The effect of bee drone brood on the motility and viability of stallion spermatozoa-an in vitro study.

Bee drone brood is a beehive by-product with high hormonal activity used in natural medicine to treat male infertility. The aim of the study was to assess the effect of drone brood on stallion spermatozoa during a short-term incubation for its potential use in the equine semen extenders. Three different forms of fixed drone brood (frozen (FR), freeze-dried (FD), and dried extract (DE)) were used. Solutions of drone brood were compared in terms of testosterone, protein, total phenolic content, and antioxidant activity. The stallion semen was diluted with prepared drone brood solutions. The computer-assisted semen analysis (CASA) method was employed to evaluate the movement characteristics of the diluted ejaculate. To determine spermatozoa viability, the mitochondrial toxicity test (MTT) and Alamar Blue test were performed. In terms of testosterone content and antioxidant activity, a close likeness between FR and FD was found whereas DE's composition differed notably. FR had a positive effect mainly on progressive motility, but also on sperm distance and speed parameters after 2 and 3 h of incubation. On the contrary, FD and DE acted negatively, depending on increasing dose and time. For the first time, a positive dose-dependent effect of fixed drone brood on spermatozoa survival in vitro was demonstrated.

The effect of training load stress on salivary cortisol concentrations, health parameters and hematological parameters in horses.

The performance of sport horses is conditioned not only by the quality of its gene pool, but also by a large number of external factors. The most dominant being nutrition, quality of breeding, level of zootechnical care and the quality of the sports rider and coach. Important factor is the process of individuals' adaptation to the training load occurring during the training itself. This study was focused on the analysis of salivary cortisol levels as well as hematological and biochemical blood parameters in relation to load to which the tested horses were subjected. In the study 14 horses of sport breeds were analyzed a all tested horses were in the same (medium) level of training load. Tested horses underwent following stages of workload - transportation, jumping training, parkour competition, treadmill training, riding training, shoeing and lunging of various intensity. Saliva samples were obtained using a tampon on a string which was inserted into horse's oral cavity, chewed by the horse and placed in a sterile tube with a closable lid. Afterwards, the samples were then stored in deep-freezing boxes at temperature of -80 °C. The EIA cortisol kit was used in this study. The absorbance was read at the wavelength of 450 nm against a reference wavelength of 620-630 nm or a blank sample. Blood samples were obtained at the beginning of the experiment, after half a year of running the experiment and at the end of the experiment from v. jugularis. Hematological analysis were carried out using automatic hematologic analyser and multiple parameters were observed. Analysis of biochemical parameters in blood serum were realized using commercial DiaSys kits and semiautomatic biochemical spectrophotometer. Sodium, potassium and chlorides were measured using automatic analyzer EasyLytePlus. In all monitored forms of exercise (transportation, jumping training, parkour competition, treadmill training, riding training, horse shoeing, lunging), an increase in cortisol concentrations immediately after the exercise was recorded, but only spotted statistically significant differences were found during the transportation of monitored horses. The levels of blood parameters were within the reference range during the experiment period. From a comprehensive evaluation of the results, it can be stated that there were no visible health changes to the horses that underwent the experimental load and that manipulation with horses is an important factor that has effect on horses' stress response. In general, the results of this study show no visible impact of training and/or load on the health status of horses over entire 12-month duration of the experiment.

Biogenic Elements and Heavy Metals in Hermann's Tortoises-Testudo hermanni: Effect on Serum Biochemistry and Oxidative Status Parameters.

BACKGROUND: Conservation of species diversity is the need of the hour for preserving life forms on Earth. Extinction of any part of the ecosystem has negative impacts on many processes and systems. The objective of this work was to analyze some biochemical and molecular indicators and their correlations to biogenic elements and heavy metals in Testudo hermanni (n = 16). METHODS: Biochemical parameters were analyzed using the commercial kit DiaSys and biochemical analyzer Randox RX Monza. Sodium, potassium, and chlorides were measured using the EasyLite analyzer. Oxidative stress was evaluated using colorimetric and luminometric methods. Quantification of chemical elements in the blood was carried out using inductively coupled plasma mass spectrometry (ICPS). RESULTS: Biochemical values of analyzed samples from Hermann's tortoises were almost the same as referential values described by multiple authors, with minor aberrations in the total protein parameter. Values of arsenic (As) and nickel (Ni) showed correlation with biochemical parameters and the parameters of oxidative stress. Cadmium (Cd) exhibited correlation with aspartate aminotransferase (AST). CONCLUSIONS: This study reports correlations among four heavy metals, and their levels were again correlated with biochemical and molecular parameters in Hermann's tortoises.

