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ULTRAWEAK LUMINESCENCE FROM DOMESTIC animals SPERMATOZOA
Marek Godlewski, Teresa Kwiecinska, Dorota Wierzuchowska
Institute of Physics, Pedagogical Academy, 30-084 Kraków, Podchorazych 2, Poland
Antropopressure results in an increased content of peroxidative factors in the natural environment. These include singlet oxygen, ozone, nitric oxide and peroxide, free radicals, heavy metal ions, electromagnetic radiation of different frequency including UV radiation, and others. These factors, together with technological factors of artificial insemination and semen cryopreservation, exert an important influence on spermatozoa, disturb spermatozoa metabolism, involve peroxidation of biological membranes. Ultraweak luminescence (UL) of farm animals spermatozoa was investigated to test whether the measurement of UL can be used as a method to study the influence of thermal and oxidative stress on spermatozoa and to broaden the knowledge about this influence.
UL was measured by means of single photon counting method. Cut-off filters were used to determine emission spectra. Measurements were performed for spermatozoa suspensions of three domestic animals species: bull, boar and ram native spermatozoa, bull and ram spermatozoa after cryopreservation cycle. Fresh ejaculates were double centrifuged (400g, 10min) and resuspended (final concentration 50-60*106 cells/ml.) in: 0.9% NaCl solution, modified Goetze solution and 5% glucose solution for bovine, ram and boar spermatozoa respectively. The same procedure was applied to cryopreserved bull and ram spermatozoa after thawing. In regular periods of time (45-60min.) UL intensity, ATP level, vitality, motility and oxygen consumption were measured for cells incubated at constant temperature ( 40oC - ram and boar, 44oC - bovine). During UL measurement cell suspension was constantly stirred and oxygenated. UL was initiated by addition of ascorbate-Fe(II) system to the spermatozoa suspension (final concentrations: 0.22mM -ascorbate, 50m M -Fe(II)) or by addition of Fe(II) (final concentration 1mM).
Addition of ascorbate-Fe(II) system to bovine spermatozoa cell suspension causes gradual increase of UL intensity; shape of the kinetic curve depends on incubation time. After subsequent addition of Fe(II) ions rapid emission quenching can be observed. In the case of ram spermatozoa there are no significant changes in UL intensity after addition of ascorbate-Fe(II) system, although its value gradually increases with incubation time. There is rapid increase and subsequent decrease of UL intensity after addition of Fe(II). UL kinetics from boar spermatozoa resemble those of bull and differs widely from ram case.
The influence of cryopreservation process on UL time course was investigated for bull and ram spermatozoa. Comparison of kinetic curves indicates that semen cryopreservation has a great influence on the UL time course.
Despite differences in the time course of emission there are no statistically significant differences between bull, ram and boar UL spectral distributions. Emission covers broad spectral region of 360-800nm with maximum at about 630nm and the intensity in the red range (600-800nm) is higher than in the blue one (350-600nm).
Simultaneously to UL measurements, ATP-level, vitality and motility values were estimated. The effect of increasing the ATP level with incubation time is observed for all investigated cells. Vitality and motility values come down as incubation time increases whereas motility falls down for each species faster than appropriate value of vitality. UL intensity negatively correlates with viability and motility.
Obtained data confirm that ultraweak luminescence is functionally related to metabolic processes which take place inside living cells and is connected with subcellular structure in which these processes occur. Results point out on possible contribution of the dismutation processes of superoxide radicals, formed as a result of prooxidative action of ascorbate+Fe(II) system, in generation of electronically excited states and subsequent radiative deactivation of these states leading to photon emission.
Conclusions from the parallel investigations of the biological and biochemical parameters indicate vital association of the UL parameters with physiological state of spermatozoa. Method of the measurement of the ultraweak luminescence allows to observe dynamic changes in the spermatozoa connected with degradation processes, in other words with aggravating quality of semen.
Investigation was done in collaboration with Department of Radio- & Photochemistry, Institute of Chemistry and Technical Electrochemistry, Poznan University of Technology, 60-965 Poznan, Poland and National Research Institute of Animal Production, Department of Physiology of Reproduction, 32-083 Balice/Kraków, Poland.
REFERENCES
Laszczka, A., M. Godlewski, T. Kwiecinska, Z. Rajfur, D. Sitko, B. Szczesniak-Fabianczyk, and J. Slawinski. Ultraweak luminescence of spermatozoa. Curr. Topics Biophys. 1995, 19: 20-31.
Guminska, M., T. Kedryna, A. Laszczka, M. Godlewski, J. Slawinski, B. Szczesniak-Fabianczyk, T. Kwiecinska, Z. Rajfur, and D. Wierzuchowska. Changes in ATP level and iron-induced ultra-weak photon emission in bull spermatozoa, caused by membrane peroxidation during thermal stress. Acta Biochim. Pol. 1997, 44: 131-138.
Slawinski, J., M. Godlewski, M. Guminska, T. Kedryna, T. Kwiecinska, A. Laszczka, B. Szczesniak-Fabianczyk, and D. Wierzuchowska. Stress-induced peroxidation and ultraweak photon emission of spermatozoa cells. Curr. Top. Biophys. 1998, 22(SB): 195-203.
Godlewski, M., T. Kwiecinska, A. Laszczka, J. Slawinski, B. Szczesniak-Fabianczyk, D. Wierzuchowska. Lipid peroxidation of domestic animals’ spermatozoa as investigated by means of ultraweak luminescence. Acta Biol. Cracoviensia. 2000, 42: 79-86.
