Osmotic tolerance and cryopreservation of Jamaican fruit bat (Artibeus jamaicensis) sperm

Abstract

Assisted reproductive technologies will become increasingly important to preserve bat genetics to prevent the extinction of bat species and provide biomedical models for viral and disease research. However, little is known regarding the ability to preserve bat genetics, including sperm. These experiments were conducted to determine some of the physiological properties of sperm from the Jamaican fruit bat (Artibeus Jamaican’s), and develop a protocol to effectively cryopreserve these sperm. Experiments were conducted to determine the osmotic tolerance of bat sperm, using flow cytometry, to determine how well bat sperm can withstand the osmotic challenges they must endure during the cryopreservation process. Additional experiments tested various cryoprotectants to determine which cryoprotectant preserved sperm most efficiently; and finally, if cryopreserved sperm maintained functionality by binding to the perivitelline membrane. Jamaican fruit bat sperm exhibit a wide osmotic tolerance; greater than 50 % of the sperm survive when exposed to very low (0 mOsm) or very high (1.2 M) osmolalities. In addition, the sperm are not sensitive to cold-shock damage, and can be cooled from room temperature to 5 ºC very quickly while retaining viability. Finally, preserving the cells, using a combination of glycerol and either methyl formamide (MF) or dimethyl formamide (DMF) as the cryoprotectant, resulted in higher percentages of motile sperm (54 %) than sperm frozen in glycerol alone (35 %; p < 0.05). Interestingly, the percentages of sperm with intact membranes following cryopreservation were similar for all cryoprotectant treatments (58–67 %; p > 0.05). Our ability to test sperm functionality after cryopreservation was limited to testing the sperm’s ability to bind the perivitelline membrane, in which higher numbers of sperm cryopreserved with glycerol + MF bound to the membrane (19.6 ± 5.5) than sperm cryopreserved in glycerol alone (12.0 ± 5.2; p < 0.05). In conclusion, Jamaican fruit bat sperm can effectively withstand membrane damage caused by osmotic issues and cold shock. In addition, bat sperm cryopreserved with a combination of glycerol and amides maintain higher percentages of motile cells, than sperm cryopreserved in glycerol alone; and cryopreserved sperm maintained their ability to bind to the perivitelline membrane.