Human-edible protein contribution of tropical beef cattle production systems at different levels of intensification.

Sustainable intensification of tropical grasslands has been identified by researchers and stakeholders as a solution to decrease greenhouse gas emissions and deforestation. However, there are concerns about food security and the role of livestock in feed-food competition between animals and humans involving land and other resources. We aimed to determine the net protein contribution (NPC), a feed-food competitiveness index, of tropical beef cattle raised on extensive systems or finished in pastures or conventional feedlots, under different levels of intensification. We modelled five scenarios, from cow-calf to slaughter, based on common beef cattle practices in Brazil, whose main production system is grazing. Scenario 1 represented the lowest level of intensification and the most extensive system. Scenario 2 represented a moderately extensive system. Scenarios 3, 4, and 5 represented different degrees and practices of intensification, with animals in cow-calf and stocker phases raised solely on well-managed permanent pastures. In Scenario 3, the animals were finished in a feedlot. In Scenarios 4 and 5, all animals in the stocker phase received a protein-energy supplement, but in Scenario 4, animals were finished in a permanent pasture with high-concentrate intake. In Scenario 5, animals were finished in a feedlot. The human-edible protein (heP) conversion efficiency (hePCE) was calculated as the ratio of heP produced (meat) to heP consumed as feed, and the NPC was the product of hePCE using the protein quality ratio, accounting for the digestible indispensable amino acid score content. An hePCE > 1 indicated that meat production did not compete with humans for food, and an NPC > 1 indicated that it contributed positively to meet human requirements. Meat production and heP intake consistently increased with intensification. The greatest hePCE values were from Scenarios 1 (9.2), 2 (2.2), and 3 (1.2), which were essentially pasture-fed systems, compared to Scenarios 4 and 5 (average of 1.0). The NPC varied from 24.1 (Scenario 1) to 2.6 (Scenario 5). The area required to produce 1 kg of carcass decreased from 147 to 45 m2, and the slaughter age decreased from 36 to 21 months from the most extensive to intensive systems. Brazilian beef cattle production contributes positively to the protein requirements of humans without limiting human food supplies. The intensification of tropical grazing beef systems is a key strategy to save land and produce more meat without limiting food for humans, playing an important role in the food security agenda.

Effects of supplementation with corn distillers' dried grains on animal performance, nitrogen balance, and enteric CH4 emissions of young Nellore bulls fed a high-tropical forage diet.

The inclusion of corn-dried distillers' grains (DDG) could be an alternative supplement to increase animal performance, nitrogen efficiency usage (NEU), and decrease enteric methane (CH4) emissions. Our goal was to determine whether DDG could replace a traditional supplement (cottonseed meal) without affecting animal performance, N balance, and CH4 emissions. The experiment was conducted during the forage growing season (December to April), with 15 d adaptation, and a 112 d experimental period. The experimental design was completely randomized with four treatments: a mineral supplement (MS), cottonseed meal supplement (CS), 50% replacement of CS by DDG (50DDG), and 100% replacement of CS by DDG (100DDG). Cottonseed meal and DDG were used as protein supplement. A total of 12 paddocks, 3 per treatment, were used to measure forage mass: morphological and chemical composition of forage, forage allowance, and animal performance. Six animals per treatment were used to evaluate DM intake, digestibility, CH4 emissions, microbial protein production (MCP), and NEU of each treatment. Eighty-one Young Nellore bulls (48 testers, 12 per treatments and 33 adjusters) with initial BW of 255 ±â€¯5 kg (10-12 months old) were supplemented with each supplement type at a level of 0.3% of BW. Pasture management was continuous stocking with a variable stocking rate (put-and-take). Enteric CH4 was measured using the gas tracer technique. The MCP was quantified using purine derivatives and the NEU mass balance. No differences were found in nutrient intake (P > 0.228). Individual animal performance and gain per area were higher in the treatments with concentrates compared with that of MS; however, there was no difference among treatments CS, 50DDG, and 100DDG. The ADG was 0.83 for MS and 1.08 kg/animal/d when supplemented (P < 0.05). Gain per hectare was 709 kg/ha for MS and 915 kg/ha when supplemented with concentrates (P < 0.05). There was no difference in CH4 production among treatments that average 180 g/animal/d; however, CH4 per kg of gain was reduced with CS. The CH4 conversion factor averaged 5.91%. There was no difference in the synthesis of MCP and NEU. Corn DDG can replace 100% of cottonseed meal as a protein source for supplementation of young Nellore bulls grazing in tropical pastures without affecting animal performance, NEU, MCP, and CH4 emissions.

