Rumen Ciliated Protozoa of the Free-Living European Bison (Bison bonasus, Linnaeus).

This study aims to perform population analysis of the rumen ciliated protozoa of the free-living European bison (wisent, Bison bonasus, Linnaeus). The samples of the rumen fluid from the 18 bison subjected to the controlled culls within the free-ranging population in the Bialowieza primeval forest in Poland were collected and examined. The examined ciliates population consisted of the species of the families Isotrichidae and Ophryoscolecidae. There were 12 genera (Isotricha, Dasytricha, Diplodinium, Elytroplastron, Entodinium, Eodinium, Epidinium, Eremoplastron, Eudiplodinium, Metadinium, Ophryoscolex, and Ostracodinium) and 32 morphospecies of the ciliates. We observed the prevalence of a type B protozoan population (56% animals) with the typical Epidinium and Eudiplodinium genera members. Other examined animals possessed the mixed A-B population with Ophryoscolex genus, distinct for type A ciliate population. The average total ciliates count was 2.77 ± 1.03 × 105/ml (mean ± SD). The most abundant genera were Entodinium, 83%, and Dasytricha, 14%. The abundance of other genera was <1% of the total count. Within the 16 Entodinium species determined, the most abundant species was Entodinium nanellum (16.3% of total ciliates count). The average Shannon-Wiener diversity index was 2.1 ± 0.39, evenness was 0.7 ± 0.11, and species richness was 24 ± 3.0 (mean ± SD). Our study is the first report on the population composition and diversity of rumen ciliates of European bison. The composition and counts of ciliate genera and species were similar to the composition and counts of the rumen ciliated protozoa of American bison and many other kinds of free-living and domestic ruminants. Our European bison ciliate population analysis has shown medium ciliate density and high diversity typical for large free-living ruminants with mixed feeding behavior.

Temporal trends (1953-2012) of toxic and essential elements in red deer antlers from northeastern Poland.

Red deer antlers have a number of advantages that make them a unique material for monitoring trace elements. As antlers are shed and regrown every year, results of toxicological investigations can be applied to a particular region and time. We analyzed the content of four toxic (Pb, Cd, Hg, As) and three essential (Cu, Zn, Fe) trace elements in 254 red deer antler samples spanning between 1953 and 2012. Age of stags did not influence concentrations of analyzed elements in antlers, except for Zn whose level increased with age. The highest concentrations of toxic elements occurred at the beginning of the analyzed period. Levels of Pb, Hg and Zn in antlers decreased over the course of the study. Levels of Cd and As were low and presented a steady trend. Variations in the levels of the analyzed elements in red deer antlers are considered to reflect levels of exposure of animals in their habitat over the sixty-year study period. The range of essential element levels did not indicate any contamination. Environmental conditions in the Mazury Region during the last decades appeared to have improved significantly, as established by declining trends of toxic elements levels in deer antlers.

Seasonal differences in the testicular transcriptome profile of free-living European beavers (Castor fiber L.) determined by the RNA-Seq method.

The European beaver (Castor fiber L.) is an important free-living rodent that inhabits Eurasian temperate forests. Beavers are often referred to as ecosystem engineers because they create or change existing habitats, enhance biodiversity and prepare the environment for diverse plant and animal species. Beavers are protected in most European Union countries, but their genomic background remains unknown. In this study, gene expression patterns in beaver testes and the variations in genetic expression in breeding and non-breeding seasons were determined by high-throughput transcriptome sequencing. Paired-end sequencing in the Illumina HiSeq 2000 sequencer produced a total of 373.06 million of high-quality reads. De novo assembly of contigs yielded 130,741 unigenes with an average length of 1,369.3 nt, N50 value of 1,734, and average GC content of 46.51%. A comprehensive analysis of the testicular transcriptome revealed more than 26,000 highly expressed unigenes which exhibited the highest homology with Rattus norvegicus and Ictidomys tridecemlineatus genomes. More than 8,000 highly expressed genes were found to be involved in fundamental biological processes, cellular components or molecular pathways. The study also revealed 42 genes whose regulation differed between breeding and non-breeding seasons. During the non-breeding period, the expression of 37 genes was up-regulated, and the expression of 5 genes was down-regulated relative to the breeding season. The identified genes encode molecules which are involved in signaling transduction, DNA repair, stress responses, inflammatory processes, metabolism and steroidogenesis. Our results pave the way for further research into season-dependent variations in beaver testes.

