Changes in the Level of DNA Methylation in Candida albicans under the Influence of Physical and Chemical Factors.

The effects of physical factors such as radiation (electromagnetic, microwave, infrared, laser, UVC, and X-ray) and high temperature, as well as chemical factors (controlled atmosphere) on the level of global DNA cytosine methylation in C. albicans ATCC 10231 cells were investigated. Prolonged exposure to each type of radiation significantly increased the DNA methylation level. In addition, the global methylation level in C. albicans cells increased with the incubation temperature. An increase in the percentage of methylated DNA was also noted in C. albicans cells cultured in an atmosphere with reduced O2. In contrast, in an atmosphere containing more than 3% CO2 and in anaerobic conditions, the DNA methylation level decreased relative to the control. This study showed that prolonged exposure to various types of radiation and high temperature as well as reduced O2 in the atmosphere caused a significant increase in the global DNA methylation level. This is most likely a response protecting DNA against damage, which at the same time can lead to epigenetic disorders, and in consequence can adversely affect the functioning of the organism.

Changes in the gene expression and methylation in chicken cecal tonsils after in ovo administration of bioactive substances.

Cecal tonsils are the main organs which generate an immune response and also the part of the GALT, thus they are in the close proximity of the intestinal microbiota and continuously exposed to microbe-associated molecular patterns. GALT developed regulatory and anti-inflammatory mechanisms which eliminate or tolerate microbiota. Bioactive substances in ovo administration ensures an early contact between the GALT and beneficial bacteria, which greatly promotes the development of tolerance. Our previous studies have shown that the administration of bioactive substances in ovo silences gene expression in the cecal tonsils. The research hypothesis assumes that negative silencing of expression is correlated with the level of methylation in the tonsils. Therefore the current study aimed to analyze the global and gene-specific DNA methylation profiles in the cecal tonsils of two distinct chicken genotypes administered in ovo with bioactive substances. Eggs of Ross 308 and Green-legged Partridgelike were stimulated on day 12 of incubation. The injected compounds were: probiotic-Lactococcus lactis subsp. cremoris, prebiotic-galactooligosaccharides, and synbiotic-combination of both. Chickens were sacrificed on d 42 post-hatching. Cecal tonsils was collected, RNA and DNA were isolated and intended to gene expression, gene methylation and global methylation analysis. Cecal tonsils changes were observed in the methylation of 6 genes: SYK, ANGPTL4, TNFRSF14, IKZF1, CYR61, SERPING. Analyzes showed that the suppression of gene expression is related to the level of methylation of individual genes. Based on the results obtained in the cecal tonsils, it can be concluded that the silencing of gene expression is of an epigenetic nature. This is another study aimed at analyzing the relationship between the host, its intestinal microbiota and the possibilities of its programming.

The use of selected herbal preparations for the disinfection of Japanese quail hatching eggs.

The aim of study was to evaluate microbial and hatchability traits as well as chicks quality after hatching eggs disinfection with aqueous solutions of ginger (GR), garlic (GC), oregano (O), and cinnamon (C) extracts. The experiment was divided into 2 stages, at preliminary in vitro stage antimicrobial susceptibility of plant extracts (PEs) was tested against reference strains from the American Type Culture Collection. O and GC extracts had the best antimicrobial properties (P < 0.05). Then in in vivo stage 2,400 Japanese quails hatching eggs were divided into 6 groups, 400 eggs each. Eggs from first group were not disinfected (NC, negative control), eggs from second group were disinfected by formalin fumigation (C, positive control), in other groups 5% aqueous solutions of plant extracts of GR, GC, O, C were applied by spraying respectively. After standard incubation fertility, hatchability and periodical embryonic mortality were calculated as well as the body weight and livability of chickens during 14 d of rearing. Egg disinfection by aqueous solution of PEs led to maintain the hatchability, chick weight at hatch and post hatch body weight and early mortality of birds. Exclusion of any fungal isolates on eggshell surface was induced by GC followed by O and C groups (P < 0.05). In case of the bacteria colonies reduction only GR extract was effective. Chosen plant extracts may be treated as safe and alternative substances to traditional disinfectants of hatching eggs.

