Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro Maturation

In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.     

L-ascorbic acid enhances apoptosis in human gastric carcinoma cell line AZ-521 cells infected with Mycoplasma hyorhinis

Mycoplasma hyorhinis (M. hyorhinis) exerts multiple effects on cell metabolisms including apoptosis mediated by their endonucleases and nitric oxide production in vitro. Although AsA is preferable to health in general because of its reactive oxygen species scavenging activity, we found that in a human carcinoma cell line AZ-521 infected with M. hyorhinis, apoptosis was enhanced by addition of L-ascorbic acid (AsA) to the cell cultures. No significant differences were evident between the AZ-521 cells with and without AsA (AsA-) after 24 hr of incubation in the mitochondrial fluorescence. M. hyorhinis-infected AZ-521 cells treated with AsA (AsA +) have developed distinct DNA ladders as compared to the control cells AsA- after 24 hr of incubation. Marked cytopathic effects were rather apparent in AsA-treated cells than in control cells AsA- after 24 hr. Our data demonstrate that AsA addition to cell cultures enhances apoptosis induced by M. hyorhinis infection. We suggest that the presence of another external apoptotic pathway by M. hyorhinis infection.     

The influence of different doses of α-tocopherol and ascorbic acid on selected oxidative stress parameters in in vitro culture of leukocytes isolated from transported calves

The objective of the study was to assess in vitro the influence of various doses of two different antioxidants, α-tocopherol and ascorbic acid, on protective mechanisms against ROS in white blood cells obtained from calves exposed to transport, and to compare these results with those obtained from non-transported animals.The concentrations of lipid peroxidation products in leukocytes and in the retained medium were assessed by determining the level of ThioBarbituric Acid Reactive Substances (TBARS). Total antioxidant status in the leukocytes and the medium were estimated using a ferric-reducing ability of plasma (FRAP) assay. Leukocyte viability was determined using the trypan blue reduction test.The study demonstrated that after bovine leukocytes (WBC) were incubated in vitro with α-tocopherol and ascorbic acid, peroxidation intensity decreased and total antioxidant capacity increased. The results of the study reveal that these antioxidants in concentrations over 0.1 mg/ml have a major impact on free radical activity on bovine white blood cells and on cell viability during transport of animals.Based on this study, we suggest that incubation of the leukocytes with antioxidants decreases the oxidative stress development, which can be helpful in protection of the immunological cells during bovine respiratory disease.     

Dietary Ascorbyl Monophosphate Depresses Lipid Peroxidation in Rainbow Trout Spermatozoa

Abstract

This study was designed to analyze lipid peroxidation in spermatozoa of rainbow trout Oncorhynchus mykiss by an optimized thiobarbituric acid (TBA) method, and to evaluate the effect of graded levels of dietary antioxidant (ascorbic acid in the form of ascorbyl monophosphate, AP) on TBA values of spermatozoa. Sperm from rainbow trout fed diets supplemented with AP at 0, 110, or 870 mg/kg were sampled during the reproductive season. The group given the unsupplemented diet had the lowest ascorbic acid concentration in seminal plasma during most of the spermiation season compared with groups fed diets with AP While the ascorbic acid concentration in seminal plasma decreased toward the end of the reproductive season, spermatozoa malondialdehyde production, indicative of increasing peroxidation, tended to increase. At the end of the season, significant differences (P < 0.05) were found in peroxidation levels in spermatozoa from fish fed different levels of ascorbic acid. The most abundant polyunsaturated fatty acid in sperm lipids, docosahexaenoic acid (C22:6), was significantly lower in lipids from spermatozoa of the AP-devoid (control) group than in the 870-mg/kg supplement group. By the end of the reproductive season, spermatozoa from the control group had significantly higher peroxidation levels than did spermatozoa from fish given AP (110 and 870 mg/kg). Thus, it is the first evidence in fish that dietary AP supplements can increase seminal plasma ascorbic acid concentrations, and it suggests that peroxidative damage to spermatozoa can be reduced during the reproductive season, which in turn may affect sperm fertilizing ability.     

