Ameliorative effect of supplementation with l-glutamine on oxidative stress,DNA damage,cell viability and hepatotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat hepatocyte cultures

The most potent of the dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a persistent and ubiquitous environmental contaminant. And the health impact of exposure to TCDD is of great concern to the general public. Recent data indicate that l-glutamine (Gln) has antioxidant properties and may influence hepatotoxicity. The objective of the present study was undertaken to explore the effectiveness of Gln in alleviating the hepatotoxicity of TCDD on primary cultured rat hepatocytes. Gln (0.5, 1 and 2 mM) was added to cultures alone or simultaneously with TCDD (0.005 and 0.01 mM). The hepatocytes were treated with TCDD and Gln for 48 h. Then cell viability was detected by [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT) assay and lactate dehydrogenase (LDH) release, while total antioxidant capacity (TAC), total glutathione (TGSH) and total oxidative stress (TOS) levels were determined to evaluate the oxidative injury. The DNA damage was also analyzed by liver micronucleus assay (MN) and 8-oxo-2-deoxyguanosine (8-OH-dG). The results of MTT and LDH assays showed that TCDD decreased cell viability but not l-glutamine. TCDD also increased TOS level in rat hepatocytes and significantly decreased TAC and TGSH levels. On the basis of increasing doses, the dioxin in a dose-dependent manner caused significant increases of micronucleated hepatocytes (MNHEPs) and 8-OH-dG as compared to control culture. Whereas, in cultures exposured with Gln alone, TOS levels were not changed and TAC and TGSH together were significantly increased in dose-dependent fashion. The presence of Gln with TCDD modulated the hepatotoxic effects of TCDD on primary hepatocytes cultures. Noteworthy, Gln has a protective effect against TCDD-mediated DNA damages. As conclusion, we reported here an increased potential therapeutic significance of l-glutamine in TCDD-mediated hepatic injury for the first time.     

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.     

Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue

For clinical utility, cardiac grafts should be thick and compact, and contain physiologic density of metabolically active, differentiated cells. This involves the need to control the levels of nutrients, and most critically oxygen, throughout the construct volume. Most culture systems involve diffusional transport within the constructs, a situation associated with gradients of oxygen concentration, cell density, cell viability, and function. The goal of our study was to measure diffusional gradients of oxygen in statically cultured cardiac constructs, and to correlate oxygen gradients to the spatial distributions of cell number and cell viability. Using microelectrodes, we measured oxygen distribution in a disc-shaped constructs (3.6 mm diameter, 1.8 mm thickness) based on neonatal rat cardiomyocytes cultured on collagen scaffolds for 16 days in static dishes. To rationalize experimental data, a mathematical model of oxygen distribution was derived as a function of cell density, viability, and spatial position within the construct. Oxygen concentration and cell viability decreased linearly and the live cell density decreased exponentially with the distance from the construct surface. Physiological density of live cells was present only within the first 128 microm of the construct thickness. Medium flow significantly increased oxygen concentration within the construct, correlating with the improved tissue properties observed for constructs cultured in convectively mixed bioreactors.     

Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia

Cryopreservation has become an integral component of any cell transplantation technique helping to overcome the issues associated with known spatial and temporal barriers between donor and recipient. The aim of this study was to develop a protocol for large quantity cryopreservation of bovine testicular germ cells. The impact of 3 different packaging methods (5 ml semen straw, 20 ml freezing bag and 1.5 ml cryovial) and varying cell densities (3 × 10⁶, 9 × 10⁶, or 18 × 10⁶ cells/ml) on the survival of testis germ cells was examined. Cells processed in 5 ml semen straws had a significantly higher viability (70.7 ± 1.2%, P < 0.05) compared to those cells in 20 ml freezing bags (46.7 ± 0.1%) or 1.5 ml cryovials (46.3 ± 2.2%). For 5 ml straws, a 20 min cooling prior to cryopreservation resulted in a higher post thaw viability (73.2 ± 0.6%) than a 10 min cooling (56.0 ± 2.2%), while the density of the cell suspension did not impact on post thaw viability. Thus cryopreservation of testicular germ cells in 5 ml straws at a density between 3 × 10⁶ and 18 × 10⁶ cells/ml in liquid nitrogen vapour for 20 min cooling appears to be a simple and practical way to preserve cells. Subsequent testing of frozen/thawed cells exhibited viable cultures and retained the ability to proliferate. The freezing protocol does not preferentially preserve type A spermatogonia. However, the cell surface properties of somatic cells appear to be affected by the freezing procedure and therefore the frozen/thawed cells are less suitable for enriching type A spermatogonia by differential plating.     

