Study on the practices of silage production and utilization on Brazilian dairy farms.

Dairy farmers across Brazil were invited to participate in a study on silage production and utilization practices. Two hundred sixty farmers filled out a questionnaire, which was made available on a website. The questionnaire consisted of 14 questions, including information about the characteristics of the herd (n=3), the crop(s) used in the ensiling process, the use of additives, the harvest (n=3), the type of silo (n=1), aspects related to sealing (n=2), and management practices applied during feed-out (n=3). Farmers were also asked a final question about the main barriers they faced when producing and using silage. The main dairy-producing regions of Brazil had a strong influence on the number of participants. The profiles of farmers were heterogeneous and divided into 5 groups, which was considered a positive attribute of the study, allowing better analysis and assessment of current circumstances. Corn was the most widely grown crop for silage. Sorghum, tropical grasses, and sugarcane were the other species most cited. Additives were used by a small number of farmers (27.7%). Approximately 40% of farmers still depended on loaned equipment or outsourced services. The pull-type forage harvester was the main piece of equipment used on dairy farms (90.4%). Only 54.6% of respondents answered that they sharpen their harvester knives daily. Horizontal silos (bunker and stack) were the structures most commonly used to store silage. Most farmers sealed silos with double-sided plastic film (black-on-white) and with soil. However, almost one-fifth of all farmers still use black plastic. Manual removal of silage from the silos was practiced at most farms (i.e., the lack of equipment was also reflected in the stage of silage utilization). Disposal of spoiled silage before inclusion in the livestock feed was not a common practice on the farms. The main barriers encountered on the farms were lack of equipment, lack of manpower, and climatic variations. The results of this research may guide researchers, industries, extension workers, and governments to seek efficiency in milk production on farms using silage in the diet of livestock throughout the year or during part of the year in Brazil.

Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits.

Neurotoxicity induced by beta-amyloid peptide (Abeta) involves glutamate toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Abeta(1-40) toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Abeta(1-40) toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Abeta(1-40) decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Abeta(1-40). Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Abeta(1-40) through changes in calcium homeostasis.

Yellow grease in sheep diets: intake and digestibility / [Óleo residual de fritura em dietas para ovinos: consumo e digestibilidade]

This study aimed to assess the effects of yellow grease supplementation on the intake, digestibility, and nitrogen balance in sheep. Twenty Santa Inês lambs with a mean age of 95 ± 10 d and body weight of 19.29 ± 3.17kg were evaluated in a completely randomized design. The diets were supplemented with oil at concentrations of 0, 20, 40, 60, and 80 gkg-1 of dry matter (DM) of the concentrate. The diets were based on roughage and concentrate (50:50). The experimental period lasted 19 d and included 14 adaptation days and five collection days for the total supplied diet, orts, feces, and urine. Supplementation with yellow grease had no significant effect on the intake of DM, crude protein (CP), neutral detergent fiber (NDF), or non-fiber carbohydrates (NFC). However, the ether extract (EE) intake increased linearly with supplementation of yellow grease. Moreover, no effect was observed for DM, CP, NDF, and NFC digestibility and nitrogen balance. EE digestibility increased linearly with the yellow grease dietary supplementation. Thus, sheep dietary supplementation with yellow grease may be used at a level of up to 80 gkg-1 of DM of concentrate without impairing nutrient intake and digestibility.(AU)

Objetivou-se, com o estudo, avaliar os efeitos do óleo residual de fritura, em dietas para ovinos, sob o consumo, a digestibilidade e o balanço de nitrogênio. Foram utilizados 20 cordeiros Santa Inês, com idade de 95 ± 10 dias e peso corporal de 19,29 ± 3,17kg, em delineamento inteiramente ao acaso. As dietas continham óleo de fritura nas concentrações de 0; 20; 40; 60 e 80gkg-1 da matéria seca (MS) do concentrado. As dietas tinham relação volumoso:concentrado de 50:50. O período experimental foi de 19 dias, incluindo 14 dias em adaptação e cinco dias de coleta do fornecido, das sobras, das fezes e da urina. A suplementação com óleo de fritura não alterou o consumo de MS, proteína bruta (PB), matéria orgânica (MO), fibra em detergente neutro (FDN) e carboidratos não fibrosos (CNF). Entretanto, o consumo de extrato etéreo (EE) aumentou com a inclusão do óleo. Não foi observado efeito na digestibilidade da MS, da PB, da FDN, dos CNF e no balanço de nitrogênio. A digestibilidade do EE aumentou com a inclusão do óleo. Assim, a inclusão de óleo de fritura em dietas para ovinos pode ser utilizada em até 80gkg-1 da MS do concentrado, sem limitar ingestão e digestibilidade dos nutrientes.(AU)

Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells.

