Phalacrocorax verrucosus during both the incubation and chick-rearing periods for the same individuals to determine the effect of sex, breeding stage, body mass and morphometrics on mean foraging metrics and their consistency. There were large differences between individuals in foraging behaviour and consistency, with strong individual specialisations in dive depths and heading from the colony. Stable isotopes revealed specialisations in feeding strategies, across multiple temporal scales. Specifically, individuals showed medium term specialisations in feeding strategies during the breeding season, as well as long-term consistency. A clustering analysis revealed 4 different foraging strategies displaying significantly different δ15N values and body masses. There were no sex or stage biases to clusters and individuals in different clusters did not differ in their morphology. Importantly, the results suggest that the different strategies emphasized were related to individual prey preferences rather than intrinsic characteristics.Individual specialisations, which involve the repetition of specific behaviours or dietary choices over time, have been suggested to benefit animals by avoiding competition with conspecifics and increasing individual foraging efficiency. Among seabirds, resident and benthic species are thought to be good models to study inter-individual variation as they repetitively exploit the same environment. We investigated foraging behaviour, isotopic niche and diet in the Kerguelen shag  According to the optimal foraging theory, an individual should select a specific foraging strategy that maximizes its net energy intake per unit of time while minimizing other costs such as predation risk . ForagerIndividual specialisations in foraging involve the repetition of specific behaviours or dietary choices over time, and have been, until recently, poorly investigated , 6, 7. IIndividual specialisations are reported across a wide range of taxonomic groups including molluscs, crustaceans, insects, fishes, reptiles, amphibians, birds and mammals . ConsistDetermining the temporal consistency of individual specialisations requires longitudinal studies involving repeated observations on individuals over time . SeabirdPhalacrocorax verrucosus (Cabanis 1875), is a member of the 13 species so-called blue-eyed shag complex [Cormorants are inshore feeders, foot-propelled pursuit-divers, feeding on a wide range of inshore benthic or pelagic prey in a limited ecological niche –23. Thei complex , 28, whi complex .Flexibility in feeding habits has been reported for various species of cormorants, and it has been argued that this flexibility plays an important role in maximizing their food intake . For exaThe aims of the present study were to (1) quantify and identify the factors influencing consistency in diving behaviour, space use, and diet in Kerguelen shags at different temporal scales, as well as to (2) study the links between foraging behaviour, consistency in foraging, diet, and morphometry. Different complementary approaches, representing different timescales, have been used to do so as no single timescale may provide a complete and accurate picture of the level of individual specialisation . Snapshoca 1% of mean body mass) for 3–6 d at the end of incubation/early chick rearing , when chicks were no older than a week. During the second session, a total of 22 birds (both partners from 11 nests) were instrumented for 3–12 d during late chick rearing , of which 10 birds had previously been sampled and were deployed only with GPS data loggers while the remaining 12 individuals were equipped both with GPS data loggers and time-depth recorders . All chick-rearing instrumented birds had a single chick, except for one nest, which had two chicks at the beginning of deployments but lost one of them within a few days. GPS loggers were programmed to sample positions every one min at incubation and every 2 min in chick-rearing. The TDR units were set to record depth and temperature at 1 s intervals.Fieldwork was conducted at the Pointe Suzanne Kerguelen shag colony , Kerguelen Island, southern Indian Ocean, during the 2014/15 breeding season. This study was approved by the ethics committee of the French Polar Institute  and therefore meets ethics guidelines. All animals in this study were cared for in accordance with its guidelines. Sampling occurred during two sessions. During the first session, a total of 20 individuals (both partners from 10 nests) were instrumented with GPS data loggers , and banded with an individually numbered coloured plastic ring on one leg and an individually numbered metal ring on the other for identification. The GPS loggers, removed from their housings and encased in heat shrink plastic for waterproofing, and the dive recorders were attached to the back feathers using waterproof tape  and cyanoacrylate glue .Individuals were recaptured 3–18 days later using the methods previously described. The data loggers were removed and individuals were weighed again and morphometric measurements  were taken with a vernier caliper and metal ruler . In addition, 3–6 dorsal dark contour feathers from between the