Effect of supplementing the hen's diet with vitamin A on the accumulation of vitamins A and E,ascorbic acid and carotenoids in the egg yolk and in the embryonic liver

1. The effect of a range of supplementations of vitamin A to the laying hen on the concentration of vitamins A, E, ascorbic acid and carotenoids in the maternal liver, the egg yolk and the embryonic liver were investigated. 2. Four groups of 25 Rhode Island Red hens were fed on standard layer-breeder diets with concentrations of supplemented vitamin A ranging from 0 to 120 mug/g retinol equivalents from 28 weeks of age. After 3 months, the concentration of vitamin A in the maternal liver was found to be greatly enhanced in proportion to the increasing rates of supplementation with the vitamin. However, the concentration of vitamin E in the maternal liver was markedly reduced by high dietary contents of vitamin A. 3. The concentration of vitamin A in the yolk of the hens' eggs was markedly increased by the dietary supplementation. However, the concentration of both vitamin E and carotenoids in the yolks were significantly reduced by high dietary contents of vitamin A. 4. The concentration of vitamin A in the liver of the embryo and the day old chick was greatly increased by the high concentrations of maternal vitamin A provision. However, the concentration of vitamin E, carotenoids and ascorbic acid in the embryonic/neonatal liver were significantly reduced by high contents of vitamin A in the maternal diet. 5. The susceptibility of the embryonic/neonatal liver to lipid peroxidation was significantly increased as a result of high provisions of maternal vitamin A. 6. It is concluded that excessive provision of vitamin A to the laying hen results in an adverse effect on vitamin E, carotenoids and ascorbic acid in the embryonic/neonatal liver and can compromise the antioxidant status of the progeny.     

High sensitivity of thymocytes of LEC strain rats to induction of apoptosis by X-irradiation

It is known that physical disruption of cell contacts induces apoptosis of thymocytes. When thymocytes from LEC and WKAH rats were incubated in vitro at 37 degrees C for 0-6 hr and then the proportion of apoptotic cells was determined using a flow cytometer, it was found that the percentages of apoptotic thymocytes from both LEC and WKAH rats increased with incubation time and that the proportion of apoptotic cells from LEC rats was significantly higher than that from WKAH rats at each incubation time. The fact that cycloheximide, an inhibitor of protein synthesis, did not show significant inhibitory effects on induction of apoptosis of thymocytes indicates that induction of apoptosis during in vitro cultivation did not require de novo protein synthesis. When thymocytes from LEC and WKAH rats were X-irradiated in vitro at 4 and 8 Gy, the percentages of radiation-induced apoptotic cells increased with post-incubation time after X-irradiation in both LEC and WKAH rat thymocytes and the proportions of apoptotic cells from LEC rats were significantly higher than those from WKAH rat cells at 2 and 4 hr post-incubation after X-irradiation. When thymocytes from LEC and WKAH rats were X-irradiated in the presence of cycloheximide, the induction of apoptosis was substantially inhibited, indicating that radiation-induced apoptosis of thymocytes from LEC and WKAH rats required de novo protein synthesis. The present results showed high sensitivities of thymocytes of LEC rats to induction of apoptosis during in vitro cultivation and by X-irradiation.     

Prevention of avian lymphoid leukosis by induction of bursal atrophy with infectious bursal disease viruses.

Five groups of genetically susceptible chickens were inoculated at hatching with lymphoid leukosis virus; four of these were given infectious bursal viruses of varying virulence at 14 days of age and one group was not inoculated (control). All chickens in the control group developed evidence of lymphoid leukosis by 180 days. Two groups given relatively virulent bursal disease viruses, which destroyed bursal lymphoid cells, did not develop lymphoid leukosis. Treatment with avirulent vaccines had no visible effect on bursal morphology and did not significantly alter the incidence of lymphoid leukosis in two other groups, although the time of development was delayed. Results of our study show that viral-induced destruction of the bursa of Fabricius eliminates the development of lymphoid leukosis but that infection without bursal destruction has little effect on lymphoid leukosis.     

Effects of ascorbic acid 2‐glucoside and alpha‐tocopherol on the characteristics of equine spermatozoa stored at 5°C

The aim of this experiment was to evaluate the effects of adding ascorbic acid 2‐glucoside (AA2G), a water‐soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat‐soluble antioxidant α‐tocopherol (α‐Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid peroxidation, the control group showed 2506.2 ± 796.4 ng malondialdehyde/10⁸ sperm, which was higher (P < 0.05) than the groups treated with antioxidants. The average value of motility in the AA2G group was 68.4 ± 18.1%, which was higher (P < 0.05) than that observed in the control group (62.1 ± 16.2%). The variables membrane integrity, chromatin fragmentation and mitochondrial activity did not show significant difference (P > 0.05) between treatments. It was concluded that the antioxidants protected sperm cells from lipid peroxidation and that AA2G was effective during the cooling process of equine semen at 5°C for72 h, providing increased levels of total motility.     

