Oxidative Stress Response of Honey Bee Colonies (Apis mellifera L.) during Long-Term Exposure at a Frequency of 900 MHz under Field Conditions.

In this study, oxidative stress and lipid peroxidation in honey bee larvae, pupae and the midguts of adult bees were investigated during a one-year exposure to radiofrequency electromagnetic fields (RF-EMFs) at a frequency of 900 MHz under field conditions. The experiment was carried out on honey bee colonies at three locations with electric field levels of 30 mV m-1, 70 mV m-1 and 1000 mV m-1. Antioxidant enzymes, glutathione-S-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS) as indicators of lipid peroxidation were measured spectrophotometrically. The GST activity within the same developmental stage showed no significant differences regardless of electric field level or sampling time. The highest GST activity was found in the pupae, followed by activity in the larvae and midguts. Both CAT activity and TBARS concentration were the highest in the midguts, regardless of field level and sampling time. The larvae showed a significantly higher TBARS concentration at the location with an electric field level of 1000 mV m-1 compared to the locations with lower levels. Our results show that RF-EMFs at a frequency of 900 MHz can cause oxidative stress in honey bees, with the larval stage being more sensitive than the pupal stage, but there was no linear relationship between electric field level and effect in any of the developmental stages.

The Distribution of Boars Spermatozoa in Morphometrically Distinct Subpopulations after In Vitro Exposure to Radiofrequency Electromagnetic Radiation at 2500 MHz and Their Motility.

Anthropogenic radiofrequency electromagnetic radiation (RF-EMR) from wireless technologies has increased dramatically. The boar semen used for artificial insemination is essential in sustaining the pig industry, and additionally it is also exposed to the effects of the RF-EMR of wireless technologies. Furthermore, there are no data on the effects of RF-EMR on semen quality, and this is the first analysis of sperm's morphometric parameters for assessing the effect of RF-EMR on the spermatozoa subpopulations of boars. This study investigated the effect of RF-EMR on in vitro exposed breeding boar semen spermatozoa motility and the proportions of spermatozoa subpopulations according to their morphometric head and tail parameters. The semen samples of 12 boars were divided into control and experimental groups. The samples in the experimental group were exposed in a gigahertz transverse electromagnetic chamber at a frequency of 2500 MHz (the frequency band used in 5G technology) and an electric field strength of 10 Vm-1 for two hours. After exposure, the spermatozoa motility was evaluated for both groups. A morphometric analysis of the semen smears was performed using SFORM software (Version 1.0; VAMS, Zagreb, Croatia). The progressive spermatozoa motility was significantly reduced in the experimental group (74.7% vs. 85.7%). PC analysis and cluster analysis revealed two spermatozoa subpopulations: S1, spermatozoa with a more regular head shape and a smaller midpiece outline, and S2, spermatozoa with a more elongated head shape and a larger midpiece outline. The experimental semen samples had a greater proportion of the S1 spermatozoa subpopulation (68.2% vs. 64.4%). The effect of RF-EMR at 2500 MHz on the in vitro exposed boar semen resulted in decreased progressive spermatozoa motility and a lower proportion of the spermatozoa subpopulation with a higher fertilizing potential.

A Survey Study of Veterinary Student Opinions and Knowledge about Pet Reptiles and Their Welfare.

Exotic pet medicine is rapidly evolving, with reptiles becoming increasingly popular pet animals. Yet, there are only a few literature reports on veterinary perception of reptiles kept as pets. The aim of the study was to assess opinions and knowledge of the Croatian veterinarians-to-be about pet reptiles and their welfare. The questionnaire survey was conducted in the academic year 2019-2020 and included students of all six years of the integrated undergraduate and graduate study at the Faculty of Veterinary Medicine, University of Zagreb. First-year students were surveyed twice, before and after having attended the compulsory course on animal welfare. Questionnaire statements were 5-point Likert scale questions, requiring the students to express their opinions about turtles, lizards and snakes as pets, issues related to their welfare, risks they pose to the health and safety of humans, other animals and the environment, and their self-reported knowledge about pet reptiles. Although expressing higher opinions after having attended the course on animal welfare, first-year student responses remained neutral to most of the statements. Such a trend continued until the end of the study. Student responses revealed that they were uncertain about their knowledge of reptiles as pets, considering different educational areas observed. Study results emphasised the need of alterations in veterinary curriculum and additional student education in reptile medicine. The results obtained have broad implications involving not only the welfare of pet reptiles in clinical practice and elsewhere but also the health and safety of humans and other animals, as well as environmental protection.

Effect of RNF4-SacII gene polymorphism on reproductive traits of Landrace × Large White crossbred sows.

The aim of the present study was to investigate whether and to what extent the RNF4-SacII gene polymorphism influences reproduction performances in hyperprolific sow lines. The study involved 101 Landrace x Large White crossbred sows, with 461 records collected on the following reproductive traits: Total Number of piglets Born per litter (TNB), Number of piglets Born Alive per litter (NBA), Number of StillBorn piglets per litter (NSB), piglet Pre-Weaning Mortality (PWM) and Number of piglets at Weaning per litter (NW). The least square method with the GLM procedure in SAS with eight effects was used to pursue the data analysis. Study results revealed that TT homozygotes and TC heterozygotes had a significantly higher (p < .05) NW than CC homozygotes for the life-span performance in all parities and first parity analysed. In the fourth parity analysed, TNB and NBA in TC genotype were significantly higher (p < .05) as compared with TT genotype. Based on the life-span performance, significant effect (p < .05) was recorded for order of parity on TNB, NBA and NW, for farrowing season on TNB and NSB, and for lactation length on PWM. In the second parity, significant effect (p < .05) was recorded for sire of boar on NSB and for gestation length on TNB. Only in the fourth parity, significant effect (p < .05) of RNF4 gene was observed on NBA. There was significant additive effect (p < .05) of the RNF4 gene polymorphism identified on NW in all parities analysed, and significant additive and dominance effects (p < .05) on NSB in the third parity analysed. In conclusion, additional research on related production pig genotypes is necessary to elucidate the effect of RNF4 gene mutation on reproductive traits.

Effects of Dietary Vibroactivated Clinoptilolite Supplementation on the Intramammary Microbiological Findings in Dairy Cows.

: The aim of this study was to determine the effects of dietary vibroactivated clinoptilolite supplementation on the intramammary microbiological findings in dairy cows, causative agents of potential intramammary infection, and their sensitivity to antibiotics. Cows (n = 78) were randomly divided into two groups: CPL-treated group that received clinoptilolite (CPL) in-feed (n = 38), i.e., 50 g natural powdered zeolite CPL, twice daily from the seventh month of pregnancy to 75 days after calving, and the control group (CON) of untreated cows (n = 40). Milk samples were taken from each cow on days 7, 25, 45, and 75 postpartum. The following causative pathogens were isolated in 86 udder quarters: Staphylococcus aureus in 5.81% of positive samples, Staphylococcus spp. 9.32%, coagulase-negative Staphylococcus (CNS) 22.09%, Streptococcus uberis 13.95%, Streptococcus agalactiae 1.16%, Streptococcus sp. 3.49%, Escherichia coli 8.13%, Enterococcus spp. 6.98%, Corynebacterium spp. 11.63%, Pasteurella sp. 10.47%, Serratia spp. 2.33%, and Arcanobacterium pyogenes, Citrobacter sp., Prototheca sp., and yeasts each in 1.16% of samples. Additionally, 3.87% of environmental microflora samples (n = 47) and 89.06% of udder samples (n = 1083) were bacteriologically negative. The most effective antibiotics were cefoperazone and amoxicillin-clavulanic acid, while cloxacillin and tetracycline were the least effective antibiotics in both groups. In the clinoptilolite supplemented (CPL) group (n = 38) of 14 cows, nine causative agents of mastitis were isolated in 27 quarters, while in the control (CON) group (n = 40) of 24 cows, 13 causative agents of mastitis were isolated in 59 quarters. Cows from the CON group had a 1.96 times higher risk of intramammary infection than cows from the CPL group during the observation period (odds ratio = 1.96, p = 0.0031; 95% CI = 1.2570-3.0770).

A New Method of Assessing Sheep Red Blood Cell Types from Their Morphology.

