Polish Journal of Veterinary Sciences

The aim of this study was to compare computer assisted sperm analysis (CASA) results of frozen thawed bull semen using three different chambers. Sixty bull frozen semen samples were thawed (37°C; 30 sec), extended in PBS (30×106 spermatozoa/mL; 37°C) and incubated (37°C; 2 min). Each semen sample was analyzed by CASA [total motility, progressive (pro)/ non-progressive/rapid/medium/slow movement spermatozoa, VCL, VSL, VAP, ALH, BCF, LIN, STR, WOB and hyperactive spermatozoa] using three different chambers: a Makler® chamber (MC; 10 μm); a Leja 4 chamber slide (LC; 20 μm); and a Glass slide covered with a coverslip (GSC; 10.3 μm). The Makler chamber gave higher values compared to both the LC and GSC for almost all examined parameters. No systematic effect was evident between LC and GSC for VCL, VSL, VAP, LIN, STR, WOB, ALH, and BCF. Method agreement between MC and LC was generally moderate, between MC and GSC poor and between LC and GSC moderate to good. In general, narrower limits of agreement were found in samples with lower values. In conclusion, the CASA outcomes could be influenced by the analysis chambers. This finding should be taken into consideration when comparing results from different laboratories.

Previous morphological studies of mammalian pancreatic islets have been performed mainly in domestic and laboratory animals. Therefore, the present immunohistochemical investigation was conducted in a wild species, the European bison, using antibodies against glucagon-like peptide-1 (GLP1), glucagon, insulin and somatostatin. Morphological analyses revealed that the mean area of the endocrine pancreas constituted 2.1±0.1% of the whole area of the pancreas, while the mean area of a single pancreatic islet was 13301.5±686.5 µm2. Glucagon-immunoreac- tive cells accounted for 22.4±1.1% and occupied 19.4±0.4% of the average islet area. As many as 14.3±1.4% of pancreatic islet cells were shown to express GLP1, which constituted 12.6±0.8% of the mean area of the islet. Insulin expression was confirmed in 67.6±0.7% of pancreatic islet cells, which represented 62.3±4.9% of the mean total area of the pancreatic islet. As many as 8.5±1.3% of cells stained for somatostatin. The somatostatin-immunoreactive cell area was 4.9±0.3% of the mean pancreatic islet area. In summary, we have determined in detail for the first time the morphometry and islet composition of the European bison pancreas. The distri- bution patterns of immunoreactivities to the substances studied in the European bison show many similarities to those described in other ruminant species.

This study was conducted to determine the serum and milk levels of thiobarbturic acid-reac- tive substances (TBARS), nitric oxide (NO), superoxide dismutase (SOD), glutathione peroxi- dase (GSH-Px), vitamin E and selenium, IL-4 and IL-6 in lactating dairy cows affected with bloody milk using commercially available ELISA kits. Milk and whole blood samples were collected from 60 cows affected with bloody milk and 20 apparently healthy cows for control. In the serum, levels of GSH-Px and SOD were significantly (p˂0.05) higher in healthy cows compared to cows affected with bloody milk while the levels of TBARS and NO were significantly (p˂0.05) higher in affected cows. In the milk, levels of SOD, TBARS and NO were significantly (p˂0.05) higher in affected cows. In the serum, levels of vitamin E were significantly (p˂0.05) lower in affected cows compared to healthy cows, while no significant changes were observed in the levels of this vitamin in the milk between healthy and affected cows. In the serum, levels of selenium were significantly (p˂0.05) lower in affected cows while in milk, selenium levels were significantly (p˂0.05) higher in affected cows compared to healthy ones. Levels of IL-4 were significantly (p˂0.05) lower in the serum and milk of affected cows compared to healthy cows while levels of IL-6 were significantly (p˂0.05) higher in both serum and milk of affected cows. Results of this study suggest a possible role of oxidative stress in the pathogenesis of bloody milk in dairy cows.