Sward structure and ingestive behavior of cows in tropical pastures managed under different forage allowances / Estrutura do dossel forrageiro e comportamento ingestivo de vacas em pastos tropicais manejados sob diferentes ofertas de forragem

This study evaluated how changing the structure of Brachiaria brizantha cv. Marandu pasture under different forage allowances (FA) of 4, 7, 10 and 13kg DM/100kg BW (body weight) affected animal behavior over a two-year evaluation period. The experiment was conducted as a randomized block design with three replications (paddock). Sward height, total forage, and stem mass were lower for pastures managed with lower FA. Lower leaf mass was observed for lower FA in the second year. In hand-plucked samples, leaf and stem percentages remained unchanged in the morning but leaf percentage increased while stems decreased in the afternoon. Permanence time decreased linearly with increasing FA. In turn, a quadratic effect was observed for displacement rate. The bite rate was similar among different FAs. The results demonstrate that FA varying between 7 and 10kg DM/100kg BW are more suitable to balance the amounts of leaves and stems in the sward. The studied FA levels do not limit forage availability. Permanence time decreases and displacement rate increases as FA increases.(AU)

O objetivo foi avaliar como mudanças na estrutura de pastos de Brachiaria brizantha cv. Marandu, sob diferentes ofertas de forragem (OF) de 4, 7, 10 e 13kg MS/100kg PC, afetam o comportamento animal durante um período de avaliação de dois anos. O delineamento utilizado foi em blocos ao acaso, com três repetições (piquetes). A altura do dossel, a massa de forragem total e de colmos foram menores em pastos manejados com menores OF. A menor massa de folhas foi observada na menor OF no segundo ano. Nas amostras de pastejo simulado, as porcentagens de folhas e colmos não diferiram pela manhã, mas a porcentagem de folhas aumentou, enquanto a de colmos diminuiu à tarde. O tempo de permanência dos animais diminuiu linearmente com o aumento das OF. Efeito quadrático foi observado para taxa de deslocamento. A taxa de bocados foi similar entre as OF. Os resultados demonstram que variar as OF entre 7 e 10kg MS/100kg PC é mais adequado para balancear as quantidades de folhas e colmos no dossel forrageiro. Os níveis de OF estudados não limitam a forragem disponível. O tempo de permanência diminuí e a taxa de deslocamento aumenta com o aumento das OF.(AU)

Effect of forage species and supplement type on rumen kinetics and serum metabolites in growing beef heifers grazing winter forage.

The objective of this study was to determine the effect of stockpiled forage type and protein supplementation on VFA production, serum metabolites, and BW in yearling beef heifers. Over 2 yr, spring-born, Angus crossbred yearling beef heifers ( = 42; 305 ± 2.9 kg initial BW) were randomly assigned to 1 of 3 forage pasture types: 1) endophyte-infected tall fescue [TF; (Schreb.) Dumort], 2) a big bluestem ( Vitman) and indiangrass ( L.) combination (BI), or 3) switchgrass (SG; L.). Each pasture was then randomly assigned to receive either 1 of 2 isonitrogenous CP treatments: 1) 0.68 kg·heifer·d of dried distiller's grains with solubles (DDGS; 28% CP and 88% TDN) or 2) 0.22 kg·heifer·d of blood meal and fish meal (BF; 72.5% CP and 69.5% TDN), resulting in a 3 × 2 factorial arrangement of treatments. Treatments were initiated in January and terminated in April in both years of the study. Body weights and blood samples were collected approximately every 28 d from initiation of grazing until the end of the trial. Heifer BW change from January to February and overall BW change were greater ( < 0.01) for TF heifers. However, BW change from March to April was not different ( = 0.84) among forage types. Supplement type did not influence ( ≥ 0.13) BW or BW change from January to February and from January to April; however, heifers fed DDGS had greater ( = 0.03) BW gain from March to April. Heifer BW change from February to March exhibited ( < 0.05) a forage type × supplement interaction, with BF-fed heifers gaining more BW on BI pastures than DDGS-fed heifers. Serum glucose concentrations, ruminal acetate, and the acetate:propionate ratio were greater ( ≤ 0.04) for SG heifers. However, circulating serum NEFA and urea N (SUN) concentrations were not different ( ≥ 0.85) among forage types. Serum glucose and NEFA concentrations were not influenced ( ≥ 0.61) by supplement type. Circulating SUN concentrations were greater ( < 0.01) in BF-supplemented heifers. Ruminal acetate tended to be greater ( = 0.09) and butyrate concentrations were greater ( < 0.01) for BF-supplemented heifers. The acetate:propionate ratio was not influenced ( = 0.15) by supplement type. These results suggest that a compensatory gain period prior to breeding would be needed for these native warm-season species to be a viable opportunity for growing and developing replacement heifers in the southeastern United States.

