Continuous monitoring of vital parameters for clinically valid assessment of human health status

Tese de mestrado integrado, Engenharia Biomédica e Biofísica (Sinais e Imagens Médicas) Universidade de Lisboa, Faculdade de Ciências, 2019The lack of devices suitable for acquiring accurate and reliable measures of patients' physiolog-ical signals in a remote and continuous manner together with the advances in data acquisition technol-ogies during the last decades, have led to the emergence of wearable devices for healthcare. Wearable devices enable remote, continuous and long-term health monitoring in unattended setting. In this con-text, the Swiss Federal Laboratories for Material Science and Technology (Empa) developed a wearable system for long-term electrocardiogram measurements, referred to as textile belt. It consists of a chest strap with two embroidered textile electrodes. The validity of Empa’s system for electrocardiogram monitoring has been proven in a clinical setting. This work aimed to assess the validity of the textile belt for electrocardiogram monitoring in a home setting and to supplement the existing system with sensors for respiratory monitoring. Another objective was to evaluate the suitability of the same weara-ble, as a multi-sensor system, for activity monitoring. A study involving 12 patients (10 males and 2 females, interquartile range for age of 48–59 years and for body mass indexes of 28.0–35.5 kg.m-2) with suspected sleep apnoea was carried out. Overnight electrocardiogram was measured in a total of 28 nights. The quality of recorded signals was assessed using signal-to-noise ratio, artefacts detection and Poincaré plots. Study data were compared to data from the same subjects, acquired in the clinical setting. For respiratory monitoring, optical fibre-based sensors of different geometries were integrated into the textile belt. Signal processing algorithms for breathing rate and tidal volume estimation based on respiratory signals acquired by the sensors were developed. Pilot studies were conducted to compare the different approaches for respiratory monitoring. The quality of respiratory signals was determined based on signal segments “sinusoidality”, evaluated through the calculation of the cross-correlation between signal segments and segment-specific reference waves. A method for accelerometry-based lying position recognition was proposed, and the proof of concept of activity intensity classification through the combination of subjects’ inertial acceleration, heart rate and breathing rate data, was presented. Finally, a study with three participants (1 male and 2 females, aged 21 ± 2 years, body mass index of 20.3 ± 1.5 kg.m-2) was conducted to assess the validity of the textile belt for respiratory and activity monitoring. Electrocardiogram signals acquired by the textile belt in the home setting were found to have better quality than the data acquired by the same device in the clinical setting. Although a higher artefact percentage was found for the textile belt, signal-to-noise ratio of electrocardiogram signals recorded by the textile belt in the home setting was similar to that of signals acquired by the gel electrodes in the clinical setting. A good agreement was found between the RR-intervals derived from signals recorded in home and clinical settings. Besides, for artefact percentages greater than 3%, visual assessment of Poincaré plots proved to be effective for the determination of the primary source of artefacts (noise or ectopic beats). Acceleration data allowed posture recognition (i.e. lying or standing/sitting, lying position) with an accuracy of 91% and positive predictive value of 80%. Lastly, preliminary results of physical activity intensity classification yielded high accuracy, showing the potential of the proposed method. The textile belt proved to be appropriate for long-term, remote and continuous monitoring of subjects’ physical and physiological parameters. It can monitor not only electrocardiogram, but also breathing rate, body posture and physical activity intensity, having the potential to be used as tool for disease prediction and diagnose support.Contexto: A falta de dispositivos adequados para a monitorização de sinais fisiológicos de um modo remoto e contínuo, juntamente com avanços tecnológicos na área de aquisição de dados nas últimas décadas, levaram ao surgimento de wearable devices, i.e. dispositivos vestíveis, no sector da saúde. Wearable devices possibilitam a monitorização do estado de saúde, de uma forma remota, contínua e de longa duração. Quando feito em ambiente domiciliar, este tipo de monitorização (i.e. contínua, remota e de longa duração) tem várias vantagens: diminui a pressão posta sobre o sistema de saúde, reduz despesas associadas ao internamento e acelera a resposta a emergências, permitindo deteção precoce e prevenção de condições crónicas. Neste contexto, a Empa, Laboratórios Federais Suíços de Ciência e Tecnologia de Materiais, desenvolveu um sistema vestível para a monitorização de