Effects of U83836E on nerve functions, hyperalgesia and oxidative stress in experimental diabetic neuropathy

Oxidative stress has been implicated to play an important role in the pathogenesis of diabetic neuropathy, which is the most common complication of diabetes mellitus affecting more than 50% of diabetic patients. In the present study, we have investigated the effect of U83836E [(-)-2-((4-(2,6-Di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl)methyl)-3,4-dihydro-2,3,7,8-tetramethyl-2H-1-benzopyran-6-ol, 2HCl], a potent free radical scavenger in streptozotocin (STZ)-induced diabetic neuropathy in rats. STZ-induced diabetic rats showed significant deficit in motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and thermal hyperalgesia after 8 weeks of diabetes induction, indicating development of diabetic neuropathy. Antioxidant enzyme (superoxide dismutase and catalase) levels were reduced and malondialdehyde (MDA) levels were significantly increased in diabetic rats as compared to the age-matched control rats, this indicates the involvement of oxidative stress in diabetic neuropathy. The 2-week treatment with U83836E (3 and 9 mg/kg, i.p.) started 6 weeks after diabetes induction significantly ameliorated the alterations in MNCV, NBF, hyperalgesia, MDA levels and antioxidant enzymes in diabetic rats. Results of the present study suggest the potential of U83836E in treatment of diabetic neuropathy.     

Influence of intracellular trehalose concentration and pre-freeze cell volume on the cryosurvival of rapidly frozen human erythrocytes

Significant interest exists in the application of trehalose, which has low permeability to the phospholipid bilayer, as a non-toxic intracellular cryopreservative for mammalian cells. Introduction of between 8 ± 3 mM and 266 ± 22 mM trehalose into human erythrocytes using the membrane permeabilizing polymer PP-50 allowed investigation of the relationship between intracellular trehalose concentration, pre-freeze cell volume, and cryosurvival. Cellular cryosurvival increased approximately linearly with pre-freeze cell volume up to the normal volume of fresh cells; diminished cell survival correlated with subnormal pre-freeze cell volume in some cases even at >100 mM intracellular trehalose concentration. Uptake of >200 mM trehalose in cells with near-normal cell volume facilitated enhancement of cellular cryosurvival by up to 15 ± 5%.     

Effects of temporary calf removal and eCG on pregnancy rates to timed-insemination in progesterone-treated postpartum Nellore cows

The objective was to evaluate the effects of temporary calf removal (TCR), eCG administration, or both, in a progesterone-based protocol. Suckled Nellore cows (40-80 d postpartum, n=443) with body condition scores from 2.0 to 3.5 (5-point scale) on three farms were all given a synchronizing protocol (PEPE). At the start (designated Day 0), cows were given an intravaginal device (1.0 g of progesterone) and 2.5mg of estradiol benzoate (EB) im. On Day 8, the device was removed and cows were given PGF(2 alpha) (150 microg of D-cloprostenol im), followed in 24h by 1.0mg EB im, and 30-36 h thereafter, fixed-time AI. The design was a 2 x 2 factorial; main effects were TCR (54-60 h; from device removal to FTAI) and eCG treatment (300 IU im, concurrent with PGF(2 alpha)). Transrectal ultrasonography was done on Days -10 and 0 to detect anestrus (absence of a CL at both examinations) and approximately 30 d after FTAI (pregnancy diagnosis). Data were analyzed by logistic regression. The following variables did not significantly affect pregnancy rates: farm, postpartum interval, cyclicity, inseminators, and semen (sire). Overall, 77% of the cows were deemed anestrus. Pregnancy rates were similar (P>0.05) among treatment groups: Control (54/108=50.0%), TCR (44/106=41.5%), eCG (63/116=54.3%), and TCR+eCG (49/113=43.4%). Pregnancy rate was higher in multiparous than primiparous cows (186/360, 51.7% vs. 24/83, 28.9%, P<0.01), but was not significantly affected by cyclicity status or body condition score. In conclusion, temporary calf removal, eCG, or both, did not significantly increase pregnancy rate to timed-insemination in a progesterone-based synchronization protocol in postpartum Nellore cows with acceptable body condition.     

