Animal Production Research Center Nitra

Cryopreservation plays a critical role in animal breeding and the conservation of endangered species, but it often compromises sperm characteristics such as morphology, motility, and viability due to oxidative stress. This study explores the antioxidative effect of Mito-Tempo (MT) and Berberine (BER) to enhance post-thaw sperm quality in rabbits. Pooled rabbit sperm samples were supplemented with different concentrations (0.0, 0.5, 5, 10, 50 µmol/L) of MT and BER. Sperm motility was evaluated using computer-assisted semen analysis, while viability, apoptosis, reactive oxygen species (ROS) levels, acrosome integrity, and mitochondrial function were assessed through flow cytometry. The results revealed that MT at 5 and 10 µmol/L and BER at 10 µmol/L significantly improved total and progressive motility, mitochondrial activity, and sperm viability compared to the control group. Furthermore, 10 µmol/L BER enhanced acrosome integrity, while both 5 µmol/L MT and 10 µmol/L BER effectively reduced ROS levels and apoptosis. This study is the first to demonstrate the protective effects of MT and BER on rabbit sperm during cryopreservation. By mitigating oxidative stress and reducing apoptosis, these antioxidants markedly improved post-thaw sperm quality, positioning MT and BER as promising agents for improving sperm cryosurvival.

In the present study, we examined the functional interrelationships between microRNAs and plant polyphenols in the regulation of ovarian cell functions. For this purpose, we compared the basic functions of porcine ovarian granulosa cells with or without transfection with miR-152 mimics that were cultured with or without quercetin. The expression levels of miR-152, cell viability, cell proliferation (accumulation of proliferating cell nuclear antigen, PCNA), apoptosis (accumulation of Bax) and the release of progesterone, estradiol, and insulin-like growth factor I (IGF-I) were analyzed by real-time quantitative polymerase chain reaction (RT‒qPCR), the Trypan blue exclusion test, quantitative immunocytochemistry, and enzyme-linked immunosorbent assays (ELISAs). Transfection of cells with miR-152 mimics increased miR-152 expression, reduced cell viability, proliferation, apoptosis, and estradiol output, and promoted the release of progesterone and IGF-I. Quercetin decreased all measured parameters. Moreover, quercetin promoted the effect of miR-152 on cell viability, apoptosis, and estradiol and mitigated the effect of miR-152 on cell proliferation and IGF-I output. For instance, miR-152 mimics promoted the effect of quercetin on cell viability, apoptosis, and estradiol but prevented the effect of quercetin on PCNA. These observations demonstrated the involvement of miR-152 and quercetin in the control of ovarian cell functions and their functional interrelationships, mainly synergism, in the regulation of these functions.

Incorporating of agro-industrial co-products into animal nutrition could represent an opportunity to lessen the environmental impact of the food production chain. One such co-product is a hempseed cake originating from cold pressing hemp seeds to extract oil for human consumption. The aim of the present study was to evaluate the action of hempseed cake in the diet on male rabbit reproductive and some non-reproductive indexes. Male rabbits were fed either a standard diet (control group; C; n = 10) or a diet enriched with hempseed cake (experimental group E5 with 5% of a hempseed cake; n = 10, and experimental group E10 with 10% of a hempseed cake; n = 10) in 100 kg of the milled complete feed mixture. Rabbit weight gain, sperm concentration, motility, progressive motility, and sperm quality were evaluated using CASA and flow cytometry. Feeding with a hempseed cake, given at both tested concentrations, had no effect on weight gain per week and the total average weight gain compared to the control group (p > 0.05). Hempseed cake addition had no effect on sperm concentration in ejaculate, sperm motility, and progressive motility (p > 0.05). Selected haematological and biochemical indexes were examined. The E5 group showed positive tendencies in hepatic profile parameters, while in the E10 group the tendencies were opposite, though within the reference values. Based on our results, no negative effects of hempseed cake feeding on rabbit reproduction and health status were found, and we can recommend the use of hempseed cake at doses up to 10% in the nutrition and feeding of rabbits. Therefore, agro-industrial co-products can decrease the feeding cost.

Honeybee venom is one of the natural substances produced by bees (Apis mellifera). Their venom gland produces venom which plays a defensive role. In this study a concentration of macro and trace elements (Ag, Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, Sb, Se, Sr, Pb and Zn) in foragers′ and honeybees′ venom was analysed by axial inductively coupled plasma optical emission spectrometry (ICP OES) with good validation parameters to differentiate the element accumulation ability in honeybee venom. Cumulative ability for some elements (As, Al, Ba, Cr, Li, Mo, Pb, and Zn) in bee venom was clearly demonstrated. Oppositely, levels of macro elements (Ca, K, Mg and Na) in venom were several times lower compared to the levels detected in foragers. Moreover, PCA analysis of bee samples showed that Cr was associated with locality Košice, and Cd with locality Krompachy; both have rich industrial history. Since some of analysed elements are potentially toxic for humans, a risk assessment for bee-stung scenario was also calculated. A new way of exposure to potentially toxic elements via honeybee stung was showed in this study. Non-carcinogenic risk assessment for humans to selected toxic elements (As, Cd, Cr, Ni, and Pb) demonstrated acceptable risk and moreover the same we may conclude for potential carcinogenic risk for beekeepers exposed to As, Cd, Ni, and Pb via venom over their whole life.

Ensuring global food security is pressing among challenges like population growth, climate change, soil degradation, and diminishing resources. Meeting the rising food demand while reducing agriculture's environmental impact requires innovative solutions. Nanotechnology, with its potential to revolutionize agriculture, offers novel approaches to these challenges. However, potential risks and regulatory aspects of nanoparticle (NP) utilization in agriculture must be considered to maximize their benefits for human health and the environment. Understanding NP-plant cell interactions is crucial for assessing risks of NP exposure and developing strategies to control NP uptake by treated plants. Insights into NP uptake mechanisms, distribution patterns, subcellular accumulation, and induced alterations in cellular architecture can be effectively drawn using transmission electron microscopy (TEM). TEM allows direct visualization of NPs within plant tissues/cells and their influence on organelles and subcellular structures at high resolution. Moreover, integrating TEM with stereological principles, which has not been previously utilized in NP-plant cell interaction assessments, provides a novel and quantitative framework to assess these interactions. Design-based stereology enhances TEM capability by enabling precise and unbiased quantification of three-dimensional structures from two-dimensional images. This combined approach offers comprehensive data on NP distribution, accumulation, and effects on cellular morphology, providing deeper insights into NP impact on plant physiology and health. This report highlights the efficient use of TEM, enhanced by stereology, in investigating diverse NP-plant tissue/cell interactions. This methodology facilitates detailed visualization of NPs and offers robust quantitative analysis, advancing our understanding of NP behavior in plant systems and their potential implications for agricultural sustainability.

Background Apricot kernels containing amygdalin (AMG) as the major cyanogenic glycoside are potentially useful as a complementary therapy for the management of several ailments including cancer. Nevertheless, little is known regarding the toxic and therapeutic doses of AMG, particularly in terms of male reproduction. Hence, this study evaluates selected qualitative characteristics of rabbit testicular tissue following in vivo administration of AMG or apricot kernels for 28 days. Methods The rabbits were randomly divided into five groups (Control, P1, P2, P3, P4). The Control received no AMG/apricot kernels while the experimental groups P1 and P2 received a daily intramuscular injection of amygdalin at a dose of 0.6 and 3.0 mg/kg of body weight (b.w.) for 28 days, respectively. P3 and P4 received a daily dose of 60 and 300 mg/kg b.w. of crushed apricot kernels mixed with feed for 28 days, respectively. Changes to the testicular structure were quantified morphometrically, while tissue lysates were subjected to the evaluation of reactive oxygen species (ROS) production, total antioxidant capacity, activities of antioxidant enzymes, and glutathione concentration. The extent of damage to the proteins and lipids was quantified as well. Levels of selected cytokines were determined by the enzyme-linked immunosorbent assay while a luminometric approach was used to assess the activity of caspases. Results Rabbits treated with 3.0 mg/kg b.w. AMG presented a significantly increased protein oxidation (p = 0.0118) accompanied by a depletion of superoxide dismutase (p = 0.0464), catalase (p = 0.0317), and glutathione peroxidase (p = 0.0002). Significantly increased levels of interleukin-1 beta (p = 0.0012), tumor necrosis factors alpha (p = 0.0159), caspase-3/7 (p = 0.0014), and caspase-9 (p = 0.0243) were also recorded in the experimental group P2 when compared to the Control. No effects were observed in the rabbits treated with apricot kernels at the oxidative, inflammatory, and histopathological levels. Conclusions Apricot kernels did not induce toxicity in the testicular tissues of male rabbits, unlike pure AMG, which had a negative effect on male reproductive structures carried out through oxidative, inflammatory, and pro-apoptotic mechanisms.

