Antioxidant status of the lower oviduct in the chicken varies with age and dietary vitamin E supplementation

Protection of sperm membranes against lipid peroxidation is a pre-requisite to prolonged sperm storage, both in vivo and in vitro. As females from avian species can store spermatozoa in the utero-vaginal junction (UVJ) for prolonged periods, we investigated the mechanisms involved in antioxidative protection of the plasma membrane of chicken sperm in this region. Comparisons of concentrations in nonenzymatic (alpha-tocopherol, ascorbic acid, and GSH) and enzymatic (GSH-Px, SOD) antioxidants among the vagina, UVJ and uterus of sexually mature chicken hens revealed tissue-specific profiles, with higher ascorbic acid content and increased GSH-Px and SOD activity in the UVJ compared to other regions of the lower oviduct (vagina, uterus). Deterioration of the antioxidant profile in the UVJ was observed in aging hens, but it was partially compensated by dietary supplementation with vitamin E (130 ppm). It is concluded that the chicken UVJ provides a complex defense barrier against lipid peroxidation of the sperm membrane during in vivo storage, which can be partially improved by dietary supplementation with vitamin E. The protective effects of this barrier decline over time during the reproductive season.     

Effect of antioxidants on adriamycin-induced microsomal lipid peroxidation

Adriamycin (25 μM) stimulated NADPH-dependent microsomal lipid peroxidation about fourfold over control values. The tested antioxidants, zinc, superoxide dismutase, vitamin E, and desferrioxamine (Desferal) inhibited Adriamycin-enhanced lipid peroxidation to varying degrees. Others antioxidants, e.g., glutathione, catalase, and selenium, were found to have no effects. Our in vitro studies suggest that adriamycin effect is mediated by a complex oxyradical cascade involving superoxide, hydroxyl radical, and small amounts of iron.     

Exercise,depletion of antioxidants and antioxidant manipulation

Strenuous physical activity is known to increase the production of reactive oxygen species (ROS), associated with depletion of antioxidant defence. In the present work we evaluated the level of lipid peroxidation and antioxidant components in blood of sportsmen under resting conditions and compared the data obtained with those in age- and sex-matched sedentary controls. A significant increase was noted in the levels of thiobarbituric acid reactive substances (TBARS) and conjugated dienes while a decrease was observed in ascorbic acid and glutathione levels in sportsmen. α-Tocopherol was unaltered in plasma of sportsmen as compared to controls. The activity of superoxide dismutase was increased (52 per cent) and glutathione peroxidase was decreased (43 per cent) in the erythrocytes of sportsmen compared to controls. Basal glutathione levels were negatively correlated with conjugated dienes and maximal oxygen uptake (VO_2max) of the subjects. Dietary supplementation with antioxidant vitamins has been shown to be beneficial in combating oxidative stress without enhancing performance while exogenous glutathione was found to influence the endurance capacity of athletes. Such studies demonstrate the critical role played by glutathione and suggest that intervention trials should include a mixture of antioxidants rather than a single antioxidant. Copyright © 1998 John Wiley & Sons, Ltd.     

Influence of N-phthaloyl gamma-aminobutyric acid on circadian rhythms of lipid peroxidation products and antioxidants

N-Phthaloyl GABA was administrated daily (50 mg/kg body weight-i.p) to Wistar rats for 21 days and circadian rhythms of thiobarbituric acid reactive substances (TBARS) and antioxidants such as reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) were studied. N-Phthaloyl GABA was found to delay TBARS and to advance GSH, CAT and SOD acrophases. Amplitude and mesor values of these rhythms were found to be altered during N-Phthaloyl GABA treatment. Since GABA is hypothesized to be involved in conveying dark information to clock, exogenous administration of P-GABA might alter the photic information received by the clock. Our study shows that P-GABA administration alters the temporal patterns of lipid peroxidation and antioxidants in Wistar rats. But the exact mechanism remains to be explored and further research is needed.     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Effect of oral methionine on blood lipid peroxidation and antioxidants in alloxan-induced diabetic rats

Supplementation of thiol compounds has been suggested to protect against the toxic effects of reduced oxygen species by contributing to the thiol pool of the cell. The present study was designed to determine whether supplementation of methionine in the diet of diabetic animals protected against the oxidative stress in diabetic pathology. Oral methionine was administered at a dosage of 330 mg/100 g feed to diabetic rats. The effect was compared with the effect of insulin administration. Levels of lipid peroxides were measured in plasma, erythrocytes, and erythrocyte membrane. Anti-oxidants were measured in plasma. Diabetic condition was associated with increased lipid peroxidation and depletion in antioxidant levels. Although methionine did not affect the level of blood glucose and some of the antioxidants, it lowered the lipid peroxide content in blood. Erythrocyte lipid peroxidation activity was unaffected by methionine treatment. Administration of insulin lowered both plasma and erythrocyte lipid peroxide levels.     

