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Piracanjuba (Brycon orbignyanus) is an endangered fish species from the Neotropical region. The establishment of a cryobank using spermatogonial stem cells (SSCs) and subsequent production of a germline chimera is thus a promising strategy for such species. In the present work, procedures for the isolation and cryopreservation of piracanjuba SSCs and subsequent transplantation into sterile recipients were established. The piracanjuba SSCs were obtained by Percoll density gradient centrifugation and differential plating. SSC fractions were evaluated by relative ddx4 expression, alkaline phosphatase activity, and light microscopy. SSC cryopreservation was performed using five cryoprotectants at three different concentrations. The mix of the cells from the 20% and 30% Percoll density gradients showed 58.35 ± 0.03% purity of SSCs. The purity of SSCs increased to 66.00 ± 0.01% after differential plating. The relative ddx4 expression was 3.5 times higher in cells from the Percoll density gradient centrifugation than in the gonad and cells after differential plating. Propanediol (1 M) was the most effective cryoprotector evaluated (P = 1.000), showing 90.75 ± 1.85% cell viability. Freshly isolated and cryopreserved cells from the Percoll density gradient centrifugation were transplanted into a sterile male adult triploid hybrid with germ cell-less gonads. SSCs were observed in the germinal epithelium of the testes of recipients 20 days after transplantation. The results are promising for obtaining functional germline chimeras in Neotropical fish. Consequently, although the number of males used for the experiment was borderline, the procedures established here can be applied in future actions for the conservation and reconstitution of the piracanjuba in case of extinction. [ABSTRACT FROM AUTHOR]

The use of model organisms is important for basic and applied sciences. Several laboratory species of fishes are used to develop advanced technologies, such as the zebrafish (Danio rerio), the medaka (Oryzias latipes), and loach species (Misgurnus spp.). However, the application of these exotic species in the Neotropical region is limited due to differences in environmental conditions and phylogenetic distances. This situation emphasizes the establishment of a model organism specifically for the Neotropical region with the development of techniques that may be applicable to other Neotropical fish species. In this work, the previous research efforts are described in order to establish the yellowtail tetra Astyanax altiparanae as a model laboratory species for both laboratory and aquaculture purposes. Over the last decade, starting with artificial fertilization, the yellowtail tetra has become a laboratory organism for advanced biotechnology, such as germ cell transplantation, chromosome set manipulation, and other technologies, with applications in aquaculture and conservation of genetic resources. Nowadays, the yellowtail tetra is considered the most advanced fish with respect to fish biotechnology within the Neotropical region. The techniques developed for this species are being used in other related species, especially within the characins class. [ABSTRACT FROM AUTHOR]

The use of model organisms is important for basic and applied sciences. Several laboratory species of fishes are used to develop advanced technologies, such as the zebrafish (Danio rerio), the medaka (Oryzias latipes), and loach species (Misgurnus spp.). However, the application of these exotic species in the Neotropical region is limited due to differences in environmental conditions and phylogenetic distances. This situation emphasizes the establishment of a model organism specifically for the Neotropical region with the development of techniques that may be applicable to other Neotropical fish species. In this work, the previous research efforts are described in order to establish the yellowtail tetra Astyanax altiparanae as a model laboratory species for both laboratory and aquaculture purposes. Over the last decade, starting with artificial fertilization, the yellowtail tetra has become a laboratory organism for advanced biotechnology, such as germ cell transplantation, chromosome set manipulation, and other technologies, with applications in aquaculture and conservation of genetic resources. Nowadays, the yellowtail tetra is considered the most advanced fish with respect to fish biotechnology within the Neotropical region. The techniques developed for this species are being used in other related species, especially within the characins class.

