Hypoxia enhances steroidogenic competence of buffalo (Bubalus bubalis) granulosa cells.

Granulosa cells (GCs) synthesize estrogens needed for follicular growth. However, the effects of hypoxia on steroidogenesis in buffalo GCs remain unclear. In this study, the impacts of hypoxic conditions (5% oxygen) on estrogen synthesis in buffalo GCs were examined. The results showed that hypoxia improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3ß-HSD) and the secretion levels of estradiol in buffalo GCs. Hypoxic conditions promoted the sensitivity of buffalo GCs to FSH. Furthermore, inhibition of cAMP/PKA signaling pathway (H89, a cAMP/PKA signaling pathway inhibitor) reduced both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3ß-HSD) and the secretion levels of estradiol in hypoxia-cultured buffalo GCs. Besides, inhibition of cAMP/PKA signaling pathway lowered the responsiveness of buffalo GCs to FSH under hypoxic conditions. The present study indicated that hypoxia enhanced the steroidogenic competence of buffalo GCs principal by affecting cAMP/PKA signaling pathway and subsequent sensitivity of GCs to FSH.

Gene expression of pregnancy-associated glycoproteins-1 (PAG-1), interferon-tau (IFNt) and interferon stimulated genes (ISGs) as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows.

The aim of this study was to determine the presence of Pregnancy-associated glycoprotein -1 (PAG-1) mRNA expression in the maternal circulation of pregnant buffaloes during the early stage of pregnancy. Contemporaneously, the mRNA expression levels of Interferon-tau (IFNt) and some Interferon stimulated genes (ISGs) (interferon stimulated gene 15 ubiquitin-like modifier interferon, ISG15; Mixoviruses resistance 1 and 2, MX1 and MX2; 2',5'-oligoadenylate synthase 1,OAS1) were evaluated in order to expand our knowledge of the molecular processes involved in the early stages of pregnancy and to identify potential biomarkers of maternal-fetal cellular interaction in buffalo. The study was conducted on 38 synchronized and artificially inseminated buffalo cows (d 0), divided ex post into 3 groups: Pregnant (n = 17), Non-pregnant (n = 15) and Embryo mortality (n = 6). Blood samples were collected on d 14, 19, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) isolation. Expression levels of mRNA of PAG-1, IFNt, ISG15. MX1, MX2 and OAS1 were measured using RT-qPCR. No significant changes were observed in IFNt and PAG gene expressions between groups, while significant differences (p < 0.001) were found for ISG15, MX1, MX2, and OAS1. Pairwise comparisons revealed that the differences between groups occurred on days 19 and 28 post-AI. ISG15 proved to have the best diagnostic performance for distinguishing between pregnant animals and animals that experienced embryo mortality with the ROC analysis. According to the results of the univariate analyses, day 19 was identified as the most indicative to discriminate between groups while the most reliable genes for this differentiation were ISG15, MX1 and MX2. MX2 proved to be the best gene for discriminating pregnant buffaloes using the discriminant analysis, while MX1 was the gene that best predicted embryo mortality. Our results showed that among PAG-1, IFNt and ISGs expression as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows, ISGs proved to be the best peripheral biomarkers for predicting pregnancy and embryonic mortality during the peri-implantation period. These insights into the mechanisms behind maternal-fetal interaction and the development of a method for the early detection of embryo distress may enable us to implement effective strategies to support embryo survival.

Enzyme histochemical characterization of orbital glands in fetuses of Indian buffalo (Bubalus bubalis).

Background: The orbital glands, viz. lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia. Methods: The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse). Results: The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved.

A new role of H89: Reduces capacitation-like changes through inhibition of cholesterol efflux, calcium influx, and proteins tyrosine phosphorylation during sperm cryopreservation in buffalo.

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 µM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 µM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process.

Structure, mechanism and lipid-mediated remodeling of the mammalian Na+/H+ exchanger NHA2.

The Na+/H+ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na+/Li+ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na+/H+ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity.

