Silvijo Vince

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n=9), and Group II was comprised of older bulls aged five to ten yrs (n=10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.

Intrauterine ozone treatment of retained fetal membrane in Simmental cows

The aim of research was to determine influence of intrauterine application of two different ozone preparations on reproductive performance in Simmental cows with retained fetal membranes (RFM). The study was performed on 143 Simmental cows aged 2-8 years and divided in three groups. Group A (n=46) consisted of cows treated with foam spray ozone applied into the body of the uterus for 5s using a sterile catheter. Group B (n=50) consisted of cows treated using six ozone pearls intrauterinely at once, as deeply and hygienically possible. Cows were observed and treated during early puerperium, 24-36h after parturition. The third group (n=47) consisted of cows without RFM (control group). To assess the reproductive performance of cows, the interval from calving to first insemination (days not pregnant to first service, DOFS), interval from calving to pregnancy (days not pregnant to pregnancy, DOP), relative pregnancy rate (%), first service conception rate (FSCR, %) and all service conception rate (ASCR, %) were measured. The estimate of hazard ratio for the Groups A and B relative to control group with DOFS were 0.423 (P=0.0006) and 0.434 (P=0.0005), and with DOP were 0.701 (P=0.003) and 0.411 (P=0.0003), respectively, implying that cows in the control group were not pregnant longer until first insemination and pregnancy. Variables that had an influence on DOFS were postpartum fever (PPF) (HR=0.458; P=0.003) and milk yield (HR=0.999; P<0.0001) and an influence on DOP were PPF (HR=0.314; P=0.001) and milk yield (HR=0.999; P<0.0001). Cows with RFM treated with intrauterine Riger spray or Ripromed ovuli O(3) have similar or enhanced reproductive performance results compared to the control group of cows demonstrating the effectiveness of therapy with intrauterine ozone products.

Effect of Preventive Intrauterine Ozone Application on Reproductive Efficiency in Holstein Cows

The aim of the research was to determine the influence of preventive application of two different ozone preparations on reproductive efficiency in dairy cows with physiological puerperium. The research was conducted on 404 Holstein cows aged 2-8 years, divided into three groups. The first group consisted of cows treated using 6 intrauterine ozone pearls (RIPROMED OVULI O(3) (®) , Novagen, Italy) at once. The second group consisted of cows treated with ozone foam spray (RIGER SPRAY(®) , Novagen, Italy) application in the body of uterus for 5 s, and the third group was the control group. Cows from the first two groups were observed and treated during early puerperium, 24-48 h after parturition. To assess the reproductive performance of Holstein cows, interval from calving to first insemination (days open to first service, DOFS), interval from calving to pregnancy (days open to pregnancy, DOP), relative pregnancy rate (%), first service conception rate (FSCR, %) and all service conception rate (ASCR, %) were measured. The estimate of hazard ratio (HR) for group B relative to group C for DOFS was 0.62 (p = 0.0002), implying that the relative rate of first service decreased in the control group by 38%. The estimate of HR for group B relative to group C for DOP was 0.65 (p = 0.0006), implying that the relative rate of pregnancy decreased in group C by 35%. Cows treated with Riger spray (group B) became pregnant earlier and had better FSCR and ASCR. It is supposed that the form of ozone preparations and the amount of active substance were decisive. A conclusion may be made that preventive ozone intrauterine application during early puerperal period, especially the foaming form of ozone preparations (spray), improved the reproductive efficacy in dairy cows.

Ligand-independent canonical Wnt activity in canine mammary tumor cell lines associated with aberrant LEF1 expression

Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1) and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand–independent mechanisms.

New trends in human and animal leptospirosis in Croatia, 2009–2014

Leptospirosis is a geographically widespread and globally underestimated zoonosis that affects humans and variety of animals. To identify trends and possible risk factors, joined medical and veterinary teams investigated epidemiology and epizootiology of leptospirosis in Croatia. Retrospective analysis of data obtained from referent diagnostic laboratories included a total of 1917 human and 123964 animal sera tested in the period from 2009 to 2014. We found high human leptospirosis average incidence rate of 1.53/100000 with clear predominance of male patients older than 40 years (sex ratio M/F:3.2; median age 51±15.1years). Statistical analysis revealed seasonal and annual variations of incidence in humans that were primarily associated with favourable weather conditions (temperature 10-19, 9°C and precipitation above 100mm/m2). Majority of infections in humans were caused by serogroups Sejroe, Australis and Icterohaemorrhagiae. Notable variations in seroprevalence and changing trends in prevailing serogroups were recorded in most of the domestic animals and during the entire period of investigation. All of the observed findings underline leptospirosis as a significant human and veterinary public health threat and emphasize the importance of continuous multidisciplinary surveillance. We also argue that only input from both professions improves our overall knowledge on leptospirosis and leads to better and more efficient prevention and control strategies.

Influence of boar breeds or hybrid genetic composition on semen quality and seminal plasma biochemical variables

The enzyme concentrations of seminal plasma are important for spermatozoa metabolism and function in boars. The need has arisen for introducing a biochemical evaluation of semen, along with the usual standard semen analyses. There are no data on the influence of boar breeds on the seminal plasma biochemical variables investigated in this study. Therefore, the objective was to determine the influence of breed and hybrid genetic composition of boars on semen quality and seminal plasma biochemical variables. Semen samples of 27 boars (Swedish Landrace, German Landrace, Large White, Pietrain and Pig Improvement Company hybrid-PIC-hybrid), aged between 1.5 and 3 years, were collected. After evaluation of semen quality, the seminal plasma was separated from the spermatozoa by centrifugation of semen. The seminal plasma was subjected to spectrophotometric analysis to determine alkaline phosphatase (ALP), acid phosphatase (ACP), γ-glutamyltransferase (GGT), creatine kinase (CK) and lactate dehydrogenase (LDH) and to atomic absorption spectrophotometric analysis to measure the concentration of calcium and magnesium. Conventional semen quality variables differed depending on breed and PIC-hybrid genetic composition, though these differences were typically insignificant. In the seminal plasma, significant differences were determined in enzyme activity (ALP, GGT, CK and LDH) and in calcium concentration among boars of different breeds. There are, therefore, differences in semen quality and significant differences in the seminal plasma biochemical variables among boars of different breeds and PIC-hybrid genetic composition. The data and differences in semen variables detected in the present study provide knowledge for enhancing evaluation and monitoring of boar reproductive potential, semen quality and explain the potential causes of boar infertility.

Extensive polymorphism of the major histocompatibility complex DRA gene in Balkan donkeys: perspectives on selection and genealogy.

The major histocompatibility complex (MHC) contains genes important for immune response in mammals, and these genes exhibit high polymorphism and diversity. The DRA gene, a member of the MHC class II family, is highly conserved across a large number of mammalian species, but it displays exceptionally rich sequence variations in Equidae members. We analyzed allelic polymorphism of the DRA locus in 248 donkeys sampled across the Balkan Peninsula (Albania, Bulgaria, Croatia, Macedonia, Greece and Montenegro). Five known alleles and two new alleles were identified. The new allele Eqas-DRA*0601 was found to carry a synonymous mutation, and new allele Eqas-DRA*0701, a non-synonymous mutation. We further analyzed the historical selection and allele genealogy at the DRA locus in equids. Signals of positive selection obtained by various tests were ambiguous. A conservative conclusion is that DRA polymorphism occurred relatively recently and that positive selection has been acting on the DRA locus for a relatively brief period.

Influence of Body Condition on Serum Metabolic Indicators of Lipid Mobilization and Oxidative Stress in Dairy Cows During the Transition Period

The objectives of this study were to examine the influence of body condition of cows on metabolic and antioxidative status, as well as to investigate the relationship between metabolic indicators of lipid mobilization and oxidative stress during transition period. The study was conducted on 24 Holstein-Friesian dairy cows divided into 2 groups according to their body condition score (BCS) as optimal (n = 12; BCS from 3.25 to 3.75) or adipose (n = 12; BCS ≥4). Metabolic status (glucose, triglycerides, total cholesterol, HDL cholesterol, NEFA and BHB), paraoxonase-1 (PON1) and apolipoprotein A-I (ApoA-I) were analysed in sera taken on days -30, -10, -2, 0, 5, 12, 19, 26 and 60 relative to parturition. Adipose cows had significantly higher glucose concentration at parturition being significantly decreased after parturition on days 12 and 19. Total cholesterol and HDL-C concentrations were the lowest at parturition and significantly higher on days 26 and 60 after parturition in both groups of cows. Both investigated groups had significantly higher NEFA concentration from parturition until day 19 after parturition, indicating energy deficit and an increased lipid mobilization after calving. There were no significant differences in BHB concentration during transition period in both groups. No significant differences were found in PON1 activity and ApoA-I concentration during transition period in both groups of cows. However, in adipose cows, although not significantly different, PON1 was decreased from calving until day 19 after parturition indicating a disturbance in antioxidative status in adipose cows. PON1 significantly positively correlated with total cholesterol and HDL-C concentrations and negatively with NEFA indicating a strong relationship of PON1 with lipid metabolism. Significant positive correlation between NEFA and BHB in both groups of cows points out on energy deficit during transition period that cows tend to overcome by lipid mobilization providing alternative source of energy needed for parturition and lactation.

Metabolic and oxidative status of Saanen goats of different parity during the peripartum period.

The aim of this study was to research changes in metabolic and antioxidative status of Saanen goats of different parity occurring during the peripartum period. Blood samples were taken on 10-7 and 3-1 d prepartally and 1-3, 14 and 28 d postpartally from goats allocated in three groups according to their parity: primiparous (PRIM), goats that kidded the 2nd or 3rd time (MID), and goats that kidded 4 or more times (MULTI)). Metabolic profile parameters (non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), glucose, triglycerides, albumin and urea) and indicators of oxidative stress ((superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA)) were determined. Intense metabolic changes associated with late pregnancy and onset of lactation were pronounced the most in MULTI goats that also had the biggest litter per goat. Significant differences were found in metabolic parameters NEFA, BHB, glucose, triglycerides within groups during peripartum period, as well as between them (the effect of parity). MDA concentrations were indicative of increased lipid peroxidation around parturition, especially pronounced in MULTI group 1-3 d prepartally, when the highest GSH-Px/SOD ratio was also found. Postpartally, antioxidant enzymes ratio in MID and MULTI group decreased while MDA concentrations remained high, suggesting antioxidant system inefficiency. Significant time × group interaction was observed for most of the parameters. The obtained results show that the goats of higher parity display higher levels of metabolism intensity and consequently, varying levels of oxidative stress during the peripartum period. Further studies should determine applicability of NEFA and BHB in periparturient metabolic profiling in dairy goats as well as establish normal ranges and cut-off levels for these biomarkers.

Influence of different periods of the year and age on the parameters of antioxidative status and oxidative stress in the blood serum of breeding bulls

The sources of variations that may cause physiological differences between blood serum biochemistry parameters of bulls have not been investigated in detail. Aim of the present study was to establish influence of different periods of the year and the age of breeding bulls on parameters of antioxidative status and oxidative stress in their serum and to correlate these monitored variables. Research was performed on two groups, each comprising 9 Simmental bulls: a younger group (YB) (aged 2-4 years) and older one (OB) (aged 5-10 years). Blood samples for biochemical analyses were collected from jugular vein in cold (CP) and warm periods (WP) of the year. Reduced glutathione (GSH), uric acid (UA), total protein (TP), albumin (ALB), thiobarbituric acid reactive substance (TBARS), and protein carbonyl content (PCC) serum concentration were determined, as well as activities of selenium-dependent glutathione peroxidase (Se-GSH-Px), total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD) and catalase (CAT). Serum values of SeGSH-Px, MnSOD, UA and TP in OB were significantly higher compared to those in YB during CP of the year. Significantly higher PCC concentration in serum of YB and OB were established in CP of the year than in WP. TBARS serum concentration in YB was significantly higher in comparison to that in OB during CP of the year. It can be concluded that both OB and YB show a great sensitivity to climate condition alterations during CP in comparison to WP of the year and that YB show even greater sensitivity.