Bees are often exposed to pesticide residues during their foraging trips in agricultural landscapes. The analysis of in-hive stored pollen reflects the spectrum of visited plants and can be almost used to link the exposure to pesticides.In the current study, bee bread samples were collected in May and July from 17 sites located in southern Slovakia. Samples were analysed using a multi-residue pesticide analysis method for a broad spectrum of active substances and microscopic for pollen identification.Our results revealed a bee bread contamination with 19 different active substances, with fungicides being predominant. Sixteen of them are authorized in the EU, but chlorpyrifos, chlorpyrifos-methyl, and chloridazon are not. The highest concentrations for pendimethalin (1400 µg/kg), fluazifop-butyl (640 µg/kg), fenpropidin (520 µg/kg), fluopyram (130 µg/kg), and difenoconazole (95 µg/kg) were detected. The total residue load in bee bread sampled in the early season (May) was significantly higher than in the late season (July). The mean residue load of insecticides analysed in July comprised 46% of May’s load, which is alarming due to the importance of bee bread in the diet for winter-rearing bees. Moreover, results from both sampling periods showed that fungicides were positively associated with plant families Apiaceae and Papaveraceae and herbicides with Aceraceae, Salicaceae, and Brassica-type/Brassicaceae.Hence, bee bread can be considered a suitable matrix and a good bio-indicator reflecting honey bee exposure to pesticides over the season.

Colony losses are frequently reported during winter and rarely during summer in Europe, the Mediterranean Sea region, and North America. The impact of air temperature on honey bee colony activities or bee pathogens suggests a potential indirect role of temperature in colony losses. Honey bees are infected by numerous viral, bacterial, and fungal diseases. However, the effects of temperature on certain bee pathogens have not been studied sufficiently. This review aims to explore the relationship between temperature and the prevalence of crucial honey bee diseases, as well as the potential role of temperature in accelerating colony losses caused by those pathogens. Herein, we only focus on the key pathogens that widely infect honey bees. The seasonal prevalence of certain pathogens has been observed, with some being more prevalent during active seasons for honey bees, while others are more prevalent during winter. Furthermore, an interactive role of temperature in the prevalence of bee pathogens and their contribution to colony losses has been suggested and discussed. Solutions to directly or indirectly reduce colony losses by controlling temperature are proposed, such as modifying hive structures and employing specialized devices and techniques to manipulate colony temperature.

Epigenetic methods to prevent the reproductive toxicity of oil-related environmental contaminants are currently unavailable. The present study aimed to examine the ability of the microRNA miR-152 to mitigate the effects of benzene on ovarian cells. Porcine ovarian granulosa cells transfected or not transfected with miR-152 mimics were cultured with or without benzene (0, 10 and 100 ng/ml). The expression of miR-152; viability; proliferation (cell proliferation and expression of mRNAs and accumulation of PCNA and cyclin B1); apoptosis (expression of mRNAs and accumulation of bax and caspase 3; and the proportion of cells with fragmented DNA); and release of progesterone, estradiol and IGF-I were analyzed via RT–qPCR; the Trypan blue exclusion test; quantitative immunocytochemistry; BrdU; XTT; TUNEL assays; and ELISA. Administration of benzene promoted the expression of apoptosis markers and reduced cell viability, all measured markers of proliferation, the release of steroid hormones and IGF-I. Overexpression of miR-152 was associated with increased cell viability, proliferation, progesterone and IGF-I release and reduced apoptosis and estradiol output. Moreover, miR-152 mitigated or prevented the effects of benzene on all the measured pa- rameters in addition to estradiol release. The present observations suggest the toxic effect of benzene and the stimulatory influence of miR-152 on ovarian cell functions. Moreover, this is the first demonstration of the ability of miRNAs to mitigate and prevent the reproductive toxicity of benzene.

