Successful formation of sperm cells from transplanted primordial germ cells in sterile interspecific avian recipients.

Primordial germ cells (PGCs) are stem cells, from which only gametes develop. In birds, the female sex is heterogametic, thus female gene conservation necessitates preservation of PGCs. PGC transplantation can generate germline chimeras in a host organism and develop into gametes. However, competition between host and transplanted PGCs hinder efficiency of germline chimera generation. We hypothezised that in sterile hybrid recipients with no germ cells of its own, transplanted donor PGCs may exclusively form gametes. Advantages of sterile hybrids as host for PGCs is compliant with many national regulations on genetically modified organisms and technically simpler procedure than the use of busulphan. Therefore, we investigated whether sterile interspecific hybrids may be suitable as recipients for supporting donor PGCs by injecting green fluorescent protein-labelled chicken PGCs into 3-day-old Guinea fowl and domestic fowl hybrid embryos and monitoring PGC development. The injected PGCs colonized almost 100% of the recipient gonads and produced mature spermatozoa after 44 weeks. However, gamete production in these hybrids was initiated much slower than in domestic fowls. This delay may be caused by suboptimal hormonal regulation of gametogenesis in the hybrids. Our results suggest that sterile interspecific hybrids may be suitable hosts for PGCs for efficient gamete production.

Creating a novel method for chicken primordial germ cell health monitoring using the fluorescent ubiquitination-based cell cycle indicator reporter system.

The most current in vitro genetic methods, including gene preservation, gene editing and developmental modelling, require a significant number of healthy cells. In poultry species, primordial germ cells (PGCs) are great candidates for all the above-mentioned purposes, given their easy culturing and well-established freezing method for chicken. However, the constant monitoring of cultures can be financially challenging and consumes large amounts of solutions and accessories. This study aimed to introduce the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) complex into the chicken PGCs. FUCCI is a powerful transgenic tool based on the periodic protein expression changes during the cell cycle. It includes chromatin licensing and DNA replication factor 1 attached monomeric Kusabira-Orange and Geminin-attached monomeric Azami-Green fluorescent proteins, that cause the cells to express a red signal in the G1 phase and a green signal in S and G2 phases. Modification of the chicken PGCs was done via electroporation and deemed to be successful according to confocal microscopy, DNA sequencing and timelapse video analysis. Stable clone cell lines were established, cryopreserved, and injected into recipient embryos to prove the integrational competency. The cell health monitoring was tested with medium change experiments, that proved the intended reactions of the FUCCI transgene. These results established the future for FUCCI experiments in chicken, including heat treatment and toxin treatment.

Investigation of Sexes and Fertility Potential of Female Russian Sturgeon (Acipenser gueldenstaedtii) and Male American Paddlefish (Polyodon spathula) Hybrids.

In the present study, 10 allotriploid (3nALT) and 10 allopentaploid (5nALP) six-month-old hybrid fish and two 3nALT and four 5nALP 40-month-old hybrid fish, which resulted by crossing female Russian sturgeon Acipenser gueldenstaedtii (Brandt and Ratzeberg, 1833) and male American paddlefish Polyodon spathula (Walbaum, 1792), were investigated. It was revealed that six-month-old 3nALT and 5nALP hybrids initially had "undifferentiated" gonads, while in the 40-month-old hybrids, only testes were observed in one case of 3nALT and one case of 5nALP hybrids. The testis of 3nALT hybrids was partially developed with spermatogonia, while the testis of one 5nALP hybrid was in the second developmental stage with low spermatogonia density. We could not determine gonad differentiation in any of the cases when the hybrid individuals had the W sex chromosome. We concluded that the gonad differentiation of these interfamilial hybrids follows a similar pattern to interspecific hybrids of different ploidy parent species of the family Acipenseridae, which is consistent with the classical Haldane's rule. However, it cannot be excluded that the testis of this/these hybrid(s) may produce fertile sperm after sexual maturity, depending on additional genetic, hormonal and environmental factors, and further research is required for its evaluation.