Effects of hydrolyzed yeast supplementation in calf starter on immune responses to vaccine challenge in neonatal calves

The effects of hydrolyzed yeast supplementation on growth performance, health and immune-physiological parameters in neonatal calves challenged with vaccine were investigated. Twelve Holstein calves were started in the experiment at 2 ± 1 days of age and were studied for 35 days. Calves were randomly assigned to each of two dietary treatments, a control (CON) and hydrolyzed yeast (HY) group. The calves in the HY group received control calf starter supplemented with 0.2% HY. All calves were given calf starter ad libitum for 5 weeks starting in week 1. Calves were also given whole milk according to a step-down milking protocol. In order to induce immune responses, all calves were challenged with Hog cholera and Erysipelothrix insidiossa live vaccines by intramuscular injection at 3 weeks of age. Growth performance and feed intake were not affected by dietary treatment throughout the experimental period, except that the HY group had significantly higher (P < 0.05) milk intake than did the CON group at 3 weeks of age. Calves in the HY group showed significantly better (P < 0.05) fecal and health scores at 3 weeks compared to those in the control group. After vaccine challenge, neutropenia, lymphophilia and thrombocytopenia were observed in the CON group, but calves in the HY group did not show significant changes of leukocytes. The average concentration of serum haptoglobin in the HY group was significantly higher (P < 0.05) at 1 and 3 days post-vaccine challenge (DPVC) than that of CON group. Feeding HY supplemented calf starter resulted in a higher (P < 0.05) relative amount of bacterial and viral - specific IgA than in the CON group at 5 DPVC. Although the percentage of CD4+ T cells was significantly (P < 0.05) higher in the HY group than in the CON group at -2 DPVC, significant differences between groups after vaccine challenge was not observed during the experimental period. These results suggest that 0.2% HY supplementation in calf starter can improve the health status and immune-related serum protein production and affect blood cell composition in neonatal calves after vaccine challenge.     

Effects of vitamin E supplementation in the extender on frozen-thawed bovine semen preservation

The maturing sperm cells discard the majority of their cytoplasm during the final stages of spermatogenesis and lose some of their defense enzymes. The purpose of this study was to investigate the effects of vitamin E supplementation on standard semen quality parameters and antioxidant activities of frozen-thawed bovine sperm. Vitamin E was added at concentrations of 0.5, 1.0, 1.5 and 2.0 mg/ml to bovine semen cryoprotective medium. The results showed that the sperm motility and VSL, STR values in the extender supplemented with 1.0 and 1.5 mg/ml of vitamin E, were significantly higher than that of other concentrations (P < 0.05). The percentages of acrosome-intact and membrane-intact sperm were significantly improved (P < 0.05) by supplementing with 1.5 mg/ml of vitamin E. In biochemical assays, the extender supplemented with vitamin E did not exhibit significant improvement in SOD (superoxide dismutase) levels, compared with the control (P > 0.05). Compared with other groups, CAT (catalase) levels were demonstrated to be greater with the supplementation of vitamin E at 1.0 and 1.5 mg/ml (P < 0.05). The extender supplemented with 1.5 mg/ml of vitamin E caused the highest levels of glutathione peroxidase (GSH-Px), compared with other groups (P < 0.05). The glutathione (GSH) activity was significantly higher with the supplementation of 0.5, 1.0 and 1.5 mg/ml of vitamin E, compared with 2.0 mg/ml in the vitamin E group and control (P < 0.05). Moreover, increasing the doses of vitamin E decreased sperm antioxidant activities, the extender supplemented with 2.0 mg/ml of vitamin E, caused the lowest levels of GSH-Px and GSH activities, compared with other treatment groups (P < 0.05). In conclusion, the beneficial effects of vitamin E noted in this study can be attributed to the antioxidant characteristics. Vitamin E supplementation in the extender reduced the lipid peroxidation potential and improved semen quality during freezing-thawing. More researches are needed to evaluate and understand the precise physiological role of vitamin E in reproduction.     

