Monday, March 16, 2009

Cryopreservation of Bee Spermatozoa

Megan Taylor, B.Sc.(H), M.Sc from the Apiculture lab at the University of Guelph who has been studying under Dr. Ernesto Guzman has finalized her thesis on the cryopreservation of honey bee spermatozoa. She graciously agreed to share the results of her study with our club.

I took notes from her talk and she kindly edited my notes and I’ve pasted her edited version below. Really the credit belongs to her since she did all the research. Thank you Megan!
The focus of her study was the question: Can bee sperm be successfully preserved long-term?

Ms. Taylor discussed the reasons why research needed to be done on the cryopreservation of bee spermatozoa - to improve the strains, preservation of current stocks, transportation of genetic material, the ability to conduct studies all year and not just in summer, and full mating control.

Mature drones embark on mating flights in the afternoon, and head to their drone congregation areas, aerially located 10-60 meters above ground, where they would wait for a queen on a mating flight. She found drones were more sexually mature from July to mid August at the age of 12 days or older. (Young drones don’t contain any semen, older ones have more. However, later in the season, the sexually mature drones appeared to contain less semen than the sexually mature drones from earlier in the season.)

Generally, by mid afternoon there would only be immature drones in the hive as the mature drones will have flown off to drone congregation areas.
(photo - a few members of the Elgin, Oxford and Middlesex Beekeeper's Assocation).
The queens will mate once they are 5 to 6 days old and she will mate with between 6 to 20 drones. When mating with the queen, after the drone has ejaculated the semen, its mucous may act as a plug inside the queen.

Drones ejaculate a very tiny amount of semen that is highly concentrated with between 8 to 11 million sperm. Drones only ejaculate and therefore mate once during their mating flight, after which they will die. The queen will save about 10% of the sperm from each drone she mates with, holding between 4 to 7 million sperm in her spermatheca. Drones produce between 8 to 11 billion sperm in one micro litre which isn't that much, but compared to other creatures it's one of the, if not the most concentrated of all.

It was noted that bee sperm left just sitting on a shelf for 3 months with no form of treatment still had sperm which could be seen moving under a microscope.

There has not been any recent studies done on honey bee spermatozoa cryopreservation since 1984 (Kaftanoglu and Peng, 1984).

Previous experiments with liquid nitrogen (-196 degrees Celsius) discovered that there was some damage to the membrane of the sperm and their tails would split (Peng et al., 1992).

In Ms. Taylor’s experiment, diluents were used containing amino acid, antibiotics and glucose for energy, and different cryoprotectants to improve the storage of the sperm. She pooled semen from multiple drones and added different amounts to test collection ratio. She experimented with 7 diluent solutions, 3 different cryoprotectants and 5 collection ratios.

Cryoprotectants and diluents did reduce cellular damage and improved the post thaw viability of sperm. A similar freezing protocol used for the cryopreservation of swine sperm was used for the bees and this was very successful. The sperm did survive the cryopreservation. The results of the cryopreservation were greater than 60% viability.

The next step was to fertilize queens with the sperm. This process proved to be very difficult and not all the queens survived (acceptance when reintroduced in the hive seemed to be an issue - and there was speculation whether the workers knew the queen was not in a good condition or was damaged in some way).

Ms. Taylor was able to successfully instrumentally inseminate one queen with cryopreserved semen and found that the sperm had migrated to the queen’s spermatheca and were even still motile when observed under the microscope. The results are very promising and further study will need to be carried out.

Ms. Taylor’s results will be published this spring in the scientific journal called “Theriogenology”.
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