Extending the viability of emu spermatozoa during in vitro storage by manipulation of temperature and diluent potassium concentration.

1. Survival of emu spermatozoa during in vitro storage is not affected by increasing the extracellular [K(+)] to the point where it does not adversely affect spermatozoa function. 2. In three experiments, the effects were studied of [K(+)] in a diluent in the range 12·5-80 mM/l on emu spermatozoa survival for up to 48 h at 5, 10 or 20°C. 3. At the end of the storage period, spermatozoa viability, motility, fertilising ability and morphology were measured. 4. In Experiment 1, spermatozoa viability and morphology were adversely affected after storage (P < 0·001) only in the diluent containing 80 mM/l [K(+)] whereas spermatozoa motility decreased as [K(+)] increased from 12·5 to 80 mM/l. 5. In Experiment 2, during storage at 5°C, the spermatozoa viability was comparable among any of the diluents (standard or modified) but morphology was better (P < 0·001) in all of the modified diluents than in the standard E3 diluent. 6. In Experiment 3, after 48 h of storage in a diluent containing 40 mM/l of [K(+)], the spermatozoa functions were better preserved at 10°C than at 5 or 20°C. 7. It is concluded that a higher than physiological level of potassium can be used in a diluent without detrimental effect on emu spermatozoa survival during 48 h storage and that the best outcome was with storage at 10°C rather than 5 or 20°C.

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/mm(2)). 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.

Sperm viability, motility and morphology in emus (Dromaius novaehollandiae) are independent of the ambient collection temperature but are influenced by storage temperature.

A protocol for storage of emu semen >6 h has not yet been optimized. The objective was to determine: a) whether sperm quality was adversely affected by sudden exposure to low temperatures (5, 10 and 20 °C) during collection; and b) the effects of three storage temperatures (5, 10 and 20 °C) on survival of emu sperm. In two experiments, each repeated three times on alternate days, ejaculates were diluted 1:1 with precooled (5, 10, or 20°C) UWA-E3 diluent and stored for up to 48 h. Collection temperature, or interaction with either the storage time or storage temperature, had no significant effect on sperm viability, motility, or morphology. Mass Motility Score (2.91-3.27 ± 0.26, mean ± SEM), and percentages of live (72.4-76.2 ± 2.4) and morphologically normal sperm (63.3-64.5 ± 2.3) were comparable among collection temperatures. Conversely, storage temperature and storage time affected (P < 0.05) sperm viability, motility, and morphology. After storage for 48 h, percentages of viable, normal, and motile sperm were higher (P < 0.001) at 5 °C (58.7% ± 1.1, 44.7% ± 1.3, and 50.7% ± 4.9, respectively) and 10 °C (62.6% ± 1.1, 54.1% ± 1.3, and 60.4% ± 4.9) than at 20 °C (27.6% ± 1.1, 20.1% ± 1.3, and 25.9% ± 4.9). Beyond 6 h of storage, the percentage of abnormal sperm was higher (P < 0.001) for storage at 5 °C compared to 10 and 20 °C. After 48 h, bacterial counts were considerably higher at 20 °C compared to 5 and 10 °C (P < 0.001). The pH of stored sperm suspension remained unaffected at 5 and 10 °C, but at 20 °C declined to 6.5 ± 0.03 after 24 h (P < 0.05) and to 6.0 ± 0.03 after 48 h (P < 0.001). We concluded that emu semen could be collected at low ambient temperatures (5-20 °C) without compromising its in vitro storage duration and that semen quality during storage for 48 h was better if it was stored at 10 °C than at 5 or 20 °C.

Response of spermatozoa from the emu (Dromaius novaehollandiae) to rapid cooling, hyperosmotic conditions and dimethylacetamide (DMA).

Three experiments conducted to improve the survival of emu sperm during cryopreservation aimed to: (1) minimize chilling injury during the cooling phase; (2) determine the osmotic effects of dimethylacetamide (DMA), sucrose and trehalose; and (3) investigate the timing and nature of cryoprotectant toxicity. We measured sperm membrane integrity, motility, morphology and egg membrane penetration. In Experiment 1, semen diluted 1:1 with a pre-cooled diluent (5°C) prevented chilling injury. In Experiment 2, semen was diluted with DMA, trehalose or sucrose (300-2400mOsm/L) in deionized water. Only added DMA decreased the percentage of morphologically normal sperm. The percentage of motile sperm was higher with DMA than with the sugars, but membrane intact sperm were comparable amongst all cryoprotectants. As for the osmotic effects, the percentage of membrane intact sperm decreased with 2400mOsm/L and sperm motility decreased with 1200-2400mOsm/L, but sperm morphology was similar at all osmolarities. In Experiment 3, sperm membrane integrity, motility and morphology were comparable at all DMA osmolarities between sperm equilibrated for 0 and 15min, and remained unchanged after removal of DMA. We conclude that: (a) loss of sperm function during the cooling phase can be avoided by using a diluent maintained at 5°C; (b) emu spermatozoa tolerate upto 1400mOsm/L; (c) DMA results in a permanent change in sperm morphology when it is dissolved in deionized water, but does not alter sperm membrane integrity and motility; and (d) equilibration time of sperm with DMA can be less than 10min.