Lipid peroxidation in human spermatozoa as relatd to midpiece abnormalities and motility

The formation of malondialdehyde (MDA), a product of lipid peroxidation (LPO), was measured in human spermatozoa from 27 subjects with normal sperm characteristics. Peroxidation of lipids in washed spermatozoa was induced by catalytic amounts of ferrous ions and ascorbate and malondiaidehyde dctermint-d by thiobarbituric method. MDA formation varied considerably from one sample to another. The studied population showed a strong correlation between lipid peroxidation potential (amount of MDA formed by 10⁸ spermatozoa after 1 hour of incubation) and 1) initial motility r = ?0.623, P = 0.001; and 2) morphologic abnormalities of the midpiece r = 0.405, P = 0.05. These results suggest that poor motility is linked with membrane fragility and that spermatozoa with midpiece abnormalities probably have membrane and/or cytoplasmic antiperoxidant system defects. Because LPO potential is related to the two most important characteristics of fertility, it seems possible that it has the potential to become a good biochemical index of semen quality.     

Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxidation in rat liver mitochondria

Mitochondria are cellular organelles where the generation of reactive oxygen species may be high. They are, however, effectively protected by their high capacities of antioxidative systems, as enzymes and either water or lipid soluble low molecular weight antioxidants.These antioxidative defence systems can be effectively regenerated after or during an oxidative stress as long as the mitochondria are in an energized state. Energization of mitochondria mainly depends on the availability of suitable respiratory substrates which can provide hydrogen for the reduction of either the glutathione- or -tocopherol-system, since GSH is regenerated by glutathione reductase with the substrate NADPH and the -tocopheroxyl-radical likely by reduced coenzyme Q. It was shown that mitochondria do not undergo damages as long as they can keep a high energy state. The delicate balance between prooxidative/antioxidative activities can be shifted towards oxidation, if experimentally prooxidants were added. After exhaustion of the antioxidative defence systems damages of rnitochondrial functions become expressed followed by membrane injuries along with the oxidation and degradation of mitochondrial lipids and proteins leading finally to the total degradation of the mitoc hondria.Extramitochondrial antioxidants may assist the mitochondrial antioxidative defence systems in a complex way, whereby particularly ascorbic acid can act both as prooxidant and as antioxidant. (Mol Cell Biochem 174: 199–205, 1997)     

Plasma lipid peroxidation and erythrocyte antioxidants status in workers exposed to nickel

The objectives were to investigate the plasma lipid peroxidation and erythrocyte antioxidants status in workers exposed to nickel. The study groups comprised 69 nickel plating workers and 50 office workers residing in the same city, but away from the place of work of the study group subjects, considered as control group. Urinary nickel concentration was determined by graphite furnace atomic absorption spectrophotometry. The plasma lipid peroxidation and erythrocyte antioxidants were measured by spectrophotmetric methods. The plasma lipid peroxidation level was significantly increased in nickel-platers and their helpers as compared with controls. Erythrocyte antioxidants were significantly decreased in the nickel-platers compared with the controls. The level of plasma lipid peroxidation was positively and erythrocyte antioxidants were negatively and significantly correlated with the urine nickel levels. Multiple regression analysis assessed the oxidative stress associated with nickel and other potential confounding factors such as body mass index, the consumption of green vegetables, coffee, tea, smoking and alcohol consumption. Analysis showed that the lifestyle confounding factors: the consumption of green vegetables, smoking and alcohol, were not significantly associated with oxidative stress. The exposure to nickel, body mass index and coffee consumption were significantly associated with oxidative stress. The results show that the increased plasma lipid peroxidation and decreased erythrocyte antioxidants levels observed in nickel-exposed workers could be used as biomarkers of oxidative stress.     

Plasma lipid peroxidation and erythrocyte antioxidants status in workers exposed to nickel

The objectives were to investigate the plasma lipid peroxidation and erythrocyte antioxidants status in workers exposed to nickel. The study groups comprised 69 nickel plating workers and 50 office workers residing in the same city, but away from the place of work of the study group subjects, considered as control group. Urinary nickel concentration was determined by graphite furnace atomic absorption spectrophotometry. The plasma lipid peroxidation and erythrocyte antioxidants were measured by spectrophotmetric methods. The plasma lipid peroxidation level was significantly increased in nickel-platers and their helpers as compared with controls. Erythrocyte antioxidants were significantly decreased in the nickel-platers compared with the controls. The level of plasma lipid peroxidation was positively and erythrocyte antioxidants were negatively and significantly correlated with the urine nickel levels. Multiple regression analysis assessed the oxidative stress associated with nickel and other potential confounding factors such as body mass index, the consumption of green vegetables, coffee, tea, smoking and alcohol consumption. Analysis showed that the lifestyle confounding factors: the consumption of green vegetables, smoking and alcohol, were not significantly associated with oxidative stress. The exposure to nickel, body mass index and coffee consumption were significantly associated with oxidative stress. The results show that the increased plasma lipid peroxidation and decreased erythrocyte antioxidants levels observed in nickel-exposed workers could be used as biomarkers of oxidative stress.     

