Sperm collection and cryopreservation for threatened newt species

Authors: GUY, EMMET - Mississippi State University; GILLIS, AMANDA - Mississippi State University; KOUBA, ANDREW - Mississippi State University; BARBER, DIANE - Fort Worth Zoo; POOLE, VICKY - Fort Worth Zoo; VANCE, CARRIE - Mississippi State University

Submitted to: Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2020
Publication Date: 5/11/2020
Citation: Guy, E., Gillis, A., Kouba, A., Barber, D., Poole, V., Vance, C. 2020. Sperm collection and cryopreservation for threatened newt species. Cryobiology. 94:1-9.

Interpretive Summary: Aspects of bird and fish reproduction can be modeled in a variety of amphibian species that exhibit similar gamete development processes and tolerances to environmental stress. Caudate amphibians exhibit internal fertilization and the thermo tolerances of caudate sperm in different natural packaging forms provides insight into how sperm structure responds to external environments, and how well fundamental principles of sperm survival transmit across species for genetic conservation. Sperm from our model species are examined for cellular structure and function using fluorescent microscopy and micro-NIR techniques to study the membrane integrity and flagellar functionality of naturally expressed sperm cells.

Technical Abstract: To determine the effectiveness of sperm cryopreservation, methods for evaluating sperm quality are necessary but underdeveloped in caudate research. 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 to identify degrees of cell viability, apoptosis and necrosis. Annexin-V marks the externalization of phosphatidylserine on the cell membrane indicating early steps in the apoptosis signaling cascade. Compromised membranes allow PI, a nucleic acid stain, access to DNA, marking cellular necrosis. SYBR-14 is a nucleic acid stain that does permeate intact membranes, labeling live cells. These fluorescent stains were assessed for marking viability and stages of cell death in post-thaw spermatozoa across three caudate species, the Eastern tiger salamander (Ambystoma tigrinum), Kweichow Emperor newt (Tylototriton kweichowensis), and Black-spotted newt (Notophthalmus meridionalis). The aims of this project were to transfer hormone-induced spermiation and sperm cryopreservation developed in the model salamander species, Ambystoma tigrinum, to three threatened newt species. Additionally, we tested if supplementation with trehalose or thawing at different temperatures impacts post-thaw sperm parameters. Hormone stimulation protocols were applied to male Notophthalmus meridionalis (N = 10), Neurergus kaiseri (N = 5) and Tylototriton kweichowensis (N = 6) with sperm collected periodically up to 24-28 hours post-spermiation dose. Samples of adequate sperm concentration (> 70%) were cryopreserved in solutions of 10% Me2SO + 1% BSA with or without a 10% trehalose cryodiluent. Frozen sperm samples were thawed at either 20°C or 40°C and examined for post-thaw motility parameters and abnormalities in head and tail structure. The spermiation response to exogenous hormone treatment was significantly different between newt species, with a success rate of 0% for N. kaiseri, 67% for T. kweichowensis, and 100% for N. meridionalis. Sperm concentration varied with time of collection after hormone administration in both T. kweichowensis and N. meridionalis. For N. meridionalis, structural abnormalities decreased in samples collected over the 24 hour period (p < 0.0001) and a thaw temperature of 40°C resulted in higher relative total sperm motility (p < 0.0001). This is the first study to describe the cryopreservation of sperm from two newt species and demonstrates the transferability of ART developed in a salamander to two newt species.