Nitric oxide (NO) is known to be a neuromodulator with dual proconvulsive and anticonvul- sive action. Valeriana officinalis (VAL) was previously believed to be antiepileptic, but is today known as a sedative and sleep regulator. Seizures may be associated with abnormal electrocardio- graphic changes and cardiac dysfunction arising from epilepsy may be related with neuronal nitric oxide (nNO). This study was aimed to investigate the effects of the neuronal nitric oxide synthase (nNOS) inhibitor 7-Nitroindazole (7-NI) and VAL on seizure behaviours and electrocar- diographic parameters in the pentylentetrazole (PTZ)-kindled seizure model.

Wistar rats were randomised into saline control, PTZ-kindled, 7-NI, VAL and VAL+PTZ, 7-NI+PTZ and VAL+7-NI+PTZ groups. Latency, stage, frequency of seizures, blood pressure (BP), heart rate (HR) and corrected QT (QTc) values were evaluated.

Frequency and stage of seizures, BP and HR increased, while seizure latency decreased and QTc was prolonged in the PTZ-kindled group. 7-NI and VAL had no effects on BP and HR variables under normal conditions, but ameliorated the seizure stage and frequency of seizures. 7-NI treatment also resulted in a reduction of the increased BP and prolonged QTc values observed in PTZ-kindled rats.

Considering these results, QTc prolongation may be used as a predictor for recurrent seizures. 7-NI and VAL exhibited different effects on seizures and ECG variables. 7-NI shows potential as an anticonvulsant drug agent in epileptic patients with cardiac dysfunctions and those additional studies including in-vivo experiments are essential.

Telomerase reverse transcriptase (TERT) vectors were transfected into bone marrow mesen- chymal stem cells (BMSCs) which were then cultured and selected to establish TERT-BMSC cell lines whilst sequencing BMSCs and TERT-BMSCs via transcriptome in this study to explore their regulatory mechanism and effect on osteogenic differentiation after TERT ectopic expres- sion in sheep BMSCs. After sequencing and analysing differential genes, PI3K/Akt signalling pathway related to osteogenic differentiation was investigated. Western blot was used before and after applying the PI3K/Akt signalling pathway inhibitor LY294002 to detect protein expression levels of AKT and p-AKT. On the twenty-first day of osteogenic differentiation, RT-qPCR and Western blot were used to detect mRNA and protein expression levels of RUNX2 and OPN and alizarin red staining was utilised to analyse calcium salt deposition. Results showed that pro- tein expression levels of AKT and p-AKT were significantly up-regulated, mRNA and protein expression levels of RUNX2 and OPN increased and calcium salt deposition increased after ectopic expression of TERT. After applying LY294002, the protein expression of AKT and p-AKT was down-regulated, mRNA and protein expression levels of RUNX2 and OPN were reduced and calcium salt deposition was reduced. These results confirmed the stable integration and expression of the exogenous TERT gene in BMSCs to promote the differentiation of BMSC osteoblasts, which may be mediated by the PI3K/Akt signalling pathway.

We investigated changes in concentrations of ADP (adiponectin), LEP (leptin), BHBA (beta-hydroxybutyric acid), NEFA (non-esterified fatty acid), Glucose (Glu) and INS (insulin) in serum of healthy perinatal dairy cows and cows with ketosis. Twenty-one healthy cows and seventeen cows with ketosis from a herd of a total 60 Holstein cows (near dry period i.e. 56 days antepartum) were selected. Blood was collected through the tail vein every 7 days, from 56 day antepartum to 56 day postpartum. Serum ADP, LEP, BHBA, NEFA, Glu, and INS concentrations were determined, and ketosis was diagnosed through serum BHBA (≥1.2 mmol/L). We showed the concentration of serum adipokines and energy balancing indices were stable during antepar- tum period. However, ADP concentration increased while LEP decreased, and there were a significant increase in cows with ketosis compared to that of in healthy cows. Serum BHBA and NEFA concentrations increased significantly at first, and then gradually decreased in both healthy cows and cows with ketosis. However, cows with ketosis showed higher concentrations of BHBA and NEFA which restored later. The serum concentration of Glu in both healthy dairy cows and cows with ketosis showed a decreasing trend. INS concentration in healthy cows was decreased while it was increased in cows with ketosis. The results reflect the extent of hypo- glycemia and lipid mobilization postpartum, suggest IR exists in cows with ketosis while serum ADP and LEP might play roles in the development of ketosis.