eletrocardiograma de longa duração. Este sistema consiste num cinto peitoral com dois elétrodos têxteis integrados. Os elétrodos têxteis são feitos de fio de polietileno tereftalato revestido com prata e uma ultrafina camada de titânio no topo. De modo a garantir a aquisição de sinais de alta qualidade, o cinto tem nele integrado um reservatório de água que liberta vapor de água para humidificar os elétrodos. Este reservatório per-mite a monitorização contínua de eletrocardiograma por 5 a 10 dias, sem necessitar de recarga. A vali-dade do cinto para a monitorização de eletrocardiograma em ambiente clínico já foi provada. Objetivo: Este trabalho teve por objetivo avaliar a validade do cinto para a monitorização de eletrocar-diograma em ambiente domiciliar e complementar o sistema existente com sensores para monitorização respiratória. Um outro objetivo foi analisar a adequação do cinto, como um sistema multisensor, para monitorização da atividade física. Métodos: Um estudo com 12 pacientes com suspeita de apneia do sono (10 homens e 2 mulheres, am-plitude interquartil de 48–59 anos para a idade e de 28.0–35.5 kg.m-2 para o índice de massa corporal) foi conduzido para avaliar a qualidade do sinal de eletrocardiograma medido em ambiente domiciliar. O sinal de eletrocardiograma dos pacientes foi monitorizado continuamente, num total de 28 noites. A qualidade dos sinais adquiridos foi analisada através do cálculo da razão sinal-ruído; da deteção de ar-tefactos, i.e., intervalos RR com um valor inviável de um ponto de vista fisiológico; e de gráficos de Poincaré, um método de análise não linear da distribuição dos intervalos RR registados. Os dados ad-quiridos neste estudo foram comparados com dados dos mesmos pacientes, adquiridos em ambiente hospitalar. Para a monitorização respiratória, sensores feitos de fibra óptica foram integrados no cinto. Al-gorítmicos para a estimar a frequência respiratória e o volume corrente dos sujeitos tendo por base o sinal medido pelas fibras ópticas foram desenvolvidos neste trabalho. As diferentes abordagens foram comparadas através de estudos piloto. Diferentes métodos para avaliação da qualidade do sinal adquirido foram sugeridos. Um método de reconhecimento da postura corporal através do cálculo de ângulos de orientação com base na aceleração medida foi proposto. A prova de conceito da determinação da intensidade da atividade física pela combinação de informações relativas á aceleração inercial e frequências cardíaca e respiratória dos sujeitos, é também apresentada neste trabalho. Um estudo foi conduzido para avaliar a validade do cinto para monitorização da respiração e da atividade física. O estudo contou com 10 parti-cipantes, dos quais 3 vestiram o cinto para monitorização da respiração (1 homem e 2 mulheres, idade 21 ± 2 anos, índice de massa corporal 20.3 ± 1.5 kg.m-2). Resultados: O estudo feito com pacientes com suspeita de apneia do sono revelou que os sinais eletro-cardiográficos adquiridos pelo cinto em ambiente domiciliar foram de melhor qualidade que os sinais adquiridos pelo mesmo dispositivo em ambiente hospitalar. Uma percentagem de artefacto de 2.87% ±4.14% foi observada para os dados adquiridos pelos elétrodos comummente usados em ambiente hospi-talar, 7.49% ± 10.76% para os dados adquiridos pelo cinto em ambiente domiciliar e 9.66% ± 14.65% para os dados adquiridos pelo cinto em ambiente hospitalar. Embora tenham tido uma maior percenta-gem de artefacto, a razão sinal-ruído dos sinais eletrocardiográficos adquiridos pelo cinto em ambiente domiciliar foi semelhante á dos sinais adquiridos pelos elétrodos de gel em ambiente hospitalar. Resul-tados sugerem uma boa concordância entre os intervalos RR calculados com base nos eletrocardiogra-mas registados em ambientes hospitalar e domiciliar. Além disso, para sinais com percentagem de arte-facto superior a 3%, a avaliação visual dos gráficos de Poincaré provou ser um bom método para a determinação da fonte primária de artefactos (batimentos irregulares ou ruído). A monitorização da aceleração dos sujeitos permitiu o reconhecimento da postura corporal (isto é, deitado ou sentado/em pé) com uma exatidão de 91% e valor preditivo positivo de 80%. Por fim, a classificação da intensidade da atividade física baseado na aceleração inercial e frequências cardíaca e respiratória revelou elevada exatidão, mostrando o potencial desta técnica. Conclusão: O cinto desenvolvido pela Empa provou ser apropriado para monitorização de longa-dura-ção de variáveis físicas e fisiológicos, de uma forma remota e contínua. O cinto permite não só monito-rizar eletrocardiograma, mas também frequência respiratória, postura corporal e intensidade da atividade física. Outros estudos devem ser conduzidos para corroborar os resultados e conclusões deste trabalho. Outros sensores poderão ser integrados no cinto de modo a possibilitar a monitorização de outras vari-áveis fisiológicas de relevância clínica. Este sistema tem o potencial de ser usado como uma ferramenta para predição de doenças e apoio ao diagnóstico

