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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. [ABSTRACT FROM AUTHOR]

The present study aimed to test the synbiotic PoultryStar® solUS delivered in ovo to evaluate its effect on hatchability, productive performance and meat quality, compared to its post-hatch administration in water. On the twelfth day of embryonic incubation, 1200 fertile eggs were divided into synbiotic groups injected with 2 mg/embryo (T1) and 3 mg/embryo (T2), a saline group injected with physiological saline and an uninjected control group (C). After hatching, 120 male chicks/group were reared and chicks from the saline group were supplemented with the synbiotic via drinking water (T3). Hatchability was low in both T1 and T2 groups. Growth performance was not affected by the treatments. However, in the second rearing phase (15–36 days), birds from the C and T3 groups were heavier than T1 birds, due to a higher feed intake and daily weight gain. Neither route of synbiotic administration influenced final body weight (at 56 days), weight and yield of the carcass or commercial cuts. Physico-chemical properties, total lipid, cholesterol and fatty acid composition of breast muscle were not affected by the treatments. Considering its exploratory nature, this study has raised many questions that need further investigation, such as the bioactive combination and the effect on embryonic development. [ABSTRACT FROM AUTHOR]

The Green-legged Partridgelike fowl is a native, dual-purpose Polish chicken. The White Leghorn has been intensively selected for several decades to mainly improve reproductive traits. Primordial germ cells (PGCs) represent the germline stem cells in chickens and are the only cells that can transfer the information stored in the genetic material from generation to generation. The aim of the study was to carry out a transcriptomic and an epigenetic comparison of the White Leghorn and Green-legged Partridgelike gonadal PGCs (gPGCs) at three developmental stages: days 4.5, 8, and 12 of the embryonic development. RNA and DNA were isolated from collected gPGCs. The RNA was further subjected to microarray analysis. An epigenetic analysis was performed based on the global methylation analysis and qMSP method for the particular silenced genes demonstrated in transcriptomic analysis. Statistically significant differences between the gPGCs from both breeds were detected on the day 8 of embryonic development. Global methylation analysis showed significant changes at the methylation level in the White Leghorn gPGCs on day 8 of embryonic development. The results suggest faster development of Green-legged Partridgelike embryos as compared to White Leghorn embryos. Changes in the levels of gene expression during embryonic development are determined by genetic and environmental factors, and this variability is influenced by breed and gender. [ABSTRACT FROM AUTHOR]

Simple Summary: Probiotics, prebiotics, and synbiotics may be used as feed additives instead of banned antibiotic-based growth promoters. These bioactive compounds applied in ovo have beneficial effects on intestinal bifidobacteria, decrease the number of detrimental bacteria in the gut, stimulate the development of gut-associated lymphoid tissues (GALT), and modulate the development of lymphoid organs. The aim of our study was to determine whether the specific in ovo-delivered prebiotics and synbiotics affected the lymphocyte subsets of the bursa of the Fabricius, thymus, and spleen in non-immunized chicken broilers and in birds immunized with T-dependent (sheep red blood cells—SRBC) and T-independent (dextran—DEX) antigens. This study demonstrated that in ovo administration of prebiotics and synbiotics is a promising approach for enhancing chicken immune system functions. We conclude that a combination of inulin and Lactococcus lactis subsp. lactis IBB SL1 was the most effective of the tested compounds in the stimulation of the chicken immune system. The effects of in ovo-delivered prebiotics and synbiotics on the lymphocyte subsets of the lymphoid organs in non-immunized 7-day-old broiler chickens and in non-immunized, sheep red blood cells (SRBC)-immunized, and dextran (DEX)-immunized 21- and 35-day-old birds were studied. The substances were injected on the 12th day of egg incubation: Prebiotic1 group (Pre1) with a solution of inulin, Prebiotic2 group (Pre2) with a solution of Bi2tos (non-digestive transgalacto-oligosaccharides), Synbiotic1 group (Syn1) with inulin and Lactococcus lactis subsp. lactis IBB SL1, and Synbiotic2 group (Syn2) with Bi2tos and Lactococcus lactis subsp. cremoris IBB SC1. In 7-day-old chicks, a decrease in T splenocytes was noticed in all groups. The most pronounced effect in 21- and 35-day-old birds was an increase in TCRγδ+ cells in Syn1 and Syn2 groups. A decrease in bursal B cells was observed in DEX-immunized Pre1 group (21-day-old birds), and in the Syn1 group in non-immunized and SRBC-immunized 35-day-old birds. An increase in double-positive lymphocytes was observed in Pre1 (35-day-old birds) and Pre2 (immunized 21-day-old birds) groups. In Pre1 and Syn1 groups (21- and 35-day-old), an increase in B splenocytes and a decrease in T splenocytes were observed. We concluded that Syn1 was the most effective in the stimulation of the chicken immune system. [ABSTRACT FROM AUTHOR]

