Isolation and identification of zinc ions and heparin binding proteins in yellow fraction of the red deer Cervus Elaphus L. semen.

During the rutting season, stag semen is accompanied by a sticky, dense secretion called yellow fraction (YF). There is little information about the role, biology, physiology, and most importantly, the composition of this fluid. The aim of this study was to isolate and identify zinc ions (ZnBPs) and heparin binding proteins (HBPs) from YF of the red deer (Cervus elaphus L.). Using liquid chromatography, the presence of 6 fractions of ZnBPs (71, 65, 55, 16, 14 and 12 kDa) and 22 fractions of HBPs (163, 140, 96, 78, 71, 65, 55, 49, 33, 31, 26, 25, 24, 22, 18, 16, 13, 12, 11, 10, 9 and 8 kDa) in YF proteome was demonstrated. By means of two-dimensional electrophoreses and MALDI-TOF/TOF mass spectrometry some of them were then identified. Amongst ZnBPs the following were identified: glutaminyl-peptide cyclotransferase, inhibitor of carbonic anhydrase-like, potassium voltage-gated channel subfamily E member 2, WD repeat-containing protein 38 isoform X4. Amongst the HBPs metalloproteinase inhibitor 2 (TIMP2), seminal plasma glycoprotein PSP-I and adseverin (scinderin) were identified. Identifying all ZnBPs and HBPs present in YF may broaden up-to-date knowledge concerning the biology, physiology and preservation of red deer semen.

Enteric nervous system in the European beaver (Castor fiber) pylorus - an immunohistochemical study.

European beaver (Castor fiber), the largest rodent species inhabiting a wide area of Eurasia, feeds mainly on dry parts of plants, bark or wood. Such kind of nourishment needs to be properly digested in each part of the gastrointestinal tract. The time of stomach digestion, which directly influences all the following steps of the digestion process, is precisely controlled by the pylorus and its innervation. However, virtually no data is available on the organization of the enteric nervous system in most of the wild animal species, including beavers. On the other hand, a pecu- liar diet consumed by beavers, suggests that the arrangement of their stomach intramural nerve elements can be atypical. Therefore, the present study investigated the distribution and chemical coding of neurons and nerve fibers in the pylorus of the European beaver. The experiment was performed on stomachs obtained from a group of 6 beavers caught in Northeastern region of Poland (due to beaver overpopulation). Pyloric wall tissue cryosections were double immunostained with a mixture of antibodies against pan-neuronal marker PGP 9.5 (to visualize enteric neurons) and ChAT (cholinergic marker), nNOS (nitrergic marker), SP, CGRP, Gal (peptidergic markers). Confocal microscopy analysis revealed that the majority of enteric nerve cells were clustered forming submucosal and myenteric ganglia and all the studied substances were expressed (in various amounts) in these neurons. We conclude, that the anatomical arrangement and chemical coding of intramural nerve elements in the beaver pylorus resemble those found in other mammalian species.

Pulmonary adiaspiromycosis in the Eurasian beaver (Castor fiber) inhabiting Poland.

Adiaspiromycosis is a rare fungal infection caused by saprophytic fungi Emmonsia spp. (type Ascomycota) occurring especially in small free-living mammals. The aim of this study was to evaluate the occurrence of histopathological lesions asscociated with adiaspiromycosis in the Eurasian beaver inhabiting Poland. In order to evaluate the presence of natural adiaspiromycosis we systematically investigated beaver populations from north-eastern Poland for adiaspores in the lungs. This study reveals for the first time the presence of pulmonary adiaspiromycosis of Eurasian beaver in Poland. As far as we know, there is no published data regarding pulmonary adiaspiromycosis in human patients in Poland.

Characteristics of Antioxidant Systems of Yellow Fraction of Red Deer's (Cervus elaphus L.) Semen During the Rutting Period.

