Instrumental Insemination
Scientific Beekeeping
This webpage will give you a simple understanding of what Instrumental Insemination (II) is and some pointers to the issues involved.

The second tab on the left describing how II should be performed is the gold standard on how to perform this procedure.

The equipment is expensive as are the courses which instruct on the technique. Having the knowedge is not the same as being an expert at this. Practice and experience will improve your technique. In addition to the equipment in the photo below syringe tips, a low heat LED light source and a low powered dissecting microscope will be required, preferably with a long arm. All the equipment must be well balanced and weighted. There are also items that need to be aquired that will be in the process on a recurring basis such as saline.

In Susan Cobey's 'Standard Methods..." it states very precise ingredients for the saline. Most people performing II on a commercial basis use ordinary sterile saline that you can buy from a pharmacy (personal communication). The extra things that are added are mostly proteins and carbohydrate(glucose). Without these extras there is no nourishment provided for the the sperm. This will shorten the shelflife of the sample. Using plain saline the semen should be used as soon as possible.

A high level of cleanliness must be used and anything in contact with the sperm or introduced into the queen, the syringe tips in particular, must be sterile.

The "Further Information" link has lots of tips about maintaining equipment along with the practicalities involved.

The use of carbon dioxide is an integral part of the process. The queen should be anaesthetised the day before insemination. She will receive her second CO2 treatment during II.
History of Instrumental Insemination
Standard methods for instrumental insemination of apis mellifera queens Susan W Cobey
In particular getting past the valvefold is difficult until you have the experience. Below is a photo of a pdf explaining this issue.
The drone is fully everted whilst sitting at the microscope. With a fully assembled syringe first take up a tiny amount of air(5µl) and then 2µl of saline soluton. Repeat this so that you have air/ saline/air/saline then touch the everted endophallus just where the brown sperm is collect it, being careful not to take in the thick mucous which will clog up the specimen and make it unusable. If it does happen try to expel the mucous plug immediately.

When collecting from the next drone push back a tiny bit of semen from the previous drone on to the end of the tip, make contact with the marbled brown sperm and draw it up thereby avoiding air bubbles being mixed up in the sperm collection. Move the everted drone to collect all the semen not the syringe. When all the semen you desire has been collected take up a tiny amount of saline (2µl) to seal the syringe tip and prevent the semen drying out.

Further information about using and cleaning equipment used in II
The queen is placed in the short piece of transparent plastic with the tip of her abdomen exposed at the narrow end. This is then slipped onto the white opaque plastic. The carbon dioxide is delivered through the central channel of the white plastic.
Harbo large capacity syringe
A syringe similar to this is preferable. It gives better manual control through tiny movements as the wheel is adjusted
Schley Standard Equipment
This is the typical arrangement for a right handed inseminator. The syringe which delivers the sperm illustrated is the Schley standard syringe
Drones must be collected from the hives that have been chosen for this purpose. The drones are kept together in this way until they are everted to collect the sperm. Once collected the semen will remain viable for up to two weeks, depending on type of saline used. The drones will get cold and hungry in the net when separated from the workers so dont collect them too far in advance.

There is no way of knowing if a drone is mature or not, that will only become apparent when the drone is everted. One way of trying to excude the evertion of immature drones is to collect them as they come back from unsuccessful mainting flights in late afternoon. If a piece of queen excluder is placed at the entrance this will slow the drones down as they try to enter the hive and they can be collected.
Bee Culture 2016
Drone Flight Box
Diagram of the queen's abdomen, showing the valvefold which must be bypassed to deliver semen into the median oviduct for proper insemination.
Credit J. Harbo USDA
Standard methods for instrumental insemination of apis mellifera queens
Partially Everted
Fully Everted
Semen on the everted endophallus. Note the brown marbled appearance, from Standard methods for instrumental insemination of apis mellifera queens
Semen contaminated on evertion when the endophallus touches the exoskeleton of the abdomen. This drone must be discarded.
Standard methods for instrumental insemination of apis mellifera queens
The semen collected like this, using the saline described in "Standard methods...." should last two weeks kept at normal room temperature (17c). Just like anything else the sooner it is used the better.
After insemmination the queens are allowed to rest for two days with a few attendant workers. This allows her to be watched for complications such as injury or infection. She should be marked with a numbering system to aid the breeder with keeping full records. If she is running around in her queen cage she is ready to go into a nucleus colony so that her laying pattern can be assessed.
Sting Hook
Ventral Hook
Sting Hook
Ventral Hook
100 ul semen storage capillaries
Pressure Forceps