Dose- and time-dependent effect of copper ions on the viability of bull spermatozoa in different media.

The purpose of this in vitro study was to determine the effect of copper (Cu) on the motility and viability of spermatozoa in the presence of different culture media. Specifically, we examined the dose- and time-dependent effect of copper ions (Cu(2+)) on the motility and viability of spermatozoa during different time periods (Time 0 h, 1 h, 24 h). The percentage of motile spermatozoa and progressive motile spermatozoa was determined after exposure to concentrations of 3.9; 7.8; 15.6; 31.2; 62.5; 125; 250; 500; 1000 µM/L of Cu(2+) using the Sperm Vision(TM) CASA (Computer Assisted Semen Analyzer) system. The cell viability was measured by the MTT (metabolic activity) assay. The initial spermatozoa motility in the presence of Cu(2+) in physiological saline solution (PS) showed slight increased values at doses <31.20 µM/L of Cu(2+) compared to the control group. The long-term cultivation (Time 24 h) reduced the average motility values in all experimental groups (P < 0.001) in comparison to the control group. Identical spermatozoa motility was detected for the percentage of progressive motile spermatozoa during all time periods. The culture medium containing 20 % bovine serum albumin (BSA), triladyl and 5 % glucose increased the overall percentage of spermatozoa motility after 1 h of cultivation. A concurrently maintained motility of spermatozoa at doses <62.50 µM/L of Cu(2+) during the long-term in vitro cultivation confirms the protective effect of albumin. The cell viability was decreased significantly (P < 0.001) in all experimental groups with copper administration. The obtained data point out that Cu(2+) at high doses is a toxic element on the spermatozoa motility, which subsequently disrupts the viability of cells. However, using a suitable culture medium containing an energy component- and protein-rich substrate, the spermatozoa motility could increase.

Impact of oxidative stress on male fertility - a review.

Oxidative stress is a state related to increased cellular damage caused by oxygen and oxygen-derived free radicals known as reactive oxygen species (ROS). It is a serious condition, as ROS and their metabolites attack DNA, lipids and proteins, alter enzymatic systems and cell signalling pathways, producing irreparable alterations, cell death and necrosis. While small amounts of ROS have been shown to be required for several functions of spermatozoa, their excessive levels can negatively impact the quality of spermatozoa and impair their overall fertilising capacity. These questions have recently attracted the attention of the scientific community; however, research aimed at exploring the role of oxidative stress and antioxidants associated with male fertility is still at its initial stages. This review summarises the current facts available in this field and intends to stimulate interest in basic and clinical research, especially in the development of effective methods for the diagnosis and therapy of semen damage caused by oxidative stress.

In vitro effect of nickel on bovine spermatozoa motility and annexin V-labeled membrane changes.

In this study the effect of in vitro culture of bovine spermatozoa with nickel (NiCl(2)) on spermatozoa motility and membrane changes was analyzed. The spermatozoa motility significantly decreased after 120 min of culture at the concentration of 1000 µM Ni ml(-1) (P < 0.05) and after 240 min of culture at the concentration of 500 and 1000 µM Ni ml(-1) (P < 0.001) as compared with control. The progressive motility was the highest in the control group and in the groups with the lowest nickel concentrations (7.8 and 125 µM Ni ml(-1)). The progressive spermatozoa motility was significantly altered even after 30 min of culture in the group with the highest nickel concentration (1000 µM Ni ml(-1)). A significant decrease in progressive motility from the concentration of 250 µM Ni ml(-1) was detected after 240 min of culture. Concentrations from 125 µM Ni ml(-1) in various time periods of culture stimulated spermatozoa motility after 30 min (P < 0.001), but later an inhibitory effect was noted. After 240 min of in vitro spermatozoa culture with 125 µM Ni ml(-1) a typical Annexin V fluorescence reaction was detected. Fluorescence was detected in mitochondrial segment of bovine spermatozoa. In spermatozoa exposed to higher nickel concentrations the Annexin V-positive reaction was detected also on the spermatozoa head membrane. In the group with the highest concentration and the longest time of exposure (1000 µM Ni ml(-1); 240 min) the apoptotic Annexin-positive regions were detected not only in the mitochondrial part, but also in the spermatozoa head (acrosomal and postacrosomal part), showing significant alteration of spermatozoa membrane integrity.

