Cryobanking the reproductive potential of Solea senegalensis

Abstract

Cryopreservation of testicular germ cells can guarantee the conservation of a given species due to spermatogonia capacity in differentiating into gametes. Moreover these cells have the ability to proliferate into a host individual after transplantation producing in their gonads functional gametes depending on the host sex determination. In fish with reproductive problems such as the Senegalese sole, this may contribute to produce a surrogate broodstock using spermatogonia xenotransplantation. For that purpose cryopreservation is a useful tool to store germ cells. Cryopreservation of testicular germ cells have been attempted in several mammalian species (Brook et al., 2001, Redden et al., 2009) and more recently in some fish (Yoshizaki et al.,2007). In the present study testicular germ cells from Senegalese sole (Solea senegalensis) were cryopreserved testing different cryopreservation solutions, packaging systems (straws or cryovials) and freezing methods (field method or programmed biofreezer). For that, testes were extracted from 18-months year old juveniles (weight: 44±14 g, length: 16.54±1.3 cm). Fish were anesthetized with a lethal phenoxyethanol doses (2,000 ppm) and the small testes were surgically removed. Two cryopreservation methods were tested both in cell suspensions obtained after testes trypsinization (n=21, 0.25% trypsin in PBS + 0.5% FBS + DNAse-I 200 units, 2 h, 22ºC) or in small testes fragments (n=35) which were trypsinized after thawing. Cellular suspensions and testes fragments were cryopreserved in PBS or L-15 based medium supplemented with 0.5% BSA and 5.5 mM glucose with 1.5 M DMSO or 1.5 M glycerol (modified from Yoshizaki et al., 2007). Cells were frozen in 0.5 ml French straws and testes fragments in cryovials. In a second experiment, testes cryopreservation was performed using a portable programmed biofreezer (Grant Asymptote) using the same conditions described before. Freezing and thawing rates were monitorized using a thermocouple inside the straws and cryovials. Cell integrity and viability were determined in postthaw samples using the dual stain IP/SYBR-14 and Calcein AM. Both cryopreservation methods provided good post-thaw viability, although freezing small testes fragments was considered a more practical procedure to preserve testicular germ cells in this species. More studies need to be conducted in order to improve protocols and transplantation technique.