wings were plucked and a blood sample (0.5–1.5 mL) was obtained by venipuncture of a tarsal vein. Spontaneous regurgitations during handling were collected for later analysis of prey remains. Handling times ranged 15–20 min during which the bird’s head was covered with a hood to reduce stress.Due to battery malfunction or flooding of loggers, GPS positions were obtained from only 36 deployments , each of which comprised 3–18 trips. Out of these deployments, 15 were carried out during incubation, and 21 during the chick-rearing period. Seven birds were used during both breeding stages, at an approximately one-month interval.13C values of seabirds indicating their foraging habitats [15N values increasing with trophic level [2 (AIR) for nitrogen. Replicate measurements of internal laboratory standards (acetanilide) indicated measurement errors < 0.15 ‰ for both δ13C and δ15N. Blood isotopic values were obtained from 32 individuals and feather values from 31 individuals .The isotopic method was validated in the southern Indian Ocean, with δhabitats , 37, andic level . Isotopiic level ) were meic level . A singlRegurgitate samples were stored frozen until processing in the laboratory. They were first defrosted and the fresh and accumulated fractions were weighed separately. The fresh fraction was sorted into different prey categories  that were weighed separately. Items were identified to species level when possible, according to  for fishdiveMove package was used to apply a speed filter to the GPS data to remove erroneous locations with a threshold of 18 m·s-1, and obtain summaries of diving metrics from TDR records (only dives deeper > 1 m were considered). Means of diving behaviour metrics and coefficients of variation (CV) per individual were calculated for dive duration, depth, and sum of vertical distance.All data analyses were conducted in the R Statistical Environment version 3.2.0 . GPS recadehabitatLT package [trip [sp [circular package.GPS records were linearly interpolated to the 1 s intervals in the  package  to provige [trip  and sp [trip [sp  were use2 = 0.74, P < 0.001). Therefore, it was considered that consistency in spatial space use was representative of consistency in foraging space use. Different grid cell sizes were used to calculate the index of consistency in space use (from 0.5x0.5 km to 5x5 km) to check the influence of grid cell size on our estimate of spatial consistency. Indices obtained, regardless of cell grid sizes, were highly correlated.An index of consistency in space use was calculated for each animal within each stage. For each trip the number of grid cells used by the individuals were identified. The number of shared grid cells for each pair of trips  was determined and the average of these calculated. This number was then divided by the average number of grid cells used per trip. To assess whether spatial information alone could reflect consistency in foraging locations, this index was compared for the GPS derived tracks and the dive locations alone. There was a significant positive correlation between the consistency index calculated from GPS data alone and diving locations obtained using GPS and TDR  were used as explanatory variables in models containing individual as a random effects and a power variance structure.To look at whether sex and stage influenced the consistency in spatial use, we used the coefficient of variation in total distance as a response variable. Indeed, this metric was correlated with the coefficients of variation in maximum distance and trip duration, with the standard deviation in heading and with the index of spatial use consistency. The model included sex, stage and their interaction as fixed effects, individual as a random effect, and a sex and stage identity variance structure. In order to quantify how specialised shags were in spatial use, we used a variance components analysis as described above. This was not done separately for each sex, however, because no difference in spatial use consistency was detected between sexes.FactoMineR package was used to determine the appropriate number of clusters [Post hoc Tukey HSD multiple comparison tests were then conducted to determine more specifically which clusters differed in the multcomp package [In order to identify differences in foraging strategies, an agglomerative hierarchical clustering analysis with Euclidian distance and Ward’s linkage criterion  was perfclusters . Data on package .13C or blood δ15N values as response variables. In the first case and second case, the random effects were individual and individual nested within pair, respectively. In order to assess dietary specialisations, linear mixed effects models were used, with either the isotope values during chick-rearing as response variables and the isotope values during incubation as explanatory variables, or feather isotope values as response variables and blood values (averaged between samples when repeat samples were obtained) as explanatory variables. Pair was included as a random effects in those 4 models. Unless stated otherwise, values presented are means ± SE.To look at the effect of sex and stage on stable isotopes, linear mixed models were used with blood δ-1 . Sex influenced the CV in maximum dive depths, with CV being higher by 0.13 ± 0.05 in males compared to females . There was substantial inter-individual variation in space use but, in general, all animals tended to dive at the section of the foraging trip most distant from the colony  from birds equipped with both GPS and TDR during the chick-rearing period. A total of 5679 dives were recorded with individuals displaying large variations in the means and CV of diving behaviour . There we colony .