The relationship between changes in mitochondrial membrane potential (Deltapsi(m)) and the failure of cytoplasmic Ca(2+) homeostasis, delayed Ca(2+)deregulation (DCD), is investigated for cultured rat cerebellar granule cells exposed to glutamate. To interpret the single-cell fluorescence response of cells loaded with tetramethylrhodamine methyl ester (TMRM(+)) or rhodamine-123, we devised and validated a mathematical simulation with well characterized effectors of Deltapsi(m) and plasma membrane potential (Deltapsi(P)). Glutamate usually caused an immediate decrease in Deltapsi(m) of <10 mV, attributable to Ca(2+) accumulation rather than enhanced ATP demand, and these cells continued to generate ATP by oxidative phosphorylation until DCD. Cells for which the mitochondria showed a larger initial depolarization deregulated more rapidly. The mitochondria in a subpopulation of glutamate-exposed cells that failed to extrude Ca(2+) that was released from the matrix after protonophore addition were bioenergetically competent. The onset of DCD during continuous glutamate exposure in the presence or absence of oligomycin was associated with a slowly developing mitochondrial depolarization, but cause and effect could not be established readily. In contrast, the slowly developing mitochondrial depolarization after transient NMDA receptor activation occurs before cytoplasmic free Ca(2+) ([Ca(2+)](c)) has risen to the set point at which mitochondria retain Ca(2+). In the presence of oligomycin no increase in [Ca(2+)](c) occurs during this depolarization. We conclude that transient Ca(2+) loading of mitochondria as a consequence of NMDA receptor activation initiates oxidative damage to both plasma membrane Ca(2+) extrusion pathways and the inhibition of mitochondrial respiration. Depending on experimental conditions, one of these factors becomes rate-limiting and precipitates DCD.

Mitochondria control ampa/kainate receptor-induced cytoplasmic calcium deregulation in rat cerebellar granule cells.

Although mitochondria mediate the delayed failure of cytoplasmic Ca(2+) homeostasis [delayed Ca(2+) deregulation (DCD)] in rat cerebellar granule cells resulting from chronic activation of NMDA receptors, their role in AMPA/KA-induced DCD remains to be established. The mitochondrial ATP synthase inhibitor oligomycin protected cells against KA- but not NMDA-evoked DCD. In contrast to NMDA-evoked DCD, no additional protection was afforded by the further addition of rotenone. The effects of KA on cytoplasmic Ca(2+) homeostasis, including the protection afforded by oligomycin, could be reproduced by veratridine. KA exposure induced a partial mitochondrial depolarization that was enhanced by oligomycin, indicating ATP synthase reversal. The nonglycolytic substrates pyruvate and lactate were unable to maintain Ca(2+) homeostasis in the presence of KA. In contrast to NMDA, KA exposure did not cause mitochondrial Ca(2+) loading. The data indicate that Na(+) entry via noninactivating AMPA/KA receptors or voltage-activated Na(+) channels compromises mitochondrial function sufficiently to cause ATP synthase reversal. Oligomycin may protect by preventing the consequent mitochondrial drain of cytoplasmic ATP.

The mechanism of mitochondrial membrane potential retention following release of cytochrome c in apoptotic GT1-7 neural cells.

The relationship is investigated between mitochondrial membrane potential (Delta Psi(M)), respiration and cytochrome c (cyt c) release in single neural bcl-2 transfected cells (GT1-7bcl-2) or GT1-7puro cells during apoptosis induced by staurosporine (STS). Bcl-2 inhibited the mitochondrial release of cyt c and apoptosis. Three different cell responses to STS were identified in GT1-7puro cells: (i) neither Delta Psi(M) nor cyt c were significantly affected; (ii) a decrease in Delta Psi(M) was accompanied by a complete release of cyt c; or (iii) cyt c release occurred independently of a loss of Delta Psi(M). The endogenous inner membrane proton leak of the in situ mitochondria, monitored by respiration in the presence of oligomycin, was increased by STS by 92% in puro cells, but by only 23% in bcl-2 cells. STS decreased respiratory capacity, in the presence of protonophore, by 31% in puro cells and by 20% in bcl-2 cells. In the absence of STS, oligomycin hyperpolarized mitochondria within both puro and bcl-2-transfected cells, indicating that the organelles were net generators of ATP. However after 15 h exposure to STS oligomycin rapidly collapsed residual mitochondrial polarization in the puro cells, indicating that Delta Psi(M) had been maintained by ATP synthase reversal. bcl-2 cells in contrast, maintained Delta Psi(M) until protonophore was added. These results indicate that the maintenance of Delta Psi(M) following release of cyt c may be a consequence of ATP synthase reversal and cytoplasmic ATP hydrolysis in STS-treated GT1-7 cells.

