Survival of emu (Dromaius novaehollandiae) sperm preserved at subzero temperatures and different cryoprotectant concentrations

Three experiments were conducted to optimize the protocol for cryopreservation of emu sperm. Ejaculates were collected from trained male emus then diluted 1:1 and pooled before allocation to treatments and measured for sperm viability, motility, egg membrane penetration ability, membrane stability, and morphology. In Experiment 1, semen was either cooled to 5 °C after dilution or diluted with a precooled to 5 °C diluent before cooling to 5 °C and then frozen at liquid nitrogen vapor temperatures of −140 °C and −35 °C, with 6% or 9% dimethylacetmide (DMA; a permeating cryoprotectant) and compared for sperm functions. The percentages of viable (42.8 ± 1.1%), normal (39.0 ± 1.3%), and motile (29.8 ± 1.3%) sperm were higher (P < 0.001) for semen frozen at −14 °C with 9% DMA (path 2) than for all other combinations. In Experiment 2, we assessed the value of combining DMA and trehalose in the diluent. Combining trehalose (3% to 9%) with DMA (3% to 9%) prior to freezing reduced (P < 0.001) the percentages of postthaw viable (by 4 to 9 ± 1.2%), normal (by 5 to 11 ± 1.3%), and motile sperm (by 13 to 17 ± 2.5%) and the number of holes on the perivitelline layer (by 27 to 29 holes/mm²). Postthaw function was best preserved with 9% DMA alone. In experiment 3, we investigated the possibility of increasing DMA concentrations from 6% to 24%. Postthaw sperm viability (52 to 55 ± 2.3%) and morphology (48 to 51 ± 1.7%) were higher (P < 0.05) with 18% and 24% than with 6% to 12% DMA and did not differ between 18% and 24% DMA. However, sperm motility (36 to 43 ± 2.9%) and the number of perivitelline holes were similar (P > 0.05) for 9% to 18% DMA (36 to 55 ± 12%). We concluded that adding 6% to 9% trehalose to the diluent offered no advantage, and that the current best practice for preserving postthaw function in emu sperm is to dilute semen with a precooled to 5 °C diluent and use 18% DMA.