Data concerning the morphometric parameters of sheep red blood cells (RBCs) obtained using computer-assisted image analysis have not yet been investigated, and there are no data on any analyses of ovine RBC subpopulations based on their morphometric parameters. The aims of this study are to determine the values of RBC haematological and morphometric size and shape parameters, to form groups according to the obtained values of haematological parameters; to determine the differences in RBC morphometric parameters between the formed groups, and to determine RBC subpopulations and their respective proportions in the formed groups. Thirty-six blood samples were collected from the jugular vein of clinically healthy Lika pramenka sheep, aged between 2 and 5 years. Haematological parameters including haemoglobin (HGB), haematocrit (HTC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and RBC distribution width were analysed using a haematology analyser. Haematological parameters were categorized into two groups: those with lower values or values below the physiological range (Groups 1) and groups with higher values or values above the physiological range (Groups 2). Morphometric parameters of RBCs were determined from stained blood smears using SFORM, a computer-assisted program. Significantly higher values of RBC area, outline, convex, minimal and maximal radius, as well as length and breadth were established in Groups 2 compared to Groups 1 of HGB, HCT, MCV, MCH, and MCHC, respectively. Based on the morphometric parameters of RBCs, three RBC subpopulations were obtained using principal component and cluster analysis: ES 1-the smallest and most elongated RBCs, ES 2-the biggest and most rounded RBCs, and ES 3-average size and shape RBCs. Significantly higher proportions of ES 2 and ES 3 subpopulations, as well as a significantly lower proportion of ES 1 subpopulation, were established in Groups 2 compared to Groups 1 of HGB, HTC, MCV, and MCH, respectively. It can be concluded that ovine RBC subpopulations, based on their morphometric parameters, can be obtained by using computer-assisted image analysis of RBC morphometry and multivariate statistical methods, including principal component and cluster analysis. RBC morphometry, including classification into subpopulations, could serve as a basis for future possibilities in the diagnostic interpretation of anaemic syndromes in veterinary medicine, especially in normocytic, macrocytic, and microcytic anaemias in sheep.

Effects of in-feed clinoptilolite treatment on serum metabolic and antioxidative biomarkers and acute phase response in dairy cows during pregnancy and early lactation.

The objective of this study was to evaluate the effects of in-feed clinoptilolite (CPL) on serum metabolic and antioxidative biomarkers, acute phase proteins and reproductive performance in cows during pregnancy and lactation. A total of 78 Holstein-Friesian cows were randomly assigned into two groups: the treatment group, cows fed CPL (n = 38) which received 50 g of powdered CPL twice a day from day 180 before parturition to day 60 postpartum; and the control group (n = 40). Blood samples were taken on days 180, 90, 60, 30 and 10 before parturition, on day of calving and on days 5, 12, 19, 26, 33, 40 and 60 postpartum, and were analysed for metabolic biomarkers: glucose, triglycerides, total cholesterol, high density lipoprotein cholesterol, non-esterified fatty acids, beta-hydroxybutyrate (BHB), antioxidative biomarkers and acute phase proteins: paraoxonase-1 (PON1), apolipoprotein A-I, haptoglobin (Hp) and serum amyloid A (SAA). CPL supplementation increased concentration of glucose and significantly decreased (P < .05) level of BHB during puerperium. The SAA concentration in CPL-fed cows was significantly decreased (P < .05) on days 33, 40 and 60 postpartum as well as Hp concentration on days 0 and 12 postpartum. The results of this study suggest that the CPL-fed cows may have improved metabolic status due to the tendency of greater glucose levels and decreased BHB values during early lactation. In addition, acute phase response was lower (P < .05) in CPL-fed cows. Such an outcome might be attributed to the effect of dietary CPL on intensity and severity of the negative energy balance and inflammatory response in dairy cows.

Influence of season, rainfall and air temperature on the reproductive efficiency in Romanov sheep in Croatia.

The aim of this study was to investigate the influence of season, rainfall and air temperature on the reproductive efficiency in the Romanov breed of sheep in continental part of Croatia during five consecutive years (2012-2016). During this period, 5379 matings resulted in 5046 successful conceptions, i.e. lambings at eight medium-scale Romanov breed sheep farms. The conception rate was 93.81%, fecundity was 195% and average preweaning mortality until 90 days of age was 12.41%. The seasonal distribution of lambings was 47.64% for ewes that delivered in winter (n = 2422), 23.37% in spring (n = 1179), 18.82% in summer (n = 950) and 9.81% in autumn (n = 495). Sexual activity was lowest during spring and early summer when air temperatures were above average (very and extremely warm), and sexual activity peaked from August to September, especially during extremely wet and very wet seasons. Litter size was greater during winter than in other seasons (1.70 vs. 1.54) and was significantly different as compared to each of selected years of the study period. There was a statistically significant difference in the number of pregnant ewes between mating seasons. Most female Romanov lambs born during winter and early spring mated in late summer or autumn and delivered at the age of 1 year or earlier. The seasonal distribution of matings and lambing was not uniform throughout the seasons over five consecutive years. Thus, it can be assumed that air temperature and rainfall during different seasons could affect the reproductive efficiency in Romanov breed of sheep in continental part of Croatia.