Effect of selenium on N-nitrosodiethylamine-induced multistage hepatocarcinogenesis with reference to lipid peroxidation and enzymic antioxidants

We have studied the relationship between antioxidant and anticancer properties of selenium (Se) in multistage hepatocarcinogenesis induced by N-nitrosodiethylamine (DEN). In this study we have observed an increased level of lipid peroxide (LPO) products and decreased antioxidant enzyme activities (superoxide dismutase and catalase) in hepatoma and surrounding liver tissues of cancer-bearing animals. Selenium (Se) was supplemented either before initiation or during initiation and selection/promotion phases of hepatocarcinogenesis and was found to be effective in altering hepatic lipid peroxidation and antioxidant enzyme activities to a statistically significant level measured either in the hepatoma or in the surrounding liver tissues. These alterations inclined towards normal in a time-dependent manner on selenium supplementation. Furthermore, increased levels of lipid peroxidation and decreased levels of antioxidants (superoxide dismutase and catalase) were also observed in distant organs of cancer-bearing rats other than the tumour-bearing site. These alterations are brought back to normal levels upon Se treatment. Our results confirm the fact that Se is particularly protective in limiting the action of DEN by its antioxidant property.     

Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

Effects of in utero and in vitro incubation on the lipid-bound fatty acids and sterols of porcine spermatozoa

Sperm-rich semen and washed porcine spermatozoa were incubated for up to 2 hr either in utero in the presence of oviduct fluid or in vitro at 37°C. Sperm lipids were extracted and separated into phospholid and neutral lipid fractions. Eleven phospholipid and five neutral lipid fatty acids were identified and quantified using GC and GC-MS. The percentage of 22:5n6, the major phospholipid fatty acid, decreased slightly but significantly during 1.5 hr of in utero incubation (41.2–38.0%), but after 2.0 hr of in utero incubation no significant difference was observed (40.0%). None of the phospholipid fatty acids changed in concentration during in vitro incubation. The mole ratio of phospholipid to phospholipid fatty acid (1.00:1.27) did not change during incubation. The levels of neutral lipid-bound 14:0 decreased (43.5% to 31.8%) and that of 18:0 increased (11.1% to 18.2%) during in utero incubation. Similar but less pronounced changes were observed during in vitro incubation. (43.5% to 36.0%; and 11.1% to 15.8%, respectively). Two major sterols, cholestrol (73%) and desmosterol (27%) were identified by gas chromatography–mass spectrometry. The mole ratio of phospholipid to sterol (2.47:1:00) did not change during incubation.     

Sperm head abnormalities are associated with excessive omega‐6 fatty acids in two finch species feeding on sunflower seeds

In a rapidly changing world, it is important to understand how urban environments impact wildlife. For example, supplementary feeding of birds, though well‐intended, might have unexpected negative effects on the health of individual animals. Sunflower seeds are commonly provided in garden bird feeders, but they contain high levels of linoleic acid (LA), an omega‐6 polyunsaturated fatty acid (PUFA). Omega‐6 PUFAs are associated with increased oxidative stress, which can damage cell membranes, and in particular sperm cells. We assessed the level of LA in the blood of two seed‐eating finch species, greenfinches Chloris chloris and hawfinches Coccothraustes coccothraustes, caught in and in environments with direct access to sunflower seed feeders (Norway), and compared these with the level of LA in a smaller number of individuals sampled in in a rural area with low incidence of sunflower seed feeders (Czech Republic). Furthermore, we investigated the relationship between the proportion of LA in the blood (as well as the proportion of 10 other fatty acids) and sperm quality (the frequency of sperm head abnormalities and sperm swimming speed). We found that both finch species, but particularly greenfinches caught near feeders, exhibited levels of LA that were considerably higher than those previously reported for other wild birds. We also found that the proportion of LA was positively correlated with the frequency of abnormal sperm heads (sperm missing the acrosome), while there was no significant effect of fatty acid composition on sperm swimming speed. Our results indicate that the sperm quality of finches may be negatively affected by a high intake of sunflower seeds, adding to a growing body of research showing that supplementary feeding may have detrimental side effects for urban animals. This is particularly relevant for the greenfinch, which is currently affected by disease and population declines.     