Induction of lipid peroxidation in mice by hexavalent chromium and its relation to the toxicity

Comparative effects of hexavalent (K2Cr2O7:Cr(VI)) and trivalent chromium (Cr(NO3)3:Cr(III)) on the development of lipid peroxidation, and the relationship between the lipid peroxidation and damage to tissues were studied using male ddY strain mice. The animals were administered with either of two chemicals at a dose of 20 mg Cr/kg by a single intraperitoneal injection. The results obtained were as follows: (1) Lipid peroxidation in the liver, as measured by the synthesis of thiobarbituric acid reactive substances (TBARS), showed a significant increase at 24 and 48 hr after Cr(VI) injection, while in the kidney it was observed only at 48 hr. In the mice administered with Cr(III), TBARS formation in the liver went down below the control levels, while no change was observed in the kidney. (2) Chromium contents in the liver and kidney showed a maximum level at 6 hr after injection of Cr(VI) and then those declined to the half of the maximum level at 48 hr, respectively. Chromium contents in the liver and kidney of the mice injected with Cr(III) were lower than those injected with Cr(VI) during the experimental period. (3) Increases of TBARS formation in the liver, chromium content in the liver and kidney, and ornithine carbamyl transferase (OCT) activity indicative of the liver cell damage, and urea nitrogen content in the serum, indicative of the kidney damage, observed at 24 hr after injection of Cr(VI) were inhibited by simultaneous injection of 100 mg/kg of L-ascorbic acid, as antichrome agent, respectively. These observations might suggest a possible causative role of lipid peroxidation in Cr(VI) toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular injury and lipid peroxidation induced by hexavalent chromium in isolated rat hepatocytes

In order to elucidate the relationship between cellular injury and lipid peroxidation induced by hexavalent chromium (CrVI), isolated rat hepatocytes treated with any one of scavengers of active oxygen species, antioxidants or antichromium agent were incubated with K2Cr2O7 as CrVI (1 mM Cr). After the incubation, the development of lipid peroxidation was determined as thiobarbituric acid (TBA)-reacting materials in total lipid extracts from the incubated hepatocytes. Cellular injury was observed as a leakage of lactate dehydrogenase (LDH) from hepatocytes into incubation medium. The contents of reduced glutathione (GSH) in hepatocytes were also assessed. Results obtained were as follows: (1) CrVI facilitated lipid peroxidation in isolated hepatocytes after 20 min of incubation. On the other hand, the cellular injury induced by CrVI was barely observed even after 60 min of incubation. (2) The CrVI-induced lipid peroxidation was inhibited by catalase and mannitol as scavengers of active oxygen species, or N,N'-diphenyl-p-phenylenediamine and alpha-tocopherol as antioxidants. However the cytotoxicity of CrVI could not be prevented by these chemicals. (3) CrVI depleted the contents of intracellular GSH and diminished the activities of glutathione reductase (GR) and glutathione-S-transferase (GST) except glutathione peroxidase. (4) The scavengers of active oxygen species and the antioxidants could not prevent the depletion of intracellular GSH induced by CrVI. (6) Ascorbic acid, antichromium agent, prevented all of the lipid peroxidation, the cellular injury and intracellular GSH depletion induced by CrVI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei,mitochondria, and plasma membranes in mice

Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylation (γH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle.     

Retinal neuronal cell is a toxicological target of tributyltin in developing zebrafish

Organotins are among the most common marine pollutants in the world, as they are widely used as antifouling paint on ships and fishing nets. It has been reported that organotin preferentially accumulates in the central nervous system, especially in the retinal neurons of marine organisms including fish. In this study, we investigated the effects of waterborne tributyltin (TBT) on early-stage developing zebrafish (Danio rerio). Below the lethal concentrations, TBT specifically increased the number of apoptotic cells in the retina as well as some cells near trigeminal neurons, detected by terminal transferase-mediated nick-end-labeling staining. Apoptosis peaked at 60 hpf and decreased by 72 hpf, which was associated with macrophage accumulation. Furthermore, the effect of TBT was markedly inhibited by antioxidants, ascorbic acid or trolox. These results suggest that TBT preferentially induces apoptosis in the retinal neuron of developing zebrafish. Oxidative stress may be involved in this toxicological response.     