Seasonal expression of insulin-like growth factor 1 (IGF-1), its receptor IGF-1R and klotho in testis and epididymis of the European bison (Bison bonasus, Linnaeus 1758).

The European bisons are the largest mammals of Europe that are still in danger of extinction. The species conservation is associated with their continuous reproduction, and bisons are characterized by the well-pronounced seasonality of reproductive processes. However, the exact mechanisms regulating their reproduction still remain unknown. Our previous studies indicated the involvement of some of the growth factors in the regulation of male seasonal reproductive activities in bison, showing expression patterns that seemed to be regulated by the length of the daylight. In the present study, using RT-PCR and Western blot approaches, we verified the expression and possible relationship between the insulin-like growth factor (IGF-1), its receptor (IGF-1R), and klotho in testis and epididymis of the European bison in pre- and post-reproductive periods, i.e., in June and in December. The observed expression of IGF-1 and IGF-1R mRNA in testis and epididymis was higher in June than in December. At the same time, klotho mRNA expression in both testis and epididymis did not differ between the analyzed seasons. However, along with the higher levels of IGF-1R protein observed in June, klotho protein levels for the membrane form and for the secrete form were higher in December than in June. Finally, the messenger and protein expression profiles presented herein indicate the importance of both the IGF-system and klotho in reproductive processes in the European bison, implying their involvement in the regulation of seasonal testicular activity in males of this threatened species.

Immunohistochemical characteristic of C cells in European bison thyroid gland.

INTRODUCTION: C cells constitute a small percentage of thyroid gland parenchyma. The number, morphology and distribution of C cells differ among species; however, data regarding their characteristics in European bison are sparse. The aim of this study was to evaluate the morphology, distribution pattern and percentage of C cells in European bison thyroid gland together with morphometric analysis. MATERIAL AND METHODS: Thyroid glands from 28 European bisons of different sex and age were collected either in autumn-winter (13/28) or in spring-summer (15/28) periods and analyzed by immunohistochemistry. RESULTS: The mean total C cell number per all endocrine (follicular and C cells) cell number (C cell concentration) was 7.33%. The tendency to increase the C cell number from periphery to the central region of thyroid lobe was observed with the mean C cell concentration of 3.95%, 7.89% and 9.97% in peripheral, intermediate and central areas, respectively. Most frequently, C cells were situated intrafolliculary whereas epifollicular and interfollicular positions were observed less often. C cells were polymorphic with long cytoplasmic processes. The mean C cell area was 61.97 µm2 and the mean C cell perimeter, length and width were: 34.92 µm, 12.85 µm and 4.91 µm, respectively. In the majority of C cells, strong immunohistochemical cytoplasmic reaction was observed with the mean color intensity of 78.32. In autumn-winter period, C cells were significantly larger with lower color intensity than during spring and summer. CONCLUSIONS: This study leads to deeper characteristics of thyroid gland C cells in European bison. The histomorphometric data suggest that in European bison production of calcitonin by C cells may differ depending on the time of the year.

Melatonin receptors subtypes (MT1 and MT2) in the uterus masculinus of mature male european bison. Biological and seasonal reproductive role.

Regulation of seasonality in reproduction is closely related to melatonin and circadian rhythms. Melatonin affects the functions of reproductive organs through membrane melatonin receptors MT1 and MT2. The current knowledge about the presence, location and function of MT1 and MT2 receptors in the reproductive tract of an adult male European bison, seasonally breeding animal, is still missing. Frequently occurring organ in the male reproductive system of the European bison is uterus masculinus, what seems to confirm its specific role in seasonal reproduction control. Taking this into account, our study aimed to evaluate the expression of mRNA and protein synthesis for both melatonin receptors in the tissues of uterus masculinus in November and December in European bison. Protein synthesis of MT1 and MT2 receptors in uterus masculinus of mature European bisons was clearly raised in November and decreased in December. The comparable results were also found for mRNA expression of MT1 and MT2 receptors, where in November the expression level was significantly higher than in December. Therefore, we suggest that melatonin is needed in the European bison's reproductive system after a period of intensive reproductive activity in November. Probably, melatonin plays a protective function in uterus masculinus of the European bison and thus regulates the seasonal reproduction.

Vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF-2) as potential regulators of seasonal reproductive processes in male European bison (Bison bonasus, Linnaeus 1758).

Growth factors: vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor (FGF-2) were reported to affect normal physiological reproductive processes in human, domestic and free living animals. Moreover, some reports suggest that VEGF-A and FGF-2 may be directly involved in the control of the annual reproductive cycle of seasonally breeding animals but detailed knowledge is still missing. Our study aimed to demonstrate the expression of mRNA and protein for both factors in the tissues of testis and epididymis (caput, corpus, cauda) at different periods of the year (March, June, November, December) in European bison as a model of seasonally breeding animal. Results suggest, that VEGF-A expression was more pronounced in testis than in epididymis and the highest expression was noted in December and June. Surprisingly, the highest protein accumulation was observed in June at the same level in all tissues analyzed. On the other hand, the highest FGF-2 mRNA expression was noted in testis in June and in epididymis in March. However, no differences in protein expression of FGF-2 were found between analyzed groups. The results indicate that both factors are necessary for proper functioning of the reproductive system and their levels differ seasonally. Perhaps, it is linked to increased need of these factors in the testis as well as epididymis during preparation for the reproductive functions. Moreover, VEGF-A and FGF-2 not only may regulate reproductive functions by affecting vascularization and cell nutrition, but it also may be possible that they possess protective functions by stabilizing the reproductive cells. Therefore, obtained results provide new insight into mechanisms underlying seasonal breeding of the male European bison.

Trace elements in the liver of captive and free-ranging European bison (Bison bonasus L.).

European bison is classified as a vulnerable species because of many threats. We analyzed the content of toxic and essential elements (Ag, Al, As, Ba, Be, Cd, Co, Cr, Cu, Hg, Mg, Mn, Na, Ni, Pb, Se, Th, Tl, U, V, and Zn) in the livers of 30 captive and free-ranging European bison from the Bison Breeding Center in Smardzewice and from Bialowieza Primeval Forest in Poland. The contents of toxic elements were lower than reported previously in European Bison and were similar to those of wild ungulates from non-polluted areas. Accumulation of Cd and Cr was related to the age of animals. We compared the mineral status between captive and free-ranging European bison to verify whether the maintenance type could affect concentrations of trace elements in the liver. The concentration of Mn and Zn differed between captive and free-ranging group. Our results were compared to the reference values of essential elements for cattle. All animals from this study were Se-deficient and more than 80% of them have Cu deficiency. Deficiency of Mn was present in 20% of captive and 37% of free-ranging animals whereas Zn in 37% and 3% respectively. Statistical analysis confirmed that Mn and Zn deficiencies were related o the maintenance of animals (p<0.05). We revealed that mineral deficiencies could be an additional threat to the Polish population of European bison. Thus, the monitoring of essential minerals is necessary and future work is required to optimize the supplementation and foddering for preventing the occurrence of mineral deficiencies.

New insight on the role of melatonin receptors in reproductive processes of seasonal breeders on the example of mature male European bison (Bison bonasus, Linnaeus 1758).

Melatonin receptors (MT1 and MT2) were shown to regulate proper functioning of reproductive system, especially in seasonally breeding animals. European bison is a unique endangered seasonal breeder and knowledge of the molecular mechanisms of its reproduction is crucial for the survival of the species. The aim of this study was to assess gene expression, protein synthesis and immunohistochemical localization of MT1 and MT2 receptors in testicular and spermatic cord vessels tissues collected in pre-rut (June) and post-rut (December) seasons from adult male European bisons in Bialowieza National Park. We confirmed the highest expression of MT1 and MT2 mRNA and protein levels in testis in December, while in spermatic cord gene expression was also highest in December, but protein amounts were comparable in both analyzed periods. Furthermore, immunohistochemical staining revealed the same amount of both receptors in arteries and veins of spermatic cord in both periods and increased amounts in December in Leydig, Sertoli and germ cells. The high level of testicular melatonin in December confirms the inhibition of spermatogenesis and increased anti-oxidant and anti-inflammatory protection. In spermatic cord vessels, it may prevent from age-related changes due to the overexploitation and ensure a constant temperature regardless of changing environmental conditions. This knowledge can contribute to finding a solution of problems associated with male infertility in general and also further explore the mechanisms regulating the proper functions of the male reproductive system.