Protective effect of zinc on amyloid-beta 25-35 and 1-40 mediated toxicity.

Amyloid beta-peptide (Abeta) is widely held to be associated with Alzheimer's disease, the insoluble aggregates of the peptide being the major constituents of senile plaques. In this study, we evaluated the effect of Zn(2+) (5, 50 and 200 microM) on Abeta induced toxicity using the human teratocarcinome (NT2) cell line. Our results proved that 50 and 200 microM Zn(2+) protected NT2 cells from Abeta 25-35 toxicity. Zinc was also shown to be effective by preventing the loss of mitochondrial membrane potential (DeltaPsi(m)) induced by Abeta 25-35, not allowing cytochrome c release from mitochondria, and subsequently, caspase 3 activation. However, when the cells were treated with Abeta 1-40, only Zn(2+) 5 microM had a protective effect. We have further observed that 5 microM Zn(2+) prevented Abeta 1-40 aggregation into a beta-sheet structure. Considering the results presented, we argue that Zn(2+) has a concentration-dependent protective effect.

Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress.

A role for the antioxidants vitamin E and idebenone in decreasing retinal cell injury, after metabolic inhibition induced by chemical ischemia and hypoglycemia, was investigated and compared with oxidative stress conditions. Preincubation of the antioxidants, vitamin E (20 microM) and idebenone (10 microM), effectively protected from retinal cell injury after oxidative stress or hypoglycemia, whereas the protection afforded after postincubation of both antioxidants was decreased. Delayed retinal cell damage, mediated by chemical ischemia, was attenuated at 10 or 12 h postischemia, only after exposure to the antioxidants during all the experimental procedure. An antagonist of the N-methyl-D-aspartate (NMDA) receptors, an inhibitor of nitric oxide synthase (NOS) or a blocker of L-type Ca2+ channels were ineffective in reducing cell injury induced by chemical ischemia, hypoglycemia or oxidative stress. Oxidative stress and hypoglycemia increased (about 1.2-fold) significantly the fluorescence of the probe DCFH2-DA, that is indicative of intracellular ROS formation. Free radical generation detected with the probe dihydrorhodamine 123 (DHR 123) was enhanced after oxidative stress, chemical ischemia or hypoglycemia (about 2-fold). Nevertheless, the antioxidants vitamin E or idebenone were ineffective against intracellular ROS generation. Cellular energy charge decreased greatly after chemical ischemia, was moderately affected after hypoglycemia, but no significant changes were observed after oxidative stress. Preincubation with vitamin E prevented the changes in energy charge upon 6 h posthypoglycemia. We can conclude that irreversible changes occurring during chemical ischemia mainly reflect the alterations taking place at the ischemic core, whereas hypoglycemia situations may reflect changes occurring at the penumbra area, whereby vitamin E or idebenone may help to increase cell survival, exerting a beneficial neuroprotective effect.

Oxidative stress in acidic conditions increases the production of inositol phosphates in chick retinal cells in culture.