-1 , while heading to most distal point was not influenced by mass, wing length or head length. Sex and stage did not influence the CV for total distances travelled. In addition, birds instrumented during both incubation and chick-rearing used the same foraging areas  than individuals from all other clusters . A small sample size and unequal representation of individuals in each cluster, prevented us from testing whether clusters differed in foraging success (mass gains of adults during deployment at incubation or mass gains of chicks during chick-rearing).13C and δ15N values averaged -16.3 ± 1.3 and 14.6 ± 1.0 ‰, respectively . Large ranges in blood isotopic values reflected foraging variation between individuals during breeding  were highly significantly positively correlated . Mass was the only morphometric measurement influencing δ13C and δ15N values , with increasing values for heavier individuals (increase at a rate of 1.23 ‰ kg-1 for both δ13C and δ15N). There were significant differences in blood δ15N values amongst the four clusters  . Large ranges in feather isotopic values were also observed, with differences between the lowest and highest values amounting to 6.7 and 3.9 ‰ in δ13C and δ15N, respectively. Importantly, both δ13C and δ15N values in blood and feathers were highly significantly positively correlated  . Post-hoNotothenia cyanobrancha, with Lepidonotothen mizops and Harpagifer kerguelensis/spinosus ranking second and third, respectively. Prey diversity was low with 85% of samples containing 1–3 prey species (maximum 7), but the dominant prey in samples differed between individuals. Repeat samples were obtained from 7 individuals; in 3 of them, the dominant prey remained consistent between the incubation and chick-rearing periods.A total of 26 dietary samples were opportunistically collected, with a mean fresh mass amounting to 85 ± 43 g. Identified prey were benthic organisms including nine fish and one octopus species, the remaining identifiable prey items being errant polychaetes . OverallUsing complementary methods corresponding to different timescales , the pr13C and δ15N values, suggesting that males and females had similar habitats and diets, respectively.Sexual dimorphism in birds is known to relate to differential niche utilization . KerguelMale and female Kerguelen shags in the present study did not differ in mean maximum dive depths, which contradicts other studies performed on species of the blue-eyed shag complex , 57, 60.True individual specialisations  were emThe Kerguelen shags in the present study exhibited consistent feeding strategies, as indicated by stable isotope values, between stages within the breeding season but also between the breeding and inter-breeding periods. Other species from the blue-eyed shag complex show dietary specialisations that can be maintained over long timescales, consistent with the ability of some animals to adopt long-term behavioural strategies , 32, 33.A large number of seabird species have been shown to exhibit significant consistency in foraging strategies . HoweverMorus bassanus), individuals might be responding differentially to environmental variables that are good proxies for prey type and abundance [Similar to other species of the blue-eyed shag complex, Kerguelen shags are benthic divers and seafloor characteristics or bathymetric features could provide them with cues to memorize the location and quality of distinct foraging areas , 30, 62.bundance . Returnibundance . Lastly,bundance , 33, 63.Rather than being explained by sex or stage, foraging strategies corresponding to different types of behaviours and levels of consistency were associated with prey of different trophic levels. Such dietary specialisations have been reported in Kerguelen and South Georgian shags  and havevice versa in birds that exhibit determinate growth. Heavier birds, such as birds from clusters 1 and 2, tended to fly farther, potentially indicating they were bringing back larger food loads, consistent with the optimal foraging theory. We suggest that the strategies identified arise as the best compromise for specific individuals given their intrinsic characteristics to respond to the variability of coastal resources [Individual morphology did not appear to influence isotopic niche and there were no significant differences in morphometric measurements between the observed foraging strategies, confirming the existence of true specialisations . As theesources .In summary, the results of the present study have demonstrated strong individual specialisations in space use and diet across multiple temporal scales in a benthic forager, the Kerguelen shag, consistent with patterns reported for different species of the blue-eyed shag complex. Within the population, different foraging strategies could be highlighted and were associated with different levels of behavioural consistency and dietary choices. Sex, breeding stage and body measurements did not influence individual strategies. We show here the usefulness of using a combination of approaches–merging spatial, behavioural and dietary analyses–to investigate the links between foraging behaviour, its consistency and diet in individuals, which is essential to understand and accurately characterise ecological processes . Future S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