Serological tests for the presence of antibodies to European brown hare syndrome virus and rabbit hemorrhagic disease virus type a and type 2 were performed on 275 samples of blood serum from the European brown hare by hemagglutination inhibition test and enzyme-linked immunosorbent assay. The presence of antibodies against European brown hare syndrome virus was 28% in the Czech Republic while in the Slovak Republic between 9-33%. Furthermore, the results showed the possibility of interspecies transmission, both rabbit hemorrhagic disease virus type a and rabbit hemorrhagic disease virus type 2 on the European brown hare. In case of RHDVa it is the first documented interspecies transmission, which has not yet been described. This study improved our knowledge about circulation of RHDV in Central European ecosystems and its possible ability to cross interspecies barriers. However, from an epizootiological point of view, it is likely European brown hare is not a significant source of RHD infection for domesticated rabbits.

Various antioxidants are tested to improve the viability and development of cryopreserved oocytes, due to their known positive health effects. The aim of this study was to find whether astaxanthin (AX), a xanthophyll carotenoid, could mitigate deteriorations that occurred during the vitrification/warming process in bovine oocytes. Astaxanthin (2.5 µM) was added to the maturation medium during the post-warm recovery period of vitrified oocytes for 3 h. Afterward, the oocytes were fertilized in vitro using frozen bull semen and presumptive zygotes were cultured in the B2 Menezo medium in a co-culture with BRL-1 cells at 38.5 °C and 5% CO2 until the blastocyst stage. AX addition significantly reduced ROS formation, lipid peroxidation, and lysosomal activity, while increasing mitochondrial activity in vitrified oocytes. Although the effect of AX on embryo development was not observed, it stimulated cell proliferation in the blastocysts derived from vitrified oocytes and improved their quality by upregulation or downregulation of some genes related to apoptosis (BCL2, CAS9), oxidative stress (GPX4, CDX2), and development (GJB5) compared to the vitrified group without AX. Therefore, the antioxidant properties of astaxanthin even during short exposure to bovine vitrified/warmed oocytes resulted in improved blastocyst quality comparable to those from fresh oocytes.

Bee bread is exceptional product of the beehive by its composition and a unique production by honeybees. Since the legislation prohibits the use of growth stimulants in animal husbandry, there is a growing interest in improving the yield of meat and eggs, and its quality parameters after applying various natural products. The impacts of bee bread supplementation on laying performance, eggshell chemical composition, serum biochemical parameters of Japanese quails were studied. Antioxidant activity, polyphenols, flavonoids, phenolic acids from bee bread, feeding mixture and combination was determined. A total of 45 female quails were involved in the experiment. The quails were divided into three groups as follows: group with 0.2% addition of bee bread into feeding mixture (E1, n = 15), group with 0.6% addition of bee bread into feeding mixture (E2, n = 15), and the control without additives (C, n = 15). The groups were kept under the standard conditions. After 180 days the animals were slaughtered, blood samples were collected. Addition of 0.6% bee bread in group E2 significantly decreased TAG level compared to group E1, without affecting laying performance. We noticed significant increase in Cd, Pb levels in eggshell in group E1 compared to control group. On the other side, 0.6% addition of bee bread caused significant reduction in Pb, Cd levels in eggshell compared to group with 0.2% addition of bee bread. The incorporation of bee bread into feeding mixture added improved antioxidant activity along with polyphenols and flavonoids. The results indicate that the effect of bee bread was dose dependent. The effective dosage estimation of additives used in feed for Japanese quails plays important role.

Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon spp. are the most common protistan parasites of vertebrates. The results show that pigeon populations in Central Europe are parasitised by different species of Cryptosporidium and genotypes of microsporidia of the genera Enterocytozoon and Encephalitozoon. A total of 634 and 306 faecal samples of captive and feral pigeons (Columba livia f. domestica) from 44 locations in the Czech Republic, Slovakia and Poland were analysed for the presence of parasites by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (18S rDNA), 60 kDa glycoprotein (gp60) and internal transcribed spacer (ITS) of SSU rDNA. Phylogenetic analyses revealed the presence of C. meleagridis, C. baileyi, C. parvum, C. andersoni, C. muris, C. galli and C. ornithophilus, E. hellem genotype 1A and 2B, E. cuniculi genotype I and II and E. bieneusi genotype Peru 6, CHN-F1, D, Peru 8, Type IV, ZY37, E, CHN4, SCF2 and WR4. Captive pigeons were significantly more frequently parasitised with screened parasite than feral pigeons. Cryptosporidium meleagridis IIIa and a new subtype IIIl have been described, the oocysts of which are not infectious to immunodeficient mice, whereas chickens are susceptible. This investigation demonstrates that pigeons can be hosts to numerous species, genotypes and subtypes of the studied parasites. Consequently, they represent a potential source of infection for both livestock and humans.

