Comparative analysis of the detrimental in vitro effects of three fusariotoxins on the selected structural and functional characteristics of rabbit spermatozoa.

In this study, we evaluated the in vitro effects of 1-50 µM zearalenone (ZEA), deoxynivalenol (DON) and T-2 toxin (T-2) on rabbit spermatozoa for as much as 8 h of in vitro exposure. Our results indicate that all sperm quality parameters were negatively affected by these fusariotoxins in a time- and dose-dependent manner. The most prominent structure affected by ZEA was the plasma membrane, exhibiting alterations consistent with the onset of apoptosis and reactive oxygen species (ROS) overproduction. This correlated with the most prominent decline of the sperm motility among all selected fusariotoxins. Significant necrotic changes and mitochondrial dysfunction were primarily responsible for the sperm damage in the presence of T-2. Finally, exposure of spermatozoa to DON led to a significant decrease in the DNA integrity. This study may provide new information on the specific mechanisms of action involved in the in vitro toxic behavior of fusariotoxins on male gametes.

The effect of brown seaweed and polyphenol supplementation in male rabbits on the blood profile and antioxidant markers.

Currently, in animal nutrition, the replacement of synthetic substances with natural ones was expected to improve animal health. The aim of the present study was to evaluate the effects of a dietary brown seaweed and plant polyphenol mixture in adult male rabbits on the haematological profile and antioxidant markers. Twenty-four adult male rabbits were divided into three experimental groups receiving a control diet (C) or diets supplemented with 0.3% (T1) and 0.6% (T2) of a feed additive containing brown seaweed (Laminaria spp.) and plant extracts of seaweed origin. The trial lasted for 90 days. A lower potassium concentration was observed at 30 days in the T2 group, compared with the T1 and C groups. An increase in the antioxidant status was observed (P < 0.05) from day 60 of the trial in the rabbits fed diets with an algae-polyphenolic supplement (T1 and T2 groups). Concluding, the diet supplementation of brown seaweed and polyphenol stimulates the antioxidant status of the blood, however, it does not affect the haematological profile.

The effect of ZnO nanoparticles on rabbit spermatozoa motility and viability parameters in vitro.

Zinc plays a very important role in various biological activities of the body. Multifaceted role of zinc is also known in testes development, spermatogenesis, capacitation and has effect on spermatozoa motility. On the other hand, the growing industry of nanotechnology has created reasonable interest of the risk assessment for nanoparticles. The aim of this study was to evaluate in vitro effect of zinc oxide (ZnO) nanoparticles on rabbit spermatozoa. Fresh semen was collected from sexually mature New Zealand rabbits. Experimental groups were prepared by diluting semen with ZnO nanoparticles in seven different concentrations (6-391 mg/mL). The experimental groups were compared with control group. Semen was assessed using computer assisted semen analysis (CASA) at intervals of 0, 1, 2 and 3 h of incubation. The mitochondrial toxicity assay (MTT) assay was used to determine cell viability. The results of monitored motility parameters in experimental groups showed a decreasing trend during whole experiment. Significant decrease (P < 0.001) of motility and progressive motility was observed after 3 h of incubation in samples cultured with higher ZnO nanoparticles in comparison to the control group. After 3 h of incubation, viability of rabbit spermatozoa showed slightly increased values in group with the lowest concentration of ZnO nanoparticles, but in other groups viability showed non-significant decrease compared to control. Similar tendency was detected for spermatozoa membrane integrity. These original data show the negative dose-dependent effect of ZnO nanoparticles on spermatozoa motility and viability parameters.

Secretome Analysis of Rabbit and Human Mesenchymal Stem and Endothelial Progenitor Cells: A Comparative Study.

