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.

Characterization of alcohol-related seizures in withdrawal syndrome.

OBJECTIVE: Alcohol-related seizures (ARS) are one of the most important consequences of alcohol withdrawal syndrome (AWS). However, demographic and clinical characteristics, and furthermore, the relationship of ARS with delirium tremens (DT), have not yet been evaluated in detail. Therefore, the aim of the present study was to reveal the correlates of ARS and examine the interaction of ARS with the occurrence of DT and with the severity of AWS. METHODS: In the retrospective study (Study 1) 2851 medical charts of inpatient admissions characterized by AWS and DT were listed. Demographic and clinical variables of ARS were assessed. In the follow-up study (Study 2), patients admitted with AWS without (N = 28) and with (N = 18) ARS were enrolled. Study 1 was performed between 2008 and 2023, and Study 2 was performed in 2019 in Hungary. To determine the severity of AWS, the Clinical Institute Withdrawal Assessment Scale for Alcohol, Revised (CIWA-Ar) was used. ARS is a provoked, occasional seizure; therefore, patients with epilepsy syndrome were excluded from the two studies. Statistical analyses were performed by the means of chi-square tests, multinomial logistic regressions, mixed ANOVA, and derivation. RESULTS: The occurrence of DT, the history of ARS, and somatic co-morbidities were found to be risk factors for the appearance of ARS. ARS was proved to be a risk factor for the development of DT. In the follow-up study, there was no difference in the decrease of CIWA-Ar scores between the groups. SIGNIFICANCE: Our present findings support the likelihood of kindling, which is one of the most important mechanisms underlying the development of ARS, but do not directly prove its presence. Additionally, our results revealed that the severity of AWS is not influenced by the presence of ARS. PLAIN LANGUAGE SUMMARY: Provoked, occasional seizures during AWS are defined as ARS. In the present study, predictors and interactions of these seizures with DT-the most severe form of withdrawal-and with the severity of withdrawal were examined in retrospective and follow-up studies. The present study shows that a history of withdrawal seizures, the occurrence of DT, and somatic comorbidities are predictors of the development of seizures. Furthermore, our findings suggest that the presence of seizures does not influence the severity of withdrawal.

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.

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.

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.