The effect of oxidative stress on the production of [3H]inositol phosphates (InsP) by retinal cells in culture was analyzed. The process of oxidation was induced by incubating the cells with ascorbic acid and ferrous sulphate, and increased extent of oxidation was obtained by varying the pH from neutral to moderate acidosis (pH 6.5). The oxidative process significantly reduced cell viability (about 15%) by decreasing the capacity of mitochondria dehydrogenases to reduce tetrazolium salts, but had no effect on the leakage of lactate dehydrogenase. The production of [3H]InsP, in the absence of receptor activation, was increased dose dependently by oxidative stress. Maximal increases to 189 +/- 7%, 197 +/- 13%, and 329 +/- 22% were observed, respectively, for inositol monophosphates (InsP1), inositol bisphosphates (InsP2), and inositol trisphosphates (InsP3), at 2.5 nmol thiobarbituric acid reactive substances (TBARS)/mg protein. The response to cholinergic receptor activation was slightly decreased in cells oxidized in acidic conditions. Antagonists of glutamate receptors failed to inhibit the enhancement in InsP that occurred upon cellular oxidation, suggesting that the effect was not mediated by activation of glutamate receptors. Cellular oxidation increased by about two fold the uptake of 45Ca2+ in the absence of agonist stimulation. However, stimulation of phospholipase C by Ca2+ did not mediate the increase in [3H]InsP upon cell oxidation in acidic conditions, because the addition of 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1-H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C-dependent processes, did not affect the production of [3H]InsP in oxidized cells. Nevertheless, U-73122 significantly inhibited carbachol- and K(+)-stimulated accumulation of [3H]InsP. Furthermore, the enhancement of [3H]InsP induced by ascorbate/Fe2+ was still observed in the absence of external Ca2+. This increase in the production of InsP did not substantially induce the release of Ca2+ from internal stores. The results suggest that both Ca(2+)-dependent and Ca(2+)-independent pathways are involved in oxidative stress-mediated InsP increment, and that the enzymes of the InsP metabolism may be affected by oxidation.

Antioxidant effect of flavonoids after ascorbate/Fe(2+)-induced oxidative stress in cultured retinal cells.

In this study, we investigated the structure-activity relationship of four flavonoids, i.e. eriodictyol, luteolin, quercetin, and taxifolin, in cultured retinal cells after ascorbate/Fe(2+)-induced oxidative stress. The relative order of antioxidant efficacy, determined by the thiobarbituric acid method, was the following: eriodictyol > quercetin > luteolin > taxifolin. Upon preincubation, the flavonoids were also effective in reducing the extent of lipid peroxidation. Oxidative stress, determined by the changes in fluorescence of 2',7'-dichlorodihydrofluorescein, was also decreased in the presence of the flavonoids, showing the following order of antioxidant efficacy: eriodictyol > taxifolin approximately quercetin > luteolin. Ascorbate/Fe(2+)-induced oxidative stress or incubation in the presence of the flavonoids did not significantly affect the viability of retinal cells. We also evaluated the degree of membrane partition of the flavonoids. In this system, the results strongly suggest that the higher antioxidant activity of the flavonoids is not correlated with the presence of a double bond at C(2)-C(3) and/or a hydroxyl group at C(3) on the C ring, but rather may depend on the capacity to inhibit the production of reactive oxygen species to interact hydrophobically with membranes. Eriodictyol was shown to be the most efficient antioxidant in protecting against oxidative stress induced by ascorbate/Fe(2+) in the retinal cells.

Influence of lipid peroxidation on [3H]ketanserin binding to 5-HT2 prefrontal cortex receptors.

The influence of lipid peroxidation on 5-HT2 receptor binding was examined in prefrontal cortex membranes from sheep brain. Lipid peroxidation was induced with ascorbic acid and ferrous sulphate and measured by the thiobarbituric acid method. In lipid-peroxidized membranes, [3H]ketanserin specific binding was inhibited. The Bmax values decreased by 80%, from 50.1 +/- 3.5 fmol/mg protein in control membranes to 10.1 +/- 2.0 fmol/mg protein in peroxidized membranes, indicating a decrease in the number of 5-HT2 binding sites. However, the KD values for the [3H]ketanserin specific binding did not significantly change. In order to further characterize [3H]ketanserin binding, the inhibition potency (IC50 values) of antagonists or agonists of serotonin and dopamine receptors for [3H]ketanserin specific binding was determined. In control membranes, the order of inhibition potency of the drugs tested was the following: ketanserin (-log[IC50]=8.56 +/- 0.70] > ritanserin (-log [IC50]=8.13 +/- 0.30)>methysergide (-log[IC50]= 7.42 +/- 0.50)> spiperone (-log[IC50]=7.23 +/- 0.18 > serotonin (-log [IC50] = 6.99 +/- 0.65)> haloperidol (-log[IC50]= 6.95 +/- 0.65) > dopamine (-log [IC50] = 5.82 +/- 0.76). After membrane lipid peroxidation, the IC50 value for ritanserin was significantly increased, suggesting a decreased capacity for displacing [3H]ketanserin specific binding. Other antagonists of 5-HT2 receptors showed apparent increases in IC50 values upon peroxidation, whereas spiperone was shown to be the most potent drug (-log[IC50]= 7.19 +/- 1.06) in inhibiting [3H]ketanserin specific binding. A decrease in polyunsaturated fatty acids, namely docosahexaenoic acid (22:6) was also observed in peroxidized membranes. These results indicate a modulating role of the surrounding lipids and of the physical properties of the membranes on the binding activity of 5-HT2 receptors upon the lipid peroxidation process, which can be involved in the tissue impairment that occurs during the aging process and in post-ischemic situations.

