Tomislav Dobranić

Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure® and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure® and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure®, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure® method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure® treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure® method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure® could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos.

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.

Concentrations of total proteins and albumins, and AST, AP, CK and GGT activities in the blood serum Boer and Saanen goats during puerperium.

The metabolism of proteins in the blood serum in Boer and Saanen goats was investigated during puerperium. Twenty Boer goats (10 primiparous and 10 pluriparous) and 10 Saanen goats (five primiparous and five pluriparous) between 2 and 5 years of age were used in this research. Blood for analysis was taken every fourth day from day 3 until day 40 post-partum. Blood samples were collected by jugular puncture. In the obtained blood serum, the concentration of total proteins (PT) and albumin (ALB), and the activity of enzymes aspartate aminotransferase (AST) [the Enzyme Commission number (EC number) 2. 6. 1. 1.], gamma-glutamyltransferase (GGT) (EC 2. 3. 2. 2.), creatine kinase (CK) (EC 2. 7. 3. 2.) and alkaline phosphatase (AP) (EC 3. 1. 3. 1.) were determined by spectrophotometry. These parameters were in physiological ranges in Boer goats and in Saanen goats, without significant differences according to number of kids per doe. According to the research results of the blood serum in goats during puerperium, there were no significant differences in the concentration of ALB. Boer goats had significant higher (p < 0.05) concentration of PT and enzyme activity of AP, CK and GGT. Saanen goats had only enzyme activity of AST significantly higher (p < 0.05). Enzyme activity of alkaline phosphatase was significant higher (p < 0.05) in pluriparous goats in both breeds than in primiparous. The obtained results may represent a contribution to a better understanding of protein metabolism during puerperium in dairy and meat goats and for diagnostic purposes.

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.

Developmental and transplacental genotoxicology: Fluconazole

Over the last 40 years mankind has been facing new types of radiochemical environmental settings with every decade. During the last decade, biomonitoring was additionally focused on assessing associations between environmental exposure(s) and both early and late biological effects in children. Despite efforts to control and avoid child exposure to genotoxic agents the incidence of childhood cancers is increasing. Some cancers in adulthood may be the consequence of a multi-step process which starts with intrauterine and childhood exposure. This highlights the importance of a comprehensive interpretation of multiple health effects, especially considering recent studies suggesting that most health disorders are related to DNA changes. When exposed to genotoxic agents, a developing organism (fetus or child) is constantly being forced to reorganize into new equilibriums in order to adjust to a xenobiotic environment. In addition, the influence of sex hormones on radiochemical sensitivity is still unknown. For this reason special attention should be paid to puberty. The results of recent studies on animal models and follow up studies on children after nuclear accidents show long-lasting cytogenetic damage even after low dose exposures and their transgenerational persistance. To evaluate age-related difference and transplacental genotoxic potency fluconazole (FC) was investigated by in vivo micronucleus (MN) assay in adult mice, young mice and in transplacentally exposed newborn pups. Compared to the baseline values, FC caused no detectable genome damage in adult animals, but there was a significant increase in MN frequency in young animals and in newborn pups. Our study thus exemplifies an age-related chemosensitivity, and argues that cancer-promoting disturbances of complex prenatal developmental mechanisms and maturation during childhood require a new approach using systems biology.

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.

Progesterone and Insulin-Like Growth Factor I Levels in Blood of Boer Goats During Puerperium Out-of-Season in a Mild Climate Region

The aims of this study were to compare insulin-like growth factor-I (IGF-I) and progesterone concentrations as indicators of cycle activity in the blood of Boer goats during puerperium out-of-season in mild climate conditions and to investigate the influence of parity and litter size on the concentration of IGF-I and progesterone in serum during the study period. Twenty Boer (8 primiparous and 12 pluriparous) goats were examined in this study. Blood samples were taken every 4 days starting on the 3rd day and up to the 40th day of puerperium. IGF-I and progesterone (P4) blood levels were determined using radio immune assay (RIA). Results indicated cyclic ovarian activity outside the breeding season in 18 of 20 Boer goats. IGF-I levels in blood sera followed oestrus and cyclic ovarian activity. IGF-I concentrations correlated significantly with P4 concentrations. The IGF-I peak preceded the P4 peak by approximately 8 days in cycling goats. IGF-I and P4 blood levels were not influenced by parity or litter size. Changes in blood serum P4 and IGF-I concentrations levels during puerperal period can assist in the evaluation of reproductive status of goats.

