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Urochloa spp. are the most important grasses for ruminants in Brazil and contain secondary metabolites, mainly saponins. Urochloa brizantha extracts (ethanolic EE and hydroalcoholic HE with 3.62 and 5.38 mg protodioscin mL−1, respectively) were developed to verify their potential as additives for ruminant nutrition. The in vitro gas production technique was used to evaluate ten treatments in a completely randomized factorial arrangement (2 × 4 + 2), where the main effects were two extracts (EE and HE); four levels (50, 100, 150, and 200 mL of the extract kg−1 of DM), plus two controls (one positive with 25 ppm of monensin and another with no additives). The extracts EXT (EE and HE) produced a higher proportion of acetate (C2) and lower propionate (C3) than CTL, reflected in a 31% higher C2:C3 ratio. However, there was no significant difference (p > 0.05) between the treatments for methane production parameters. Archaea and Ruminococcus’ relative gene expressions were higher in EE than in HE; however, the protozoa opposite occurred, HE was higher than EE. Fibrobacter succinogenes were 33% lower in EXT than in CTL. The addition of these extracts in a sheep diet increased the production of SCFA and decreased Fibrobacter succinogenes without altering the methane and archaeal population.

Essential oils (EOs), as rumen additives, decreased CH4 emissions in in vitro trials but results from in vivo studies are still limited. We investigated the effects of Origanum vulgare (OEO) and Thymus vulgaris (TEO) EOs on in vivo methane emissions from Nellore beef cattle. Six adult rumen-cannulated Nellore cattle were used in a double 3 × 3 Latin square design. Treatments consisted of three diets containing either 3 mL OEO per kg of concentrate, 3 mL TEO/kg of concentrate, or no EO addition. The experimental period consisted of three 21 d feeding periods and methane production was measured using the sulfur hexafluoride (SF6) technique from Day 16 to Day 21 of each feeding period. Intake, total apparent digestibility (dry matter as well as neutral and acid detergent fiber), and rumen parameters (pH, ammoniacal nitrogen concentration, and short-chain fatty acids) were also evaluated. The EOs did not decrease CH4 emissions and had no effect on rumen parameters.

Twenty-four adult Beagles were utilised to evaluate the partial replacement of wheat bran with corn gluten feed without steep water on digestibility and characteristics of faeces. The treatments were 0 (no substitution), 30, 60 or 90 g/kg of corn gluten without steep water. There was no effect (p > .05) on the digestibility coefficients (g/kg) of dry matter (0.771), organic matter (0.806), crude protein (0.813), ether extract (0.798), crude fibre (0.393), neutral detergent fibre (0.425), acid detergent fibre (0.286) and crude energy (0.812), whilst there was effect (p .05) was found on the remaining faecal characteristics: excretion for 100 g of food (56.77 g), excretion (129.6 g/day and 49.0 g dry matter/day), score (3.90), dry matter excretion for 100 g of food (22.86 g), buffering capacity (BC) at pH 5 (57.81), ammonia nitrogen (1.46 g/kg of faecal dry matter) and water balance (333.25 mL/day), in vivo and in situ gas production (p > .05). Corn gluten feed without steep water can be utilised to replace up to 90 g/kg of wheat bran without causing negative effect on the digestibility and characteristics of faeces.

Abstract: The objective of this work was to evaluate the distribution of imidazolinone-resistant (IMI-R) red rice (Oryza sativa) populations, the frequency of alleles conferring resistance to IMI, and the adoption of agronomic practices applied to red rice control, across growing seasons and production regions of the state of Rio Grande do Sul (RS), Brazil. In the experiment, 1,008 red rice populations were screened for resistance to IMI, 760 IMI-R red rice plants were genotyped for the acetolactate synthase (ALS) alleles conferring resistance to IMI, and 40 'Clearfield' rice growers were surveyed. IMI-R red rice populations were widespread throughout RS since the 2006/2007 growing season, with a higher initial frequency in the Depressão Central and Fronteira Sul production regions. The occurrence of IMI-R red rice ranged from 1.6 to 3.5 years after 'Clearfield' rice release. Gly654Glu was the most frequent ALS mutation in IMI-R red rice populations, which shows a gene flow from the most used 'Clearfield' rice cultivars to the red rice plants. Crop rotation systems and certified seed were used by only 30% of the surveyed growers of 'Clearfield' rice, with lower percentages in the production regions where IMI-R red rice appeared faster.

Currently, the use of alternative energy ingredients for horses has increased because these foods contain "superfibers", making safer diet for these animals. To study the influence of diets containing increasing levels of citrus pulp on albumin, triglycerides, cholesterol, glucose, insulin and short chain fatty acid (SCFA) concentrations in the blood, five animals were used, with an average age of 3.5 years and a live weight of 460.66±76.86 kg, they were fed twice a day, at 7:00 am and 4:00 pm. The diets were formulated to meet the requirements of the animals at maintenance. 60% of the energy was obtained from forage, and 40% from concentrate, containing the following inclusion levels of citrus pulp (0, 7, 14, 21 and 28%). The area under the curve (AUC) and the glucose and insulin peaks were calculated, and no differences were observed between the treatments. There was no effect of diet on the blood concentrations of the evaluated parameters tested, and there was no effect of the time of collection on the following variables: SCFAs, cholesterol, triglycerides and albumin. However, the diet did have quadratic effect on the glucose (Ŷ =-0.5327X²+4.2130X+84.5276) and insulin (Ŷ=-0.1002X² +0.8233X + 1.6336) concentrations. Up to 28% of the concentrate can be composed of citrus pulp in horse diets without causing any alterations on the concentrations of the parameters analyzed in the blood. High-fiber diets with easily fermentable fibers are beneficial because they maintain the glucose and insulin curves close to the baseline levels.