Aluminum bioaccumulation and tolerance in Plantago algarbiensis Samp. and Plantago almogravensis Franco, assessed by using in vitro cultures and micropropagated plants

This thesis aims to understand the aluminum (Al) bioaccumulation capacity, toxicity effects and tolerance mechanisms in two rare species endemic from Portugal, Plantago algarbiensis Samp. and Plantago almogravensis Franco. Firstly, two protocols were developed to propagate in vitro these species (Chapter 2). Afterwards it was investigated the influence of low pH on in vitro growth (Chapter 3). The seed germination requirements and the effects of Al on the germination and early development of seedlings were then evaluated in Chapters 4 and 5, respectively. The impact of low pH and Al on physiological responses, oxidative stress status and capacity for Al accumulation in micropropagated shoots and plantlets was investigated in the subsequent chapters (Chapters 6, 7 and 8). Finally, in Chapter 9 the possible implications of organic acids in Al detoxification were evaluated. Both species were successfully micropropagated showing high shoot multiplication rates and rooting frequencies and the micropropagated shoots of both species were able to tolerate and grow in low pH conditions. The seeds of both species germinate readily at 15 ºC and Al showed no impact on the germination percentage in either species but oxidative stress during early seedling development was detected. Although, both species accumulated considerable Al amounts and showed to be moderately tolerant to low pH and Al, P. almogravensis appeared to be more able to manage the oxidative stress and, therefore, adapted to maintain cellular physiology and growth under these stress conditions. Results suggested that Al detoxification in P. almogravensis implies both secretion of organic acids from roots and Al intracellular chelation by organic acids, while in P. algarbiensis only the internal mechanism seems to be involved. This research contributes to the understanding of the mechanisms underlying P. algarbiensis and P. almogravensis adaptation to acidic Al-rich conditions.O alumínio (Al) é o metal mais abundante na crosta terrestre, onde permanece maioritariamente insolúvel. Quando os solos se tornam ácidos, como é o caso de cerca de 40% do solo arável, o Al, dependendo do pH, pode hidrolisar-se em vários complexos que podem provocar diferentes efeitos biológicos. O complexo Al(H2O)6 3+ (ou simplesmente Al3+) é o mais fitotóxico, afetando o crescimento e o desenvolvimento das plantas. A sua toxicidade é explicada pela elevada afinidade do Al3+ para as paredes celulares, membranas plasmáticas e metabolitos, interferindo em importantes processos morfológicos, fisiológicos e moleculares. Apesar disso, algumas plantas desenvolveram mecanismos para combater o stress causado pelo Al sendo capazes de crescer em áreas com elevado teor deste metal. As espécies selvagens podem apresentar fenótipos mais tolerantes que as plantas cultivadas, e ser úteis para compreender os mecanismos de tolerância ao Al, fornecendo informação fundamental para aumentar a tolerância das espécies cultivadas.Universidade do Algarve, Faculdade de Ciências e Tecnologi

Cold thermal priming of Laminaria digitata (Laminariales, Phaeophyceae) gametophytes enhances gametogenesis and thermal performance of sporophytes

