3.3. Insemination of the queen

Inseminate queens between 5 and 12 days post-emergence. Carbon dioxide is used to anesthetize the queen during the procedure and also stimulates oviposition. Queens can be emerged in a queenless bank or, preferably, in their own colonies (typically small hives each with several hundred adult workers with a single virgin queen, called mating nuclei). If mating nuclei are used, cage the queen cells (so queens emerge into a cage) or be sure that the hive entrances are covered with queen excluder material to prevent unwanted natural mating flights. Queenless banks, mating nuclei, and caging queens cells are all discussed in detail in Büchler et al., 2013.

 Procedure for inseminating queens:

  1. Two CO2 treatments are usually required. Give the first CO2 treatment, a 1- to 4-minute exposure, one or two days before the insemination procedure. The dose can be applied by individually caging queens and placing them in a jar or plastic bag filled with CO2. The second treatment is administered during the procedure.
  2. Align the syringe and queen holder on the instrument stand at a 30º to 45º angle (dependent upon the instrument used) to facilitate bypassing the valvefold (Figs 13 and 18).
  3. Place the queen in the holding tube abdomen first, ventral side up, with her abdomen protruding several segments (Fig. 13), and administer a slow continuous flow of CO2.
  4. Separate the abdominal plates to expose the vaginal orifice using a pair of hooks or forceps (Fig. 14).
  5. Lift the sting structure dorsally, to expose the vaginal cavity (Figs 15-17).
    a. During this manipulation, position the ventral hook only to stabilize the queen.
  6. Position the syringe tip dorsally above the "V", defining the vaginal orifice. Insert the tip into the vaginal orifice 0.5 to 1.0 mm, slightly forward of the apex of the "V" (Fig. 18).
  7. Insert the tip further, another 0.5 to 1.0 mm, while using the tip to lift the valvefold ventrally (Fig. 18). Use a slight “zigzag” movement to bypass the valvefold.
    a. The valvefold, a stretchy flap of tissue covering the median oviduct, must be bypassed or semen will backflow out of the vaginal orifice.
    b. Correctly inserted, the tip slips easily past the valvefold without resistance.
    c. As outlined in section 3.2., it is useful to leave a tiny air bubble between the saline and semen and release some of the saline for lubrication before inserting the tip in the queen.
  8. Deliver a measured amount of semen directly into the median oviduct (Fig. 19).
    a. The standard dosage is 8 to 12 µl per queen. When giving a 12 µl semen dose, release the queen directly into her mating nucleus colony to promote sperm migration or give two 6 µl semen doses 48 hours apart.
    b. With practice, the insertion of semen is preformed quickly and precisely, requiring only seconds per queen.
  9. After insemination, remove the syringe tip, collect a small air space and small drop of saline, (~0.5 µl) to precede the next insemination.
    a. Keep a drop of saline in the tip to prevent any residual semen from drying and to initiate subsequent semen collection.
    b. If inseminating queens with different drones where precise genetic crossings are paramount, rinse the insemination tip with distilled water then saline to completely cleanse the syringe of semen from the previous drone.
  10. Release the queen from the holder, place her in a cage, and return her to her nucleus colony.

Fig. 13. Virgin queen positioned in the holding tube. The syringe and queen holder are aligned at a 45º angle on the device to facilitate bypassing the valvefold. 


Fig. 14. Separating the abdominal plates of the queen to expose the sting structure using the perforated sting hook. The ventral hook is on the left while the perforated sting hook (seen with small hole to accommodate the sting) is on the right.


Fig. 15. Threading the sting through the perforated sting hook (on right). The ventral hook is on the left and is used only to stabilize the queen.


Fig. 16. Lifting the sting structure to expose the vaginal cavity, using a perforated sting hook (on right). 


Fig. 17. Lifting the sting structure, using Schley pressure grip forceps (on right).  


Fig. 18. Bypassing the valvefold. To bypass the valvefold, position the syringe tip dorsally above the "V", defining the vaginal orifice. Insert the tip about 0.5 to 1.0 mm, slightly forward of the apex of the "V". Insert the tip another 0.5 to 1.0 mm lifting the valvefold ventrally, using a slight “zigzag” movement to manoeuvre around the valvefold. 



Fig. 19. Insertion of semen in to the median oviduct. Positioned correctly, the tip slips easily past the valvefold without resistance.