Effects of lycopene supplementation on antioxidant status,oxidative stress,performance and carcass characteristics in heat-stressed Japanese quail

Lycopene, a carotenoid present predominantly in tomatoes, is one of the most efficient antioxidants. This experiment was conducted to evaluate the effects of dietary lycopene supplementation on performance, carcass characteristics, biomarkers of oxidative stress (malondialdehyde (MDA) and homocysteine), and concentrations of vitamins C, E, A, cholesterol, triglyceride, and glucose in Japanese quails (Coturnix coturnix Japonica) exposed to high-ambient temperature of 34 °C. Two hundred and forty Japanese quails (10 day-old) were randomly assigned to eight treatment groups consisting of 10 replicates of three birds. The birds were kept at a temperature-controlled room at 22 °C (Thermoneutral, TN groups) or 34 °C (for 12 h/day; 09.00 am–05.00 pm; Heat stress, HS groups). Birds were fed either a basal (control) diet (TN and HS) or the basal diet supplemented with 50, 100 or 200 mg of lycopene/kg of diet. Lycopene supplementation linearly increased feed intake (P=0.05$P=0.05$), live weight gain (P=0.01$P=0.01$), feed efficiency (P=0.01$P=0.01$) and cold carcass weight (P=0.01$P=0.01$) and yield (P=0.05$P=0.05$) under heat stress conditions but did not show the same effect at thermoneutral conditions (P>0.05$P>0.05$). The interaction Serum vitamin C, E, and A (P=0.01$P=0.01$) concentrations increased linearly in birds reared at high temperature while non-significant changes occurred at TN groups. Homocysteine level in serum and malondialdehyde (MDA) levels in serum, liver, and heart (P=0.001$P=0.001$) linearly decreased in all birds of both TN and HS groups as dietary lycopene supplementation increased. Heat stress-induced increase in serum cholesterol (P=0.01$P=0.01$), triglycerides (P=0.05$P=0.05$) and glucose (P=0.01$P=0.01$) concentrations were linearly reversed by lycopene supplementation. Supplementation of lycopene increased the HDL concentration whereas, the VLDL and LDL concentrations reduced with lycopene supplementation (P=0.01$P=0.01$, linear), particularly at a dietary concentration of 200 mg/kg. Lycopene could not be detected in control birds while a linear increase was observed in the sera of lycopene supplemented birds The results of the study indicate that lycopene supplementation attenuated the increase in oxidative stress and depletion in antioxidants caused by heat stress in Japanese quails.     

Effects of Lonomia obliqua caterpillar venom upon the proliferation and viability of cell lines

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology, the caterpillar Lonomia obliqua has become the focus of toxicological studies due to recent findings about its venom constituents. The objective of this study was to investigate the effects of L. obliqua venom upon the viability and the proliferation of different cell lineages and to propose mechanisms for the herein observed induction of cell proliferation in glioma cell lines. MTT analyses indicate that L. obliqua venom increases the viability of tumor cell lines U138-MG and HT-29; on the other hand, it inhibits the viability of V-79 nontumor cells. Cell count based on the trypan blue exclusion method suggests a proliferating activity of the venom upon U138-MG cells. Exposure of U138-MG to crude venom extract led to a decrease in the production of nitric oxide, and activation of the cAMP signaling pathway inhibited the effects of the venom, indicating that these mechanisms may influence cell proliferation triggered by the venom. Despite the proliferative effects of crude venom on U138-MG and HT-29 cell cultures, a protein purified from L. obliqua hemolymph previously shown to have cytoprotective activity had no effect on U138-MG and HT-29; however, this same protein increased the viability of V-79 cells that had previously been exposed to the cytotoxic activity of the crude venom extract. This study indicates that the venom and the antiapoptotic protein act differently and have different effects on cell cultures, depending on the cell line analyzed. Biomolecules displaying either mitogenic or cytotoxic activities are of great biotechnological interest. Further studies encompassing the purification of active principles from L. obliqua venom are necessary to further elucidate its effects on different cell types.     

Effect of Echinacea augustifolia extract on cell viability and differentiation in mammary epithelial cells

Echinacea spp. are popularly used as an herbal medicine or food supplement for enhancing the immune system and activating biological property in different tissues. In this study we show the biological effect of Echinacea augustifolia extract on cell viability and cell differentiation in mammary epithelial cell lines. These effects have been observed in two different cell line derived from mouse (HC11) and bovine (BME-UV). Echinacea extract enhanced cell liability from 100 to 1000 ng/ml in association with growth factors, epidermal growth factor (EGF) or insulin, but also without EGF (p<0.05) up to 37% vs. control. This effect may be modulated by MAPK and Akt activation that Echinacea extract treatment increased and/or by a reduction of caspase 3 activity, showed a dose–response decrease after Echinacea treatment. Finally Echinacea extract was able to increase (p<0.05) at 100 ng/ml β-casein expression in association with PRL (5 μg/ml). These data demonstrate that Echinacea angustifolia extract can stimulate mammary epithelial cell physiology and may be considered a candidate to support mammary gland activity during a mammogenetic and lactogenetic state.     

Effects of osmotic pressure on motility, plasma membrane integrity and viability in fresh and frozen-thawed buffalo spermatozoa

In the first experiment, osmotic pressure of semen and seminal plasma in a semen sample from each of the 20 mature Nili-Ravi buffalo bulls was determined. In the second experiment, effects of osmotic pressure on motility (%), plasma membrane integrity (%) and viability (%) in fresh and frozen-thawed semen samples from each of the seven mature Nili-Ravi buffalo bulls was determined. In the first experiment, seminal plasma was harvested by centrifuging semen at 400 × g for 10 min at 37°C and osmotic pressure was determined using an osmometer. In the second experiment, motility (%) was assessed in fresh and frozen-thawed (37°C for 30 s) semen samples using a phase-contrast microscope (×400). Plasma membrane integrity (%) was determined by mixing 50 μl each of fresh and frozen-thawed semen with 500 μl of solution having an osmotic pressure of 50, 100, 150, 190 or 250 mOsm/l (hypotonic treatments of fructose + sodium citrate) and incubating at 37°C for 1 h. Viability (%) of fresh and frozen-thawed spermatozoa before and after challenging them to osmotic pressure (hypotonic treatments) was assessed using supravital stain under a phase-contrast microscope (×400). In the first experiment, the mean ± s.e. osmotic pressures of the buffalo semen and seminal plasma were 268.8 ± 1.17 and 256.0 ± 1.53 mOsm/l, respectively. In the second experiment, motility (%) decreased (P < 0.05) in frozen-thawed semen samples as compared with fresh semen (60.1 ± 1.34 v. 81 ± 1.57, respectively). The plasma membrane integrity (%) and magnitude of osmotic stress in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50, 100, 150 and 190 mOsm/l as compared with 250 mOsm/l. Loss of viability (%) in fresh and frozen-thawed semen samples was higher (P < 0.05) at 50 mOsm/l (59% in fresh, 70% frozen thawed) as compared with other osmotic pressures, while it was lowest at 250 mOsm/l (4.1% for fresh, 9.7% frozen thawed). In conclusion, osmotic pressure of Nili-Ravi buffalo semen and seminal plasma is determined. Furthermore, variation in osmotic pressure below 250 mOsm/l is not favorable to fresh and frozen-thawed buffalo spermatozoa.     

Effects of Onosma armeniacum root extract on ethanol-induced oxidative stress in stomach tissue of rats

This study investigated the effects of Onosma armeniacum K. (Boraginaceae) root extract (AR-1) on ethanol-induced stomach ulcers, and on some oxidant and antioxidant parameters, in stomach tissue in rats. The results obtained showed that AR-1 significantly inhibited ethanol-induced ulcers at 25, 50, 100 and 200 mg/kg doses. We found that 50, 100 and 200 mg/kg doses of AR-1 inhibited ulcers more effectively than did ranitidine. AR-1 at doses of 25, 50, 100 and 200 mg/kg significantly prevented the decrease in total glutathione (tGSH) level which occurs in damaged stomach tissues of rats given ethanol (control group). Only a 100 mg/kg dose of AR-1 significantly increased the glutathione S-transferase (GST) level in stomach tissue compared to the control. All doses of AR-1 except the 25 mg/kg dose eliminated the decrease in the superoxide dismutase (SOD) level in the stomach tissue of rats given ethanol. While all doses of AR-1 decreased malondialdehyde (MDA) levels significantly; all doses AR-1 except 25 mg/kg decreased myeloperoxidase (MPO) levels significantly compared to the control. The effect of AR-1 on catalase (CAT) activity was insignificant at all doses. AR-1 significantly increased nitric oxide (NO) levels at 50, 100 and 200 mg/kg doses compared to the control. Our results indicate that the protection of some antioxidant mechanisms and the inhibition of some oxidant mechanisms have a role in AR-1's antiulcer effect mechanism.     

