Somatic cells derived from haploid larvae are feasible as donors for nuclear transplant in zebrafish. Preliminary results.

Summary 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.

Ultraviolet radiation and handling medium osmolarity affect chimaerism success in zebrafish.

The effects of a predefined ultraviolet radiation dose (0.529 mW/cm2 for 30s) together with two different micromanipulation medium osmolarities (30 mOsm/kg vs 300 mOsm/kg) were tested on embryo survival at different developmental stages and on the somatic (skin) and germ-line chimaerism rates. Somatic (13%, 6/47 adults) and germ-line chimaerism (50% pigmented F1 larvae) were detected only in the UV-treated recipient embryos micromanipulated in a 300 mOsm/kg medium.

Transplantation of adult fibroblast nuclei into the central region of metaphase II eggs resulted in mid-blastula transition embryos.

Recently, a novel technical method to perform somatic nuclear transplantation (NT) in zebrafish using nonactivated eggs as recipients without the need to detect the micropyle was developed in our lab. However, the use of spermatozoa as an activating agent prevented to know whether the inserted nucleus compromised embryonic and early larval developmental ability. The aim of the present work was to test the developmental ability of the embryos reconstructed by transplanting adult fibroblast nuclei into the central region of the metaphase II egg but subsequently activated by only water.

Micromanipulation medium osmolarity compromises zebrafish (Danio rerio) embryo and cell survival in chimaerism experiments.

In zebrafish chimaerism experiments, the cell injection can involve intra-embryonic cell lyses by osmolar effects. Moreover, the donor cells can be injured during manipulation due to osmolar changes into the transplant pipette. Therefore, the present study aimed to assess the effects of manipulation medium osmolarity on embryonic survival and donor cell viability.

Ultraviolet radiation dose to be applied in recipient zebrafish embryos for germ-line chimaerism is strain dependent.

Germ-line chimaerism is a powerful technique that has proved to be useful to produce viable gametes when transplanted blastomeres colonize the germinal ridges in recipient embryos and obtaining offspring from such transplanted cells. In fish, ionizing radiations were commonly used for embryo penalization to cancelling the cell participation of recipient embryos in development and in gamete production. The ultraviolet (UV) radiation when compared with other radiation types is cheaper, easier and no special installations are required for its use.

Definition of three somatic adult cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization.

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.

Effect of gametes aging on their activation and fertilizability in zebrafish (Danio rerio).

The zebrafish represents an important model organism for biological research. In this context, in vitro collection and fertilization of zebrafish gametes are basic and widely used techniques for many topical research works. In this work, the fertilization ability and normal embryo development of gold-type zebrafish sperm and eggs were re-evaluated after being stored for different times at 8 degrees C in a modified medium (Hanks' saline supplemented with 1.

Osmolarity and composition of cell culture media affect further development and survival in zebrafish embryos.

With the aim of carrying out chimaerism and somatic cell-midblastula transition (MBT) embryos co-culture experiments in freshwater fish species, we evaluated the effect of osmolarity and composition of two media commonly used in cell fish culture on MBT zebrafish embryos and their further development and survival. To this end, wild zebrafish dechorionated embryos in midblastula stage were cultured for 6 days (Experiment 1: 189 embryos) or 1 h (Experiment 2: 150 embryos) in three different media: Hanks' 10% (H-10), 35 mOsm; Hanks' 100% (CH), 315 mOsm; and L-15 with serum (L-15: 315 mOsm). High osmolarity affected the survival rate (6 days: L-15: 45.

Severe oligoasthenoteratozoospermias, secretory and obstructive azoospermias: motility as a criterion of sperm viability.

Purpose: Comparison of pregnancy rates in cases of Secretory Azoospermias (SA), Obstructive Azoospermias (OA) and severe Oligoasthenoteratozoospermias (OATZ). Evaluation of sperm motility as a quality criterion.

Methods: In SA cases (n = 35), 9 samples were cryopreserved.

Vitrification of caudal fin explants from zebrafish adult specimens.

No data on vitrification of tissue samples are available in fishes. Three vitrification solutions were compared: V1: 20% ethylene glycol and 20% dimethyl sulphoxide; V2: 25% propylene glycol and 20% dimethyl sulphoxide, and; V3: 20% propylene glycol and 13% methanol, all three prepared in Hanks' buffered salt solution plus 20 percent FBS, following the same one step vitrification procedure developed in mammals. Caudal fin tissue pieces were vitrified into 0.