhnert actually showed the flexible insemination technique (forceps technique) is about 18% faster and it is easier for most students to learn. I have to agree. I really like using forceps because it allows for a greater amount of flexibility and the instruments are almost always simple to use. In addition, I like the forceps, because one the tip is inserted, I can release the sting for a better "seal" around the tip.

Joe


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## HVH (Feb 20, 2008)

JSL said:


> I am biased...
> 
> I learned on a Mackensen using the old spoon shaped hook, then used a Schley with a perforated sting hook. Now my instrument designs are based on the use of forceps. The work by Laidlaw and Kuhnert actually showed the flexible insemination technique (forceps technique) is about 18% faster and it is easier for most students to learn. I have to agree. I really like using forceps because it allows for a greater amount of flexibility and the instruments are almost always simple to use. In addition, I like the forceps, because one the tip is inserted, I can release the sting for a better "seal" around the tip.
> 
> Joe


I'm all set then and have no excuses.

Thanks


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## beekuk (Dec 31, 2008)

Hi. All really interesting,i am also new to II, and no doubt will be asking many questions,and this looks like a really good place to get some advice,how important is the saline ph and what do most use to test it with.


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## HVH (Feb 20, 2008)

beekuk said:


> Hi. All really interesting,i am also new to II, and no doubt will be asking many questions,and this looks like a really good place to get some advice,how important is the saline ph and what do most use to test it with.


Phosphate buffered saline (PBS) is normally at pH 7.4. Saline made without a buffer could vary quite a bit depending on your source of water. If the semen has a good buffering capacity of its own it probably isn't that important. If anyone could find details about bee semen pH and buffering capacity that would help, but I would guess the experts on this site probably won't think its that important?????
I think I will use PBS just to play it safe. A pH meter is needed to get a relatively accurate pH. pH papers aren't very accurate. Again - it may not matter. We will have to wait on the pro's for a response.


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## beekuk (Dec 31, 2008)

Thank you HVH, digital ph meter i will get. My next question is the ammount of drones to use from different colonys for each insemination. Say rear queens from one,and drones from eight others,all good, but as unrelated as poss.Is it better to collect the semen all from one colony,or say one drone from each of ten per insemination. I realize the way to go is mixing semen,but thats going to be a bit further down the line,as i want to get good at just inseminating to start with.Is using more different drones per insemination better for the genetic diversity,and less likely to get recessive genes,spotty brood.All advice much appreciated.


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## JSL (Sep 22, 2007)

I agree with HVH... I am not sure that the pH really matters that much. The saline solutions published for use with II all have slightly different pH recommendations and yet the authors claim good success with their solution. I am almost embarrassed to admit it, but I have not checked the pH of saline in many years.

I once had a conversation with John Harbo and he said plain water worked as long as the semen was used promptly and you were not homogenizing the semen. I think the osmolarity is more important than the pH, but again I try to use as little saline as possible.

As for genetic diversity and collecting drones to produce semen… Homogenized semen is the best way to go for maintaining diversity in a population over time. However, the next best option is to collect drones from as many different colonies as possible. The more the better!

Why not raise queens from eight and collect drones from eight, provided they are all good quality? Diversity is very important in the honey bee mating strategy and you must be very careful, especially when you have such tight genetic control using II.


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## fatscher (Apr 18, 2008)

I read in my ABC & XYZ of Bee Culture (new 41st edition) that back in the 1920's young Harry Laidlaw Jr. actually held virgin queens between his fingers and manually mated sexually mature drones to them with the other hand. Has anybody done this technique since then? Obviously this isn't as efficient and effective as II, but I'm curious if anybody out there has done this.


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## beekuk (Dec 31, 2008)

Joe, thank you for your helpful advice,and i also like your website and products.so can see me in contact with you in the future.And yes i can see the point of rearing queens as well from many different colonys,i do have plenty to choose from. Hope to just get as good as i can to start with rearing drones and collecting semen,as this seems key to the whole project of II,and the most difficult part.mixing semen will follow in time.Thank you for the answer to my drone question.The saline mix i intend to use is the same as used by sue cobey,but was not sure about how critical the ph bit had to be.
pete.


