The Cryopreserved Sperm Traits of Various Ram Breeds: Towards Biodiversity Conservation.

The aim of our research was to compare three Slovak sheep breeds in the quality parameters of cryopreserved sperm. The ejaculates of Slovak Dairy (SD), Native Wallachian (NW), and Improved Wallachian (IW) sheep rams (n = 12) were collected by electro-ejaculation. Heterospermic samples were created from suitable ejaculates, separately for each breed (at least 90% of total and 80% of progressive motility). Samples were equilibrated in a Triladyl® diluent and frozen by automated freezing. Sperm samples were subjected to the motility, morphology, (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), fertilizing capability (penetration/fertilization test (P/F) in vitro) and acrosomal status (transmission electron microscopy) assays before freezing and after thawing. It was found that there were no significant differences (p < 0.05) between the evaluated breeds in motility, viability, apoptosis, morphological properties, and fertilizing ability of cryopreserved sperm. Significant differences occurred in acrosomal status. Our results demonstrate that the use of the selected cryopreservation protocol is suitable for at least three different sheep breeds, which can greatly benefit the biodiversity protection and simplifies the creation of an animal genetic resources gene bank.

The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role?

Bacterial contamination of semen is an often overlooked, yet important, factor contributing to decreased sperm vitality. Understanding the impact of bacterial presence on sperm structural integrity and functional activity may assist the development of effective strategies to prevent, or manage, bacteriospermia in the breeding practice. The aim of this study was to describe the bacterial profiles of ram semen (n = 35), and we also focused on the associations between bacteriospermia, sperm structure, and function, as well as oxidative and inflammatory characteristics of semen. For a better insight, the samples were divided into three groups, according to the breeds used in the study: native Wallachian (NW), improved Wallachian (IW), and Slovak dairy (SD) breeds. The results showed a significantly lower motility and membrane integrity in the NW group in comparison to the IW and SD groups, which was accompanied by a significantly higher concentration of leukocytes, increased reactive oxygen species (ROS) generation, and subsequent oxidative insults to the sperm lipids and proteins. Accordingly, the NW group presented with the highest bacterial load, in which Staphylococcus and Escherichia were the predominant representatives. The Pearson correlation analysis uncovered positive relationships amongst the bacterial load and leukocytospermia (r = 0.613), the extent of lipid peroxidation (r = 0.598), protein oxidation (r = 0.514), and DNA fragmentation (r = 0.638). Furthermore, positive correlations were found between the bacterial load and pro-inflammatory molecules, such as the C-reactive protein (r = 0.592), interleukin 1 (r = 0.709), and interleukin 6 (r = 0.474), indicating a possible involvement of the immune response in the process of bacteriospermia. Overall, our data indicate that ram semen quality may be equally affected by the bacterial load and diversity. Furthermore, we can assume that the presence of bacteria in ejaculates triggers inflammatory processes, causes ROS overproduction, and, thereby, contributes to alterations in the sperm structure, while at the same time compromising the fertilization ability of male gametes.

Ram semen quality can be assessed by flow cytometry several hours after post-fixation.

Ram spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.

Impact of Ergothioneine, Hercynine, and Histidine on Oxidative Degradation of Hyaluronan and Wound Healing.

A high-molecular weight hyaluronan is oxidatively degraded by Cu(II) ions and ascorbate-the so called Weissberger biogenic oxidative system-which is one of the most potent generators of reactive oxygen species, namely •OH radicals. Ergothioneine, hercynine, or histidine were loaded into chitosan/hyaluronan composite membranes to examine their effect on skin wound healing in ischemic rabbits. We also explored the ability of ergothioneine, hercynine, or histidine to inhibit hyaluronan degradation. Rotational viscometry showed that ergothioneine decreased the degree of hyaluronan radical degradation in a dose-dependent manner. While histidine was shown to be potent in scavenging •OH radicals, however, hercynine was ineffective. In vivo results showed that the addition of each investigated agent to chitosan/hyaluronan membranes contributed to a more potent treatment of ischemic skin wounds in rabbits compared to untreated animals and animals treated only with chitosan/hyaluronan membranes.

