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.

Identification of Honey Bee Sperm Structures Following the use of Various Staining Techniques.

Introduction: Bees are currently artificially inseminated on a large scale for breeding and research purposes. The sperm of bees has a complex and varied structure, and determination of specific morphological defects in it is very difficult. Its comprehensive analysis by inspecting morphology and morphometry is an important tool for improving honey bee lines. The staining technique should interfere with the cells as little as possible while clearly showing the boundaries of the head and other elements. In this study, a comparative analysis of the morphometry of sperm was performed with various techniques for staining drone semen. Material and Methods: Semen was collected from 150 sexually mature Buckfast bee drones by artificially everting the copulatory organ. The morphology and morphometry of the sperm were assessed on slides prepared by three staining methods according to the protocols described online, using the Sperm Class Analyzer system. The lengths of the acrosome, nucleus, head in total, midpiece, tail without midpiece, tail with midpiece, and entire sperm were measured. Results: The most details of the drone sperm structure could be seen when stained with the eosin-nigrosin complex. This method made it possible to identify all structures and revealed the uneven distribution of sperm proteins in different parts of the tail. With the Sperm Stain method fewer details of the sperm structure were recognisable, and the fewest were with SpermBlue. Conclusion: The staining method, and thus the chemical reagents used, affect the dimensions of drone sperm. Given the great research potential of modified spermatozoa of insects, a standard for slide preparation for the evaluation of morphological and morphometric semen parameters should be established, as this would facilitate result comparison between laboratories and increase the value of morphological analysis of sperm for predicting and assessing fertility.

Assessment of the Morphometry of Heads of Normal Sperm and Sperm with the Dag Defect in the Semen of Duroc Boars.

INTRODUCTION: The Dag defect is one of the primary morphological defects in sperm correlating with reduced fertility. This defect is found in the spermatozoa of many livestock species. The aim of the study was to assess the morphometry of the heads of normal sperm and sperm with the Dag defect in the semen of Duroc breeding boars. MATERIAL AND METHODS: Sperm morphology was examined in ten ejaculates each from 12 Duroc boars. In total, 3,600 morphologically normal sperm and 838 sperm with the Dag defect were evaluated. The area, perimeter, length and width of the sperm head were measured and these basic morphometric parameters were used to calculate four additional shape indices characterising the sperm head, i.e. ellipticity, elongation, roughness and regularity. RESULTS: Sperm with this defect had markedly smaller heads, 0.32 µm shorter and 0.19 µm narrower than the heads of sperm with normal morphological structure. The heads of sperm with the Dag defect also had a 1.1µm smaller perimeter and a 2.5 µm2 smaller surface area than the heads of morphologically normal sperm. CONCLUSIONS: The Dag defect is found in boar sperm irrespective of the age of the individual. It affects the morphology of the sperm head.

Evaluation of sperm morphometry of rabbits (Oryctolagus cuniculus f. domesticus).

Sperm morphology and morphometry are considered parameters in fertility diagnosis. They are especially important in the case of species for which there is no standard with respect to morphometric sperm parameters. It is then crucial to apply the staining technique that has the least influence on the sperm structure and provides the most detailed image, so as to enable measurements. The aim of the research was to assess the morphometric parameters of rabbit sperm using silver nitrate staining. The staining process revealed a detailed image of the spermatozoon head and tail, thus enabling precise measurements. From these basic morphometric parameters, four additional shape indices characterizing the sperm head were calculated: ellipticity, elongation, roughness and regularity. These parameters more precisely characterize the shape of the sperm head. Silver nitrate staining can be used as an independent technique in assessment of sperm structure or to supplement routine diagnostics.

Morphometric Characteristics of the Spermatozoa of Blue Fox (Alopex lagopus) and Silver Fox (Vulpes vulpes).