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## HVH (Feb 20, 2008)

fatscher said:


> I read in my ABC & XYZ of Bee Culture (new 41st edition) that back in the 1920's young Harry Laidlaw Jr. actually held virgin queens between his fingers and manually mated sexually mature drones to them with the other hand. Has anybody done this technique since then? Obviously this isn't as efficient and effective as II, but I'm curious if anybody out there has done this.


I am pretty sure that I read about this and it didn't work because of the valve fold. I think this is what led to II.


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## beekuk (Dec 31, 2008)

This is a saline i assume for mixing with semen,does this seem about the normal mix,or are there others even more easy to make up.also when using this to mix semen what would be the minimum ammount of fertile drones to use from obviously as many different colonys as possible.If using the semen say within 48 hours can a different saline be used. homogenising,thats what i mean,not mixing.



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


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## JSL (Sep 22, 2007)

This is actually the recipe designed by Harbo and another gentleman whose name escapes me at the moment. Yes, that will work just fine for homogenizing semen. When homogenizing semen in a separate vial, I like to use say 100 microliters of semen and approximately 20 microliters of saline. I have no data to support this, just experience, but I do not like to use more than 20% saline for homogenized semen. 

I do not know of any more simple formulas that you can mix on your own. There was an interesting older paper by Camargo et al. that looked at the use of Coconut Milk as a saline and I know some of the large animal breeders have actually used milk as a semen extender with good success. I would run all of these through a BactriFilter before use... 

I will make a separate post for this next point, but I am just finishing a new instrument and syringe that will homogenize semen in one step without additional handling!


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## beekuk (Dec 31, 2008)

Thank you Joe, will be very interested in your new instrument.I allready have bacterial filters,i assume by using these there is no need to heat sterilize any of the saline.


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## HVH (Feb 20, 2008)

beekuk said:


> Thank you Joe, will be very interested in your new instrument.I allready have bacterial filters,i assume by using these there is no need to heat sterilize any of the saline.


The only exception I can see to this would be if you were worried about a viral contamination. The bacterial filters are usually either .22 or .45 micron and cannot eliminate viruses.
As far as semen homogenization I wonder if Grace's insect media with non-essential amino acids added would be a good substitute for the Harbo recipe. You can purchase Grace's media and NEAA from Invitrogen. Once you start down this path, though, you are asking for trouble if you don't have a sterile hood to work in and an understanding of sterile technique. This type of media will support bacterial and fungal growth very well (especially if you add fetal calf serum). I don't like to use antibiotics/antimycotics (ABAM) in most cell culture work unless absolutely required. I have kept honeybee cells (harvested from eggs) growing for months in this media without ABAM. Grace's is likely to support semen homogenization very well but I haven't even done II yet so I cannot guarantee results.


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## beekuk (Dec 31, 2008)

Hi HVH at least we are together on this as we are both learning the II. Would you advise to sterilize the saline and use a bacterial filter,as i do realize that this process requires very hygenic/sterile conditions,someone said 10 times more hygenic than human surgery,don't know how correct that would be,or even possible.


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## HVH (Feb 20, 2008)

beekuk said:


> Hi HVH at least we are together on this as we are both learning the II. Would you advise to sterilize the saline and use a bacterial filter,as i do realize that this process requires very hygenic/sterile conditions,someone said 10 times more hygenic than human surgery,don't know how correct that would be,or even possible.


There is the anal scientist type in me that says ya! Let’s use a level 4 facility. The thing is, though, Sue Cobey is a pioneer in this field and doesn't even wear gloves. I've watched her video many times and it is anything but sterile technique. This is not a criticism; it is probably just the reality of doing II. Let's not forget that sex is not done with sterile technique. With that said, I would still keep a reasonable sterile field, with judicious applications of alcohol to equipment coming into contact with the queens working end, wash hands frequently and use common sense. I might even consider using the simpler saline recipes because they won't promote bacterial growth. Saline can be made 10X and diluted 10 fold before using which would be another way to restrict any bacterial growth. Saline is normally about 150-160 mM of NaCl or a mix of NaCl and KCl. A 10X stock would be closer to 1.5 molar which is bacteriostatic. If you make 500mls of 10X it will last a very long time and it can be autoclaved in a pressure canner 15 PSI for 15 min. If you pressure can some DI water at the same time then you have diluent to make your 1X saline. 
Lots to choose from. Again, I am the last person to ask because I have zero experience with II.


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