The influence of various storage conditions on cell viability in amniotic membrane

Up to now freeze-dried, gamma-sterilised or glycerol-preserved amniotic membranes (AMs) have widely been used in the field of ophthalmology and wound care (e.g. leg ulcers, burns). After some preservation processes in use, like freeze-drying or glycerol-preserving, the cells in the AM are no longer viable. Within this study we evaluated the influence of different short-term and long-term storage conditions on cell viability in AM. Therefore AMs from cesarean section placentae were washed and biopsied to evaluate the microbiological status and to determine the viability of the tissue. Additionally, viability under various storage conditions was examined by assessment of mitochondrial activity. Preservation included temperatures above and below 0°C as well as various media compositions. As expected, cell viability in amnion decreases during storage, in fact the effect was more pronounced when stored frozen, but the higher viability of amnion obtained by storage above 0°C with medium is associated with the limitation to a short period of storage of about 28 days. The evaluated preservation methods are the basis for future non-clinical in-vivo studies in which the possible benefit of amnion as a viable biomaterial in wound healing will be investigated. A part of this work was presented at the World Congress on Tissue Banking in Rio in May 2005 and was honoured by the Poster Award Commission.     

Effects of non-esterified fatty acids on bovine granulosa cells and developmental potential of oocytes in vitro

Cryopreservation of bull semen is sub-optimal, causing cell death of a majority of spermatozoa. Even the surviving cells are affected post-thaw, either structurally or functionally. The aim of this study was to investigate the sequence of events that take place when sperm plasma membrane and acrosome deteriorate during a 4 h incubation period post-thaw, with special attention paid to the acrosome status of dying cells. Frozen-thawed semen of six AI dairy bulls was used. Three straws per batch were pooled and incubated at 37 °C. Sub-samples were taken at 30 min intervals and stained with SYBR 14, propidium iodide (PI) and phycoerythrin-conjugated peanut agglutinin (PE-PNA). Plasma membrane and acrosome integrity were measured by flow cytometry. The experiment was repeated three times. Immediately after thawing, only 3.45% of the dying cells showed acrosomal exocytosis. This number increased dramatically during incubation, reaching 67% after 4 h. Within the intact cell population, the overall decrease in viability and acrosome integrity was kept at five percentage points. Flow cytometry and the triple fluorochrome combination presented a detailed picture of the time course in plasma membrane and acrosome deterioration of frozen-thawed bull semen. The results are expected to be useful for monitoring new cryopreservation protocols.     

The effect of iron and/or zinc diet supplementation and termination of this practice on the antioxidant status of the reproductive tissues and sperm viability in rats

AimsThe aim of this study was to investigate the effect of iron or/and zinc supplementation and termination of this treatment on the antioxidant defence of the male reproductive system and sperm viability in rats.MethodsThe study consisted of 3 stages: I) 4-week adaptation to the diets (C-control or D-iron deficient); II) 4-week iron and/or zinc supplementation (10-times more than in the C diet of iron: CSFe, DSFe; zinc: CSZn, DSZn; or iron and zinc: CSFeZn, DSFeZn; and III) 2-week post-supplementation period (the same diets as during stage I). Parameters of antioxidant status (total antioxidant capacity and SOD, GPx, and CAT activiy), oxidative damage (lipid and protein peroxidation), and sperm viability were measured.ResultsSimultaneous iron and zinc supplementation compared to iron supplementation (CSFeZn vs CSFe) increased SOD activity in the testes and decreased the level of malondialdehyde in the epididymis after stage II, and increased the percentage of live sperm after stage III. After discontinuation of the iron and zinc supplementation and a return to the control diet, the following was observed a decrease of SOD activity in the testes and GPx activity in the epididymis, and a increase malondialdehyde concentration in prostates. After stage III, in DSFeZn vs DSFe rats, an increase of SOD and CAT activity in the epididymis was found.ConclusionZinc supplementation simultaneous with iron may protect the male reproductive system against oxidative damage induced by high doses of iron and may have a beneficial effect on sperm viability. The effect of this supplementation was observed even two weeks after the termination of the intervention.     