A similaridade botânica do capim-annoni com as espécies forrageiras nativas ou cultivadas dificultam o seu controle em pastagens naturais. O objetivo deste trabalho foi avaliar o efeito de associações entre herbicidas e protetores sobre o controle de capim-annoni e a seletividade em pastagem natural. Foram realizados dois experimentos a campo em área de pastagem natural típica do Bioma Pampa avaliando-se herbicidas aplicados em pré e pós-emergência. Outro experimento foi conduzido em casa de vegetação com as espécies capim-annoni e grama forquilha onde foram avaliados os herbicidas glufosinate-ammonium e imazethapyr. Em ambos os experimentos os herbicidas foram avaliados isolados ou em associação com os protetores anidrido naftálico, dietholate e fluxofenim. Os experimentos a campo demonstraram que os herbicidas aplicados em pré ou pós-emergência em interação com os protetores não proporcionaram controle satisfatório do capim-annoni e nem aumento da seletividade à pastagem natural. Por outro lado, o experimento em condições controladas indicou a ocorrência de sinergismo do herbicida glufosinate-ammonium em interação com os protetores anidrido naftálico, dietholate e fluxofenim sobre o capim-annoni. As diferenças relacionadas a estádio de desenvolvimento e densidade de infestação estão relacionadas as variações das respostas nos estudos a campo e em condições controladas. A utilização de herbicidas em associações com protetores apresenta potencialidade de controle seletivo de capim-annoni quando aplicado em plantas em estádios iniciais de desenvolvimento.The botanical similarity of the invasive weed Eragrostis plana Nees (South African lovegrass) difficult its selective control in natural pastures. The aim of this study was to evaluate the effect of the association between herbicides and safeners on the control of E. plana and on the selectivity to the natural pastures. Two experiments were conducted at field conditions in area of natural grassland of the Biome Pampa where several herbicides were evaluated in pre and post-emergence. A third experiment was carried out in greenhouse evaluating the herbicides glufosinate-ammonium and imazethapyr and the species E. plana and Paspalum notatum. In both experiments, the herbicides were evaluated isolated and in association with the safeners anhydride naphtalic, dietholate and fluxofenim. The field experiments indicated that the evaluated safeners did not increase E. plana control and native grassland selectively. The greenhouse experiment indicated the occurrence of synergism on E. plana for the herbicide glufosinate-ammonium in mixture with the safeners anhydride naphtalic, dietholate and fluxofenim. The different results in the field and greenhouse experiments are related to the plant stage and density. The use of herbicides in association with safeners indicated a potentiality for selective control of E. plana when applied to plants in the early stages of development.

Aiming at evaluating hibiscus (Hibiscus rosa-sinensis) and sacarina as supplements for young bulls, four crossbred animalswere displayed in a Latin square experimental design. The following treatments were performed: 1 – 75% pasture + 25% concentrate; 2 – 50% pasture + 25% concentrate + 25% hibiscus; 3 – 50% pasture + 25% concentrate + 25% sacarina; and 4 – 25% pasture + 25% concentrate + 25% hibiscus + 25% sacarina. Diet composition, rumen fermentation, DM and CP degradation rates were evaluated. Rumen pH values varied from 5.82 to 6.98, for diets supplemented with hibiscus, at 9h post-feeding, and with sacarina, at the feeding moment. Ammonia N contents did not present great changes when only hibiscus or sacarina were added to the diet, but there was a significant effect when both were added P

Diversos biótipos de sagitária (Sagittaria montevidensis Cham. & Schlecht) foram constatados como resistentes a herbicidas inibidores da enzima ALS no Brasil e em outros locais do mundo. Os objetivos deste trabalho foram isolar e sequenciar o gene ALS de sagitária de forma a identificar a ocorrência de mutações relacionadas ao mecanismo de resistência de insensibilidade do local da ação de herbicidas. O material vegetal consistiu de sementes de sagitária coletadas em lavouras de arroz irrigado localizadas na região Sul do Estado de Santa Catarina. Nove pares de sequências nucleotídicas iniciadoras foram desenhadas para a amplificação dos domínios C, A e D do gene ALS. As sequências nucleotídicas dos fragmentos amplificados com quatro pares de oligonucleotídeos inciadores resultaram em amplicons com 360 a 393 nucleotídeos. Essas sequências apresentaram homologia com o gene ALS padrão de Arabdopsis thaliana e arroz, possuindo apenas três mutações de ponto. Uma dessas alterações foi a mutação Pro197Phe, que é relacionada à ocorrência de resistência à herbicida em diversas espécies vegetais. As sequências obtidas evidenciaram que o gene ALS avaliado encontrava-se em heterozigose. Esses resultados sugerem a ocorrência de insensibilidade do local de ação como mecanismo de resistência a herbicidas inibidores de ALS em sagitária. A prevenção e o controle da resistência a herbicidas inibidores da ALS em sagitária devem ser fundamentados na rotação de herbicidas com diferentes mecanismos de ação.California arrowhead (Sagittaria montevidensis Cham. & Schlecht) is an aquatic weed often found in rice paddy fields. Several biotypes of California arrowhead resistant to ALS-inhibiting herbicides were found in Brazil and in several rice fields worldwide. The objective of this study was to sequence the ALS gene of California arrowhead and to identify the occurrence of mutations related to target site insensitivity as the mechanism of herbicide resistance. The plant material consisted of seeds collected in rice paddy fields located in the state of Santa Catarina, Brazil. Nine pairs of primers were designed for the amplification of the domains C, A and D of the ALS gene. PCR reactions with four pairs of primers resulted in amplification of fragments with size close to expected, ranging from 360 to 393 nucleotides. These sequences showed high homology to the standard ALS gene Arabdopsis thaliana and rice having only three point mutations. One of these mutations was Pro197Phe that is related with herbicide resistance in several weeds. The sequences obtained indicated that the ALS gene evaluated was heterozygous. These results suggest the occurrence of insensitivity of the site of action as the mechanism of resistance to ALS-inhibitors herbicides in California arrowhead. The prevention and control of resistance to ALS-inhibitors in California arrowhead should be based on the rotation of herbicides with different mechanisms of action.

Aiming to study the limiting factors for degradation of sugarcane fiber to be used as cattle feed, sugarcane fibrous components were fractioned in neutral- and acid-detergent fibers (respectively, NDF and ADF). Whole sugarcane and its fibers were incubated with rumen bacteria, in presence or absence of fibrolytic enzymes (Fibrozyme, Alltech Inc.). Microbial growth and total bacteria count were determined, and the growth rate, generation rate, lag time, and degradability were also determined. Results were analyzed in randomized block design, with a 2x3 factorial arrangement, with two doses of enzymes, and three substrates, using four replications for incubation. The highest microbial growth was observed for whole sugarcane, and the lowest for its fibers. The in vitro degradability was 0.701, 0.392 and 0.191, respectively for whole sugarcane, NDF and ADF (P

The objective of this work was to evaluate the dry matter rumen degradability of three distinct quality hays, using in situ and in vitro techniques. The animals used to incubate the bags in and as inoculum donors were six Santa Ines sheep, male, castrated, weighting 40±5.7 kg LW, all with rumen cannulas. Three forage hays were chosen for this experiment, based on their crude protein content. The studied hays were alfalfa (Medicago sativa) (ALF), signalgrass (Brachiaria decumbens) (SIG), and Tifton-85 (Cynodon sp.) (TIF). The in situ rumen degradability kinetics was determined using the nylon bag technique, and the in vitro degradability was accessed by the semi-automated gas production technique. There was difference between the three hays for CP (P < 0.01), ADIP (P < 0.01), DM, (P < 0.05) OM (P < 0.05), NDF (P < 0.05), hemicellulose (P < 0.05), and cellulose (P < 0.05) contents. Feeds SIG and TIF did not differ (P > 0.05) among them for ADF, ADL and silica contents, but both differed of feed ALF for the same constituents (P < 0.05). There were noticed differences between in situ and in vitro degradabilities, mainly for readily soluble fraction and degradation rates. The in situ and in vitro techniques to estimate the degradation kinetics were consistent, except for data on the initial solubility and lag time. The in vitro technique showed strong dependence of the quality of inoculum; the inoculum from animals better fed presented the best results. When the focus was to determine the lag time, the first-order exponential model underestimated the observed results. The fermentative kinetics (gas production) described in greater fidelity the feed degradation due to microbial action.

Forages usually fed to sheep in tropics have low values of crude protein (CP) content and/or low availability of protein. The aim of this work was to identify different profiles of microbial protein (MP) yield for sheep fed hays with distinct CP levels. Three hays (LUC - Lucerne, SIG - signalgrass and TIF - Tifton-85), six animals (Santa Inês wethers fitted with rumen and duodenum cannulas) placed in a double Latin square were used to evaluate transit rates of microbial protein and in vivo microbial synthesis. In vitro microbial protein synthesis was evaluated in a complete factorial statistical design. The parameters studied were rumen pH and ammonia-N, microbial protein transit using 15N as marker and microbial protein synthesis by in vivo (purine derivatives urinary excretion) and in vitro (32P incorporation) techniques. Rumen pH values did not present significant differences for treatments (hays) and these values were in a level considered as satisfactory to good microbial development. As studied hays presented great variations on CP levels, the concentration of ammonia-N was higher for animals fed LUC (191 g CP/kg DM) and lower for animals fed SIG (29 g/kg DM), therefore presented no difference for ammonia-N concentration (P > 0.05) between SIG and TIF (75 g CP/kg DM). The nitrogen assimilation rate (k1) by rumen microorganisms was much faster (P < 0.01) for treatment LUC, which showed that, in this treatment, nitrogen was more available than in others. MP produced from LUC had a faster passage (k2) from rumen to duodenum (P < 0.01). Animals fed LUC were those that presented higher MP supply (P < 0.05). Although there was no difference (P = 0.12) between both treatments with lower protein levels (TIF and SIG), there is a visible trend of that with lower protein content, the results were even more unsatisfactory, regarding the MP flow. The analysis of variance of the results of net MP production after 8 h incubation with 32P showed that there was an effect of inoculum (P = 0.0089), but there was no effect neither of substratum nor of substratum*inoculum interaction (P > 0.05). Feeds with low proteic content had prejudicial effect on utilization of nutrients by sheep and on maintenance of a healthy rumen environment. This fact was highlighted by variations on rumen ammonia-N concentrations. The microbial synthesis capacity was identified more efficiently by purine derivatives urinary excretion technique. The in vitro technique of 32P incorporation did not distinguish microbial growths from tested feeds degradation. In vitro technique showed strong dependency of inoculum quality.

The nutritive value, tannins bioactivity and methane production of some tropical plants e.g. Acacia (Acacia saligna), Leucaena (Leucaena leucocephala), Atriplex (Atriplex caseneace), Eucalyptus (Eucalytus globules), and Alfalafa (Medicago sativa) hay were evaluated by chemical composition and gasp production (GP) technique in vitro. Chemical analysis indicated that crude protein content was ranged from 95.4 to 204.0 g kg-1DM for eucalyptus and leucaena, respectively. The leucaena and alfalfa had the highest neutral detergent fibre (NDF) content (543.1 and 546.7g kg-1DM), respectively. Atriplex had the lowest content (173.8 g kg-1DM) of acid detergent fibre (ADF) and acid detergent lignin (ADL, 85.89 g kg-1DM). Alfalfa, atriplex and eucalyptus had negligible condensed tannins (CT) content (0.2, 0.3, 4.9 eq-g leucocyanidin kg-1DM), while acacia and leucaena had the highest CT (61.4 and 32.5 eq-g leucocyanidin kg-1DM). The acacia and leucaena produced less GP in the absence of PEG. Inclusion of PEG in fermentation of tropical browses resulted in a significant increase of GP (28.1 and 87.4 1%) particularly the plants rich in condensed tannins. The methane production was decreased significantly (P alfalfa > acacia > eucalyptus > leucaena. Partitioning factor (PF) values were differed significantly (P

Estudou-se o metabolismo de cálcio em eqüinos em crescimento recebendo dietas com diversos níveis desse mineral. Utilizaram-se 12 machos, com média de 12 meses de idade e peso vivo médio de 221,0 kg, mantidos com três dietas com 0,15; 0,45; ou 0,75 de cálcio e 0,23% de fósforo, constituindo um delineamento inteiramente casualizado, com três níveis de cálcio e quatro repetições. Foram injetados em cada animal, via jugular direita, os volumes de uma seringa, correspondentes a 7,4 MBq de 45Ca (CaCl2). Após a injeção, foram colhidas amostras (10 mL) de sangue, via jugular esquerda, aos 5 minutos, 1, 2, 4, 6, 24, 48, 72, 96, 120 e 144 horas. As fezes foram colhidas diariamente pela manhã, a partir de 24 horas após a aplicação do radionuclídeo até o sétimo dia e uma alíquota de 5% do total diário excretado foi colhida e armazenada em congelador para posterior análise. O mesmo procedimento das fezes foi adotado para a urina, quando foram colhidas amostras de 1% do volume total diário. Os níveis de cálcio plasmático, cálcio total na urina e cálcio endógeno nas fezes não diferiram entre os níveis de cálcio com valores médios de 11,76; 5,54 e 20,86 mg/kgPV/dia, respectivamente. A disponibilidade biológica de cálcio (média de 81,71%) também não diferiu entre os níveis de cálcio. As regressões lineares entre níveis de cálcio na dieta e cálcio total ingerido, cálcio absorvido, cálcio total nas fezes e cálcio retido foram expressas, respectivamente pelas equações: y=10,7+2,78x r²=1,0; y=12,41+2,24x r²=0,99; y=5,97+0,92x r²=0,93 e y=6,4+1,70x r²=0,94, com médias de, respectivamente, 135,80; 113,21; 47,37 e 82,90 mg/kgPV/dia.The objective of this work was to study the calcium metabolism in growing equines receiving diets with different calcium levels. Twelve male were used, with average age of twelve months and average alive weight of 221,0 kg. The treatments consisted of three diets, composed for increasing calcium levels (low = 0.15%-N15; standard = 0.45%-N45; high = 0.75%-N75), and standard phosphorus level (0.23%). Each animal was injected, through the right jugular vein, the volumes of a syringe, which corresponded to about 7.4 MBq of 45Ca (CaCl2). After the injection, blood samples (10 mL) were collected through left jugular vein at five minutes, 1, 2, 4, 6, 24, 48, 72, 96, 120 and 144 hours. The feces were daily collected in the morning, 24 hours after the application of the radionuclide until the seventh day, and an aliquot of 5% of the daily total excreted was collected and stored in freezer for posterior analysis. The same procedure of excrements was adopted for urine, when samples of 1% of the daily total volume had been collected. The experimental techniques used a completely randomized design with three treatments and four replications. No effects were observed on the plasma calcium, total calcium in urine and endogenous calcium in feces, with average values of 11,76; 5,54 and 20,86 mg/kgLW/day, respectively. No effects were observed for the biological calcium availability, with 81.67%, on average. Linear regressions between calcium levels in the diet and total calcium ingested, absorbed calcium, total calcium in feces and restrained calcium have been respectively verified, which were expressed according to the following equations: y=10.7+2.78x r²=1.0; y=12.41+2.24x r²=0.99; y=5.97+0.92x r²=0.93 and y=6.4+1.70x r²=0.94, with averages of 135.80; 113.21; 47.37 and 82.90 mg/kgLW/day, respectively.