The objective of this study was to make the preliminary characterization of the antioxidant defence systems of the yellow fraction (YF) of red deer's (Cervus elaphus L.) semen during the rutting period. The semen was collected using artificial vagina (AV). The studies included spectrophotometric determination of antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). We also analysed the contents of low-molecular antioxidants such as L-glutathione (GSH + GSSG), L-ascorbate (ASC) and total antioxidant status (TAS). Additionally, the samples were subjected to PAGE and stained for SOD and GPx activities. It was demonstrated that the yellow fraction exhibited activities of SOD and GPx, with the highest activities in September and October. CAT activity was not detected. Staining for the SOD and GPx activities confirmed three protein bands with SOD activity and one protein band with GPx activity. The content of GSH + GSSG was similar in trials dating from October to December contrary to the content of ASC which was high in samples from September and October. The stable rate of TAS was observed during the whole rutting period. The results of this study showed that the YF of red deer semen is equipped with basic battery of antioxidant enzymes comprising SOD and GPx, with the supporting role of GSH + GSSG and ASC. Moreover, the samples obtained at the peak of the rutting period occurring from September to October had the highest enzymatic activity in comparison with remaining months of the rutting period, which contributed to the high quality of the semen by preventing it from the formation of oxidative stress during the short period of intense sexual activity of male red deer. The better understanding of the mechanisms of antioxidant defence systems in the YF of deer's semen may contribute to the potential use of this fraction in technology of wild ruminant semen preservation.

Production of prostaglandins in placentae and corpus luteum in pregnant hinds of red deer (Cervus elaphus).

Prostaglandins (PGs) are synthesized from arachidonic acid by prostaglandin synthase 2 (PTGS2) and specific terminal PG synthases such as PGES and PGFS. The role of PGs in the reproductive processes of domestic ruminants is well recognized, whereas in cervidae, it is almost unknown, although it is noteworthy because some species of this family are valued in meat production and trophies. The aim of this study was to determine an effective marker of pregnancy and investigate the production and secretion of PGs in placenta and CL tissue in pregnancy. In the preliminary experiment, the levels of progesterone and 17-ß estradiol (RIA; N = 14 divided into seven pregnant and seven nonpregnant hinds) were measured in the peripheral blood. In the main experiment, a comparison of messenger RNA (real-time polymerase chain reaction) and protein expression (Western blotting) of PTGS2, PGES, and PGFS, the level of prostaglandin E2 (PGE2) and PGF2α in the placentae and CL in pregnant hinds (aged 3-4 years, ca. 100 days of pregnancy, N = 6). In pregnant hinds, the level of progesterone in the blood was higher than that in nonpregnant hinds (P < 0.05), whereas the level of E2 was similar in all animals (P > 0.05). The highest messenger RNA expression of PTGS2, PGES, and PGFS was observed in the placentae than in the CL (P < 0.05). The protein expression of PTGS2 and PGES was elevated in the placentae compared with the CL (P < 0.05). The PGE2 output was the highest in cotyledonary tissue (P < 0.05). Pregnancy development in hinds around 100 days is regulated by arachidonic acid metabolites, especially PGE2 produced by the placentae, which production increases in pregnancy. Further studies are required to unravel the mechanisms involved in the regulation of PG and biosynthetic enzymes in uteroplacental and ovarian tissues during pregnancy in red deer females.

Persistent Müllerian duct syndrome (PMDS) in the Polish free-ranged bull populations of the European bison (Bison bonasus L.).

This study describes the diversity of vestigial male uteri of the European bison (Eb) examined for: (1) morphology, (2) glycoprotein localization, (3) total protein and glycoprotein profiles, (4) steroid concentrations, and (5) PMDS based on the mutation of AMH and AMHR2 genes. Uteri of adult bulls (5-12 years old) were compared to a uterus of a juvenile female (6 months old). Male uterine proteins were analyzed in parallel to secretory endometrial proteins of pseudo-pregnant pig (PsEND) and BSA used as profile-controls. Hematoxylin/eosin-staining revealed the diversity of male uterine morphology, including lumen size/shape, endometrial (END) gland density, luminal knob-like epithelial structures and multiple intrauterine cells proliferating within the lumen. PAS-staining revealed the presence of glycoproteins restricted to luminal epithelial cells and END glands. Heterologous total protein PAGE-profiles (20-66kDa) revealed two dominant fractions (66 and 45kDa), similar to PAS-profiles (67 and 47kDa) in male and female uterine tissues. In male uterine tissues, androstendione and progesterone, but not testosterone, estrone or estradiol concentrations were lower than in the female. Sequencing of AMH- and AMHR2-like amplicons allowed identification of these gene mutations in Eb. Our results provide novel data regarding PMDS, demonstrating the diversity of uterine morphology, glycoprotein mass/profile, steroid concentration and AMH/AMHR2 mutations in Eb bulls.

Histological Evaluation of Selected Organs of the Eurasian Beavers (Castor fiber) Inhabiting Poland.