1. A series of experiments were conducted to examine the developmental potential of cryopreserved gonadal germ cells (GGCs) recovered from both males and females on embryo day 7 (7 d-GGCs) using the PBS(-) method. Germline chimeras were produced by transferring 200 frozen/unfrozen 7 d-GGCs recovered from female/male Rhode Island Red (RIR) embryos into the dorsal aorta of 2-day-old female and male white leghorn (WL) embryos. 2. Germ-cell recipient embryos were hatched and raised to sexual maturity and progeny testing was conducted by mating with RIR of the opposite sex. Brown-feathered progeny chicks were hatched in all eight possible progeny testing combinations, except for male GGC recipients produced by transferring female GGCs. Furthermore, brown-feathered progeny chicks were hatched when frozen-thawed sperm from male germline chimeras, produced by transferring unfrozen 7d-GGCs, were inseminated in normal female RIR and female WL germline chimeras. 3. The results indicated that cryopreserved female/male GGCs from 7-day-old chick embryos, recovered using the PBS(-) method, were fully capable of developing into normal spermatozoa and ova in the gonad of recipient embryos under appropriate GGC donor/recipient combinations. [ABSTRACT FROM AUTHOR]

The germplasm is a resource and tool for the conservation of genetic diversity in animals, including birds. Securing germplasm is limited in most bird species due to difficulties in semen collection and germ cell isolation, lack of germ cell-specific markers, and in vitro culture systems. Here, we report the production of germline chimeric quails by transplant of cryopreserved testicular cells (TCs) into the developing embryo. The testicular germ cell properties were maintained after freeze-thaw, with no significant reduction in cell viability irrespective of storage length. Cryopreserved TCs were transferred into Hamburger Hamilton (HH) stage 14–17 quail embryos, and were demonstrated to migrate into the embryonic gonads with similar efficiency to freshly isolated TCs. Twenty of 81 recipient embryos yielded hatchlings from cryopreserved TCs and the germline transmission efficiency was similar to that of freshly isolated cells. In conclusion, cryopreserved adult quail TCs are capable of (de)differentiation into functional gametes in recipient quail gonads and can generate donor TCs-derived progenies. This system is feasible for the isolation of sufficient germplasm resources from various bird species for conservation purposes. • Quail testicular cells (TCs) retain germ cell characteristics after cryopreservation. • Cryopreserved TCs can migrate into gonads of developing embryos. • Reversible transplantation of cryopreserved whole TCs resulted in a successful production of germline chimeras. [ABSTRACT FROM AUTHOR]

The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22°C, 26°C, and 30°C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26°C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3'UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera. [ABSTRACT FROM AUTHOR]

Contents: Birds obtained by embryo engineering are used to study embryo development and to produce transgenic birds. As this method of producing birds still generate strong emotions of the public opinion head ornaments, testes and semen characteristics of sex chimera roosters were examined to check whether they differ from chickens obtained by non‐manipulated methods. Measurements of head ornaments, testes and semen were correlated with each other. Semen quality factor (SQF) was calculated, as well as the level of fluctuating asymmetry (FA) of bilateral traits (wattles and testes). Positive correlation was found for comb width and wattle length and comb thickness and sperm concentration. Semen characteristics and FA did not exceed the level encounter in other chicken lines. Results obtained indicate that germline chimeras are similar in appearance of secondary sexual traits, and semen and testes characteristics to chickens produced in non‐manipulated way. [ABSTRACT FROM AUTHOR]

Chicken embryo manipulation, especially germline manipulation, recently has progressed greatly by devising important experimental techniques, such as embryo culture and primordial germ cell (PGC) manipulation. Embryogenesis and embryonic development in chickens are affected by gravity at various developmental stages. Freshly collected PGC population seems to contain germline-competent PGCs in opposite-sex germline chimeric chickens, but these PGCs probably disappear from the population during in vitro culture. Long-term culture of chicken PGCs makes it possible to transfer foreign DNA into the germline and also contributes to preserving avian genetic resources. Propagation of endangered avian species could become possible via interspecies germline chimeras. Development of techniques producing somatic cell-derived offspring could also be useful to avian germline manipulation.

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.

Testicular gonocytes differentiate into spermatogonia and spermatogonial stem cells could be established by culturing spermatogonia in vitro. Spermatogonial stem cells differentiate exclusively into spermatozoa and thus they are very useful for genetic manipulation in chickens. The present study was carried out to examine the possibility of in vitro proliferation of testicular and ovarian gonocytes in chickens. Gonads were obtained from 19-day incubated chicken embryos and the dissociated gonadal cells were cultured in vitro. Then, non-adherent cells containing gonocytes were collected and cultured further. It was confirmed that a part of the testicular gonocytes proliferated in vitro, when analyzed by anti-CVH antibody. The proliferated testicular gonocytes occasionally formed cell colonies. The cultured testicular gonocytes were successfully implanted in the gonads by transferring them into the coelomic epithelium that corresponds to the future gonadal region of recipient embryos. However, no apparent proliferation was observed in the ovarian gonocytes in vitro. These results suggest that testicular goncytes can be cultured in vitro and are one of the candidate cells for genetic manipulation in chickens.

