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A split-plot design was utilized to assess the motility of the two pre-spermatophore sample types at two temperatures, 0\u00b0C and 20\u00b0C (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\u00b0C) or on the benchtop (20\u00b0C). 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\u00b0C 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<jats:italic>in-vitro<\/jats:italic>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.<\/jats:p>","DOI":"10.1371\/journal.pone.0245047","type":"journal-article","created":{"date-parts":[[2021,1,12]],"date-time":"2021-01-12T20:21:25Z","timestamp":1610482885000},"page":"e0245047","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":16,"title":["Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time"],"prefix":"10.1371","volume":"16","author":[{"given":"Amanda B.","family":"Gillis","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmet L.","family":"Guy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew J.","family":"Kouba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter J.","family":"Allen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruth M.","family":"Marcec-Greaves","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8466-6077","authenticated-orcid":true,"given":"Carrie K.","family":"Kouba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,1,11]]},"reference":[{"key":"pone.0245047.ref001","unstructured":"IUCN. 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