As fermentation could reduce the negative effects of bran on final cereal products, the utilization of whole-cereal flour is recommended, such as brown rice flour as a functional food ingredient. Therefore, this study aimed to investigate the effect of fermented brown rice flour on white rice flour, white rice batter and its steamed bread qualities. Brown rice batter was fermented using commercial baker’s yeast (Eagle brand) according to the optimum conditions for moderate acidity (pH 5.5) to obtain fermented brown rice flour (FBRF). The FBRF was added to white rice flour at 0%, 10%, 20%, 30%, 40% and 50% levels to prepare steamed rice bread. Based on the sensory evaluation test, steamed rice bread containing 40% FBRF had the highest overall acceptability score. Thus, pasting properties of the composite rice flour, rheological properties of its batter, volume and texture properties of its steamed bread were determined. The results showed that peak viscosity of the rice flour containing 40% FBRF was significantly increased, whereas its breakdown, final viscosity and setback significantly decreased. Viscous, elastic and complex moduli of the batter having 40% FBRF were also significantly reduced. However, volume, specific volume, chewiness, resilience and cohesiveness of its steamed bread were significantly increased, while hardness and springiness significantly reduced in comparison to the control. These results established the effectiveness of yeast fermentation in reducing the detrimental effects of bran on the sensory properties of steamed white rice bread and encourage the usage of brown rice flour to enhance the quality of rice products. The past two decades have seen a rapid increase in consumer demand for healthy foods, which has prompted recent research to find methods for production of healthy and functional foods. The usage of whole grain cereal instead of milled cereals is one such trend for production of healthy and functional foods, as consumption of whole grain foods has shown a reduction in the risk of several diseases, such as cardiovascular diseases, obesity, diabetes and some types of cancers ,2. One oRice is a unique crop due to its colorless, soft taste, low sodium levels, easy digestible carbohydrates and hypoallergenic properties. Therefore, its flour is an attractive food material to be used for making gluten free foods . SteamedGluten free materials such as white rice flour do not have the required characteristics for production of leavened foods, since their proteins have no ability to develop a viscoelastic network such as gluten. In addition, lack of nutritional value has existed as a health problem of gluten free products specifically produced from white rice flour. Consequently, in recent years, there has been an increasing interest in using different food materials such as, gums, hydrocolloids and starches to enable developing a similar gluten network . MoreoveModification of whole-cereal flour prior to its usage by using simple food processing, such as fermentation, could eliminate the negative effects of the bran. Pre-fermentation of the whole-flour might increase fiber solubility due to enzyme reactions on the cell wall structure. The addition of pre-fermented wheat bran to wheat dough caused an increase in the bread volume of high fiber wheat bread . The addRecently, fermentation became a trend for production of healthy foods from whole grain cereals. Industrial application of the biotechnology of fermentation for the production of gluten free baked products is a promising innovation in the health foods industry. Thus, the objectives of the current study is to investigate the effect of fermented brown rice flour on white rice flour, white rice batter and its steamed bread (Apam) characteristics, which could encourage the usage of whole rice grains.Brown rice grains (MR219) and commercial baker’s yeast  were bought from a local supermarket in Selangor, Malaysia. Brown rice grains were ground using a Cyclotech™ (1093) grinder  and a 500 µm sieve to obtain brown rice flour (BRF), which was used for the fermentation process. Brown rice grains were milled using a rice miller  to obtain white rice grains that were ground to flour in a Cyclotech™ (1093) grinder  and sieved to produce white rice flour (WRF). Flour samples were packaged in polyethylene bags and stored at 4 °C. Fermentation conditions  were optimized to achieve moderate acidity (pH 5.5) of fermented brown rice batter using Minitab 14 