Objetivou-se estudar o metabolismo de cálcio (Ca) e fósforo (P) nos eqüinos em crescimento que receberam diferentes níveis de suplementação de Ca: 0,15; 0,45 e 0,75% na dieta, utilizando-se o modelo determinístico e compartimental. Foram utilizadas informações sobre o estudo de metabolismo e cinética de Ca e P em tecidos, obtidas pela técnica da diluição isotópica. Constatou-se que os níveis dietéticos de Ca tiveram influência na absorção real do Ca, sendo menor para o nível 0,15% (4,97 g Ca/dia). As trocas de Ca entre o sangue e o trato digestivo foram menores para o nível 0,15%. A mobilização entre o sangue e os ossos e sangue e tecidos moles não foi influenciada pelos tratamentos, mas o balanço nos ossos e tecidos foi menor para o nível 0,15%. Os níveis de Ca dietéticos tiveram influência no P eliminado através da urina, sendo este valor maior para o tratamento 0,15% (2,49 g/animal/dia). A absorção real média do P foi de 83%, não havendo diferenças para os tratamentos. Verificou-se deposição óssea média de 9,69 g P/animal/dia, indicando que a quantidade de P fornecida foi adequada em relação à tolerância permissível à amplitude na relação Ca:P para a espécie na categoria animal estudada. Não houve diferenças significativas entre os fluxos de P nos diversos compartimentos, por intermédio do modelo de metabolismo. A ingestão de níveis crescentes de Ca afetou o metabolismo e a cinética deste elemento, entretanto, a proporção Ca:P é o fator predominante para determinar a excreção, retenção e absorção de Ca. A deposição de Ca no osso foi influenciada pela quantidade ingerida deste mineral. O metabolismo de P em eqüinos em crescimento não foi afetado pelos teores de Ca. O fornecimento de 13,4 g P/animal/dia foi suficiente para manter o metabolismo de P nos padrões considerados normais.This experiment was carried out to study calcium (Ca) and phosphorus (P) kinetics of growing horses fed diets with different Ca levels: 0.15%, 0.45, and 0.75%, using a deterministic and compartmental model. The information on metabolism and kinetic of Ca and P in tissues was obtained by the isotopic dilution technique. Dietary Ca levels influenced Ca true absorption, showing lower value (4.97 g Ca/day) for the Ca level of 0.15%. The changes of Ca between the blood and digestive tract were lower for 0,15% Ca. The flows between the blood and bone and blood and soft tissues did not show differences between treatments, but bone and tissue balance was lower for the level of 0.15%. Dietary Ca levels influenced on P in urine and values were higher for 0.15% (2.49 g P/anim.day). The mean true P absorption was of 83%, with no differences among treatments. Bone deposition of 9.69g P/anim.day was observed, showing that the amount of P fed was adequate in relation to the allowed amplitude for Ca:P ratio for the specie in the studied animal category. No differences between the P flows in the diverse compartments, for interference of the metabolism model. The ingestion of increasing levels of Ca affected the metabolism and the kinetic of this element, however Ca:P ratio is the predominant factor to determine the excretion, retention and absorption of Ca. The Ca deposition in bone is influenced by the amount ingested of this mineral. The P metabolism in growing horses was not affected by Ca levels. The amount of 13.4 g P/anim.day in the diet was sufficient to maintain P metabolism at normal standard levels.

The aim of this work was to evaluate the voluntary intake, apparent digestibility and digesta kinetics of three protein-level forages fed to Santa Inês sheep. Six sheep (LW = 40 ± 5.7 kg) fitted with rumen cannulas were used. Lucerne (LUC), signalgrass (SIG) and Tifton-85 grass (TIF) hays were chosen due to their crude protein (CP) content (respectively, 191, 29 and 75 g kg-1 DM). Treatments and animals were fitted in a double Latin square (3 treatments, 3 periods, 6 animals). To measure the voluntary intake, animals were kept in individual stall with free access to water and food, during the first phase of each period. The feed consisted exclusively of LUC, SIG or TIF and mineral supplementation. Apparent digestibility was determined by total faeces collection. Digesta kinetics was estimated using Co-EDTA and Cr mordant fibre as markers to liquid and solid phases, respectively. Means were compared by Tukey test. Dry matter voluntary intake of treatment LUC was higher (P < 0.05) than treatments SIG and TIF and it reflected the strong relationship between intake and protein content in the diet. Dry matter and organic matter apparent digestibility coefficients were superior (P < 0.05) for LUC. Crude protein apparent digestibility showed differences (P < 0.05) between the three treatments, being LUC (0.694) the highest observed followed by TIF (0.500). Crude protein apparent digestibility of SIG was practically null (0.001), indicating intense protein deficit. Dry matter voluntary intake and digestibility of tested feeds were influenced by chemical composition and digestion kinetics. Low protein feeds had a prejudicial effect in nutrient apparent digestibi-lity, decreasing until nullity for protein of treatment SIG. Key-words: Lucerne, marker, nutrition, ruminant, signalgrass, Tifton. O objetivo deste trabalho foi avaliar o consumo voluntário, a digestibilidade aparente e a cinética diges-tiva de três forragens, com teores protéicos distintos, em ovinos. Utilizaram-se seis ovinos da raça Santa Inês (PV = 40 ± 5,7 kg), machos adultos, castrados e providos de cânulas ruminais. Optou-se pelo uso de fenos de alfafa (ALF), de braquiária (BRA) e de capim Tifton-85 (TIF) pelos seus teores de proteína bruta (PB), respectivamente, 191, 29 e 75 g kg-1 MS. Alocaram-se os tratamentos e os animais em delineamento de dois quadrados latinos (três tratamentos, três períodos e seis animais). As dietas consistiam exclusivamente de ALF, BRA ou TIF e mistura mineral, oferecida separadamente. O ensaio de consumo voluntário foi realizado em baias individuais e os de digestibilidade e cinética digestiva, em gaiolas de metabolismo. Determinou-se a digestibilidade aparente por coleta total de fezes. Estimou-se a cinética digestiva mediante o uso de Co-EDTA e fibra Cr-mordantada como marcadores das fases líquida e sólida, respectivamente. Compararam-se as médias por teste de Tukey. O consumo voluntário de MS do tratamento ALF foi maior (P < 0,05) que os tratamentos BRA e TIF, o que refletiu a forte relação entre consumo e teor de proteína na dieta. Os coeficientes de digestibilidade aparente da MS e da MO também foram superiores (P < 0,05) para ALF. A digestibilidade aparente da proteína bruta mostrou diferenças (P < 0,05) entre os três tratamentos, sendo ALF (0,694) a mais alta que se observou, seguida por TIF (0,500). Para o tratamento BRA, essa variável foi praticamente nula (0,001), indicando déficit protéico intenso. O consumo voluntário e a digestibilidade aparente dos alimentos testados receberam influência da composição química e da cinética digestiva. Alimentos com baixo teor protéico prejudicaram a digestibilidade aparente de nutrientes, diminuindo, próximo à nulidade, para a proteína do tratamento BRA. Palavras-chaves: Alfafa, braquiária, marcadores, nutrição, ruminantes, Tifton.

