Index Of Membrane Integrity Research Articles

Alleviating Chilling Injury in Stored Pomegranate Using a Single Intermittent Warming Cycle: Fatty Acid and Polyamine Modifications.

Pomegranate is a perishable superfruit with important human health-promoting phytochemicals. The use of cold storage is inevitable for its long-term preservation. As pomegranate is sensitive to temperatures below 5°C, it is therefore necessary and worthwhile to introduce a postharvest technique that is safe, applicable, and commercially acceptable to maintain the fruit quality under a cold storage condition. The efficacy of intermittent warming (IW) in the form of a single warming period (1 day at 20°C with 70% relative humidity (RH) before returning the treated fruit to storage) during the cold storage of ‘Rabab-e-Neyriz' pomegranate (70 days at 2 ± 0.5°C and 90 ± 5% RH) was evaluated. To find the best treatment time, warming was performed at 4 temporary interruption points in storage (after 15, 25, 35, or 45 days of storage). For each interruption date, the treated fruit were compared to the controls twice, once immediately after treatment and once at the end of the storage period. It was founded that a single warming period at the right time during cold storage (before irreversible damage occurs) activated multiple mechanisms and physiological responses in pomegranate fruit peel that are significantly responsible for alleviating the severity of chilling damage to this commodity. In other words, warming on the 15th day was the most efficient treatment, resulting in better preservation of unsaturated fatty acids from peroxidation, lower malondialdehyde (MDA) production, and preservation of the unsaturated/saturated fatty acids (UFAs/SFAs) ratio (membrane integrity index) in the peel during storage and lower chilling injury symptoms. Moreover, the content of spermine (Spm) and putrescine (Put) (as important antioxidants acting as membrane safety agents) was significantly increased immediately after treatment, followed by a continuous increase in Spm and a higher level of Put compared to control until the end of storage.

Proton spectroscopy study of the dorsolateral prefrontal cortex in youth with familial depression.

Structural, functional, and metabolic changes in the dorsolateral prefrontal cortex (DLPFC) are implicated in the pathogenesis of major depressive disorder (MDD). We used proton magnetic resonance spectroscopy ((1) H-MRS) to examine the metabolite choline (glycerophosphocholine plus phosphocholine), which is used as an index of membrane integrity in the left DLPFC, in adolescents and young adults with MDD who were treatment-resistant and had a positive family history compared to healthy controls. Differences in the choline resonance indicate an imbalance between synthesis and degradation activity of neuronal and glia membrane phospholipids. Seventeen adolescents with MDD and 11 healthy controls underwent (1) H-MRS. A short echo point-resolved spectroscopy (echo time = 30 ms, repetition time = 2000 ms) protocol was used with a voxel (4.5cm(3) , 128 averages) placed within the left DLPFC. There were significantly increased choline (P = 0.04) and creatine concentrations (P = 0.005) in the left DLPFC of the MDD group compared to controls. In MDD participants, choline concentration correlated with scores on the Beck Depression Inventory (r = 0.41, P = 0.03). Increased left DLPFC choline and creatine levels in depressed adolescents may be biomarkers for the disorder. The increased choline levels may indicate abnormalities in neuronal membrane integrity, and the increased creatine could be reflective of altered energy demands and metabolism.

Protective effect of Phyllanthus fraternus against mitochondrial dysfunction induced by co-administration of cisplatin and cyclophosphamide

