Genotoxicity of physical silver nanoparticles, produced by the HVAD method, for Chinchilla lanigera genome.

Each year, growing demand for silver nanoparticles (AgNP) contributes to the search for alternative methods of their production. Stable AgNP with antibacterial properties, low toxicity to the environment and living organisms are especially valued. In the study presented here, an attempt was made to assess the toxicity of two AgNP solutions produced using the HVAD method to the Chinchilla lanigera genome. The AgNO3 solution was the indicator and reference for the harmfulness of AgNP. The study was carried out in vitro on bone marrow cells isolated from Chinchilla lanigera bones. The genotoxicity was assessed by comet assay, following the treatment of cells with three silver solutions: unstable and sodium citrate-stabilized silver nanoparticles, as well as silver nitrate at three concentrations (5, 10 and 20 µg/L), after 3, 6 and 24 h. Based on the percentage of the DNA content in the comet tail and the tail moment, an increase in cell DNA integrity disruption was demonstrated in all tested variants: of solution, exposure time and concentration, compared to the control sample. A statistically significant correlation was determined between the level of induced DNA breaks and the concentration of the active solutions and the duration of their activity. A solution of silver nanoparticles stabilized with sodium citrate was shown to have the most harmful effect on bone marrow cells. Silver nitrate demonstrated a level of toxicity similar to these particles. Further studies are necessary to directly compare the genotoxic properties of AgNP produced using the HVAD method and the chemical method under the same conditions.

Chromosomal Instability at Fragile Sites in Blue Foxes, Silver Foxes, and Their Interspecific Hybrids.

A cytogenetic assay based on fragile sites (FS) enables the identification of breaks, chromatid gaps, and deletions. In healthy individuals, the number of these instabilities remains low. Genome stability in these species is affected by Robertsonian translocations in the karyotype of the blue fox and by B chromosomes in the silver fox. The aims of the study were to characterise the karyotype of blue foxes, silver foxes, and their hybrids and to identify chromosomal fragile sites used to evaluate genome stability. The diploid number of A chromosomes in blue foxes ranged from 48 to 50, while the number of B chromosomes in silver foxes varied from one to four, with a constant number of A chromosomes (2n = 34). In interspecific hybrids, both types of karyotypic variation were identified, with the diploid number of A chromosomes ranging from 40 to 44 and the number of B chromosomes varying from 0 to 3. The mean frequency of FS in foxes was 4.06 ± 0.19: 4.61 ± 0.37 in blue foxes, 3.46 ± 0.28 in silver foxes, and 4.12 ± 0.22 in hybrids. A relationship was identified between an increased number of A chromosomes in the karyotype of the hybrids and the frequency of chromosomal breaks. The FS assay was used as a biomarker for the evaluation of genomic stability in the animals in the study.

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.

Determination of cytogenetic markers for biological monitoring in coypu (Myocastor coypu).

Cytogenetic tests are used to assess the influence of physical and chemical factors with potential mutagenic and genotoxic properties on the animal organism. The test results make it possible to eliminate mutagens, as well as helping predict possible genetic consequences in animal cells and assess animal resistance. The aim of this study was to examine, using cytogenetic tests, the spontaneous chromosome and DNA damage in coypu lymphocytes. Four tests: fragile site (FS), bleomycin (BLM), micronucleus, (MN) and comet were used for the first time in coypu cells. The averages with standard deviations obtained in the research were as follows: 3.30 ± 0.80 fragile sites/cell; 0.63 ± 0.80 BLM damage/cell; 6.10 ± 0.53% binucleated cells with MN; and 3.24 ± 0.63% DNA in tail. The present analysis showed high interindividual variation in spontaneous chromosomal and DNA damage levels. In the case of micronucleus, fragile sites, and comet assays, the differences between animals were statistically significant. The data suggest that these assays are sensitive enough to detect some effects on an individual animal and can be proposed as tools for coypu biomonitoring.

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.

Cytogenetic Karyotype Analysis in Selected Species of the Erinaceidae Family.

INTRODUCTION: Comparative analysis of the karyotype structure was made in two hedgehog species: the northern white-breasted hedgehog (Erinaceus roumanicus) and the African pygmy hedgehog (Atelerix albiventris). MATERIAL AND METHODS: The cytogenetic analysis used differential staining techniques (DAPI, Ag-NOR, and C-banding/DAPI) and sequential QFQ/FISH banding with NOR20 and TEL20 probes which showed 45S rDNA and (TTAGGG)n repeat sequences, respectively, on hedgehog chromosomes. RESULTS: It was confirmed that the somatic cells of the hedgehogs have a constant number of chromosomes (2n = 48,XY). Differences were observed in the NOR number between the species. NORs were identified on three autosome pairs in the northern white-breasted hedgehog and on only two pairs in the African pygmy hedgehog. Chromosome analysis by C-banding/DAPI showed large segments of heterochromatin rich in A-T pairs on three autosome pairs in both the northern white-breasted and African pygmy hedgehogs. The heterochromatin segments encompassed large fragments of the longer arm of chromosome pairs 13, 14 and 20. The (TTAGGG)n repeat sequences on the hedgehog chromosomes were only observed in the terminal position of all the chromosomes in both species. CONCLUSION: Our observations provide new information on the level of diversity within the Erinaceidae family.