O resíduo úmido de cervejaria é um subproduto disponível ao longo do ano, podendo ser utilizado como um substituto das forragens durante os períodos críticos. O objetivo deste estudo foi avaliar a utilização do resíduo úmido de cervejaria como substituto do feno em dietas exclusivas de volumoso para ovinos. Mantiveram-se seis ovinos machos da raça Santa Inês em gaiolas de metabolismo em um delineamento experimental de quadrado latino múltiplo de 3 x 3, com dietas experimentais 100% de feno de Tifton 85 capim-bermuda (Cynodon ssp) (FT), 67% de FT + 33% de resíduo de cervejaria (RUC33) e 33% de FT + 67% de resíduo de cervejaria (RUC67). O ensaio avaliou o consumo voluntário, a digestibilidade aparente in vivo da matéria seca, da matéria orgânica, da proteína bruta, e das fibras em detergente neutro e em detergente ácido, pH ruminal e perfil de nitrogênio (N) amoniacal. O consumo voluntário foi de 1.090, 1.129 e 737 (EP = 66,9) g MS dia-1 nas dietas FT, RUC33 e RUC67, respectivamente. A digestibilidade aparente da matéria seca não foi afetada pelos tratamentos, mas a inclusão do resíduo aumentou a digestibilidade da proteína bruta. Os perfis de N-NH3 no rúmen foram semelhantes em todos os tratamentos e o pH ruminal diferiu entre os tratamentos (6,26; 5,98 e 6.28 (EP = 0,05 ) no FT, RUC33 e RUC67, respectivamente). Os animais apresentaram diminuição no consumo quando o resíduo foi oferecido nas quantidades de 67% da MS. PALAVRAS-CHAVE: Avaliação de alimentos, subprodutos, suplementação para ruminante Wet brewers’ grain (RUC) is an available by-product throughout the year and its fiber fractions could replace forage fibers during critical periods. The aim of this study was to evaluate wet brewers’ grain as a substitute for hay in exclusive forage diets for sheep. Six Santa Inês male sheep were kept in metabolic cages in a 3x3 multiple Latin square, in which the experimental diets were 100% Tifton 85 Bermudagrass (Cynodon ssp) hay (FT), 67% FT + 33% wet brewer’s grain (RUC33) and 33% FT + 67% wet brewers’ grain (RUC67). The study evaluated voluntary intake, in vivo apparent digestibility of dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF), rumen pH and ammonia-N profiles. Voluntary intake was 1090, 1129 and 737 (SE = 66.9) g DM day-1 for diets FT, RUC33 and RUC67, respectively. Dry matter apparent digestibility was not affected by treatments, but the brewers’ grain inclusion increased crude protein digestibility. Rumen ammonia-N content profiles were similar for all treatments and pH differed among treatments (6.26, 5.98 and 6.28 (SE = 0.05) for FT, RUC33 and RUC67, respectively). By-product inclusion reduced DM intake at level of 67%. KEY WORDS: By-products, feed evaluation, ruminant supplementation

Este trabalho foi realizado com o objetivo de estudar, por meio de dois modelos matemáticos, o metabolismo do cálcio (Ca) em ovinos recebendo dietas suplementadas com diferentes fontes de Ca. Foram utilizados 20 cordeiros mestiços Santa Inês (6 a 8 meses de idade) alimentados com uma dieta basal composta de milho, farelo de soja, bagaço de cana hidrolisado, uréia, fosfato monoamônio (MAP) e mistura mineral suplementada com cinco fontes de cálcio: polpa cítrica (PC), feno de alfafa (FA), calcário calcítico (CC), farinha de conchas de ostras (FC) e fosfato bicálcico (FB). Os animais receberam, via jugular, solução contendo 7,7 MBq de 45Ca. Durante sete dias, realizaram-se coletas de fezes, sangue e urina. Após o período de coleta, os animais foram sacrificados para retirada de amostras de tecidos (fígado, rins, coração, músculo e 12ª costela) para análises. O metabolismo de Ca foi avaliado pelos modelos de Vitti et al. (2000) e Fernandez (1995). O delineamento experimental foi o inteiramente casualizado e as médias foram comparadas pelo teste Duncan. Não houve diferença significativa para o consumo de Ca entre os tratamentos. A excreção fecal foi menor para o CC. O balanço de Ca foi negativo para FA e PC. A absorção de Ca pode ter sido prejudicada pela presença de pectina e oxalato na polpa cítrica e no feno de alfafa, respectivamente. Os fluxos entre o compartimento central e o trato digestivo indicaram maior absorção de Ca para o CC. As fontes CC, FB e FC apresentaram maior disponibilidade cálcio, com valores respectivos de 65,38; 34,12 e 32,43%. A comparação entre os modelos comprovou diferenças nos fluxos entre o sangue e os tecidos e ossos, todavia, o balanço entre esses compartimentos foi similar. As diferenças entre os modelos possivelmente foram ocasionadas pela sua estrutura.This research was conducted to compare the accuracy of two mathematical models to predict calcium (Ca) metabolism in sheep supplemented with different Ca sources. Twenty Brazilian male sheep averaging 7 to 8 months of age were fed a basal diet containing corn, soybean meal, hydrolyzed sugarcane bagasse, urea, mono-ammonium phosphate, mineral mixture and one of the following Ca sources: limestone (LM), alfalfa hay (AH), dicalcium phosphate (DP), oyster shell meal (SM), and citrus pulp (CP). On day 22 each animal received an intravenous injection of 7.7 MBq of radio-calcium (45Ca) followed by collection of blood, feces, and urine every 24 h for 7 days. Total Ca and radioactivity were measured in all samples. At the end of the collection period, animals were slaughtered and samples of liver, heart, kidney, muscles and 12th rib were taken for analysis. Calcium metabolism was evaluated by the models proposed by Vitti et al. (2000) and Fernandez (1995). A completely randomized design was used and means were compared by the Duncan test. No significant differences were observed for Ca intake among treatments. However, total fecal excretion of Ca differed and was lowest in animals fed LM. Calcium balance was negative in sheep receiving AH and CP because Ca absorption might have been compromised by the high contents of pectin and oxalate in these two supplements, respectively. Flows of Ca between the central pool and the digestive tract showed the greatest absorption of Ca on animals fed LM. Diets supplemented with LM, DP, and SM had highest Ca availability with values of 65.38, 34.12 and 32.43%, respectively. The models differed in the predictions of Ca flows among blood, bone and soft tissues but showed similar values for Ca balance in the different compartments. These discrepant results may be explained by structural difference between models.