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) have been studied several years for their immunomodulatory effect through the paracrine mechanism and cytokine secretion. In combination with endothelial progenitor cells (EPCs), MSCs have great therapeutical potential for the repair of endothelium and wound healing. However, little is known about the cytokine profile of rabbit AT-MSCs or even EPCs. The aim of this study was to analyze the secretomes of these rabbit stem/progenitor cells. A large-scale human cytokine array (up to 80 cytokines) was used to identify and compare cytokines secreted into conditioned media of human and rabbit AT-MSCs as well as HUVECs and rabbit EPCs. Few cytokines were highly expressed by human AT-MSCs (TIMP-2, TIMP-1), HUVECs (MCP-1, TIMP-2, GRO, Angiogenin, IL-8, TIMP-1), or by rabbit EPCs (TIMP-2). Several cytokines have moderate expression by human (MCP-1, GRO, Angiogenin, TGF-ß 2, IL-8, LIF, IL-6, Osteopontin, Osteoprotegerin) and rabbit AT-MSCs (TIMP-2, TGF-ß 2, LIF, Osteopontin, IL-8, IL-5, IL-3) or by HUVECs (IL-6, MIF, TGF-ß 2, GCP-2, IGFBP-2, Osteoprotegerin, EGF, LIF, PDGF-BB, MCP-3, Osteopontin, Leptin, IL-5, ENA-78, TNF-ß) and rabbit EPCs (TGF-ß 2, Osteopontin, GRO, LIF, IL-8, IL-5, IL-3). In conclusion, the proposed method seems to be useful for the secretome analysis of rabbit stem/progenitor cells.

Effects of dietary plant polyphenols and seaweed extract mixture on male-rabbit semen: Quality traits and antioxidant markers.

Feed additive consisting of polysaccharides from brown seaweeds plus phenolic acid, hydroxycinnamic acids, tannins, and flavonoids from plant extracts, was used as rabbit food supplement. Main aim of the study was to determine the effect of natural mix of marine and freshwater seaweed polyphenols on selected reproductive characteristics of male rabbits during the 90-days experiment. Natural mix was incorporated in feed-pellets for rabbits in two different concentrations - 0.3% (T1 group) and 0.6% (T2 group), compared with a control group (C group). In experimental groups a significant increase of concentration of calcium during first 30 days of supplementation was found. An increase of alanine aminotransferase, glutathione peroxidase and ferric reducing ability of plasma and a decrease in aspartate aminotransferase after 90 days were recorded in the same groups. Except for that we noticed decrease of semen distance of curved line and velocity of curved line after 30 days though only while being supplemented with 0.6% proportion of seaweed polyphenols in feed mixture. Based on the results it can be stated that the natural mix in the tested levels do not show adverse effect on male rabbit reproductive parameters, and an improvement of antioxidant status was observed. The feed additives can have a very important effect on growth, health and development of animals in general as it supplies the with the much-needed minerals, nutritional substances and antioxidants, on which we focused in our study.

The effect of different sample collection methods on rabbit blood parameters.

Nowadays great deal of research is physiological field is conducted on experimental animals and there is a lot of criticism from the wide public on methods used. Therefore, recently there is a lot of effort focused on the welfare of the animals. Main aim of this study is to determine the effect of experimental sample collection method on the selected parameters of stress. In the experiment two sample collections of rabbit blood from marginal ear vein were realized - first using standard method with one person fixing the animal and other collecting the blood using gently fixating the animal. In the second groups experimental method of inserting the experimental animal into a sack and further collection in dark was realized. During the experiment the levels of cortisol - main stress indicator in organism and other health parameters of animals including mineral profile and haematological parameters were observed. Our results show no significant changes in levels of cortisol but also a decreasing tendency in the sample from the second (dark) collection. Haematological parameters were generally in the reference values and any significant changes except levels of lymphocytes and percent of lymphocytes which shown significant increase in the second collection period were found. Also the levels of mean corpuscular haemoglobin and percent of neutrophils unveiled a significant decrease in values. Values of mineral profile parameters have indicated no significant changes except the levels of phosphorus. Based on the result we can state that the experimental sample collection has no effect on blood parameters of the animals but we spectated a statistically insignificant decrease in the levels of cortisol which can suggest that the dark collection is possibly less stressful to the animals.

Corneal UV Protective Effects of a Topical Antioxidant Formulation: A Pilot Study on In Vivo Rabbits.

This study aimed to evaluate the protective effect of a topical antioxidant and ultraviolet (UV) shielding action formulation containing riboflavin and D-α-tocopherol polyethylene glycol succinate (TPGS) vitamin E against corneal UV-induced damage in vivo rabbit eyes. In vivo experiments were performed using male albino rabbits, which were divided into four groups. The control group (CG) did not receive any UV irradiation; the first group (IG) was irradiated with a UV-B-UV-A lamp for 30 min; the second (G30) and third (G60) groups received UV irradiation for 30 and 60 min, respectively, and were topically treated with one drop of the antioxidant and shielding formulation every 15 min, starting one hour before irradiation, until the end of UV exposure. The cornea of the IG group showed irregular thickening, detachment of residual fragments of the Descemet membrane, stromal fluid swelling with consequent collagen fiber disorganization and disruption, and inflammation. The cornea of the G30 group showed edema, a mild thickening of the Descemet membrane without fibrillar collagen disruption and focal discoloration, or inflammation. In the G60 group, the cornea showed a more severe thickening, a more abundant fluid accumulation underneath the Descemet membrane with focal detachment, and no signs of severe tissue alterations, as were recorded in the IG group. Our results demonstrate that topical application of eye drops containing riboflavin and TPGS vitamin E counteracts UV corneal injury in exposed rabbits.

