Short-term storage and management of sperm in vitro is an easy and economical process in which suitable extenders can be utilized to extend the storage period and prevent sperm function impairment. Therefore, the current study aimed to evaluate the effect of suitable extenders during the short-term storage of sterlet sperm and determine their fertilizing capacity and hatching success. Three extenders containing a composition of 16, 20, and 24 mM NaCl, 1 mM KCl, 0.1 mM CaCl2, 10 mM Tris, pH 8.0 with osmolarity of 46, 55, and 62 mOsm/kg, were used to dilute the sperm of four sexually mature sterlet males (n = 4). Using a CASA system, the motility and velocity of undiluted and diluted sperm with extenders (E1 - E3) were assessed over 6 days at 0-2 °C. The short-term stored diluted sperm was then used in the fertilization and hatching assay, and undiluted fresh and stored sperm was used as a control. A two-way factorial analysis of variance (ANOVA) model confirmed significant effects on sperm motility, curvilinear velocity (VCL), and straight-line velocity (VSL) (P < 0.001), as well as their interaction with the extender. The model was decomposed into a one-way ANOVA to examine the impacts of extenders and storage time. With increasing storage periods, the sperm motility and velocity gradually decreased for diluted sperm with three extenders (E1-E3) but sharply decreased for undiluted sperm (Control). The motility of undiluted sperm was found 3.77 ± 4.09% at 4 days, whereas sperm diluted with extenders showed 57.57 ± 12.33% (E1), 64.34 ± 11.86% (E2), and 61.40 ± 12.41% (E3) motility at 6 days. This study explored extenders optimized with higher osmolarity (39-62 mOsm/kg) and lower K+ (1 mmol/L) as the most suitable medium for storing sterlet sperm for 6 days. After 6 days post storage, sperm diluted with extenders E1-E3 achieved a fertilization rate of 31.29 ± 14.2%, 31.66 ± 8.84%, and 30.67 ± 10.02%, respectively, and hatching success of 29.58 ± 13.4%, 30.50 ± 7.89%, and 27.95 ± 9.62%, respectively with freshly ovulated eggs.

• Klíčová slova
• CASA, Fertilization rate, Hatching rate, Sperm motility, Sperm short-term storage, Sperm velocity,
• MeSH
• fertilizace * účinky léků   MeSH
• motilita spermií * účinky léků   MeSH
• ryby * fyziologie   MeSH
• spermie * fyziologie   účinky léků   MeSH
• uchování spermatu * veterinární   metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Sturgeons are the most endangered species group and their wild populations continue to decrease. In this study, we apply intracytoplasmic sperm injection (ICSI), an assisted reproductive technology, for the first time in endangered and critically endangered sturgeons. Using various egg-sperm species combinations we performed different ICSI experiments with immobilized pre- or non-activated spermatozoa, single or many, fresh or cryopreserved. Then we evaluated the fertilization success as well as the paternity of the resultant embryos and larvae. Surprisingly, all experimental groups exhibited embryonic development. Normal-shaped feeding larvae produced in all egg-sperm species-combination groups after ICSI using single fresh-stripped non-activated spermatozoa, in one group after ICSI using single fresh-stripped pre-activated spermatozoa, and in one group after ICSI using multiple fresh-stripped spermatozoa. ICSI with single cryopreserved non-activated spermatozoa produced neurula stage embryos. Molecular analysis showed genome integration of both egg- and sperm-donor species in most of the ICSI transplants. Overall, ICSI technology could be used as an assisted reproduction technique for producing sturgeons to rescue valuable paternal genomes.

• Klíčová slova
• assisted reproduction, embryo, intracytoplasmic sperm injection, larva, sturgeon,
• Publikační typ
• časopisecké články MeSH

Spermatogenesis is a continuous and dynamic developmental process, in which a single diploid spermatogonial stem cell (SSC) proliferates and differentiates to form a mature spermatozoon. Herein, we summarize the accumulated knowledge of SSCs and their distribution in the testes of teleosts. We also reviewed the primary endocrine and paracrine influence on spermatogonium self-renewal vs. differentiation in fish. To provide insight into techniques and research related to SSCs, we review available protocols and advances in enriching undifferentiated spermatogonia based on their unique physiochemical and biochemical properties, such as size, density, and differential expression of specific surface markers. We summarize in vitro germ cell culture conditions developed to maintain proliferation and survival of spermatogonia in selected fish species. In traditional culture systems, sera and feeder cells were considered to be essential for SSC self-renewal, in contrast to recently developed systems with well-defined media and growth factors to induce either SSC self-renewal or differentiation in long-term cultures. The establishment of a germ cell culture contributes to efficient SSC propagation in rare, endangered, or commercially cultured fish species for use in biotechnological manipulation, such as cryopreservation and transplantation. Finally, we discuss organ culture and three-dimensional models for in vitro investigation of fish spermatogenesis.

• Klíčová slova
• fish, florescence-activated cell sorting (FACS), germ cell culture, magnetic-activated cell sorting (MACS), spermatogenesis, spermatogonial stem cell (SSC),
• MeSH
• buněčné kultury * MeSH
• kmenové buňky cytologie   ultrastruktura   MeSH
• ryby metabolismus   MeSH
• separace buněk * MeSH
• spermatogeneze MeSH
• spermatogonie cytologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Several steps of sturgeon somatic cell nuclear transfer (SCNT) have been recently established, but improvements are needed to make it a feasible tool to preserve the natural populations of this group of endangered species. The donor cell position inside the recipient egg seems to be crucial for its reprogramming; therefore by injecting multiple donor somatic cells instead of a single cell with a single manipulation, we increased the potential for embryo development. Using the Russian sturgeon Acipenser gueldenstaedtii as a multiple cell donor and sterlet Acipenser ruthenus as the non-enucleated egg recipient, we obtained higher proportion of eggs developing into embryos than previously reported with single-SCNT. Molecular data showed the production of a specimen (0.8%) contained only the donor genome with no contribution from the recipient, while two specimens (1.6%) showed both recipient and donor genome. These findings are the first report of donor DNA integration into a sturgeon embryo after interspecific cloning. In all, we provide evidence that cloning with the multiple donor somatic cells can be feasible in the future. Despite the fact that the sturgeon cloning faces limitations, to date it is the most promising technique for their preservation.

• MeSH
• embryonální vývoj * MeSH
• genom * MeSH
• klonování DNA metody   MeSH
• ohrožené druhy statistika a číselné údaje   MeSH
• ryby embryologie   genetika   MeSH
• techniky jaderného přenosu * MeSH
• zachování přírodních zdrojů metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH