Polish Journal of Veterinary Sciences

Phosphorothioate CpG oligodeoxynucleotides (ODN) are reported to be recognized by the membrane-bound TLR9 and trigger the MyD88-dependent up-regulation of Type I interferons and pro-inflammatory cytokines. Whether plasmids containing multiple CpG motifs stimulate the same signaling pathway is yet to be determined. The present results show that the CpG motifs enrich plasmid pUC18-CpG stimulates RAW 264.7 in vitro, mainly through the TBK1-mediated signaling pathway, causing the up-regulation of IFN-β, and pro-inflammatory cytokines TNF-α and IL-6. When pUC18-CpG is co-administered with the recombinant Echinococcus granulosus antigen, the antigen-specific antibody titers are markedly increased compared to the Quil-A adju- vanted group. Antigen specific cytokine quantification shows that cytokine profiles from the pUC18-CpG adjuvanted-group are switched to a Th1-biased immune response.

Matrix metalloproteinases 2 and 9 (MMP2 and MMP9) are proteolytic enzymes involved with extracellular matrix degradation. They play a role in tumor invasion and metastases. Be- cause of their ability to degrade signaling molecules presented in extracellular matrix, MMPs contribute to tumor proliferation and apoptosis. The aim of this study was to evaluate expression of MMP2 (latent and both active and latent forms) and MMP9 (active, latent, active and latent forms) in different subtypes of canine lymphomas and their relationship with proliferative (mi- totic index and percentage of Ki67-positive cells) and apoptotic (apoptotic index) markers. Ex- pression of MMPs was assessed immunohistochemically using an immunoreactive score system. Expression of both MMPs was found in all 20 examined lymphomas belonging to six subtypes. Most cases showed a moderate level of all analyzed forms of MMP2 and MMP9. High expres- sion of MMPs was found in single cases. Except for a positive correlation between the active form of MMP9 and the mitotic index for all lymphoma cases, no other correlations between any remaining forms of MMPs and neither proliferative nor apoptotic markers were found, irrespec- tive of whether the analysis encompassed all cases or the most numerous lymphoma subtypes i.e. centroblastic and Burkitt-like. Our results were not able to clearly confirm the influence of MMPs on the proliferation and apoptotic activity of canine lymphoma cells. However, further studies examining MMPs activity by zymography, expression of their inhibitors and other factors in- volved in activation of cell proliferation and apoptosis inhibition are needed to clarify the role of MMPs, especially the active form of MMP9, in the behavior of canine lymphoma cells.

Fatty acids are very important biological substances due to their metabolic, structural and signal- ing functions. Omega-3 has different beneficial, harmful and neutral effects on adipokines. Adi- pokines have autocrine, paracrine and endocrine effects on metabolism. In the study 54 German Fawn x Hair crossbred goats were synchronized using intravaginal sponges. During the first pe- riod (mating-75 days), all animals were fed a diet supplemented with protected fat and during the second period of pregnancy (76 days-kidding), one of the groups was fed a diet supplemented with fish oil and other was fed a diet supplemented with protected fat. Serum leptin, ghrelin, adi- ponektin and omentin levels were measured by ELISA system. Distributed fed (roughage and concentrate) were sampled and dry matter, crude protein, fat, and ash were determined by AOAC (1988) analysis methods. The Acid Detergent Fiber (ADF) and Neutral Detergent Fiber (NDF) analysis were conducted using heat stable α-amylase and sodium sulphite. Fat source (fish oil or protected fat) affected feed consumption and the highest feed consumption was found in the group fed with protected oil first half of the pregnancy and with fish oil in the second half of the pregnancy and in the fish oil group during the pregnancy. It was determined that the use of fish oil during pregnancy did not affect ghrelin, leptin and omentin concentrations in serum. Adipokine levels of fish oil fed animals during any period of pregnancy were found to be high and it was also found that serum adiponectin levels in goats fed with diet containing fish oil in the first half of pregnancy and protected fat in the second half were statistically significantly high in adipokines.

In our previous Genome-wise Association Study we found that Cystic Fibrosis Transmem- brane Conductance Regulator gene (CFTR) is a candidate gene for sperm motility in fresh semen of Holstein-Friesian bulls. Since in cows thawed semen is commonly used for the artificial insem- ination (AI) we have decided to find out whether functional polymorphism within CFTR gene coding sequence is associated with selected parameters of thawed sperm, including their motility evaluated by computer-assisted sperm analysis (CASA), the activity of three antioxidant enzymes: glutathione peroxidase (GPx) catalase (CAT), superoxide dismutase (SOD), ATP con- tent and integrity of sperm membranes. One hundred twenty Holstein Friesian bulls kept in uni- form environmental conditions (one AI company) were included in the study. Significant associ- ations between genotypes of missense mutation within exon 11 of the CFTR gene (Met468Leu) and the activity of antioxidant enzymes and sperm mitochondrial function were revealed. No effect of CFTR genotypes on sperm motility was observed. Significant differences in CAT and SOD activity were found between AA and TT homozygous individuals. Bulls with TT genotype had the lowest activity of both antioxidant enzymes. The same bulls also showed the lowest num- ber of sperm with active mitochondria. Our results demonstrate that missense mutation Met468Leu within CFTR gene is associated with antioxidant enzyme activity and mitochondrial function of bovine thawed sperm without affecting their motility.