Low dose exposure of patulin and protective effect of epicatechin on blood cells in vitro.

In the present study, we aimed to assess antioxidant status in erythrocytes in vitro after patulin (PAT) and epicatechin exposure by measuring antioxidant enzymes (superoxide-dismutase - SOD, glutathione peroxidase - GPx and catalase - CAT) and parameters associated with oxidative stress (malondialdehyde - MDA and ROS). We also investigated the effect of PAT on viability and count of lymphocytes and lymphocyte subpopulations in rabbit blood in vitro. Whole blood of rabbits was used for analysis of antioxidant changes in rabbit erythrocytes after epicatechin and PAT treatment (separately or in combination, at concentrations of 0.2; 2; 20; 200 µg mL-1 of epicatechin and 0.5; 5; 10 µg mL-1 of PAT). Whole blood of rabbits was also used for analysis of count and viability of lymphocytes after PAT treatment at concentrations of 10; 25 and 50 µg mL-1. Results from our experiment confirmed the ability of epicatechin to protect cells against oxidative stress and lipoperoxidation. Our findings indicate that mycotoxin PAT in low concentrations did not affect the activity of antioxidant enzymes in erythrocytes of rabbits significantly. Only slight non-significant changes in lymphocytes count after treatment with low doses of PAT in rabbit blood were observed.

The occurrence and dynamics of polychlorinated hydrocarbons in roe deer (Capreolus capreolus) in South-western Slovakia.

The goal of this study was to determined polychlorinated biphenyls (PCBs) and organochlorine pesticides in the depot fat of roe deer (Capreolus capreolus) coming from south-western Slovakia. The mutual correlations of the organic pollutants were analyzed. The study included dichlorodiphenyltrichloroethane (DDT), hexachlorobenzen (HCB), alpha-hexachlorocyclohexane and beta-hexachlorocyclohexane (α + ß-HCH), gamma-hexachlorocyclohexane (γ-HCH), and polychlorinated biphenyls (PCB-delor). The gas chromatograph with an electron capture detector ECD was used for analysis. The accumulations of organic pollutant in depot fat of roe deer were in following order: DDT > PCB-delor > α + ß-HCH > HCB > γ-HCH. Among all pollutants, DDT was accumulated significantly in the highest level in the samples. The significantly higher content of DDT, HCB, α + ß-HCH, and γ-HCH was detected in the adult animals when compared to the juveniles. Some strong positive correlations among pollutants, between HCB and DDT, α + ß-HCH and HCB, α + ß-HCH and HCB, between γ-HCH and other pollutants, and between PCB-delor and γ-HCH were found. Game animals are a part of human food chain and monitoring of the environment pollution by PCBs and other organic pollutants are worthy to study.

Potential toxicity of cyanogenic glycoside amygdalin and bitter apricot seed in rabbits-Health status evaluation.

Amygdalin is one of the most studied secondary metabolites of Prunus genus. It is a cyanogenic glycoside which was initially obtained from the bitter almonds seeds and is a major component of the seeds of plants, such as apricots, almonds, peaches, apples and other rosaceous plants. The views of scientists on the use of amygdalin have been contradictory for many years, partly because toxicokinetics and metabolism of amygdalin still have not been adequately explored. The present in vivo study was designed to reveal whether pure amygdalin intramuscularly injected or apricot seeds oral consumption induce changes in overall health status of rabbit as a biological model. A total of 60 adult rabbits were randomly divided into five groups. The control group received no amygdalin while the two experimental groups E1 and E2 received a daily intramuscular injection of amygdalin at doses 0.6 and 3.0 mg/kg bw. The experimental groups E3 and E4 were fed crushed bitter apricot seeds (Prunus armeniaca L.), at doses 60 and 300 mg/kg bw, mixed with commercial feed for rabbits. Blood collection was carried out after 14 days. Biochemical, haematological and antioxidant enzymes activity analysis were performed and statistically evaluated. A short-term amygdalin administration had negligible impact on biochemical parameters-mainly level of urea, bilirubin, cholesterol. Haematological profile of rabbits was influenced very slightly-non-significant platelet count and platelet percentage increase, erythrocytes count and haemoglobin decrease. SOD activity of rabbits decreased significantly (p > 0.05) after apricot seeds consumption (102.3 U/ml) in comparison to control (117.4 U/ml). Differences might be connected to diverse metabolism by different administration routes and at the same time by the presence of other substances in apricot seeds (phytosterols, polyphenols, fatty acids). However, a short-term consumption had only slight effect on health status of rabbits and at recommended doses did not represent risk for their health.

Assessment of rabbit spermatozoa characteristics after amygdalin and apricot seeds exposure in vivo.

This study evaluates rabbit spermatozoa motility parameters after in vivo administration of amygdalin and apricot seeds during a 28-day period. Apricot seeds are potentially useful in human nutrition and amygdalin is the major cyanogenic glycoside present therein. The rabbits were randomly divided into the five groups (Ctrl-Control, P1, P2, P3, P4) with 4 males in each group. Control group received no amygdalin/apricot seeds while the experimental groups P1 and P2 received a daily intramuscular injection of amygdalin at a dose 0.6 and 3.0 mg/kg b.w. respectively during 28 days. P3 and P4 received a daily dose 60 and 300 mg/kg b.w. of crushed apricot seeds mixed with feed during 28 days, respectively. CASA system was used to evaluate for motility, progressive motility, curvilinear velocity, amplitude of lateral head displacement and beat cross frequency. Intramuscular application of amygdalin resulted in a significant time- and dose-dependent decrease of spermatozoa motility as well as progressive motility. On the other hand, oral consumption of apricot seeds had no significant effect either on the rabbit spermatozoa motility or progressive motility over the entire course of the study. The analysis of the other motion characteristics revealed a similar trend depicting a continuous, time- and dose-dependent decrease of all parameters following intramuscular AMG administration, with significant differences particularly for the dose 3.0 mg AMG/kg b.w. On the other hand, oral administration of apricot seeds had no significant impact on spermatozoa motility parameters. The present study suggests that short-term intramuscular application of amygdalin decreased rabbit spermatozoa motility in vivo. Whereas, consumption of apricot seeds did not induce any change in rabbit spermatozoa in vivo. Our findings suggest dose-dependent negative effect of pure amygdalin, but not apricot seeds on the rabbit spermatozoa parameters.

MitoQ Loaded Chitosan-Hyaluronan Composite Membranes for Wound Healing.

Two self-associating biopolymers, namely chitosan (Ch) and a high-molar-mass hyaluronan (HA), were used to prepare membranes with the aim to protect and to enhance the healing of injured skin. A mitochondrially-targeted antioxidant-MitoQ-was incorporated into the mixture of biopolymers prior to their self-association. These three-component membranes were evaluated in detail utilising surface roughness measurements, contact angle measurements, hemocompatibility, and thrombogenicity analyses. Furthermore, in vivo application of Ch/HA/MitoQ membranes was assessed on injured rabbit and rat skin utilizing histological methods. The results showed that the prepared thrombogenic Ch/HA/MitoQ membranes had higher roughness, which allowed for greater surface area for tissue membrane interaction during the healing processes, and lower cytotoxicity levels than controls. MitoQ-loaded composite membranes displayed superior healing properties in these animal models compared to control membranes.

Seasonal, age and sex fluctuations in aflatoxin B1 content in the liver and kidney of brown hares (Lepus europaeus Pall).

The goal of this study was to monitor the accumulation of aflatoxin B1 in the liver and kidney of brown hares (Lepus europaeus Pall) in the region of south-western Slovakia. A total of 65 samples were involved for analysis by RIA method. Brown hares were divided into the groups according to age, sex and season (month). The sex was determined visually after shooting, and the age was assigned from dried eye lens. The average concentration of AFB1 in the liver of hares was 0.54 ± 0.053 µg/kg, and lower values were measured in the kidney (0.41 ± 0.038 µg/kg). The significantly (P < 0.05) higher values were found in winter months when compared to summer months. According to the age, juvenile animals showed significant higher accumulation of B1 in both organs than adults (P < 0.05). Wild animals can serve as a good model of real environmental contamination. Thus, monitoring of risk factors such as mycotoxins in the environment is important with regard to public health, as game animals constitute an important part of food chain for humans.

The occurrence and dynamics of polychlorinated hydrocarbons in brown hare (Lepus europaeus) in south-western Slovakia.

This study aimed at obtaining the data on the occurrence, levels and correlations of organic pollutants present in game animals (n = 75, Brown hare, Lepus europaeus Pall.) in the region of south-western Slovakia. The analyses performed included dichlorodiphenyltrichloroethane (DDT), hexachlorobenzen (HCB), alpha- and beta hexachlorocyclohexane (α+ß-HCH), gamma-hexachlorocyclohexane (γ-HCH), and polychlorinated biphenyls (PCB-delor, commercial mixture of PCB congeners). A gas chromatograph with an electron capture detector was used for the analysis. PCB-delor and DDT were accumulated significantly in the highest level (0.105 ± 0.059 mg/kg; 0.070 mg/kg) in depot fat in brown hares; however maximum permissible limits for the observed pollutants were not exceeded. Significantly higher concentrations of DDT, HCB, γ-HCH, and PCB-delor were found in adult animals when compared with juvenile hares. Gender and season had no effect on the accumulation of observed pollutants. Moderately positive correlation was found between PCB-delor and DDT (r = 0.59). Monitoring of environmental pollution with polychlorinated hydrocarbons is important with regard to public health, as game animals constitute an important part of food chain also for humans.

Environmental levels of cadmium, lead and mercury in brown hares and their relation to blood metabolic parameters.

The purpose of this study was to examine concentrations of selected heavy metals in the liver and kidney of brown hares (Lepus europaeus). In addition, correlations between heavy metals and biochemical parameters in blood plasma were determined. The average concentrations of heavy metals (mmol/L) +/- SD were as follows: liver: Pb 0.221 +/- 0.189, Cd 0.160 +/- 0.140, Hg 0.021 +/- 0.030, kidney: Pb 0.115 +/- 0.125, Cd 1.570 +/- 1.103, Hg 0.030 +/- 0.053. The average concentrations of biochemical parameters in the blood plasma were as follows: Ca 3.16 mmol/L, P 2.19 mmol/L, Mg 1.40 mmol/L, Na 148.71 mmol/L, K 8.12 mmol/L, glucose 6.56 mmol/L, total proteins 56.49 g/L, urea 5.00 mmol/L, total lipids 1.40 g/L, bilirubin 3.97 micro mol/L, cholesterol 1.53 mmol/L, aspartate aminotransferase (AST) 6.06 micro kat/L and alanine aminotransferase (ALT) 1.94 micro kat/L. Average levels of hormones (ng/mL) were as follows: testosterone 2.94, androstendiol 0.13, estradiol 501.59, progesterone 6.63, oxytocin 328.60. Tissue analysis showed an accumulation of lead, cadmium and mercury in the liver and kidney of brown hares. There were no significant correlations between levels of heavy metals in liver, kidney, and biochemical parameters.

New variants of European brown hare syndrome virus strains in free-ranging European brown hares (Lepus europaeus) from Slovakia.

