Long term storage of bee semen – a six month assessment of cryopreserved semen quality using motility as an index

Bee (Apis mellifera Linn.) spermatozoa is known for its ability to remain viable for at the least two years within the spermatheca as attested to by the queens’ continued laying of fertile eggs without remating  over two to three (Woyke 1960; Winston 1987). However, the semen collected from the drones by artificial means also remains viable for months with refrigeration or even at room temperatures (Collins 2000). This characteristic could be utilized for conservation, transportation, storage and comprehension of aging and dormancy physiology in plants and animals. We have initiated studies to understand the factors that might play a role in the semen longevity. Our primary focus is to retain the above described characteristic of the bee spermatozoa even after cryopreservation.

Bee semen was received from the USDA facility in Baton Rouge, LA. One hundred microliters of the semen was collected in a glass capillary from drones returning to the hive after an unsuccessful mating flight. Semen was proportioned into 10 µl samples in PCR vials. All the transfers and treatments were done aseptically and in dust-free conditions in a laminar flow. Three types of storage procedures were studied. These include, storage at room temperature (22±2.7°C), storage at 7±0.2°C and cryostorage (-196°C). The data presented here is for a period of six months of storage. The difference between the room temperature and 7°C stored samples versus the cryopreserved samples was the use of an extender medium as a diluent to carry and to remove the cryoprotectant before and after the procedure. A non-capacitating extender medium based on Hank’s balanced salt medium was tested for this study. Post-storage assessments were conducted by 50x dilution of the spermatozoa with 0.2 µm filtered water and estimating the proportion of spermatozoa showing full motility. Spermatozoa showing vibratory motility were excluded. Semen in 7.5% dimethyl sulphoxide in the extender medium was frozen using a Planer Kryo 10 programmable freezer (Planer PLC, Sunbury-On-Thames, UK) at a cooling rate of 2.5°C/min to -35°C and then transferred to liquid nitrogen for storage. The freeze ramp was interrupted for a minute for auto-seeding at -7°C. Cryopreserved semen was thawed in a 36°C water bath. The semen was diluted with the extender medium and centrifuged at 1000x for 12 minutes (Wegener et al. 2014). Three different formulations of the artificial extender media were investigated (ed#1-3). Studies in this report were done using ed#3.The supernatant was removed and the sample was re-suspended in 12 µl of the diluent ed#3 and stored either at room temperature or at 7°C until further assessment.

After six months of storage (since the summer of 2014), the following observations were made. The control semen that was received contained 76±3.05% (n=12) motile spermatozoa. The untreated control estimates were made within a period of 7 days after the receipt of the sample during which it was stored at 7°C. The semen aliquoted and stored at room temperature contained 48.9±20.4% (n=8) motile spermatozoa. Aliquoted semen stored at 7°C however contained 24% more motile spermatozoa (73.1±10.9%; n=10) [p>|z=2.934|=0.0034]. After cryopreservation, the reduction in motile spermatozoa was approximately 9%. More than 85% of the spermatozoa survived the procedure. Since the interest in this study was to assess the viability on storage, cryopreserved semen was thawed, washed and stored either at 7°C or at room temperature. Six months of storage of the cryopreserved spermatozoa at 7°C resulted in negligible reduction in the viability (69.1±17.63%; n=11) compared to untreated control (76±3%), the untreated spermatozoa stored at 7°C (73.1±10.9%) as well as the freshly thawed spermatozoa (67.6±10.9%; n=10). Semen reconstituted after cryostorage when stored at room temperature contained approximately 70.9±11.8% (n=10) motile spermatozoa. Statistically, this was not unlike the untreated controls or the cryopreserved controls or the untreated but chilled semen at 7°C. However it was significantly higher than the controls that were aliquoted and stored at the room temperature. Studies are in progress to determine the reasons for this difference.

References: (1) Collins A (2000) Survival of honey bee (Hymenoptera: Apidae) spermatozoa stored at above freezing temperatures. Journal of Economic Entomology 93: 568-571; (2) Wegener J, May T, Kamp G, Bienefeld K (2014) New methods and media for the centrifugation of honey bee (Hymenoptera: Apidae) drone semen. Journal of Economic Entomology 107: 47-53; (3) Winston ML (1987) The biology of the honey bee. Harvard University Press. Cambridge, MA, 281p; (4) Woyke J (1960) Natural and artificial insemination of queen honey bees. Pszczelnicze Zeszyty Nauwoke 4:183–273