Performance and methane emissions of grazing Nellore bulls supplemented with crude glycerin.

Supplementation of grass-fed cattle with low-cost feeding alternatives may be an attractive way to improve efficiency of cattle production. We hypothesized that inclusion of crude glycerin (CG) in supplements provided to grass-fed cattle could improve feed conversion without negative effects on growth performance while reducing methane emissions. Our hypothesis was tested using Nellore bulls grazing tropical pasture ( = 50; initial BW of 427 ± 19.41 kg; age of 17 ± 2 mo) supplemented with increasing concentrations (0, 70, 140, 210, and 280 g/kg DM basis of supplement) of CG and corn gluten replacing corn grain. A second experiment was conducted using 10 ruminally cannulated Nellore steers (490.1 ± 47.8 kg BW; age of 25 mo) to assess the impact of different concentrations of glycerin in the supplement on ruminal VFA concentration. Inclusion of CG did not affect total DMI ( = 0.53), DMI of forage ( = 0.41), supplement DMI ( = 0.47), organic matter intake ( = 0.50), crude protein intake ( = 0.24), NDF intake ( = 0.49), GE intake ( = 0.50), NDF digestibility ( = 0.17), final BW ( = 0.17), LM area ( = 0.50), rib fat thickness ( = 0.87), or carcass gain ( = 0.13). The inclusion of CG in the supplement linearly increased ( < 0.001) the molar proportion of propionate, butyrate, and valerate; linearly decreased acetate ( = 0.001); and did not affect the molar proportion of isovalerate ( = 0.31) and isobutyrate ( = 0.63), thereby reducing the acetate to propionate ratio ( < 0.001). The increase of CG supplementation of young bulls in pasture had a quadratic effect on BW gain ( = 0.002), with lower BW gain with 140 g/kg DM of CG in the supplement and tended ( = 0.06) to improve G:F. Inclusion of CG did not affect ruminal CH emission expressed in kilograms per year ( = 0.74), grams per kilogram of DMI ( = 0.69), and grams per kilogram of carcass gain ( = 0.48). Crude glycerin supplementation was not effective as a strategy to reduce CH emission in grass-fed cattle. However, CG can be effectively used as a partial energy source in supplement of grazing cattle, promoting an improvement in feed efficiency.

The endocannabinoid system in renal cells: regulation of Na(+) transport by CB1 receptors through distinct cell signalling pathways.

BACKGROUND AND PURPOSE: The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70-80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na(+) in LLC-PK1 cells. EXPERIMENTAL APPROACH: Changes in Na(+) /K(+) -ATPase activity were assessed after treatment with WIN55,212-2 (WIN), a non-selective lipid agonist, and haemopressin (HP), an inverse peptide agonist at CB1 receptors. Pharmacological tools were used to investigate the signalling pathways involved in the modulation of Na(+) transport. KEY RESULTS: In addition to CB1 and CB2 receptors and TRPV1 channels, the mRNAs encoding for enzymes of the ECS were also expressed in LLC-PK1. WIN (10(-7) M) and HP (10(-6) M) altered Na(+) re-absorption in LLC-PK1 in a dual manner. They both acutely (after 1 min) increased Na(+) /K(+) -ATPase activity in a TRPV1 antagonist-sensitive way. WIN's stimulating effect persisted for 30 min, and this effect was partially blocked by a CB1 antagonist or a PKC inhibitor. In contrast, HP inhibited Na(+) /K(+) -ATPase after 30 min incubation, and this effect was attenuated by a CB1 antagonist or a PKA inhibitor. CONCLUSION AND IMPLICATIONS: The ECS is expressed in LLC-PK1 cells. Both CB1 receptors and TRPV1 channels regulate Na(+) /K(+) -ATPase activity in these cells, and are modulated by lipid and peptide CB1 receptor ligands, which act via different signalling pathways.

