Somatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.

Zebrafish somatic nuclear transplant has only been attempted using preactivated eggs. In this work, three methods to carry out the nuclear transplant using adult cells before, during and after the egg activation/fertilization were developed in zebrafish with the aim to be used in reprogramming studies. The donor nucleus from somatic adult cells was inserted: (method A) in the central region of the egg and subsequently fertilized; (method B) in the incipient animal pole at the same time that the egg was fertilized; and (method C) in the completely defined animal pole after fertilization. Larval and adult specimens were obtained using the three methods. Technical aspects related to temperature conditions, media required, egg activation/fertilization, post-ovulatory time of the transplant, egg aging, place of the donor nucleus injection in each methodology are presented. In conclusion, the technical approach developed in this work can be used in reprogramming studies.