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.

Antibiotics Versus Natural Biomolecules: The Case of In Vitro Induced Bacteriospermia by Enterococcus Faecalis in Rabbit Semen.

Male subfertility is a global issue in human reproduction as well as in animal reproduction. Bacterial infection and semen contamination are still widely overlooked. As the collection of ejaculates is not a sterile process, it is necessary to add antimicrobial agents to avoid a possible depreciation of semen samples. As traditionally used antibiotics have been questioned because of an ever-increasing bacterial resistance, natural bioactive molecules could offer an alternative because of their antibacterial and antioxidant properties. As such, we decided to compare the effects of selected natural biomolecules (resveratrol-RES, quercetin-QUE and curcumin-CUR) with routinely used antibiotics in animal biotechnologies (penicillin-PEN, gentamicin-GEN and kanamycin-KAN) on the rabbit sperm vitality in the presence of Enterococcus faecalis. Changes in the sperm structural integrity and functional activity were monitored at 0, 2, 4 and 6 h. Computer-assisted sperm analysis (CASA) was used for the assessment of spermatozoa motility. Production of reactive oxygen species (ROS) was evaluated using chemiluminiscence, while the mitochondrial membrane potential (ΔΨm) was examined using the JC-1 dye. Finally, the sperm chromatin dispersion (SCD) test was used to assess DNA fragmentation, and changes to the membrane integrity were evaluated with the help of annexin V/propidium iodide. The motility assessment revealed a significant sperm motility preservation following treatment with GEN (p < 0.001), followed by PEN and CUR (p < 0.01). QUE was the most capable substance to scavenge excessive ROS (p < 0.001) and to maintain ΔΨm (p < 0.01). The SCD assay revealed that the presence of bacteria and antibiotics significantly (p < 0.05) increased the DNA fragmentation. On the other hand, all bioactive compounds readily preserved the DNA integrity (p < 0.05). In contrast to the antibiotics, the natural biomolecules significantly maintained the sperm membrane integrity (p < 0.05). The microbiological analysis showed that GEN (p < 0.001), KAN (p < 0.001), PEN (p < 0.01) and CUR (p < 0.01) exhibited the strongest antibacterial activity against E. faecalis. In conclusion, all selected biomolecules provided protection to rabbit spermatozoa against deleterious changes to their structure and function as a result of Enterococcus faecalis contamination. Therefore, administration of RES, QUE and/or CUR to rabbit semen extenders in combination with a carefully selected antibacterial substance may be desirable.

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.

Benefits of combinative application of probiotic, enterocin M-producing strain Enterococcus faecium AL41 and Eleutherococcus senticosus in rabbits.

This study presents the effects of the probiotic and enterocin M-producing strain Enterococcus faecium AL41 on microbiota, phagocytic activity (PA), oxidative stress, performance and biochemical parameters when applied individually or in combination with Eleutherococcus senticosus in rabbits. The novelty of the study lies in the use of our non-rabbit-derived strain (AL41 = CCM8558) which produces new enterocin M. Ninety-six post-weaned rabbits (Hyplus breed) aged 5 weeks were divided into three experimental groups, 24 in each: E. senticosus (ES, 30 g/100 kg) in feed, E. faecium AL41 (10(9) CFU/mL marked by rifampicin to differentiate it from other enterococci) in water, and ES + AL. AL41 colonized sufficiently in rabbits to reduce coliforms, staphylococci, pseudomonads and clostridia. Slight decrease in bacteria was also found in the caecum and appendix. Phagocytic activity was significantly increased in the experimental groups compared to the control group (CG) (p < 0.001; p < 0.05). Applications did not evoke oxidative stress. Biochemical parameters in blood and caecal organic acids were slightly influenced. Average daily weight gain was slightly higher in ES and AL + ES. Combinative application of E. faecium with E. senticosus can be beneficial in rabbits. AL41 strain alone and in combination with ES produced reduction in spoilage bacteria; the highest stimulation of PA was in the AL41 + ES group.

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.