Thermal characteristics of kelp species have been studied in many ways, but potentially persistent effects of temperature across generations are yet poorly understood. In this context, the effect of thermal priming on fertility and growth of the N-Atlantic kelp species Laminaria digitata was investigated within and across life cycle generations in a two-step common garden experiment. Using vegetative clonal gametophytes from cold (5°C) and warm (15°C) pre-experimental cultivation (3 years), we first quantified gametogenesis and recruitment over two weeks at a common temperature of 10°C. Then, recruited sporophytes were transferred to a temperature gradient spanning the tolerance range of the species from 0°C to 20°C. We hypothesized that a warm gametophyte preexperimental cultivation promotes performance of sporophytes at warm temperatures and vice versa. Interestingly, gametogenesis speed and sporophyte recruitment were higher in gametophytes following cold compared to warm pre-experimental cultivation, which indicates carry-over effects of temperature within the gametophyte generation. Compared to warm pre-experimental cultivation of gametophytes, a cold preexperimental cultivation enhanced growth of juvenile Laminaria digitata sporophytes by more than 69% at the extreme low and high temperatures of 0 and 20°C. This is the first evidence for a cross-generational effect between gametophyte parents and offspring sporophytes. As cold gametophyte cultivation increased the trait performance of gametogenesis, recruitment and thermal tolerance of juvenile sporophytes, priming of early life cycle stages may be used to increase resilience and productivity of kelps in marine forest restoration efforts and kelp mariculture

Thermal traits for reproduction and recruitment differ between Arctic and Atlantic kelp Laminaria digitata

The plasticity of different kelp populations to heat stress has seldom been investigated excluding environmental effects due to thermal histories, by raising a generation under common garden conditions. Comparisons of populations in the absence of environmental effects allow unbiased quantification of the meta-population adaptive potential and resolution of population-specific differentiation. Following this approach, we tested the hypothesis that genetically distinct arctic and temperate kelp exhibit different thermal phenotypes, by comparing the capacity of their microscopic life stages to recover from elevated temperatures. Gametophytes of Laminaria digitata (Arctic and North Sea) grown at 15°C for 3 years were subjected to common garden conditions with static or dynamic (i.e., gradual) thermal treatments ranging between 15 and 25°C and also to darkness. Gametophyte growth and survival during thermal stress conditions, and subsequent sporophyte recruitment at two recovery temperatures (5 and 15°C), were investigated. Population-specific responses were apparent; North Sea gametophytes exhibited higher growth rates and greater sporophyte recruitment than those from the Arctic when recovering from high temperatures, revealing differential thermal adaptation. All gametophytes performed poorly after recovery from a static 8-day exposure at 22.5°C compared to the response under a dynamic thermal treatment with a peak temperature of 25°C, demonstrating the importance of gradual warming and/or acclimation time in modifying thermal limits. Recovery temperature markedly affected the capacity of gametophytes to reproduce following high temperatures, regardless of the population. Recovery at 5°C resulted in higher sporophyte production following a 15°C and 20°C static exposure, whereas recovery at 15°C was better for gametophyte exposures to static 22.5°C or dynamic heat stress to 25°C. The subtle performance differences between populations originating from sites with contrasting local in situ temperatures support our hypothesis that their thermal plasticity has diverged over evolutionary time scales.FCT: PTDC/MAR-EST/6053/2014/ UID/Multi/04326/2019/ BIODIVERSA/0004/2015/ SFRH/BPD/122567/2016/ DL 57/2016/CP1361/CT0039/ SFRH/BSAB/150485/2019info:eu-repo/semantics/publishedVersio

Editorial: Opening the black box of kelps: Response of early life stages to anthropogenic stressors

Kelps form marine forests along world’s coastlines, providing valuable ecosystem goods and services, either directly as a source offood or medicinal products, or indirectly as biogenic habitats or carbon sink agents (Teagle et al., 2017; Wernberg et al., 2019). However, kelp forests are currently under threat due to anthropogenic climate change with latitudinal range shifts and large-scale declines at a global scale (Smale et al., 2019; Wernberg et al., 2019). Most studies on the impact of anthropogenic stressors on kelps have focused on the macroscopic sporophyte stage of the haploid-diploid life cycle (Schiel and Foster, 2006; Veenhof et al., 2022). However, the microscopic stages considered as the “black box” of kelps due to the complexity of studying them in situ, have been suggested to play a crucial role in the persistence of populations that experience sporophyte mortality after large-scale disturbances (McConnico and Foster, 2005; Barradas et al., 2011) as they can persist as “seed bank” analogues under adverse conditions (Hoffmann and Santelices, 1991; Veenhof et al., 2022). This Research Topic is a collection of 8 articles contributing to opening the “black box” of kelps by providing greater insight into how microscopic life stages of kelps are affected by anthropogenic climate change, helping to predict the persistence of these foundation species and therefore the fate of ecosystems and coastal communities. These studies highlight that the response of kelp early life stages to stressors can be strongly dependent on the population and thermal history.Australian Research Council DP190100058, DP200100201info:eu-repo/semantics/publishedVersio