There is an increased interest in using automatic milking systems (AMS) to indirectly assess the welfare of dairy cows, but knowledge on analyzing the association between lameness, milk yield characteristics, and reproductive performance in cows is still insufficient. The main aims of this study were to evaluate the influence of lameness on several AMS variables and reproduc- tive performance indicators during the early stage of lactation and estrus in Lithuanian Black and White dairy cows, as well as to assess the associations between lameness, productivity and repro- ductive efficiency. A total of 418 milking cows (50.3±1.2 d postpartum) without any apparent reproductive disorder were monitored for hoof health status. Cows were assigned to two groups on the basis of visual locomotion scoring: “non-lame“cows (group 1; 74.20%) and cows presen- ting “lameness“ (lame cows) (group 2; 25.80%).

Productive and milking performances of dairy cows were recorded from 50 to 100 days in milk (DIM) and 1 day after the first estrus. The lameness was predominantly localized on the hind feet (79.60%) and less frequently - on the front feet (20.40%; p<0.001). Furthermore, the lameness had a tendency to decrease milk production (4.24%; p<0.05) and increase the diffe- rence in milk yield between rear and front quarters of the udder (1.20%; p<0.05). The frequency of milking (5.19%) was lower in lame cows (p<0.05). The lame cows during estrus showed a more pronounced decrement in milk yield and milking frequency (p<0.05), and also higher milk progesterone concentration values (1.55-1.76 time’s; p<0.001), and an increasing number of inseminations (11.69%; p<0.05) were observed. The results highlighted that analysis of data from AMS programs can be a successful tool for reducing risk factors related to the effective management of reproductive performance and hoof health of dairy cows.

The aims of the study were to (1) compare the serum concentration of anti-Müllerian hormone (AMH) with the number of follicles in ovaries and (2) determine the serum AMH con- centration before and after ovariohysterectomy in dioestrus and anoestrus bitches. Sixteen bitches were divided into two groups: Group I (n=8) consisted of dioestrus and group II (n=8) anoestrus bitches. The blood samples for AMH assesment were taken before ovariohysterectomy (day 0) and on day 1, 5 and 10. Both in group I and II, serum AMH concentrations on day 1 and 5 were significantly different compared to day 0 (p<0.05). However, the concentrations at day 10 were under the minimum detectable concentration (1.0 ng/mL) and this finding revealed that ovaries are the only source of AMH synthesis. Follicle counts were not statistically different between the groups (p>0.05). Significantly positive correlation in serum AMH with secondary follicle num- bers (r=.942, p<0.01), as well as negative correlation with antral follicle numbers (r=-.765, p<0.05) were determined in the group I. In the group II, positive correlations between serum AMH concentration and secondary follicle numbers (r=.960, p<0.01) and early antral follicles (r=.726, p<0.05) were noted. Assesment of AMH concentration seems to not only provide the diagnosis of the presence of ovaries but also correlate with the number of secondary follicles in young dioestrus and anoestrus bitches.

