Bacterial contamination of semen has become an important contributor to the reduced shelf life of insemination doses in the poultry industry, which is why antibiotics (ATBs) are an important component of semen extenders. Due to a global rise in antimicrobial resistance, the aim of this study was to assess the efficiency of selected commercially available semen extenders to prevent possible bacterial contamination of rooster ejaculates. Two selected extenders free from or containing 31.2 μg/mL kanamycin (KAN) were used to process semen samples from 63 healthy Lohmann Brown roosters. Phosphate-buffered saline without ATBs was used as a control. The extended samples were stored at 4 °C for 24 h. Sperm motility, viability, mitochondrial activity, DNA integrity and the oxidative profile of each extended sample were assessed following 2 h and 24 h of storage. Furthermore, selective media were used to quantify the bacterial load and specific bacterial species were identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The results indicate that semen extenders enriched with KAN ensured a significantly higher preservation of sperm quality in comparison to their KAN-free counterparts. Bacterial load was significantly decreased in diluents supplemented with ATBs (p ≤ 0.001); however, KAN alone was not effective enough to eradicate all bacteria since several Escherichia coli, Enterococcus faecalis, Enterococcus faecium and Micrococcus luteus were retrieved from samples extended in KAN-supplemented commercial extenders. As such, we may suggest that more focus should be devoted to the selection of an optimal combination and dose of antibiotics for poultry extenders, which should be accompanied by a more frequent bacteriological screening of native as well as extended poultry semen.
- Publikační typ
- časopisecké články MeSH
This study aimed to characterize the bacterial profiles and their association with selected semen quality traits among two chicken breeds. Thirty Lohmann Brown and thirty ROSS 308 roosters were selected for semen quality estimation, including sperm motility, membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. The oxidative profile of the semen, including the production of reactive oxygen species (ROS), antioxidant capacity, protein, and lipid oxidation, were assessed as well. Moreover, the levels of pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukins 1 and 6 (IL-1, IL-6) and C-reactive protein, as well as the concentrations of selected antibacterial proteins (cathelicidin, β-defensin and lysozyme) in the seminal plasma were evaluated with the enzyme-linked immunosorbent assay. The prevailing bacterial genera identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were Citrobacter spp., Enterococcus spp., Escherichia spp. and Staphylococcus spp. While the bacterial load was significantly higher in the ROSS 308 line (p < 0.05), a higher number of potentially uropathogenic bacteria was found in the Lohmann Brown roosters. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains, particularly to ampicillin, tetracycline, chloramphenicol, and tobramycin. Furthermore, Lohmann Brown ejaculates containing an increased proportion of Escherichia coli presented with significantly (p < 0.05) elevated levels of TNF-α and IL-6, as well as ROS overproduction and lipid peroxidation. Inversely, significantly (p < 0.05) higher levels of β-defensin and lysozyme were found in the semen collected from the ROSS 308 roosters, which was characterized by a higher quality in comparison to the Lohmann Brown roosters. In conclusion, we emphasize the criticality of bacteriospermia in the poultry industry and highlight the need to include a more complex microbiological screening of semen samples designated for artificial insemination.
- Publikační typ
- časopisecké články MeSH
Artificial insemination, as an essential pillar of the modern poultry industry, primarily depends on the quality of semen collected from stud roosters. Since the collection and storage of ejaculates is not a sterile process, antimicrobial agents have become essential supplements to semen extenders. While the use of traditional antibiotics has been challenged because of rising bacterial resistance, natural biomolecules represent an appealing alternative because of their antibacterial and antioxidant properties. As such, this study strived to compare the effects of 50 μmol/L curcumin (CUR) with 31.2 μg/mL kanamycin (KAN) as a conventional antibiotic on rooster sperm quality in the presence of Salmonella enterica, Escherichia coli and Pseudomonas aeruginosa. Changes in sperm structural integrity and functional activity were monitored at 2 and 24 h of culture. Computer-assisted semen analysis revealed significant sperm motility preservation following treatment with KAN, particularly in the case of Salmonella enterica and Pseudomonas aeruginosa (p < 0.001) after 24 h. On the other hand, CUR was more effective in opposing ROS overproduction by all bacteria (p < 0.05), as determined by luminol-based luminometry, and maintained sperm mitochondrial activity (p < 0.001 in the case of Salmonella enterica; p < 0.05 with respect to Escherichia coli and Pseudomonas aeruginosa), as assessed by the fluorometric JC-1 assay. The TUNEL assay revealed that CUR readily preserved the DNA integrity of rooster sperm exposed to Salmonella enterica (p < 0.01) and Escherichia coli (p < 0.001). The bacteriological analysis showed higher efficiency of KAN in preventing the growth of all selected bacterial species (p < 0.0001) as opposed to CUR. In conclusion, CUR provided protection to rooster spermatozoa against alterations caused by uropathogens, most likely through its antioxidant activity. Hence, CUR supplementation to poultry semen extenders in combination with properly selected antibacterial substances may become an interesting strategy in the management of bacterial contamination during semen storage.
