Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time

Authors: GILLIS, AMANDA - Mississippi State University; GUY, EMMET - Mississippi State University; KOUBA, ANDREW - Mississippi State University; ALLEN, PETER - Mississippi State University; MARCEC-GREAVES, RUTH - Pelican River Watershed District; VANCE, CARRIE - Mississippi State University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2020
Publication Date: 1/11/2021
Citation: Gillis, A.B., Guy, E.L., Kouba, A.J., Allen, P.J., Marcec-Greaves, R.M., Vance, C.K. 2021. Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time. PLoS ONE. 16(1):e0245047. https://doi.org/10.1371/journal.pone.0245047.
DOI: https://doi.org/10.1371/journal.pone.0245047

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 (salamanders) 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 over extended periods of time and under different environmental circumstances with the aim of maiximizing the sperm survivability, using fluorescent microscopy and micro-NIR techniques to study the membrane integrity and swimming capacity of naturally expressed sperm cells to optimize fertilization. The most effective mode of sperm storage for transportation and genetic management is different than found for frog-like species, and higher cellular content that is more stable in extreme salt-and sugar environments. This new-knowledge provides a novel approach to storing aqua-cultural and agricultural genetic lines of species in biobanking and germ plasm repository science.

Technical Abstract: The aims of this project were to characterize tiger salamander (Ambystoma tigrinum) spermatozoa motility over time, when excreted as either milt or spermic urine prior to packaging into a spermatophore, and to determine the effect of temperature on sperm motility. A splitplot design was utilized to assess the motility of the two pre-spermatophore sample types at two temperatures, 0°C and 20°C (n = 10 for each treatment). Spermiation was induced through exogenous hormone treatment of luteinizing hormone releasing hormone analog in order to collect both milt and spermic urine, which were evaluated for motility, divided into two separate aliquots, and subsequently stored in either an ice-bath (0°C) or on the benchtop (20°C). The decay rate of sperm motility was assessed by reevaluating subsamples at 0.5, 1, 2, 3, 5, 7, and 24 hours following the initial assessment. Results showed that sperm stored at 0°C had significantly higher progressive, non-progressive, and total motility for both sperm collection types over time. An interaction was found between collection type and time, with milt exhibiting lower initial motility that was more sustainable over time, compared to spermic urine. For both milt and spermic urine, motility decreased rapidly with storage duration, indicating samples should be used as soon as possible to maximize motility for invitro fertilization and cryopreservation. This is the first study to describe the differences in sperm motility between milt and spermic urine from an internally fertilizing caudate and demonstrates the benefits of near freezing temperatures on sperm longevity.