software (data not shown). Fermented brown rice batter at the optimum conditions was dried in an air oven drier at 50 °C for 3 h, ground to flour sieved in a 500 µm sieve and verified as fermented brown rice flour (FBRF).The formula of 100 g of WRF which contains 2% sugar, 2% salt, 3% baker’s yeast and 93% volume water based on the flour weight was used to prepare steamed white rice bread (SWRB). The SWRB formula of 60% white rice flour substituted with 40% FBRF achieved the highest bread volume and the overall acceptability of the tested sensory properties among the other ratios that included 0%, 10%, 20%, 30%, and 50% FBRF (data not shown) . To prepPasting properties of WRF and WRF substituted with 40% FBRF was determined using a rapid viscos analyser  following the method of AACC 61-02 . Peak viThe dynamic rheological test was performed using a HAAKE Rheowin 600 rheometer,  at 32 °C using parallel plate geometry (35 mm diameter and 1 mm gap). Batter samples were prepared as mentioned earlier, but without adding the yeast to avoid interference of air cells formation. After placing the batter samples between plates; batter samples were allowed to rest for 2 min to relax the residual stress. The linear viscoelastic part was measured by performing stress sweep experiment at 1 Hz frequency prior to determination. Based on the linear viscoelastic region, frequency sweep test was carried out at 1% strain between 0.1 and 10 Hz. Storage modulus (Gʹ), loss modulus (Gʺ), complex modulus (G*) and tan delta (δ) values were obtained from the Rheowin software (3.3). All the rheological tests were done in five replicates and their average values were stated in the results.et al. [1) and filled with sago pearl. After that, the volume of sago pearl used was recorded (V2). Bread volume (V) was calculated based on the following formula:The volume of steamed breads was measured after 1 h of steaming according to the seed displacement method described by Hallén, et al.  using sa3/g).Specific volume was determined by dividing the volume of the bread by its weight  equipped with P/75 mm plate, 30 N load cell and trigger force of 5 g. After cooling at room temperature for 1 h, bread samples were prepared with approximately 2.5 × 2.5 × 2.5 cm dimensions taken from the crumb and compressed to 40% of its thickness , at a poOne way analysis of variance (ANOVA) and Tukey’s multiple range tests with a confidence interval of 95% were used to report the significant differences between the obtained results.et al. [et al.  who repoet al. [et al. [The viscoelastic properties of the WRB and WRB containing 40% FBRF were studied using a dynamic oscillatory test. The mechanical spectra of the WRB sample indicated that both elastic (G′) and viscos module (G′′) values were higher than that of WRB with FBRF at all the tested frequency ranges . In addiet al.  reported [et al.  also ind [et al. .3) and specific volume (2.46 cm3/g) in comparison to the reference sample . This finding supports previous research, where the addition of fermented barley flour to whole barley dough improved the overall quality of its bread. Its bread volume was significantly higher than that of the control [2 produced during fermentation, consequently increase the loaf volume [ control . Dough af volume . Howeverf volume . The modet al. [et al. [2 retention during dough proofing, and as a result increased the bread volume and the crumb softness [et al.  reportedet al. . Also, fet al. . Accordi [et al.  additionsoftness ,22.Addition of fermented brown rice flour to steamed white rice bread significantly improved rheological and textural properties as well as volume of the bread as indicated in this study. Currently, investigation of the effect of fermented brown rice flour on the nutritional value of steamed white rice bread is under way.


Three-dimensional photonic body surface scanners (3DPS) feature a tool to estimate total body volume (BV) from 3D images of the human body, from which the relative body fat mass (%BF) can be calculated. However, information on validity and reliability of these measurements for application in epidemiological studies is limited.® device using equations by Siri and Brozek. Three scans were obtained per participant . Validity was evaluated based on the agreement of 3DPS with ADP using Bland Altman plots, correlation analysis and Wilcoxon signed ranks test for paired samples. Reliability was investigated in a separate sample of 18 participants  aged 25–66 years using intraclass correlation coefficients (ICC) based on two repeated 3DPS measurements four weeks apart.Validity was assessed among 32 participants  aged 20–58 years. BV and %BF were assessed using a 3DPS (VitusSmart XXL) and air displacement plethysmography (ADP) with a BOD PODMean BV and %BF were higher using 3DPS compared to ADP, , yet the disagreement was not associated with gender, age or body mass index (BMI). Reliability was excellent for 3DPS BV  and good for 3DPS %BF . Results were similar for the standard scan and the relaxed scan but somewhat weaker for the exhaled scan.Although BV and %BF are higher than ADP measurements, our data indicate good validity and reliability for an