The evolving role of mitochondria, in mediating chemotherapy-induced apoptosis motivated us for the studies described here. The combination of cisplatin and cyclophosphamide is widely used in treating various types of cancers. The purpose of our study was to understand the mechanism of the toxicity induced by the co-administration of cisplatin and cyclophosphamide, on mitochondrial bioenergetics, and to study the protective effect of prior administration of the medicinal plant extract Phyllanthus fraternus. Our results reveal that co-administration of cisplatin (12 mg/kg, i.p) and cyclophosphamide (150 mg/kg, oral) to wistar rats (100 g) significantly alters mitochondrial structure and hence function. The rate of mitochondrial respiration was decreased significantly with both NAD + and FAD-linked substrates. The respiratory control ratio, an index of membrane integrity and the P/O ratio, a measure of phosphorylating efficiency also were decreased significantly accompanied by elevation in the lipid peroxide levels in liver, kidney homogenate and liver mitochondria respectively. Also, the phospholipid content of the mitochondrial membrane, showed a significant decrease, indicating mitochondrial membrane changes. Prior administration of an aqueous extract of P. fraternus (100 mg/kg) to rats, showed protection on all parameters investigated. Administration of P. fraternus alone did not show any significant changes on mitochondrial membrane bioenergetics. Thus, we propose, that the toxic side effects of cisplatin (+) cyclophosphamide, are due to a chain of interconnected events, within the mitochondrial inner membrane, ultimately leading to hepatotoxicity and nephrotoxicity. Further, our work also suggests that administration of aqueous extract of P. fraternus can enhance the therapeutic potential of anticancer drugs by reducing drug related toxicity.

91 TREATMENT OF GOAT SPERM WITH CATALASE TO IMPROVE POST-THAW QUALITY

Semen cryopreservation is extremely important to the use of reproductive biotechnologies in goats. However, studies indicate that cryopreservation may lead to increased oxidative stress which may cause structural damage to biomolecules, DNA, lipids, carbohydrates, and proteins, as well as other cellular components. Previous studies performed by our group indicate fresh goat sperm is highly susceptible to the attack of hydrogen peroxide. Therefore, the treatment with hydrogen peroxide scavengers would be an alternative to improve post-thaw sperm quality in goats. The aim of the present study was to evaluate the efficiency of catalase, an important hydrogen peroxide scavenger, to improve post-thaw quality in cryopreserved goat semen samples. Semen samples from 5 adult goats were collected and cryopreserved (Botu-Bov®, Biotech Ltda.). After thawing, samples were incubated (2 h, 37°C) with 0, 60, 120, and 240 UI mL–1 of catalase. Samples were then analysed for motility using the computer assisted sperm analysis (CASA); the 3–3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin nigrosin stain, as an index of membrane integrity; the simple stain (Fast green/Bengal rose), as an index of acrosome integrity; sperm chromatin structure assay, as an index of DNA fragmentation; and the measurement of thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). Results showed that catalase treatment after thawing played a role on improving mitochondrial activity. Samples treated with 240 UI mL–1 showed lower percentage of sperm showing low mitochondrial activity when compared with samples treated with 0 and 120 UI mL–1 of catalase (6.5 ± 2.3, 17.2 ± 3.5, and 10.0 ± 1.3%, respectively). However, no effect of catalase was observed on any of the other variables studied. Results indicate that catalase, despite its beneficial effect on mitochondrial activity, does not influence positively on sperm quality after thawing. A hypothesis to explain such results would be that because of seminal plasma dilution with the extender, the antioxidants were also diluted. Therefore, the antioxidant protection would be impaired and the most deleterious reactive oxygen species, as observed in fresh semen, would also be different depending on the semen extender used because sperm are extremely dependent on the extracellular environment due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in the membrane. A study performed by our group confirms such a hypothesis. Possibly, the treatment with catalase would be more effective if performed before cryopreservation. Also, it is possible that the use of different antioxidants would provide better results. Thanks to Nutricell for the media used and CAPES for financial support.