Simple Summary: Chicken primordial germ cells are specialized cells that are formed outside the developing embryo, from where they migrate into the gonad, and give rise to the gametes. There are two types of those cells: Circulating blood primordial germ cells and gonadal primordial germ cells. They can be isolated only in low numbers from the bloodstream or gonads of donor embryos. Hence, efficient in vitro cultivation systems are required to increase their quantity through proliferation. Here, we provide a single culture system that can be used to cultivate both cell types. We also present a novel, easy-to-train, and non-invasive method to identify live primordial germ cells in the culture. In this analysis, chicken primordial germ cells obtained from embryonic blood or gonadal regions were cultured in vitro on feeder cell layers derived from embryonic chick cells. The use of the chicken origin feeder layer allowed reducing the xenogenic animal factors in the culture. We demonstrated a feasible and cost-efficient technique to routinely assess the cultivated primordial germ cells on the basis of their morphological characteristics and using the optical features of cells in darkfield illumination. This method is especially useful to distinguish primordial germ cells during co-cultivation with other cell types. The present study had two aims: (1) To develop a culture system that imitates a normal physiological environment of primordial germ cells (PGCs). There are two types of PGCs in chicken: Circulating blood (cPGCs) and gonadal (gPGCs). The culture condition must support the proliferation of both cPGCs and gPGCs, without affecting their migratory properties and must be deprived of xenobiotic factors, and (2) to propose an easy-to-train, nonlabeling optical technique for the routine identification of live PGCs. To address the first aim, early chicken embryo's feeder cells were examined instead of using feeder cells from mammalian species. The KAv-1 medium at pH 8.0 with the addition of bFGF (basic fibroblast growth factor) was used instead of a conventional culture medium (pH approximately 7.2). Both cPGCs and gPGCs proliferated in vitro and retained their migratory ability after 2 weeks of culture. The cultivated cPGCs and gPGCs colonized the right and/or left gonads of the recipient male and female embryos. To address the second aim, we demonstrated a simple and rapid method to identify live PGCs as bright cells under darkfield illumination. The PGCs rich in lipid droplets in their cytoplasm highly contrasted with the co-cultured feeder layer and other cell populations in the culture. [ABSTRACT FROM AUTHOR]

Simple Summary: Poultry production plays a major role in providing meat products to global markets. Hence, there is a continued interest of researchers in the possibilities of improving the production parameters of broiler chickens. A direct response from muscles and their metabolism to in ovo synbiotic stimulation on day 12 of egg incubation has already been widely documented. However, it is necessary to analyze the molecular mechanisms determining the phenotypic effects. The present research aimed to explain the molecular background of the quality of broiler chicken meat after injection of synbiotics based on Lactobacillus strains into the air chamber of the egg. Characterization of the meat quality is based on the signature of the metabolic gene expression closely related to muscles and basic physiological parameters. To better understand the effects of synbiotics administered at early stages of embryonic development in poultry, it is necessary to analyze direct effects (meat quality) and the molecular background. The molecular interpretation of poultry meat properties after in ovo administration of synbiotics remains to be reported. The purpose of the present study was to analyze the molecular background of meat quality based on gene expression and basic physiological parameters. Eggs were injected with (S1) Lactobacillus salivarius with galacto-oligosaccharides or (S2) Lactobacillus plantarum with raffinose family oligosaccharides. The pectoral muscle was collected at two time points (day 7 and day 42) and subjected to RNA isolation. Gene expression analysis was performed by RT-qPCR for a panel of eight genes associated with metabolism. The concentration of glucose and hormones (insulin, glucagon, and leptin (S1 p = 0.04)) was also increased. The obtained results showed that metabolic gene expression in the muscle was more differential due to synbiotic stimulation on day 7 (FST in S1 p = 0.03; PDK4 in S1 p = 0.02 and S2 p = 0.01; CEBPB in S1 p = 0.01 and S2 p = 0.008; PHKB in S1 p = 0.01; PRKAG3 in S1 p = 0.02) than on day 42 (PDK4 in S1 p = 0.04). On the basis of the results obtained, it can be concluded that in ovo stimulation with S1 triggered the most potent and favorable changes in the pectoral muscle gene expression in broiler chickens. [ABSTRACT FROM AUTHOR]