Interactions of daylength, temperature and nutrients affect thresholds for life stage transitions in the kelp Laminaria digitata (Phaeophyceae)

Kelp beds worldwide are under pressure from ongoing climate and environmental change. Along European coastlines increases in seawater temperature and changes in nutrient conditions occur where upwelling events are disrupted and also along eutrophicated coasts. In addition, seaweed responses to change may interact with seasonal daylength cycles. We performed a factorial experiment to examine the combined effects of seawater temperatures, nutrient regimes and photoperiod (long and short days) in order to better understand how latitudinal or seasonal differences in daylengths affect the sensitivity of transient microscopic kelp stages of Laminaria digitata from the North Sea to warming and eutrophication. While the optimal temperature range for vegetative gametophyte growth was 10 degrees C-18 degrees C under long summer photoperiod conditions, gametogenesis was induced at lower temperatures between 5 degrees C and 15 degrees C, with maximum sporophyte development under long photoperiods and enriched nutrient regimes, which represent local late spring conditions. Although gametogenesis was fastest at 10 degrees C-15 degrees C, sporophyte recruitment was highest at 5 degrees C. As these particular early life cycle processes in L. digitata have different temperature optima, this may drive the seasonal cycle of recruitment in the field. Increasing summer temperatures due to global warming will increase gametophyte size due to enhanced vegetative growth and inhibition of gametogenesis. This will probably lead to delayed but enhanced recruitment of new sporophytes under cooler autumn to spring conditions over a wide geographical scale, preventing the formation of juvenile sporophytes under stressful summer conditions and possibly changing annual recruitment patterns.STSM Grant from the COST Action "Phycomorph" [FA1406]; Portuguese Science Foundation (FCT) programs [EXCL/AAG-GLO/0661/2012, UID/Multi/04326/2013]; BiodiVERsA [Biodiversa/0004/2015, PTDC/MAR-EST/6053/2014]info:eu-repo/semantics/publishedVersio

Sex-dependent and-independent transcriptional changes during haploid phase gametogenesis in the sugar kelp Saccharina latissima

In haplodiplontic lineages, sexual reproduction occurs in haploid parents without meiosis. Although widespread in multicellular lineages such as brown algae (Phaeophyceae), haplodiplontic gametogenesis has been little studied at the molecular level. We addressed this by generating an annotated reference transcriptome for the gametophytic phase of the sugar kelp, Saccharina latissima. Transcriptional profiles of microscopic male and female gametophytes were analysed at four time points during the transition from vegetative growth to gametogenesis. Gametogenic signals resulting from a switch in culture irradiance from red to white light activated a core set of genes in a sex-independent manner, involving rapid activation of ribosome biogenesis, transcription and translation related pathways, with several acting at the post-transcriptional or post-translational level. Additional genes regulating nutrient acquisition and key carbohydrate-energy pathways were also identified. Candidate sex-biased genes under gametogenic conditions had potentially key roles in controlling female- and male-specific gametogenesis. Among these were several sex-biased or -specific E3 ubiquitin-protein ligases that may have important regulatory roles. Females specifically expressed several genes that coordinate gene expression and/or protein degradation, and the synthesis of inositol-containing compounds. Other female-biased genes supported parallels with oogenesis in divergent multicellular lineages, in particular reactive oxygen signalling via an NADPH-oxidase. Males specifically expressed the hypothesised brown algal sex-determining factor. Male-biased expression mainly involved upregulation of genes that control mitotic cell proliferation and spermatogenesis in other systems, as well as multiple flagella-related genes. Our data and results enhance genome-level understanding of gametogenesis in this ecologically and economically important multicellular lineage.FCTprograms UID/Multi/04326/2019, GENEKELP-PTDC/MAR-EST/6053/2014,MARFOR-Biodiversa/0004/2015, FRH/BPD/122567/2016. Swedish Research Council Formas, Naturvardsverket French National Research Agency (ANR) Spanish Government, FRCT, German Research Foundation (DFG), Pew Marine Fellowship, STSM Grant from the COST Action "Phycomorph" FA1406info:eu-repo/semantics/publishedVersio

