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Surrogate broodstock technology is emerging as a promising biotechnology for xenogeneic production of gametes of highly valuable fish species. In this study, we produced gametes of landlocked Atlantic salmon using triploid rainbow trout as recipient through germ cell transplantation technique in embryos. Spermatogonial cells were obtained from Atlantic salmon juveniles and transplantation conducted into the coelomic cavity of 150 mixed-sex triploid embryos of rainbow trout. Colonization efficiency assessed in larvae four weeks post-transplantation showed presence of PKH-26-labeled cells in 61.1% of individuals. Analysis in sexually mature adults revealed presence of donor-derived sperm in four transplanted males (10%), whereby one of them reached sexual maturity in the first year. Transplanted females became mature after the second year and donor-derived oocytes were detected in four individuals (12.1%). Milt produced by transplanted triploid males were used for artificial insemination. Genetic analysis of hatchlings revealed that progeny was composed by pure Atlantic salmon and therefore were donor-derived. In conclusion, this study shows that surrogate broodstock can be used to produce oocytes and spermatozoa using species from different genus. The production of surrogate gametes using species with a shorter life cycle represents an efficient approach to streamline the development of improved strains in aquaculture industry.

Made available in DSpace on 2020-12-10T19:59:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-27 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Albinism is the most common color variation described in fish and is characterized by a white or yellow phenotype according to the species. In rainbow trout Oncorhynchus mykiss, aside from yellow-albino phenotypes, cobalt blue variants with autosomal, recessive inheritance have also been reported. In this study, we investigated the inheritance pattern and chromatophores distribution/abundance of cobalt blue trouts obtained from a local fish farm. Based on crosses with wild-type and dominant yellow-albino lines, we could infer that cobalt blue are dominant over wild-type and co-dominant in relation to yellow-albino phenotype, resulting in a fourth phenotype: the white-albino. Analysis of chromatophores revealed that cobalt blue trouts present melanophores, as the wild-type, and a reduced number of xanthophores. As regards to the white-albino phenotype, they were not only devoid of melanophores but also presented a reduced number of xanthophores. Cobalt blue and white-albino trouts also presented reduced body weight and a smaller pituitary gland compared to wild-type and yellow-albino phenotypes. The transcription levels of tshb and trh were up regulated in cobalt blue compared to wild type, suggesting the involvement of thyroid hormone in the expression of blue color. These phenotypes represent useful models for research on body pigmentation in salmonids and on the mechanisms behind endocrine control of color patterning. Agencia Paulista Tecnol Agronegocios, Secretaria Agr, Unidade Pesquisa & Desenvolvimento Campos Jordao, Salmonid Expt Stn Campos Jordao, Sao Paulo, Brazil Univ Sao Paulo, Sch Vet Med & Anim Sci, Dept Surg, Sao Paulo, Brazil Sao Paulo State Univ, Grad Program Biol Sci Genet, Inst Biosci Botucatu, Botucatu, SP, Brazil Tokyo Univ Marine Sci & Technol, Dept Marine Biosci, Tokyo, Japan Puriaqua Limitada, Sao Paulo, Brazil Agencia Paulista Tecnol Agronegocios, Secretaria Agr, Sao Paulo Fisheries Inst, Sao Paulo, Brazil Sao Paulo State Univ, Grad Program Biol Sci Genet, Inst Biosci Botucatu, Botucatu, SP, Brazil FAPESP: 2013/17612-9 FAPESP: 2014/00569-6

Parabens are widely used as preservatives in personal care products, medicines and foods, resulting in substantial human exposures, even though some harmful effects, such as endocrine-disrupting activity, have been reported. Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are members of the nuclear receptor superfamily, regulate the metabolism of endogenous substrates including hormones. Therefore, we hypothesized that parabens may alter hormone-metabolizing activities by acting on these receptors, and such changes could contribute to the endocrine-disrupting activity. To test this idea, we systematically examined the effects of 17 parabens on these receptors using reporter gene assays. Nine parabens significantly activated human and rat PXR. Parabens with C2–C5 (linear and branched) side chains were most active. Butylparaben and isobutylparaben also significantly activated rat CAR. We found that long-side-chain (C7–C12) parabens showed up to 2-fold activation of PPARα at 10 μM. Furthermore, pentylparaben and hexylparaben showed rat PXR antagonistic activity and rat CAR inverse agonistic activity. The activity of butylparaben towards PXR and CAR was lost after carboxylesterase-mediated metabolism. These findings confirm that parabens influence the activities of PXR, CAR and PPARα, and thus have the potential to contribute to endocrine disruption by altering hormone metabolism.