Specific features of in vivo and in vitro sperm storage in birds

This review focuses on some of the main features of sperm selection and storage in birds mainly on the basis of studies performed in poultry species, with emphasis on the initial selection of sperm at the female vagina level prior to migration towards the sperm storage tubules. Sperm originating from low-quality males or subjected to inappropriate in vitro storage conditions are rapidly discarded, resulting in impaired fertility in corresponding flocks. In the absence of accessible and appropriate technology for matching the 'storing' potential of sperm in the oviduct, conditions for prolonged sperm storage under a liquid (through the use of semen extenders) or a solid state (cryopreservation) have received only limited attention, despite their potential interest to facilitate male and female management in poultry flocks. Despite this, technology for short-term liquid storage is currently used in turkeys, guinea fowl and muscovy ducks and also in progress in chickens. In addition, technology for cryopreservation of avian semen has become available for some species (chicken, goose) to facilitate the management of genetic resources, including the preservation of rare and economically important breeds.     

Antioxidant effect of vitamin E and glutathione on lipid peroxidation in boar semen plasma

The protective effect of vitamin E and reduced glutathione (GSH) against lipid peroxidation in boar semen plasma was studied. The lipid peroxidation, measured by the test for thiobarbituric acid reactive substances (TBARS), doubled in the presence of the lipid peroxidation Fe²⁺-sodium ascorbate-inducing system. The ascorbate-induced TBARS were inhibited by about 62% through the water-soluble vitamin E analog (TROLOX) and about 57% by GSH. In the in vivo experiments, 7 wk of oraldl-α-tocopherol acetate (1000 IU/d/animal) administration caused a significant fall in the level of the semen plasma TBARS, from 2.2±0.09 to 1.2±0.13 nmol MDA/mL. The semen plasma superoxide dismutase (SOD) and GSSG tended to increase with the time of vitamin E administration, but the increment did not reach a significant level by the seventh week. The vitamin E supplementation significantly increased the number of spermatozoa per 1 cm³ of ejaculate. The protective role of vitamin E and GSH with respect to boar semen against fatty acid peroxidation and a positive influence of vitamin E supplementation on semen quality have been evidenced.     

Iron-induced luminescence as a method for assessing lipid peroxidation of frozen-thawed goat spermatozoa

Freezing/thawing procedures induce enhanced reactive oxygen species (ROS) formation in mammalian sperm and these ROS may be a cause for the decrease in sperm function following cryopreservation. In the present study, we used a chemiluminescence method to detect ROS-induced damage in goat spermatozoa. Iron-induced luminescence of fresh and frozen/thawed sperm cells was assessed using a luminometer. It was shown that the freezing/thawing procedure had a significant effect on some luminescence parameters. Semen freezing significantly increased the values of integral, peak max, T.half (rise) and T.max (peak) parameters. A significant correlation was observed between the percentage of motile spermatozoa and integral, peak max and T.half (rise) parameters. In conclusion, the results of the present study indicate that measurement of induced luminescence can be an alternative, sensitive and relatively simple method for assessing the effect of cryopreservation on oxidative damage to spermatozoa.     

Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation

Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H₂O₂. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H₂O₂ on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1-5 mM H₂O₂, while the rate in rabbit sperm is unaffected by H₂O₂. The enhancement of lipid peroxidation, the rate of reaction of H₂O₂ with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H₂O₂ due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H₂O₂. This implies that H₂O₂ by itself at 1-5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H₂O₂ or mercapto-succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O₂ as the rate-determining step.     

Computerized analysis of motility, motility patterns and motility parameters of spermatozoa of carp following short-term storage of semen

A computer-aided semen analysis system was used to assess the % motile cells following storage of carp semen in 11 different buffers at 2, 5 or 22° C. BWW and TLP were the most suitable storage buffers because carp semen stored at 5° C in these buffers following activation showed no significant decrease in % motile spermatozoa up to 24 h. But, in most of the other buffers (Fish Ringer, Cytomix, Cortland, FRT, Mannitol, FPS, NAS and TSM) the motility potential was lost by 2 h. Storage was best at pH 6–9 and at 5° C. Carp spermatozoa exhibit three distinct motility patterns, namely 'linear', 'circular' and 'haphazard', the proportion of spermatozoa with a particular motility pattern depending on storage buffer and time. All spermatozoa with a linear trajectory had high VSL, STR and LIN; those moving in circles had low VSL, STR, LIN and BCF and those with a haphazard trajectory were distinct in that they had the highest ALH and their VSL, STR, LIN and BCF were higher than the circular moving spermatozoa and lower than the spermatozoa exhibiting linear trajectory. The study also demonstrates a pronounced time-dependent decrease in VCL, VAP, VSL and ALH of carp spermatozoa following activation with water or low osmolality solutions. This study provides for the first time data related to seven motility parameters of carp spermatozoa and demonstrates how these parameter values could be used to evaluate quality of carp milt following storage in different buffers. It confirms that carp spermatozoa exhibit linear or circular trajectories and provides evidence for a third type of trajectory described as haphazard. All three motility patterns could be discriminated objectively on the seven motility parameters.