33 Cryopreservation of Jamaican fruit bat (Artibeus jamaicensis) spermatozoa

Bats are integral parts of balanced ecosystems while also being reservoir hosts of many viruses that cause significant diseases in humans; thus, there is a need for assisted reproductive technologies (ART) in bats for conservation and infectious disease research purposes. Little research has been done on ART in bats. Two experiments were performed to develop a cryopreservation protocol for epididymal sperm from Jamaican fruit bats (Artibeus jamaicensis [Aj]). In Experiment 1, we compared the effectiveness of glycerol and combinations of glycerol with methylformamide (MF) or dimethylformamide (DMF) as cryoprotectants for Aj bat sperm. Seven mature male bats were selected from a captive colony and killed 1 h before sperm were collected by mincing epididymides in TALP. Each sample was assessed for total and progressive sperm motility using a computer-assisted sperm analysis (CASA) system, and sperm concentration was determined by using a hemocytometer. If sperm motility and concentration were good, sperm from two males were pooled and diluted to 40 × 10⁶ sperm/mL in egg-yolk TEST (EYT) diluent containing one of five cryoprotectant combinations (5% glycerol, 1% glycerol + 4% MF, 2% glycerol + 3% MF, 3% glycerol + 2% MF, and 2% glycerol + 3% DMF). Straws were loaded with 100 mL diluted sperm, cooled to 5°C over 2 h, and frozen in LN₂ vapour for 15 min before plunging into LN₂ for storage. Straws were thawed in 37°C water for 30 s and the sperm was analysed for total and progressive motility, sperm velocities, and linearity with the CASA and sperm viability using flow cytometry. Treatment differences were determined using ANOVA, and treatment means separated using SNK. Sperm viability was similar in all treatments (58–67%; P > 0.05). However, sperm treated with MF and DMF showed higher percentages of progressively motile sperm (22–35%) compared with sperm frozen in glycerol alone (15%; P < 0.05). In Experiment 2, osmotic tolerance limits of bat sperm were determined. Epididymal sperm from 10 individual males were assessed for concentration and motility and diluted to 100–120 × 10⁶ sperm/mL in TALP. For each male, 10 mL of sperm were added to 75 mL of TALP with varying osmolalities (final concentrations of 0, 50, 75, 150, 225, 270, 300, 350, 370, 425, 600, 1200 mOsm). Sperm were incubated in these solutions for 5 min at 22°C, then diluted back to 300 mOsm and analysed for viability as described above. Results indicate that Aj bats have a wide osmotic tolerance, maintaining viability in all aniosmotic solutions; however, sperm at 0 and 1200 mOsm exhibited lower viability (<43%) than treatments between 50–600 mOsm (58–74%; P < 0.05). Collectively, these results indicate that MF and DMF in combination with glycerol result in higher post-thaw viability than glycerol alone for Aj bat sperm and that bat sperm may be able to withstand exposure to a wide variety of cryoprotectants. This information is valuable for optimising cryopreservation of bat spermatozoa for use in future ART applications.

We thank the LAR staff, Maria Alexandra Marquez, and Carolina Acevedo for their help with these experiments.