The results presented in this study are the first such extensive characterization of the sperm morphometry of the blue fox (Alopex lagopus) and silver fox (Vulpes vulpes), as representatives of the family Canidae. Canine spermatozoa, especially the sperm of farmed foxes, are not often described in studies on reproduction. The aim of the study was a detailed comparison of the morphometric dimensions and shape of the sperm of two fox species: silver fox and blue fox. Semen collected from 10 silver foxes and 10 blue foxes was used for the study. The specimens were stained with silver nitrate. Measurements were performed of the length, width, perimeter, and area of the head; the area of the acrosome and its coverage; the length of the midpiece and its coverage; the length of the tail; and the length of the end piece of the tail. In addition, four head shape indices were calculated: ellipticity, elongation, roughness and regularity. The following values for the morphometric parameters and shape indices were obtained for blue fox and silver fox, respectively: head length-6.72 µm and 6.33 µm; head width-4.54.µm and 4.21 µm; head perimeter-18.11 µm and 17.37 µm; head area-21.94 µm2 and 21.11 µm2; acrosome area-11.50 µm2 and 10.92 µm2; midpiece length-12.85 µm and 12.79 µm; tail end piece length-3.44 µm and 3.28 µm; tail length-65.23 µm and 65.09 µm; acrosome coverage-52.43% and 52.83%; midpiece coverage-19.71% and 19.65%; sperm length-71.95 µm and 71.42 µm; ellipticity-1.49 and 1.52; elongation-0.19 and 0.20; roughness-0.84 and 1.88; regularity-1.09 and 0.99. The significance of differences between species was verified by Tukey's test at p ≤ 0.05. Statistically significant differences between species were found for the following parameters: head length, width, perimeter and area; acrosome area; tail, end piece, and total sperm length; roughness and regularity. The differences in the size and shape of sperm can be used to establish reference patterns for fox sperm enabling more accurate species identification.

Comparison of the structure of chinchilla sperm isolated from semen and from the tail of the epididymis.

Sperm cells isolated from the tail of the epididymis and from the semen of the same individuals were analysed. The use of silver nitrate to stain sperm cells isolated from the tail of the epididymis made it possible to identify structures that were not visible in the sperm from semen. Silver nitrate very clearly distinguished the acrosomal and distal parts of the sperm head. Following silver nitrate staining, the sperm isolated from the tail of the epididymis were characterized by dark 'collars' in the distal part of the head. These 'collars' are not visible in the sperm cells isolated from semen. The results of the study indicate differences in the dimensions of sperm isolated from the tail of the epididymis and sperm in semen. Sperm isolated from the tail of the epididymis had smaller heads, despite their longer length, and had longer midpieces and tails than ejaculate sperm. Silver nitrate staining is a simple and fast technique. Silver nitrate makes it possible to identify the acrosome and post-acrosomal region of the sperm head and to clearly identify the midpiece. Therefore, it can be successfully used to supplement routine techniques for evaluating sperm morphology or as an independent technique.

Influence of the age of the individual on the stability of boar sperm genetic material.

Routine evaluation of the sperm of livestock animals involves detection of morphological abnormalities. However, most sperm defects that reduce fertilizing capacity are a result of anomalies in spermatogenesis. The aim of the study was to evaluate the effect of a boar's age on the stability of the genetic material of its sperm. The age of the boar was found to have a significant effect on sperm DNA stability and chromatin structure. The highest percentage of spermatozoa with DNA fragmentation was found in the oldest group of boars (0,61%), while the highest proportion of spermatozoa with abnormal histone retention (8,01%) and protamination (9,78%) was found in the youngest group of boars. Aniline blue (AB), chromomycin A3 (CMA3) and acridine orange (AO) staining should be routinely used in individuals used for artificial insemination especially young animals at the start of their exploitation for breeding, as well as older individuals with an age-related decrease in the stability of genetic material. Earlier diagnosis based on additional tests would allow for stricter selection and elimination of males with fertility disorders from breeding, to be replaced by breeders of full value. It was also demonstrated that all three staining methods mentioned above can be used in classical morphological analysis, because they clearly distinguish the sperm head from the background of the slide. Chromomycin staining clearly reveals the midpiece and thus can be used as a specific staining method for its evaluation. Staining with aniline blue is a fast and simple test whose result can be analysed under a light microscope. This staining technique can be recommended for use at insemination stations.

Content of selected inorganic compounds in the eggs of hens kept in two different systems: organic and battery cage.

Recent years have seen increased interest in the influence of bioactive dietary components on human genes and gene expression. A good source of many bioactive substances is the chicken egg. The egg is considered to be an excellent food provided by nature. It is a good source of nutrients such as vitamins A, B2, B6, B12, D, E and K, as well as elements including phosphorus, selenium, iron, zinc, magnesium and calcium. The research material use in this study consisted of eggs from hens kept in two different systems: organic and battery cages. The content of calcium (Ca), magnesium (Mg) and zinc (Zn) was determined in the egg contents - in the yolk and white respectively. The content of elements was determined by atomic absorption spectrometry (AAS) using an AA280 FS spectrometer with the automatic dilution of standards and samples. The eggs from the organically raised hens had a higher calcium, magnesium and zinc content. The greater variation in the Ca, Mg and Zn content in the organic eggs is due to the more individualized feeding system. The rearing system of the hens significantly affects the concentration of elements in the egg. The results of this research indicate that eggs from organic farming systems have a richer chemical composition in terms of the content of nutrients such as calcium, magnesium and zinc compared with eggs obtained from caged hens. Therefore, consumers purchasing eggs should consider the system in which the hens were reared, as eggs can be a valuable source of these elements in the diet.

