Quality evaluation of sperm from livebearing fishes: Standardized assessment of sperm bundles (spermatozeugmata) from Xenotoca eiseni (Goodeidae)

Abstract

Standardized evaluation of sperm quality is essential for research, commercial-scale cryopreservation, and induced spawning. However, standardized methods for evaluation of sperm bundles (spermatozeugmata or spermatophores) have not been established. The purpose of the present study was to use Redtail Splitfin (Xenotoca eiseni) as a model for freshwater livebearing fishes to establish initial standardized methods to collect sperm bundles, and quantitatively and qualitatively evaluate quality-related attributes. No sperm or sperm bundles were able to be collected by stripping. Testes were removed, rinsed, weighed, placed in 50 μL of buffer solution on a glass slide, and crushed gently 3-5 times with angled spade-tip forceps. Sperm bundles were released into the buffer solution and collected with a pipette into 1.5-mL centrifuge tubes. To quantify size and shape, images of bundles were captured with a CCD camera connected to a microscope, and measured with computer software. There was no significant correlation between body wet weight and major bundle axis length (P = 0.6759), minor axis length (P = 0.5658), average axis length (P = 0.5869), aspect ratio (P = 0.7839), and observed area (P = 0.5727). The concentrations of sperm bundles, estimated with the three methods (Makler counting chamber, a hemocytometer, and direct counting) were significantly different (P < 0.0001). Hemocytometers were suitable for estimation of bundles from X. eiseni. To evaluate activation of sperm, bundles were viewed with a microscope, and classified into one of five phases by evaluating morphology of the bundles and motion of sperm within the bundles as Phase 0 through Phase 4 that represented early through late activation stages. The frequencies and duration of each activation phase were used to evaluate dissociation of sperm bundles and motility capability of sperm within the bundles. Within 180 min of activation, all five phases were observed. Overall, this study for the first time established standardized methods to collect and evaluate quality-related attributes of sperm bundles. These standardized evaluations provide a basis for further modification, standardization, and generalization, which are useful in research on livebearing fishes involving male gametes, such as studies on cryopreservation, artificial insemination, and in development of germplasm repositories for imperiled species including goodeids.

Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids

“…Livebearing fishes from the family Poeciliidae and Goodeidae each produce sperm bundles, however the independent evolution of the viviparity for these two groups resulted in different reproductive characteristics. For example, milt can be collected by stripping from poeciliids but not from goodeids [23]. As such, males were euthanized in the present study.…”

“…Previous studies showed sperm from X. eiseni could not be collected by stripping [23]. As such, fish were euthanized by exposure to 300 mg/l Tricaine methanesulfonate (MS-222, Western Chemical, Inc. WA, USA) followed by severing of the spinal column behind the head.…”

“…For example, after testes were crushed, bundle dissociation occurred rarely (in less than 10% of males) [24]. To date, there are no reports on cryopreservation of fish sperm in the form of bundles, although methods to evaluate quality of sperm within bundles have been developed previously [23]. The goal of the present study was to develop protocols for cryopreservation of sperm bundles from endangered livebearing goodeids using three species as models, Redtail Splitfin ( Xenotoca eiseni , listed as endangered in the 2005 report, but not assessed by the Red List), Blackfin Goodea ( Goodea atripinnis , of least concern in the 2005 report and the Red List), and Striped Goodeid ( Ataeniobius toweri, critically endangered in the 2005 report, and endangered in the Red List) [6, 21].…”

Testicular structure and spermatogenesis of the oviparous goodeids Crenichthys baileyi (Gilbert, 1893) and Empetrichthys latos Miller, 1948 (Teleostei, Cyprinodontiformes)

“…The viviparous goodeids discharge semen in large aggregates of cysts filled with spermatozoa, called spermatozeugmata, into the deferent ducts (spermiation) whereas empetrichthyins release “free sperm.” The presence of spermatozeugmata in goodeins has been documented previously (Grier et al, ; Uribe et al, , ). In species of the subfamily Goodeinae, as in Ilyodon whitei (see Grier et al, ; Grier and Uribe, ), Xenotoca eiseni (see Grier and Uribe, ; Liu, Torres, & Tiersch, , ; Uribe et al, ), Goodea atripinnis (see Liu et al, ; Uribe et al, ) and Ataeniobius toweri (see Liu et al, ), large groups of cysts with spermatozoa are bundled in spermatozeugmata in the testis and in the deferent ducts. The spermatozeugmata are formed by an enveloping layer of mucotic substance secreted by the Sertoli cells (Fishelson, Gon, Holdengreber, & Delarea, ).…”

Activation of free sperm and dissociation of sperm bundles (spermatozeugmata) of an endangered viviparous fish, Xenotoca eiseni

“…Each of a total of 5–15 spermatozeugmata distributed across the viewing area were categorized into one of five phases by evaluating morphology of the spermatozeugmata and motion of sperm within the spermatozeugmata as Phase 0 (P 0 ) through Phase 4 (P 4 ), representing early through later dissociation stages. The frequencies of each dissociation phase (FDP) were referred as FDP 0 through FDP 4 and were used to evaluate dissociation of spermatozeugmata and motility capability of sperm within the spermatozeugmata (Liu et al, 2017). At P 0 , the spermatozeugmata were quiescent; At P 1 , sperm were activated within spermatozeugmata but remained associated; At P 2 , the spermatozeugmata dissociated and sperm within them swam away; At P 3 , sperm stopped swimming away from the spermatozeugmata but remained vibrating in place; At P 4 , all sperm movement ceased.…”