240 FUNCTIONAL TRAITS OF CAT SPERM DURING DISTINCT MATURATION STATUS

Sperm recovery from the caudae epididymides can be advantageous for preserving semen of endangered animal species. In this context, the domestic cat is a suitable model for the study of sperm physiology in endangered feline species and the research on epididymal sperm preservation combined with the use of reproductive biotechnologies including intracytoplasmic sperm injection (ICSI). The aim of the present study was to examine the sperm collected from the cauda and caput of the cat epididymis using functional tests. Testicles and epididymides from 5 adult tomcats were collected by orchiectomy and maintained at 4°C for 4 h, until semen collection. Semen samples were collected from the epididymal tail and head by careful dissection. Samples were then analysed for motility by computer assisted sperm analysis (CASA; only for the caudal sperm). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). No motility was observed in samples collected from the epididymal head, whereas samples from the tail showed 50.0 ± 4.2% motile spermatozoa. Surprisingly, more spermatozoa with high mitochondrial activity were found in the epididymal head than in samples from the tail (74.0 ± 3.5 v. 50.0 ± 4.3%, respectively). Similarly, samples collected from the head showed a higher susceptibility against the attack of ROS (31.9 ± 5.5 v. 16.3 ± 7.1 ng of TBARS/106 sperm, respectively). Furthermore, epididymal head sperm showed a lower percentage of sperm with intact membrane and a higher percentage of sperm with intact acrosome (44.9 ± 3.3 and 78.4 ± 1.8 v. 66.4 ± 4.2 and 56.7 ± 4.4%, respectively). Our results demonstrate that, during maturation, feline sperm are subjected to high oxidative stress, as shown by the lipid peroxidation assay, which would lead to structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components, such as mitochondria, and acrosomal impairment. Similar results were found in humans, in which higher levels of oxidative stress occurred in the post-testicular environment. The plasma membrane seems to be more resistant to damages. This may be due to the described rearrangement in the lipid profile occurring during maturation, but studies to test this hypothesis are still underway.

75 INFLUENCE OF CRYOPRESERVATION ON THE SUSCEPTIBILITY OF GOAT SPERM AGAINST DIFFERENT REACTIVE OXYGEN SPECIES

Semen quality after cryopreservation is one of the main limiting factors for the success of artificial insemination in goats. Previous studies indicate that cryo-injuries may be related to the oxidative stress which is caused by the reactive oxygen species (ROS) and leads to structural and functional damages to the sperm. The understanding of sperm oxidative mechanisms in goats may provide information on possible treatments to improve semen quality post cryopreservation. The aim of the present study was to verify the resistance of cryopreserved goat spermatozoa to different reactive oxygen species. Semen samples from 5 adult goats were collected and cryopreserved (Botubov®, Biotech Ltda.). After thawing, samples were washed twice with PBS and incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical) with and without the addition of seminal plasma. Samples were analysed for motility using computer-assisted sperm analysis (CASA); the 3–3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin nigrosin stain, as an index of membrane integrity; the simple stain (Fast green/Bengal rose), as an index of acrosome integrity; sperm chromatin structure assay as an index of DNA fragmentation; and the measurement of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). Results showed that cryopreserved goat sperm after thawing is highly susceptible to the hydroxyl radical. No differences were found on CASA variables between the different ROS. On the other hand, lipid peroxidation and DNA fragmentation were higher for samples treated with hydroxyl radical when compared to samples treated with the other ROS. Furthermore, sperm showing low mitochondrial activity were lower also for samples treated with hydroxyl radical. Negative correlations were found between lipid peroxidation, and most of the variables evaluated by the CASA. A positive correlation was found between the percentage of sperm showing low mitochondrial potential and DNA fragmentation, indicating that impaired mitochondrial activity may be related to an increase on DNA fragmentation. Previous studies indicate that fresh goat semen is highly susceptible to the attack of hydrogen peroxide. We observed that after thawing there is a shift towards a higher susceptibility to the hydroxyl radical. This may indicate that seminal plasma in goats may be an important source of hydroxyl radical scavengers and that, due to the dilution of the seminal plasma with the extender, such antioxidant protection may be impaired. Therefore, an alternative to improve semen quality in cryopreserved goat semen would be the treatment with hydroxyl radical scavengers such as vitamins E and C, reduced glutathione, and other non-enzymatic antioxidants. Thanks to CAPES for financial support.