application of 3DPS in epidemiological studies. Obesity is a major risk factor for non-communicable diseases like type-2-diabetes mellitus and cardiovascular diseases, and accounts for a substantial proportion of disability-adjusted life years and mortality worldwide .The definition of obesity is based on the body mass index (BMI) , 3; howeThree-dimensional photonic body surface scanners (3DPS) provide an opportunity for a standardized acquirement of data on total body volume (BV). This is promising particularly for large-scale epidemiological studies, where standardized, fast, accurate, and precise assessment methods are key issues for a valid estimation of disease risk. In contrast to the abovementioned methods to assess the body composition, the 3DPS examination does not require the participants to be in a fasting state or to restrict physical activity beforehand. In Germany, measurement of body size has been recently conducted for the clothing industry in more than 13,000 individuals using a laser-based 3DPS  . Within The aim of this study was therefore to assess the validity of 3DPS total BV and %BF measures against air displacement plethysmography (ADP) measures and to identify factors potentially contributing to a measurement disagreement in the general adult population. Further, as we have already shown a good short-term reliability of the 3DPS , we examParticipants were recruited from February 2016 to June 2016 as part of the larger-scaled MetSScan study, which targeted a sample size of 500 participants and was conducted by the Molecular Epidemiology Research Group of the Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin. The MetSScan study aims to develop algorithms for the assessment of parameters of the metabolic syndrome [Participants for the main study were recruited based on institution's internal mailing lists, local press releases, newspaper articles, and advertisements as well as public postings. Inclusion criteria were age 18–79 years, sufficient German language skills and the ability to give informed consent. Exclusion criteria were current pregnancy or breastfeeding, known hemophilia, anticoagulant medication, dependency on medical appliances for stand and movement, or current wearing of a plaster cast.For assessment of validity of total BV measurements, ADP measurements were performed on 32 participants in addition to the main study protocol. ADP measurements were not taken in persons with claustrophobia.To assess longer-term reliability  of BV and %BF, the 3DPS measurements were repeated after approximately four weeks in an independent sample of 18 participants under identical conditions.The study was approved by the Ethics Committee of the Charité—Universitätsmedizin Berlin and by the local data protection officer. Informed written consent was obtained from all participants.All participants completed a study protocol at the study center comprising measurement of height and weight, the 3DPS and ADP examination. Due to the original aim of the MetSScan study, we needed a fasting blood sample to determine blood glucose. Thus participants were in a fasting state for at least 8 hours prior to the investigation. All examinations were taken on the same day with a maximum interval of one hour between the 3DPS and ADP measurements.For all examinations, participants wore minimal, tight-fitting, unpadded underwear, no jewelry or eyeglasses and a bathing cap. The measurement of body height followed standardized procedures according to WHO guidelines  using a 2 within a scan time of 10 seconds. Based on the 3D point cloud, 153 anthropometric measures are computed by the software according to international standard DIN EN ISO 20685 [The 3DPS examination was performed with a VitusSmart XXL and the software Anthroscan Professional version 3.3.0 . The measurement principle is based on optical triangulation, whereby the scanner uses eight eye-safe laser sensor heads and cameras to create a 3D point cloud of the human body surface. The measuring range covers 2100 mm height x 1000 mm width x 1200 mm depth. The accuracy is within a level of ±1 mm and the scanner reaches a density of 27 points/cmSO 20685 . FurtherThe study protocol for the 3DPS measurement consisted of three consecutive scans in three different postures: 1. standard position: upright posture with legs shoulder-wide apart and, if possible, no contact of thighs; arms slightly bend and away from the body; hands making a fist with thumbs showing forward; and head positioned compliant with the Frankfort horizontal plane (StdScan), 2. relaxed position: legs hip-wide apart, arms hanging relaxed without contact to the body, hands making a fist, head aligned in Frankfort horizontal plane (RlxScan), and 3. relaxed position after maximum expiration (ExhScan). Except for the ExhScan, participants were asked to breathe normally during the scan procedure. All scan images were immediately visually checked by the study personnel. A scan was repeated if the scan quality did not reach pre-defined standards, e.g. with regard to image artifacts, deficient resolution, or if the posture was not correct. The scan measurement series was cancelled in case the quality remained insufficient after three repeated attempts in the same individual. The 3DPS was calibrated prior to the first examination of each day with a cylinder tube of defined height and width provided by the manufacturer.