O objetivo deste trabalho foi avaliar o consumo voluntário, a digestibilidade aparente e a cinética diges-tiva de três forragens, com teores protéicos distintos, em ovinos. Utilizaram-se seis ovinos da raça Santa Inês (PV = 40 ± 5,7 kg), machos adultos, castrados e providos de cânulas ruminais. Optou-se pelo uso de fenos de alfafa (ALF), de braquiária (BRA) e de capim Tifton-85 (TIF) pelos seus teores de proteína bruta (PB), respectivamente, 191, 29 e 75 g kg-1 MS. Alocaram-se os tratamentos e os animais em delineamento de dois quadrados latinos (três tratamentos, três períodos e seis animais). As dietas consistiam exclusivamente de ALF, BRA ou TIF e mistura mineral, oferecida separadamente. O ensaio de consumo voluntário foi realizado em baias individuais e os de digestibilidade e cinética digestiva, em gaiolas de metabolismo. Determinou-se a digestibilidade aparente por coleta total de fezes. Estimou-se a cinética digestiva mediante o uso de Co-EDTA e fibra Cr-mordantada como marcadores das fases líquida e sólida, respectivamente. Compararam-se as médias por teste de Tukey. O consumo voluntário de MS do tratamento ALF foi maior (P < 0,05) que os tratamentos BRA e TIF, o que refletiu a forte relação entre consumo e teor de proteína na dieta. Os coeficientes de digestibilidade aparente da MS e da MO também foram superiores (P < 0,05) para ALF. A digestibilidade aparente da proteína bruta mostrou diferenças (P < 0,05) entre os três tratamentos, sendo ALF (0,694) a mais alta que se observou, seguida por TIF (0,500). Para o tratamento BRA, essa variável foi praticamente nula (0,001), indicando déficit protéico intenso. O consumo voluntário e a digestibilidade aparente dos alimentos testados receberam influência da composição química e da cinética digestiva. Alimentos com baixo teor protéico prejudicaram a digestibilidade aparente de nutrientes, diminuindo, próximo à nulidade, para a proteína do tratamento BRA. Palavras-chaves: Alfafa, braquiária, marcadores, nutrição, ruminantes, Tifton.

O resíduo úmido de cervejaria é um subproduto disponível ao longo do ano, podendo ser utilizado como um substituto das forragens durante os períodos críticos. O objetivo deste estudo foi avaliar a utilização do resíduo úmido de cervejaria como substituto do feno em dietas exclusivas de volumoso para ovinos. Mantiveram-se seis ovinos machos da raça Santa Inês em gaiolas de metabolismo em um delineamento experimental de quadrado latino múltiplo de 3 x 3, com dietas experimentais 100% de feno de Tifton 85 capim-bermuda (Cynodon ssp) (FT), 67% de FT + 33% de resíduo de cervejaria (RUC33) e 33% de FT + 67% de resíduo de cervejaria (RUC67). O ensaio avaliou o consumo voluntário, a digestibilidade aparente in vivo da matéria seca, da matéria orgânica, da proteína bruta, e das fibras em detergente neutro e em detergente ácido, pH ruminal e perfil de nitrogênio (N) amoniacal. O consumo voluntário foi de 1.090, 1.129 e 737 (EP = 66,9) g MS dia-1 nas dietas FT, RUC33 e RUC67, respectivamente. A digestibilidade aparente da matéria seca não foi afetada pelos tratamentos, mas a inclusão do resíduo aumentou a digestibilidade da proteína bruta. Os perfis de N-NH3 no rúmen foram semelhantes em todos os tratamentos e o pH ruminal diferiu entre os tratamentos (6,26; 5,98 e 6.28 (EP = 0,05 ) no FT, RUC33 e RUC67, respectivamente). Os animais apresentaram diminuição no consumo quando o resíduo foi oferecido nas quantidades de 67% da MS. PALAVRAS-CHAVE: Avaliação de alimentos, subprodutos, suplementação para ruminante

O propósito deste estudo foi determinar uma metodologia mais prática para a extração de progesterona (P4) das fezes e observar se os perfis plasmáticos de progesterona (P4) durante a gestação de coelhas era refletida na concentração total de P4 nas fezes. Esta espécie animal foi utilizada como modelo para as diferentes metodologias. O plasma e as fezes foram coletadas de 11 coelhas, durante período de 42 dias. Três métodos diferentes de extração de P4 foram testados. A P4 total foi medida com auxílio de radioimunoensaio de fase sólida (RIE) utilizando 125I-P4 como traçador. Os resultados sugerem que foi possível extrair P4 total das fezes de coelhas com metanol e éter de petróleo. Os teores de P4 plasmática e fecal foram comparados entre animais gestantes e ovariectomizados. Foi possível a diferenciação (p

ABSTRACT: This study aimed to evaluate the effects of substituting wheat bran (WB) by corn gluten feed without steep water (CGF) in cats feeds regarding the nutritional value of the diet, stool characteristics, time of food passage through the gastrointestinal tract (GIT), intestinal gas production and concentrations of short chain fatty acids (SCFA) in blood and faeces. Twenty eight cats were distributed in a randomized block design with four treatments (0, 2.6, 5.3 or 8.0% CGF, based on dry matter, substituting for WB) and seven replicates. Substitution of WB by CGF decreased (P0.05) the digestibility of other nutrients and apparent metabolizable energy. There was also no effect (P>0.05) on faeces moisture, faecal pH and score, food transit time in the GIT, intestinal gas area and SCFA concentrations in faeces, while blood concentrations of propionate were influenced differently by the different feeds and sampling times (P