Evaluation of antioxidant and neuroprotective effect of Hippophae rhamnoides (L.) on oxidative stress induced cytotoxicity in human neural cell line IMR32

Aim and objectiveHippophae rhamnoides is an edible, nutrient rich plant found in the northern regions of India. It belongs to the family Elaeagnaceae and is well known for its traditional pharmacological activities. The present study was aimed to investigate the antioxidant and neuroprotective activities of H. rhamnoides.MethodologyThe hydroalcoholic extract of H. rhamnoides was evaluated for free radical scavenging activity using DPPH, hydroxyl radical scavenging and ferric thiocyanate assays. In vitro neuroprotective activity was assessed on human neuroblastoma cell line-IMR32 against hydrogen peroxide (H₂O₂) induced cytotoxicity. The neuroprotective effect was determined by measuring the cell viability through tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reducing assay and propidium iodide (PI) staining. Also the intracellular reactive oxygen species (ROS) activity was assessed using dichloro-dihydro-fluorescein diacetate (DCFDA) assay by flowcytometer.ResultsThe results of the study demonstrated that H. rhamnoides extract possesses potential free radical scavenging activity. The IC₅₀ value for DPPH and OH radical scavenging assay was 70.92 μg/ml and 0.463 mg/ml, also the extract was also found to have considerable level of lipid peroxidation activity. The neuroprotective effect of H. rhamnoides was confirmed by its cell viability enhancing capacity against hydrogen peroxide induced cell cytotoxicity. The extract acted on IMR32 cells in a dose dependent manner as observed through PI and MTT assays. The percentage intracellular ROS activity was reduced by 60–70% in treated cells compared to H₂O₂ control.ConclusionThus the outcome of the study suggests that H. rhamnoides acts as a neuroprotectant against oxidative stress induced neurodegeneration.     

Effect of metallothionein on cell viability and its interactions with cadmium and zinc in HEK293 cells

Metallothioneins (MTs) are thought to participate in a wide variety of physiological roles, but the mechanisms involved are still unclear. The study was designed to examine the possible factors related to these mechanisms. Methods, including transfection, MTT assay and flow cytometry, were used to investigate the effect of MTs on cell viability and their interactions with cadmium and zinc in HEK293 cells. The results showed that transient overexpression of human MT1A, MT2 and MT3 genes dynamically affected cell viability, and the effect was influenced by zinc and cadmium ions. Overexpressed MTs with added zinc showed a greater inhibitory effect on cell viability. Overexpressed MTs protected cells against low concentrations of cadmium ions (10 microM), but increased cell death in response to high concentrations (20-50 microM). Out of the three MTs, MT1A was more efficient than MT2 and MT3 in its resistance to cadmium (10 microM), and MT3 together with zinc showed more cell growth inhibition than MT1 and MT2. These results indicate that both of the divalent metal ions that could bind MTs, as well as the individual MT isoforms, affect the role of MTs on cell viability, which may explain in part why the comprehensive effect of MTs on the cells was elusive.     

Inhibition of monoterpene biosynthesis accelerates oxidative stress and leads to enhancement of antioxidant defenses in leaves of rubber tree (Hevea brasiliensis)

This paper mainly studies the possible antioxidant of monoterpene and effects of its absence on other antioxidant defense. The leaves of rubber tree (Hevea brasiliensis) were fed with fosmidomycin through transpiration stream, in the dark, at room temperature for 2 h, and were then exposed to bright illumination (1,500 μmol m⁻² s⁻¹) and moderately high temperature (30°C) for 1 h. The results showed that monoterpene biosynthesis in leaves was considerably inhibited by fosmidomycin, and the elevated levels of both hydrogen peroxide and malondialdehyde were observed in the leaves fed with fosmidomycin (LFF). Compared to the control leaves (CK), ∆F/F _m′ in the LFF was markedly lower during the first 20 min; however, there were no significant differences in non-photochemical quenching and photosynthetic pigments (chlorophylls and carotenoids). In contrast, the activities of antioxidant enzymes (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase) were enhanced in the LFF. Meanwhile, the contents of antioxidant metabolites (ascorbate and glutathione) were also elevated in the LFF, when compared with the CK. The results obtained here suggest that monoterpene may be very effective molecule in protecting plants against oxidative stress, the absence of monoterpene leads to the increased responses of the enzymatic and non-enzymatic antioxidant defenses to oxidative stress, and the enhancement of the enzymatic and non-enzymatic antioxidant defenses may, in part, compensate for the loss of antioxidant conferred by monoterpene.