Influence of different therapy approaches - with or without manual extraction - of retained placenta on reproductive performance in simmental cows

The aim of the research was to determine the influence of different methods used in the therapy of retained placenta on conception rate and duration of days open in cows. Research was performed on 241 Simmental cows aged 2-8 years divided in three groups. The first group of cows (group A) (n=82) with retained placenta was treated with manual extraction of placenta 12-24 h after parturition combined with application of intrauterine antibiotics. The second group (group B) (n=79) with retained placenta was treated with intrauterine antibiotics only 12-24 h after parturition and repeated 2-3 times in 48 h intervals. The third group (group C) (n=80) was the control group and had physiological puerperium (without retained placenta). To assess the reproductive performance of Simmental 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 for DOFS in group A relative to cows in group B was 2.20 (p<0.0001), implying that cows in group B had oestrus earlier and were open shorter until the first insemination, that is, the relative rate of first service decreased by 54.5% in the group A while holding all other variables constant. The variable that had a significant influence on DOFS was lactation (HR=0.99; p<0.0001). The estimate of hazard ratio for DOP in group A relative to cows in group B and C was 3.53 (p<0.0001) and 1.73 (p<0.0001), respectively, implying that cows in group B and C were open longer until pregnancy, that is, the relative rate of pregnancy in group A decreased by 71.6% and 42.2% in comparison with group B and C while holding all other variables constant. Variables that had a significant influence on DOP were lactation (HR=0.99; p<0.0001) and peripartal diseases (HR=0.48; p=0.02). The estimate of hazard ratio for DOFS and DOP between group B and C was not significant. Cows treated with intrauterine foaming tablets after RFM became pregnant earlier and had a better conception rate in contrast to cows treated with manual extraction of placentae.