Health disparities are driven by unequal conditions in the environments in which people are born, live, learn, work, play, worship, and age, commonly termed the Social Determinants of Health (SDoH). The availability of recommended measurement protocols for SDoH will enable investigators to consistently collect data for SDoH constructs. The PhenX (consensus measures for Phenotypes and eXposures) Toolkit is a web-based catalog of recommended measurement protocols for use in research studies with human participants. Using standard protocols from the PhenX Toolkit makes it easier to compare and combine studies, potentially increasing the impact of individual studies, and aids in comparability across literature. In 2018, the National Institute on Minority Health and Health Disparities provided support for an initial expert Working Group to identify and recommend established SDoH protocols for inclusion in the PhenX Toolkit. In 2022, a second expert Working Group was convened to build on the work of the first SDoH Working Group and address gaps in the SDoH Toolkit Collections. The SDoH Collections consist of a Core Collection and Individual and Structural Specialty Collections. This article describes a Basic Protocol for using the PhenX Toolkit to select and implement SDoH measurement protocols for use in research studies. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: Using the PhenX Toolkit to select and implement SDoH protocols., (© 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Introduction: Social determinants are structures and conditions in the biological, physical, built, and social environments that affect health, social and physical functioning, health risk, quality of life, and health outcomes. The adoption of recommended, standard measurement protocols for social determinants of health will advance the science of minority health and health disparities research and provide standard social determinants of health protocols for inclusion in all studies with human participants., Methods: A PhenX (consensus measures for Phenotypes and eXposures) Working Group of social determinants of health experts was convened from October 2018 to May 2020 and followed a well-established consensus process to identify and recommend social determinants of health measurement protocols. The PhenX Toolkit contains data collection protocols suitable for inclusion in a wide range of research studies. The recommended social determinants of health protocols were shared with the broader scientific community to invite review and feedback before being added to the Toolkit., Results: Nineteen social determinants of health protocols were released in the PhenX Toolkit (https://www.phenxtoolkit.org) in May 2020 to provide measures at the individual and structural levels for built and natural environments, structural racism, economic resources, employment status, occupational health and safety, education, environmental exposures, food environment, health and health care, and sociocultural community context., Conclusions: Promoting the adoption of well-established social determinants of health protocols can enable consistent data collection and facilitate comparing and combining studies, with the potential to increase their scientific impact., (Published by Elsevier Inc.)

Purpose: To increase the proportion of dialysis patients using native arteriovenous fistulae (AVF), improved selection of the most appropriate procedure must be coupled with early access surveillance to determine which access would likely mature and when intervention might lead to access salvage. This study was aimed at auditing pre-operative vessel mapping and post-operative access surveillance against the primary outcome measure of AVF maturation., Methods: Between January 2006 and August 2007, 113 AVF created in 101 patients were studied. Data on pre-operative vessel mapping, type of AVF, post-operative surveillance scans were analyzed against the outcome AVF., Results: Pre-operative mapping and post-operative scanning were carried out in 86% and 91%, respectively. A maturing fistula on post-operative scan highly correlated with a satisfactory outcome (p<0.001). The sensitivity and specificity of the post-operative scan were 100% and 85%, respectively. There were 79 brachiocephalic and 34 radiocephalic fistulae with a primary failure rate of 23% and 47%, respectively, giving an overall failure rate of 30%. Nine fistulae had further intervention (angioplasty or thrombolysis) and five (56%) were salvaged. Seventy-two AVF matured satisfactorily giving a primary cumulative patency of 71% (72/102)., Conclusion: This study shows that preoperative vessel mapping provides useful information regarding the choice of AVF. Access surveillance duplex scanning at 6-8 weeks post-operatively is viable and has a high sensitivity and specificity for final outcome of fistula. Identifying AVF with potential problems early means that further intervention or surgery can be planned earlier, which will have a positive impact on patients.

Final results of an investigation into whether oxygen saturation of tissues (StO2, measured by spectrophotometry) could predict surgical site infections (SSI) after major abdominal surgery are presented. StO2 was measured on the arm and wound site pre-operatively and then at 12, 24 and 48 hours post-operatively. A Whitland Research RM200 was employed as the visible lightguide spectrophotometer. StO2 measurements using this machine were designated SSO2 (skin SO2). A Hutchinson Inspectra Model 325 was used for the near infrared spectroscopy (NIS) measurements. StO2 measurements using this machine were designated MSO2 (muscle SO2). Of 59 patients (38 males, 21 females), 42 healed uneventfully and 17 developed SSI. The overall infection rate was 28.8%. No significant differences were seen in wound SSO2 between outcome groups at any stage. At 12 hours there was a significant difference between the two groups with respect to mean wound MSO2 (A = 58.3 +/- 21.6%, B = 42.2 +/- 16.6%, p = 0.005, 95% confidence interval = 5.26, 26.98). A receiver operating characteristic curve showed that when a wound MSO2 of 53% was chosen as the threshold to classify potential infection a sensitivity of 71% and a specificity 73% (chi-squared test, p = 0.002) was achieved. The use of the near-infrared spectrophotometry as a tool to predict wound infections should be further evaluated and advocated.

Background: Surgical-site infections (SSIs) are common after major abdominal and groin bypass surgery. Tissue oxygen tension has been shown to predict these infections accurately. This study assessed whether a non-invasive measurement of tissue oxygenation, tissue oxygen saturation as measured by spectrophotometry, was as accurate., Methods: Fifty-nine patients having major abdominal or groin bypass surgery had tissue oxygen saturation measured by near-infrared spectrophotometry at the incision site and in the arm before operation, and at 12, 24 and 48 h after surgery. Masked outcome assessments for SSI were made at 7 and 30 days after operation., Results: In this retrospective analysis, 17 patients (29 per cent) developed an SSI. At 12 h after operation there was a significant difference in tissue oxygen saturation at the surgical site between patients who developed an SSI and those who did not (mean(s.d.) 43.4(18.1) versus 55.8(22.0) per cent; P = 0.032). These oxygen saturation readings were found to be more specific and sensitive in predicting SSIs than the National Nosocomial Infection Surveillance system., Discussion: There is a difference in postoperative surgical-site oxygen saturation between patients who subsequently develop SSIs and those who do not. Prediction of SSIs provides opportunities for intervention and prevention., (Copyright 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

The BamHI restriction-modification system contains a third gene, bamHIC, which positively regulates bamHIR. Similar small genes from other systems were tested in vivo for their ability to cross-complement. C.BamHI protein was identified, purified, and used to raise polyclonal antibodies. Attempts to detect other C proteins in cell extracts by cross-reactivity with C.BamHI antibodies proved unsuccessful.