The prevalence of virulence determinants in methicillin-resistant Staphylococcus aureus isolated from different infections in hospitalized patients in Poland.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for hard-to-treat infections. The presence of 19 virulence genes in 120 MRSA isolates obtained from hospitalized patients and genetic relationships of these isolates were investigated. The eno (100%) and ebps (93.3%) genes encoding laminin- and elastin binding proteins, respectively, were ubiquitous. Other adhesion genes: fib (77.5%), fnbB (41.6%), bbp (40.8%), cna (30.8%) encoding proteins binding fibrinogen, fibronectin, bone sialoprotein and collagen, respectively, and map/eap (62.5%), encoding Eap, were also frequent. The etB and etD genes, encoding exfoliative toxins, were present in 15.6% and 12.5% isolates, respectively. The splA, splE and sspA, encoding serine protease were detected in 100%, 70.8% and 94.2% isolates, respectively. The tst gene, encoding toxic shock syndrome toxin-1 was found in 75% isolates. The cna, map/eap and tst genes were the most common in wound isolates and much less common in blood isolates. We identified 45 different spa types, t003 (21.7%) and t008 (18.8%) being the most common. The t003 was the most frequent among isolates from the respiratory tract (35.5%), while t008 in blood isolates (40%). Identification of virulence factors of MRSA is important for evaluation of pathogen transmission rate and disease development.

Effectiveness of various methods of DNA isolation from bones and teeth of animals exposed to high temperature.

In the event of fires, natural disasters, and other events associated with high temperature, bones and teeth are the only source of genetic material for identifying human or animal carcasses. To obtain reliable final results of identification tests, the use of appropriate nucleic acid extraction methods is crucial. Therefore, the main objective of this research was to evaluate the effectiveness of selected methods of DNA isolation from animal burnt bones and teeth. In addition, the effect of the duration of high temperature on the stability of nuclear and mitochondrial DNA in these tissues was determined, as well as the possibility of using the genetic material obtained for species identification of remains of unknown origin. Bones and teeth collected during necropsy of dogs were burnt in a laboratory oven at 400 °C (752 °F; 673.15 K) for 5, 10, 15, 30, 45 and 60 min. DNA was isolated according to four different protocols, using three commercial kits, i.e. the PrepFiler® Forensic DNA Extraction Kit from Applied Biosystems, the QIAamp® DNA Investigator Kit from QIAGEN, and the DNA Mini Kit from Syngen, as well as a classic organic method. The effectiveness of these methods was compared by assessing the amount of isolated DNA using Real-Time PCR and its purity using a NanoDrop™ spectrophotometer. Each isolate was also subjected to PCR with primers designed to amplify fragments of dog mitochondrial DNA. The effectiveness of species identification was assessed for the method showing the best DNA recovery and for the organic method, considered the gold standard for analysis of difficult material. The QIAamp® DNA Investigator Kit showed the highest efficiency of DNA isolation from bones and teeth burnt for 15 min (the longest burning time for which DNA could still be recovered from bones and teeth). The results of the experiment clearly indicate that DNA stability in hard tissues depends on how long they burn. In the case of exposure to 400 °C, reliable genetic testing, including species identification, is possible when the burning time does not exceed 15 min. Among the hard tissues examined, bones proved more suitable than teeth for identification purposes. It was also concluded that identification of bone remains with extreme heat damage should be based on mitochondrial DNA analysis.

Post-mortem analysis of gunshot wounds to the head and thorax in dogs by computed tomography, radiography and forensic necropsy.

In view of the scarcity of literature data on the use of radiological imaging techniques in forensic veterinary medicine, while at the same time the number of reported crimes against animals involving the use of firearms is rising, this paper attempts to assess the usefulness of radiography and computed tomography (CT) in the post-mortem diagnosis of gunshot wounds (GSW) in comparison to classic necropsy. The design of the experiment was as follows: preparation of the research material (13 dog carcasses), shooting of the material from different distances (1.5 and 12 m, plus one contact shot to the head) and using different types of ammunition, followed by X-rays and CT scans in each case to examine the injuries resulting from the shot. The final steps of the experiment were photographic documentation and autopsy by the Virchow method. In the examined material, post-traumatic bone lesions and the presence of metallic foreign bodies were successfully imaged by both radiography and CT. GSW analysis using CT provided much better data quality and some additional information. Two general conclusions can be drawn from the results of the experiment. First, damage caused by gunshots is correlated with the calibre, initial velocity and kinetic energy of the projectile, as well as the distance from the muzzle of the gun to the object shot. Second, radiological examination is useful in preparing forensic veterinary opinions. Used as a complement to classic necropsy, they increase the possibility of an accurate post-mortem diagnosis of shooting victims.

Influence of the Treatment Used in Inflammatory Bowel Disease on the Protease Activities.