The aim of this study was to identify the proteoforms of albumin and kallikrein in stallion seminal plasma (SP), and to determine their correlations with sperm motility parameters. The experimental material consisted of ejaculates from 8 stallions, which were collected during the breeding and non-breeding seasons (BS and NBS, respectively). SP proteins were identified by 2-D PAGE and mass spectrometry (MALDI TOT/TOF MS). Sperm motility parameters were analyzed using the CASA system. Protein expression (integrated optical density-IOD) of albumin proteoforms 1 (ALB 1) and 2 (ALB 2) and kallikrein proteoforms 1 (KAL 1) and 2 (KAL 2) was correlated (p<0.05) with sperm motility parameters (total motility and progressive motility) during the BS. No significant correlations were found between the expression of albumin or kallikrein and sperm motility parameters during the NBS. The presence of correlations between the expression of ALB 1, ALB 2, KAL 1, KAL 2 and selected sperm motility parameters could suggest that the analyzed components of the SP belong to the group of fertility-associated pro- teins (FAPs).

Allergic skin diseases in cats are amongst the most prevalent dermatological conditions in this species. The objectives of this study were to evaluate different types of skin barrier measurements in healthy cats and cats with non-flea non-food hypersensitivity dermatitis (NFNFHD). 24 clinically healthy and 19 NFNFHD cats were included in this clinical trial. In each animal, the transepidermal water loss (TEWL) and skin hydration (SH) were assessed on six clipped body sites by VapoMeter SWL 4605 and Corneometer ®CM 825, respectively. Results of TEWL measurement were , significantly higher in one of the six examined body sites, namely on the lumbar area (p=0.0049). Furthermore, a statistically significant difference was found between the average TEWL values (p=0.019). Statistically notable differences were mea- sured at least in one certain body site for SH: in the groin (p=0.02), where the values in the affect- ed cats were lower than in the healthy individuals. These results may suggest that in NFNFHD cats transepidermal water loss is higher than in healthy cats. Skin hydration is, at least, in certain body sites, lower in atopic feline patients than in healthy individuals.

The purpose of the study was to assess the effect of autologous activated platelet-rich plasma on healing of a bone substitute - tricalcium phosphate hydroxyapatite in experimental long bone defects using an animal model.

The experiment involved an animal model of femoral defect. 24 Termond white rabbits were used in the study. We evaluated the effect of autologous platelet-rich plasma on tricalcium phos- phate using classical radiology, micro-CT studies, strength tests and histological evaluation.

Radiological and histological assessment did not show a beneficial effect of PRP together with a bone substitute in comparison to filling the defects only with bone replacement material. The only benefit of adding platelet-rich plasma to a bone substitute was shown in microCT imaging.

Autologous, activated platelet-rich plasma combined with hydroxyapatite tricalcium phos- phate has a positive effect on the remodeling of the newly formed bone tissue, increasing its density.

The aim of this study was to determine to what extent the ions present in hard water (125 mg/L of MgCl2 and 500 mg/L of CaCl2) may intensify the feed-induced decrease in oxytetracycline (OTC) absorption rate in broiler chickens after single oral administration at a dose of 15 mg/kg. Drug concentrations in plasma were determined by liquid chromatography-tandem mass spectrometry and combined, compartmental and non-compartmental approach was used to assess OTC pharmacokinetics.

The administration of feed decreased the absolute bioavailability (F) of OTC from 12.70%±4.01 to 6.40%±1.08, and this effect was more pronounced after the combined administration of OTC with feed and hard water (5.31%±0.90). A decrease in the area under the concentration- time curve (AUC0-t), (from 10.18±3.24 μg·h/ml in control to 5.13 μg·h/ml±1.26 for feed and 4.26 μg·h/ml±1.10 for feed and hard water) and the maximum plasma concentration of OTC (Cmax) (from 1.22±0.18 μg/ml in control, to 1.01 μg/ml ±0.10 for hard water, 0.68 μg/ml±0.10 for feed and 0.61 μg/ml±0.10 for feed and hard water) was observed. The results of this study indicate that feed strongly decreases F, AUC0-t and Cmax of orally administered OTC. The ions present in hard water increase this inhibitory effect, which suggests that, therapy with OTC may require taking into account local water quality and dose modification, particularly when dealing with outbreaks caused by less sensitive microorganisms.