The bamHIC gene, controlling the BamHI restriction-modification (R-M) system can functionally be replaced by providing pvuIIC or smaIC in trans. C.BamHI, the protein product encoded by bamHIC, has been purified and shown to bind a 345-bp DNA fragment within the BamHI R-M system.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.

Several strains of Bacillus subtilis, e.g., 168 derivatives and R, were found to carry a single copy of a tetracycline-resistance (TcR) determinant (named tetBS908) at a site close to the origin of replication on the chromosome. This gene is highly homologous (80% identical) to the TcR determinant of plasmids widely dispersed among aerobic spore-forming bacilli. B. subtilis RM125 (168 strain) transformants which carry a varying number of tetBS908 sequences in a tandem array on the chromosome were constructed and examined for their TcR level. A nearly proportional relationship between the TcR level and copy number of tetBS908 existed.

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.

Endogenous chromosomal DNA amplifications with associated tetracycline resistance (Tcr) in Bacillus subtilis were first described by C. R. Wilson and A. E. Morgan (J. Bacteriol. 163:445-453, 1985). We have confirmed and extended their results, and we show that fusion of protoplasts from Tcs B. subtilis 168 trpC2 with polyethylene glycol and regeneration on medium containing 20 micrograms of tetracycline per ml induces Tcr regenerants that contain amplified DNA. This phenomenon appeared to be recE dependent and requires the addition of polyethylene glycol. Along with three regenerants kindly provided by Wilson and Morgan (RAD1, RAD6, and RAD7), we characterized three strains (CLI20, CLI22, CLI30) isolated in this laboratory. All six contain an amplified region of DNA which was independently cloned on plasmid pCIS7. Integration of pCIS7 into the wild-type (Tcs) B. subtilis chromosome and amplification of the plasmid sequences generated a Tcr phenotype, even though the DNA on pCIS7 was cloned from Tcs B. subtilis KS162 (Ives and Bott, J. Bacteriol. 171:1801-1810, 1989). The amplified DNA also showed homology (through hybridization analysis) with pAM alpha 1 delta 1, a gram-positive Tcr plasmid, indicating that B. subtilis normally contains a silent integrated copy of the gene whose amplification confers Tcr. The amplifications were determined to lie between purA and gyrB on the B. subtilis chromosome, and the endpoints were mapped. RAD6 and CLI30 may share the same left-hand endpoint, but the other endpoints are different in each isolate. The amplified DNAs of RAD1, RAD6, CLI20, and CLI30 end near known DNA membrane binding sites. The number of amplified units of DNA was determined through dot blot analysis to do approximately 80 to 100 copies per cell, with corresponding increases in transcription of RAD1, RAD6, CLI20, CLI22, and CLI30.

Plasmid pCIS7, containing 11.5 kilobases (kb) of Bacillus subtilis DNA, was isolated from a Tn917 transposon insertion in tetracycline-sensitive B. subtilis KS162. When integrated into the chromosome of B. subtilis 168, this plasmid conferred tetracycline resistance upon reiteration of the plasmid DNA sequences in the chromosome. Deletions and subclones of pCIS7 were constructed and introduced into an Escherichia coli in vitro transcription-translation system. A 72-kilodalton protein was localized to a 3.1-kb PstI-EcoRI fragment of the plasmid. Amplification of the 3.1-kb PstI-EcoRI fragment was required for expression of tetracycline resistance in B. subtilis 168. By hybridization to previously characterized clones, the 11.5-kb fragment was localized to the origin region of the chromosome. Through contour-clamped homogeneous electric field electrophoresis, this cluster of clones was shown to reside on a 200-kb NotI fragment bridging SfiI fragments of 150 and 250 kb and was oriented with respect to the purA and guaA loci, developing an accurate physical map of the region surrounding the origin of replication.

A system has been developed for subjecting endothelial cell monolayers to prolonged steady flow while maintaining normal culture conditions. Cloned bovine endothelial cells were grown to confluence on one wall of a square glass tube which was then incorporated in the flow circuit. Flow rates of 19-21 ml/min were sustained for periods of 6-45 hr, subjecting the cells along the center line of the wall of the tube to a maximum shear stress of 34 dyn/cm2. The cells in all the experiments remained attached and viable when subjected to this shear stress. Photographic data from experimental runs were qualitatively assessed for changes in cell morphology, confluence, and orientation and were compared to data from matched stationary controls. Five experiments were chosen for quantitative morphometric analysis. In three experiments, the cells showed elongation with their long axes aligned with the direction of flow in 6.5, 21, and 22 hr. In the other experiments, either the cells formed a swirling pattern or no change in morphology was apparent. Although cell shape (form) changed in response to shear stress, cell area remained unaffected by exposure to flow.

Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function.

Endothelial cell seeding of expanded polytetrafluoroethylene (e-PTFE) arteriovenous prostheses was performed to compare seeding with homologous vs. autologous cells and to study the effect of homologous seeding with a larger vs. a smaller number of cells. Sixteen dogs were randomly assigned to four equal groups: I, control; II, light homologous seeded; III, heavy homologous seeded; and IV, autologous seeded. Bilateral femoral arteriovenous loop grafts were inserted in all. Efficiency of seeding was assessed by cell counts of instilled vs. retained cells. All grafts remained patent and were harvested approximately 8 weeks after implantation. Samples were evaluated grossly by scanning electron microscopy and light microscopy. There was a direct correlation between number of cells instilled and number retained for each graft; group III received and retained the largest number (p less than 0.001 and p less than 0.05, respectively). The amount of thrombus deposition on the lumen of all grafts was grossly the same. Endothelium was demonstrated in samples obtained from the midgraft of groups III and IV; in contrast no endothelium was seen in groups I and II. The percentage of endothelialized surface was not determined. No immunologic cellular reaction was detected in any of the samples. We conclude that in the animal laboratory it is possible to seed e-PTFE arteriovenous prostheses successfully with homologous cells and to improve the efficiency of seeding by implanting a larger number of cells obtained from an endothelial cell bank. The potential applications of this technique to the clinical field are discussed herein.

Endothelial cell functions, such as arachidonic acid metabolism, may be modulated by membrane stresses induced by blood flow. The production of prostacyclin by primary human endothelial cell cultures subjected to pulsatile and steady flow shear stress was measured. The onset of flow led to a sudden increase in prostacyclin production, which decreased to a steady rate within several minutes. The steady-state production rate of cells subjected to pulsatile shear stress was more than twice that of cells exposed to steady shear stress and 16 times greater than that of cells in stationary culture.