The effects of monthly air temperature and rainfall variations on the reproductive performance and lambing distribution of the Jezersko-Solcava sheep.

The aim of this study was to investigate the effects of monthly air temperature and rainfall variations on the reproductive performance and lambing distribution of the Jezersko-Solcava breed in northwestern Croatia during 7 years period from 2010 until 2016. At 11 medium-scale sheep farms, the sheep were kept on a pasture in a semi-intensive environment with access to the stables. The anomalies of rainfall observed on a monthly basis during monitoring were statistically significant. Non-parametric test showed that the percentages of lambing differ among the months during the year (P = 0.0001). By using regression analysis, it was found that the percentages of pregnant sheep were linearly associated with the sequential number of the month. The seasonal distribution of lambings were 53.85% of ewes delivered in winter (n = 1296), 21.13% in spring (n = 509), 8.91% in summer (n = 215) and 16.11% in autumn (n = 387). Sexual activity was lowest during spring and early summer (from March to May) with a peak from the end of July to October. The conception rate during year 2011 was significantly lower (89.74%), and the litter size was the lowest (1.05) than in other years of the observation. Average conception rate was 92.85% and average litter size 1.21. Despite being a seasonally polyoestrous, the distribution of matings and lambings of this breed of sheep was not uniform throughout the seasons. There were established a positive or negative correlations between the air temperature and rainfall precipitation variations with tested reproductive performances of Jezersko-Solcava breed sheep in the Northwestern part of Croatia.

Exogenous melatonin influences distribution of French Alpine buck spermatozoa in morphometrically distinct subpopulations during the non-breeding season.

The aim of this study was to establish subpopulations of spermatozoa in bucks using the principal component (PC) and cluster analysis according to morphometric head and tail variables, and to determine differences in proportions of subpopulations between exogenous melatonin-treated and control bucks. The bucks (n = 12) were assigned to two groups comprising six bucks each. By the end of March, four melatonin implants were inserted in the bucks in the experimental group. Semen was collected weekly using an artificial vagina from March to May (the non-breeding season). Analyses were performed in stained smears by SFORM computer-assisted program for eight head and five tail variables. The PC analysis revealed four components with the most important value for each (head outline, head ellipticity, mid-piece length and width). Cluster analysis indicated there were three subpopulations (average-sized spermatozoa- C_1; small and less-elliptic - C_2; big and elliptic - C_3). Melatonin-treated bucks had a greater proportion of C_1 spermatozoa and a lesser proportion of C_2 spermatozoa during May (P < 0.05). To the best of our knowledge, this is the first study where PC and cluster analyses were performed on buck semen with head and tail variables analyzed together, in the same analysis, to evaluate spermatozoa population. Also, this is the first analysis of morphometric variables for assessing the influence of melatonin on spermatozoa subpopulations. The positive effect of melatonin on the proportions of spermatozoa in subpopulations could have been a consequence of the decreasing the proportion of the subpopulation with the least head and tail sizes and ellipticity.

Risk Factors Associated with Black Tooth Stain

The aim of the study was to show whether there is any influence of food, drink or drug intake on the formation of tooth discoloration. A total of 500 patients aged 15-25 years were examined to take part in the study. Of these, 60 patients were selected and divided into two groups of 30 patients each. Group 1 included patients with black pigmentation on vestibular/oral tooth surfaces. Group 2 included patients without discoloration (control). Data were recorded in a questionnaire. Atomic absorption spectrometry was used to determine elements in discoloration samples. The Caries Risk Test (CRT) buffer was used to assess buffer capacity of saliva, while CRT bacteria were used to determine the presence of Streptococcus mutans and Lactobacillus spp. Statistically significant between-group differences were found for the intake of collard greens and beets (p<0.05), but not for other vegetables. As for drink consumption, patients with pigmentation reported less wine intake (p<0.05) than those without pigmentation. There was no difference according to drug intake between patients with and without pigmentation. Patients with pigmentation were older, smoked and had lower saliva pH with lower presence of Streptococcus mutans than those without pigmentation (p<0.05). In tooth discoloration samples, there were traces of calcium, magnesium, iron, copper and zinc. The appearance of tooth discoloration is influenced by many factors, among which diet and saliva seem to be very important. Our study showed that patients with black pigmentation used to take more beets, while patients without pigmentation were taking more collard greens and red wine.