Bee pollen is a natural apicultural product that is becoming popular among health-conscious consumers due to its wide variety of nutrients and bioactive substances. However, only a limited number of countries have established requirements for the quality and safety of the product so far. Pollen loads may become contaminated by the environment with pesticides, toxic elements, metalloids, and mycotoxin-producing molds. In addition, pollen of certain plant species initially contains hepatotoxic pyrrolizidine alkaloids in relatively large concentrations. Based on literature data, a risk assessment was conducted for the most common pesticide active substances (chlorpyrifos, fluvalinate, carbendazim, thiacloprid), toxicologically important elements (arsenic, cadmium, mercury, lead), common mycotoxins (aflatoxin-B1, ochratoxin-A, fumonisins, zearalenone, deoxynivalenol, T-2 toxin) and pyrrolizidine alkaloids. Only a few countries possess legislation on bee pollen, such as Poland, Bulgaria, Switzerland, Brazil, and Argentina. Currently, there is no European standard document for beekeeping products. The Technical Committee for Food Products of the International Organization for Standardization established a subcommittee on beekeeping products, which has recently begun standardizing the product. At present, however, due to the lack of regulations, bee pollen of different origins can be characterized by heterogeneous nutritional properties and food safety risks.

The aim of the chapter was to describe what microorganisms are typical for pollen, including factors affecting their presence and activities. Pollen is a natural reservoir of microorganisms, which originate from the surrounding. Moreover, bee-collected pollen (BCP) is influenced by bees. Bee-stored pollen (BSP), known as bee bread (perga), is additionally affected by hive environment. Total plate count (TPC) in BCP is usually at level 5 log CFU/g. Present microbiota consists mainly of Enterobacteriaceae and related bacterial families, sporulating bacteria, lactic acid bacteria (LAB), yeasts as Candida spp., Zygosaccharomyces spp. or Metschnikowia spp., microscopic filamentous fungi as Cladosporium spp., Penicillium spp., Alternaria spp., Aspergillus spp. or Fusarium spp. and viruses. Most of present microorganisms behave according to strength of bee colony or human body condition because some of them could act as a pathogen. On the other side, contact with microbes is important for immunity. Microbial counts and diversity decrease by transformation of BCP to BSP. LAB (e.g., Apilactobacillus kunkeei) and yeasts are important in term of this pollen change and belong to main microorganisms in BSP. Decrease of microbial counts was observed also during the dehydration and fermentation of BCP. Increase of microbial counts can occur with inappropriate processing.

In this study, response of ovarian cells (human granulosa cell line HGL5, and human adenocarcinoma cell line OVCAR-3) to short-term pomegranate peel extract (PPE) treatment (for 24 hours in cell culture) was evaluated in vitro. Quantitative and qualitative screening of polyphenols revealed punicalagins α and β as major polyphenolic components. Total phenolic content (TPC) was 93.76 mg GAE/g d.w. with a high antioxidant activity of 95.30 mg TEAC/g d.w. In OVCAR-3, PPE treatment inhibited the metabolic activity, and increased cyclin-dependent kinase 1 (CDKN1A, p21) level at the highest dose, but not in HGL5. Flow cytometry analysis could not detect any significant difference between proportions of live, dead, and apoptotic cells in both cell lines. Reactive oxygen species (ROS) revealed an antioxidant effect on HGL5, and a prooxidant effect by stimulating ROS generation in OVCAR-3 cells at the higher doses of PPE. However, in contrast to HGL5, PPE treatment decreased release of growth factors – TGF-β2 and EGF at the highest dose, as well as their receptors TGFBR2 and EGFR in OVCAR-3 cells. PPE also influenced steroidogenesis in granulosa cells HGL5 by stimulating 17β-estradiol secretion at higher doses. In conclusion, the present study highlighted the bioactive compounds in pomegranate peels and the possible mechanisms of action of PPE, shedding light on its promising role in ovarian cancer (chemo)prevention and/or management.