A Combination of Taurine and Caffeine in Stallion Semen Extender Positively Affects the Spermatozoa Parameters.

This study was aimed to determine the impact of different taurine and caffeine combinations on the motility, viability, and oxidative markers of chilled stallion spermatozoa. Each stallion semen sample was diluted in a ratio of 1:2, with various taurine and caffeine concentrations (2.5-7.5 mg/mL taurine + 0.625-1.25 mg/mL caffeine) dissolved in a conventional extender. The control samples (CON) were prepared by diluting ejaculate only using the conventional extender. The motility was analyzed using a CASA system at different time intervals (0, 6, 12, 24, and 30 h) and the viability was evaluated using a mitochondrial toxicity test (MTT) performed at the end of the incubation at 5 °C. The liquid part of experimental samples was separated by centrifugation after 30 h of incubation and underwent the evaluation of oxidative stress via the quantification of markers ferric reducing ability of plasma (FRAP) and total oxidant status (TOS). The samples that were treated with a combination of taurine and caffeine significantly improved the motility parameters, mainly after 12, 24, and 30 h of incubation. Samples extended with combination of taurine and caffeine neither compromise viability nor alterations of redox status. The results of this study describe the combination of taurine and caffeine as an optimal supplement for improving the quality of stallion semen during chilled storage.

Microbiota, Phagocytic Activity, Biochemical Parameters and Parasite Control in Horses with Application of Autochthonous, Bacteriocin-Producing, Probiotic Strain Enterococcus faecium EF 412.

The beneficial influence of bacteriocin-producing, probiotic, mostly non-autochthonous bacteria has already been reported in various animals. However, their use in horses provides limited information, and results with autochthonous bacteria have not been reported. Therefore, the main objective of this model study was to test the effect of autochthonous, bacteriocin-producing faecal strain Enterococcus faecium EF 412 application in horses. One gram of freeze-dried EF 412 strain (109 CFU/mL for 21 days) was applied to horses in a small feed ball. Clinically healthy horses (12), Slovak warm-blood breed of various ages (5-13 years), were involved in a 35-day-long experiment, also functioning as control for themselves. They were stabled in separate boxes (university property), fed twice a day (hay, whole oats or grazed) with water access ad libitum. Sampling was performed at the start of the experiment, i.e. at days 0/1, 21 (3 weeks of EF 412 application) and at day 35 (2 weeks of EF 412 cessation). EF 412 colonized GIT of horses was 3.54 ± 0.75 CFU/g (log 10) at day 21. The eggs of the nematode Strongylus spp. were not found in horses after EF 412 application, and Eimeria spp. oocysts were similarly not found. The other microbiota were not reduced as evaluated by the use of standard method. Using next-generation sequencing, at phylum level, phyla Bacteroidetes and Firmicutes dominated and at family level, they were Bacteroidales BS11 and S24-7 gut goups and Lentisphaerae. In horses, the increasing tendency in phagocytic activity was noted after EF 412 application. Biochemical parameters were in the physiological range. Total protein value was significantly decreased at day 21 compared with day 0/1 as well as with day 35 (P < 0.05). Cholesterol and triglycerides were influenced (decreased) at day 21 compared with day 0/1 and day 35. Neither nematode eggs Strongylus spp. nor Eimeria spp. oocysts were found in faeces after EF 412 application. Autochthonous, faecal strain E. faecium EF 412 showed promising application potential.

Changes in haematological and biochemical parameters in blood serum of horses during exposition to workload stress.

Health state of animals undergoing experimental procedures is an important topic nowadays, as even the small changes can influence the outcome of entire outcomes. Main aim of the study was to evaluate the influence of horse training on variety of blood parameters including mineral profile, energy profile, hepatic profile and haematology. In the experiment, the studied group of horses underwent training programme which consisted of transportation, lounging, riding, jumping, racing, treadmill training and shoeing. Blood samples were collected and later evaluated at the beginning, in the middle and at the end of 1-year lasting process. Our results show multiple significant changes in blood parameters, including changes to multiple minerals, such as Ca, K, Na as well as significant changes of total proteins, urea and certain hepatic profile parameters. Haematology results have also been affected in individual sample collections. Based on results of our study we can state that there have been changes to the internal milieu of the horse but also that there have not been any visible changes of the health status of the animals over the duration of the experiment.

Sperm Quality Affected by Naturally Occurring Chemical Elements in Bull Seminal Plasma.