The effect of the staining technique on morphological and morphometric parameters of boar sperm.

Sperm morphology and morphometry are important parameters in predicting fertility. Sperm are considered to be normal if the shape and size of the head, midpiece and tail fall within the classification for a given species. It is important to select the appropriate technique for staining the semen of a given species, because, as many authors have pointed out, some methods work well for one species but are not suitable for analysing another. The aim of the study was to assess the morphometric parameters of boar sperm following the use of different staining techniques and to verify the hypothesis that the staining technique affects the morphometric parameters of sperm. The staining method was found to significantly affect the dimensions of the boar sperm head. The semen stained by the SpermBlue technique had the closest morphometric sperm head parameters to those of the unstained sperm, so this technique, rather than the routinely used eosin and gentian complex, should be the leading technique in the evaluation of boar sperm morphometry. Silver nitrate staining reveals the structure of the sperm in the most detail; this method can be considered universal, and can be used independently or to supplement routine diagnostics. As the staining technique should interfere as little as possible with the structure of the sperm, while revealing its morphology in as much detail as possible, it is crucial to establish the natural dimensions of the unstained sperm head before determining the optimal technique and its reference values. The recommended or most commonly-used techniques are not always the best options for the staining and analysis of sperm of a given species.

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.

The effect of selected staining techniques on bull sperm morphometry.

Sperm morphometry has some value as an indicator of reproductive capacity in males. In laboratory practice a variety of slide-staining methods are used during morphological evaluation of semen to predict male fertility. The aim of this study was to determine the effect of staining of semen using four different techniques on the morphometry of the bull sperm cell. The material for the study consisted of semen collected from test bulls of the Black-and-White variety of Holstein-Friesians. The results obtained in the study indicate differences in the dimensions of bull sperm heads when different slide staining techniques were used. The most similar results for sperm head dimensions were obtained in the case of SpermBlue(®) and eosin+gentian violet complex, although statistically significant differences were found between all the staining techniques. Extreme values were noted for the other staining techniques - lowest for the Papanicolaou and highest for silver nitrate, which may indicate more interference in the cell by the reagents used in the staining process. However, silver nitrate staining was best at identifying the structures of the sperm cell. Hence it is difficult to determine which of the staining methods most faithfully reveals the dimensions and shape of the bull sperm.

Frequency of Cytoplasmic Droplets Depends on the Breed and Age of Insemination Boars.

In this study an attempt was made to analyse morphological changes in sperm with particular attention to sperm with a cytoplasmic droplet, taking into account the age and breed of the boar. The material for the study consisted of ejaculates of insemination boars of five breeds. Morphological examination of sperm was carried out in 30 randomly selected boars--6 individuals from each breed. The morphology of 500 spermatozoa was evaluated in each slide. The percentage of sperm with normal morphology in the semen of the boars varied between breeds and was dependent in varying degrees on the age of the boar. The primary defects in sperm occurred more frequently in Duroc and Pietrain boars than in other breeds and were more dependent on the age of the boar. The high percentage of primary defects in the sperm of young Duroc boars was determined by the frequency of sperm cells with a proximal cytoplasmic droplet on the midpiece. A particularly high percentage of sperm cells with secondary defects was noted in the initial stage of the reproductive life of the boars. The high number of sperm cells with secondary defects noted in the semen of the Duroc and Pietrain boars and the changes occurring with age in the frequency of secondary morphological defects were mainly determined by the frequency of sperm cells with a distal protoplasmic droplet in the middle piece and to a lesser degree by that of sperm cells with a pseudodroplet.

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.

[The comet assay as a method of identifying chromosomes instability]. / Test kometowy jako metoda identyfikacji niestabilnosci chromosomów.