Antioxidant protection and lipid peroxidation in testes and different parts of epididymis in boars.

The antioxidative ability of testes and epididymis to protect spermatozoa from detrimental effects of oxidation processes induced by excessive generation of reactive oxygen species has not been previously studied in detail in boar reproductive tissues. The aim of this study was to investigate differences in level of antioxidative protection, intensity of lipid peroxidation, and values of biochemical parameters in testes and different parts of epididymis in sexually mature boars. The study was performed on five Swedish landrace boars from the same litter aged 10 months kept under same ambient conditions. After slaughtering performed at the end of November; tissue samples of testes and the head, body and tail of epididymis were taken. The activity of glutathione peroxidase, glutathione reductase, superoxide dismutase, γ-glutamyltransferase, acid phosphatase, and lactate dehydrogenase, and concentrations of triacylglycerol, phospholipids, cholesterol, free fatty acids were determined in obtained supernatants from homogenized tissues spectrophotometrically; the concentration of malondialdehyde was determined by high-performance liquid chromatography. Significantly higher activities of glutathione peroxidase and glutathione reductase (P < 0.05) were found in testes compared with epididymis. In testes, a significantly higher activity of superoxide dismutase was found than in the head and tail of epididymis (P < 0.05). Malondialdehyde concentration in head of epididymis was significantly higher than in testes, or the body and tail of epididymis (P < 0.05). Simultaneously, malondialdehyde concentration in testes was significantly higher than in body and tail of epididymis (P < 0.05). In tail of epididymis, significantly higher activities of γ-glutamyltransferase, acid phosphatase, and lactate dehydrogenase were recorded than in testes. Significantly lower concentrations of triacylglycerol and free fatty acids were recorded in epididymis tail in comparison to epididymis head (P < 0.05). It could be concluded that high activities of antioxidative enzymes in testes of boars are essential for the appropriate protection of spermatozoa and cells of testes tissue against oxidative damages. The tissues of testes and epididymis head in boars were more susceptible to lipid peroxidation. Results of the present study indicated physiological importance of antioxidative enzymes in reproductive system in boars, and thus may serve for better understanding the mechanisms of male infertility.

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.

Differences in seminal plasma and spermatozoa antioxidative systems and seminal plasma lipid and protein levels among boar breeds and hybrid genetic traits.

The objectives of this study were to determine the influence of breed and hybrid genetic traits of boars on lipid and protein concentrations and antioxidative system variables in seminal plasma (SP) and spermatozoa and their correlations with semen quality variables. Semen samples from 27 boars: Swedish Landraces (SL), German Landraces (GL), Large Whites (LW), Pietrains (P) and Pig Improvement Company hybrids (PIC-hybrid), aged from 1.5 to 3 years old, were collected. SP was spectrophotometrically analyzed to determine total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triacylglycerol (TAG), total protein (TP), albumin, and zinc concentrations. The antioxidative system in SP and spermatozoa was established spectrophotometrically by determining total antioxidative status (TAS), total superoxide dismutase (TSOD) and glutathione peroxidase (GSH-Px) parameters, as well as copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activity in spermatozoa. The hybrid boars had higher (P<0.05) SP concentrations of: TC, LDL-C and TAG than P and GL; HDL-C than P, GL and SL; and TP than P and LW. PIC-hybrid had lower values (P<0.05) in spermatozoa of: TAS and CuZnSOD than SL; TSOD and GSH-Px than SL and P; and MnSOD than SL and LW. Differences in SP and spermatozoa antioxidative system variables and the significant differences in SP protein and lipid variables exist among boars of different breeds and hybrid. Novel data and observed differences in semen variables among boar breeds and hybrids and their correlations with semen quality parameters in this study could contribute to better assessment of boar semen quality.

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.