239 POSSIBLE MECHANISMS OF SPERM DAMAGE CAUSED BY HEAT STRESS IN EUROPEAN BULLS RAISED IN TROPICAL ENVIRONMENTS

Previous studies have indicated that semen of heat-stressed bulls shows impaired mitochondrial activity and high levels of oxidative stress, which may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components (Nichi et al. 2006 Theriogenology 66, 822–828). Disruption of the sperm mitochondria could have a potential damaging effect not only on an individual sperm cell but also on the surrounding cells, especially regarding the sperm membrane, possibly due to the release of a high amount of reactive oxygen species (ROS) produced in this environment (rich in electrons) that would then lead to oxidative stress. To test this hypothesis, semen samples of 11 Simmental bulls kept in tropical environments were collected during the summer months. Semen was evaluated as follows: the 3-3′ diaminobenzidine stain (DAB) as an index of mitochondrial activity, the hypo-osmotic swelling test (HOST) as an index of membrane integrity, measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation, and measurement of the enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase activities. For correlation analysis, the Pearson test was used (variables were transformed when necessary), and for nonparametric variables, the Spearman rank test was used. A high positive correlation was found between sperm cells with highly active mitochondria (DAB class I) and the percentage of cells with intact membrane by HOST (r = 0.93; P < 0.05), and a negative correlation between the latter and the percentage of inactive mitochondria (r = –0.91; P < 0.05), indicating that the higher the percentage of cells showing impaired mitochondrial activity, the higher the percentage of cells with damaged membrane. There was also a positive correlation between TBARS and the percentage of cells with disrupted mitochondria (r = 0.86; P < 0.05), indicating that the higher the percentage of sperm with impaired mitochondrial activity, the higher the oxidative stress. No correlation existed between the enzymatic antioxidants and any of the variables studied. The results indicate that heat stress may lead to an increase in testicular ROS levels, overcoming the seminal antioxidant protection. This, in turn, may cause damage of the mitochondria and a subsequent release of more pro-oxidative substances, and an exponential increase of oxidative stress. Understanding these mechanisms may lead to more tailored antioxidant therapies in the future. The authors thank FAPESP for the scholarship and financial support.

232 INFLUENCE OF SEMINAL PLASMA ON THE SUSCEPTIBILITY OF DOG SPERM AGAINST DIFFERENT REACTIVE OXYGEN SPECIES

Due to the importance of dogs to humans, there is increasing interest in breeders in the use of reproductive biotechnologies. However, most of the biotechnologies would require the removal or dilution of the seminal plasma, which is known to exert both beneficial and deleterious effects on sperm quality. One of the beneficial effects of seminal plasma would be the antioxidant protection because sperm are particularly susceptible to oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in their membrane. An alternative to overcome the injuries caused by oxidative stress is the antioxidant treatment, which requires the identification of those reactive oxygen species (ROS) that are the most deleterious. The aim of this study was to identify the most harmful ROS to dog semen. Semen samples from 6 adult dogs were collected and centrifuged. Seminal plasma (SP) was removed and samples were incubated (1 h, 37°C) with 4 ROS-inducing mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical) alone or with additional SP. Samples were analysed for motility by computer assisted sperm analysis (CASA). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, sperm chromatin structure assay (SCSA) as an index of DNA fragmentation, and measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). Results showed that dog sperm is differentially modulated depending on the presence of SP. In addition, damage to the different sperm structures depended on the different ROS. Samples incubated with SP showed no differences concerning TBARS (1 233 in SP, 1 260 in Tris; P = 0.99). On the other hand, samples incubated without SP showed higher lipid peroxidation when treated with hydroxyl radical compared with the other ROS. Furthermore, although hydroxyl radical mostly altered mitochondrial activity in samples incubated with SP (DAB IV = 4.3%; P < 0.05 against all other ROS), the most significant ROS in samples incubated without SP was hydrogen peroxide (DAB IV = 4.7%; P < 0.05 against all other ROS). Superoxide anion was less harmful to acrosome integrity in samples incubated with SP and to motility in samples incubated without SP. The present results suggest that seminal plasma may play an important role in the susceptibility of dog sperm to oxidative stress. Moreover, the results indicate that different sperm compartments are susceptible to different ROS. It is concluded that the quality of frozen–thawed dog semen may be improved by treating with a combination of different antioxidants to destroy the chain reaction causing the oxidative stress. FAPESP is acknowledged for financial support.