Can red deer antlers be used as an indicator of environmental and edible tissues' trace element contamination?

Venison is an attractive product for consumers concerned with healthy lifestyle; however, it can contain high levels of toxic elements, and therefore, it is a possible source of hazardous contaminants in human diet. Antlers are suitable bioindicators of environmental metal contamination, and herein, we assessed the ability of trace element levels in antlers to indicate levels in edible soft tissues. We determined the concentrations of lead (Pb), cadmium (Cd), mercury (Hg), arsenic (As), copper (Cu), zinc (Zn), and iron (Fe) in the liver, kidney, muscle, and antlers of 14 free-ranging red deer (Cervus elaphus) from northeastern Poland using atomic absorption spectrometry. We found the highest concentrations of Pb (0.321 ± 0.165 mg/kg), As (0.045 ± 0.074 mg/kg), Zn (105.31 ± 16.33 mg/kg), and Fe (220.92 ± 117.18 mg/kg) in antlers; of Cd (4.974 ± 1.90 mg/kg) and Hg (0.048 ± 0.102 mg/kg) in kidney; and of Cu (7.29 ± 7.02 mg/kg) in the liver. A positive relationship between concentrations in antlers and muscle was found only for Cu (p = 0.001), and it therefore appears that red deer antlers cannot be used as an index for element concentrations in soft tissues. While our results confirm that the Mazury region is little polluted, consumption of red deer offal from this area should be limited according to extant legal limits set for livestock consumption.

Morphological and neuroanatomical study of the mammary gland in the immature and mature European beaver (Castor fiber).

This study investigated general morphology and immunohistochemical properties of nerve fibres supplying the mammary gland (MG) in the European beaver. The microscopic analysis of the beaver mammary gland revealed the presence of morphological structures which are characteristic for mammals. There were no distinct differences in the morphological features of the mammary gland between the juvenile and non-pregnant mature beaver. The nerve fibres were visualized using antibodies against protein gene product 9.5 (PGP) and biologically active substances including ß-hydroxylase tyrosine (DßH), neuropeptide Y (NPY), calcitonine gene-related peptide (CGRP) and substance P (SP). The study has revealed that the MG in the juvenile and mature beaver is richly supplied with PGP-immunoreactive (PGP-IR) nerve fibres. The most abundant innervation was observed in the nipple and less numerous nerve terminals supplied the glandular tissue. Double-labelling immunohistochemistry disclosed that the majority of PGP-IR nerve fibres associated with blood vessels and smooth muscle cells in both the nipple and glandular tissue were also DßH-IR. However, these nerve terminals were less numerous in the glandular tissue than in the nipple. Most of the DßH-IR axons associated with arteries and smooth muscle cells in the entire gland also stained for NPY. Small number of DßH/NPY-IR fibres supplied veins. CGRP-IR fibres were more abundant than those expressing SP. No distinct differences in the distribution and immunohistochemical characteristic of nerve fibres were observed between the juvenile and adult animals. The distribution and immunohistochemical properties of nerve fibres supplying the gland in the beaver remind those previously described in other mammalian species.

Determination of perfluorinated sulfonate and perfluorinated acids in tissues of free-living European beaver (castor fiber L.) by d-SPE/ micro-UHPLC-MS/MS.

Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the main representatives of an rising class of persistent organic pollutants (POPs), perfluorochemicals (PFCs). In this study, determination of selected PFCs concentration in liver, brain, tail, adipose and peritoneum tissues of free-living European beaver (Castor fiber L.) was addressed. Tissue samples, collected from beavers living in Masurian Lakeland (NE Poland), were analyzed by dispersive Solid Phase Extraction (d-SPE) with micro-UHPLC-MS/MS system. In a group of ten selected pefrluorinated compounds only two perfluorinated acids (PFOA and PFNA) and one perfluorinated sulfonate (PFOS) were quantified. PFOA was detected in all analysed tissue samples in both female and male beavers in a range from 0.55 to 0.98ngg(-1) ww whereas PFOS was identified in all analyzed female beaver tissues and only in liver, subcutaneous adipose and peritoneum tissues of male beavers at the concentration level from 0.86 to 5.08ngg(-1) ww. PFNA was only identified in female beaver tissues (liver, subcutaneous adipose and peritoneum) in a range from 1.50 to 6.61ngg(-1) ww. This study demonstrated the bioaccumulation of PFCs in tissue samples collected from beavers living in area known as green lungs of Poland. The results provided in this study indicate for the increasing risk of PFCs occurrence in the environment and the level of PFCs in tissue of free-living European beavers may serve as bioindicator of environmental pollution by these compounds.

Light and electron microscopy of the European beaver (Castor fiber) stomach reveal unique morphological features with possible general biological significance.

Anatomical, histological, and ultrastructural studies of the European beaver stomach revealed several unique morphological features. The prominent attribute of its gross morphology was the cardiogastric gland (CGG), located near the oesophageal entrance. Light microscopy showed that the CGG was formed by invaginations of the mucosa into the submucosa, which contained densely packed proper gastric glands comprised primarily of parietal and chief cells. Mucous neck cells represented <0.1% of cells in the CGG gastric glands and 22-32% of cells in the proper gastric glands of the mucosa lining the stomach lumen. These data suggest that chief cells in the CGG develop from undifferentiated cells that migrate through the gastric gland neck rather than from mucous neck cells. Classical chief cell formation (i.e., arising from mucous neck cells) occurred in the mucosa lining the stomach lumen, however. The muscularis around the CGG consisted primarily of skeletal muscle tissue. The cardiac region was rudimentary while the fundus/corpus and pyloric regions were equally developed. Another unusual feature of the beaver stomach was the presence of specific mucus with a thickness up to 950 µm (in frozen, unfixed sections) that coated the mucosa. Our observations suggest that the formation of this mucus is complex and includes the secretory granule accumulation in the cytoplasm of pit cells, the granule aggregation inside cells, and the incorporation of degenerating cells into the mucus.

Co-expression pattern of dopamine beta-hydroxylase (DßH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.).

Co-expression of dopamine ß-hydroxylase (DßH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45-120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DßH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DßH immunoreactive nerve fibers (DßH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DßH, while some DßH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DßH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DßH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.

Divergent evolution in the cytoplasmic domains of PRLR and GHR genes in Artiodactyla.

BACKGROUND: Prolactin receptor (PRLR) and growth hormone receptor (GHR) belong to the large superfamily of class 1 cytokine receptors. Both of them have been identified as candidate genes affecting key quantitative traits, like growth and reproduction in livestock. We have previously studied the molecular anatomy of the cytoplasmic domain of GHR in different cattle breeds and artiodactyl species. In this study we have analysed the corresponding cytoplasmic signalling region of PRLR. RESULTS: We sequenced PRLR gene exon 10, coding for the major part of the cytoplasmic domain, from cattle, American bison, European bison, yak, sheep, pig and wild boar individuals. We found different patterns of variation in the two receptors within and between ruminants and pigs. Pigs and bison species have no variation within GHR exon 10, but show high haplotype diversity for the PRLR exon 10. In cattle, PRLR shows lower diversity than GHR. The Bovinae PRLR haplotype network fits better the known phylogenetic relationships between the species than that of the GHR, where differences within cattle breeds are larger than between the different species in the subfamily. By comparison with the wild boar haplotypes, a high number of subsequent nonsynonymous substitutions seem to have accumulated in the pig PRLR exon 10 after domestication. CONCLUSION: Both genes affect a multitude of traits that have been targets of selection after domestication. The genes seem to have responded differently to different selection pressures imposed by human artificial selection. The results suggest possible effects of selective sweeps in GHR before domestication in the pig lineage or species divergence in the Bison lineage. The PRLR results may be explained by strong directional selection in pigs or functional switching.