Simple Summary: The immune system of chickens matures in the course of embryonic development. Early in ovo supplementation with bioactive substances leads to the long-term maintenance of a high level of intestine bifidobacteria, reduces the number of detrimental microorganisms in the gut, modulates the central and peripheral lymphatic organ development in broilers, and stimulates gut-associated lymphoid tissues (GALT) development after hatching. In this investigation, we studied whether the early in ovo application (at the 12th day of embryo incubation) of selected bioactives (prebiotics and synbiotics) influences the humoral immune response against experimental antigens, and the delayed-type hypersensitivity (DTH) skin reaction to experimental mitogen. This study demonstrated that the in ovo application of bioactives did not significantly influence the humoral immune response against T-dependent and T-independent model antigens. Prebiotics in chickens immunized with T-dependent antigen (SRBC) protected them from a retarded rise of the IgG concentration. Bioactives reduced the mortality of birds, markedly with inulin (−6.4%), and the DTH reaction to phytohemagglutinin on the 7th and 21st day after hatching. The effect of the in ovo application of selected prebiotics and synbiotics on the humoral immune response against T-dependent (SRBC) and T-independent (dextran) antigens and delayed-type hypersensitivity (DTH) to phytohemagglutinin was studied. On the 12th day of incubation, 800 eggs (Ross 308) were divided into five groups and injected into the egg air chamber with prebiotic inulin (Pre1), Bi2tos (Pre2), a synbiotic composed of inulin and Lactococcus lactis subsp. lactis IBB SL1 (Syn1), a synbiotic composed of Bi2tos and L. lactis subsp. cremoris IBB SC1 (Syn2), and physiological saline (control group; C). The chickens were immunized twice at the 7th and 21st day of life with SRBC and dextran. A DTH test was performed on the 7th, 21st, and 35th day. The application of prebiotics and synbiotics had no significant effect on the humoral immune response. SRBC-immunized in ovo Pre1- and Pre2-treated chickens showed significantly higher serum IgG levels than the control. A significant effect on the DTH reaction was detected on the 7th (Pre1 < C) and 21st (Pre2 > Syn2) day. However; Bi2tos may transiently stimulate the cellular immune response on the 21st day. It may be concluded that the application of inulin in an egg air chamber on the 12th day of incubation may stimulate the secondary immune response. The inulin-treated group exhibited a lower mortality rate than the control group. [ABSTRACT FROM AUTHOR]

Simple Summary: In the face of a changing climate, antibiotic resistance and uprising outbreaks of 'forgotten' diseases, there is an urgent need for new, safe strategies and natural immunomodulatory products in intensive broiler production. So far, many prebiotic and synbiotic preparations have been explored to influence the gut microbiota composition and the host immune system. However, the effects of bioactive compounds are not always found to be consistent. Global analysis allows us to define at least several reasons for those discrepancies: different chemical composition and origins of the oligosaccharides, interaction with other feed ingredients, and unfavorable environmental impact, where the two latter seem to be most important. The in ovo strategy to automatically inject prebiotics at day 12 of egg incubation has been elaborated to eliminate some of those factors and provide a fully controlled modulation of the host biology. Here, the prebiotic modulatory effects were reflected in the improved gut structure and resistance to opportunistic pathogens in the final weeks of broiler rearing, showing lifelong effects. The in ovo strategy allows for the earliest possible immunomodulatory treatments with the use of naturally sourced bioactive compounds, one of them being legume raffinose oligosaccharides. The aim was to investigate the impact of an automatic in ovo injection of the raffinose family oligosaccharides (RFO) extracted from the seeds of Lupinus luteus L, on the chicken performance and resistance in a production environment. At day 12 of incubation, a total of 57,900 eggs (Ross 308) were divided into two groups: 1/ Control, injected with 0.9% NaCl and 2/ RFO group, injected with 1.9 mg/egg of the lupin seed extract, dissolved in 0.2 mL NaCl. The performance parameters, biochemical indices (lipid profile, hepatic parameters), gut histomorphology and duodenum structure, oxidative stability of the meat and microbiological counts of the major commensal microbiota species were analyzed. Mortality, body weight, and feed conversion ratio (FCR) were not affected. By day 42, several health indices were improved with RFO and were reflected in a beneficial lipid blood profile, increased villi surface and better combating opportunistic pathogens through reduction of Clostridia and decreased coccidia counts. The RFO increased meat oxidation, but only at the beginning of the storage. The RFO sourced from local legumes can be considered a promising prebiotic for broiler chickens. In ovo delivery of prebiotics and/or synbiotics should be further optimized as an important strategy for the earliest possible modulation of chicken resistance. [ABSTRACT FROM AUTHOR]