Investigations regarding European brown hare syndrome virus (EBHSV) in European brown hares (Lepus europaeus) in Slovakia were undertaken in order to detect the possible presence of EBHSV and to evaluate its phylogenetic position. Liver and/or serum samples were obtained from 135 European brown hares shot by hunters in eight regional hunting areas. From 36 animals corresponding liver and serum samples were available; from the remaining 49 and 50 animals only liver or serum samples were examined, respectively. Samples were tested for antibodies against EBHSV and for viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and RT-PCR products were subsequently sequenced. Additionally, matrilinear hare haplotypes were analyzed in order to detect potential familial susceptibility to EBHSV. Sixty-three of 86 sera contained antibodies against EBHSV, whereas 15 of 85 liver samples were PCR positive. Of the latter, 14 were sequenced, revealing three new strains of EBHSV. Fifteen different matrilinear haplotypes were identified, but no correlation was found between haplotype and susceptibility to EBHSV infection. Our findings confirmed the existence of EBHSV in Slovakia and reinforce the need for determining EBHSV status when importing hares for restocking.

The effect of hFVIII transgene on the chromosomal aneuploidy rate in rabbits.

The aim of this study was to compare the chromosomal aneuploidy rate between transgenic and non-transgenic rabbits derived from the F4 generation. Chromosomal analysis was carried out on bone marrow samples of New Zealand White transgenic (carrying human factor VIII gene) and non-transgenic rabbits (F4 generation) each having a different genetic background (female no. 1-3-5 line I and female no. 1-9-7 line II). C-metaphase plates were obtained from the bone marrow lymphocytes synchronized by the addition of 0.25 microg/ml colcemide. No significant difference in chromosomal aneuploidy between transgenic (61%) and non-transgenic (51.27%) rabbits of line I was observed. A higher but non-significant aneuploidy rate between transgenic and non-transgenic rabbits was found in line II, on the other hand a significant difference (P<0.05) was observed in diploidy rate. In conclusion, chromosomal aneuploidy rates in this experiment were higher than published previously in other reports.

Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos.

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

Effect of ghrelin on activities of some lysosomal hydrolases in rabbits.

OBJECTIVES: Observing the changes of activity of some lysosomal enzymes in blood serum of female rabbits subjected to injection of 10 microg of ghrelin/kg of body weight. METHODS: In the blood serum the activity of cathepsins D and L, alanine aminopeptidase, acid phosphatase, lysosomal lipase and lysosomal esterase was determined. RESULTS: As a result of ghrelin injection the activity of all the enzymes examined in blood serum increased markedly. CONCLUSION: Changes of lysosomal enzymes activities in the blood serum caused by the effects of ghrelin should be regarded as the response of the lysosomal system.

Distribution of cadmium and lead in liver and kidney of some wild animals in Slovakia.

The content of cadmium and lead, as risk factors of environment, in liver and kidneys of wild animals as brown hare (Lepus europaeus), yellow-necked mouse (Apodemus flavicollis), wood mouse (Cleithrionomys glareolus), and red deer (Cervus elaphus) were studied. Samples were analyzed by the atomic absorption spectrophotometry method (AAS). The highest levels of cadmium were found in kidneys (0.213-2.387 mg/kg) of all animal species. The concentration of cadmium in liver was 0.032-0.258 mg/kg. The analysis of lead showed that the concentration of this element was higher in kidneys of yellow-necked mouse and wood mouse (0.503-0.780 mg/kg) than in liver (0.177-0.268 mg/kg). In brown hare and red deer a higher accumulation of lead in liver (0.221-1.904 mg/kg) in comparison with kidneys (0.115-0.561 mg/kg) is reported.

Concentration of selected metals in liver, kidney, and muscle of the red deer (Cervus elaphus).

Concentration of cadmium, lead, chromium, zinc, copper, and manganese in liver, kidney, and muscle of red deer was investigated. For analysis of the content of these trace elements an AAS method was used. The concentration of cadmium was significantly (p < 0.05) higher in kidney in comparison with liver and muscle. The level of lead was the highest in muscle (p < 0.05). Lower values were detected in liver and in kidney. The concentration of chromium was very similar in all studied tissues. A higher concentration of zinc was found in muscle, followed by kidney and liver. The level of copper was significantly higher in liver (p < 0.05), and lower in kidneys and muscle. In evaluation of the concentration of manganese in red deer, the highest concentration of this element in liver was detected.