The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Unidirectional hybridization between American paddlefish Polyodon spathula (Walbaum, 1792) and sterlet Acipenser ruthenus (Linnaeus, 1758).

Interspecific hybridizations among sturgeon species are feasible and often bidirectional. The American paddlefish (Polyodon spathula) from Family Polyodontidae and sturgeon species from Family Acipenseridae were reported capable of hybridization, but viable hybrids have been described only in crosses with the American paddlefish as paternal parents. In the reciprocal cross, the hybrids were not viable however embryos start to develop and reach late gastrula and early neurula stages. The goal of this study was to examine the hybridization between the sterlet sturgeon (Acipenser ruthenus) and the American paddlefish. Hybrid and purebred crosses were produced by artificial fertilization. Viable hybrid offspring were harvested (three month old) and verified in the families produced by female sterlet crossing with male American paddlefish. In the reciprocal hybrid crosses with female American paddlefish and male sterlet, the embryos development did not pass over 120 h post fertilization, indicating the unidirectional hybridization between American paddlefish and sterlet. Chromosome counting showed for the same ploidy level of viable hybrid and parent species. Analysis of three microsatellite markers confirmed the unidirectional hybridization between the American paddlefish and the sterlet species. Overall, the inferred genetic cause suggests that unidirectional hybridization between American paddlefish and sterlet may be the case not only for these two species but likely also between American paddlefish and other sturgeon species.

The Effects of Freezing Media on the Characteristics of Male and Female Chicken Primordial Germ Cell Lines.

Recently, in vitro gene preservation has gained ground thanks to its lower cost and higher stability compared to in vivo techniques. One of the methods that can preserve female-specific W chromosome-linked genes is primordial germ cell (PGC) freezing. PGCs can be isolated from Hamburger-Hamilton stage 14-16 embryos via blood sampling. In our experiment, we used two newly established Black Transylvanian naked neck chicken cell lines and four cell lines from our gene bank. We compared two different freezing media (FAM1 and FAM2) in this study. The cell number and viability of the PGCs were measured before freezing (BF) and after thawing on Day 0, Day 1, and Day 7 of cultivation. We analyzed the germ cell-specific chicken vasa homologue (CVH) expression profile in PGCs using RT-qPCR. We found that on Day 0, immediately after thawing, the cell number in cell lines frozen with the FAM2 medium was significantly higher than in the FAM1-treated ones. On Day 1 and Day 7, the cell number and viability were also higher in most cell lines frozen with FAM2, but the difference was insignificant. The freezing also affected the chicken vasa homologue gene expression in male lines treated with both freezing media.

Effects of Hydrostatic Pressure Treatment of Newly Fertilized Eggs on the Ploidy Level and Karyotype of Pikeperch Sander lucioperca (Linnaeus, 1758).

We studied the effect of different magnitudes (7000 PSI (48.26 MPa), 8000 PSI (55.16 MPa), and 9000 PSI (62.05 MPa)) of hydrostatic pressure on the ploidy of pikeperch larvae. Pressure shock was applied 5 min after the fertilization of eggs at a water temperature of 14.8 ± 1 °C. A 7000 PSI pressure shock was applied for 10 or 20 min, while 8000 and 9000 PSI treatments lasted for 10 min. Each treatment with its respective control was completed in triplicate, where different females' eggs served as a replicate. In the treatment groups exposed to 7000 PSI for 10 min, only diploid and triploid larvae were identified, while 2n/3n mosaic individuals were found after a 20-min exposure to a 7000 PSI pressure shock. The application of 8000 or 9000 PSI pressure shocks resulted in only triploid and mosaic individuals. Among larvae from eggs treated with 8000 PSI, three mosaic individuals with 2n/3n karyotype were identified (4.0 ± 6.9%), while a single (2.0 ± 3.5%) 1n/3n mosaic individual was found in the 9000 PSI-treated group. To our knowledge, this is the first report that demonstrates the induction of a haplo-triploid karyotype by hydrostatic pressure shock in teleost fish. The dominance of triploid individuals with a reasonable survival rate (36.8 ± 26.1%) after 8000 PSI shock supports the suitability of the hydrostatic pressure treatment of freshly fertilized eggs for triploid induction in pikeperch.

