Spermatozoa must possess many attributes to fertilize an egg but few laboratory methods can assess all of these attributes simultaneously and objectively. Most laboratory methods used to assess the quality of semen in veterinary andrology can be inaccurate and time-consuming. Laboratory techniques which evaluate only one sperm attribute, often provide results that have a weak correlation with fertility. Therefore, semen used for artificial insemination should not be assessed based on the results of one method only but rather on the comprehensive results of several laboratory tests. Flow cytometry is a modern method of analysing diJ169fferent types of cells, including sperm cells. It is based on the scattering of light and fluorescence, the outcome of which yields results that are recorded and evaluated by computer technology which makes an objective assessment. Flow cytometry in combination with fluorescence microscopy and fluorescent probes provides a comprehensive, accurate, objective, and rapid analysis of the ejaculate. In a short time frame it allows us to test thousands of sperm for their structure and properties, even with a minimal amount of semen. This method enables the evaluation of several indicators simultaneously in a population of sperm as a whole or for each sperm individually. It informs us about the selected indicators of sperm quality in the sample by examining the membrane integrity, DNA, mitochondria, acrosome, oxidative stress, and other properties. Flow cytometry has become an important method of evaluating the functional and morphological properties of sperm and is used for research in veterinary science as well as for a routine assessment of the semen quality.
The aim of our study was to find the most appropriate way of sample collection from cattle feet as well as to assess simple and effective sample processing, including DNA extraction for reliable diagnosis of bacteria Dichelobacter nodosus and Fusobacterium necrophorum. 11 clinically healthy cows were included in the study, from which swabbing samples (2 types: surface swab and deep swab) were taken. Two isolation methods were used for DNA extraction: 1. freezing and boiling the samples, 2. commercial kit (Roche). PCR analysis of the samples has not shown any variations in the detection ratio of D. nodosus and F. necrophorum at different swabbing methods. The highest sensitivity of the detection of both bacteria was reached with a cultivation of samples in AB with subsequent extraction of DNA with freezing and boiling. The cultivation in anaerobic broth resulted in the detection rate of D. nodosus and F. necrophorum in over 95% and 27%, respectively. To conclude, the simple 'surface' swab is sufficient to detect studied pathogens, the most appropriate method of DNA extraction has proven to be freezing and boiling of the sample. ARTICLE HISTORY
The aim of the experiment was to determine the effect of a single subcutaneous application of Selevit inj. on the volume and density of ejaculates, sperm viability, level of oxidative stress (OS) and apoptosis in semen by using flow cytometry. Ten rams were divided into two groups. The experimental group, (EG; n=6) was injected one time subcutaneously with the Selevit inj. at a dose of 5 ml per animal (11 mg of sodium selenite). The control group, (K; n=4) was subcutaneously administered with physiological solution at a dose of 5 ml per animal. Samples of blood and semen were collected from each ram prior to application of Se and at day one, 14, 26, 38, 50, 62 after selenium injection. Results showed, Se concentration in the blood of EG was significantly higher, but short-term. Se concentration in the semen of the EG was significantly higher during the whole duration of the experiment (62 days). The level of OS was significantly reduced at day one, 14 and 60 after application of Selevit injection. The number of dead spermatozoa was significantly lower in the EG only at day 14 and 26 after Se application. There was only a slight increase in the percentage of sperm in the early phase of apoptosis in EG. There were no significant differences in ejaculate volume and sperm concentration between groups. Single subcutaneous injection of Selevit is sufficient enough to maintain a significant long-term increase of Se concentration in semen and has a positive effect on the level of OS, but there was no substantial influence on the quality of ejaculates.
A Simmental dairy cattle, aged seven years old, was presented with a history of foul smelling discharge from the external genitalia. According to the history, during the latest parturition the foetus had died in the uterus and was partially removed, but parts of the foetus had remained in the uterus for the following fourteen months. Gynaecological examination confirmed an open cervix. Trans-rectal palpation and ultrasound examination revealed extension, thickening and tension of the uterine wall and the presence of putrid parts of the foetus in the uterus. Attempts to remove the foetus by prostaglandin injections were futile, hence left side low flank hysterotomy was performed under cranial epidural anaesthesia and local infiltration anaesthesia. The foetal bones were removed and the other pathological contents were also removed. The cow gained weight and could subsequently be sent for slaughter. It was concluded that left flank hysterotomy can be useful for removal of macerated foetus from cows.
The aim of this study was to determine the effects of sodium selenite on the level of oxidative stress and viability of spermatozoa in fresh bull ejaculate in in vitro conditions at different temperatures. Samples of the bull's ejaculates with a concentration of 7 × 105 spermatozoa per ml were placed into the commercial semen extender containing 0 (control), 1 (1Se), 3 (3Se) and 5 (5Se) µg.ml–1 of sodium selenite. The following analyses were performed by flow cytometry after 1, 3, 6, 8, 24, 48 and 72 hours of incubation at 4 °C and 37 °C. All analyses were carried out in triplicate. The level of oxidative stress at both temperatures were significantly lower in the experimental groups in comparison to the control group. However, a significant decline of live sperm concentration and a rise of damaged sperm concentration were recorded, especially in groups 1Se and 3Se in comparison to the control group. Only in group 5Se was there observed a positive effect on the damaged spermatozoa level in comparison with groups C, 1Se and 3Se at 4 °C. In conclusion, the applied concentrations of sodium selenite had a positive effect on the level of oxidative stress in all experimental groups, but mainly at concentration of 5 µg.ml–1 of sodium selenite, especially at 4 °C. However, the effect of selenium was not sufficient for improving the sperm viability.
The aim of the experiment was to determine the effect of a single subcutaneous administration of selenium (Se) + vitamin E on the ejaculate volume, sperm count and viability, level of apoptosis and oxidative stress (OS) and Se concentration in ejaculates and blood of rams with respect to the time course of the spermatogenic cycle. The experimental group (EG; n = 6) was treated with a single injection of Selevit at a dose of 5 ml per animal (11 mg of sodium selenite and 125 mg of vitamin E/per animal). The control group (CG; n = 4) was treated in the same way, only with saline. Samples of blood and ejaculates were collected from each ram prior to application of Se and on the 1st, 14th, 26th, 38th, 50th and 62nd day after injection. Results showed that the Se concentration in the blood of EG was significantly higher only 24 h after application. Selenium concentration in the ejaculates of the EG was higher during the whole duration of the experiment (62 days), but significantly so only until day 14. The level of OS was significantly reduced on day 1, 14, and 62 after application of Selevit. There were no significant differences in the other analysed indicators. The results showed that one subcutaneous injection of Selevit had a positive effect on Se concentration and OS level in ejaculates, but was not sufficient to improve other monitored sperm quality indicators.
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