I inseminate approximately 250-300 queens per year. This is purely for research reasons in my case. We inseminate queens with known numbers of drones for genetic comparisons and for studying task specialisation.
Comercially in Australia insemination is used by a consortium of queen breeders to maintain pure strains collected for breeding stock (in some lines have existed for 50 plus years). They maintain over 30 lines.
Queen breeders generally have about 2 or 3 inseminated breeder queens that they raise all their queens from. The progeny is then mated in yards (often isolated) so it will mate with selected drone parent colonies. The difference is that the queens fly to the nearest DCA (drone congregation area which can contain 100's of thousands of drones. So it is very difficult to get a pure mating. In this case at least a good proportion of the progeny carry the charecteristics of the breeder.
The reason for the cost is that beekeepers don't requeen all their hives with these, they simply breed from them. Often they are from lines kept for many years with generations of breeding and selection. So they are just going to give the genes away.
Many will tell you that A.I. is hard, queens are inferior etc, etc. Most of this is mythical, it's purely dependant on your level of skill, hygeine and experience and ability to follow scientifically proven protocols.
It's like anything, "practice makes perfect" and if your interested go and give it a try. You can cross whatever you want to in that case. Make your own selections from queens. However you must understand a bit of genetics which can be learned from bee breeding books.
There are many methods, apparatus types, and syringes. Like cars all have pluses and minuses.
On the point of storing drone sperm. This has not been perfected by any means. It loses viability quickly when frozen. So far if stored in the correct buffer it can remain almost 100% viable for 72 hours. This means you generally collect it freshly every time.
The following links give some background on insemination and courses. If your interested in learning Sue Coby runs great courses and I highly reccomend her as a teacher.
If your interested I can outline my equipment, procedures, hints tips, drone selection etc, etc however it is fairly long winded.http://maarec.cas.psu.edu/bkCD/HBBiology/breeding_genetics.htmhttp://www174.pair.com/birdland/Breeding/
The following may seem trivial however they make the procees so much smoother.
Preparation:- Drones generally have to be prepared first, as they take a long time to develop and mature. You need to aim to have drones between 16-30 days old to cut down time in sperm collection.
You can simply guess by taking drones from outside combs, but it generally takes 4-5 drones to get a good one. We confine the queen to a drone comb using a frame confinement cage made from metal queen exluder in the colony we wish to breed from. The colony should be fed protein supplements to ensure they want to rear drones. This works well when bees are on good conditions.
The queen is released after approximately 4-5 days, this is enough time to get her to lay numerous drones. The whole colony is shaken out and made to walk through an exluder (except the queen of course). This removes any foreign drones that have drifted in. This excluder remains on until you have completed the process. The colony is only opened early morning to prevent the loss of drones from flying.
Everyone has their own methods for raising queens so I won't go into that. I simply use the grafting method into cell starter colonies and the cell builder colonies.
The queens need to emerge in an exluded nucleus colony or stronger to ensure the queens are looked after. Never underestimate the importance of this. The queen is looked after well, fed groomed and cleaned. This can make all the difference.
A virgin queen is ready for insemination at approximately 7 days of age. We catch virgins very early (to prevent flying) on day 6 and place them in LABELLED queen cages for their preparatory CO2 treatment. CO2 does something to trigger the activation of ovaries. The scientific mechanism is still unclear. They get treated either the day before or after insemination and during insemination.
The queens are taken to the lab, factory wherever the CO2 bottle is. Queens are placed into plastic jar with loose fitting plugs in the lid. The bottle is filled with CO2 until queens fall down in cages (unconcious). The plugs are then loosely put in place. You do not want to suffocate the queens but simply keep them asleep. They are given 7 minutes like this.
The queens are removed from the container their right wing clipped and thorax marked (with queen tag) and allowed to wake up in the cages. They are returned to their colonies and hung in the colony as so the bees have good access to the queen. Our cages are designed with a mesh that allows good contact with the workers.
Now the queens are ready for insemination the next day.
Equipment:- I personally learnt on a schley apparatus. This is flexible and easy to learn on. It relies on two hooks (dorsal and ventral) and a syringe. After years of practice I now use the same apparatus using only the ventral hook and a pair of jewellers forceps to grab the sting. This is much similar to the Latshaw instrument.
