Cryopreservation of sperm bundles (spermatozeugmata) from endangered livebearing goodeids

Abstract

More than half of fishes in the family Goodeidae are considered to be endangered, threatened, or vulnerable. Sperm cryopreservation is an effective tool for conserving genetic resources of imperiled populations, but development of protocols with livebearing fishes faces numerous challenges including the natural packaging of sperm into bundles. In this study the cryopreservation of sperm bundles (spermatozeugmata) of three goodeids species was evaluated. Sperm quality was evaluated by activation with NaCl-NaOH solution (at 300 mOsmol/kg and pH 11.8), and analysis of dissociable bundles and dissociation duration. Using Redtail Splitfin (Xenotoca eiseni) as a model, the effects of cryoprotectants (dimethyl sulfoxide, methanol, and glycerol) with different concentrations (5-15% v/v %), equilibration exposure times (1-60 min), cooling rates (5-40 °C/min), concentrations (4 × 10-4 × 10 bundles/ml), buffers (HBSS, PBS and NaCl), and buffer osmolalities (200-400 mOsmol/kg) were investigated. After cooling and thawing, sperm bundles maintained their packed form. A specific protocol was developed (10% dimethyl sulfoxide, 20-min equilibration, 10 °C/min cooling rate, 4 × 10 bundles/ml, and 300 mOsmol/kg HBSS). This protocol yielded 89 ± 5% of post-thaw dissociable bundles with 209 ± 10 s of dissociation duration for X. eiseni, 96 ± 9% with 814 ± 14 s for Blackfin Goodea (Goodea atripinni), and 66 ± 2% with 726 ± 25 s for Striped Goodeid (Ataeniobius toweri). This is the first study of cryopreservation of sperm within bundles for livebearing fishes and provides a basis for establishment of germplasm repositories for goodeids and other livebearers.

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, ).…”

“…One generalization for teleosts is that viviparous species form spermatozeugmata and that oviparous species release “free sperm” (Grier et al, ; Jamieson, ; Liu et al, ). Yet this is not true in all cases (e.g., Mann, ).…”

Production of live young with cryopreserved sperm from the endangered livebearing fish Redtail Splitfin (Xenotoca eiseni, Rutter, 1896)

“…Previous studies (Liu et al, 2018a) compared the effects of the cryoprotectants dimethyl sulfoxide (DMSO), methanol, and glycerol on quality of sperm bundles, showing that sperm within bundles had greatest post-thaw survival rates when mixed with 10% DMSO, equilibrated for 20 min, and cooled at 10 °C/min. Two trials were used in the present study to assess whether the protocol developed for bundles would be applicable to free sperm.…”

“…Previous studies have resulted in reports of sperm survival within bundles of goodeids after cryopreservation (Liu et al, 2018a), but no protocols have been developed for use with free sperm outside bundles and no live young have been produced with thawed sperm for goodeid fish. The goal of the present study was to investigate the feasibility to produce live young of viviparous goodeids with cryopreserved sperm, using an endangered species, the Redtail Splitfin ( Xenotoca eiseni, Rutter, 1896) as a model.…”

>b<Avaliação dos efeitos do tempo de equilíbrio nos protocolos de criopreservação do sêmen suíno>/b<: um estudo fisiológico e morfofuncional

“…Considerando as condições adequadas, quando uma solução é refrigerada abaixo do seu ponto de congelação, a água pura se cristaliza (GRAHAM, 1996;NEILD et al, 2002;MEYERS et al, 2003), e os solutos na fração de água líquida remanescente aumentam a pressão osmótica no espaço extracelular. ALMEIDA et al, 2017;GURLER et al, 2018;MURPHY et al, 2018), bubalinos (SHAHVERDI et al, 2014) equinos (JIMENEZ et al, 1987;BRISKO et al, 2000;PUGLIESI et al, 2012), suínos (YI et al, 2002SCHAFER et al, 2017), caprinos (DUTTA et al, 1996PRADEE et al, 2014) e cães (DU BOIS et al, 2012;BELALA et al, 2016), como também em espécies de vida selvagem e aquática (LÓPES-UREÑA et al, 2014;LIU et al, 2018;VIYAKARN et al, 2018…”