Plantas do Gênero Xylopia: Composição Química e Potencial Farmacológico / Plants of the Xylopia Genus: Chemical Composition and Pharmacological Potential

RESUMO A família Annonaceae possui representantes de grande interesse medicinal e o gênero Xylopia é um dos que merecem destaque. Composta por aproximadamente 160 espécies distribuídas na América do Sul, América central, África e Ásia, as espécies desse gênero podem ser arbustivas ou arbóreas. No Brasil são encontradas nas regiões Norte, Nordeste, Centro-Oeste e Centro Sul. Este gênero produz uma variedade de metabólitos incluindo alcalóides, amidas, lignóides, acetogeninas e terpenóides e têm sido investigados como fonte potencial de acetogeninas, compostos esses que apresentam uma ampla variedade de propriedades biológicas com destaque para: citotóxica, antitumoral, antiparasitária, antimicrobial, inseticida e antimalarial. Neste estudo, efetuou-se uma revisão das principais espécies de Xylopiaencontradas no Brasil, já estudadas e descritas na literatura, abordando os aspectos químico-farmacológicos, destacando os constituintes químicos isolados bem como a ação farmacológica evidenciada.

ABSTRACT The family Annonaceae has representatives of great medical interest, and the Xylopia species deserves attention. The Xylopia genus is composed by approximately 160 species, with geographic distribution in tropical and subtropical regions of America, Africa and Asia. This genus can present shrubs or trees. In Brazil, they can be found at the North, North-west, Central-West and Central-South Regions. The phytochemical investigations resulted mainly in the isolation of alkaloids, diterpenos, quinolines and acetogenins, with the latter presenting very interesting biological properties such as the cytotoxic, antiprotozoal and the insecticide activities.This study aimed to review the botanical, chemical and pharmacological aspects of the Xylopia genus found in Brazil, highlighting the chemical components, as the well-known pharmacological effect .

Effects of feeding corn silage inoculated with microbial additives on the ruminal fermentation, microbial protein yield, and growth performance of lambs.

This study aimed to examine the effects of feeding corn silage inoculated without or with either Lactobacillus buchneri (LB) alone or a combination of LB and Lactobacillus plantarum (LBLP) on the apparent digestibility, ruminal fermentation, microbial protein synthesis, and growth performance of lambs. Thirty Santa Inês×Dorper crossbred intact males lambs weighing 20.4±3.8 kg were blocked by weight into 10 groups. Lambs in each group were randomly assigned to 1 of the following 3 dietary treatments: untreated (Control), LB, and LBLP silage. Lambs were fed experimental diets for 61 d. The apparent digestibility was indirectly estimated from indigestible NDF measured on d 57 to 59. Spot urine samples were collected from all animals on d 59 to estimate microbial protein synthesis. Lambs were slaughtered for carcass evaluation on d 61 when they weighed 32.4±5.2 kg. Six additional ruminally cannulated Santa Inês×Dorper crossbred wethers weighing 40.5±1.8 kg were used to examine dietary effects on ruminal fermentation. Average daily gain was increased when lambs were fed LBLP silage (P<0.05) but not LB silage. The LBLP silage had the highest (P<0.05) lactic acid concentration and both inoculated silages had greater acetic acid concentrations than the Control silage (P<0.05). Inoculation of corn silage increased intakes of DM, OM, CP, NDF, total carbohydrate (CHO), and GE by the lambs but decreased digestibility of DM, OM, CP, total and nonstructural carbohydrates, and concentration of GE and ME. (P<0.05). Nevertheless, lambs fed inoculated silages had greater microbial N supply than those on the Control treatment (P<0.05). The acetate to propionate ratio was lower in ruminal fluid of wethers in LBLP treatment than LB and Control treatment (P<0.05) and ruminal pH tended to be greater in LB lambs than in LBLP and Control wethers (P<0.10). Finally, the inoculation with both bacteria combined enhanced the silage fermentation. The intakes of DM, OM, CP, NDF, and GE were improved in the lambs fed corn silage inoculated with L. buchneri alone or combined with L. plantarum. The microbial N supply was enhanced in the lambs fed corn silage inoculated with L. buchneri. The inoculation of L. buchneri combined with L. plantarum reduced the acetate to propionate ratio in ruminal fluid and improved the ADG of lambs.

