Australia has a rich diversity and high endemism (93%) of reptilian species, the largest family being Scincidae (252 species), yet there continues to be a paucity of reports for gamete cryopreservation in reptilians (Clulow and Clulow 2016 Reprod. Fertil. Dev. 28, 1116-1132, DOI: 10.1071/RD15466). The goal of this study was to collect sperm from a locally abundant skink species (Eulamprus quoyii; Eastern water skink) to examine sperm sensitivity to cryopreservation. Wild-caught males (n=50; snout-vent length=103-126mm) were held in seminatural conditions for the duration of the study. Semen was collected during the breeding season (September-October) using an adapted ventral-massage technique (Molinia et al. 2010 Herp. Cons. Biol. 5, 311-319; retrieved from http://www.herpconbio.org). Sperm metrics (volume, concentration, progressive and total motility, and membrane integrity) were assessed subjectively under light or fluorescence microscopy. Results were compared using ANOVA. Sperm volume (9.43±5.18μL) and concentration (7.79×108±5.32×108 sperm/mL) did not correlate with snout-vent length in adult males (r2=0.025 and r2=0.15, respectively). Due to small volumes, sperm samples (motility >80%) from 4 to 6 males were pooled before allocation across treatments. For all cryopreservation experiments, sperm samples (5-10μL) were loaded into 0.2-mL French straws and frozen using a controlled-rate freezer (Cryobath; −6°C per minute), then plunged into LN. Sperm were thawed in a water bath at 35°C for 10s. In year 1, pooled samples (n=3) were maintained at room temperature (21±1.5°C) either raw, or diluted in PBS, Tris-citrate glucose, TLHepes, or Ham’s F-10 and assessed at 0, 1, 3, 16, 40h. Tris-citrate-glucose-diluted sperm had significantly lower total motility from 3h (36.7±11.5%; P<0.05), decreasing to 1% motility by 16h. Up to 70% motility could be maintained for 16h in all other treatment groups. In year 2, pooled samples (n=3) were cooled to 4°C over a period of 2h, then gradually diluted 1:1 with buffer to a final concentration of either 0.6, 1.35, or 2.7M cryoprotectant (CPA; dimethyl sulfoxide, dimethyl acetamide, glycerol) in PBS or no CPA. Sperm diluted in 1.35 and 2.7M dimethyl sulfoxide in PBS had significantly (P<0.05) higher kinetic rating and proportion of live sperm than control or dimethyl acetamide treatments; 1.35 and 2.7M glycerol were intermediate. In year 3, dilution and cryopreservation using 1.35M CPA in complex diluents, Tris-yolk buffer (20.1±2.6% live) and Beltsville poultry semen extender (29.7±2.0% live), did not significantly improve sperm survival compared to PBS (26.4±2.7% live); however, post-thaw progressive motility (5±1.1%) was significantly (P < 0.05) higher with 1.35M dimethyl sulfoxide Tris-yolk buffer than all other treatment groups. In conclusion, dimethyl sulfoxide yields promise for sperm cryopreservation in a skink, but further studies are required.
Sperm Cryopreservation in American Flamingo (Phoenicopterus Ruber): Influence of Cryoprotectants and Seminal Plasma Removal