- Publikační typ
- časopisecké články MeSH
The aim of this study was to evaluate the impact of diabetes mellitus type 2 (DM2) on the male endocrine system of Zucker Diabetic Fatty (ZDF) rats. Sexually mature ZDF rats were divided to a lean (control) and obese group, and had diabetes confirmed by blood tests. For the in vivo experiment, fasting blood was collected to obtain blood plasma. In case of the in vitro experiments, testicular fragments were cultured for 24 h, and the culture medium was collected. The concentrations of testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA-S), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were quantified in the blood plasma and the medium by the ELISA method, while cholesterol (CHOL) was assessed spectrophotometrically. A significant decline of T (36.31 %), A4 (25.11 %) and FSH (26.99 %) as well as a significant increase of CHOL and E2 (36.17 %) was observed in the blood plasma of obese ZDF rats in comparison to the control. Under in vitro conditions, a significant decrease of FSH (23.35 %) accompanied by an increase of E2 was observed in the obese group compared to the control. In the case of CHOL, LH, T, DHEA and A4 no significant differences were observed. Our results suggest that except for FSH and E2 all steroid biomolecules were synthetized normally by the testicular tissue, however a dramatic endocrine disturbance was observed at the system level. We may conclude that DM2 has negative effects on systemic hormone secretion and these alterations are more pronounced in combination with obesity.
- MeSH
- dehydroepiandrosteron MeSH
- diabetes mellitus 2. typu * MeSH
- estradiol MeSH
- folikuly stimulující hormon MeSH
- krysa rodu rattus MeSH
- luteinizační hormon * MeSH
- obezita MeSH
- potkani Zucker MeSH
- testosteron MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Arsenic trioxide (As2O3) poisoning and associated potential lesions are of a global concern. Inversely, riboflavin (vitamin B2, VB2) as a component of flavoproteins could play a vital role in the spermatogenic enzymatic reactions. Thus, this research aimed to explore potential beneficial roles of VB2 during As2O3-injured-toxicity. Rats were randomly allocated into 4 groups (n=8/group) and challenged as follows (for 30 days continuously): Group 1 received normal saline; Group 2 was treated with 3 mg As2O3/L; Group 3 received 40 mg VB2/L; Group 4 received 3 mg As2O3/L + 40 mg VB2/L. Both As2O3 and VB2 were dissolved in deionized water. Malondialdehyde (MDA), Glutathione Peroxidase (GSH-Px), Superoxide dismutase (SOD), and Catalase (CAT) were assessed for the oxidative profile, while TAS (Total Antioxidative Status) levels were evaluated for the antioxidant system, in both serum and testicular tissue. P<0.05 was considered statistically significant. The results show that As2O3 significantly decreased the body weight, testicular weight and testis volume, semen quality and testicular cell count (p<0.05). Furthermore, MDA content in the testicular tissue of the As2O3 group rats was significantly higher in comparison to the vehicle group (p<0.05). Likewise, TAS and the activities of GSH-Px, CAT and SOD were reduced (p<0.05) when compared to the control. As(2)O(3) induced testicular damage and seminiferous tubular atrophy. Monodansylcadaverine assays mirrored the histopathology observations. Meanwhile, As2O3 upregulated the expression of mitophagy-related genes including PINK1, Parkin, USP8, LC3-I, Fis1 and Mfn2. The p38 gene, responsible to stress stimuli, was also upregulated by As2O3 administration. Meanwhile, exposure to VB2 led to a significant decrease of the expression levels of mitophagy related genes. Our study revealed that VB2 supplementation protected testicular structures against As2O3-induced injury via a dual inhibition of oxidative changes and a regulation of the PINK1-mediated pathway.
- MeSH
- antioxidancia farmakologie MeSH
- mitofagie účinky léků MeSH
- oxid arsenitý toxicita MeSH
- oxidační stres účinky léků MeSH
- potkani Wistar MeSH
- proteinkinasy genetika metabolismus MeSH
- proteiny spojené s autofagií genetika metabolismus MeSH
- riboflavin farmakologie MeSH
- signální transdukce MeSH
- spermatogeneze účinky léků MeSH
- spermie účinky léků enzymologie patologie MeSH
- testis účinky léků enzymologie patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5; 75; 150 and 300 μg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5-carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 μg/ml (109.0±4.3%) followed by a decline at 300 μg/ml (89.4±2.3%). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 μg/ml (88.8±11.66%; 87.4±6.0%) and 300 μg/ml (86.2±9.3%; 84.1±4.6%). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 μg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 μg/ml. Finally, the disturbance of GJIC was significantly affected at 300 μg/ml of Levisticum officinale (82.5±7.7%) and Calendula officinalis L. (79.8±7.0%). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.
