Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility≥60%), and were used for cryopreservation studies. Semen were frozen in TEST+glycerol, TEST+DMSO, HEPT+glycerol, or HEPT+DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0±4.3%; EM2: 26.0±17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5±8.1%; EM2: 46.3±6.4%) cryopreserved in TEST+glycerol were significantly higher than (P<0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST+glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.