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors.

Caffeine, a methylated derivative of xanthine and widely consumed psychoactive substance, acts in several targets in the nervous system. We investigated its role in retinal explants of chick embryo analyzing the role of purinergic receptors in [(3)H]-GABA release induced by d-aspartate (d-asp). d-Asp increases GABA-release 4.5-fold when compared to basal levels from 13-day-old chick embryo retinal explants. Caffeine 500µM elevated d-asp-induced GABA release in 60%. The release was inhibited in the presence of NNC-711, a GABA transporter-1 (GAT-1) blocker or by MK-801, an N-methyl-d-aspartate receptor (NMDAR) antagonist. Caffeine did not modify [(3)H]-GABA uptake carried out for 5, 10, 30 and 60min and did not increase the release of d-asp or glutamate at basal or stimulated conditions. The caffeine effect was mimicked by the adenosine A1 receptor antagonist DPCPX and by the adenylyl cyclase (AC) activator forskolin. It was also blocked by the protein kinase A (PKA) inhibitor H-89, tyrosine kinase inhibitor genistein or by the src family kinase (SFK) inhibitor PP1. Forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels were reduced in the presence of the A1 receptor agonist CHA. Western blot analysis revealed that 500µM caffeine increased phosphoGluN2B expression levels in approximately 60% when compared to total GluN2B levels in embryonic E13 retina. The GluN2B subunit-containing NMDAR antagonist ifenprodil inhibited the caffeine effect. Our results suggest that caffeine potentiates d-asp-induced GABA release, which is mediated by GAT-1, via inhibition of adenosine A1 receptor and activation of the PKA pathway. Regulation of NMDAR by phosphorylation of GluN2B subunit by a SFK may also be involved in the effect promoted by caffeine.

Fatty acid profile, carcass and meat quality traits of young Nellore bulls fed crude glycerin replacing energy sources in the concentrate.

Carcass and meat quality traits of 60 Nellore young bulls fed diets without crude glycerin (CG); with CG replacing corn (CGc; 10% of dry matter - DM) in the concentrate; and with CG replacing soybean hull (CGsh; 10% of DM) in the concentrate were evaluated. Diets were evaluated at two concentrate levels (CLs). The CL did not affect cold carcass weight (CCW; P=0.6074), cold carcass dressing (CCD; P=0.9636), rib fat thickness (RFT; P=0.8696) and longissimus muscle area (LMA; P=0.7524). Animals fed diets with CGc or CGsh showed meat with greater deposition of monounsaturated fatty acid (MUFA; P=0.0022) and CLA (18:2 cis-9, trans-11) contents (P=0.0001) than animals fed diets without CG. The inclusion of 10% of CG in diets CGc or CGsh does not affect the carcass and meat quality traits; however, it increases the MUFA and CLA contents in beef, although these changes are very small in nutritional terms.

Oxidative stress on cardiotoxicity after treatment with single and multiple doses of doxorubicin.

The mechanism of doxorubicin (DOX)-induced cardiotoxicity remains controversial. Wistar rats (n = 66) received DOX injections intraperitoneally and were randomly assigned to 2 experimental protocols: (1) rats were killed before (-24 h, n = 8) and 24 h after (+24 h, n = 8) a single dose of DOX (4 mg/kg body weight) to determine the DOX acute effect and (2) rats (n = 58) received 4 injections of DOX (4 mg/kg body weight/week) and were killed before the first injection (M0) and 1 week after each injection (M1, M2, M3, and M4) to determine the chronological effects. Animals used at M0 (n = 8) were also used at moment -24 h of acute study. Cardiac total antioxidant performance (TAP), DNA damage, and morphology analyses were carried out at each time point. Single dose of DOX was associated with increased cardiac disarrangement, necrosis, and DNA damage (strand breaks (SBs) and oxidized pyrimidines) and decreased TAP. The chronological study showed an effect of a cumulative dose on body weight (R = -0.99, p = 0.011), necrosis (R = 1.00, p = 0.004), TAP (R = 0.95, p = 0.049), and DNA SBs (R = -0.95, p = 0.049). DNA SBs damage was negatively associated with TAP (R = -0.98, p = 0.018), and necrosis (R = -0.97, p = 0.027). Our results suggest that oxidative damage is associated with acute cardiotoxicity induced by a single dose of DOX only. Increased resistance to the oxidative stress is plausible for the multiple dose of DOX. Thus, different mechanisms may be involved in acute toxicity versus chronic toxicity.