235 MANGALARGA STALLION SPERM IS HIGHLY SUSCEPTIBLE TO THE HYDROXYL RADICAL

Mangalarga, due to its marching abilities, is the mostly widespread and numerous equine breed in Brazil. Furthermore, previous studies indicate that the semen of these horses is particularly susceptible to cryo-injuries. Therefore, the use of chilled semen is crucial when employing reproductive biotechnologies. However, previous studies indicate that chilled semen is highly impaired by the oxidative stress, which is caused by reactive oxygen species (ROS). An alternative to overcome the injuries caused by oxidative stress is antioxidant treatment, which requires the identification of those ROS that are the most deleterious. The aim of this study was to identify the most harmful ROS to Mangalarga sperm. Semen samples from 4 horses were collected, mixed with chilling media (Equimix®, Nutricell) and transported to the laboratory at 15°C. Samples were then incubated (1 h, 37°C) with 4 ROS inducing mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical). Samples were analysed for motility using computer assisted sperm analysis (CASA). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, sperm chromatin structure assay (SCSA) as an index of DNA fragmentation, and the measurement of thiobarbituric acid reactive substances (TBARS) an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). Results showed that Mangalarga sperm is highly susceptible to the hydroxyl radical. Samples treated with this ROS showed a lower percentage of sperm with high mitochondrial activity then samples treated with hydrogen peroxide (24.6 ± 5.9 v. 43.7 ± 6.8%, respectively). Similarly, lipid peroxidation (TBARS) was higher in samples treated with hydroxyl radical when compared with those treated with both superoxide anion and hydrogen peroxide (2037.7 ± 154.8, 681.2 ± 170.1, and 789.4 ± 124.5 ng/106 sperm). In addition, for all variables analysed using CASA except for ALH and BCF, samples treated with hydroxyl radical showed decreased quality when compared with the other samples. A positive correlation was found between TBARS and mitochondrial activity, indicating that the higher the sperm susceptibility of sperm against oxidative stress, the lower the mitochondrial activity. Level of TBARS also correlated negatively with most of the variables evaluated by CASA. The present results suggest that Mangalarga sperm is highly susceptible to the hydroxyl radical, a mechanism apparently related to the mitochondrial activity. Therefore, an alternative to overcome the deleterious influence of oxidative stress in semen of Mangalarga stallions would be the treatment with hydroxyl radical scavengers such as vitamins C and E, reduced glutathione, and other nonenzymatic antioxidants. The authors acknowledge Nutricell for the media used and CAPES for financial support.