Effect of ascorbic acid supplementation on the immune response of chickens vaccinated and challenged with infectious bursal disease virus

One-day-old chickens were divided into two groups and reared under similar conditions. One group was fed a diet supplemented with 1000ppm ascorbic acid and the other group was fed an identical diet, but not supplemented with ascorbic acid. Both groups were vaccinated against infectious bursal disease (IBD) at 7 days of age and challenged orally with 4x10(5) of 50% embryo-lethal-dose IBDV 14 days later. The number of anti-IBDV antibody secreting cells, production of interleukin-2 (IL-2) by splenocytes, number of CD4(+), CD8(+) and IgM(+) cells in spleen and IgM(+) cells in bursa of Fabricius were compared between the two groups at 7 days (prior to vaccination), 21 days (14 days post-vaccination and prior to challenge) and 31 days (10 days post-challenge) of age. The number of CD8(+) in spleen at 7 days of age and IgM(+) cells in bursa at 7, 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Production of IL-2 by splenocytes was higher as indicated by higher stimulation indices in ascorbic acid supplemented group. The number of anti-IBDV IgG antibody secreting cells in spleen at 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Dietary supplementation of ascorbic acid may ameliorate the immunosuppression caused by IBDV vaccination and improve humoral and cellular immune responses.     

Effects of vitamin C supplementation on plasma ascorbic acid and oxalate concentrations and meat quality in swine

Two experiments were conducted to determine the effects of vitamin C supplementation 48 h before slaughter on plasma ascorbic acid and oxalate concentrations and its effect on pork quality. In Exp. 1, 16 pigs (87.8+/-2.13 kg BW) were blocked by sex and weight and assigned randomly within block to one of three vitamin C treatments: 1) control; 2) 1,000 mg/L; or 3) 2,000 mg/L supplemented in the drinking water for a 48-h period. This was then followed by an additional 48-h period without supplemental vitamin C. Vitamin C increased plasma ascorbic acid concentrations (11.6, 19.5, and 23.4 microg/mL for 0, 1,000, and 2,000 mg/L of vitamin C; P < 0.05) within 6 h of supplementation. Plasma ascorbic acid concentrations from treated pigs decreased and did not differ from those of control pigs (13.7, 18.2, and 18.6 microg/mL for 0, 1,000, and 2,000 mg/L of vitamin C; P = 0.30) within 2 h of ending supplementation. No differences in plasma ascorbic acid concentrations were found between the two levels of supplementation. Vitamin C did not affect plasma oxalate or cortisol; however, cortisol tended to increase quadratically (P = 0.077) with vitamin C after 96 h. In Exp. 2, 30 pigs (107.5+/-0.54 kg BW) were blocked by sex and weight and assigned randomly within block to one of three vitamin C treatments: 1) control; 2) 500 mg/L; or 3) 1,000 mg/L supplemented in the drinking water 48 h before slaughter. Pigs were slaughtered 4 to 5 h after vitamin C supplementation ended, and loin samples were collected for meat quality measurements. At the time of slaughter, no differences in plasma ascorbic acid or cortisol were observed, but oxalate tended (P = 0.074) to increase quadratically with increasing vitamin C. Muscle ascorbic acid at slaughter and lactic acid in muscle at 0 and 1.5 h after slaughter were not different; however, lactic acid increased (P = 0.048) quadratically at 24 h after slaughter. Vitamin C did not affect initial or ultimate pH. Initial fluid loss (P = 0.041), and fluid loss on d 4 (P = 0.014) and 8 (P = 0.076) of simulated retail display; L* on d 0 (P = 0.038), 4 (P = 0.010), and 8 (P = 0.051); a* on d 0 (P = 0.021); and b* on d 0 (P = 0.006), 4 (P = 0.035), and 8 (P = 0.017) were negatively affected in a quadratic manner when vitamin C was supplemented. Vitamin C tended (P = 0.086) to increase oxidation in chops on d 0, but not d 4 or 8. Results indicate that on-farm supplementation of vitamin C was generally not effective in improving pork quality, which may be related to timing relative to slaughter.     