Albinism is the most common color variation described in fish and is characterized by a white or yellow phenotype according to the species. In rainbow troutOncorhynchus mykiss, aside from yellow-albino phenotypes, cobalt blue variants with autosomal, recessive inheritance have also been reported. In this study, we investigated the inheritance pattern and chromatophores distribution/abundance of cobalt blue trouts obtained from a local fish farm. Based on crosses with wild-type and dominant yellow-albino lines, we could infer that cobalt blue are dominant over wild-type and co-dominant in relation to yellow-albino phenotype, resulting in a fourth phenotype: the white-albino. Analysis of chromatophores revealed that cobalt blue trouts present melanophores, as the wild-type, and a reduced number of xanthophores. As regards to the white-albino phenotype, they were not only devoid of melanophores but also presented a reduced number of xanthophores. Cobalt blue and white-albino trouts also presented a more elongated body shape and, most remarkably, a smaller pituitary gland compared to wild-type and yellow-albino, suggesting that the allele for blue color is somehow linked with this abnormal pituitary phenotype. These phenotypes represent interesting models for research on body pigmentation in salmonids and on the mechanisms behind endocrine control of color patterning.

Cre/loxP-mediated cell targeting is considered to be a powerful tool for biotechnology in farmed fish. As a first step toward establishing cell targeting in salmonids, we analyzed the functionality of the Cre/loxP system in rainbow trout. We first established stable transgenic strains carrying the DsRed gene, which was flanked by loxP sites and further spliced with the EGFP gene. By microinjecting Cre complementary RNA (cRNA) into fertilized eggs of the transgenic trout, the functionality of the Cre/loxP system was evaluated. The results showed that all of the embryos exhibited green fluorescence in at least some of their cells. While 19 out of 20 embryos comprised cells showing both green and red fluorescence, the remaining embryo showed only green fluorescence. Polymerase chain reaction (PCR) using primers designed to recognize sequences outside of the two loxP sites revealed that, in addition to long intact fragments, the 19 individuals carried short fragments that were equivalent in length to the loxP-excised fragments. The remaining green embryo carried only this short fragment. DNA sequencing of the short fragment revealed that it lacked the DNA fragments flanking the loxP sites and the spliced fragments did not contain any sequence rearrangements. These results suggest that the Cre/loxP system is functional in rainbow trout.

Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.

Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.

Cre/loxP-mediated DNA excision in germ cell lineages could contribute substantially to the study of germ cell biology in salmonids, which are emerging as a model species in this field. However, a cell type-specific Cre/loxPsystem has not been successfully developed for any salmonid species. Therefore, we examined the feasibility of Cre/loxP-mediated, germ cell-specific gene excision and transgene activation in rainbow trout. Double-transgenic (wTg) progeny were obtained by mating a transgenic male carryingcrewith a transgenic female carrying thehsc-LRLGgene;crewas driven by rainbow troutvasaregulatory regions and thehsc-LRLGgene was made up of the rainbow troutheat-shock-cognate71promoter, theDsRedgene flanked by twoloxPsites, and theEgfpgene. PCR analysis, fluorescence imaging, and histological analysis revealed that excision of theloxP-flanked sequence and activation ofEgfpoccurred only in germ cells of wTg fish. However, progeny tests revealed that the excision efficiency ofloxP-flanked sequence in germ cells was low (≤3.27%). In contrast, the other wTg fish derived from two differentcre-transgenic males frequently excised theloxP-flanked sequence in germ cells (≤89.25%). Thus, we showed for the first time successful germ cell-specific transgene manipulation via the Cre/loxPsystem in rainbow trout. We anticipate that this technology will be suitable for studies of cell function through cell targeting, cell-linage tracing, and generating cell type-specific conditional gene knockouts and separately for developing sterile rainbow trout in aquaculture.