Edible snails are kept in farms in many countries worldwide. As farm animals, they are an object of interest of veterinary studies and applied biology. There is a large demand for tests which would help identify their health and well-being. The objective of this study was to assess the usefulness of determining the concentration of urea in hemolymph as a marker of health of the Lissachatina fulica and Cornu aspersum edible snails. The observation covered snails from four farms marked from A to D, in which numerous deaths (farm A) and decreased body weight gain (farms B and C) were observed. In experimental farm D we observed a group of snails subjected to stress and a control group maintained in correct conditions. High concentrations of urea were found in the hemolymph of all farm animals from farms A, B and C, as well as in those subjected to food deprivation in farm D (on average from 96 mg/dl in farm D to 320 mg/dl in farm A). On the other hand, in controls from group D, the concentration of the parameter in question was much lower (< 2.0 mg/dl). The results obtained indicate that the urea concentration is a non-specific marker of pathological conditions in snails, and that the continuous monitoring of this parameter makes it possible to demonstrate irregularities in farming and introduce appro- priate and early measures to eliminate such disturbances.

The present study was undertaken to highlight the influence of simvastatin administration on hepatocyte morphology, proliferation, and apoptosis. The study included 48 gilts aged 3 months (weighing ca. 30 kg) divided into groups I (control; n=24) and II, receiving 40 mg/animal simvas- tatin orally (simavastatin; n=24) for 29 days. The animals were euthanized on days subsequent to the experiment. The livers were sampled, fixed, and processed routinely for histopathology, histochemistry, and immunohistochemistry (for proliferating cell nuclear antigen, Bcl-2, and caspase-3). Apoptosis was visualized by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL). Simvastatin administration caused acute hepatocyte swelling, glycogen de- pletion, hyperaemia, multifocal hepatocyte proliferation with occasional pseudoacinar formation, connective tissue hyperplasia, eosinophil infiltration, and interface hepatitis. The proliferating cell nuclear antigen index, mean diameter of argyrophilic nucleolar organizer regions, and Bcl-2 immunoexpression were lower compared to control, and mean caspase-3 immunoexpression was higher in group II compared to control. On day 25 and 29 single hepatocytes in the simvasta- tin-treated group were TUNEL-positive. Simvastatin caused morphological alteration which became more intense over time. The results from the present study suggest that simvastatin treat- ment may cause glycogen, lipid metabolism and cell membrane permeability distortion, fibrosis, interface hepatitis, reduction in hepatocyte proliferation and transcriptional activity, and enhanced vulnerability to apoptosis. Summing up the results, it can be concluded that simvastatin caused liver damage with similar morphological changes seen in autoimmune-like liver injury, which may indicate that simvastatin may induce autoimmune-like drug induced liver injury.

This study aimed to determine the levels of milk cell total protein (TP), reduced nicotinamide adenine dinucleotide phosphate (NADPH), total glutathione (tGSH), activities of glucose-6-phos- phate dehydrogenase (G6PD) and glutathione peroxidase (GPx) in subclinical mastitic cows. Milk from each udder was collected and grouped by the California Mastitis Test. Then, a somatic cell count (SCC) was performed, and the groups were re-scored as control (5–87 × 103 cells), 1st group (154–381 × 103 cells), 2nd group (418–851 × 103 cells), 3rd group (914–1958 × 103 cells), and 4th group (2275–8528 × 103 cells). Milk cell TP, NADPH, tGSH levels, G6PD, and GPx ac- tivities were assessed. Microbiological diagnosis and aerobic mesophyle general organism (AMG, cfu/g) were also conducted. In mastitic milk, TP, NADPH, and tGSH levels, and G6PD and GPx activities were significantly reduced per cell (in samples of 106 cells). In addition, milk SCC was positively correlated with AMG (r=0.561, p<0.001), NADPH (r=0.380, p<0.01), TP (r=0.347, p<0.01) and G6PD (r=0.540, p<0.001). There was also positive correlation between NADPH (r=0.428, p<0.01), TP (r=0.638, p<0.001) and AMG. NADPH was positively correlated with TP (r=0.239, p<0.05), GPx (r=0.265, p<0.05) and G6PD (r=0.248, p=0.056). Total protein was positively correlated with tGSH (r=0.354, p<0.01) and G6PD (r=0.643, p<0.001). There was a negative correlation between tGSH and GPx activity (r=-0.306, p<0.05). The microbiological analysis showed the following ratio of pathogens: Coagulase-Negative Staphylococci 66.6%, Streptococcus spp 9.5%, Bacillus spp 9.5%, yeast 4.8%, and mixed infections 9.5%.

As a conclusion, when evaluating the enzyme and oxidative stress parameters in milk, it is more suitable to assign values based on cell count rather than ml of milk. The linear correlation between the SCC and AMG, milk cell NADPH, TP and G6PD suggests that these parameters could be used as markers of mastitis.