There is a general scarcity of data on the histological structure of major organs in the Eurasian beaver (Castor fiber). This study presents the histological characteristics of beaver organs such as the liver, spleen, cardiac muscle, lungs and kidneys. Tissue samples were collected from 21 beavers and analysed. Selected samples of tail tissue were additionally examined. Tissue samples were placed in neutral buffered formalin and embedded in paraffin. 4-µm-thick sections were stained with haematoxylin and eosin, and other staining techniques were also used. Scant amounts of inter-lobular connective tissue were found in the liver. Ion or copper deposition was not observed, but scattered cytoplasmic glycogen deposits were present in hepatocytes. Our results suggest that beavers have defensive rather than storage spleens. Interestingly, the presence of melanin in splenic red pulp was noted. The histological structure of the examined organs closely resembled that of other rodent species. According to our knowledge, this is the first study evaluating the histological structure of beaver organs. Nevertheless, precise characterization of the evaluated organs requires further work with the involvement of accurate and reliable techniques, such as molecular biology or electron microscopy methods.

Immunoexpression of aromatase in immature and adult males of the European bison (Bison bonasus, Linnaeus 1758).

Based on recent literature dealing with the role of oestrogens in the male gonad, attempts were undertaken to reveal the site of aromatization within the testis of the European bison (Bison bonasus). Testes were collected from culled animals living in free-ranging populations in Bialowieza Forest, Poland (nine males aged 8 months to 10 years). Moreover, to check for any alterations in the expression of testicular aromatase between American bison (Bison bison) and European bison, testes from one adult 10-year-old individual were also chosen for this study. For immunohistochemistry, 4% formaldehyde fixative was used. Both qualitative and quantitative evaluations of immunohistochemical staining were performed. Leydig cells, Sertoli cells and germ cells exhibited a positive immunoreaction for aromatase in testes of immature and sexually mature bison. A marked increase in aromatase expression was observed in three adult European individuals with impaired spermatogenesis. Consistent with recent data and those of our own, it might be suggested that the strong expression of aromatase negatively affects spermatogenic function in bison testes and may serve as a possible explanation of specific sperm defects observed in European bison bulls. On the contrary, one cannot exclude that differences in the aromatase immunoexpression levels are attributed to the homozygosity, the cause of frequent disease in European bison.

Identification of multiple pregnancy-associated glycoproteins (PAGs) purified from the European bison (Eb; Bison bonasus L.) placentas.

This paper describes the first identified chorionic PAGs in the European bison (Eb), named EbPAGs, predominantly expressed during early and mid-pregnancy (45-120 day post-coitum; dpc). Many EbPAGs were extracted from various cotyledonary tissues, precipitated, chromatographed (DEAE and VVA: Vicia villosa agglutinin), electrophoresed (1D- and 2D-PAGE), analysed by heterologous (cross-species) Western blotting and then micro-sequenced by Edman degradation. Finally, twelve selected VVA-purified isoforms (Ip 3.7-7.4) were entirely characterised. Nine identified NH(2)-terminal micro-sequences were found to be PAGs. On 45 dpc, three identified forms were named: EbPAG(67AkDa) (RGSNLTHPLRNIGDLFYVGN), EbPAG(55BkDa) (RGSNLTHPL) and EbPAG(50CkDa) (SQISLRGSNLTI). On 60 dpc, the next three forms were named: EbPAG(71DkDa) (RGSNLTIHPLRNIIDLFYVG), EbPAG(55EkDa) (RGSNLTHPLRNI) and EbPAG(50FkDa) (SQISLRGS). On 120 dpc, three other forms were named: EbPAG(71GkDa) (RGSNLTHPLRNIRDLFYVG), EbPAG(60HkDa) (RGSNLTTHPLRNIKDLVVYM) and EbPAG(50IkDa) (SGSNLTTV). These EbPAG ((A-I)) sequences are unique, as they are not identical to any other PAGs purified previously in related species of the Bovidae family. However, the EbPAGs (A-I forms) have some sequence resemblance to internal sequences of various full-length polypeptide PAG precursors (in silico translated from cloned cDNAs) identified in domestic cattle. Three other novel native isoforms (J1, J2 and K): EbUPG(45kDa) J1 (SKDNYKNYIPLIVPFAT), EbUPG(45kDa) J2 (SKDNQKNYIPLIVPFAT) and EbUPG(76kDa) K (SPEFTV), were temporarily named 'unknown placental glycoproteins' (UPGs), due to their efficient VVA-purification (specific for glycoproteins only) and a lack of considerable consensus to previously sequenced placental glycoproteins in the Bovidae family. This is the first study identifying NH(2)-terminals of multiple/diverse EbPAGs and some EbUPGs purified from the synepitheliochorial cotyledonary placenta of the endangered Bison bonasus (Red List).