The basic method for analyzing the degree of DNA fragmentation caused by genotoxic factors is gel electrophoresis of single cells (single cell gel electrophoresis), also called the comet assay. The comet assay enables the analysis of the level of several different DNA modifications. The basic testing procedure has been only slightly modified. This method helps identify single-strand and double-strand DNA cracks, as well as any chemical and enzymatic modifications that can potentially turn into cracks in DNA or chromatids. The comet assay makes it possible to detect DNA damage at the level of single cells. It can be employed in analyses of any tissues which provide cellular suspensions. Analysed cells are submerged in agarose on a microscope slide. DNA is what is left after proteins have been broken down. The slide is then subjected to electrophoresis and stained with a fluorescent dye. A "comet-like" image is obtained. The "head" is the cell fixation site prior to lysis; the "tail" represents damaged DNA fragments. The extent of DNA damage is reflected in the length of the tail and the amount of DNA contained in it. The assay finds research applications in the following fields: genetic toxicology, monitoring of DNA repair following chemotherapy and radiotherapy, ecotoxicology, animal and human nourishment, biomonitoring of genotoxicity, epidemiology and assessment of material deposited in sperm and blood banks.

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.

Structure of the nucleoli in domestic cattle spermatocytes.

The work was aimed at determining the number and morphology of nucleoli in the prophase of the first meiotic division in domestic cattle males. The use of AgNO3 staining, commonly applied in cytogenetics for the identification of nucleolar organiser regions, made it possible to identify nucleoli in first-order spermatocytes. One nucleolus was identified in each analysed cell. Considerable morphological differentiation of the nucleoli during the prophase of the first meiotic division, particularly in leptotene, unobserved in other farm animal species, was noticed. Dark-hued grain-like structures were found within the disintegrating nucleoli, corresponding approximately or exactly to the number of the nucleolar organiser regions in the domestic cattle karyotype. Dark areas were identified in the selected prometaphase chromosomes. Their number corresponded with the number of active NORs defined in the domestic cattle karyotype.

Number and size of nucleoli in the spermatocytes of chicken and Japanese quail.

Nucleoli are the product of nucleolus organizing region activity (NOR) of specific chromosomes. Their basic function is to synthetise ribosomal RNA precursors and promote the maturation and assemblage of preribosomal RNP molecules. Information on rRNA-coding gene activity can be provided by the analysis of the number and size of nucleoli in the prophase of the first meiotic division. The morphology and ultrastructure of a nucleolus depends, among others, on the species and cell growth cycle as well as the physiological and pathological state of an organism. The purpose of this research was to determine the number and size of nucleoli in the spermatocytes of the domestic chicken and the Japanese quail. Diverse numbers and sizes of nucleoli in the cells of the analysed birds were observed. 1-4 nucleoli were identified in chicken cells (1.91 +/- 0.63 on average) and 1-2 in quail cells (1.13 +/- 0.33 on average). For the total of 957 nucleoli observed in Gallus cells, 329 were classified as large and 628 as small. In Coturnix cells, 563 nucleoli were identified (66 large and 497 small ones). An analysis of the numbers and sizes of nucleoli can be performed at the cytogenetic level and serve as an alternative source of information on rRNA encoding gene and nucleolus organising region (NOR) activities.

Identification and structure of lampbrush sex bivalents prior to and after the reproductive period of the European domestic goose Anser anser.

Lampbrush chromosomes (LBCs) present in bird oocytes are a new model in cytogenetics with particular significance for bird chromosome analysis. The fact that female birds are heterogametic makes it possible to observe both sex chromosomes in the form of decondensed structures typical of lampbrush chromosomes. A change in transcription activity associated with physiological processes in geese prior to and after the reproductive season is reflected in chromosome morphology. Lampbrush chromosomes obtained after the reproductive period have reduced side loops, sites of intensive transcriptional activity. However, noticeable characteristics in the chromosomes include inactive chromomeres. Chiasms, PBs, large side loops (ML) and telomeric loops (T, TLL, and GLL) are structures that undergo degradation latest after the termination of reproduction, and as a result, constitute the basis of identification of individual bivalents in different periods of the cell's transcriptional activity.

The structure of the synaptonemal complexes in meiocytes of European domestic goose (Anser anser).

The synaptonemal complex (SC) is a protein structure which binds two homologues during prophase of the first meiotic division and assures the correct course of genetic recombination. The demonstration and identification of SCs in European domestic goose Anser anser was the aim of the current research. Standard techniques of SC identification do not allow for an analysis of their molecular structure. In meiotic cells of one-day-old nestlings and 17-week-old geese the haploid number of bivalents, darkly stained kinetochores and subtelomeric regions may be evidence for the presence of SCs. Experimental chromosome staining with the DAPI fluorochrome permitted the observation of the characteristic ladder-like structure of SCs and the course of synapsis formation within homologues from early leptotene to their degradation in late pachytene. The detailed molecular structure of synaptonemal complexes can be analysed with an electron or scanning microscope only. Because the bivalent is a direct result of the complex's presence, in the literature the presence of bivalents is equivalent to the term "synaptonemal complex". However, the bivalent and the SC are two different structures.