In this investigation, the effects of genistein (GEN) on the expression of steroidogenic genes such as steroidogenic acute regulatory protein (StAR), side-chain cleavage enzymes (P450scc) and cytochrome P450 aromatase (CYP19) were assessed. For this study, forty young female Sprague Dawley (SD) rats at aged 2-3 months (200±20 g) and forty aged female SD rats aged 10-12 months (490±20 g) were selected. Also, based on weight they were divided into a negative control group (NC), three different GEN dose groups, which received GEN of 15, 30, 60 mg/kg, and a positive control group (PC). The experiment lasted 30 days. Concentrations of serum hormones were determined by Enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of StAR, P450scc and CYP19 were determined by Real-Time PCR and western blot techniques. It was observed that 30-60 mg/kg GEN could increase the expression of androgen generating key enzymes in the young rat ovary. GEN also significantly increased progesterone and E2 levels in the serum of aged rats and reduced the levels of LH and FSH in the serum of both young and aged rats. Compared with young rats, the effect of GEN on the ovary of aged rats was stronger and a lower dose of GEN (15 mg/kg) showed an obvious effect on these indicators. GEN influenced both estrogen level and indicators associated with estrogen and androgen transformation processes, which indicates that GEN can impair the growth and maturation of the ovary.

Canine parvovirus (CPV) causes acute gastroenteritis in domestic dogs, cats, and several wild carnivore species. In this study, the full-length VP2 gene of 36 CPV isolates from dogs and cats infected between 2016 and 2017 in Beijing was sequenced and analyzed. The results showed that, in dogs, the new CPV-2a strain was the predominant variant (n = 18; 50%), followed by the new CPV-2b (n = 6; 16.7%) and CPV-2c (n = 3; 8.3%) strains, whereas, among cats, the predominant strain was still CPV-2 (n = 9; 25%). One new CPV-2a strain, 20170320-BJ-11, and two CPV-2c strains, 20160810-BJ-81 and 20170322-BJ-26, were isolated and used to perform experimental infections. Multiple organs of beagles that died tested PCR positive for CPV, and characteristic histopathological lesions were observed in organs, including the liver, spleen, lungs, kidneys, small intestines, and lymph nodes. Experimental infections showed that the isolates from the epidemic caused high morbidity in beagles, indicating their virulence in animals and suggesting the need to further monitor evolution of CPV in China.

Artemisinin is a powerful antimalarial drug, useful in the treatment of many diseases, includ- ing chickens coccidiosis. Its toxic effects have been well studied in humans and experimental animals, but not sufficiently in broiler chickens. Therefore, in the present study, we aimed to assess the side effects of artemisinin in chickens, by measuring the serum level of proteins and enzymes (ALT, AST, GGT, ALP, CK), by histopathological examination and by the evaluation of relative weight of organs (liver, kidney, heart). Artemisinin was administered in the standard feed for chickens in three different concentrations: 5, 50 and 500 ppm.

Each concentration of artemisinin increased the total serum proteins, gamma-globulins and the serum activity of CK and decreased the serum ALP level. The values of ALT and GGT were higher in the chickens treated with 50 and 500 ppm of artemisinin. Multifocal liver necrosis and inflammatory infiltrate were detected in the chickens that received the 50 and 500 ppm dosage of artemisinin. Minimal tubular necrosis, renal tubular epithelium vacuolation, multifocal interstitial nephritis and mild uric nephrosis were detected in chickens treated with the drug. Artemisinin administration produced no significant changes in the organs relative weight.

Artemisinin, at a concentration of 5 mg/kg of feed is well tolerated by broiler chickens, but the concentrations of 50 and 500 mg/ kg feed can produce toxic effects.

Aim: The aim of this study was to analyze the effect of bovine follicular fluid on the survival, morphology and kinetic parameters of bovine thawed spermatozoa under laboratory conditions.

Materials and methods: The semen from 5 bulls of proven fertility was incubated in follicular and physiological fluid for 8 hours. During this time assessment using the CASA system was performed. At the beginning and the end of incubation process evaluation by flow cytometry was conducted.

Results: The results of the sperm motility assessment showed a significant decrease in the analyzed parameters both in the follicular and physiological fluid. A significant reduction in all parameters characterizing movement properties in the semen incubated in the follicular fluid was found. In the physiological fluid, a similar trend was demonstrated only for the following proper- ties: VAP, VSL, VCL, ALH, BCF. A significant difference was found for both fluids in: VCL (p=0.026), ALH (p=0.038) and LIN (p<0.001) at the beginning of incubation. The results of the plasma membrane integrity assessment showed a statistically significant increase in the percent- age of dying sperm at the 8th hour of the incubation in the follicular fluid. In the case of semen incubation in physiological fluid, a statistically significant decrease in the percentage of live non-damaged cells was found with a simultaneous increase in the subpopulation of undamaged dead cells.

Conclusions: Follicular fluid rapidly accelerates the capacitation process. The results of flow cytometry support the hypothesis concerning the ability of follicular fluid to prolong sperm sur- vival.