® , which shows high short-term reliability [® was calibrated each day with a 50 l calibration cylinder, allowing for a maximum standard deviation (SD) of 50 ml in a series of five volume measurements. Body weight was measured with an integrated electronic scale  connected to the BOD POD®, which was calibrated every 14 days with two pre-specified 10 kg weights. Body height was taken from the above described stadiometer measurement. Directly before each measurement, an obligate 2-step calibration was carried out: first, the volume of the chamber was measured without participant inside and then again with the 50 l calibration cylinder placed inside. After the participant entered the chamber, two measurements of raw BV were executed. If these two measurements differed by 150 ml or 0.2% of BV, a third measurement was performed and the two closest measurements within these agreement criteria were averaged. The estimate of raw BV was adjusted for thoracic gas volume (TGV) predicted from gender, age, and height by the software (8) and a body surface area artifact, which arises due to the apparent negative volume of air under isothermal conditions related to the skin surface [corr), body density was calculated as:2) [2) [The assessment of BV by ADP was performed with a BOD PODiability –22, and  surface . Using t0 kg/m2)  or with /m2) [2) :Siri:%B® software.All calculations were automatically executed by the BOD PODThe 3DPS derived BV was corrected for TGV for the StdScan and RlxScan, and residual volume (VR) for the ExhScan, respectively. TGV was calculated according to the following equations by Brozek et al.  and CrapVR was calculated with equations developed by Crapo et al. :Men:VR=%BF was calculated for all corrected BVs of 3DPS scans using Eqs  and (3).2) and was used to define non-overweight , overweight (BMI≥25 to <30 kg/m2) and obesity (BMI≥30 kg/m2) [Data are presented as mean ± standard deviation (SD). BMI was calculated as body weight in kilogram (kg) divided by body height in meter squared (m0 kg/m2) . The com0 kg/m2) , 28. Q-Q0 kg/m2) . Since BMultiple regression analyses were used to predict ADP BV based on 3DPS BV including gender, age, BMI, and an interaction term of 3DPS BV*gender. The final models retained only the significant factors after a stepwise exclusion procedure.For the assessment of reliability, the differences in the duplicated 3DPS measurements (BV and %BF), calculated as scan at date 1 –scan at date 2, and the intraclass correlation coefficients (ICC) ± 95%CI were calculated.P-values presented are 2-tailed and p<0.05 were considered statistically significant. The analyses were performed using SPSS 18  and SAS Enterprise Guide 4.3. .No technical issues or refusal of participants occurred during the completion of the study and quality checks revealed formal plausibility of the data obtained. Basic characteristics of the two subsamples including 32 and 18 participants are summarized in For the comparison of %BF, one participant was excluded from the analysis due to invalidly low %BF (<1% BF) given by ADP. %BF based on 3DPS StdScan and on ADP were highly correlated  as reference methods. A study from Wells et al. with 22 adults using a Hamamatsu Bodyline Scanner reported an underestimation of 0.3 ± 2.7 L against ADP and overestimation of 0.5 ± 2.4 L for UWW, respectively . SimilarTaken together, the calculation rather than measurement of TGV  could have resulted in discrepancies from actual TGV values on an individual level, for which we could not account for. Though this similarly affected both ADP and 3DPS, the ongoing approaches to adjust for TGV differed between methods, e.g. ADP BV was adjusted for 40% of TGV, whereas it was completely deducted from 3DPS BV.The 3DPS does not follow the standardized WHO measurement protocol for assessment of height. In this regard, we previously showed that the VitusSmart XXL 3DPS overestimates body height by on average 0.9 cm compared to manual measurements on basis of standardized WHO guidelines . HoweverFurthermore, other factors independent of the scanner’s ability to validly estimate BV could have contributed to the overestimation of BV. 3DPS visually scans the body surface and thus, any body-enlarging object, like clothing and hair, amplifies BV. Even though this influence was minimized by wearing tight-fitting underwear and a bathing cap, it could not be completely eliminated, e.g. if the participant had a large hair volume, a beard, thick body hair, or padded underwear. In addition, participants may have had the tendency to hold their breath or breathe flatly during the scanning procedure in order to not blur the picture, which could have led to a higher air volume in their lungs.While we observed an acceptable difference between 3DPS and ADP for BV, the agreement between methods found for %BF was lower. A non-zero intercept and non-identity slope in the Bland Altman plots reflected the estimation of %BF on the basis of 3DPS BV to yield