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. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.

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. From the results obtained, we concluded that the conditions cited above were the most suitable to improve somatic and germ-line chimaerism rates in zebrafish.

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. So, the aim of this work was to establish the optimal UV radiation dose to be applied in zebrafish embryos at mid-blastula transition stage of development, in order to use them as penalized recipient embryos in futures chimaerism assays. A UV germicide lamp was used as radiation source (0.529 mW/cm(2)). Four exposure levels and three exposure times of UV radiation were tested. The survival rates obtained with the non-dechorionated embryos without lid group suggested that it could be the optimal exposure level to achieve the objective proposed. With the obtained results, we concluded that this UV radiation dose for 60 and 30 s are optimal parameters to penalize recipient wild and gold strain zebrafish embryos, respectively in chimaerism assays, but without involving their survival and apparently normal development.

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.In Experiment I, 0.1 microl to 0.15 microl approximately of an isosmolar solution (300 mOsm) was injected into recipient embryos, which were kept at 300 (E1) or 30 mOsm (E2). Survival at day 1 was significantly higher in the E2 group than in E1 (E1: 68% vs E2: 81%, p < 0.05), but after 5 days embryo survival in the E1 group was slightly higher. In Experiment II, donor cells from zebrafish embryos were exposed (or not) to a possible osmolarity change (inner pipette medium: 300 mOsm vs external medium: 30 or 300 mOsm) using two different micropipette outer diameters, 40-50 and 60-70 microm. Cell mechanical damage was detected in the 40-50 microm pipette (p < 0.05), but not by the handling medium osmolarity. Results recommend the use of a 300 mOsm manipulation medium and bore-sized pipettes adjusted as closely as possible to the donor cell size.

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. 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.

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: 45.1% v. CH: 72.34% v. H-10: 100%, P < 0.05; after 6 days: 0% both in L-15 and CH) and slowed their developmental timing. Embryos showed tail deformation (curly) as well as body paralysis at 48 h when they showed tail movements at 28 h. Differences in tail deformation were observed between high-osmolarity groups (CH: 85.10% v. L-15: 98.04%; P < 0.05). In Experiment 2, no effects on survival rate were observed. Teratogenic effects were only observed in L-15 (L-15: 12.98% v. CH: 0%; P< 0.05). Loss of motility was not detected in any group at 48 h. Optimum osmolar condition for cultured cells and also embryonic cells is around 315 mOsm and so, during chimaerism experiments (usually practised at MBT stage), present results indicate that midblastula embryos can acceptably bear the effects caused by 315 mOsm (CH) for 1 h, even though this involves a certain delay in developmental timing.

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. In OA cases (epididymal aspiration: n = 91; testicular biopsy: n = 206), all samples were cryopreserved. 596 OATZ ejaculates were included. RESULTS: In SA cases, 2 pregnancies were achieved from 9 ICSI cycles. In OA, motile sperm rates were higher in testicular biopsies. After thawing sperm motility was not different between testicular and epididymal origin. 2 pregnancies were achieved with immotile testicular sperm after thawing, but none with immotile epididymal sperm. In OATZ cases, one pregnancy was obtained from the 9 cryopreserved ejaculates and 35.3% with fresh motile sperm. CONCLUSIONS: In SA cases, the use of donor sperm is recommended due to the lower pregnancy rate achieved. Motility, before and after cryopreservation, as a criterion of sperm viability is discussed and its use should be reconsidered in some cases.

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 0.25 ml plastic straws in 30s and stored in liquid nitrogen for 3 days minimum, warmed (10s in nitrogen vapour and 5s in a 25 degree C water bath) and cultured (L-15 plus 20% FBS at 28.5 degree C). At the third day of culture, both attachment and outgrowing rates were recorded. V3 led to the worst results (8% of attachment rate). V1 and V2 allow higher attachment rates (V1: 63% vs V2: 50%. P < 0.05) but not significantly different outgrowing rates (83% to 94%). Vitrification of caudal fin pieces is advantageous in fish biodiversity conservation, particularly in the wild, due to the simplicity of procedure and equipment.