Enterococcus faecium AL 41: Its Enterocin M and Their Beneficial Use in Rabbits Husbandry.

Enterococci are ubiquitous microbiota constituting a large proportion of autochthonous microflora in animals. Some produce bacteriocins mostly enterocins; some of bacteriocin-producing strains also possess probiotic properties. Enterococcus faecium AL 41, Ent M-producing strain was tested for beneficial effect in rabbits. Five-week-old animals (72, Hycole) were divided into experimental groups (E1, E2) and control (C); 24 animals in each. Rabbits in E1 were administered AL 41 (500 µl per animal/day, 10(9) cfu/ml) in water for 21 days; rabbits in E2 were administered Ent M (50 µl/animal/day, activity 12,800 AU/ml) in water for 21 days. Rabbits in C fed a commercial diet. The experiment lasted 42 days. Sampling of faeces and blood was provided on day 0-1 and 21, 42; 3 animals per group were slaughtered. Caecum and appendix were separated. AL 41 colonized rabbits intestines <1.0 (log10) cfu/g, but stimulation of immunity was noted (P < 0.01; P < 0.001). Antimicrobial activity of both was noticed in faeces and/or caecum against pseudomonads. Significant decrease of coliform bacteria in faeces of E1 was noted on day 42 comparing with E2 (P < 0.05). On day 21, S. aureus cells were not detected in E1, E2. On day 42, S. aureus was not found in E2; in E1 their counts were <1.0 cfu/g, while in C it was in the count more than 1.0 cfu/g. In appendix, on day 21, significant decrease of not specified bacteria was found in E1, E2 comparing with C (P < 0.01). Administration of additives has not evoked oxidative stress. Biochemical parameters were not influenced. Higher average daily weight gains were detected by both, AL 41 and Ent M.

Elimination of apoptotic spermatozoa from rabbit insemination dose using annexin V associated with the MACS technique. A preliminary study.

The aim of this study was to verify whether the separation and elimination of the apoptotic fraction in rabbit semen using a MACS technique may improve sperm fertility potential and consequently rabbit kindling rate. Semen samples from 25 New Zealand White (NZW) rabbit males were collected using an artificial vagina and evaluated using the CASA system for concentration and motility. For artificial insemination the best 11 bucks were chosen based on motility parameters. Their ejaculates were mixed to make a heterospermic pool and routinely diluted in a commercial insemination diluent (MiniTüb, Tiefenbach, Germany) at a ratio of 1:6. Diluted heterospermic spermatozoa were filtered through a Sartorius filter to wash out seminal plasma, re-diluted in binding buffer (Annexin V Microbead Kit, Miltenyi Biotec, Germany) at a ratio of 1:3.66 and divided into two groups: an experimental group intended for MACS separation and control group without MACS separation. Then hormonally treated females of NZW rabbits were inseminated with fresh doses of filtered heterospermic semen (n = 27; 0.5 ml I.D. per female) and MACS separated semen (n=28; 0.5 ml I.D. per female). Separation and subsequent elimination of apoptotic spermatozoa (positive selection) from the insemination dose (after negative MACS selection) was verified under in vivo conditions on the basis of increased kindling rate in the experimental group in comparison with kindling rate in the control group (81.3% vs. 73.8%). In conclusion, elimination of apoptotic spermatozoa by the use of the MACS technique results in a slight improvement in kindling rate of rabbit does.

Influence of a 50 hz extra low frequency electromagnetic field on spermatozoa motility and fertilization rates in rabbits.

Effects of a 50 Hz extra-low frequency electromagnetic field (ELF EMF) on in vitro rabbit spermatozoa motility were analyzed, as well as the effect on fertilization rates after insemination. Pooled semen samples and a control were exposed to 50 Hz ELF EMF. The difference of the samples of the test groups G1 and G2 with the control group CG (75.56%) for spermatozoa motility were found to be significant (P < 0.01). Differences were significant (P < 0.01) for curvilinear velocity (VCL) between the test group G3 (122.38 microm/s) and the control group CG (112.02 microm/s). Hormonally stimulated adult (9-12 months) females (n = 140) were inseminated with semen samples from G1, G2, G3 and CG (0.88 x 109 spermatozoa/0.5 mL average insemination portion) immediately after ELF EMF exposure and fertilization (kindling) rates were calculated. For the G2 it was 54.28% data indicate 50 Hz ELF EMF induced alterations of spermatozoa motility and kindling rate in rabbits, therefore influencing fertility.