Whatever apparatus you use you must feel comfortable or you will never succeed.
I'll continue this evening, better get back to the research work. Other topics I'll cover include preparation around apparatus, sterilisation, drone husbandry in the lab, diluent - buffer solution, semen collection - dose, insemination process, post insemination care of queens, follow up treatments, methods of checking your success, record keeping.
Before you inseminate you must have your work bench well layed out, and all equipment sterile. Laboratory grade tissues and are very handy for cleaning up contamination.
Sterility is of the upmost importance, any contamination can give the queen and infection and she will surely die. Glass tips, tweezers, forceps etc are autoclaved (this can actually be done in a pressure cooker if no autoclave available).
You try to get yourself in the habit of moving always from left to right so every task is carried out methodically.
Drone Husbandry in the Lab:-
Once you have collected your mature drones in drone cages (wooden cage with queen exluders either side, about 100 per cage), they must be nurtured. Drones without workers get cold very fast, they become hard to evert as well as many simply will drop dead.
The best method is to make a drone flight box with a heated lamp. A box with an elastic sleeve inside is a cage containing workers with a candy food source. You simply release the drones inside and secure the sleeve. This box serves many purposes. Firstly as the drones were excluded in the colony they were unable to fly and defecate (which is what they like to do) In the cage they can do this, so the drones are very clean and this reduces a possible contamination source. Secondly they fly around then return to the cage surface containing nurse bees and are fed. This keeps them energetic at all times resulting in an easy eversion during the semen collection period.
Buffer solution:- This is a saline based solution containing an antibiotic. There are many different recepies for this. It is personal preference. The purpose of the solution is a backing solution inside the semen collection syringe and tip. You need it to prevent drying of the semen and to assist semen delivery by capillary action. Some people also collect a drop of this solution between drones to prevent drying from the other end. This is then expelled from the tip when collecting from the next drone.
Drone semen collection:-
Once you have mastered this you are well on your way. The process requires you to firstly achieve the partial eversion of the drone and then the full eversion using pressure.
The drone must be tense to achieve this properly. A good way to achieve this is by turning the drone upside down in one hand and grip it between the thumb and forefinger. Apply pressure to the abdomen and remove or crush the head on the drone Shocked with your other hand. This helps the drone tense. By now you should have acheived partial eversion. Maintain pressure and gently roll your fingers downwards until the full eversion takes place. Many drones will pop fast, it is important to learn to control the speed of the full eversion. If the semen sac bends right over and touches the body of the drone, this is another potential contamination. These drones are simply discarded into your prepared rubbish bin. I EXPECT YOU ALL TO GIVE THIS A TRY, AFTER ALL WHEN A DRONE MATES IT'S WHOLE APPENDAGE IS TORN OFF AND THE SIMPLY BLEED TO DEATH. CRUSHING THE HEAD IS MORE HUMANE IN MY EYES.
Once you have the full eversion it's time to collect the semen. You must have your apparatus and prepared syringe under a microscope with sufficient power. You will see a ball of pinky white liquid on the surface of a white fluffy mucus layer. The idea is to skim the pink semen with the use of capillary action from the mucus layer. Mucus in the tip can be devestating, It clogs the tip and is a possible contaminent.
Firstly you wind the buffer solution up and down and then all the way out of the tip and make sure you have good capillary action. This is vital to make semen collection smooth. You retract this solution about 5mm back up the tip to maintain an airspace between the buffer and the semen. You make contact with the largest portion of the pink layer, wind foward slightly to make contact and then begin retracting removing semen until the majority is removed. A big tip, don't try to take all of it. you are destined to get mucus. A good mature drone contains approximately 1 microlitre of semen. 8 microlitres are used to inseminate carniolans and 10 microlitres for italians (it varys throughout the world).
The process is repeated untill you fill a glass tip. You wind the sperm down the tip to make contact with the next drone and then repeat capillary action. The glass tips I use contain enough sperm for inseminating 4 queens. I collect all semen first this may be multiple tips. The filled tips can be removed and remain perfectly viable for up to 48 hrs. However the fresher the better. Never attempt to do more queens than you can handle.