The purpose of the study was to obtain values of acid-base balance and basic biochemical parameters in neonatal kids of the White Shorthaired goat depending on colostrum intake. The research was focused on changes in acid-base balance parameters and basic biochemical parameters in neonatal kids before and two hours after colostrum intake. Total of 66 blood samples were taken from 33 neonatal kids. Blood pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), bicarbonate concentration (cHCO3 -), base excess (BE), oxygen saturation (cSO2), total carbon dioxide (TCO2), sodium (Na+), potassium (K+), calcium (Ca2+), chloride (Cl-), glucose (Glu), lactate (Lac), creatinine (Crea), hematocrit (Hct) and haemoglobin (Hgb) were measured. There were no statistically significant differences in acid-base balance parameters such as pO2, pCO2, TCO2, cSO2 and biochemical parameters such as Na+, K+, Ca2+, lactate between the two groups - before colostrum intake (group BF) and after colostrum intake (group AF). There were statistically significant differences in acid-base balance parameters such as pH, BE, cHCO3 - between these groups. Differences in acid-base values of pH, BE and cHCO3 - were statistically significant (p˂0.05). Differences in biochemical values of creatinine and glucose were statistically significant (p˂0.05). Differences in values of hematocrit and haemoglobin were statistically significant (p˂0.05). The present results are important for veterinary practice and can improve the neonatal care especially for impaired kids.

MDAP-2 is a new AMP with high inhibitory activity on Salmonella gallinarum, which may be developed as an antimicrobial agent in the agricultural industry and food preservation. To investigate the underlying the action mechanism of MDAP-2 on Salmonella gallinarum, impacts of MDAP-2 on the growth curve and bacterial morphology of Salmonella gallinarum were studied. iTRAQ-based proteomics analysis was also performed on proteins extracted from treated and untreated Salmonella gallinarum cells. The differentially expressed proteins were then analyzed using the KEGG and GO databases. Finally, the function of some differentially expressed proteins was verified. The results showed that 150 proteins (41 up-regulated and 109 down-regulated) were found differentially expressed (fold > 1.8, p<0.05). The results indi- cate that MDAP-2 kills Salmonella gallinarum mainly through two mechanisms: (i) direct inhibi- tion of cell wall/ membrane/ envelope biogenesis, energy production/ conversion, carbohydrate transport/ metabolism, and DNA transcription/ translation through regulation of special protein levels; (ii) indirect effects on the same pathway through the accumulation of Reactive oxygen species (O2 ▪-, H2O2 and OH▪-).

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis mainly in domestic and wild ruminants; paratuberculosis is also known as Johne’s disease. This disease is endemic all over the world generating significant economic losses, especially in dairy herds, although, MAP is the cause of infection in many other species including primates. Currently, MAP mycobacteria are recognized as pathogens transmitted by food. They are a potential threat to animal and human health. Infected animals excreting mycobacteria with faeces are the main source of MAP. The development of control strategies and disease control are based on determi- nation of the genetic diversity of the MAP strains causing Johne’s disease. This study describes 43 strains isolated from a herd of dairy cows located in northern Poland. The types of MAP were determinted based on the polymorphism analysis of two insertion fragments: IS900 and IS1311. The polymorphism of IS900 was analyzed with the use of a PCR multiplex according to Collins’ method and the IS1311 polymorphism with the use of the PCR-REA method. Based on the diffe- rences observed, the strains isolated were classified into two MAP types, cattle (C-type) and sheep (S-type), with the predominance of the cattle type.

An eukaryotic expression system of Congjiang pigs IFN-λ1 was constructed to obtain its expression in CHO-K1 cells and the inhibition effect of Congjiang pig IFN-λ1 on PRRSV proliferation was verified. The eukaryotic expression plasmid pEGFP-PoIFN-λ1 was constructed from the pig IFN-λ1 gene fragment and transfected into CHO-K1 cells. Expression was detected by fluorescence microscopy and Western blotting. The influence on the proliferation of PRRSV was assessed. The results of the study showed that the recombinant plasmid pEGFP-PoIFN-λ1 was constructed correctly. After transfection, green fluorescent signal was detected in CHO-K1 cells by fluorescence microscopy. Western blot analysis revealed that in cells at different time periods after transfection, porcine IFN-λ1 was expressed, with the highest expression observed 36 h after transfection. The antiviral activity of the supernatant after 36 h of transfection was determined by the micro cytopathic inhibition method, and the biological activity was 2.1×103 U/mL. Quantitative PCR was used to detect the proliferation of PRRSV, and the results showed that Congjiang pigs IFN-λ1 significantly inhibited the mRNA expression of PRRSV and viral proliferation in a dose- and time-dependent manner. This study established a Congjiang pig IFN-λ1 eukaryotic expression system, and the quantitative PCR method showed that it has a significant inhibitory effect on the proliferation of PRRSV, which lays a foundation for the future production of antiviral drugs and clinical application.