Successful cryopreservation and regeneration of a partridge colored Hungarian native chicken breed using primordial germ cells.

Primordial germ cells (PGCs) are the precursors of germline cells that generate sperm and ova in adults. Thus, they are promising tools for gene editing and genetic preservation, especially in avian species. In this study, we established stable male and female PGC lines from 6Hungarian indigenous chicken breeds with derivation rates ranging from 37.5 to 50 percent. We characterized the PGCs for expression of the germ cell-specific markers during prolonged culture in vitro. An in vivo colonization test was performed on PGCs from four Hungarian chicken breeds and the colonization rates were between 76 and 100%. Cryopreserved PGCs of the donor breed (Partridge color Hungarian) were injected into Black Transylvanian Naked Neck host embryos to form chimeric progeny that, after backcrossing, would permit reconstitution of the donor breed. For 24 presumptive chimeras 13 were male and 11 were female. In the course of backcrossing, 340 chicks were hatched and 17 of them (5%) were pure Partridge colored. Based on the backcrossing 1 hen and 3 roosters of the 24 presumptive chimeras (16.6%) have proven to be germline chimeras. Therefore, it was proven that the original breed can be recovered from primordial germ cells which are stored in the gene bank. To our knowledge, our study is a first that applied feeder free culturing conditions for both male and female cell lines successfully and used multiple indigenous chicken breeds to create a gene bank representing a region (Carpathian Basin).

Effect of Post-Hatch Heat-Treatment in Heat-Stressed Transylvanian Naked Neck Chicken.

Although numerous studies reported the effects of heat stress in chickens, it was not investigated in the Transylvanian Naked Neck breed. In our research, Transylvanian Naked Neck chickens, 24 h after hatching, were heat-treated at 38.5 °C for 12 h. We compared the control and heat-treated adult chickens' productivity parameters following 12 weeks of heat-stress at 30 °C. We found that the heat-treated layers had significantly higher egg production in heat stress, but in cockerels, the sperm quality did not differ significantly between the two groups. To detect the effect of heat-treatment on a molecular level, the expression of two heat-shock proteins and four heat-shock factors were analysed in the gonads of control and heat-treated chickens. We found that the expression level of HSP90 and HSF4 increased significantly in heat-treated female chicken gonads. Still, in adult females, the expression of HSF2 and HSF3 were substantially lower compared to the control. In adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads compared to the control. We think that the presented significant differences in egg production might be related to the increased expression level of HSP90 and HSF4 in heat-treated female gonads.

Effect of miR-302b MicroRNA Inhibition on Chicken Primordial Germ Cell Proliferation and Apoptosis Rate.

The primordial germ cells (PGCs) are the precursors for both the oocytes and spermatogonia. Recently, a novel culture system was established for chicken PGCs, isolated from embryonic blood. The possibility of PGC long-term cultivation issues a new advance in germ cell preservation, biotechnology, and cell biology. We investigated the consequence of gga-miR-302b-5P (5P), gga-miR-302b-3P (3P) and dual inhibition (5P/3P) in two male and two female chicken PGC lines. In treated and control cell cultures, the cell number was calculated every four hours for three days by the XLS Imaging system. Comparing the cell number of control and treated lines on the first day, we found that male lines had a higher proliferation rate independently from the treatments. Compared to the untreated ones, the proliferation rate and the number of apoptotic cells were considerably reduced at gga-miR-302b-5P inhibition in all PGC lines on the third day of the cultivation. The control PGC lines showed a significantly higher proliferation rate than 3P inhibited lines on Day 3 in all PGC lines. Dual inhibition of gga-miR-302b mature miRNAs caused a slight reduction in proliferation rate, but the number of apoptotic cells increased dramatically. The information gathered by examining the factors affecting cell proliferation of PGCs can lead to new data in stem cell biology.