Pharmacokinetics of tulathromycin after subcutaneous injection in North American bison (Bison bison).

Tulathromycin is approved for the treatment of respiratory disease in cattle and swine. It is intended for long-acting, single-dose injection therapy (Draxxin), making it particularly desirable for use in bison due to the difficulty in handling and ease of creating stress in these animals. The pharmacokinetic properties of tulathromycin in bison were investigated. Ten wood bison received a single 2.5 mg/kg subcutaneous injection of Draxxin. Serum concentrations were measured by liquid chromatography-mass spectrometry (LC-MS) detection. Tulathromycin demonstrated early maximal serum concentrations, extensive distribution, and slow elimination characteristics. The mean maximum serum concentration (Cmax) was 195 ng/mL at 1.04 h (tmax) postinjection. The mean area under the serum concentration-time curve, extrapolated to infinity (AUC0-inf ), was 9341 ng · h/mL. The mean apparent volume of distribution (Vd /F) and clearance (Cls/F) was 111 L/kg and 0.4 L/h/kg, respectively, and the mean half-life (t1/2) was 214 h (8.9 days). Compared to values for cattle, Cmax and AUC0-inf were lower in bison, while the Vd /F was larger and the t1/2 longer. Tissue distribution and clinical efficacy studies in bison are needed to confirm the purported extensive distribution of tulathromycin into lung tissue and to determine whether a 2.5 mg/kg subcutaneous dosage is adequate for bison.

The PUFA-enriched fatty acid profiles of some frozen bison from the early Holocene found in the Siberian permafrost.

Knowledge concerning the availability of n-3 fatty acids for humans in prehistoric times is highly relevant in order to draw useful conclusions on the healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen bison found in the permafrost of Siberia (Russia). A total of 3 bison were included in this study, all them very close to the early Holocene (8,000; 8,200; and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass spectrometry (GLC-MS) and GLC flame-ionization detection (GLC-FID). Fat samples from two bison showed two well-differenced areas, i.e. brown and white, the latter being saturated fatty acid enriched, corresponding to an intermediate stage of adipocere formation, while the brown ones yielded α-linolenic acid in higher percentages than found in present-day bison. As demonstrated in this work, the subcutaneous fat of bison consumed by Mesolithic hunters contained amounts of n-3 fatty acids in higher quantities than those found in current bison; thus, the subcutaneous fat of bison could have contributed to meet today's recommended daily intake of essential fatty acids for good health in the Mesolithic to a greater extent than previously thought.

Magnesium concentrations in the tissues of free-ranging European bison.

The European bison (Bison bonasus) is the only living species of the bison genus in the Old World. It is the largest, wild, herbivorous mammal living in Europe. As a result of the efforts of biologists, the European bison has been saved as a species. In Poland, they were reintroduced into the natural conditions of the Bialowieza Forest. By 2011, this herd of free-living bison comprised more than 700 animals. The aim of the present work was to determine tissue levels of magnesium in free-living bison. Samples for the investigations were collected during the winter, from 20 European bison aged from five months to five years culled as part of the annual management programme. Segments of rib, muscle, liver, kidney, hoof and hair were collected. With regards to the gender and age of the animals studied, magnesium content in particular tissues was as follows: in the liver, magnesium content was significantly higher in the group of males; in rib, muscle and kidney there were no statistically significant differences between groups; in hair and hoof wall, a significantly higher magnesium content was found in the group of calves.

Historic, pre-European settlement, and present-day contribution of wild ruminants to enteric methane emissions in the United States.