The chronic degenerative endometritis (CDE) is recognised as directly related to age and infertility in the mare. In this study, the 14 days post ovulation (PO) pregnancy rate was assessed in 60 barren mares affected by CDE, submitted to platelet-rich-plasma intrauterine infusion (PRPI) 24 hours PO. Data showed a significant positive effect of PRPI on the chance to become pregnant in mares affected by Kenney I-II CDE. The overall 14 days PO pregnancy rate was 75%. Sixty-nine % of the overall pregnancies was achieved with the 1st PRPI, but an additional 31% was obtained with the 2nd PRPI at the subsequent heat, increasing significantly the number of pregnancies. Although the 1st PRPI lead to a higher pregnancy rate than the 2nd PRPI (52 vs 23%), the repetition of the treatment had a significant positive effect on the overall pregnancies. Mare’s young age positively influenced the chance to become pregnant and was associated to endometritis Kenney I. Mares with endometritis Kenney I had better chance to become pregnant than those with endometritis Kenney II. The PRPI proved, therefore, to be beneficial in barren mares with CDE, and resulted in high overall pregnancy rate achievement in barren mares.

Bactericidal activity of caprylic acid (CA) and hydrogen peroxide (HP) was investigated in this study in order to design a suitable formulation for use in the food-processing industry. Antibacterial effects of the two chemicals were tested in vitro against the reference strains of Salmonella enterica subsp. enterica serotype Enteritidis CCM 4420, Escherichia coli CCM 3988, Listeria monocytogenes CCM 5578 and Staphylococcus aureus CCM 4223, as well as against the wild bacterial strains obtained from various food commodities (poultry meat, rabbit meat, raw milk sheep cheese ‘Bryndza’) and potable water. First, suspension test was carried out to determine the minimum bactericidal concentrations for individual chemical compounds. While most Gram-negative bacteria tested were effectively inhibited by HP at a 0.5% concentration, the growth of Gram-positive bacterial strains was stopped by a 2% solution. CA showed similar antibacterial effect on all bacterial strains tested except for Staph. aureus showing the same sus- ceptibility as Gram-negative bacteria. The wild strains generally had higher resistance to both chemicals than the reference strains. Combination of HP and CA at concentrations of 0.01%; 0.05% and 0.1% was further tested by the suspension test, carrier test, and carrier test with simul- taneous exposure to UV light. The total bactericidal activity against selected foodborne pathogens was already observed at a concentration of 0.1% and the efficiency was significantly increased by the use of UV radiation. A novel disinfectant based on the combination of HP with CA appears to be a suitable binary formulation for potential use in the food sector.

This study investigated changes in serum levels of hepatic, bone, and intestinal alkaline phosphatase (ALP) isoenzymes (ALP2, ALP3, and ALP5, respectively) in Holstein cows around parturition. Tartrate-resistant acid phosphatase 5b (TRAP5b) activity and calcium (Ca) concen- trations were also measured. We analyzed blood samples from 11 late-pregnant heifers (primipa- rous group) and 13 multiparous (2-4 lactations; multiparous group) cows at 3 weeks (18-24 days prepartum; -3 weeks), 2 weeks (17-11 days prepartum; -2 weeks), and 1 week (10-4 days prepar- tum; -1 weeks) before parturition; the day of calving (within 12 h post-calving; day 0); and 5 days postpartum (5 days). ALP3 activity was significantly higher in the primiparous group than in the multiparous group, whereas the activities decreased significantly in both groups after 5 days. ALP2 and ALP5 activities did not change, whereas ALP2 activity was significantly higher in the primiparous group than in the multiparous group. TRAP5b activity was significantly higher in the primiparous group than in the multiparous group and showed a transient significant increase at day 0. Ca concentration significantly decreased at day 0 in both groups; the Ca level at day 0 was significantly higher in the primiparous group than in the multiparous group. These data show that ALP3 activity in serum may indicate a change in osteoblastic bone forma- tion after calving, but further study is needed to determine the clinical application for measuring ALP isoenzymes in bovine medicine.