Hybridization of Russian Sturgeon (Acipenser gueldenstaedtii, Brandt and Ratzeberg, 1833) and American Paddlefish (Polyodon spathula, Walbaum 1792) and Evaluation of Their Progeny.

Two species from the families Acipenseridae and Polyodontidae, Russian sturgeon (Acipenser gueldenstaedtii, Brandt and Ratzeberg, 1833; functional tetraploid) and American paddlefish (Polyodon spathula, Walbaum 1792, functional diploid) were hybridized. The hybridization was repeated using eggs from three sturgeon and sperm from four paddlefish individuals. Survival in all hybrid family groups ranged from 62% to 74% 30 days after hatching. This was the first successful hybridization between these two species and between members of the family Acipenseridae and Polyodontidae. Flow cytometry and chromosome analysis revealed two ploidy levels in hybrids. The chromosome numbers of the hybrids ranged between 156-184 and 300-310, in "functional" triploids and "functional" pentaploids, respectively. The hybrid origin and the ploidy levels were also confirmed by microsatellite analyses. In hybrids, the size and the number of dorsal and ventral scutes correlated with the ploidy levels as well as with the calculated ratio of the maternal and paternal chromosome sets. An extra haploid cell lineage was found in three hybrid individuals irrespective of the ploidy level, suggesting polyspermy. Although the growth performance showed high variance in hybrids (mean: 1.2 kg, SD: 0.55), many individuals reached a size of approximately 1 kg by the age of one year under intensive rearing conditions.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes.

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane's rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

Enhancement of chicken primordial germ cell in vitro maintenance using an automated cell image analyser.

Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.

Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines.

Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.

Successful chimera production in the Hungarian goose (Anser anser domestica) by intracardiac injection of blastodermal cells in 3-day-old embryos.

The conservation of genetic resources of avian species has become increasingly important over the past decade. The aim of the present study was to develop a genome preservation technique for the Hungarian goose Anser anser domestica. To this end, we developed a novel approach combining the simplicity of isolating a blastodermal cell suspension, which includes forming primordial germ cells (PGCs), with the efficiency of targeting future gonads by injecting these cells into the cardiac vein of the developing host embryo. First, we determined that the migratory period of PGCs in goose embryos was between 69 and 84h of development. Then, we injected the blastodermal cell suspension into the bloodstream of recipient embryos at this stage of development and monitored donor cell transmission into the genital tract. In all, 249 embryos were injected; three were found to be chimeras in gonadal tissues, whereas only one was a chimera in other tissues. Based on these results, it is concluded that this method is suitable for producing chimeras in the domestic goose. The optimal time of cell injection was found to be between 74 and 76h. The present study is the first report of the generation of chimeras in the domestic goose using intracardiac transplantation of embryonic cells.

Vitrification of early avian blastodermal cells with a new type of cryocontainer.

Although cryopreservation of avian semen is only applicable for singlegene traits, cryopreservation of avian blastodermal cells could facilitate preservation of the entire genome of endangered or rare-breed poultry. Slow freezing methods result in acceptable survival rates; however, there are apparently no reports regarding the use of vitrification. The aim of the study was to establish methods for chicken embryonic cell vitrification, including development of a container which supported cryopreservation of large numbers of cells (to increase the probability of chimera production). Based on a preliminary study, vitrification seemed to be practical for avian blastodermal cell preservation. Pieces of mosquito net as carrier increased live cell rates compared to pellet form in media containing two macromolecules. Furthermore, we concluded that fetal calf serum in the vitrification medium could be replaced by polyvinylpyrrolidone, a chemically defined substance free of unwanted growth factors and potential pathogens.