The objectives of this analysis were to estimate historic (pre-European settlement) enteric CH(4) emissions from wild ruminants in the contiguous United States and compare these with present-day CH(4) emissions from farmed ruminants. The analysis included bison, elk (wapiti), and deer (white-tailed and mule). Wild ruminants such as moose, antelope (pronghorn), caribou, and mountain sheep and goat were not included in the analysis because their natural range is mostly outside the contiguous United States or because they have relatively small population sizes. Data for presettlement and present-day population sizes, animal BW, feed intake, and CH(4) emission factors were adopted from various sources. Present-day CH(4) emissions from livestock were from recent United States Environmental Protection Agency estimates. The most important factor determining CH(4) emissions from wild ruminants in the presettlement period was the size of the bison population. Overall, enteric CH(4) emissions from bison, elk, and deer in the presettlement period were about 86% (assuming bison population size of 50 million) of the current CH(4) emissions from farmed ruminants in the United States. Present-day CH(4) emissions from wild ruminants (bison, elk, and deer) were estimated at 0.28 Tg/yr, or 4.3% of the emissions from domestic ruminants. Due to its population size (estimated at 25 million), the white-tailed deer is the most significant present-day wild ruminant contributor to enteric CH(4) emissions in the contiguous United States.

Assessment of S100 protein expression in the epididymis of juvenile and adult European bison.

In our study, we decided to compare S100 protein expression in the material obtained from the epididymes of 5- and 12-month-old calves, and adult European bison, and to detect any differences in S100 expression according to the animal age and size of the organ examined. We used the epididymes obtained from 6 adult European bison aged 6-12 years, from 6 at the age of 12 months and 6 calves aged 5 months. Immunocytochemical reactions were performed using the avidin-biotinylated-peroxidase (ABC) technique according to HSU. Specific polyclonal rabbit antiserum against bovine S100 protein (Bio Genex Laboratories) at a dilution at 1:400 was applied. We found the expression of S100 protein in endothelial cells of arteries, veins and lymphatic vessels in all the study animals. At the same time, we found no differences in the expression of S100 protein in vascular endothelial cells. Our observations seem to indicate that S100 expression in endothelial cells of European bison epididymis is not correlated with age or maturity of the organ tested. We found S100 protein in smooth muscle cells of arteries and veins in all European bison specimens examined. Interestingly in the current study, in young 5-month-old sexually immature European bison specimens we observed weaker expression of S100 protein in smooth muscle cells of small vessels as compared to the same cell type both in large vessels in these animals and in small vessels in adult specimens.

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).

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.

Free-ranging European bisons accumulate more cadmium in the liver and kidneys than domestic cattle in north-eastern Poland.

It has been shown that free-ranging big game animals accumulate several-fold more cadmium (Cd) in the liver and kidneys than domestic animals. To examine possible reasons for this difference, in the present work we determined the concentrations of Cd in the liver and kidney cortex of European bisons (n=23) from Bialowieza Forest (north-eastern Poland) and domestic cattle (n=15) from the same region; in addition, analyses of Cd in the grasses and soil as well as of soil pH were carried out. The accumulation of Cd in liver and kidney cortex of the female bisons correlated significantly with the age up to 7 years, but stabilized thereafter. The 7-12-year-old bisons had 2.14- and 2.25-fold higher concentrations of Cd in the liver and kidney cortex, respectively, than the age-matched domestic cattle. Notably, the Cd levels in the liver and kidneys of the 8-12-year-old cattle were comparable to those found in the 2-year-old and 4-6-year-old bisons, respectively. The content of Cd in the grasses from Bialowieza Forest appeared to be 2.1-fold higher than that in the plants from the pastures. Similarly, the concentration of water-extractable Cd in the soil was 2.7-fold greater in Bialowieza Forest than in the pastures, despite the fact that nitric acid-extractable Cd (total Cd) was similar in the soils from the two sites. The concentration of water-extractable Cd in the soil as well as the content of Cd in the grasses inversely correlated with soil pH, which appeared to be significantly lower in Bialowieza Forest. These data indicate that soil pH is probably responsible for the higher concentrations of Cd in the feed and tissues of bisons as compared with those of domestic cattle.