317 RESISTANCE AGAINST DIFFERENT REACTIVE OXYGEN SPECIES IN BOVINE EPIDIDYMAL SPERM UNDER DISTINCT MATURATION STATUS

Oxidative stress is caused by reactive oxygen species (ROS) that may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components. Evidence indicates that oxidation products are also deleterious to biological systems. Spermatozoa are particularly susceptible the oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in its membrane. The mechanisms by which sperm acquire antioxidant capacity are still not completely elucidated. The aim was to study the resistance of sperm derived from different epididymal compartments (caudae and head) to the different ROS and to the lipid peroxidation product malondialdehyde (MDA). Epididymal sperm samples from 4 testicles were collected from the head and caudae epididymides. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical), and MDA. Samples were analyzed for 3-3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin nigrosin stain, as an index of membrane integrity; the simple stain (fast green/Bengal rose), as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Pearson correlation). Results showed that immature sperm (head epididymides) were significantly more susceptible to the MDA and to the hydroxyl radical in all studied variables, especially acrosomes, membranes, and mitochondrial potential. Semen derived from the caudae epididymides was more susceptible to the hydrogen peroxide and to the MDA, especially regarding mitochondrial potential. In semen from the epididymal head, a positive correlation was found between TBARS and sperm showing no mitochondrial potential (r = 0.66, P = 0.01). On the other hand, negative correlations were found between TBARS and sperm with damaged acrosome and membrane (r = -0.63, P = 0.01 and r = -0.58, P = 0.02, respectively) in samples collected from the caudae epididymides. The present results suggest that sperm susceptibility to the attack of ROS is different throughout maturation. Although immature sperm are more susceptible to the hydroxyl radical, mature sperm are more susceptible to the hydrogen peroxide. Furthermore, MDA, a product of lipid peroxidation, is also deleterious to the sperm, indicating that once oxidative stress starts, further damage may be caused by their products. The authors thankNutricell for the media used in the experiment andFAPESP for financial support (process #06/05736-1).

34 OVINE SPERM IS HIGHLY SUSCEPTIBLE TO ATTACK BY HYDROGEN PEROXIDE

Ram sperm membrane displays a particular lipid composition, especially regarding the high quantity of polyunsaturated cholesterol. This trait improves membrane fluidity; however, the spermatozoa become more susceptible to the attack of reactive oxygen species (ROS), which may lead to structural and functional damage, impairment or even impeded fecundity. The aim of the present experiment was to study the resistance of ovine spermatozoa to different ROS. Sperm samples from 4 rams were collected using an artificial vagina. Sperm samples were then incubated (1 h, 37°C) with four ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produces hydroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were analysed using the 3-3′ diamino benzidine (DAB) stain as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity; the simple stain (fast green/Bengal rose) as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Results showed that acrosome and membrane integrity as well as mitochondrial potential were highly impaired by hydrogen peroxide, which was not the case for the other ROS (Table 1). Surprisingly, TBARS production was higher in samples incubated with ascorbate and ferrous sulfate (hydroxyl radical). Furthermore, sperm showing impaired mitochondrial potential were negatively correlated with membrane and acrosome integrities (r = -0.83, P < 0.0001 and r = -0.62, P = 0.01, respectively). Results of the present experiment suggest that semen of rams is extremely susceptible to attack by hydrogen peroxide. However, the mechanism by which this substance impairs sperm quality apparently does not involve oxidative stress, because no increase in TBARS was observed. Despite the necessity of further studies to investigate how hydrogen peroxide negatively influences sperm function, the use of catalase and glutathione peroxidase, important hydrogen peroxide scavengers, appears to be an alternative to improve the quality of ram sperm. Table 1.Effect of different reactive oxygen species in semen of rams The authors thank Nutricell for the media used in the experiment and CAPES for financial support.