In previous studies, two types of offspring were generated from germline chimera between NH-413 strain (donor) and White Leghorn L-M strain (recipient). Phenotype and symptom of type-I offspring were quite similar to that of the NH-413 strain. In the other offspring of type-II, feather color showed mixture of white and brown and the symptom was not dominantly indicated.In the present studies, sexually matured males and females of the type-I were mated each other. Form these mating, chickens manifesting completely same phenotype to that of the donor NH-413 strain; brown feather color and symptoms of muscular dystrophy, were regenerated. Therefore, complete regeneration of the muscular dystrophy chickens could be achieved by mating males and female offspring derived from the germline chimeras. Fertility, hatchability and survival rate of these regenerated offspring were significantly improved as compared to that of the original NH-413 strain.The established strategies should be one of the useful systems to regenerate chickens with muscular dystrophy.

A novel strategy has been developed to generate muscular dystrophy chickens by means of germline chimeras. Donor embryos were obtained from the New Hampshire chicken; NH-413 strain which have genes responsible for Fukuyama type muscular dystrophy (Saito et al., 2005). Donor cells were isolated from the center of area pellucida of the blastoderms. Recipient embryos were obtained from White Leghorn chicken; Line-M. The generated chimeric chickens had the donor derived brown plumage in the down in some extent, suggesting that the cells containing muscular dystrophy were introduced into the chimeras. These chimeric chickens have been raised until sexual maturity. The chimeric chickens were back-crossed to donor strain; the NH-413 strain. The phenotype of some of the offspring was very similar to that of the donor strain. The offspring showed some characters typical to the muscular dystrophy.It was suggested that the donor derived NH-413 strain offspring was generated. The established system should be one of the powerful strategies for breeding and regeneration of the muscular dystrophy chickens.

This is the first successful report on producing the endangered domestic fowl, Kureko Dori (KD), which is a Kumamoto prefectural natural treasure, using germline chimeras transplanted from primordial germ cells (PGCs) in practice. The attempt was made to produce germline chimeras between KD fowl, as a model of rare and/or endangered poultry, and common White Leghorn (WL) for the proliferation of KD individuals. The PGCs of KD were collected from embryonic blood at stages 13 to 16 (H & H) and preserved in liquid nitrogen until injection. A certain number of frozen-thawed PGCs were picked up under a phase contrast microscope, and injected into the peripheral vein of WL embryos (common poultry) at stages 12 to 14. We obtained one male and 6 females as the putative germline chimera, they were raised until sexual maturity, and the progeny test was performed by mating them with KD of the opposite sex to analyze the efficiency of germline chimera. The average rate of offspring originating from transplanted PGCs of KD was only 1.4%, and the maximum was 5.3% from all 577. These KD offspring derived from the KD-WL germline chimeras also achieved fertility as normal. These results show that rare and/or endangered birds can proliferate through producing germline chimeras between the same species of bird.

The migratory ability of germ cells collected from gonads at different stages of embryonic development was investigated in the domestic chicken. In Experiment 1, primordial germ cells (PGCs) were collected from 2.5-day-old embryo. Also gonadal germ cells (GGCs) were collected from 6.5-, 10.5-, 14.5-, 18.5-, and 20.5-day-old embryos. PGCs and GGCs were labeled with the fluorescent dye PKH-26, and 20 fluorescently labeled PGCs or GGCs were injected into the vascular systems of 2-day-old recipient embryos. The total number of fluorescently labeled cells recovered from both the left and right gonads of the recipient embryos was counted 5 days after injection. The proportion of recipient embryos carrying fluorescently labeled cells was 100% when the donor GGCs were collected from 6.5-, and 10.5-day-old embryos. The proportion decreased when the donor GGCs were from embryos incubated for longer than 14.5 days. An inverse relationship was observed between the total number of fluorescently labeled cells in the recipient gonads and the age of the donor embryo. In Experiment 2, gonadal cell suspensions were prepared from the left gonads of newborn chicks and adult chickens. Gonadal cell suspensions were labeled with the fluorescent dye PKH-26 and injected into the vascular systems of 2-day-old recipient embryos. The proportions of recipient embryos with fluorescently labeled cells in their gonads were 100% (3/3), 100% (3/3), 100% (3/3), and 67% (2/3) when the injected gonadal cell suspension was from male newborn chicks, female newborn chicks, adult roosters, and adult hens, respectively. These results indicate the presence of migratory germ cells in the gonads of developing embryos until day 20.5 of incubation. Furthermore, these results also suggest the presence of migratory cells even in the sexually matured testes or ovaries.

Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii , using 0.3% trypsin (2 hours, 23 °C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell–specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate–labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P < 0.05). The efficiency of transplantation was 60% (counted 90 dpt). [ABSTRACT FROM AUTHOR]

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka ( Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model. [ABSTRACT FROM AUTHOR]

Diseases such as avian influenza can de-stroy turkey flocks, potentially resulting in the loss of valuable or rare genetic material. Consequently, there is an urgent need to develop a means to archive such germplasm. Germline chimeras produced by intravas-cular transfer of primordial germ cells (PGC) have been reported in other avian species but not turkeys. This study examined the feasibility of both establishing an archive of frozen PGC, and producing germline chime-ras by injecting the thawed PGC into host embryos. To meet these aims, the following experiments were per-formed: (1) PGC identification within turkey embryos; (2) development of an efficient method for isolation of turkey PGC; (3) demonstration that PGC can be cryopreserved, recovered, and retain viability; (4) re-injection into embryos and detection of injected PGC. Primordial germ cells were identified using periodic ac-id-Schiff reagent and the immunological marker OLP-1. Bloodstream PGC were isolated using Ficoll density gradient centrifugation with PGC recovery peaking at stages 13, 14, and 15 with 32 ± 4.9, 33 ± 6.4, and 26 ± 5.4 PGC recovered, respectively. Primordial germ cells were frozen using Dulbecco's modified Eagle medium, 20% fetal calf serum, and 10% dimethylsulfoxide and demonstrated 90 ± 1.7% viability after 3 mo frozen in liquid nitrogen. Freshly isolated and frozen thawed Dil- and Q-Tracker-labeled PGC repopulated stage 30 gonads after vascular transfer into ex ovo cultured embryos. The Dil-labeled cells repopulated gonads less frequently, with 36 ± 13.2% of gonads containing the Dil-labeled PGC, and 7 ± 3.8% of reinjected PGC reaching the gonads of positive embryos. The Q-track-er-labeled cells were detected more frequently in embry-os, with 67 ± 21.1% having positive signals, and 44 ± 4.9% of reinjected Q-tracker-labeled PGC colonized the gonads of positive embryos. This study demonstrated the feasibility of using turkey PGC to archive turkey germplasm from different strains because frozen PGC reintroduced into host embryos can colonize the host gonads, suggesting the possibility of producing turkey germline chimeras. [ABSTRACT FROM AUTHOR]

The use of model organisms is important for basic and applied sciences. Several laboratory species of fishes are used to develop advanced technologies, such as the zebrafish ( Danio rerio ), the medaka ( Oryzias latipes ), and loach species ( Misgurnus spp . ). However, the application of these exotic species in the Neotropical region is limited due to differences in environmental conditions and phylogenetic distances. This situation emphasizes the establishment of a model organism specifically for the Neotropical region with the development of techniques that may be applicable to other Neotropical fish species. In this work, the previous research efforts are described in order to establish the yellowtail tetra Astyanax altiparanae as a model laboratory species for both laboratory and aquaculture purposes. Over the last decade, starting with artificial fertilization, the yellowtail tetra has become a laboratory organism for advanced biotechnology, such as germ cell transplantation, chromosome set manipulation, and other technologies, with applications in aquaculture and conservation of genetic resources. Nowadays, the yellowtail tetra is considered the most advanced fish with respect to fish biotechnology within the Neotropical region. The techniques developed for this species are being used in other related species, especially within the characins class., Competing Interests: GY and JS were employed by Peixetec Biotecnologia Em Organismos Aquáticos LTDA. NV was employed by China Three Gorges Corp. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yasui, Ferreira do Nascimento, Pereira-Santos, Santos Silva, Coelho, Visintin, Porto-Foresti, Okada Nakaghi, Vianna, Carvalho, Monzani, López and Senhorini.)

