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

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 split-plot 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 in-vitro 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.

Establishment of a Practical Sperm Cryopreservation Pathway for the Axolotl (Ambystoma mexicanum): A Community-Level Approach to Germplasm Repository Development

“…The large size of axolotl sperm (among the largest of vertebrates) prevents the use of typical settings in CASA systems, and thus motility was classified without CASA in this work. Previously published reports classified salamander sperm motility into two categories: 'motile' (circular movement) or 'not motile' (undulating and static) [9], or 'motile' (circular plus undulating) and 'non-motile' (static) [15]. In the present study, motility was categorized into three classes.…”

“…The two common methods of noninvasive sperm collection for salamander species are the collection of spermatophores [13,14] and the collection of sperm fluid by stripping [9,15]. The collection of spermatophores and stripping have each been used in axolotls [7,9,14] and other Ambystoma species [15,16].…”

“…The two common methods of noninvasive sperm collection for salamander species are the collection of spermatophores [13,14] and the collection of sperm fluid by stripping [9,15]. The collection of spermatophores and stripping have each been used in axolotls [7,9,14] and other Ambystoma species [15,16]. Each provides unique benefits and constraints that may affect the potential for medium-or high-throughput processing necessary for repository production and management.…”

Oral administration of GnRH via a cricket vehicle stimulates spermiation in tiger salamanders (Ambystoma tigrinum)

“…For reproductive management, anuran species have traditionally been given GnRH through intraperitoneal, intramuscular, dorsal lymph sac, and subcutaneous injections, with the intraperitoneal method being used most frequently [21][22][23][24]. In comparison, the attempts to administer GnRH to caudates have generally been via epaxial intramuscular injections [19,[25][26][27]. Many caudate species can be challenging to administer hormones to, not only because of their size (many below 10 grams), but also their quick movements during handling.…”

Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking

“…Amphibian semen can be refrigerated (4°C) for temporary holding or cryopreserved (−196°C) for long-term storage ( Browne et al 2002 , 2019 ). For some species, semen has been held at 4°C for 30 days with retained viability ( Browne et al 2001 ), and refrigerated semen has been used in over 40 amphibian species, with outcomes including retrieval of motile sperm and fertilisation in vitro ( Browne et al 2001 , Keogh et al 2017 , Gillis et al 2021 a ). Refrigerated and cryopreserved semen are the two most successful ARTs for amphibians, with semen capable of storage in whole testes and sectioned testicular strips, as well as spermic urine ( Poo & Hinkson 2019 ).…”