Glutamate-mediated inhibition of oxidative phosphorylation in cultured retinal cells.

Glutamate is an excitotoxin responsible for causing neuronal damage associated with mitochondria dysfunction. We have analyzed the relationship between the mitochondrial respiratory rate, the membrane potential (delta psi) and the activity of mitochondrial complexes in retinal cells in culture, used as neuronal models. Glutamate (10 microM-10 mM) dose-dependently decreased the O2 consumption and the membrane potential. A linear correlation was found between these parameters, suggesting that the mitochondrial respiratory function was affected. Exposure to glutamate (100 microM) for 10 min, in the absence of Mg2+, inhibited the activity of complex I (26.3%), complexes II/III (22.2%) and complex IV (26.7%). MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), a non-competitive antagonist of the NMDA (N-methyl-D-aspartate) receptors, completely reversed the effect exerted by 100 microM glutamate at the level of complexes I, II/III and IV. These results suggest that NMDA receptor-mediated inhibition of mitochondrial respiratory chain complexes may be responsible for the alteration in the respiratory rate of chick retinal cells submitted to glutamate.

Adenosine modulation of D-[3H]aspartate release in cultured retina cells exposed to oxidative stress.

In this study we evaluated the role of adenosine receptor activation on the K+-evoked D-[3H]aspartate release in cultured chick retina cells exposed to oxidant conditions. Oxidative stress, induced by ascorbate (3.5 mM)/Fe2+ (100 microM), increased by about fourfold the release of D-[3H]aspartate, evoked by KCl 35 mM in the presence and in the absence of Ca2+. The agonist of A1 adenosine receptors, N6-cyclopentyladenosine (CPA; 10 nM), inhibited the K+-evoked D-[3H]aspartate release in control in oxidized cells. The antagonist of A1 adenosine receptor, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM), potentiated the release of D-[3H]aspartate in oxidized cells, and reverted the effect observed in the presence of CPA 10 nM. However, in oxidized cells, when DPCPX was tested together with CPA 100 nM the total release of D-[3H]aspartate increased from 5.1 +/- 0.4% to 11.4 +/- 1.0%, this increase being reverted by 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), an antagonist of A2A adenosine receptors. In cells of both experimental conditions, the K+-evoked release of D-[3H]aspartate was potentiated by the selective agonist of A2A adenosine receptors, 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680; 10 nM), whereas the antagonist of these receptors, DMPX (100 nM), inhibited the release of D-[3H]aspartate in oxidized cells, but not in control cells. Adenosine deaminase (ADA; 1 U/ml), which is able to remove adenosine from the synaptic space, reduced the K+-evoked D-[3H]aspartate release, from 5.1 +/- 0.4% to 3.1 +/- 0.3% in oxidized cells, and had no significant effect in control cells. The extracellular accumulation of endogenous adenosine, upon K+-depolarization, was higher in oxidized cells than in control cells, and was reduced by the inhibitors of adenosine transporter (NBTI) and of ecto-5'-nucleotidase (AOPCP). This suggests that adenosine accumulation resulted from the outflow of adenosine mediated by the transporter, and from extracellular degradation of adenine nucleotide. Our data show that both inhibitory A1 and excitatory A2A adenosine receptors are present in cultured retina cells, and that the K+-evoked D-[3H]aspartate release is modulated by the balance between inhibitory and excitatory responses. Under oxidative stress conditions, the extracellular accumulation of endogenous adenosine seems to reach levels enough to potentiate the release of D-[3H]aspartate by the tonic activation of A2A adenosine receptors.

Drugs of abuse induce apoptotic features in PC12 cells.