Substituição de monensina sódica por bicarbonato de sódio em dietas de novilhas confinadas / Evaluation of the replacement of Monensin with Sodium Bicarbonate in diets for confined bovines

Avaliaram-se os efeitos da substituição da monensina sódica pelo bicarbonato de sódio em dietas de novilhas em confinamento em relação ao desempenho animal, à conversão alimentar e ao parâmetro de fermentação ruminal. Foram utilizadas 56 novilhas mestiças, confinadas por 112 dias, submetidas a dietas com duas porcentagens de concentrado, 50% e 80% da matéria seca, com adição de monensina sódica ou bicarbonato de sódio, em fatorial 2x2. O volumoso utilizado para compor as dietas testadas foi composto de silagem de milho e cana-de-açúcar na proporção de 60:40, respectivamente. O nitrogênio amoniacal foi maior nos animais alimentos com dietas com alto grão. A monensina, na dieta 80:20, proporcionou maiores valores de nitrogênio amoniacal, uma e nove horas após o consumo da ração. O uso de bicarbonato promoveu os maiores valores de pH ruminal na dieta com 50% de concentrado, de seis a 18 horas após o consumo. Não houve efeito do aditivo sobre o desempenho animal. A alta inclusão de grãos na dieta propiciou aumento do desempenho animal e da eficiência alimentar. Conclui-se que o uso de bicarbonato de sódio em dietas para terminação de animais em confinamento é uma opção viável em substituição à monensina sódica para a manipulação da fermentação ruminal.

The effects of replacing monensin with sodium bicarbonate in diets for confined heifers on weight gain (WG), dry matter intake (DMI), feed:gain ratio (FGR) and rumen fermentation parameters were evaluated. Fifity six heifers were utilized (Santa Gertrudis x Braunvieh). Diets were formulated with two levels of concentrate (50% and 80% concentrate) with sodium monensin or sodium bicarbonate. The DMI was higest in heifers with an intake of 80% concentrate. There was not difference when the same concentrate levels were evaluated in relation to the DMI. The heifers at 80% concentrate showed highest WG. There was no significant difference in relation to WG between monensin and bicarbonate treatments and levels concentrate proportions. There were significant effects regarding FGR between treatments. The heifers on 80% concentrate showed lowest FGR. In the evaluation of N-NH3, the highest values were observed, on the whole, at 80:20 diets in relation to 50:50 diets. In the additive use, the monensin on 80:20 diets promoted the highest values of N-NH3 at 1 for 9 hours after feed intake. In the values of ruminal pH, the bicarbonate increased the values on 50:50 at 6 for18 hours after feed intake, in relation to the 80:20 diet. In this case, it was concluded that the use of sodium bicarbonate in diets for confined finishing heifers is a viable option for the replacement of monensin sodium.

Functional identification of cell phenotypes differentiating from mice retinal neurospheres using single cell calcium imaging.

Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) stimulated Ca(2+) transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response to muscimol. Ca(2+) fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal diseases.

Digestibilidade aparente da dieta com capim-elefante ensilado com diferentes aditivos / Apparent digestibility of diet with elephant grass ensiled with different additives

Foram estudados os efeitos da adição de 15 por cento de diferentes aditivos - casca de café, farelo de cacau e farelo de mandioca - à forragem verde de capim-elefante (peso/peso), no momento da ensilagem, sobre a digestibilidade aparente de dietas. Foram usados 20 ovinos machos, não castrados, com média de 22,27±3,24kg de peso corporal, em um delineamento inteiramente ao acaso, com quatro tratamentos e cinco repetições. Os tratamentos foram: T1 = capim-elefante ensilado sem aditivo; T2 = capim-elefante ensilado com 15 por cento de casca de café; T3 = capim-elefante ensilado com 15 por cento de farelo de cacau; T4 = capim-elefante ensilado com 15 por cento de farelo de mandioca. Os animais receberam dieta isoproteica (10 por cento de proteína bruta) em proporção de 60 por cento de volumoso e 40 por cento de concentrado, na base da matéria seca. Utilizou-se o método de coleta total de fezes durante sete dias. Os coeficientes de digestibilidade aparente da matéria seca, proteína bruta, fibra em detergente neutro e fibra em detergente ácido e de digestibilidade verdadeira da proteína bruta foram maiores no capim-elefante ensilado sem aditivo. O coeficiente de digestibilidade do extrato etéreo foi mais alto na silagem sem aditivo e na silagem com farelo de cacau. Os coeficientes de digestibilidade da matéria orgânica e dos carboidratos totais foram mais elevados na silagem sem aditivo e na silagem com farelo de mandioca. Os maiores valores de nutrientes digestíveis totais foram observados nas dietas que continham silagem sem aditivo e silagem com farelo de mandioca. A silagem de capim-elefante com 15 por cento de farelo de mandioca, bem como a silagem sem aditivo apresentaram melhor digestibilidade dos nutrientes.

