Characterization, cryopreservation and genetic manipulation of semen from sole Paralichthys orbignyanus (Teleostei: Paralichthyidae)

Author: Carlos Frederico Ceccon Lanes (Currículo Lattes)
Supervisor: Dr Luis Fernando Fernandes Marins
Co-supervisor: Dr Luís André Nassr de Sampaio


Paralichthys orbignyanus sole is an important fishing resource in the South Atlantic Ocean and is considered a species with great potential for aquaculture. Semen quality is an important variable in the reproductive management of cultivated species, directly influencing the production of viable eggs. In addition, knowledge of the physical and chemical characteristics of semen is an important factor in the development of semen cooling and cryopreservation protocols. The objective of this work was to characterize the semen of P. orbignyanus throughout the reproductive season and to use this information to assist in the semen cryopreservation process and in the development of a sperm-mediated gene transfer protocol (TGME). In the first stage of this study,semen samples from wild flounder were collected throughout the breeding season. Through the obtained results it was verified that the sperm production and the progressive motility increase significantly as the reproductive season progresses to its end (P <0.05). The progressive sperm motility lasted an average of 10 min, but the total motility time reached about 100 min. With regard to pH, osmolality and the concentration of K +, Cl- and Mg + ions measured in seminal plasma, as well as, for the percentage and mobile cells, no difference was verified throughout the reproductive season. In the middle of the season a decrease in the concentration of the Ca2 + ion was observed, which coincided with the shorter sperm motility time.The concentration of Na + increased over the monitored periods, reaching the highest concentration at the end of the reproductive season (P <0.05). In the second part of this study, cryopreservation of the semen of the sole P. orbignyanus was performed. For this, two cryoprotective solutions were tested: one containing glycerol diluted in a salt-based solution and another containing DMSO (dimethylsulfoxide) diluted in a sucrose-based solution. The data of fertilization, hatching and larval viability demonstrated that the two solutions used are efficient in the cryopreservation of sole semen. In the last stage of this study, some limiting factors in TGME in fish were evaluated and a protocol for incorporating exogenous DNA into sperm was developed. The results obtained in this part of the work demonstrated that the seminal plasma of P.orbignyanus has strong DNase enzyme activity. However, the activity of this enzyme can be eliminated or reduced by washing the semen with solutions containing EDTA. In addition, it was found that an amount of exogenous DNA similar to or less than 50 ng / 106 sperm should be used in TGME in fish. Finally, it has been shown that sperm from the P. orbignyanus sole are able to incorporate exogenous DNA spontaneously after eliminating DNases activity. This was evident through the amplification of exogenous DNA through PCR. Thus, the results obtained in this study can assist in the reproductive management of this species and, consequently,in the implementation of genetic improvement programs both in the classic way through the cryopreservation of the semen of the breeders that present the best phenotypic characteristics, as well as through modern techniques such as TGME.