Drugs of abuse induce the release of dopamine in the central nervous system, particularly in the mesolimbic-mesocortical pathway. As dopamine may act as a neurotoxin, in this study, we analyzed the effects of the drugs of abuse, cocaine, heroin, and amphetamine, on the neurodegeneration of PC12 cells, a dopaminergic cell line, by evaluating the activity of caspase-3 and mitochondrial cytochrome c release. All the drugs were shown to induce caspase-3 activation, similarly to staurosporine, a classical inducer of apoptotic cell death. Furthermore, like staurosporine, the drugs of abuse induced a decrease in mitochondrial cytochrome c content, suggesting the involvement of the mitochondrial apoptotic pathway.

Toxic effects of opioid and stimulant drugs on undifferentiated PC12 cells.

Cell death and reactive oxygen species production have been suggested to be involved in neurodegeneration induced by the drugs of abuse. In this study we analyze the toxicity of the following drugs of abuse: heroin, morphine, d-amphetamine, and cocaine in undifferentiated PC12 cells, used as dopaminergic neuronal models. Our data show that opioid drugs (heroin and morphine) are more toxic than stimulant drugs (d-amphetamine and cocaine). Toxic effects induced by heroin are associated with a decrease in intracellular dopamine, an increase in DOPAC levels, and the formation of ROS, whereas toxic effects induced by amphetamine are associated with a decrease in intracellular dopamine and in ATP/ADP levels. In contrast with cocaine, both amphetamine and heroin induced features of apoptosis. The data suggest that the death of cultured PC12 cells induced by the drugs of abuse is correlated with a decrease in intracellular dopamine levels, which can be associated with an increased dopamine turnover and oxidative cell injury.

Developmental profile of excitatory GABA(A) responses in cultured rat cerebellar granule cells.

GABA induced a transient increase in cytosolic free Ca2+ in cerebellar granule cells, which decreased from 3 to 8 days in vitro (DIV). Cytosolic Ca2+ changes induced by glutamate/glycine were comparable at 3 and 7 DIV. The GABA response was ascribed to GABA(A)-receptor mediated depolarization activating L-type Ca2+ channels since the response was inhibited by bicuculline or nifedipine. GABA-mediated Ca2+ rise at 4 DIV was potentiated by pentobarbital or by the neurosteroid 5beta-pregnan-3alpha-ol-20-one, or by decreasing the extracellular Cl- concentration. Neurons cultured for > 7 DIV showed no rise in intracellular Ca2+ in response to GABA regardless of the Cl- gradient. GABA(A) receptor-mediated cytosolic Ca2+ rise suggests an important role for the excitatory activity of GABA in developing cerebellar granule neurons.

Influence of gamma-aminobutyric acid on retinal cells excitotoxicity upon glucose deprivation.

The role of extracellular endogenous gamma-aminobutyric acid (GABA) in rescuing retinal cells in culture from the decrease in viability induced by Glu under metabolic inhibition is analyzed. Glutamate (10 microM-10 mM) dose-dependently decreased the intracellular GABA content, but increased the extracellular accumulation of GABA. In the absence of glucose, Glu (10-100 microM) decreased the intracellular GABA (2-fold), whereas the extracellular accumulation of GABA was increased by about 4-fold. Glu-mediated decrement in cell survival was not affected by inhibiting the GABA(A) receptors with bicuculline (1 or 10 microM) or by blocking the Na+ -dependent release of GABA with 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (SKF89976-A). Data suggest a non-protective role of endogenous GABA release after metabolic deprivation of retinal cells submitted to Glu.

Glutamate regulates the viability of retinal cells in culture.

In this study, we show that glutamate regulates the viability of cultured retinal cells upon transient glucose deprivation. At low concentrations (10-100 microM) glutamate decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction to about 50% of control and decreased intracellular ATP levels (about 4-fold) after transient glucose removal. Under these conditions, the decrease in MTT reduction was associated with the activation of NMDA (N-methyl-D-aspartate) receptors. Upon exposure to high (10 mM) glutamate and transient glucose deprivation, the intracellular levels of glutamate increased. High glutamate significantly counteracted the decrease in MTT reduction and ATP production observed in the presence of low glutamate concentrations. AOAA (aminooxyacetic acid), a non-specific inhibitor of mitochondrial transaminases, enhanced the intracellular glutamate levels, but did not largely affect glutamate-mediated changes in MTT reduction or ATP production. Furthermore, the intracellular levels of pyruvate were not significantly altered, suggesting that changes in ATP production were not due to an increase in glycolysis. Thus, the recovery from glucose deprivation seems to be facilitated in retinal neuronal cells that had been exposed to high glutamate, in comparison with low glutamate, suggesting a role for high glutamate and glucose in maintaining retinal cell function following conditions of glucose scarcity.

