Abstract
Three experiments were performed to evaluate the effects of osmolarity, temperature, media and cryoprotectants on white spotted bamboo shark (Chiloscyllium plagiosum) spermatozoa. Semen was collected from 7 different males using a massage technique with the animals held in tonic immobilization with gills submerged. A Computer Assisted Sperm Analysis (CASA) system was used to assess sperm motility. Experiment one evaluated the effects of Hank’s balanced salt solution (HBSS) with different osmolarities (200, 400, 800 and 1000 mOsm Kg1) and incubation temperatures (5 °C and 24 °C [room temperature]) over time (6 h, 12 h and 24 h). Membrane integrity and morphology were degraded (P < 0.05) over time; however, sperm extended with HBSS 1000 and HBSS 800 were least affected. Higher total motility (TM) and average path velocity (VAP, P > 0.05) sperm parameters were observed with HBSS 1000 and HBSS 800, respectively. In addition, TM, VAP, and curvilinear velocity (VCL) were all reduced (P < 0.05) compared to sperm held at 5 °C. Experiment two compared the effects of sperm diluted in crystal reef water (CRW), elasmobranch ringers (ER) or raw held at 5 °C. Results indicated that CRW had improved viability, TM, VAP, VSL, VCL, and straightness (STR) when compared to sperm diluted in ER or Raw at 5 °C. The final experiment compared different cryoprotectants (dimethyl sulfoxide [DMSO] 10%, DMSO 20%, Ethylene-glycol [EG] 10%, EG 20%, and Trehalose [Tre] 10%, Tre 20%) during incubation at 5 °C. Across all time points, sperm incubated in Tre (10 or 20%) had increased (P < 0.05) membrane integrity and normal morphology compared to all other media and raw. Across all time points, sperm exposed to Tre (10% and 20%) and 10% DMSO had significantly improved TM (P < 0.05) compared to EG (10% and 20%) and 20% DMSO which were similar to raw. Although sperm collected from WSBS are robust and appear to tolerate a wide range of osmotic conditions and media, media made at 1000 mOsm Kg1 should provide the best in-vitro osmotic pressure, while storing at cooler temperatures should improve storage survival. For future cryopreservation studies, media using TRE and DMSO as cryoprotectants should be evaluated.
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