The effect of increased ozone concentrations in the air on selected aspects of rat reproduction.

Five-month-old male rates were exposed to 0.5 ppm ozone for 50 days, 5 hours a day. A week before the completion of ozone exposure, a biological test was performed to determine the fertilization rate and the survival rate of newborns in both ozone-exposed and control animals. After 50 days, the rats were sacrificed with an overdose of halotane, and testes were collected to assess the morphology and motility of spermatozoa. Neither the morphology of spermatozoa nor motility parameters determined by the CASA (computer-assisted sperm analysis) system showed statistically significant differences between ozone-exposed and control males. The number of successful matings and the survival rate of newborns per litter within one year postpartum were also similar in both groups. However, sperm concentration was by 17% lower in ozone-exposed rats, compared with the control animals.

Chorionic mRNA expression and N-glycodiversity of pregnancy-associated glycoprotein family (PAG) of the European bison (Bison bonasus).

Placental PAG mRNA expression and N-glycodiversity of multiple PAG proteins secreted in vitro by trophectoderm (chorion epithelium) of wild pecoran Bovidae taxons was not examined previously. The study on European bison (Eb) aimed: (1) to determine placental PAG mRNA expression by in situ hybridisation; (2) to identify a profile of pecoran PAG protein family secreted in vitro by cotyledonary (CT) explants; (3) to examine N-glycodiversity of the PAG proteins in this wild taxon. In addition, we compared (4) a profile and N-glycodiversity of the PAG protein family secreted in vitro by CT and interCT-trophectoderm (intCT-TRD) explants of domestic ruminants. Cotyledonary sections of the Eb were used for in situ hybridisation (ISH) with (35)S-labelled probes produced with porcine PAG cDNA as templates. Various CT and intCT-TRD explants were long-term cultured in vitro. Chorionic proteins were isolated from media, ultra-filtrated (>10 kDa MWCO) and analysed by PAGE-Western blotting with various polyclonal anti-PAG sera. Protein samples with or without enzymatic deglycosylation were examined after different times of explant cultures. Released chorionic proteins were deglycosylated by N-glycanase F (PNGase F+) and compared to glycosylated forms (PNGase F-). This is the first paper demonstrating the PAG-like mRNA transcript expression (by ISH) and N-glycodiversity of immuno-reactive PAG-like proteins (produced in vitro by chorionic explants) of European bison. Various PAG proteins of Eb (EbPAG) were secreted by CT explants during long-term in vitro studies. Major approximately 78, approximately 67 and approximately 65 kDa EbPAG-like proteins were reduced by enzymatic deglycosylation (at least by 10 kDa). Considerably smaller amounts of approximately 45 kDa EbPAG-like proteins were also observed. In addition, we have found that various PAG proteins (30-73 kDa) were secreted by bovine CT explants, during long-term in vitro cultures. Corresponding amounts of PAG proteins, similar in M(r), were also secreted by intCT-TRD explants, whose tissues were not utilised for PAG protein extraction during other scientists' previous studies. It seems that the M(r)-heterogeneity and N-glycodiversity of the PAG protein family can play very important role during feto-placental interactions in Bovidae species.

Fractions of the semen of red deer (Cervus elaphus)--their occurrence and characteristics in different periods of season.

During a complete season 145 ejaculates were collected from 4 red deer stags (Cervus elaphus) using a modified artificial vagina. Differences found in characteristics of ejaculates allowed to identify four different types of ejaculates: pre-mating, mating, transitory, and post-mating ejaculate. These types of ejaculates occurred exclusively during corresponding periods of the season in the sequence mentioned above. Three fractions of ejaculates were identified: grey, white, and yellow. Pre- and post-mating ejaculates were homogenous and grey. The mating- and transitory ejaculate consisted of two fractions; the mating ejaculate consisted exclusively of white and yellow fractions and the transitory one contained exclusively the white and grey fraction. Respective fractions of ejaculates differed also in consistency. Pre- and post-mating ejaculates as well as the grey fraction of the transitory ejaculate were watery, the white fraction of the mating and transitory ejaculate were milky and the yellow fraction was honey-like. All respective fractions of ejaculates contained spermatozoa, except the yellow fraction. Volume, sperm concentration and pH of the respective fractions of ejaculates were examined. The yellow fraction had the largest volume and contributed most to differences between the ejaculates as concerns the volume. Sperm concentration was highest in the white fraction of the mating and the transitory ejaculate. The objective of this report was to confirm the previous findings about occurrence of distinguishable fractions and to describe their characteristics in respect to different periods of the season.