Twenty eight male Sprague Dawley rats (aged 3 months) were used in the study. The animals were given feed and water as ad libitum. Sprague dawley rats were randomly divided into 4 groups as 7 rats in each group. Except for the control one, aflatoxin B1 (7.5 μg / 200 g), resvera- trol (60 mg / kg) was administered to rats of 3 other groups. At the end of the 16th day, blood, semen and tissue specimens were taken by decapitation under ether anesthesia. When we evaluate the spermatological parameters, it is understood that resveratrol has a statistically significant difference in terms of sperm motility and viability (membrane integrity) compared to the control group and aflatoxin B1 administration groups, indicating a protective effect on spermatological parameters. In terms of pathological parameters - histopathological examination - in the control and resveratrol groups, seminiferous tubules were observed to be in normal structure. In the group treated with aflatoxin, the regular structure of the spermatogenic cells deteriorated and the seminiferous tubules became necrotic and degenerative. In the group treated with Afb1 + res, the decreasing of necrotic and degenerative changes were determined compared with in the group treated with aflatoxin. As immunohistochemical examination, cleaved caspase 3 expression was found to be very low in the control and resveratrol groups. Cleaved caspase 3 expression was severely exacerbated in seminiferous tubules in aflatoxin group but cleaved caspase 3 expression level decreased in Afb1 + res. In the biochemical direction, resveratrol has been shown to inhibit the adverse effects of aflatoxin on antioxidant levels and to show a protective effect. For this purpose, the use of resveratrol with antioxidant activity was investi- gated in preventing or ameliorating damage to aflatoxin B1. It has been concluded that resveratrol effectively prevent the aflatoxin-induced testicular damage and lipid peroxidation. It has also been shown that resveratrol has protective effects on sperm motility and viability.

Introduction: Arthrocentesis has been used in the temporomandibular joint (TMJ) to analyze components of the synovial fluid or as a therapeutic procedure associated or not with the admini- stration of a drug. The rabbit is one of the most commonly used animal species as a model for pathologies that affect the TMJ. The aim of this study was to propose a specific technique to perform arthrocentesis on the rabbit TMJ, emphasizing descriptions of reference points and measurements for a successful puncture without complications. Materials and methods: Fourteen adult rabbits (Oryctolagus cuniculus) were used. The project was approved by the Scientific Ethics Committee of the Universidad de La Frontera (File Nº083/2016). Results: The description of the technique was divided into three steps: 1) Location of the rabbit TMJ, 2) Positioning of the needles in the TMJ, and 3) Passage of fluid through the TMJ. Conclusions: This arthrocen- tesis technique could help to simplify the procedure and give the investigator a guide for joint washing and extraction of synovial fluid in the rabbit TMJ.

Effects of fermented extruded rye flour supplements with Lactobacillus sakei KTU05-6 or Pediococcus pentosaceus BaltBio02 on milk production and composition, as well as ruminal parameters, were determined in Lithuanian Black & White dairy cows. Also, determination of antimicrobial activities of tested lactic acid bacteria (LAB) against a variety of pathogenic and opportunistic bacterial strains previously isolated from diseased cattle was performed. The highest antimicrobial activity was demonstrated in L. sakei against S. aureus, and in P. pentosaceus against P. aeruginosa and S. aureus. The count of LAB in the supplements after 72 h of fermentation of extruded rye flour with L. sakei and P. pentosaceus was 9.6±0.4 log10 CFU/g and 9.5±0.3 log10 CFU/g, respectively. All cows (n=60) were fed the same basal diet. The treatment differences were achieved by individually incorporating (65 d.) one of the supplements: L. sakei KTU05-6 (group B; n=20) or P. pentosaceus BaltBio02 (group C; n=20). The control group A (n=20) was on the basal diet only. A supplement fermented with L. sakei does not have a significant influence on dairy cattle milk production and rumen fluid parameters. The type of LAB used has a significant influence (p<0.0001) on microbiological parameters of the rumen (TCM, TCL, TCE). The milk yield was increased (p≤0.05) using P. pentosaceus BaltBio02 supplement, and further research is needed to identify w hat is the main mechanism of the positive action.

The aim of this study was to assess sand accumulation in the gastrointestinal tract and fecal sand excretion in Silesian foals using three diagnostic methods and taking into account the sex and age of the animals. Another aim of the study was to compare the three diagnostic methods. The study was carried out on 21 clinically healthy Silesian foals (10 females and 11 males) from 9-28 weeks old grazed on permanent pasture. The sand intake was assessed using a sedimentation test, abdominal ultrasonography and a quantitative evaluation of sand per 100 g of stool. In the sedimentation test, the sand was palpable in the stool of 57.1% of the horses, and clearly visible in 42.9% of the animals. The ultrasound examination revealed the presence of sand in the gastrointestinal tract in 66.7% of the horses. It was limited to a single location in 60% of the horses, while it was present in several regions in 40% of the horses. The mean amount of sand was 0.14 ± 0.33 g per 100 g of stool. It did not exceed 0.1g in 71.4% foals, while it ranged from 0.1-0.5 g in 23.8% foals. In 4.8% of the animals, it amounted to 1.6 g per 100 g of stool. There was no correlation between age and gender and the results. There was a positive correlation between the ultrasound examination and the sedimentation test.

Sand may be accumulated in the gastrointestinal tract of foals without any clinical signs. The amount of sand excreted in the stool is not an indicator of the amount of sand accumulated in the gastrointestinal tract. An abdominal ultrasound examination should be combined with a sedimentation test for more specific results.

