ABSTRACT 


	In this thesis there are various experimental studies. They are primarily conducted in zebrafish and in the Pacific oyster as experimental models, and pursue the development of relevant techniques in the field of biomedicine, toxicogenomics, environmental risk assessment and aquaculture. 

	In zebrafish, there have been developed and tested:
?	Vitrification techniques of caudal fin tissue, blastomeres (in microvolumes) and of adult testicular tissue. 
?	The germ-line chimaerism technique at MBT stage of embryo development, with a previous penalization of recipient embryos by ultraviolet radiation.
?	Chimaerism technique at larval stage (48-72 h) with previously criopreserved testicular cells as donors obtained from adult individuals.
?	Nuclear transplantation technique using cell cultures from both adult somatic diploid cells and parthenogenetic haploid larvae as nuclei donors.
?	Electroactivation technique of zebrafish oocytes (in ionic medium).

	In Pacific oyster, there have been developed and tested: 
?	Assessment of seasonal evolution of gonadal maturation, gametes quality and fertilizability of Pacific oyster.
?	Electrofusion technology of Pacific oyster zygotes obtained by in vitro fertilization.









Vitrification techniques of caudal fin tissue, blastomeres (in microvolumes) and of adult testicular tissue.

	In this set of techniques, to highlight their different difficulty level as well as the results achieved. In this way, the vitrification of zebrafish caudal fin explants did not led to unexpected problems. In fact, efficacies achieved were in line with those achieved for the epthelial tissue cryobanking from five different mammalian species. This shows the versatility of this basic vitrification technique. In contrast, the blastomere cryopreservation has been a real challenge. The cell permeability patterns in aquatic species is very different from those in mammals. Therefore their final cryopreservation achievement came from an innovative approach: the blastomere vitrification in microvolumes (0.25µl) without their requirement of cryoprotectant permeation.
	Regarding the testicular tissue cryopreservation, there were tested three procedures, two of conventional freezing and a vitrification one. This last was finally selected for its best results acomplished. Also, their vitrification procedure became more efficient when it was applied previous to the tissue trypsinization.


Germ-line chimaerism technique at MBT stage of embryo development, with a previous penalization of recipient embryos by ultraviolet radiation.

	Regarding to the germ-line chimaerism at MBT stage various issues should be addressed. Firstly, the recipient embryo penalization with UV radiation is important but the not only requirement to chieve the best germ-line chimaerism results (50%). In fact, this penalization treatment must be completed with the embryo manipulation in a 300 mOsm/kg medium to avoid negative osmolar effects throughout the chimaerism procedure. Secondly, the importance of different embryo sensitivity to UV radiation according to the zebrafish strain used, being the gold strain more sensitive than the wild.
	With respect to chimaerism using previously vitrified blastomeres in microvolumes, many problems must be solved in future. An example was the morphologic disruption caused by the blastomere volume increasment after thawing and the subsequent requirement of using micromanipulation pipettes of increased size.


Chimaerism technique at larval stage (48-72 h) with previously criopreserved testicular cells as donors, obtained from adult individuals.

	This technique development was the logical consequence of a previous testicular tissue cryopreservation development indicated above. This technique is required to achieve the testicular cell integration, concretely spermatogonia, into the germ-line of recipient specimens. This procedure needed the use of 48-72 h age larvae, assuming that their immune system is not fully established and therefore it will not interfere with transplanted cells. Technical results on micromanipulation, larval survival and further development to adulthood were fully satisfactory. Despite these, no germ-line chimaerism was finally detected in any adult specimen. Among all reasons which would explain this, the more plausible by authors would be the possible dissynchrony between temporal patterns of spermatogonia development and those from testicular tissue, maybe faster than the first. 


Nuclear transplantation technique using cell cultures from both adult somatic diploid cells and parthenogenetic haploid larvae as nuclei donors.

	This group is perhaps the most original in techniques and the more forward looking from all provided in this thesis. Various items can be cited. Firstly, it was achieved a nuclear transplantation technique for non-activated oocytes. Moreover, none of the three nuclear transplantation (NT) developed techniques required the micropile detection as well as the previous recipient oocyte dechorionation. Secondly, it has been possible to compare the subsequent embryonic development effects among the three NT techniques assayed and with the fact that the oocyte was previously activated by either sperm fertilization (genetically inactivated or not) or by water stimuli. To this respect, the pipette puncture and water stimuli did not achieve further parthenogenetic development in any case when no cell nucleus was injected. This allows us to affirm the relevant participation of transplanted nucleus in the further embryo developmental ability. Thirdly, the recipient (egg) aging as a useful promoting factor of egg activation was rejected in contrast with the possitive results commonly observed in mammals.
	In this section, the use of cell nuclei donors from gynogenetic haploid larvae cultures in nuclear transplant of non-enucleated oocytes acquires a special mention. This technology would serve for genomic imprinting studies and even for a plausible alternative to the transgenic animal obtaining. The potentiality importance of this technique has only been evaluated preliminary in this thesis, although to date new experiments are being carried out.


Electroactivation technique of zebrafish oocytes (in ionic medium).

	The most relevant aspect of the procedure is perhaps the easiness of using ionic media for electroactivation, especially when non-ionic media are the most commonly used. To other respect, the best electroactivation procedure (one DC square pulse for 20µs at 5.4V and applied at 0, 10 and 20min post-activation in system water) allowed the activation of 68% non-manipulated oocytes. Unfortunately, when the best electroactivation sequence (that cited above) was assayed in nuclear transplanted eggs, it produced their immediate lysis. These aspects will be the key of future works to allow the coupling of egg electroactivation and NT techniques.


Assessment of seasonal evolution of gonadal maturation, gametes quality and fertilizability of Pacific oyster.

	This study do not refers the development of any micromanipulation technique. Despite this, it was essential for establishing the period of gametes availability throughout the year as well as their quality and allowed us to work, precisely, in the development of embryologic and micromanipulation techniques in this species. Results of this research were conclusive: only the period comprised from July to October assured the both gametes availability.


Electrofusion technology of Pacific oyster zygotes obtained by in vitro fertilization.

	Based on results reached above, zygotic electrofusion assays were carried out in the period from July to October for their possible potentiality in tetraploid oyster obtaining. In this regard, many interesting results came to light. On the one hand, it was observed that electric pulses (pursuing in this case the cell fusion) can be applied in both highly conductor (ionic) and high osmolarity media (in our case seawater, 1130 mOsm/kg). On the other hand, the fusion efficacy of Pacific oyster zygotes was high, and also they showed very low mortality rates as well as high normal developmental rates to D-larval stage (reached after 24 h culture post-electrofusion treatment). Also, it was observed that the electric treatment did not alter in a significant way the further cell cleavage patterns, assessed 25-30 min post-zygotic fusion. This would indicate the virtual damage absence on early embryos caused by the electric pulse.
	To other respect and because of the unavailability of specific installations and food required, treated embryos did not reach other key stages as “spat” or even the adulthood. At this moment, the ploidy assessment from obtained specimens at an individualized level is unavailable.