INTRODUCTION: There is growing evidence that intestinal proteases have a role in the pathogenesis of gastrointestinal inflammatory diseases. Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), has an additional source of proteases represented by infiltrated and activated inflammatory cells. The aim of our study was to determine proteolytic system activity in patients with CD and UC. We limited the number of proteases tested by determining proteases active in acidic, neutral and alkaline pH. MATERIALS AND METHODS: The study included 40 patients with IBD - 20 CD patients and 20 UC patients. The control group consisted of 20 healthy subjects. Among the 20 CD patients, 17 were treated with aminosalicylates, 14 with azathioprine, and 4 with corticosteroids, while 8 patients were undergoing biological treatment. Among the 20 UC patients, 19 were treated with aminosalicylates, 8 with azathioprine, and 3 with corticosteroids. The total protein concentration was assayed by the Lowry method. The optimal pH was assayed in pH from 2.2 to 12.8, separated by 0.2 intervals. Proteolytic activities were determined against different substrates (gelatine, haemoglobin, ovalbumin, albumin, cytochrome C, and casein), and haemoglobin was the optimal substrate. Protease activities were determined according to Anson method. Determination of the activities of natural inhibitors of acidic, neutral and alkaline proteases is based on the Lee and Lin method. RESULTS: Decreases were observed in the activities of acid proteases (pH 5), alkaline proteases (pH 7), and neutral proteases (pH 7.6 and 8.6) in the groups of CD patients in remission in comparison with the active phase. In the group of patients with biologically treated CD patients, acid protease activity (pH 5.0) was lower than in CD patients not receiving biological treatment. Activities of neutral (pH 7.0) and alkaline (pH 7.6 and 8.6) proteases in the plasma of patients with UC in remission were lower in comparison to the active phase. Activities of acid (pH 5.0) and alkaline (8.6) protease inhibitors were higher in CD patients in the active phase in comparison to remission. In UC patients with exacerbation of the disease, the activity of alkaline (pH 8.6) protease inhibitors was increased compared to remission. CONCLUSION: 1. Our research may suggest that the immunomodulatory treatment used in IBD, aimed at reducing the level of leukocytes and reduction of inflammation, may contribute to a reduction in protease activity. 2. The decrease of protease activities in patients with CD and UC in remission may be a marker suggesting the patients' response to the treatment.

Use of reactive oxygen species (ozone, hydrogen peroxide) for disinfection of hatching eggs.

The sample consisted of 480 hatching eggs of Japanese quails and was divided into 4 groups. Before the transfer to the incubator, the first group was not disinfected (negative control). In the second group, eggs were disinfected by means of formaldehyde fumigation (positive control). In the third and fourth group, reactive oxygen forms were used for disinfection- perhydrol (H2O2) and ozone (O3), respectively. Eggs were incubated under standard conditions. On the 14th D, eggs were candled, and proportions of fertilized eggs and died embryos were calculated. In addition, samples were collected for microbiological examination. After 17.5 D, the results of the whole hatching were evaluated. Chicks were reared for 14 D. Their survivability and body weight gain were recorded. Disinfection by means of reactive oxygen forms did not prove to be more effective in reducing the number of bacterial colonies on the shell. Reduced hatching and significantly increased mortality in the O3 group may indicate the negative impact of this gas on developing embryos. The results of hatching from eggs disinfected with H2O2 did not differ from those obtained in control groups. The biggest chicks were obtained from O3 disinfected eggs. However, during rearing, their growth did not match the one observed for birds in the remaining groups. Chicks hatched from eggs disinfected with H2O2 were characterized by the largest survivability. Disinfection with reactive oxygen forms did not significantly improve the hygiene of hatching eggs, hatching performance, and quality of hatched chicks. Hydrogen peroxide, whose application offered satisfactory hatching results, may be the recommended disinfectant. On the other hand, O3 appears to be undesirable because of its negative impact on bird embryos.

Time of sexual maturity and early egg quality of Japanese quails affected by in ovo injection of medicinal plants.