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enablingchicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containingPGCs obtained from 2.5-day-old chicken embryos accordingto the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced duringculture in the basic medium containingeither basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defininga culture condition enablingPGCLCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline. [ABSTRACT FROM AUTHOR]

Over the past two decades numerous reports have demonstrated that the genetic modification of poultry genomes has great potential for improving poultry production; moreover, it may be used as a powerful tool for the production of industrial proteins. To date, transgenic techniques have been established for generating transgenic birds that express recombinant human proteins in hen eggs, as well as tissue-specific genes as an animal model. The production of transgenic birds is a promising approach that could have practical applications in agriculture and biopharmacology, in addition to advancing our understanding of avian biology. Finally, germ cell-mediated transgenesis could provide a more efficient strategy for creating gene-targeted insertions and deletions in avian species. [ABSTRACT FROM AUTHOR]

Abstract: Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. We previously visualized the PGCs of several teleostean embryos by injecting RNA synthesized from constructs encoding green fluorescent protein (GFP) fused to the 3''UTR of the zebrafish (Danio rerio) nanos1 gene (nos1). However, this technique was not always suitable for visualizing PGCs in embryos from all teleost species. In this study, we compared the visualization of PGCs in common carp (Cyprinus carpio) embryos using two artificial constructs containing GFP fused to the 3''UTR of nanos from either common carp or zebrafish. Visualization was better using GFP fused to the 3''UTR of the nanos gene from common carp, compared with that from zebrafish. The visualized PGCs successfully migrated toward the gonadal ridge after transplantation into goldfish host embryos, suggesting that they maintained normal migratory motility. These techniques could be useful for the production of inter-specific germline chimeras using common carp donor PGCs. [Copyright &y& Elsevier]

Abstract: Interspecific hybrids provide insights into fundamental genetic principles, and may prove useful for biotechnological applications and as tools for the conservation of endangered species. In the present study, interspecies hybrids were generated between the Korean ring-necked pheasant (Phasianus colchicus) and the White Leghorn chicken (Gallus gallus domesticus). We determined whether these hybrids were good recipients for the production of germline chimeric birds. PCR-based species-specific amplification and karyotype analyses showed that the hybrids inherited genetic material from both parents. Evaluation of biological function indicated that the growth rates of hybrids during the exponential phase (body weight/week) were similar to those of the pheasant but not the chicken, and that the incubation period for hatching was significantly different from that of both parents. Primordial germ cells (PGCs) of hybrids reacted with a pheasant PGC-specific antibody and circulated normally in blood vessels. The peak time of hybrid PGC migration was equivalent to that of the pheasant. In late embryonic stages, germ cells were detected by the QCR1 antibody on 15 d male gonads and were normally localized in the seminiferous cords. We examined the migration ability and developmental localization of exogenous PGCs transferred into the blood vessels of 63 h hybrid embryos. Donor-derived PGCs reacted with a donor-specific antibody were detected on 7 d gonads and the seminiferous tubules of hatchlings. Therefore, germ cell transfer into developing embryos of an interspecies hybrid can be efficiently used for the conservation of threatened animals and endangered species, and many biotechnological applications. [Copyright &y& Elsevier]

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2–4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days. [Copyright &y& Elsevier]

ABSTRACT The development of chicken embryo culture techniques, from single-cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro . Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single-cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non-viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary. [ABSTRACT FROM AUTHOR]

