16. Transplantation of cryopreserved testicular germ cells in flatfish species: a useful technique for reproductive biotechnology

Abstract

Cryopreservation of testicular germ cells plays an important role for biotechnology in fish reproduction. Cryopreserved spermatogonias can be transplanted between closely related species without losing the ability to differentiate in gametes into the host gonads. Due to this capacity, spermatogonia xenotransplantation is becoming a useful tool to produce surrogated broodstocks. These biotechnological practices can favour production for fish species that are not completely domesticated. The Senegalese sole ( Solea senegalensis ) is an important commercial species, nevertheless its reproduction in captivity still presents a challenge for researchers and farmers. On the contrary, turbot ( Scophthalmus maximus ) is one of the most produced flatfish species in European aquaculture and has a high potential to be a recipient species for xenotransplantation. Our main goal was to produce xenotransplanted turbot fish using cryopreserved spermatogonia from Senegalese sole. Vasa gene was cloned and used as a molecular marker for studying germ cell development in early stages of embryonic and larval development and in adult gonads by means of qPCR, in situ hybridization and inmunohistochemistry (vasa proteins). Testicular fragments obtained from young donors (12 months old) were cryopreserved in cryovials according to Cabrita et al. (Cryobiology 61 (2010) 405) in a portable N 2 -free controlled rate biofreezer (Asymptote EF600, Grant). Cell recovery, viability and DNA damage (comet assay) were assessed. To perform both inter and intraspecific transplantations, testes fragments obtained using the best cryopreservation protocol were thawed, dissociated, marked using PKH26 and successfully transplanted into recipient 6–20 DAH (days after hatch) Senegalese sole larvae and 18–42 DAH turbot larvae with no effects on the survival rate up to three weeks after microinjection. Previously several trials were performed to develop a method to deplete endogenous germ cells in recipient fish. Busulfan (40 mg/Kg) treated fish were depleted of germ cells since vasa expression decreased in gonads from female fish demonstrating to be a useful tool for recipients’ preparation. S . vasa qPCR expression demonstrated the presence of S. senegalensis cryopreserved germ cells into S. maximus larvae after 21 days post-transplantation.