Conservation of genetic resources by semen cryopreservation is essential for biodiversity conservation and storage of rare poultry breeds. Despite the widespread use of this method not all individuals presentia similar capacity for semen to be used after defrosting. The aim of the current study was to identify SNP markers and linked candidate genes potentially associated with rooster (Gallus gallus) sperm motility after cryopreservation. Genome-wide association studies were performed using 33 roosters from four breeds genotyped using Illumina Chicken 60K SNP BeadChip Calculations were performed using PLINK and EMMAX software. Significant SNP associations rs15557972 (p<1.36E-07) on chromosome 10 in the LOXL1 gene and rs15751385 (p<6.10E-06) on chromosome 6 in the intron of the ENSGALG00000052127 gene were identified. These findings associated with sperm motility SNPs will help to develop strategies for the selection of valuable individuals and the efficient conservation of the gene pool.

Staphylococcus pseudintermedius (SP) and methicillin-resistant SP (MRSP) is one of the most important veterinary pathogens in the dog. Herein, from a total of 126 S. pseudintermedius strains, 23 MRSP (18%) were identified. Multi-Locus Sequence Typing (MLST) revealed that most of MRSP strains belonged to ST71 (26%), which have been already reported in Italy and other countries. Interestingly, nine new sequence types (39%), from 1053 up to 1061, were described for the first time. Moreover, the isolated MRSP strains showed relevant antibiotic resistance profiles. This report highlights the circulation of new sequence types of MRSP in Italy and underlines the need of a global epidemiological surveillance to limit the increasing spread of multidrug-resistant MRSP strains worldwide, since they may represent a considerable concern for dog’s health.

Canine babesiosis is a tickborne, protozoal, haemoparasitic disease. Babesia organisms are frequently classified as either large (B. canis) or small (B. gibsoni). The aim of this study was an attempt to detect B. gibsoni DNA in blood samples taken from dogs suspected of suffering from tick-borne diseases. 216 samples were tested using PCR, of which, in 99 of them B. canis DNA was detected, whereas in 3 of them B. gibsoni was detected. Positive PCR results for B. gibsoni were confirmed using a Qube MDx real-time analyzer. The results indicate that infections with this B. gibsoni should be taken into account and included in the differential diagnosis of vector-borne diseases in dogs in Poland, and that the accurate identification of the species of parasite causing the infection is crucial for developing the correct treatment regimen and prognosis.

The main objective of this study was to assess the concentration of various minerals (Ca, P, Mg, Cu, Zn, Fe) in the blood of sheep, followed by biochemical analysis in order to reveal possible associations of season and breed. The study was conducted by sampling four herds: Suffolk (n=20), Merino (n=20), Lithuanian blackhead (n=20) and Charolaise (n=7). The first blood collection was conducted in April and the last one was performed in February. The highest level of Ca was estimated in Suffolk ewes, lowest (12.61%) in Merino breed; the highest Mg content was found in Lithuanian blackhead breed, lowest (5.26%) in Charolaise; highest P content was determined in Merino, lowest (24.18%) in Suffolk breed (p<0.05). Evalua- tion of the biochemical parameters during different seasons showed a possible environmental effect on the health of the animals. The difference among minerals content showed the highest level in Ca, Mg, Fe in the autumn, P - in the summer, Cu and Zn - in the winter. The lowest differences between seasons were observed in content of Mg (1.24-4.03% from total average of all seasons) and Ca (0.59-8.18%), the highest – in Cu (2.52-18.36 %) and Zn (4.33-24.33%) (p<0.05). The significance of this work is the possible use of the data in the prevention of metabolic and production diseases.