Effects of vitamin E and selenium supplementation on blood lipid peroxidation and cortisol concentration in dairy cows undergoing omentopexy

Twenty dairy cows with left abomasal displacement were used to investigate the effects of vitamin E and selenium treatment on thiobarbituric acid reactive substances (TBARS) and blood cortisol in dairy cows stressed by omentopexy. The cows were randomly divided into two groups. Ten hours before surgery 6 g of DL‐α‐tocopheryl acetate (6 mg/kg) and 67 mg of natrium selenite (0.1 mg/kg) in volume of 40 ml (Vitaselen^®) were administered subcutaneously to 10 cows; the control animals (n = 10) received an equivalent volume of injectable water (40 ml). The injection of vitamin E and selenium produced a rapid rise (p < .05) in blood α‐tocopherol and selenium concentrations. The serum vitamin E increased several times 10 hr after vitamin E and Se injection and raised continuously to the highest average concentration 21.6 mg/L at hr 24 after the surgery. The highest selenium concentration was seen 10 hr after selenium administration with holding the increased concentrations in comparison with initial ones during the whole study. Two‐way ANOVA did not show significant treatment effect on plasma concentrations TBARS in the study. The plasma concentrations of thiobarbituric acid reactive substances reached the maximum value of 0.18 μmol/L in the control group 5 hr after the surgery. Twenty‐four hours after the surgery, the TBARS values returned to the initial ones. Serum cortisol increased in both groups after surgery. The highest cortisol concentrations were reached at 1 hr after surgery in the experimental and control group (56.7 ± 28.8 and 65.3 ± 26.1 μg/L respectively). A return to the levels similar to the initial ones was recognized 24 hr after the surgery. The ANOVA revealed a significant effect of vitamin E and selenium injection on plasma cortisol (p < .05). In conclusion, we have demonstrated that abdominal surgery resulted in typical stress changes with no significant effects of a single vitamin E/Se injection on blood lipid peroxidation. In addition, a weaker cortisol response to the abdominal surgery was recognized in animals treated with vitamin E and selenium.     

日粮维生素E,抗坏血酸水平对肉仔鸡生长及免疫功能的影响

试验用ＡｒｂｏｒＡｃｒｅ肉仔鸡６７２只，随机分成７个处理，每处理９６只，雌雄各半。２×３因子设计，其中维生素Ｅ２个水平分别为２０、８０ｍｇ／ｋｇ；抗坏血酸３个水平依次为２００、４００和８００ｍｇ／ｋｇ。另外设一个不添加维生素Ｅ和抗坏血酸的对照组。试验期分为０～２周龄和３～４周龄两个阶段。试验结果表明，提高日粮维生素Ｅ水平（８０ｍｇ／ｋｇ）对肉仔鸡体内抗坏血酸的合成有促进作用，抗坏血酸对维生素Ｅ的作用不明显。随着日龄的增加，肉仔鸡血清维生素Ｅ的含量呈下降趋势，日粮尽早添加维生素Ｅ可以减缓血清维生素Ｅ的下降；肉仔鸡体内可以合成一定量的抗坏血酸，合成能力随日龄的增加而增强。日粮高水平的维生素Ｅ（８０ｍｇ／ｋｇ）可提高２８日龄肉仔鸡血液淋巴细胞转化率和血清新城疫抗体滴度，抗坏血酸对肉仔鸡细胞及体液免疫功能影响不显著。     

Effect of chronic oxidative/corticosterone-induced stress on ascorbic acid metabolism and total antioxidant capacity in chickens (Gallus gallus domesticus)

The consequences of chronic corticosterone-induced stress (CCIS) on ascorbic acid (AsA) metabolism in chickens, an animal that syntheses the vitamin, are not known. This study was conducted to determine whether CCIS alters AsA synthesis, as measured by l-gulonolactone oxidase (GLO) activity, tissue AsA, lipid peroxides and tissue total antioxidant capacity (TAC). Stress was induced by dietary administration of corticosterone from 2 to 4 weeks of age and measurements were made at 0, 7 and 14 days post-treatment. Ascorbic acid synthesis was not influenced by CCIS but hepatic, cardiac, renal, bursal and duodenal AsA concentrations were significantly decreased and plasma TAC and uric acid concentrations were significantly elevated. Stress caused significant hepatomegaly and hepatic lipidosis but hepatic peroxides were not elevated despite the slight decrease in hepatic TAC. Tissue TAC varied in different organs. It was markedly elevated in the kidney, reduced by 49% in the spleen, and changes were not detected in the heart and duodenum even though AsA concentration was significantly decreased in all tissues. We conclude that CCIS caused a significant reduction in tissue AsA concentration but did not inhibit GLO activity. The change in AsA concentration was associated with increase, decrease or no change in TAC in tissues examined. The findings suggest that CCIS may alter AsA recycling, influx or turnover in different tissues of chickens.     

