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The purpose of this study was to characterize the testicular profile of Zucker diabetic fatty (ZDF) rats presenting with type 2 diabetes mellitus (DM2) in the absence or presence of obesity. To achieve this, testes were collected from 270-day-old male Wistar (n = 15), ZDF nonobese (n = 15), and ZDF obese rats (n = 16). Changes to the testicular structure were quantified morphometrically, while immunocytochemistry was employed to assess caspase-3 activity. Reactive oxygen species (ROS) production, fluctuations of major antioxidant molecules, and the extent of damage to the proteins and lipids were assessed in tissue lysates. Levels of selected interleukins (ILs) were determined by enzyme-linked immunosorbent assay. The results reveal significant alterations to the testicular structure accompanied by caspase-3 overexpression, particularly in ZDF obese rats. The most notable disruption of the oxidative balance, characterized by ROS overproduction, antioxidant deficiency, protein, and lipid deterioration was recorded in ZDF rats suffering from both DM2 and obesity. Accordingly, the highest concentrations of pro-inflammatory IL-1, IL-6, and IL-18 accompanied by reduced levels of the anti-inflammatory IL-10 were found in testicular tissue collected from ZDF obese rats. This study highlights the vulnerability of male gonads to pathophysiological changes caused by hyperglycemia, which are further exacerbated by excessive adipose tissue.

Bacterial colonization of male reproductive tissues, cells, and fluids, and the subsequent impact of bacteria on the sperm architecture, activity, and fertilizing potential, has recently gained increased attention from the medical and scientific community. Current evidence strongly emphasizes the fact that the presence of bacteria in semen may have dire consequences on the resulting male fertility. Nevertheless, the molecular basis underlying bacteriospermia-associated suboptimal semen quality is sophisticated, multifactorial, and still needs further understanding. Bacterial adhesion and subsequent sperm agglutination and immobilization represent the most direct pathway of sperm–bacterial interactions. Furthermore, the release of bacterial toxins and leukocytic infiltration, associated with a massive outburst of reactive oxygen species, have been repeatedly associated with sperm dysfunction in bacteria-infested semen. This review serves as a summary of the present knowledge on bacteriospermia-associated male subfertility. Furthermore, we strived to outline the currently available methods for assessing bacterial profiles in semen and to outline the most promising strategies for the prevention and/or management of bacteriospermia in practice.

The aim of our study was evaluation of potential antioxidant effects of marigold (Calendula officinalis) extract and assessment of its in vitro impact on the selected quality parameters of bovine spermatozoa. Marigold is medicinal herb from the family Asteraceae native to southern Europe, and it commonly used in pharmacology and medicine. Its well-known positive properties include antioxidant, antibacterial, antiflammatory, antiviral, antifungal, antihelmintic and wound-healing activities. In this study, the flower extracts were subjected to high performance liquid chromatography (HPLC), which identified especially phenolic acids (rosmarinic and chlorogenic acids) and polyphenols (rutin, kaempferol, resveratrol, quercetin and apigenin). These substances are known for their antioxidant activity and protective effects against oxidative stress. For our experiments, 10 samples of semen from sexually mature Holstein bulls were collected on a single day by using an artificial vagina, diluted in physiological saline solution and exposed to solutions with different concentration of marigold flower extract (75, 150 and 300 µg/mL). Selected quality parameters (motility, mitochondrial activity, production of reactive oxygen species – ROS, protein oxidation and lipid peroxidation – LPO) were analyzed after 0, 2 and 24 hours of in vitro culture. The motility evaluation was performed by using the computer-assisted sperm analysis (CASA) method. This method revealed that 75 and 150 µg/mL extract had positive effects and increased the motility (P

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen–thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 μM KAE and compared to native ejaculates (negative control—CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control—CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 μM KAE (p < 0.01). At the same time, supplementation with 25 μM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 μM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze–thaw process. [ABSTRACT FROM AUTHOR]

This study assessed the potential efficiency of selected biologically active substances on the motility behavior of rabbit spermatozoa subjected to in vitro induced E. faecalis contamination. Semen samples were collected from 10 male rabbits and the presence of E. faecalis was confirmed using MALDI-TOF Mass Spectrometry. For the in vitro experiments rabbit spermatozoa were resuspended in the presence of 0,3 McF E. faecalis and different concentrations of selected biomolecules (resveratrol - RES, quercetin - QUE, curcumin - CUR, epicatechin - EPI, isoquercitrin - IZO). Sperm motility was assessed using the computer-aided sperm analysis at 0h, 2h, 4h, 6h and 8h. The presence of E. faecalis significantly decreased the motility (P

This study was designed to describe and compare the time- and dose-dependent in vitro effects of selected trichothecenes (deoxynivalenol-DON, zearalenone-ZEA and T-2 toxin) on the motility behavior of rabbit spermatozoa. The rabbit semen was diluted in PBS supplemented with different concentrations (1, 5, 10, 50, 100 μmol/L) of DON, ZEA or T-2 while the Control carried no mycotoxin. At culture times of 0h, 2h, 4h and 8h, the spermatozoa motility was assessed using the computer-aided sperm analysis (CASA) with the help of the IDENT stain and fluorescent illumination. The motility assessment revealed different behavior patterns, specific and unique to each of the studied mycotoxins. DON exhibited the ability to temporarily increase the sperm motility, followed by its rapid decline at later stages of the experiment (P

