Isolation and cryopreservation of early stages of germ cells of tench (Tinca tinca)

Abstract

A practical technique for isolation and cryopreservation of tench (Tinca tinca) (Cyprinidae, Teleostei) early stages of germ cells (GC), including spermatogonia and spermatocytes, is reported for the first time. The germ-line cells possess the ability to differentiate into functional gametes of both sexes. These early stages of germ cells are small enough to be well-suited to cryopreservation, which, together with their high level of plasticity, makes their preservation a promising tool for maintaining genetic resources. Testicular cells were distinguished and separated by Percoll gradient, with the highest proportion of GC (62.2%) obtained from the 30% layer. The concentration and viability of GC were determined, and specific staining (DDX4) for germ cells was used to distinguish GC from somatic cells. Early stages of germ cells were cryopreserved in an extender composed of phosphate buffered saline (pH 8) with 0.5% BSA, 50mM d-glucose, and containing 1.5M cryoprotectant in the pre-programmed PLANER Kryo10 series III using a cooling protocol from +10°C to -80°C at a rate of 1°C/min. The effect of six cryoprotectants -methanol, dimethyl sulfoxide, dimethyl sulfoxide + propanediol (1 : 1), glycerol, ethylene glycol, and dimethylacetamid was assessed, and the results were evaluated by comparing the percentage of viable frozen/thawed GC by ANOVA, Tukey's HSD test (P < 0.05). Almost the same viability rates were obtained with no significant differences among tested cryoprotectants, indicating high stability of GC in cryoprotectants. Nevertheless, glycerol at a concentration of 1.5M was associated with the highest survival rate of thawed tench GC (57.69 ± 16.85%).

Cryopreservation of Fish Spermatogonial Cells: The Future of Natural History Collections

“…This increased the percentage of sperm and reduced the overall percentage of large spermatogonial cells in the testes, constraining our analysis of post-thaw large cells. Previous studies have used genetic constructs with Green Florescent Protein (GFP) to mark their viable spermatogonia cells 22 , 23 post-thaw, used Percoll gradients to separate cells 34 or increased their overall numbers of large cells in the testes by using immature males 33 . Some freshwater fishes showed cross-reactivity in VASA antibody staining of their spermatogonial cells, but A .…”

Equilibration and freezing interactions affecting bull sperm characteristics after thawing

“…Extenders used serve as protection during cooling and freezing (Linhartova et al 2014) and preserve spermatozoa motility after subsequent thawing (Muchlisin et al 2015). Nevertheless, the most suitable interactions of extender vs equilibration length still are not determined sufficiently (Shahverdi et al 2014).…”

Cryopreservation of testicular tissue from Murray River Rainbowfish, Melanotaenia fluviatilis

“…Early stages of the germ line, specifically the oogonia and spermatogonia, are large, round cells found in the gonads of female and male fish, respectively. These cells have been successfully cryopreserved in various fish species including tench (Tinca tinca) 11 , tiger puffer (Takifugu rubripes) 12 , brown trout (Salmo trutta) 13 , rainbow trout (Oncorhynchus mykiss) 14 , manchurian trout (Brachymystax lenok) 15 , marine goby (Asterropteryx semipuctata) 16 , Siberian sturgeon (Acipenser baerii) 17 , sterlet (Acipenser rethenus) 18 and Chinese rosy bitterling (Rhodeus ocellatus ocellatus) 19 . Spermatogonia are a flexible target for biobanking compared to spermatozoa, as these cells can be collected from a fish at any life stage, without having to target adult fish during spawning periods.…”