Use of fluorescence-activated flow cytometry to determine membrane lipid peroxidation during hypothermic liquid storage and freeze-thawing of viable boar sperm loaded with 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid

Part of the reduction in boar sperm motility and fertility associated with hypothermic liquid storage and cryopreservation may be due to membrane lipid peroxidation. Lipid peroxidation was monitored by the shift from red to green fluorescence emission of the lipophilic probe 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, C(11)BODIPY(581/591) (BODIPY), as measured by fluorescence-activated flow cytometry in live sperm (negative for propidium iodide). Experiments were conducted with Percoll-washed sperm to determine the specificity of BODIPY oxidation in the presence of different reactive oxygen species generators and metal chelators. Compared with no FeSO(4) and Na ascorbate, the combination of FeSO(4) and Na ascorbate (FeAc) increased (P < 0.01) the percentage of sperm containing oxidized BODIPY from 70% and increased (P < 0.05) BOD-IPY fluorescence intensity/cell by 5- to 10-fold after a 30-min incubation. Motility was depressed (P < 0.05) after exposure to FeAc, but viability was not affected. Of the reactive oxygen species generators tested, BODIPY oxidation was specific for FeAc, because menadione and H(2)O(2) had little or no effect. The oxidization of hydroethidine to ethidium was specific for menadione and H(2)O(2); FeAc had no effect. The presence of the metal chelators EDTA or deferoxamine mesylate at 3 and 9 muM inhibited FeAc-induced BODIPY oxidation and maintained motility. Experiments were conducted to determine the effect of liquid storage at 17 degrees C for 1 and 5 d and the effect of freeze-thawing on basal and FeAc-induced BODIPY oxidation. Basal BODIPY oxidation (no FeAc) was low in liquid stored and thawed viable sperm (1.3 and 3.4%, respectively). Although the incidence of basal or spontaneous membrane lipid peroxidation was low during liquid storage and after freeze-thawing, viable boar sperm were susceptible to FeAc-induced lipid peroxidation.     

Oxidative damage to the membrane of canine erythrocytes with inherited high Na, K-ATPase activity.

Oxidative damage to the membrane in canine erythrocytes with inherited high Na, K-ATPase activity (HK cells) was compared with that in normal canine cells (LK cells). When 30 mM beta-acetylphenylhydrazine (APH) was applied to HK and LK cells, lipid peroxidation and hemoglobin denaturation occurred. Lipid peroxidation determined from malondialdehyde (MDA) formation was significantly lower in HK than in LK cells so far as endogenous glutathione (GSH) concentration was maintained at appropriate levels. With the depletion of GSH, MDA formation was accelerated and difference between HK and LK cells was not significant. Denatured hemoglobin bound to the membrane protein was less in HK than in LK cells. During incubation with APH, osmotic fragility increased markedly in LK cells, while HK cells showed very little change. The amounts of total lipid, total and free cholesterol, glycolipid, phospholipid and fatty acids were essentially the same in both cell types. Fatty acid compositions showed very small differences. The membrane of HK cells thus appear to have greater protection against oxidative damage induced by APH, owing to the presence of excess GSH in HK cells. The capability of HK cells to withstand oxidative damage would not be due to differences in membrane lipid compositions.     

Determination of plasma vitamin C concentration in fattening cattle

Plasma vitamin C (ascorbic acid + dehydroascorbic acid) concentration is a good index of the nutritional status of vitamin C. However, the methodologies for storage and analyses have not been investigated in bovine plasma. The validity of an analytical method for bovine plasma using high performance liquid chromatography (HPLC) with a spectrophotometric detector was examined. Exogenous dehydroascorbic acid was almost completely converted to ascorbic acid during the preparation for analysis with a reducing reagent, dithioerythritol. The analytical recoveries of ascorbic acid were high. Ascorbic acid was not detected after treatment with ascorbic acid oxidase. Thus, the specificity of this method is considered to be high. Although vitamin C was stable in plasma treated by dithioerythritol at ?20°C for 6 days, vitamin C in untreated plasma significantly decreased during 3‐day storage at ?20°C. These results indicate that the HPLC method is suitable for the determination of plasma vitamin C in cattle and that the storage conditions are important for determination of plasma vitamin C. Plasma vitamin C concentration ranged between 1.49 mg/L and 3.33 mg/L in fattening cattle. This result suggests that fattening cattle show large individual variation in plasma vitamin C concentration.