Age-dependent pattern of connexin43 expression in testes of European bison (Bison bonasus, L.).

Morphology and the expression of connexin43 (Cx43) was investigated in testes of immature, prepubertal, and adult European bison bulls by means of routine histology, immunohistochemistry, and Western blotting, respectively. Testes were collected from culled animals living in Bialowieza and Borecka Forests, Poland. Histological examination of testicular tissue of immature and prepubertal males revealed normal structure, whereas of adult individuals either normal testicular structure with advanced spermatogenesis or varying degrees of tubule and interstitial tissue abnormality were seen. Immunohistochemical studies revealed Cx43 signal mostly at Leydig cell membrane appositions. In testes of immature males heterogeneous staining was observed; its intensity markedly increased in prepubertal males reaching almost two times more intense staining in adults. Strong Cx43 signal between Leydig cells was also observed in testes with structural alterations; however, sporadically it was of weak linear pattern. In the tubules of the latter, the intensity of Cx43 staining was weak to moderate or it was even absent. Immunohistochemical qualitative analysis was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density of diaminobenzidine deposits. Data from Western blot analyses confirmed the results obtained by immunohistochemistry; immunodetectable Cx43 protein as a band of 43 kDa was detected in all testes samples. Overall, the increase in Cx43 level in testes along the bison postnatal development may be capable of a better exchange of metabolites and coordinating Leydig cell activity during maturation. A relationship between homozygosity occurring in European bison and altered intercellular communication is suggested.

Cellular localisation of the pregnancy-associated glycoprotein family (PAGs) in the synepitheliochorial placenta of the European bison.

This paper describes the cellular immuno-localisation of the PAG family in synepitheliochorial (cotyledonary) placenta of the European bison (Eb). Uteri were harvested from pregnant wild Eb (n=4; 45-150 days post coitum-dpc); and additionally from cattle (30, 45 dpc) and pigs (42 dpc)--both domestic species were used as positive controls for cellular PAG immunodetection. Placentas were sectioned, fixed, dehydrated and subjected to double fluorescent immunohistochemistry (dF-IHC) with the use of Alexa 488 fluorochrom (A488) and propidium iodide (PI). Native positive EbPAG signals were detected by heterologous (ht; cross-species) dF-IHC with primary rabbit anti-PAG polyclonals against native or recombinant porcine PAG antigens (anti-pPAG); then visualised with secondary anti-rabbit goat immunoglobulins--conjugated to A488. Our htdF-IHC indicated an unequivocal localisation to the mono- and bi-nuclear trophectoderm (chorionic epithelium) cells expressing the PAGs (A488-green) among all placental cells, in which PI (red) stained nuclei. This is the first paper reporting the EbPAG family expression examined by htdF-IHC at the feto-maternal interface in wild Pecoran species. The cross-reactivity of anti-pPAG polyclonals with the EbPAGs suggests that shared epitopes are present in these molecules. It seems that the EbPAG family, which is robustly expressed in mono- and bi-nucleated trophectoderm cells, is associated with events taking place during placenta development. Our study also provided a proficient ht-system to identify various PAGs that could be useful as prenatal protein markers for pregnancy diagnoses, which is essential for effective reproductive management of endangered mammals.

Sperm morphology of the Eurasian beaver, Castor fiber: an example of a species of rodent with highly derived and pleiomorphic sperm populations.

The structural organization of the spermatozoon from the Eurasian beaver, Castor fiber (Family: Castoridae), was determined and compared to that of other sciuromorph rodents. The beaver spermatozoon has a head, which is variable in form but usually paddle-shaped, with a small nucleus and very large acrosome, and a tail that is relatively short compared to that of most other rodents. Transmission electron microscopy indicates that in most testicular spermatozoa the acrosome projects apically, although in a few it becomes partly flexed. During the final stages of maturation, however, the acrosome becomes highly folded so that the apical segment comes to lie alongside part of the acrosome that occurs lateral to the nucleus, with, in some cases, fusion taking place between the outer acrosomal membranes. The sperm nucleus is wedge-shaped, being broader basally and narrowing apically with an occasional large nuclear vacuole occurring. This spermatozoon structure is markedly different from that found in the other species of Geomyoidea, which is the sister group of the Castoridae. The findings thus emphasize the highly divergent nature of the beaver spermatozoon and demonstrate that, within the proposed Infraorder Castorimorpha, very large differences in sperm structure have evolved.