Marine fish primary hepatocyte isolation and culture: New insights to enzymatic dissociation pancreatin digestion

PTDC/CTA-AMB/29173/2017 UIDB04292/2020 UIDP/04292/2020 UIDB04378/2020 UIDP/04378/2020 UIDB/04138/2020 UIDP/04138/ 2020 PTDC/CTA-AMB/29173/2017, NO-VAID39 grant 2020.09005. BD. DL57/2016/CP1376/CT002 CEECINST/00102/2018Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.publishersversionpublishe

Uso da cinza insolúvel em ácido para determinar o consumo de forragem dos bovinos

Six young crossbred bulls were used to determine dry matter intake by cattle fed a tropical grass hay (Cynodon dactylon, Coastcross 1). Digestion trial lasted for 23 days being the last 7 days for feed and feces sample collections. Dry matter intake was calculated by use of adequate formulas and indicator method. Dry matter intake was calculated by acid insoluble ash HCl 2N and concentrated HCl and also by direct consumption. There was no statistical difference among methods for dry matter consumption.Foram utilizados seis bovinos mestiços holandês/zebu, machos inteiros, com idade variando entre 16 e 33 meses. O alimento foi o feno de Cynodon dactylon hibrido Coasteross - 1. O período experimental teve duração de 23 dias, sendo os últimos sete dias para colheita das amostras de alimento e de fezes. O feno foi fornecido picado e à vontade em duas refeições, diariamente. O consumo de matéria seca pelo método do indicador, foi calculado usando fórmulas adequadas, delineamento estatístico foi o inteiramente cercalizado. Foram comparados os consumos de matéria seca pelos métodos da cinza insolúvel em ácido HCl concentrado, HCl 2N e também pelo consumo observado no experimento. Não houve diferença estatisticamente significativa nos consumos médios de matéria seca g/kg 0,75 /dia, entre os três métodos

Uso da cinza insolúvel em ácido como indicador natural para determinação da digestabilidade em bovinos

Six crossbred young bulls were used in a digestibility trial during 23 days being the last 7 days for feed and feces sample collections. Cynodon dactylon grass hay was the sole ration. Nutrient digestibility coefficients were calculated by total collection method and by HCl 2N and concentrated HCl, acid insoluble ash methods which were statistically different among them for dry matter, crude protein, crude fiber, crude energy and nitrogen-free extract when calculated by SCHNEIDER &amp; FLATT (1975) or LUCAS (1952) formulas. By use of total feces collection, digestibility coefficients were not statistically different. These results show the validity of acid insoluble ash methods to determine digestibility of nutrients for tropical grass hay.Foram utilizados seis bovinos mestiços Holandês/Zebu, machos inteiros, com idade entre 16 e 33 meses, para ensaio de digestibilidade em gaiolas, que teve duração de 23 dias, sendo os 7 dias finais, para colheita das amostras de fezes e do alimento. O alimento constituiu-se de feno de Cynodon dactylon, hibrido Coastcross 1, o que foi fornecido picado à vontade, em dois arraçoamentos, diariamente, sendo que junto ao primeiro destes era acrescentada mistura mineral completa na base de 40 g para cada animal. Os cálculos dos coeficientes de digestibilidade dos nutrientes foram feitos de dois modos: pela fórmula de SCHNEIDER e FLATT (1975) e pela fórmula de LUCAS (1952). O delineamento experimental foi inteiramente casualizado, com aplicação do teste de Duncan. A recuperação fecal foi alta para os dois métodos aplicados: HCl 2N com 109,46% e HCl concentrado com 103,20%. Houve diferença estatisticamente significativa nos coeficientes de digestibilidade  da matéria seca, da proteína bruta, da fibra bruta, dos extrativos não nitrogenados e da energia bruta quando foram comparados os métodos de cinza insolúvel em ácido  HCl 2N , HCl concentrado e pelo método de colheita total de fezes. Apenas a fração de extrato etéreo não mostrou diferença estatística significativa nos coeficientes de digestibilidade, entre os métodos .Usando a recuperação de fezes observada no experimento, para o cálculo dos coeficientes de digestibilidade, não houve diferença estatisticamente significativa para qualquer dos nutrientes; comprovando assim, a validade do uso da cinza insolúvel em ácido, como indicador para determinar a digestibilidade dos nutrientes de gramínea tropical fornecida a bovinos