The aim of this study was to evaluate the time to sexual maturity and quality of initial eggs of Japanese quail affected by in ovo injection of plant extracts: ginger (GR), garlic (GC), oregano (O) and cinnamon (C). In total, 2400 eggs of Japanese quails were divided into six groups on the fifth day of incubation. Group I was the control group (NC), which was not injected. Other eggs were injected with 0.1 mL of liquid: group II - the positive control (PC) - with distilled water, group III with 1 % solution of GR, group IV with GC, group V with O and group VI with C. After hatching, the birds were reared in a cage system and fed with balanced mixtures, and 24 h lighting was used. The time at which birds reached sexual maturity was registered, while in the seventh week of rearing, 120 eggs were subject to quality evaluation. The traits of a whole egg (shape index as the ratio of egg width to egg length, weight, specific gravity), shell (strength, weight, thickness and density), albumen (weight, height), yolk (color, weight, index) were evaluated. At the earliest, on 36th day of life, eggs were laid by birds from the GC group, followed by C (37th day), O and NC (38th day), GR (39th day), and PC (41st day). During the first 2 weeks significantly more eggs were collected from the GC than from the other groups. The heaviest eggs derived from GC and GR groups, whereas the lightest came from the C group. Eggs from the GC group had the best shell strength and the greatest proportion of yolk. The use of medicinal herbs by injection in ovo may considerably modify both time of sexual maturity and quality of the initial eggs of Japanese quail.

Imbalance of Controlled Death in Peripheral Blood Lymphocytes in Crohn's Disease and Ulcerative Colitis.

Background and objectives: Inflammatory bowel disease (IBD) mainly includes Crohn's disease (CD) and ulcerative colitis (UC). Both conditions are associated with an exacerbated intestinal immune response to harmless stimuli, leading to upregulation of pro-inflammatory mediators. Materials and Methods: The subjects of the study were 55 patients with IBD. The control group consisted of 35 healthy subjects. The researched material consisted of peripheral blood lymphocytes collected from the subjects. Expression of the genes BAX, BCL2, CASP3 and CASP9 was assessed at the mRNA level in the peripheral blood lymphocytes of patients with ulcerative colitis and Crohn's disease relative to the healthy subjects. The expression of the genes was determined by rtPCR using TaqMan probes specific for these genes. Results: The group of patients diagnosed with CD had statistically significantly higher expression of the genes BAX (p = 0.012), BCL2 (p = 0.022), CASP3 (p = 0.003) and CASP9 (p = 0.029) than healthy subjects. Expression of BAX, BCL2, CASP3 and CASP9 in UC patients in the active phase of the disease was significantly lower than in patients in remission: BAX (p = 0.001), BCL2 (p = 0.038) and CASP9 (p = 0.007). In patients with UC, the BAX/BCL2 ratio was significantly correlated (r = 0.473) with the duration of the disease. In the group of CD patients treated biologically, a significantly lower BAX/BCL2 ratio was demonstrated than in patients that were not biologically treated. Conclusions: Our research has shown a simultaneous increase in the expression of the anti-apoptotic BCL2 gene and the proapoptotic BAX gene, which suggests the dysregulation of apoptosis mechanisms in IBD. Significantly higher expression of BAX and BCL2 in UC patients in remission as compared to CD may suggest differences in these diseases in terms of prognosis and treatment. Our results may suggest that an underlying imbalance in factors controlling apoptosis in peripheral blood lymphocytes may be the response of the immune system to inflammation of the intestinal mucosa. Modulation of apoptosis may become an important therapeutic mechanism in IBD.

Molecular markers used in forensic genetics.

Forensic genetics is a field that has become subject to increasing interest in recent years. Both the technology and the markers used for forensic purposes have changed since the 1980s. The minisatellite sequences used in the famous Pitchfork case introduced genetics to the forensic sciences. Minisatellite sequences have now been replaced by more sensitive microsatellite markers, which have become the basis for the creation of genetic profile databases. Modern molecular methods also exploit single nucleotide polymorphisms, which are often the only way to identify degraded DNA samples. The same type of variation is taken into consideration in attempting to establish the ethnicity of a perpetrator and to determine phenotypic traits such as the eye or hair colour of the individual who is the source of the genetic material. This paper contains a review of the techniques and molecular markers used in human and animal forensic genetics, and also presents the potential trends in forensic genetics such as phenotyping.

Oxidative Stress and Effect of Treatment on the Oxidation Product Decomposition Processes in IBD.

Oxidative stress plays an important role in IBD because chronic intestinal inflammation is associated with the overproduction of reactive oxygen species (ROS) leading to oxidative stress, which has been implicated in IBD. Many lines of evidence suggest that IBD is associated with an imbalance between ROS and antioxidant activity which generates oxidative stress as the result of either ROS overproduction or a decrease in antioxidant activity. Our study was to evaluate the influence of oxidative stress and antioxidants on the course of the disease and treatment of IBD patients. Our results show that an increase of LOOH levels positively correlates with an increase in MDA levels; therefore, MDA may be a marker indicating lipid peroxidation. Also, being the decomposition product of oxidation processes, MDA may be applied as a useful biomarker for identifying the effect of endogenous oxidative stress in Crohn's disease patients. The anti-inflammatory efficacy of AZA drugs may be the result of a reduction of the amount of lipid peroxides in the intestinal mucosa cells in CD patients and facilitate mucosal healing.