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.

Multiple forms of Pregnancy-Associated Glycoproteins released in vitro by porcine chorion or placentomal and interplacentomal explants of wild and domestic ruminants.

Characterization of the Pregnancy-Associated Glycoproteins (PAG) is important for studies of reproduction of various eutherian domestic, wild and endangered mammals. Distinct chorionic PAG genes are expressed in embryo-origin cells: pre-placental trophoblast (TR) and in placental trophectoderm (TRD) of various entherians. This study demonstrates in vitro production of the PAG proteins during long-term cultures of various chorionic explants: porcine TR or TRD, cotyledonary (CT) of European bison (Eb), and CT or intercotyledonary (intCT)-TRD of the cattle. Chorionic proteins isolated from media were analyzed by homologous or heterologous Western immunoblotting with anti-PAG sera, raised against cellular bovine or secretory porcine antigens. Used anti-PAG sera identified diverse molecular forms of released PAG proteins: 43-69 kDa for EbPAG proteins, 40-85 kDa for bovine PAG (bPAG), and 43-73 kDa for porcine PAG (pPAG). Immunoblotting revealed also that both CT and intCT-TRD explants secreted equivalent amounts of bPAG proteins. This useful system of in vitro protein production can provide native chorionic PAG proteins with placental unique carbohydrate chains. The PAG proteins are required as standard markers for diagnostic tests of pregnancy in domestic and wild mammals, in which seasonal reproductive processes are relatively difficult to control.

The effect of cyproterone acetate on the antler cycle in red deer (Cervus elaphus L.).

The aim of the study was to test the effect of antiandrogen, cyproterone acetate (CA) on the antler cycle in the red deer (Cervus elaphus). CA was administered to three adult red deer stags (Edward, Fuks and Gacek) in weekly intervals. Edward and Fuks were given 600 mg + 600 mg of CA, whereas Gacek was given 600 mg + 300 mg. CA was injected during the hard antler phase: in mid-October (Edward), at the end of November (Fuks) and at the end of January (Gacek). CA caused the antler casting 17 to 22 days after the first injection. In all stags, the casting of antlers was followed by a period of intensive growth of new antlers. Edward was given CA at the end of October. This treatment was responsible for occurrence of the two antler cycles in the year of the experiment. When CA was administered during the middle of the hard antler phase an additional short antler cycle occurs followed by new antler growth. CA treatment in the later part of hard antler phase may cause a prolonged antler cycle.

Effect of season on characteristics of red deer /Cervus elaphus L./ semen collected using modified artificial vagina.

During three reproductive seasons, 572 ejaculates were collected from five farmed red deer stags using a modified artificial vagina. The ejaculates were evaluated in terms of their quality and quantity by standard procedures applied for domestic animals. The total length of sexual activity was 245 days, from August 4 to April 6. Significant changes in the semen parameters were noted over this period, divided into three stages: pre-mating, mating, post-mating. During mating season the following values of sperm parameters were recorded: 1/ volume of white and yellow fractions -0.18 and 2.03 ml, respectively; 2/ pH of white and yellow fraction -6.80-7.41 and 6.65-7.45, respectively; 3/ sperm concentration -2.27 mln/mm3; 4/ sperm motility -47% (with 80% of them showing steady progressive motion at moderate speed); 5/ duration of sperm motility -6 hours 39 minutes, and 6/ low percentage of major and minor sperm defects (<5% and <10%, respectively). In summary, measures of semen quality including fraction volume, pH, sperm concentration and sperm motility change gradually during the pre-mating, mating and post-mating seasons of red deer. The period of greatest libido (from the end of September until the end October) coincides with highest semen quality.