The effects of adding 15 percent of different additives (coffee hulls, cocoa meal and, cassava meal) to green elephant grass forage (weight/weight) at the time of ensiling on the apparent digestibility of diets were studied using 20 male sheep in a completely randomized design, with four treatments and five replications. The treatments were: T1 = elephant grass ensiled without additive; T2 = elephant grass ensiled with 15 percent coffee hulls; T3 = elephant grass ensiled with 15 percent cocoa meal; and T4 = elephant grass ensiled with 15 percent cassava meal. The animals were fed isonitrogen diet (10 percent crude protein) in 60 percent roughage and 40 percent concentrate rate, on dry matter basis. Total feces collection method was used for seven days. The apparent digestibility coefficients of dry matter, crude protein, neutral detergent fiber, and acid detergent fiber and the true digestibility coefficient of crude protein were higher in elephant grass ensiled without additive. The ether extract digestibility coefficient was higher in the silages without additive and with cocoa meal. The digestibility coefficients of organic matter and total carbohydrates were higher in the silage without additive and in the silage with cassava meal. The highest values of total digestible nutrients were observed in diets containing silages without additive and with cassava meal. The elephant grass silage with 15 percent cassava meal, together with the silage without additive, showed better nutrients digestibility.

Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells.

The P2X(7) receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X(7) receptor is induced by increasing the intracellular Ca(2+) concentration (Faria et al., Am J Physiol Cell Physiol 297:C28-C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X(7) large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca(2+)-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X(7) receptor large-channel formation in 2BH4 cells and peritoneal macrophages.

Degradabilidade ruminal da matéria seca, da proteína bruta e da fração fibrosa de silagens de milho, de sorgo e de Brachiaria brizantha / Ruminal degradability of dry matter, crud protein, and fiber fraction of silages of corn, sorghum, and Brachiaria brizantha

Avaliou-se a degradação da matéria seca (MS), da proteína bruta (PB), da fibra em detergente neutro (FDN), da fibra em detergente ácido (FDA) e da hemicelulose das silagens de milho, de sorgo e de Brachiaria brizantha. Foram utilizados três novilhos Nelore, machos, fistulados no rúmen, com média de peso de 200kg. Em cada animal foram incubadas amostras das três silagens nos tempos de 0, 6, 12, 24, 36, 48, 72 e 96 horas. Para as degradabilidades potencial e efetiva da MS (taxa de passagem no rúmen de 5 por cento/h), a silagem de milho foi a que apresentou valores mais altos, 80,1 e 56,7 por cento, respectivamente, seguida da silagem de sorgo, 73,0 e 45,3 por cento, e da silagem de Brachiaria, 60,8 e 33,7 por cento. Para a PB, o maior valor de degradabilidade foi verificado para a silagem de milho, 87,0 por cento, seguida das silagens de sorgo, 81,8 por cento, e de Brachiaria, 75,2 por cento. A degradabilidade da FDN foi maior para as silagens de milho e sorgo. Para FDA, a silagem de sorgo apresentou perfil de degradação menor que a silagem de milho. Os resultados sugerem melhor qualidade da silagem de milho, seguida pela do sorgo e pela de Brachiaria.

The experiment evaluated the degradation of the dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose of silages of corn, sorghum and Brachiaria brizantha. Three steers averaging 200kg bw were rumen fistulated. In each animal, the three silages were incubated for 0, 6, 12, 24, 36, 48, 72, and 96 hours. For the potential degradability and the effective degradability (passage rate 5 percent/h) the corn silage presented the highest values, 80.1 and 56.7 percent, respectively, followed by the sorghum silage (73.0 and 45.3 percent), and the Brachiaria silage (60.8 and 33.7 percent). For CP, the highest degradability value was also verified for the corn silage (87.0 percent), followed by the sorghum silage (81.8 percent), and Brachiaria silage (75.2 percent). For NDF, the highest degradability values were also verified for the corn and sorghum silages. However, for ADF, the sorghum silage presented degradation profile lower than the corn silage. The results suggest a better quality of the corn silage, followed by sorghum and Brachiaria silages.

