23 Viability staining techniques for cryopreserved spermatozoa in 3 caudata species

Salamanders are the most threatened vertebrate taxa; thus, conservation-based research including spermatozoa cryopreservation and other assisted reproductive technologies is essential to their survival. To determine the effectiveness of sperm cryopreservation, methods for evaluating sperm quality are necessary but underdeveloped in caudate research. Evaluating motility has been the primary analysis for sperm viability but is difficult to perform due to the scythe-like morphology, slow rotating progression, and minute undulations of the tail membrane. Estimating apoptosis is a new approach to evaluating caudate spermatozoa survival through cryostress. Fluorescent dyes, such as SYBR-14, annexin-V, and propidium iodide (PI), are valuable tools for identifying degrees of cell viability, apoptosis, and necrosis. Annexin-V marks the externalization of phosphatidylserine on the cell membrane indicating early steps in the apoptosis signalling cascade. Compromised membranes allow PI, a nucleic acid stain, access to DNA, marking cellular necrosis. The SYBR-14 is a nucleic acid stain that permeatesssss intact membranes, labelling live cells. These fluorescent stains were assessed for marking viability and stages of cell death in post-thaw spermatozoa across 3 caudate species: the Eastern tiger salamander (Ambystoma tigrinum), Kweichow Emperor newt (Tylototriton kweichowensis), and black-spotted newt (Notophthalmus meridionalis). For each species, spermic urine samples were acquired by hormone treatment and frozen based on protocols developed in A. tigrinum, yielding an average of 18.2% relative motility recovered at thaw. Straws were thawed for 5 min at 20°C. Viability was tested by staining 5μL 1:1 with a 1:50 dilution of SYBR-14 and 2 μL of PI. Stages of cell death were evaluated by staining 10 μL with 2 μL of annexin-V and 2 μL of PI. Cell viability was assessed immediately under a fluorescence microscope. For each of the 3 species, 2 samples were stained with both assays in triplicate. Sperm stained with SYBR-14 alone were considered viable, and sperm stained with any annexin-V or PI were considered not viable. Visible dynamic shifting from SYBR-14 to PI staining was observed in real time, indicating rapid necrosis. Morphological abnormalities, not observed in unstained samples, were prevalent across all species following staining, signifying a possible cytotoxic effect of the dyes. High mortality and abnormality rates suggest that fluorescent dyes have elevated toxicity and permeability in caudate sperm. Caudate spermic urine has a very low osmolality, implying high permeability, which could lead to rapid staining and toxicity effects. Shorter incubation times may be required for accurate staining. Results may also indicate that cryopreservation protocols need to be species specific and do not transfer well across taxa. This is one of the earliest studies to evaluate the use of fluorescent stain protocols on measuring cell viability in caudate sperm and indicates that further refinement is required.