This study monitored the chemical and biochemical composition of bovine seminal plasma (SP). Freshly ejaculated semen (n = 20) was aliquoted into two parts. The first aliquot was immediately assessed to determine the sperm motion parameters. Another motility measurement was performed following an hour-long co-incubation of spermatozoa with SP at 6 °C. The other aliquot was processed to obtain the SP. Seminal plasma underwent the analyses of chemical composition and quantification of selected proteins, lipids and RedOx markers. Determined concentrations of observed parameters served as input data to correlation analyses where associations between micro and macro elements and RedOx markers were observed. Significant correlations of total oxidant status were found with the content of Cu and Mg. Further significant correlations of glutathione peroxidase were detected in relation to Fe and Hg. Furthermore, associations of chemical elements and RedOx markers and spermatozoa quality parameters were monitored. The most notable correlations indicate beneficial effects of seminal Fe on motility and Mg on velocity and viability of spermatozoa. On the contrary, negative correlations were registered between Zn and sperm velocity and seminal cholesterol content and motility. Our findings imply that seminal plasma has a prospective to be developed as the potential biomarker of bull reproductive health.

Effects of Selected Metal Nanoparticles (Ag, ZnO, TiO2) on the Structure and Function of Reproductive Organs.

Various studies have shown that the reproductive organs are highly sensitive to toxic elements found in the environment. Due to technological progress, the use of nanoparticles has become more common nowadays. Nanoparticles are used for drug delivery because their dimensions allow them to circulate throughout the body and enter directly into the cell. Antimicrobial properties are increasingly used in the manufacture of medical devices, textiles, food packaging, cosmetics, and other consumer products. Nanoparticles provide several benefits, but aspects related to their effects on living organisms and the environment are not well known. This review summarizes current in vivo, and in vitro animal studies focused on the evaluation of toxicity of selected metal nanoparticles (Ag, ZnO, TiO2) on male and female reproductive health. It can be concluded that higher concentrations of metal nanoparticles in the male reproductive system can cause a decrease in spermatozoa motility, viability and disruption of membrane integrity. Histopathological changes of the testicular epithelium, infiltration of inflammatory cells in the epididymis, and prostatic hyperplasia have been observed. Nanoparticles in the female reproductive system caused their accumulation in the ovaries and uterus. Metal nanoparticles most likely induce polycystic ovary syndrome and follicular atresia, inflammation, apoptosis, and necrosis also occurred.

The effects of caffeine on the motility and viability of stallion spermatozoa at different temperature conditions.

The purpose of this study was to evaluate the dose- and time-dependent effect of caffeine treatment on the motility and viability of stallion spermatozoa at different temperatures. Six dose groups (A to F) were established with changing caffeine concentrations (from 0.625 to 10 mg/mL). The control samples were prepared by diluting the ejaculate only with physiological salt solution. The samples were examined after 0, 1, 2 and 3 h of incubation at 5 °C and 37 °C. The motility parameters were evaluated by Computer Assisted Semen Analyzer (CASA) system, and the viability was assessed by the mitochondrial toxicity test at the end of the incubation. A positive effect of the lowest tested caffeine concentration on the motility parameters was observed throughout the incubation period at 5 °C. At the end of the 3h incubation, the viability in every sample in these groups, treated with any caffeine concentration, showed lower values compared to the control. At the higher incubation temperature (37 °C), caffeine positively affected the motility in samples B (P < 0.05) and D, E, F (P < 0.001) after 3 h of incubation; however, the viability showed a slightly decreasing tendency. Our results suggest that caffeine, in an optimal concentration, may be used as a component of stallion semen extenders.

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.

Composition of Stallion Seminal Plasma and Its Impact on Oxidative Stress Markers and Spermatozoa Quality.

The composition of seminal plasma of individual sires varies and so does the fertilizing ability. Micro and macro elements along with seminal enzymes, hormones, proteins, and lipids contained in seminal plasma are essential for the proper physiological function of spermatozoa. However, elevated levels against the normal physiological values, especially in the case of trace metals, result in the production of reactive oxygen species. The deficiency of antioxidants in the seminal plasma that could scavenge free radicals causes an impairment of spermatozoa quality. Ejaculates were obtained from 19 stallions. The fresh semen was analyzed to evaluate qualitative parameters of spermatozoa in terms of the motility, viability, and integrity of DNA. Separated seminal plasma underwent the assessment of the chemical and biochemical composition and RedOx markers. Based on the obtained concentrations of individual chemical elements, the correlation analysis suggested a negative impact of Cu in seminal plasma on the SOD, GPx, and LPO. Contrary, positive correlation was detected between FRAP and motility features. While Cu negatively correlated with sperm motion parameters, the adverse effect on viability was suggested for Cd. Our data suggest that seminal plasma has a potential due to its availability to become the potential biomarker of the reproductive health of farm animals.