Evaluation of propolis extract as a disinfectant of Japanese quail (Coturnix coturnix japonica) hatching eggs.

This study was carried out to evaluate the effectiveness of an alcoholic propolis extract (15%) as a disinfectant for Japanese quail (Coturnix coturnix japonica) hatching eggs. A total of 600 eggs were randomly divided into four experimental groups: 1) negative control (NC), without disinfection, 2) control (C), fumigated with formaldehyde gas, 3) (A), sprayed with 96% alcohol, and 4) (P), sprayed with 15% alcoholic propolis extract. The eggs were incubated artificially in a BIOS hatching apparatus under standard conditions. On the 14th day, the eggs were candled to determine the number of infertile eggs and dead embryos and samples were collected for microbial analysis. After 17.5 d, fertility, hatchability, embryonic mortality, and eggshell conductance were calculated. Fertile eggs sprayed with propolis were shown to have a lower eggshell conductance constant (egg weight loss) than eggs from groups C and A. Total microbial activity on the eggshells did not differ significantly between groups, but Staphylococcus aureus, Micrococcus spp., Bordetella spp., and Chryseobacterium meningosepticum isolates were significantly affected by the propolis treatment. There were no significant differences between treatments for total hatchability, embryonic mortality, and chick body weight on the 1st, 7th, and 14th days of life. The total chick survivability during the first 2 wk was significantly higher in group P than in the other groups. The results indicate that spraying hatching eggs with 15% propolis as a disinfectant can be recommended as a safe and natural sanitizer in place of formaldehyde, with no negative effect on quail chicks.

Single Nucleotide Polymorphisms in Selected Genes in Inflammatory Bowel Disease.

INTRODUCTION: Inflammatory bowel disease (IBD) is a complicated, multifunctional disorder characterized by chronic, recurring inflammation of the digestive tract. The two main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD). The aim of the study was to determine single nucleotide polymorphism in fragments of the genes CARD15/NOD2 and DLG5 in patients from the Lublin Voivodeship. PATIENTS AND METHODS: The study was carried out in Lublin (Poland) in 2016. 27 individuals participated in the research. The research group comprised 9 patients with a diagnosis of Crohn's disease and 9 with ulcerative colitis, aged 20 to 48, and 9 healthy volunteers. RESULTS: No SNPs were confirmed for the CARD15/NOD2 gene fragment, but a substitution (T>C) was found in the DLG5 gene in a Crohn's disease patient. CONCLUSION: Absence of extraintestinal symptoms in patients with Crohn's disease may be associated with the absence of CARD15/NOD2 SNPs. The study suggests that SNPs (T>C substitution) affect the function of the DLG5 protein and thus play a role in the development of IBD, in particular Crohn's disease. The analysis presented is a pilot study due to the small number of samples.

Impact of lymphocyte culture media on the number of metaphases and chromosome band resolution.

A special type of differential staining of chromosomes is replication banding. This staining technique reveals the band pattern characteristic of each homologous pair of chromosomes, which is a reflection of heterogeneous euchromatin structure. Banding enables identification of homologous chromosomes and detection of chromosomal aberrations, both structural and numerical. Slide preparation requires knowledge of many techniques, and the procedure is often different for each laboratory. The aim of the study was to determine the effect of selected media for lymphocyte cultures on the number of metaphases and the band resolution of chromosomes. The study was carried out using cell cultures from whole peripheral blood. The slides were stained by the GTG method. After their removal from the water bath they were immersed in trypsin solution, then rinsed in PBS solution and stained in Giemsa solution. After staining they were rinsed again and left to dry. The study confirmed the effect of selected commercially available cell media on the number of metaphases and band resolution of chromosomes, which have not previously been described. In all of the tests performed, the cell culture, fixation, slide preparation (automatic method), staining, and number of reagents were identical.

Analysis of cases of forensic veterinary opinions produced in a research and teaching unit.