Drone quality is extremely variable. Good prepared drones like I've mentioned make the semen collection process smoother. Some drones just simply don't have any sperm. It can often take 15-50 drones per queen depending on you level of preparation.
Once semen has been collected it's time to A.I. The queen holder on the apparatus is hooked up to the CO2 bottle. This CO2 is filtered through a bottle containing water. A tube then runs from the bottle into the queen holder. The idea is to get a steady flow of gas that relaxes the queen during the process.
A queen is removed from the holding cage and placed into a back up tube. This tube is held against the queen holder and the queen walks backwards into the conical shaped holder. The queen holder is then placed onto the apparatus and gentle pushed downwards onto the CO2 entry tube. This pushes the the posterior of the queen so it is protruding out of the end of the queen holder. If possible you try and make sure the hind legs stay in the holder. They only get in the way if they are protruding. Shortly after being positioned the queen will relax and practically go to sleep.
Depending on what technique you use, you will have one or two hooks. In both techniques a ventral hook is used. This hook is inserted into the rear of the queen and moved gently to the left. This exposes the inside tissue and entrance into the oviducts. The apparatus will hold this hook in place. You will notice at this stage a "V" shapes piece of tissue. At the bottom end of the "V" is the entrance into the queen. The internal passage into the oviduct is zigzag shaped. This must be by-passed without damaging the tissue with the glass tip. Using the dorsal hook method, this is either threaded onto the sting or simply just stretches the dorsal part of the opening outward and upward.
Positioning is the key, if the queen is at the wrong angle you will never succeed. This takes practice. Unfortunately if this step takes too long (10 plus minutes) the CO2 is generally lethal. Once the queen is correctly positioned the tip of the syringe is gently lowered a short way into the entrance. The tip is then moved slightly to the left to by-pass the valve fold (dorsal hook adjusted upwards if required) and then the tip is lowered into the oviduct entrance. The semen is then released into the queen (10 microlitres) by winding the syringe tip. There is a measurement on the tip (using tygon tubing). If there is resistance and the semen does not flow in smoothly, the tip is likely to be in too deep and pressing against tissue, in this case you retract the tip ever so slightly and try again. If semen is visible out of the valve fold this is what is known as (welling back). This is an unsuccessful insemination.
The method I now use differs slightly. The dorsal hook is replaced by the set of Jewellers tweezers. This requires a slightly steadier hand. You grab the sting between the palp like structures that surround it. Once you have a firm grip you pullthe sting upwards. With this technique the "V" tissue opens right up and allows easier access through the valve fold. Once the tip is in you can let go of the sting and release the semen.
At the conclusion of the insemination the queen is placed back into the holding cage and then taken to her colony. A small ball of candy is placed in the entrance of the cage to slow release her as she wakes up. She is best looked after by nurse bees.
It good when you are practising to do a few extra queens for dissection
To check how your insemination wet recapture the queen after 48hrs. This give the sperm enough time to migrate into the spermathaeca. To check the contents of the spermathaeca simply grab the last few segments of the abdomen with your fingernails and tear it right off the queen. Inside you will see a ball shaped mass. This is the spermathaeca. It is covered in tissue. To remove this you simply rub it in a circular motion between your thumb and forefinger. If the insemination was successful the spermathaeca will appear pinkish in colour. Repeat the same to some virgin queens and you will notice they are clear.
Follow up treatments:-
After 7-10 days, queens need to be checked for laying. In colder weather it may take a bit longer. If the queen is not laying she needs to be treated with CO2 again. She is simply placed in a cage gassed for 7 minutes and then returned to the colony with candy. She should then start laying in another few days. It rarely takes another gassing after this.
You must also check the queen about 3 weeks after laying commenced. You need to examine the capped brood to determine if the queen is laying worker brrod. If the cell caps are significantly raised you have a drone layer queen which means the insemination was unsuccessful and she needs to be replaced.
Extremely important so you understand the genetic principles behind why you are doing this. Your records should contain facts such as Colony number, AI number, Queen hatching date, Date of first CO2 second CO2 etc, Queen mother, Drone Mother, Date of AI, Date of lay, Remarks any important facts or problems with AI.