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.

Exposure to non-ionizing electromagnetic radiation of public risk prevention instruments threatens the quality of spermatozoids.

The use of artificial insemination in cattle breeding has evolved to global extent, and insemination doses are often shipped via air transport which requires strict radiation-based examinations. For the determination of effect of non-ionizing radiation (NIR), to which are beings frequently exposed due to protection of airport or cultural event security, freshly ejaculated and cryopreserved bovine spermatozoa were used as experimental model. Following radiation with hand-held metal detector in various exposition times (0, 10 s, 15, 30 and 60 min-groups FR, FR10, FR15, FR30 and FR60) the spermatozoa underwent motility and DNA fragmentation analyses. Study on cryoconserved semen treated with NIR was performed in time intervals 0, 10 s, 1 and 5 min (insemination doses radiated before cryoconservation-CB, CB10, CB1, CB5; samples radiated after freezing-CA, CA10, CA1 and CA5). Fresh semen and insemination doses radiated after cryoconservation showed significantly lower total and progressive motility. No effect on motility parameters was detected in semen extended with cryopreservative medium and radiated prior to freezing. Surprisingly, NIR showed a potential to stimulate spermatozoa velocity; however, the effect was modulated throughout the post-thawing incubation. Based on the DNA fragmentation assay, sperm DNA stayed intact. Present study underlines the potential harm of NIR, which is frequently used in everyday life, with overall adverse impact on human and animal reproduction. Current study also points out on interesting short-term spermatozoa stimulation induced by NIR.

Blood concentration of copper, cadmium, zinc and lead in horses and its relation to hematological and biochemical parameters.

Environmental pollution results in serious health hazards to animals and blood analysis serves as a good alternative for health status assessment. The target of this study was to analyze the concentration of selected metals in equine blood, to analyze the blood parameters and to find possible correlations. Blood samples were collected from the vena jugularis of healthy adult horses. The highest concentration of all elements was found in whole blood (Cu 3.84 ± 0.90 mg L(-1); Cd = 0.81 ± 0.90 mg L(-1); Zn 26.67 ± 14.12 mg L(-1); Pb 9.33 ± 5.76 mg L(-1)). Higher concentrations of copper, cadmium, zinc and lead were detected in blood clots compared to blood sera (44.04%). A similar tendency was found for cadmium (50%), zinc (13.08%) and lead (46.02%), which showed generally higher concentrations in blood clots (cells). Correlation analysis proved some relations between analyzed elements. In blood clots there is a strong positive correlation between Cd - Pb (r = 0.93) and Zn - Pb (r = 0.71) was detected. For biochemical and hematological parameters mainly medium correlations were detected. Obtained results prove different correlations of analyzed elements in blood components as well as the effect on parameters of blood biochemical and hematological profiles.

Concentration of copper, iron, zinc, cadmium, lead, and nickel in boar semen and relation to the spermatozoa quality.

The concentration of copper, iron, zinc, cadmium, lead, and nickel as well as its relation to spermatozoa quality was investigated. The semen samples were analyzed by atomic absorption spectrophotometry (AAS). The concentration of copper in boar semen was 1.64 +/- 0.28 mg kg(-1) and of iron 16.14 +/- 10.35 mg kg(-1). The concentration of zinc in boar semen reached an average value of 171.74 +/- 64.72 mg kg(-1) and the level of cadmium reached 0.01-0.16 mg kg(-1) with the average value of 0.05 mg kg(-1). The analysis of lead showed that the concentration of this element in boar semen was 0.02 +/- 0.03 mg kg(-1) and the average level of nickel was 0.06 +/- 0.08 mg kg(-1). The total percentage of pathological spermatozoa was 9.82 +/- 1.47%. Detail analysis determined 3.18% of separated flagellum, 2.26% knob twisted flagellum, 0.88% flagellum torso, 0.85% flagellum ball, 0.42% broken flagellum, 0.23% retention of the cytoplasmic drop, 0.14% small heads, 0.03% large heads, and 1.83% forms other of pathological changes. Correlation analysis showed significant (p < 0.05) positive correlation between copper and lead (r = 0.52). High correlation between small head and knob twisted tail (r = 0.67), small head and broken flagellum (r = 0.88) as well as between small head and total number of pathological spermatozoa (r = 0.73) was determined.