Influence of vitamin E succinate on retinal cell survival.

In this study, we analyzed the influence of vitamin E succinate (5-80 microM), supplemented in the culture medium, on the survival of cultured retinal cells. The release of lactate dehydrogenase (LDH) was decreased in the presence of low concentrations (10-20 microM) of vitamin E succinate, whereas high concentrations (80 microM) induced a significant increase (about 2-fold) in the release of LDH, indicating a reduction of plasma membrane integrity. Supplementing with vitamin E succinate (80 microM) greatly enhanced its cellular content, as compared to vitamin E acetate (80 microM), and the membrane order of the retinal cells, as evaluated by the fluorescence anisotropy (r) of TMA-DPH (1-(4-(trimethylammonium)-phenyl)-6-phenylhexa-1,3,5-triene), was not altered. Furthermore, vitamin E succinate was more potent than vitamin E acetate in reducing thiobarbituric acid reactive substances (TBARS) formation upon ascorbate-Fe2+-induced oxidative stress (TBARS formation after cell oxidation decreased by about 15-fold or 1.6 fold, respectively, in the presence of 20 microM vitamin E succinate or 20 microM vitamin E acetate). A decrease in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction induced by supplementing with vitamin E succinate (80 microM), to 35.99 +/- 1.96% as compared to the control, but not by vitamin E acetate (80 microM), suggests that vitamin E succinate may affect the mitochondrial activity. Vitamin E succinate also reduced significantly the ATP:ADP ratio in a dose-dependent manner, indicating that vitamin E succinate-mediated cytotoxic effects involve a decrement of mitochondrial function.

Apparent selectivity of sheep in deferred marandu palisadegrass pastures with variable initial heights / Seletividade aparente de ovinos em pastos de capim-marandu diferidos com alturas iniciais variáveis

The objective of this study was to evaluate the apparent selectivity of sheep in marandu palisadegrass (Urochloa brizantha cv. Marandu) pastures with four heights at the beginning of the deferment period (15, 25, 35 and 45cm). The deferment period was 92 days and started on 03/21/2014. Evaluations occurred in the beginning (first week), middle (45th day) and end (92nd day) of the grazing period, in winter (06/21/2014 to 09/21/2014). Deferred pastures with 15 and 25cm presented lower forage mass (FM), but higher live leaf (LL) percentage in FM than deferred pastures with 35 and 45cm. The live stem percentage in the FM and the apparent selectivity index (ASI) of the LL were superior in the deferred pasture with 45cm. The dead stem (DS) percentage in the grazing simulation (GS) and the ASI of this morphological component were lower in the pasture with 15cm, compared to the deferred pasture with 45cm. The FM and the LL percentages in FM and in the GS sample decreased, while the DS percentages in FM and in GS sample increased with the grazing period. Marandu palisadegrass with 15cm at beginning of the deferment period improves the morphology of the deferred pasture. Selective grazing is difficult during the grazing period.(AU)

Objetivou-se avaliar a seletividade aparente de ovinos em pastos de capim-marandu (Urochloa brizantha cv. Marandu) com quatro alturas no início do diferimento (15, 25, 35 e 45cm). O período de diferimento foi de 92 dias e iniciou em 21/03/2014. As avaliações ocorreram no início (primeira semana), meio (45° dia) e fim (92° dia) do período de pastejo, no inverno (21/06/2014 a 21/09/2014). Os pastos diferidos com 15 e 25cm apresentaram menor massa de forragem (MF), mas maior percentual de folha viva (FV) na MF do que os pastos diferidos com 35 e 45cm. O percentual de colmo vivo na MF e o índice de seletividade aparente (ISA) da FV foram superiores no pasto diferido com 45cm. O percentual de colmo morto (CM) na simulação de pastejo (SP) e o ISA desse componente morfológico foram menores no pasto diferido com 15cm, em comparação ao diferido com 45cm. A MF e os percentuais de FV na MF e na amostra de SP se reduziram, enquanto os percentuais de CM na MF e na amostra de SP aumentaram com o período de pastejo. O capim-marandu com 15cm no início do período de diferimento melhora a morfologia do pasto diferido. O pastejo seletivo é dificultado no decorrer do período de pastejo.(AU)