The aim of the study was to present the results of necropsies carried out in the years 2000-2014 in the Department of Pathological Anatomy, Faculty of Veterinary Medicine, University of Life Sciences in Lublin. The material used for the analysis consisted of expert opinions prepared on the basis of a decision by a judicial body to admit an expert opinion as evidence. An increase was observed in the demand for the services of veterinary forensic experts, beginning in 2006 and persisting through 2014. The response to the growing popularity of veterinary forensic examinations should be systematization of knowledge and exchange of experience, which would enable the further development of this interdisciplinary science.

Structure of nucleoli in first-order spermatocytes of selected free-living animal species.

Nucleoli are the product of the activity of nucleolar organizer regions (NOR) in certain chromosomes. Their main functions are the formation of ribosomal subunits from ribosomal protein molecules and the transcription of genes encoding rRNA. Nucleoli are present in the nuclei of nearly all eukaryotic cells because they contain housekeeping genes. The size and number of nucleoli gradually decrease during spermatogenesis. Some of the material originating in the nucleolus probably migrates to the cytoplasm and takes part in the formation of chromatoid bodies (CB). Nucleolus fragmentation and CB assembly take place at the same stage of spermatogenesis. CB are involved in the formation of the acrosome, the migration of mitochondria to the midpiece, and the formation of the sperm tail fibrous sheath. The aim of the study was to characterize the nucleoli in the early prophase of spermatogenesis in the wild boar and the roe deer. The roe deer cells have larger nucleoli and a larger cell nucleus than the wild boar cells. The area of the nucleolus as a percentage of the total area of the nucleus was larger as well. The coefficients of variation for all parameters were higher in the roe deer. In the wild boar cells the nucleoli were mainly regularly shaped. The size of the nucleolus and the nucleus of the spermatocyte is a species-specific trait associated with karyotype and the number of nucleolar organizer regions in a given species.

Estimation of global content of 5-methylcytosine in DNA during allantoic and pulmonary respiration in the chicken embryo.

DNA methylation is an epigenetic modification that plays an important role in the proper development and functioning of an organism. The DNA methylation level is species-, tissue- and organelle-specific, and the methylation pattern is determined during embryogenesis. A correlation between methylation and age is also observed. Epigenetic phenomena are an enormously interesting research subject, not only from the perspective of pure science, but also due to their possible applications in medicine. The aim of this study was to determine the global DNA methylation level in relation to the developmental stage of the embryo. The global level of 5-methylcytosine in the DNA during pulmonary respiration was found to be higher than during allantoic respiration. The analysis shows a clear dependence between the stage of individual development and the global DNA level of 5-methylcytosine. In the future, methylation level may be a determinant of age and perhaps even a tool for predicting life expectancy. Abnormalities in the methylation process result in premature ageing at the cellular and individual level.

Age-dependent change in the morphology of nucleoli and methylation of genes of the Nucleolar Organizer Region in Japanese quail Coturnix japonica) model (Temminck and Schlegel, 1849) (Galliformes: Aves).

Nucleoli are the product of the activity of nucleolar organizer regions (NOR) in certain chromosomes. Their main functions are the formation of ribosomal subunits from ribosomal protein molecules and the transcription of genes encoding rRNA. The aim of the study was to determine the shape of nucleoli and analyse methylation in the gene RN28S in the spermatocytes of male Japanese quail (Coturnixjaponica) in two age groups. Nucleoli were analysed in cells of the first meiotic prophase. Their number and shape were determined and they were classified as regular, irregular or defragmented. In the cells of the young birds no defragmented nucleoli were observed, with regular and irregular nucleoli accounting for 97% and 3%, respectively. In the cells of older birds no regular nucleoli were observed, while irregular and defragmented nucleoli accounted for 37% and 67%, respectively. MSP (methylation-specific PCR) showed that the gene RN28S is methylated in both 15-week-old and 52-week-old quails. In recent years an association has been established between nucleolus morphology and cellular ageing processes.

DNA methylation analysis of the gene CDKN2B in Gallus gallus (chicken).

Methylation is an epigenetic modification of DNA affecting gene expression without changing the structure of nucleotides. It plays a crucial role in the embryonic and post-embryonic development of living organisms. Methylation level is tissue and species-specific and changes with age. The study was aimed at identifying the methylation of the CDKN2B gene situated at locus bar in Polbar chickens on the 6th and 18th day of embryonic development using the MSP (methylation-specific PCR) method. Methylation was not detected in the promoter region of gene CDKN2B on the 6th and 18th day of embryonic development. As one of the five genes responsible for melanine activity in melanocytes and highly active, it can contribute to the production of this pigment. The present research broadens the current knowledge of the chicken epigenome and the mechanism of autosexing in birds.