In vitro copper toxicity on rabbit spermatozoa motility, morphology and cell membrane integrity.

In this in vitro study the effects of copper sulphate on the motility, morphology and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated by CASA method and Annexin analysis was used for detection of structural changes. For analysis of morphology samples of rabbit semen were fixed with Hancock's solution and stained with Giemsa, and for each sample at least 500 spermatozoa were evaluated. The concentration of copper in the medium varied from 3.57 to 4.85 microg CuSO4/mL. At Time 0 the highest motility was detected in the control group (57.78 +/- 3.90%). Motility in groups with copper administration was lower in comparison to control. Significant differences were detected in groups with 3.70-4.85 microg CuSO4/mL (P<0.05) at Time 0. After 1 h of incubation with copper sulphate the motility significantly decreased almost in all experimental groups. However, at Time 2 h significant increase of total motility was observed in groups with lower concentrations of copper (3.57 and 3.63 microg CuSO4/mL). After 24 and 48 h of incubation almost all the spermatozoa were dead recording no motility at all concentrations. The concentration- dependent decrease of spermatozoa motility up to 50% of control was detected for the group receiving highest copper administration (4.85 microg CuSO4/mL) at Times 1 and 2 h. Progressive motility had an identical trend to that of motility in all experimental groups, at all culture times and for all concentrations. Evaluation of distance and velocity parameters indicated that a sort of stress tolerance developed in lower concentrations (3.57 and 3.63 microg CuSO4/mL). At lower concentrations, an increase was noted for distance parameter DCL and velocity parameter VCL, indirectly confirming the significant motility and progressive motility increase. Other motility parameters (straightness index, linearity index, wobble and amplitude of lateral head displacement) revealed decrease in the group with the highest copper concentration (4.85 microg CuSO4/mL) in comparison to the control group after 2 h of incubation, only. No significant alteration was noted for these parameters in comparison to control at Times 0 and 1 h. The total percentage of morphologically abnormal spermatozoa was significantly higher (P<0.05) in the group with the highest copper concentration (46.20+/-5.54%) in comparison to control (30.60+/-2.91). Predominant morphological abnormalities were acrosomal changes, knob-twisted flagellum and small heads. Detection of spermatozoa with disordered membrane was carried out for groups with higher copper concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the copper-exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In copper-exposed groups positive reaction proved alteration in anterior part of head (acrosome) and in connection segment (mid-piece) of spermatozoa. Detected data evidently confirm adverse effects of high copper sulphate concentrations in rabbit semen on parameters of spermatozoa motility, morphology and membrane integrity. This paper also indicates the lowest possible toxic concentration of copper (3.70 microg CuSO4/mL) to rabbit spermatozoa in relation to motility.

Effect of beta-carotene supplementation on plasma and yolk IgY levels induced by NDV vaccination in Japanese quail.

Newly hatched Japanese quail (Coturnix coturnix japonica) chicks were fed diets containing different levels of retinoids (vitamin A) or beta-carotene. Group A received a commercial diet containing 10,000 IU vitamin A per kilogram. The diets of Groups B, C, and D contained no vitamin A but were supplemented with 1-, 2.5-, and 5-fold retinol equivalents of beta-carotene. Each group contained 16 quails in a 1:1 sex ratio. At 8 weeks of age the quails were immunized orally with Newcastle disease virus (NDV) vaccine according to the manufacturer's recommendations. Boosters were given three times at two-week intervals. Blood samples were taken at two-week intervals until 14 weeks of age. The anti-NDV IgY titre was determined by a locally developed direct enzyme-linked immunosorbent assay (ELISA). Groups A and B showed nearly the same antibody response. This indicates that the preformed vitamin A and the equivalent beta-carotene have the same immunomodulatory effect. Groups receiving higher doses of beta-carotene (Groups C and D) exhibited significantly higher plasma IgY levels compared to Groups A and B. The results indicate that elevated doses of beta-carotene have a slight effect on the adaptive immune response in Japanese quail.