Interactions between spermatozoa and bacterial species in ejaculates comes along with deleterious effects on the structure and function of male gametes. However, the effect of bacterial load on the seminal plasma remains unexplored. Therefore, this study was focused on the identification of bacterial species present in Duroc boar semen (n=18), quantification of the bacterial load (CFU) and alterations to the biochemical parameters of seminal plasma. The computer-assisted semen analysis (CASA) analysed the progressive motility (PRO; motility=25µm/s) of spermatozoa, their concentration and kinematic parameters. The MALDI Biotyper was used for the bacterial characterization of fresh ejaculates. Seminal plasma was separated and biochemical analysis was carried out using the Randox RX Monza analyzer. Calcium (Ca), magnesium (Mg), total proteins (TP), aspartate transaminase (AST), alanine transaminase (ALT), cholesterol (CHOL), bilirubin (BR), albumins (ALB) and uric acid (UA) were subjected to the Pearson correlation with CFU and PRO. The bacterial identification showed the prevalence of Escherichia coli and Pseudomonas aeruginosa. The correlation analysis showed that the progressive motility of spermatozoa was strongly dependent (-0.799; P<0.01) on the seminal bacterial load. Moreover, a negative significant correlation (-0.810; P<0.001) was detected between CFU and Mg concentration. Also ALT (-0.647; P<0.01) and ALB (-0.484; P<0.05) were negatively affected by the bacterial load. Altogether, the presence of bacterial species may adversely affect the progressive motility as well as the biochemical composition of seminal plasma and thereby create unfavorable conditions for the sperm survival. Further analyses are necessary to reveal interactions between semen and bacteria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study served to elucidate the effects of three selected doses of epicatechin (EPI; 25, 50 or 100 µmol/L) on the motion and oxidative profile of bovine spermatozoa subjected to the process of cryopreservation. Furthermore, we focused on the impact of EPI on the expression patterns of heat shock proteins 90 and 70 as well as the BAX and Bcl-2 proteins involved in the process of apoptosis. The experimental samples were compared with a native control as well as specimens cryopreserved in the absence of any supplement. Our dada indicates that particularly 50 and 100 µmol/L EPI assured a higher preservation of the sperm motility (p<0.001) in comparison with the cryopreserved control. The best protection of the sperm DNA (p<0.05), lipids (p<0.05) and proteins (p<0.01) were observed in the samples exposed to 100 µmol/L EPI in comparison to untreated frozen samples. The presence of higher EPI concentrations in the cryopreservation medium led to significant stabilization of the heat shock protein 90 (p<0.01 in case of 50 µmol/L EPI; p<0.001 with respect to 100 µmol/L EPI) as well as a higher maintenance of the BAX:Bcl-2 ratio (p<0.001) when compared to the untreated frozen control. In summary, we may assume that EPI exhibits antioxidant properties which enable the molecule to prevent excessive damage to biomolecules essential for the cell survival and offer an additional layer of protection to spermatozoa exposed to low temperatures, all of which may be translated into a higher post-thaw sperm motility and activity. [ABSTRACT FROM AUTHOR]

The presence of bacterial contamination in bovine insemination doses may lead to unexpected economic loss. Currently used antimicrobial supplements are often ineffective due to antibiotic resistance of the common bacterial contaminants. Staphylococcus aureus belongs to the frequent inhabitant of bovine semen with a deleterious effect on spermatozoa quality. Bioactive substances including naringenin, curcumin and isoquercitrin were previously studied with various beneficial effects on spermatozoa. Therefore, the study aimed to simulate bacteriospermia in vitro using S. aureus, which was previously isolated from the bovine ejaculate, and to test if the bioactive substances mentioned above would bring some protective properties against the bacterial damage. Density gradient centrifugation was used to avoid contamination by naturally present bacteria. S. aureus was inoculated in BGM-3 medium, and co-incubated with pre-washed spermatozoa and bioactive substances. At times of 0, 2, and 4 h, progressive motility, the membrane potential of mitochondria, reactive oxygen species (ROS) formation, and sperm DNA damage were evaluated. The results showed that S. aureus significantly increased ROS production, sperm cells with damaged mitochondrial membrane potential, and sperm DNA integrity which led to a decrease in the progressive movement of spermatozoa. On the other hand, all three bioactive substances significantly scavenged elevated ROS. Especially, the groups treated with naringenin and isoquercitrin showed preserved mitochondrial membrane, DNA integrity inside the sperm head, and improved sperm progressive movement under bacteriospermia in vitro. [ABSTRACT FROM AUTHOR]

The goal of our research was to evaluate the impact of cryopreservation on the antioxidant activity, lipid peroxidation and protein carbonylation as well as its connection with the progress of cryocapacitation in bovine spermatozoa. As biological material we used semen obtained from 20 sexually mature Holstein bulls. Each ejaculate was divided into three equal aliquots as follows: the first part or control (CTRL) was incubated in physiological saline solution while the second part (CAP) was incubated in a capacitation medium at specific conditions. The third part of each sample was cryopreserved (CRYO) and stored in liquid nitrogen at -196°C for further analysis. The motility of spermatozoa was assessed with CASA (computer assisted sperm analysis), while the capacitation status was evaluated by the chlortetracycline (CTC) fluorescent staining. Total antioxidant capacity (TAC) and the level of lipid peroxidation (LPO) was measured with a combined spectro-fluoro-luminometer. The presence of protein oxidation was detected by the traditional DNPH (2,4-dinitrophenylhydrazine) method and evaluated spectrophotometrically. Based on our data, the motility was significantly decreased (P<0.0001; P<0.01) in the CRYO group against CAP and CTRL. There was a significant increase (P<0.01; P<0.05) of acrosome-reacted spermatozoa (AR-pattern) in the CRYO group when compared to CTRL and CAP, while TAC was statistically decreased (P<0.05) between the CRYO and CAP experimental group. In the case of protein oxidation and LPO, both parameters were significantly higher (P<0.0001) in the CRYO group when compared to CAP or CTRL. In summary, cryopreservation induces capacitation-like changes and promotes oxidative damage in frozen-thawed bovine spermatozoa. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]