323 POSSIBLE BENEFICIAL EFFECT OF REACTIVE OXYGEN SPECIES ON BOAR SPERM FUNCTION

Boar spermatozoa are sensitive to the attack of reactive oxygen species (ROS) due to the high content of unsaturated fatty acids in its plasma membrane and the relative low antioxidant capacity of boar seminal plasma. The benefical effect of antioxidant supplementation to the semen of boars is still a matter of debate, with different results in the literature, probably due to the empirical methods used to determine the more adequate antioxidant, and to the physiological role or the ROS. The aim of the present experiment was to study which ROS is the most deleterious to the boar sperm. Sperm samples from 4 boars were collected by digital manipulation. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produced hydroxyl radical), and malondialdehyde (lipid peroxidation product). Samples were analyzed for the 3-3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin/nigrosin staining, as an index of membrane integrity; the simple stain (fast green/Bengal rose), as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation. Results showed that the boar sperm is relatively resistant to all ROS studied in the present experiment. Only for the percentage of sperm showing intact membrane, a difference was found between incubation with superoxide anion and hydroxyl radical (Table 1). The TBARS production was higher in samples incubated with hydroxyl radical, indicating that these samples were more susceptible to the attack of this ROS. Surprisingly, a positive correlation was found between TBARS and membrane integrity (r = 0.51, P = 0.04), indicating that samples more susceptible to the oxidative stress showed higher membrane integrity. A hypothesis to explain such conflicting results would be the physiological role of the ROS on several spermatic events, especially in this particular species. It is well known that boar semen, in order to achieve ideal motility, requires aerobic conditions. Further studies are necessary to study the effect of each ROS on each physiological or pathological event on boar sperm function. Table 1.Effect of different ROS in boar semen The authors thank Nutricell for the media used in the present experiment.

Protective effect of Phyllanthus fraternus against allyl alcohol-induced oxidative stress in liver mitochondria

The effect of administration of allyl alcohol on the oxidative stress and the protective effect due to administration of an aqueous extract of Phyllanthus fraternus against allyl alcohol-induced damage in liver mitochondria were studied. When rats were treated with allyl alcohol, the rate of mitochondrial respiration was decreased significantly with both NAD +- and FAD-linked substrates. The respiratory control ratio, an index of membrane integrity and the P/O ratio, a measure of phosphorylation efficiency also decreased significantly. There was a significant increase in the lipid peroxide level and the protein carbonyl content. A significant decrease was observed in the total sulphydryl groups and a significant increase in the generation of superoxide radicals. Administration of rats with an aqueous extract of Phyllanthus fraternus (100 mg/kg) prior to allyl alcohol administration showed protection of 72, 40 and 80% using glutamate + malate (NADH oxidation) and 77, 54 and 20% using succinate as substrate on state 3, RCR and P/O ratio, respectively. The protection on lipid peroxide level was 88 and 91% in homogenate and mitochondria, respectively. In case of protein carbonyls, total sulphydryl groups and on the generation of superoxide radicals the protection was 99, 59 and 53%, respectively.

Correlation between sperm membrane destabilization by heparin and aniline blue staining as membrane integrity index.

Acidic aniline blue stain (AAB) was studied in relation to sperm membrane destabilization and nuclei decondensation by heparin. Untreated spermatozoa smears stained with AAB or vital stain shows 28.4% of stained and 71.6% of unstained nuclei. This behavior was also observed when incubation was done in the presence of 5 mM glutathione (GSH) used alone. In the presence of 21.6 microM heparin, staining of sperm cells commenced 10 min after heparin addition and was dependent on the incubation time. During the experiment 12.3% of the total cholesterol content and 20 micrograms protein/10(8) sperm cells were released. In the presence of 21.6 microM heparin-5 mM/GSH, swelling of sperm nuclei reach 95% after 150 min incubation. When this experiment was run along with AAB, the same average (45%) was seen in the first 30 min, which gives plenty of time to trigger the nuclei's decondensation mechanism. The percentage of stained cells was of 71%, indicating that the histone is not completely replaced, and insuring a positive reaction with AAB stain. It would appear that AAB stain can be used as a membrane integrity index to confirm the destabilization effect of heparin on the sperm membrane.

Protective Effect of Phyllanthus fraternus against Thioacetamide-Induced Mitochondrial Dysfunction.