Tight junction proteins are important for the maintenance and repair of the intestinal mucosal barrier. The present study investigated relationships among tight junction protein gene expres- sion, porcine epidemic diarrhea virus (PEDV) infection, and intestinal mucosal morphology in piglets. We compared the expression of six tight junction proteins (ZO-1, ZO-2, Occludin, Claudin-1, Claudin-4, and Claudin-5) between seven-day-old piglets infected with PEDV and normal piglets, as well as in PEDV-infected porcine intestinal epithelial cells (IPEC-J2). We also evaluated differences in mucosal morphology between PEDV-infected and normal piglets. The expression of six tight junction protein genes was lower in PEDV-infected piglets than in the normal animals. The expression of ZO-1, ZO-2, Occludin, and Claudin-4 in the intestine tissue was significantly lower (p<0.05) in PEDV-infected than in normal piglets. The expression of Claudin-5 in the jejunum was significantly lower in PEDV-infected piglets than in the normal animals (p<0.01). The expression of Claudin-1 and Claudin-5 genes in the ileum was signifi- cantly higher in PEDV-infected piglets than in normal piglets (p<0.01). Morphologically, the intestinal mucosa in PEDV-infected piglets exhibited clear pathological changes, including breakage and shedding of intestinal villi. In PEDV-infected IPEC-J2 cells, the mRNA expression of the six tight junction proteins showed a downward trend; in particular, the expression of the Occludin and Claudin-4 genes was significantly lower (p<0.01). These data suggest that the expression of these six tight junction proteins, especially Occludin and Claudin-4, plays an important role in maintaining the integrity of the intestinal mucosal barrier and resistance to PEDV infection in piglets.

Primiparous and multiparous lactating crossbred dairy cows with a mature corpus luteum and a follicle with >10 mm in diameter were treated with cloprostenol. Those cows that showed oestrus within 5 days after treatment were inseminated (Group P). The other cows (Group PG) were treated with GnRH 2 days after cloprostenol treatment and timed artificial insemination (AI) was performed on the consecutive day, or were inseminated (Group G) after detected oestrus and treated with GnRH immediately after AI. The control cows (Group C) after detected oestrus were only inseminated. All of the AIs using frozen semen were done between 6 and 7 a.m. while the ultrasonographic examinations after AI were performed between 4 to 6 p.m. The ovaries of each cow were scanned by means of transrectal ultrasonography from the day of AI until ovulation. Daily blood samples were collected for progesterone measurements. The ovulation and preg- nancy rates among the groups changed between 84.6% and 95.5%, as well as 44.4% and 60%, respectively, however the differences were not statistically significant.

All the cows were evaluated according to date of ovulation after AI and the pregnancy rate was 55.4% (Group 1: ovulation occurred between AI and 9-11 h after AI), 54.5% (Group 2: ovulation occurred between 9-11 h and 33-35 h after AI) and 35.5% (Group 3: ovulation occurred between 33-35 h and 57-59 h after AI), respectively. There was a trend (P=0.087) for 2.2 greater odds of staying open among cows inseminated between 33 to 35 h and 57 to 59 h before ovulation compared to cows inseminated within 9 to 11 h before ovulation. If ovulation occurred before AI, the pregnancy rate was only 22.2%, therefore determination of optimal time for AI is of great importance.

The objectives of this study were to examine the option of being able to use rumination time (RT) as a form of stress indicator in the first thirty days after calving, and to determine the rela- tionship between rumination time, blood cortisol levels, and lactate concentration levels in dairy cows during the first thirty days after calving.

Ninety cows which produced milk (DIM) within 1-30 days were selected and categorised into the following groups: the first group (1) fell within 1-7 days after parturition (dpp) (n=30); the second group (2) fell within 8-14dpp (n=30); and the third group (3) fell within 15-30dpp (n=30) after calving. The cows were milked using Lely Astronaut® A3 milking robots with free traffic. The blood samples were tested using the fluorescence enzyme immunoassay method for cortisol analysis. Lactate concentrations were tested with a Lactate Pro2 ®.

The RT increased during all of the exploratory periods (with readings between 1.12-4.90%). A decrease was also observed in the lactate levels (by 1.10 times) and cortisol levels (by 1.98 times, p<0.05) of cows which fell within the 8-14dpp group, when compared to an average of 1-7dpp in the previous study period (15-30dpp). However, lactate concentrations increased (by 1.84 times, p<0.05) as well as cortisol levels (by 2.09 times, p <0.01) when compared with a figure between 8-14 dpp on the average. The results obtained indicate that, RT increased during all exploratory periods, while a decrease by 1.10 times and 1.98 times was observed in lactate levels and cortisol levels, respectively. During the entire period of the study RT was positively correlated with the lactate concentration levels, and negatively correlated with cortisol levels. Within a period of 1-14 days, a negative correlation was determined with lactate levels along with a 15-30dpp-positive correlation coefficient. In conclusion, RT can be used as a kind of stress indicator for cows in the first thirty days after calving; however, further research is required to ascertain this conclusion.