Evaluation of chromosomal instability in somatic cells of farmed foxes.

The micronucleus (MN) test is a common tool used to evaluate cellular genetic instability at the chromosomal level. It determines the effect of physical, chemical and environmental factors on DNA, and thus the body's individual resistance to harmful substances. The karyotypes of blue and silver foxes and their interspecific hybrids are characterized by morphological and structural variation. This variation is partly attributable to the presence of chromosomal polymorphism, which may significantly influence the stability of genetic material in the cells of these species. The objective of the study was to evaluate genetic material stability in selected Canidae species. To this end, analyses using the MN test were performed. Binucleated cells (BNCs) were analysed in microscopic preparations, and the number of micronuclei was determined within these cells. For the proportions of both MN and BNCs, highly significant differences were observed between the fox species. The interspecific hybrids differed from the other fox species in MN percentage. The lowest average was noted in blue foxes (3.33) and the highest in interspecific hybrids (15.21).

The Effect of Bromodeoxyuridine on Spontaneous Sister Chromatid Exchange Frequency in Rabbit (Oryctolagus cuniculus) and Coypu (Myocastor coypu) Chromosomes.

The sister chromatid exchange test is regarded as a highly sensitive cytogenetic assay. It measures chromosome sensitivity to particular damage factors and provides information on control and repair mechanism performance. It is instrumental in the early identification of the effects of noxious factors present in the habitat. This investigation was aimed at identifying sister chromatid exchange sites in coypu and rabbit chromosomes, as well as determining the spontaneity of the process by applying different BrdU doses. The chromosomes were obtained from an in vitro culture of blood lymphocytes, supplemented with 4 different BrdU doses: 0.25/0.5/1.0/2.5 µg/ml in order to identify spontaneous sister chromatid exchanges in both animal species. The chromosomes were stained according to the FPG method. Spontaneous SCEs were observed in coypu at a concentration of 1.0 µg/ml, and in rabbits at 0.5 µg/ml. The mean SCE/cell incidence was 1.41±1.15 in coypu, and 2.69±2.14 in rabbits. Differences in SCE incidence were identified between the analysed animal species and the applied BrdU doses.

Influence of Selected Extenders for Liquid Storage at 4 degrees C of Breeding Chinchilla (Chinchilla lanigera) Semen on Sperm DNA Integrity.

The influence of two commercial and two laboratory oriented extenders on survival rate and DNA integrity of chinchilla (Chinchilla lanigera) sperm was determined during liquid storage. Semen was collected using an electroejaculator from 6 adult male chinchillas. Ejaculates (n = 16) were diluted with extenders to obtain a concentration of 40 x 10 (3) sperm/5 µl. After dilution the semen samples were stored at 4"C. The percent motility, progressive motility, and morphology were assessed conventionally, whereas DNA integrity was evaluated by Single Cell Gel Electrophoresis (comet) assay at 0 (just after dilution), 24, 48 and 72 h. Conventional assessment of sperm quality showed that commercial extenders are characterized by the lowest sperm survival parameters out of the investigated extenders. In commercial extenders spermatozoa lost their capacity for progressive motility compared to laboratory extenders. After 24 h storage, from 21.67% to 30% of motile sperms were observed in commercial extender whereas total sperm motility was 63.33% (41.67% with progressive motility) in samples in which stallion semen extender was used. After 72 h storage, 10% of sperm were motile in stallion semen extender while no sperm movement was observed in tubes containing the commercial extender. Furthermore, a lower percentage of damaged spermatozoa in laboratory oriented extenders was demonstrated. It was also stated that along with the extended time of semen storage at 4 degrees C, commercial extenders lost their protective action. An analysis of DNA content in the heads of sperm cells and tail moment (TM) showed that the most useful extender for liquid preservation of chinchilla semen was the extender for stallions.

Season controlled reproduction of undomesticated animals.

The following article is a summary of research on the influence of season on the reproductive processes in undomesticated animals. The results presented below show: a/ an annual hormonal profile of domestic pig and wild boar crossbreed and the antioxidant blood system in the different seasons, b/ the possibility of gonadptropic hormone stimulation in chinchillas which are in diestrus or infertile, c/ the possibility of using bison's semen (collected post mortem from the epididymis) for cryoconservation.