94 CRYOPRESERVATION OF HONEY BEE SPERMATOZOA

Abstract

Our project investigated a new method for the cryopreservation of honey bee (Apis mellifera) sperm cells (SC). Few methods have been developed and none achieve normal sex ratios in progeny. Recently, honey bee colonies have been decimated by colony collapse disorder and infestation by varroa bee mites. A bank of preserved SC might enable the creation of a seed stock for restoration of genetic diversity through AI (Cobey 1983 Am. Bee J. 123, 389–395). We investigated two freezing rates using two diluents and their effect on post-thaw survival of the SC. The slower freezing rate was chosen from a report with the highest success to date (Harbo 1983 Ann. Entomol. Soc. Am. 76, 890–891). The rapid freezing rate was a method developed by us. We reasoned that the small volumes of ejaculate made it potentially suitable for vitrification. The SC were frozen either in 40% Harbo's DMSO diluent containing 25% DMSO, 25% egg yolk, 50% buffer (1.1% NaH₂PO₄·H₂O (w/v) and 0.845% Na₂HPO₄·2H₂O (w/v)), and 60% semen; or in 50% glycerol-based diluent containing 9% glycerol, 24% egg yolk, 67% buffer (5.9% Tris (w/v), 0.8% glucose (w/v), and 3.2% citric acid (w/v)), and 50% semen. Ejaculates were collected by applying bilateral pressure to the abdomens of the drones causing endophallus eversion. About 1 µL (8 × 10⁶ SC) of ejaculate was drawn into siliconized 50-µL capillary tubes fitted to a Hamilton threaded-plunger syringe preloaded with Fluorinert (Sigma, St. Louis, MO, USA). Micro-glass cryostraws (µC) were constructed by pulling Pasteur pipettes to a 230-µm internal diameter and keeping what was the tip end of the pipette as the large end of the µC. The large end was fitted with Silastic tubing to act as a bulb for drawing and expelling fluid. Three µC per treatment were filled with 5 µL of diluted ejaculate and sealed with Critoseal. The µC were inserted into 500-µL Cassou straws (IMV Technologies, L'Aigle, France), immersed in a water bath, and cooled from 21°C to 5°C over 2 h. A Freeze Control^® programmable cryochamber (CryoLogic Pty. Ltd., Mulgrave, Victoria, Australia) was used to cool samples slowly from 5°C to –40°C at 3°C min^–1. At –40°C, the cryostraws were plunged into liquid N₂ (LN₂). Rapid freezing was done by plunging µC into a LN₂ vortex, created using a magnetic stir bar. The µC were reinserted into the Cassou straws, while still under LN₂, for storage in an LN₂ tank. The µC were thawed by removal from the Cassou straws and immediate immersion in a 35°C H₂O bath. Survival rates were evaluated using a dual fluorescent staining system (Molecular Probes, Eugene, OR, USA) and fluorescent microscopy. The largest portion of live staining cells (93.18%) were treated with DMSO diluent using the rapid freezing. The remaining treatments ranked as follows: slow freezing with DMSO (78.84%), rapid freezing with glycerol (38.9%), and slow freezing with glycerol (26%). All treatments differed significantly (P < 0.01). Other studies state that queens inseminated with greater than 50% viable SC have a good probability of producing normally throughout a season. Therefore, our technique of rapid freezing in DMSO diluent might be useful to apiculturists.