Despite many phytochemical and pharmacological investigations, to date, there are no reports concerning the antibabesial activity of extracts of A. millefolium against B. canis. This study was aimed at investigating the biological activities of A. millefolium against the Babesia canis parasite and to identify its chemical ingredients. The water (WE), ethanol (EE) and hexane/acetone (H/AE) extracts of plant aerial parts were screened for total phenolic content (TPC), total flavo- noid compound (TFC), DPPH free radical-scavenging activity and its antibabesial activity assay. In this study, imidocarb diproprionate was used as a positive control. The H/AE and EE extracts were analysed using gas chromatography–mass spectroscopy (GC–MS).

In the EE extract, the main compounds were 17.64% methyl octadec-9-ynoate, 16.68% stigmast-5-en-3-ol(3α,24S) and 15.17% hexadecanoic acid. In the H/AE extract, the main com- pounds were 34.55% 11-decyldocosane, 14.31% N-tetratetracontane, 8.22% β-caryophyllene, and 7.69% N-nonacosane. Extract of EE contained the highest content of phenolics followed by H/AE and WE. The concentration of flavonoids in EE, H/AE and WE extracts showed that TFC was higher in the EE samples followed by H/AE and WE. The antioxidant activities were highest for AA, followed by EE, WE and H/AE. The antibabesial assay showed that the WE, EE and H/AE extracts of A. millefolium were antagonistic to B. canis. At a 2 mg/mL concentration, it showed 58.7% (± 4.7%), 62.3% (± 5.5%) and 49.3% (± 5.1%) inhibitory rate in an antibabesial assay, respectively.

Considering these results, the present findings suggest that A. millefolium extracts may be a potential therapeutic agent and that additional studies including in vivo experiments are essential.

Early embryonic death (EED) is one of the causes of infertility in the mare. We compared endometrial environment in 9 mares with EED and 13 mares in diestrus phase. Cotton swab (CS), cytobrush (CB) and uterine biopsy (B) samples were obtained for the cytological, bacteriological and histopathological examinations. In the first step we compared CS and CB methods to biopsy as a reference method, as B revealed the highest number of positive results in cytological and bacteriological examinations in both groups. In turn, we also compared cytological, bacteri- ological and histopathological findings between EED and control animals using the B sampling. Although the differences between these groups were not statistically significant (p≥0.05), there was a tendency to a higher prevalence of subclinical endometritis in the control group, than in the EED group (62% vs 22%). In general, positive bacteriological results were similar in both groups (62% vs 55%), whereas positive cytological results were higher in the control group (62% vs 22%; p≥0.05). In histopathological examination in EED mares endometrial degeneration was better expressed (all mares were with grades IIB and III on the Kenney-Doig scale); however, the differences between both groups were not statistically significant (p≥0.05). We could not confirm a clear difference in uterine environment between the two groups. Moreover, the uterine biopsy seemed to be the most reasonable sampling method for diagnosis of endometrial state.

The aim of this study was to evaluate the safety and anti-obesity effects of the Korean red ginseng extract in dogs. To this end, we fed healthy beagles a Korean red ginseng diet and/or snack for 8 weeks. The dogs were submitted to a thorough physical examination, complete blood count, serum biochemistry analysis, analysis of adipose tissue activity, and body fat-con- tent analysis by computed tomography (CT). At the end of the study period, the dogs that were fed the ginseng extract-diet/snack showed a significant decrease in body weight, body condition score and leptin levels relative to the baseline value. The CT findings revealed a decrease in body fat content in dogs fed the ginseng extract diet but not in those fed the ginseng-extract snack. The results of blood analysis did not show any meaningful changes in any of the dogs. All dogs tolerated the diet/snack well, and there were no adverse events. Our results suggest that the Korean red ginseng extract diet can potentially serve as an anti-obesity diet for reducing fat mass in dogs.

Telomeres are repetitive sequence structures at the ends of chromosomes. They consist of the double stranded DNA repeats followed by the short single stranded DNA. In humans and other verterbrates the telomeric sequence is composed of tandem of TTAGGG repeats. With each cells division telomeres shorten by up to 200 base pairs. Telomerase is an enzyme responsible for continuous cell growth and is repressed in most somatic cells, except proliferating progenitor cells, but in more than 85% of cancer cells telomerase expression is observed. Tumour cells with metastatic potential may demonstrate a high telomerase activity, allowing cells to escape from the inhibition of cell proliferation due to shortened telomeres. Determination of telomerase expres- sion was performed with the use of PCR ELISA in samples isolated from bovine leukaemia virus (BLV) infected cows. Telomerase activity was found in almost all investigated samples. The relative telomerase activity (RTA) was higher in infected cows than in healthy animals and the differences were statistically significant (α=0.05). In blood lymphocytes of BLV-infected cows the mean values of telomerase expression determined in real-time PCR were 3534.12 copies, in the healthy group there were 1010.10 copies and these differences were also statistically significant. For telomere length evaluation the Telomere PNA/FITC FISH and Telomere PNA/FITC FISH for flow cytometry were used. The mean fluorescence intensity of telomere sequences calculated on the surface of interphase nuclei of leukaemic blood lymphocytes was lower than that in the control animals and the difference was statistically significant. The mean length of telomeres in BLV- infected and healthy cows was 31.63 ± 12.62 and 38.4 ± 4.03, (p=0.112), respectively.