- MeSH
- Apium * chemie MeSH
- buněčná membrána účinky léků patologie MeSH
- buněčné linie MeSH
- Leydigovy buňky účinky léků metabolismus patologie MeSH
- libeček * chemie MeSH
- měsíček * chemie MeSH
- mezerový spoj účinky léků metabolismus patologie MeSH
- mezibuněčná komunikace účinky léků MeSH
- myši inbrední BALB C MeSH
- pohlavní steroidní hormony biosyntéza MeSH
- rostlinné extrakty izolace a purifikace farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.
- MeSH
- buněčné linie MeSH
- Leydigovy buňky účinky léků metabolismus MeSH
- listy rostlin chemie účinky léků MeSH
- myši inbrední BALB C MeSH
- myši nahé MeSH
- myši MeSH
- progesteron biosyntéza MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rostlinné extrakty farmakologie MeSH
- šalvěj lékařská chemie MeSH
- techniky in vitro MeSH
- testosteron biosyntéza MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Isoquercitrin is a dietary bioflavonoid used as a food supplement. We studied the mechanism underlying its effect in human ovarian cancer cells using OVCAR-3 cell line. Viability, survival, apoptosis, release of human transforming growth factor-β1 (TGF-β1) and TGF-β1 receptor, and intracellular reactive oxygen species (ROS) generation by OVCAR-3 cells were examined after isoquercitrin treatment at concentrations 5, 10, 25, 50, and 100 μg mL-1. AlamarBlue assay revealed that isoquercitrin did not cause any significant change (P > 0.05) in cell viability as compared to control. Apoptotic assay using flow cytometry did not find any significant change (P > 0.05) in the proportion of live, dead and apoptotic cells as compared to control. ELISA also showed that the release of human TGF-β1 and TGF-β1 receptor were not significantly (P > 0.05) affected by isoquercitrin as compared to control. Chemiluminescence assay demonstrated that lower concentrations (5, 10, and 25 μg mL-1) were able to exhibit beneficial effects by inhibiting the generation of intracellular ROS. In contrast, elevated concentrations of 50 and 100 μg mL-1 led to oxidative stress (P < 0.05). We concluded that the beneficial effect of isoquercitrin on ovarian cancer cells may be mediated by an antioxidative pathway that involves inhibition of intracellular ROS generation, thereby limiting oxidative stress.
- MeSH
- antioxidancia farmakologie MeSH
- apoptóza účinky léků MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků farmakoterapie genetika metabolismus patofyziologie MeSH
- oxidační stres účinky léků MeSH
- quercetin analogy a deriváty farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- signální transdukce účinky léků MeSH
- transformující růstový faktor beta1 genetika metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Exogenous substances altering the function of the endocrine system and exhibiting adverse health effects on the organism are defined as endocrine disruptors. Nonylphenol is one of the most abundant alkylphenol ethoxylate derivatives, being detected in food products. Diverse studies have classified nonylphenol as hazardous to the health, especially to male reproduction. This in vitro study aimed to examine the effects of 4-nonylphenol on androstenedione and testosterone production as well as on the viability of Leydig cells of NMRI mice. The cells were cultured for 44 h with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 μg/ml of 4-nonylphenol and compared to the control. Quantification of testosterone and androstenedione directly from aliquots of the medium was performed by enzyme-linked immunosorbent assay. Cell viability was measured by the metabolic activity assay for mitochondrial functional activity. Androstenedione production significantly (P < 0.001) increased with 1.0; 2.5 and 5.0 μg/ml 4-nonylphenol. Although cAMP-stimulated testosterone production was not significantly affected by 4-nonylphenol, a tendency to attenuate the level of testosterone in the Leydig cells treated with 2.5 and 5.0 μg/ml 4-nonylphenol was observed. The viability of mouse Leydig cells was slightly increased at the lowest doses of 4-nonylphenol (0.04 and 0.2 μg/ml). We also observed an increase at higher concentrations of the substance (1.0; 2.5 and 5.0 μg/ml), but this increase was not significant. Further investigations are required to establish the biological significance and possible reproductive implications.
- MeSH
- AMP cyklický metabolismus MeSH
- androstendion biosyntéza MeSH
- fenoly farmakologie MeSH
- hormony biosyntéza MeSH
- kultivované buňky MeSH
- Leydigovy buňky cytologie účinky léků MeSH
- myši MeSH
- testosteron biosyntéza MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