This study aims to describe the early development of Piaractus mesopotamicus in detail at 22 °C , 26 °C , and 30 °C and describe primordial germ cell migration in vivo by using artificial GFP- nos1 3'UTR mRNA for future application in surrogate technology. The highest hatching rate was observed at 26 °C with 52.9 ± 18.9%, which was considered the best incubation temperature. At 22 °C , the embryos survived only until the blastula stage. The first visualization of PGCs occurred in the segmentation period when the embryos presented between 6 and 16 somites. The number of GFP-positive cells varied from 7 to 18 per embryo (mean 11.2; n = 15). After hatching, the GFP-positive cells ranged from 7 to 22 per embryo (mean 14.6; n = 15). The PGCs were traced until the 9th day after hatching. The obtained data allow for the incubation temperature manipulation and early development and traceability of PGCs, which are important for reproductive biotechniques, establishment of cryobanks, and subsequent reconstitution through a germline chimera. • Different incubation temperatures influence embryonic development. • The highest hatching rates and normality occurred at 26 °C. • PGCs were viewed from the segmentation period to the ninth dph. [ABSTRACT FROM AUTHOR]

Common carp (Cyprinus carpio) is the fourth most-produced species in worldwide aquaculture. Significant efforts are invested in breeding and preservation of genetic integrity of this important species. However, maintaining a carp gene bank in situ can be demanding due to the large body size of the fish. Moreover, ex situ gene banking in common carp is as in other fish species limited to sperm cryopreservation. Recent progress in reproductive biotechnology in fishes has allowed the transfer of germ stem cells (gamete precursors) between individuals or even species. After maturation, the recipients are producing gametes of the donor. Surrogacy can serve as a valuable tool in germplasm banking to recover cryopreserved germ stem cells. Some unfavourable characteristics of the donor species such as long maturation time or large body size hampering its reproduction can be overcome by choosing a surrogate with more convenient characteristics. Efficient protocols for cryopreservation of common carp male and female germ stem cells have been recently developed in our laboratory. The next step has been to assess the potential of smaller goldfish (Carassius auratus) surrogates to produce donor-derived gametes of common carp after intraperitoneal transplantation of testicular cells. High transplantation success was achieved when 44% of the surviving goldfish produced pure donor-derived gametes of common carp at the age of 3 years giving rise to viable progeny. Donor-derived identity of the offspring was confirmed by genotyping and the production of a typical phenotype corresponding to the donor species. Reproductive performance of chimeras was similar to goldfish controls. Assessment of gamete characteristics showed that the size of donor-derived eggs is between control common carp and goldfish eggs. Interestingly, flagellum length in donor-derived spermatozoa was comparable to the common carp flagellum and significantly shorter than goldfish flagellum. Genotyping of the Y chromosome locus in chimeric goldfish provided a novel insight on the fate of transplanted cells, where only genotypic females were capable of producing eggs, while both genotypic males and females were capable of producing sperm. The present technology can be combined with cryopreservation procedures to ease needs for common carp breeds preservation and their recovery using many times smaller goldfish surrogates but also serve as a convenient model to study interspecific surrogacy in cyprinids. • Production of common carp derived progeny from goldfish surrogates is reported. • First successful donor-derived egg production after inter-genus transplantation • Goldfish surrogates were able to reproduce repeatedly. • Donor-derived eggs were obtained from transdifferentiated spermatogonia. • Donor-derived spermatozoa have carp-like flagellum. [ABSTRACT FROM AUTHOR]

Surrogate propagation is a systematic approach to producing donor-derived gametes using germline chimeras. In fish, the use of germline chimeras to study the development of germ cells was first conducted in the 1990s in the model fish species medaka (Oryzias latipes) and zebrafish (Danio rerio). More recently, surrogate propagation has been actively investigated as a means of efficient gamete production not only in model fish species but also in aquaculture species and endangered species. Surrogate propagation has the following components: combination of the donor and host species, host sterilization, donor cell preparation, transplantation of germ cells, and gametogenesis and gamete production in surrogate fish. In this review, we first provide a general overview of previous studies related to germ cell transplantation and the methodologies developed for different species, and consider how these have been applied in practice. Second, we consider the development of primordial germ cells in fish embryos, particularly the molecular biological approaches used for the visualization of germ cells and sterilization of host embryos. Finally, we discuss sex control in germline chimeras, which may be a key component of the use of surrogate production in aquaculture. We focus on techniques to produce sterile fish, as these are crucial to the exclusive production of donor gametes in a surrogate host. The advantages and disadvantages of various aspects of surrogate propagation are discussed, the potential use of surrogate propagation as a seedling production system is considered, and future perspectives for aquaculture are suggested., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Germ cell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germ cell transplantation systems have been developed, including sperm, oocyte, or germline stem cell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germ cell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos.