The effect of administration of thioacetamide on rat liver mitochondrial functions and the protective effect of an aqueous extract of Phyllanthus fraternus against thioacetamide-induced damage were studied. When rats were treated with thioacetamide, the rate of mitochondrial respiration was decreased significantly with both NAD+ linked and FAD linked substrates, and the respiratory control ratio, an index of membrane integrity and the P/O ratio, a measure of phosphorylation efficiency, decreased significantly. Also, there was a significant decrease in the activities of NADH dehydrogenase, succinate cytochrome c reductase, and cytochrome oxidase; whereas succinate dehydrogenase was not affected. A significant decrease was seen in membrane potential and in the level of mitochondrial ATP. There was a significant decrease in the levels of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin by thioacetamide treatment. The lipid peroxide level increased significantly in thioacetamide-treated rats. Administration of P. fraternus prior to thioacetamide treatment relieved the inhibition of all the parameters studied, and brought down the lipid peroxide levels significantly in liver homogenates and mitochondria. This study shows that P. fraternus protects against thioacetamide-induced toxicity by its ability to suppress the elevated lipid peroxide levels in the mitochondrial membrane.

Proline Accumulation as Drought-tolerance Selection Criterion: its Relationship to Membrane Integrity and Chloroplast Ultrastructure in Nicotiana tabacum L.

The value of proline accumulation as a criterion in selecting for drought-tolerance, was evaluated in four tobacco cultivars of differing drought-tolerance. Proline determination, measurement and calculation of a membrane integrity index (MII), and ultrastructural observations were conducted simultaneously under controlled environmental conditions during the stress period. Water stress of increasing severity (0.2-0.3 MPad-1) that ranged from light (-0.52MPa) to severe (-2.51 MPa) was induced by withholding water. A substantial accumulation of proline was observed in all four cultivars, the extent of which correlated positively with their individual drought-tolerance. Ultrastructural observation indicated that the drought-tolerant cultivars mobilized the, more than adequate, store of starch to a greater extent than the drought-sensitive cultivars during stress. Water stress-induced membrane damage occurred earlier and was much more severe in the drought-sensitive cultivars, as their MII-values already rose to between 20.4% and 23.4% at a ΨL of -1.27 MPa, while those of the drought-tolerant cultivars only reached values of 18.5% and 20.9% at a ΨL of -1.67 MPa. The ΨL at which proline levels start to increase dramatically (the shorter the response time the better) and the end concentrations of proline accumulated, are advocated as criteria to be used in selecting for drought-tolerant tobacco genotypes as early as the F1- or F2-generations.

Mucosal glutamine utilization after small-bowel transplantation: An electrophysiologic study

A short course of FK 506 after small bowel transplantation averts rejection in the rat and achieves indefinite survival of the recipient whose nutritional status is dependent on the function of the intestinal graft. Ex vivo electrophysiologic studies using the Ussing Cell were conducted to delineate functional competence of the graft by evaluating mucosal ion transport and glutamine utilization. Orthotopic small-bowel transplantation was performed in Lewis (LEW) rats as recipients of either Brown-Norway (BN) allografts or LEW syngeneic grafts. Allograft recipients received FK 506 either as a short course (2 mg/kg on Day 0–4 after transplantation) or continuously (2 mg/kg Day 0–4, then 0.5 mg/kg weekly). Ileal mucosa was harvested from small bowel grafts 9 and 60 days after transplantation and mounted in the Ussing Cell containing Hanks' balanced salt solution with/without l-glutamine (20 m M). Transmembrane potential difference (PD), which represents mucosal active ion transport, and mucosal resistance, an index of membrane integrity, were recorded. Nine days after transplantation, mucosal PD was the same in the ileum from syngeneic grafts, allografts treated with FK 506 and normal LEW and BN rats, and the addition of glutamine increased PD equally in all groups. In comparison, PD was markedly decreased in allografts undergoing rejection, and the glutamine response was blunted. Sixty days after transplantation, mucosal PD was reduced in allografts treated with a short course of FK 506, but normal in allografts receiving continuous immunosuppression with FK 506 and in syngeneic grafts. A decrease of mucosal resistance was not a feature of rejection nor a sequel of limited FK 506 therapy. Our data indicate that allograft rejection results in a significant decrease in mucosal PD and a poor response to glutamine. Control of rejection by FK 506 preserves normal electrophysiologic responses of the allograft mucosa.