5.1. Homogenizing honey bee semen

To homogenize or mix honey bee semen from numerous drones requires dilution, mechanical movement and reconstitution of semen. Current techniques using centrifugation result in a high percentage of damaged spermatozoa, although 50% viability of spermatozoa is sufficient to produce normal brood patterns (Collins, 2000). Semen is very dense, tends to clump, and the long, fragile tails of spermatozoa are subject to damage during processing and some components of the seminal fluid are removed.

Migration of sperm from the oviducts into the spermatheca is a complex process involving contraction of muscles mediated by the specialized composition of fluids in the semen and the oviduct as well as active sperm movement (Koeniger, 1986). Queens are very active after natural mating which also promotes sperm migration; therefore, use a direct queen introduction release method (Büchler et al., 2013).

Procedure by centrifugation:

  1. Collect semen into glass capillary tubes (section 3.2).
    a. Collect 10 µl for each queen to be inseminated.
  2. Add diluent (the recipe can be found in Table 4) to Eppendorf tube.
    a. Ratio should be 10 parts diluent to 1 part semen by volume.
    b. Up to 700µl of semen can be added per Eppendorf tube.
  3. Expel semen into an Eppendorf tube.
  4. Mix semen by inversion, gently shaking, until suspension is uniform.
    a. Strong mixing with a Pasteur pipette may damage spermatozoa.
  5. Centrifuge at a 45º angle until a semen pellet is formed.
    a. Use speeds of 82 or 250 g at 20–30 or 10–20 min, respectively.
    b. Higher speeds can damage tails (see Collins, 2003).
  6. Carefully remove supernatant.
  7. Draw semen into syringe and use immediately.
  8. Inseminate queens with the required dosage.


Table 4. Recipe for making desired volumes of diluent for counting sperm. Mix the ingredients together and add distilled water to achieve the final volume.

 

Desired Volume

Reagents

2 ml

5 ml

10 ml

20 ml

1M HEPES

20 µl

50 µl

100 µl

200 µl

NaCl

0.017 g  

0.0425 g  

0.085 g  

0.17 g

BSA

0.20 g

0.50 g

1.0 g

2.0 g

add water to bring total volume to:

2.0 ml

5.0 ml

10.0 ml

20.0 ml