MODULATION OF SYSTEMIC INNATE AND ADAPTIVE IMMUNE PARAMETERS IN WEANED PIGS BY SINGLE ORAL APPLICATION OF IMMUNOBIOTICS

The aims of this study were to evaluate the effects of peroral treatment of 4-week old pigs at weaning (Day 0) with a single dose of levamisole (LEVA) or polyoxyethyle-polyoxypropylene (POE-POP), well known immunobiotics (IBC) and promising alternatives to dietary antibiotic growth promoters (AGP), on systemic innate and adaptive immunity by determining phagocytosis (PHC) and microbicidity (MBC) efficiency of their monocytes (MO) and granulocytes (GR), changes of serum C-reactive protein (CRP) and haptoglobin (HpG) profiles and kinetics of CD4+ CD8+ T cells expression during 5 weeks following the treatments. Levels of CRP were decreased by either POE-POP or LEVA (P<0.05) at Day 7 and 21, respectively. LEVA-treated pigs had increased levels of HpG (P<0.05) at Day 14 and 21, whereas POE-POP-treated pigs had decreased and increased levels (P<0.05) at Day 7 and 21, respectively. Both IBC stimulated in vitro PHC of GR (from Day 7 to 35) and MBC of MO (at Day 35) from treated pigs (P<0.05). MO from POE-POP-treated pigs exhibited increased PHC (P<0.05) at Day 35, whereas GR from LEVA- or POE-POP-treated pigs showed increased MBC (P<0.05) at Day 7 and 35, respectively. The pigs treated by LEVA or POE-POP had higher proportions of CD4+ CD8+ T cells (P<0.05) from Day 14 to 35 or Day 28 to 35, respectively. Tested IBC showed capability of stimulating particularly cellular components of nonspecific and specific immunity during early postweaning period before pigs reach adult immunocompetence values, and thus could be considered as potent immunostimulators in swine production. Key words: synthetic immunobiotics; innate/adaptive immunity; weaned pig VPLIV NA SISTEMSKO PRIROJENO IN PRIDOBLJENO IMUNOST PRI ODSTAVLJENIH PUJSKIH PO ENKRATNEM DODATKU IMUNOBIOTIKOV PREKO PREBAVNEGA TRAKTA Namen raziskave je bil ugotoviti vpliv lavamisola (LEVA) in polyoxyethylen-polyoxypropylena (POE-POP), znanih imunobiotikov, ki bi jih lahko uporabljali v prasicereji za boljsi prirast namesto antibiotikov, na sistemsko prirojeno in pridobljeno imunost pri odstavljenih, 4 tedne starih pujskih. Vsem živalim smo LEVA in POE-POP dajali preko ust (peroralno) in tedensko v odobju petih tednov dodajanja imunobiotikov ugotavljali stopnjo fagocitoze in protimikrobno aktivnost monocitov in granulocitov, raven serumskega C-reaktivnega proteina (CRP), profil haptoglobina (HpG) ter raven prisotnosti limfocitov T, ki izražajo gena CD4 in CD8 v krvi. Rezultati so pokazali, da je bila raven serumskega CRP znižana pri pujskih, ki so prejemali tako LEVA kot POE-POP 7. in 21. dan raziskave (p < 0,05). Pujski, ki so prejemali LEVA, so imeli zvisan HpG 14. in 21. dan raziskave (p < 0,05), medtem ko so imeli pujski, ki so prejemali POE-POP, 7. dan raziskave HpG znižan, 21. dan pa zvisan (p < 0,05). Oba imunobiotika sta v pogojih in vitro dvignila stopnjo fagocitoze pri granulocitih in protimikrobno aktivnost monocitov na 35. dan raziskave (p < 0,05). Pujski, ki so prejemali en ali drugi imunobiotik, so imeli 35. dan raziskave visje vrednosti limfocitov T, ki so izražali CD4 in CD8, od vrednosti na 14. ali 28. dan raziskave (p < 0,05). Rezultati raziskave torej kažejo, da sta oba imunobiotika pozitivno vplivala na izbrane kazalce delovanja imunskega sistema in sta potencialno zanimiva za uporabo v prasicereji za spodbujanje delovanja imunskega sistema in s tem boljsi prirast prasicev. Kljucne besede: odstavljeni pujski; umetni imunobiotiki; prirojena imunost; pridobljena imunost;

Ultrasound Examination of Cows During the Puerperium in Everyday Veterinary Practice

Regular control of cows during puerperium allows as detecting and treating pathological processes in the early phase, to optimise duration of service and calving period followed with the occurrence of physiological cycles. The application of ultrasound in daily practice enables precise detection of follicles ability for ovulation and identification of functional corpus lutei, early diagnose of pregnancy, determination of foetal sex as well as an early and accurate identification of pathological processes (metritis, endometritis and pyometra). This survey included 54 cows: experimental group consist of 32 animals (26 of Simmental and 6 of Holstein-Friesian breed) that were under continuous control of the veterinarian (routine gynaecological and ultrasound examination), while remaining 22 animals in the barn (12 Simmental and 10 Holstein-Friesian cows) served as a control group – inseminated only at the invitation of the owner. Insemination index (1.69) and duration of service period (82.27 days) in the experimental group of Simmental cows were significantly lower (P<0.001) than in the control group (3.25 and 155.17 days, respectively). Although smaller insemination index was observed in experimental group of Holstein-Friesian cows (2.17) compared to the control (3.10) this difference wasn’t significant. Also, service period (100.67 days) in the experimental group was significantly shorter (P<0.05) compared to the control animals (180.30 days). These results showed that application of ultrasound improved certain fertility indicators in cows, i.e. minus insemination index and shortened duration of service period.