Pharmacological properties of a pore induced by raising intracellular Ca2+.

Recent studies on the P2X(7) receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca(2+) concentration induces a pore opening similar to P2X(7) receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca(2+) concentration is associated to P2X(7) receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X(7) receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10 microM) increased intracellular free Ca(2+) concentration and induced pore formation with conductance of 421 +/- 14 pS, half-time (t(1/2)) for ethidium bromide uptake of 118 +/- 17 s, and t(1/2) for Lucifer yellow of 122 +/- 11 s. P2X(7) receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca(2+) concentration. However, 5-(N,N-hexamethylene)-amiloride, a P2X(7) receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca(2+) at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca(+2) induces a novel membrane pore pharmacologically different from the P2X(7) associated pore and hemigap-junction pore.

Trophic activity derived from bone marrow mononuclear cells increases peripheral nerve regeneration by acting on both neuronal and glial cell populations.

A rat model of complete sciatic nerve transection was used to evaluate the effect of bone marrow mononuclear cells (BMMC) transplanted to the injury site immediately after lesion. Rats treated with BMMC had both sensory and motor axons reaching the distal stump earlier compared to untreated animals. In addition, BMMC transplantation reduced cell death in dorsal root ganglia (DRG) compared to control animals. Transplanted BMMC remained in the lesion site for several days but there is no evidence of BMMC differentiation into Schwann cells. However, an increase in the number of Schwann cells, satellite cells and astrocytes was observed in the treated group. Moreover, neutralizing antibodies for nerve growth factor (NGF) (but not for brain-derived neurotrophic factor and ciliary-derived neurotrophic factor) added to the BMMC-conditioned medium reduced neurite growth of sensory and sympathetic neurons in vitro, suggesting that BMMC release NGF, improve regeneration of the sciatic nerve in the adult rat and stimulate Schwann and satellite cell proliferation or a combination of both.

Herbimycin A induces sympathetic neuron survival and protects against hypoxia.

We have examined how herbimycin affects the survival and neuritogenesis of avian sympathetic neurons. Herbimycin promoted sympathetic neuron survival and neuritogenesis. At higher concentrations (> or = 100 ng/ml), herbimycin still enhanced neuron survival but blocked neuritogenesis. Addition of herbimycin (10-30 ng/ml) to neurons cultured in the presence of NGF or retinal conditioned medium altered neuronal morphology, with an increase in the number of neurites. Addition of NGF during hypoxia rescued 52% of the neurons compared to 14% survival in control conditions. Herbimycin alone rescued about 50% of the neurons. In the presence of NGF and 100 ng/ml herbimycin, 81% of the neurons survived hypoxia. Our results show that herbimycin promotes survival of chick sympathetic neurons and potentiates the effects of NGF.

Dopaminergic retinal cell differentiation in culture: modulation by forskolin and dopamine.

We examined the effects of dopamine and cAMP on the differentiation of dopaminergic retinal cells in the chick retina, using an in vitro system and tyrosine hydroxylase immunocytochemistry. Tyrosine hydroxylase-positive cells were detected in cultures prepared from embryonic day 10 retinas. These increased in number as a function of time in vitro and by treatment for 4 days with forskolin. Besides causing a 3.4-fold increase in the tyrosine hydroxylase-positive population, forskolin also caused these cells to developed morphogenetic features of more mature cells. As opposed to forskolin, cultures treated with dopamine exhibited a 55% reduction of the tyrosine hydroxylase-positive cell population, as compared to untreated cultures. Quinpirole was able to mimic the dopamine effect. This dopamine effect could only be blocked by clozapine, whereas raclopride and eticlopride were ineffective. Our results suggest the existence of a narrow window during development when undifferentiated dopaminergic cells are capable of being influenced by specific signals, possibly via cAMP production. The data also indicate that dopamine may act as a regulatory factor limiting the tyrosine hydroxylase-positive population in the retina.