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Ing. Eva Tvrdá, PhD.
Identification number: 49880
University e-mail: eva.tvrda [at]
University scientific researcher - Department of Animal Physiology (FBFS)

     Final thesis
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Basic information

Basic information about a final thesis

Type of thesis: Dissertation thesis
Thesis title:Spermatozoa defense mechanisms against oxidative stress
Written by (author): Ing. Eva Tvrdá, PhD.
Thesis supervisor:
Opponent 1:
prof. Ing. Róbert Toman, Dr.
Opponent 2:Dr.h.c. prof. MVDr. László Bárdos, PhD.
Opponent 3:
prof. Simon Stephanus du Plessis, MBA
Final thesis progress:
Final thesis was successfully defended.

Additional information

Additional information about the final thesis follows. Click on the language link to display the information in the desired language.

Language of final thesis:English

Slovak        English

Title of the thesis:
Spermatozoa defense mechanisms against oxidative stress
INTRODUCTION: Oxidative stress in the male germ line has a significant impact on male fertility and as well as normal embryonic development. Current research is actively exploring the impact of such stress in spermatozoa and evaluating the possible use of antioxidants to ameliorate this condition. AIMS: The in vivo part was designed to investigate the mutual relationships between selected chemical elements (sodium-Na, potassium-K, iron-Fe, copper-Cu, magnesium-Mg, zinc-Zn, lead-Pb, cadmim-Cd), basic motility characteristics (motility-MOT and progressive motility-PROG), markers of the oxidative balance (total antioxidant status-TAS, ferric reducing ability of plasma-FRAP, total iron binding capacity-TIBC, superoxide dismutase-SOD, catalase-CAT, glutathione-GSH, albumin-ALB, malondialdehyde-MDA) and biochemical parameters (uric acid-UA, urea-U, bilirubin-BILI) in two separate fractions of bovine semen -- the seminal plasma and spermatozoa. The purpose of the in vitro experiments was to assess the impact of ferrous (Fe2+) or ferric (Fe3+) ion on the motility, viability, oxidative balance and biochemical status of bovine spermatozoa at specific time intervals of a 24h cell culture. Furthermore, the in vitro part was focused on examining the effects of five natural substances (resveratrol-RES, quercetin-QUE, a-tocopherol/vitamin E-VIT E, curcumin-CUR, lycopene-LYC) on bovine spermatozoa in the presence or absence of a potent prooxidant (ferrous ascorbate-FeAA) during a short term cell culture (6h). MATERIAL AND METHODS: Semen samples from 36 breeding bulls were used in the in vivo experiments. Motility analysis was carried out using the Computer Assisted Sperm Analysis (CASA) system. The samples were centrifuged, fractions of seminal plasma and spermatozoa were separated, lysates were prepared from the sperm cell fractions. Mineral concentrations were determined by the voltammetric method and flame absorption spectrophotometry; prooxidant, antioxidant and biochemical markers were evaluated by UV/VIS spectrophotometry. In the in vitro part, bovine spermatozoa were incubated in physiological saline solution supplemented with different concentrations (1000, 500, 200, 100, 50, 10, 5, 1, 0 umol/L) of divalent (FeCl2) or trivalent iron (FeCl3). Spermatozoa motility (via the CASA system), viability (using the mitochondrial metabolic test-MTT), superoxide production (via the nitroblue tetrazolium test-NBT) and oxidation/reduction potential (ORP; using an ORP electrode) were assessed in each sample at cultivation times of 0h, 2h, 8h, 16h and 24h. Furthermore, the cells were collected, lysed and examined for changes in the prooxidant, antioxidant and biochemical markers. In the second in vitro part, bovine spermatozoa were diluted in 2.90% sodium citrate, divided into equal fractions and subjected to treatment based on a specific concentration of RES (50, 25, 10, 5, 0 umol/L), QUE (100, 50, 25, 7.5, 0 umol/L), VIT E (500, 100, 50, 10, 0 umol/L), CUR (50, 25, 10, 5, 0 umol/L) or LYC (2, 1, 0.5, 0.25, 0 mmol/L) in the presence or absence of an oxidative stress inducer (FeAA). Spermatozoa motility (using the CASA system), superoxide production (via NBT) and ORP (using an ORP electrode) were assessed in each sample at 0h, 2h and 6h of culture. After 6h, the cells were collected, lysed and assessed for FRAP, SOD, CAT, GSH, ALB, UA and MDA. RESULTS: In vivo. Concentrations of chemical elements in both seminal fractions were in the following descending order: Na > K > Zn > Mg > Fe > Cu> Pb > Cd. Higher amounts of all minerals and nonenzymatic antioxidants were detected in the seminal plasma (P<0.001 in case of TAS, GSH, UA and BILI; P<0.01 for FRAP, ALB, Fe, Cu, Mg and Zn; P<0.05 in case of Na and Pb), while higher MDA concentration and activity of enzymatic antioxidants were recorded in the cell lysates (P<0.05 in case of SOD; P<0.01 for CAT and P<0.001 in case of MDA). Na, Fe, Cu, Mg, and Zn were positively correlated with the motility and antioxidant parameters. Inversely, K, Pb and Cd exhibited positive associations with malondialdehyde (P<0.05 for K; P<0.01 for Pb and Cd), while being negatively associated with spermatozoa motility and antioxidant status (P<0.05 for Cd; P<0.01 for Pb). In vitro: iron. Both ferrous and ferric iron had a doseand time-dependent impact on the spermatozoa physiology and oxidative balance. Concentrations >50 umol/L FeCl2 and >100 umol/L FeCl3 led to a significant (P<0.001 in case of 200 - 1000 umol/L FeCl2/FeCl3 at 2h - 24h) decrease of spermatozoa motility and mitochondrial activity, accompanied by membrane potential changes related to cell death and oxidative stress development, associated with a significant (P<0.001 in case of 100 - 1000 umol/L FeCl2/FeCl3 at 2h - 24h) superoxide production, inactivation of antioxidant proteins, as well as a significant (P<0.001 in case of 50 - 1000 umol/L eCl2 and 200 - 1000 umol/L FeCl3 at 2h - 24h) rise of lipid peroxidation. The ability of the cell to process free iron was significantly (P<0.001 in case of 200 - 1000 umol/L FeCl2 at 8h - 24h and 500 - 1000 umol/L FeCl3 at 16h - 24h) impaired too. Concentrations below 10 umol/L FeCl2 and 50 umol/L FeCl3 proved to be stimulating to the spermatozoa activity, as shown by a significantly (P<0.001 in case of 5 - 10 umol/L FeCl2 at 16h - 24h and P<0.05 in case of 5 - 10 umol/L FeCl3 at 2h - 16h) increased antioxidant capacity along with a significant (P<0.001 in case of 5 - 10 umol/L FeCl2 at 24h and P<0.05 in case of 5 - 10 umol/L FeCl3 at 8h - 24h) preservation of enzymatic and non-enzymatic antioxidants. In a direct comparison, ferrous iron has been shown to be more toxic than the ferric iron. In vitro: natural substances. The addition of natural substances into the culture medium was able to slow down the deleterious effects of in vitro-associated free radical production. All natural substances were able to significantly (P<0.05 for 25 - 50 umol/L RES; P<0.01 in case of 50 - 100 umol/L QUE and 2 - 1 mmol/L LYC; P<0.001 for 100 - 500 umol/L VIT E and 25 - 50 umol/L CUR) prevent the decline of spermatozoa physiology, functional activity and antioxidant capacity as a consequence of FeAA-associated oxidative stress development. Each natural substance showed to be effective in a different aspect of spermatozoa physiology protection, and at a different concentration. CONCLUSIONS: the in vivo investigation revealed a complex intercellular and intracellular network of interactions and associations between the chemical elements, markers of spermatozoa quality, and antioxidant profile existing within the bovine semen. Any mineral and/or biochemical imbalance in semen may have a negative impact on spermatozoa abnormalities, and therefore may be considered as a risk factor for male fertility complications. The in vitro data suggest that 50 umol/L FeCl2 and 100 umol/L FeCl3 may be considered as the critical in vitro concentrations of iron balancing between being an essential trace metal and a serious prooxidant substance. At the same time we may suggest that each of the natural substances may be a suitable supplement for the in vitro management and preservation of male gametes.
Key words:
free radicals, reactive oxygen species, oxidative stress, antioxidants, iron, male fertility, spermatozoa, bull

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