Primordial germ cells (PGCs), the precursors of sperm or ova, could be used to generate transgenic animals and interspecies germ-line chimeras, which would facilitate the recovery of endangered species by making their access and manipulation in vitro easier. During early embryogenesis in avian species, PGCs are transported via the bloodstream to the gonadal anlagen. PGCs of most avian species-particularly wild or endangered birds-are not readily isolated from the embryonic bloodstream because germ-cell markers have not yet been defined for them. Here, we report a rapid, efficient, and convenient method for PGC isolation from various avian species. Blood PGCs were isolated based on the difference in size between PGCs and other blood cells, using a microporous membrane. The efficiency of this size-dependent isolation for the White Leghorn chicken was not significantly different from that of magnetic-activated cell sorting, and the isolated cells expressed chicken PGC-related genes and PGC-specific markers. The utility of the method was then verified in Japanese quail (Coturnix japonica), Mallard duck (Anas platyrhynchos), and Muscovy duck (Cairina moschata). Immunocytochemistry and an in vivo migration assay indicated that this method was able to enrich for true embryonic blood PGCs without specific antibodies, and could be applied to the development of avian interspecies chimeras for restoration of wild or endangered species., (© 2017 Wiley Periodicals, Inc.)

Cloning (somatic cell nuclear transfer) in avian species has proven unachievable due to the physical structure of the avian oocyte. Here, the sexual differentiation of primordial germ cells with genetic sex ZZ (ZZ PGCs) was investigated in female germline chimeric chicken hosts with the aim to produce uniparental offspring. ZZ PGCs were expanded in culture and transplanted into the same and opposite sex chicken embryos which were partially sterilized using irradiation. All tested chimeric roosters (ZZ/ZZ) showed germline transmission with transmission rates of 3.2%-91.4%. Unexpectedly, functional oogenesis of chicken ZZ PGCs was found in three chimeric hens, resulting in a transmission rate of 2.3%-27.8%. Matings were conducted between the germline chimeras (ZZ/ZZ and ZZ/ZW) which derived from the same ZZ PGCs line. Paternal uniparental chicken offspring were obtained with a transmission rate up to 28.4% and as expected, all uniparental offspring were phenotypic male (ZZ). Genotype analysis of uniparental offsprings was performed using 13 microsatellite markers. The genotype profile showed that uniparental offspring were 100% genetically identical to the donor ZZ PGC line, shared 69.2%-88.5% identity with the donor bird. Homozygosity of the tested birds varied from 61.5% to 84.6%, which was higher than the donor bird (38.5%). These results demonstrate that male avian ZZ PGCs can differentiate into functional ova in an ovary, and uniparental avian clones are possible. This technology suggests novel approaches for generating genetically similar flocks of birds and for the conservation of avian genetic resources., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Abstract: The use of genetically modified germ cells is an ideal system to induce transgenesis in birds; the primordial germ cell (PGC) is the most promising candidate for this system. In the present study, we confirmed the practical application of this system using lentivirus-transduced chicken gonadal PGCs (gPGCs). Embryonic gonads were collected from 5.5-d old Korean Oge chickens (black feathers). The gPGC population was enriched (magnetic-activated cell sorting technique) and then they were transduced with a lentiviral vector expressing enhanced green fluorescent protein (eGFP), under the control of the Rous sarcoma virus (RSV) promoter. Subsequently, the eGFP-transduced PGCs were transplanted into blood vessels of 2.5-d-old embryonic White Leghorn (white feathers). Among 21 germline chimeric chickens, one male produced transgenic offspring (G1 generation), as demonstrated by testcross and genetic analysis. A homozygous line was produced and maintained through the G3 generation. Based on serum biochemistry, there were no significant physiological differences between G3 homozygotes and non-transgenic chickens. However, since eGFP transgene expression in G3 chickens varied among tissues, it was further characterized by Western blotting and ELISA. Furthermore, there were indications that DNA methylation may have affected tissue-specific expression of transgenes in chickens. In conclusion, the PGC-mediated approach used may be an efficient tool for avian transgenesis, and transgenic chickens could provide a useful model for investigating regulation of gene expression. [ABSTRACT FROM AUTHOR]