The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic proto- col which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5′ - and 3′-untranslated regions (5’UTR, 3’UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were tran- scribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excel- lent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.

Function of duck (Anas platyrhynchos) major histocompatibility complex class I (Anpl-MHC I) molecules in binding peptides is through the peptide binding groove (PBG), which is thought to be influenced by the high polymorphism of α1 and α2 domains. However, little is known about the polymorphism of Anpl-MHC I peptide binding domain (PBD), especially in the domestic duck. Here, we analyzed the polymorphism of forty-eight Anpl-MHC I α1 and α2 domains from domestic duck breeds previously reported. All sequences were analyzed through multiple sequence alignment and a phylogenetic tree was constructed. The coefficient of variance of the peptide binding domains (PBDs) from WS, CV, JD, and SX duck breeds was estimated based on the Wu-Kabat variability index, followed by the location of the highly variable sites (HVSs) on reported crystal structure models. Analysis of α1 and α2 domains showed common features of classical MHC class I and high polymorphism, especially in α1 domain. The constructed phylogenetic tree showed that PBDs of domestic ducks did not segregate based on breeds and had a close phylogenetic relationship, even with wild ducks. In each breed, HVSs were mostly located in the PBG, suggesting that they might determine peptide-binding characteristics and subsequently influence peptide presentation and recognition. The combined results of sequence data and crystal structure provide novel valuable insights into the polymorphism and diversity of Anpl-MHC I PBDs that will facilitate further studies on disease resistance differences between duck breeds and the development of cytotoxic T-lymphocyte (CTL) epitope vaccines suited for preventing diseases in domestic ducks.

The pharmacokinetics of a diclofenac sodium was investigated in swine. A single intravenous (i.v.) or intramuscular (i.m.) injection of 5% diclofenac sodium (concentration = 2.5 mg · kg-1) was administered to 8 healthy pigs according to a two-period crossover design. The pharmacokinetic parameters were calculated by non-compartmental analysis with DAS2.1.1 software. After a single i.v. administration, the main pharmacokinetic parameters of diclofenac sodium injection in swine were as follows: the elimination half-time (T1/2β) was 1.32±0.34 h; the area under the curve (AUC) was (55.50±5.50 μg · mL-1 h; the mean residence time (MRT) was 1.60±0.28 h; the apparent volume of distribution (Vd) was 0.50±0.05 L · kg-1; and the body clearance (CLB) was 0.26±0.04 L · (h · kg)-1. After the single i.m. administration, the pharmacokinetic parameters were as follows: peak time (Tmax) was 1.19±0.26 h; and peak concentration (Cmax) was 11.61±5.99 μg mL-1. The diclofenac sodium has the following pharmacokinetic characteristics in swine: rapid absorption and elimination; high peak concentration; and bioavailability.

Combined retrograde tracing and double-labelling immunofluorescence were used to investi- gate the distribution and chemical coding of neurons in aorticoerenal (ARG) and testicular (TG) ganglia supplying the urinary bladder apex (UBA) in the juvenile male pig (n=4, 12 kg. body weight). Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the bladder apex under pentobarbital anesthesia. After three weeks all the pigs were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde. TG and ARG were collected and processed for double-labelling immunofluorescence. The presence of tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), nitric oxide synthase (NOS) and vesicular acetylcholine transporter (VAChT) were inves- tigated. The cryostat sections were examined with a Zeiss LSM 710 confocal microscope equipped with adequate filter blocks.

The TG and ARG were found to contain many FB-positive neurons projecting to the UBA (UBA-PN). The UBA-PN were distributed in both TG and ARG. The majority were found in the left ganglia, mostly in TG. Immunohistochemistry disclosed that the vast majority of UBA-PN were noradrenergic (TH- and/or DBH-positive). Many noradrenergic neurons also contained immunoreactivity to NPY, SOM or GAL. Most of the UBA-PN were supplied with varicose VAChT-, or NOS- IR (immunoreactive) nerve fibres.

This study has revealed a relatively large population of differently coded ARG and TG neu- rons projecting to the porcine urinary bladder. As judged from their neurochemical organization these nerve cells constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

The aim of the study was to evaluate the visualization of the rabbit common calcanean tendon and adjacent structures in the high-field magnetic resonance imaging (MRI) of 1.5 T field strength and to compare the results with those previously obtained for the low-field MRI (0.25 T). Eight New Zealand rabbits were used in the post-mortem study and the results indicate that the high-field MRI provides more detailed images only in transverse scans, where the outer outline of the tendon was visualized more accurately. Other analysed structures were imaged with a resolution comparable to the low-field MRI.