higher values than estimation based on ADP. Similar graphical findings were also reported in a study by Wang et al. using underwater weighing as comparison method to evaluate validity of 3DPS based BV and %BF . Bearing2 (as well as for persons with BMI >30.0 kg/m2) Brozek’s [Furthermore, in contrast to BV, for %BF we found a correlation between the difference and the mean of 3DPS and ADP measurements, with higher estimates based on 3DPS than for ADP observed for lower %BF, while with increasing %BF the opposite was true. A similar dependency of the difference between methods and the mean %BF was also found by Wang et al. . This skBrozek’s  instead Brozek’s ; using tIn respect to the reliability of the 3DPS, it should be mentioned that a single assessment of a biomarker, as has often been done in baseline examinations of large-scale epidemiological studies, may be susceptible to short-term variation and not reflect true long-term exposure. Therefore, random measurement error generally tends to decrease correlation and regression coefficients in epidemiological studies toward 0 and bias relative risks toward 1. However, in our analysis, the 3DPS scanner revealed an excellent reliability after a time period of four weeks. To our knowledge, reliability over a longer-time period has not been analyzed previously; however, other studies with consecutive measurements showed very good short-term reliability , 30–32. Our study has some strengths and limitations. ADP has been shown to be an accurate method for assessment of BV, and it is probably the method that is most often used in experimental studies –39. HoweIn conclusion, our study showed good agreement between 3DPS and ADP for measurement of BV and %BF, although measurements were slightly higher for 3DPS compared to ADP. Further, we found high ICCs for BV and %BF when measured with 3DPS four weeks apart. These data indicate good validity and excellent four-week reliability for an application of 3DPS to assess BV and %BF in an epidemiological study.


Treatment of animals with CIS alone significantly increased the ALDH1 CSC population in tumors, suggesting that CIS targets cancer cells but spares cancer stem cells, which undergo amplification. WFA and CIS combination suppresses the expression of securin an “oncogene”, suggesting that securin may serve as a downstream signaling gene to mediate the antitumor effects of WFA.Ovarian cancer is the fifth leading cause of deaths due to cancer among women in the United States. In 2017, 22,440 women are expected to be diagnosed with ovarian cancer and 14,080 women will die with it. Currently used chemotherapies (Cisplatin or platinum/taxane combination) targets cancer cells, but spares cancer stem cells (CSCs), which are responsible for tumor relapse leading to recurrence of cancer. Aldehyde dehydrogenase I (ALDH1) positive cancer stem cells are one of the major populations in ovarian tumor and have been related to tumor progression and metastasis. In our studies, we observed expression of ALDH1 in both ovarian surface epithelium (OSE) and cortex with high levels of expression in OSE in normal ovary and benign (BN) tumor, compared to borderline (BL) and high grade (HG) ovarian tumors. In contrast, high levels of expression of ALDH1 were observed in cortex in BL and HG tumors compared to normal ovary and BN tumor. Withaferin A (WFA) alone or in combination with cisplatin (CIS) significantly inhibited the spheroid formation  of isolated ALDH1 CSCs  Ovarian cancer is the most lethal of all the gynecological malignancies, affecting over 22,000 women annually in the United States alone . Due to There are several factors reported for the development of cisplatin resistance and recurrence of cancer including changes in DNA repair mechanisms, expression of drug transporters and multidrug resistance genes. While the ‘classical’ stochastic model of cancer development holds that any cell may become source of malignant transformation, however, emerging evidence supports the view that only a minor subpopulation of cancer cells have the potential to initiate cancer growth. These cells are known as cancer stem cells (CSCs) and have the ability to undergo self-renewal, and propagate to tumorigenesis , 8. CSCs+ dependent oxidation of various aldehydes. Despite 19 isoforms of ALDH and each isoform having its own specific function, ALDH1 is the most often correlated with cancer stem cells and is found in various tissues [Recently, it has been reported that aldehyde dehydrogenase (ALDH) activity to be a very attractive CSC marker in many cancers such as lung, breast, prostate, thyroid, head and neck, and ovarian -39. ALDH tissues . In epit tissues -47 have There is increasing evidence for the existence of CSCs in solid tumors, which are reported to be responsible for chemo-resistance as well as recurrence of cancer . In ovarin vitro, which describes the tumorigenic potential of CSCs [The CSCs have the capability to form spheroid on ultralow attachment plate  of CSCs , 48. To  of CSCs . ApproxiIn our previous studies, we showed that treatment of mice bearing orthotopic tumors generated by injection of ovarian cancer cells (A2780) directly into ovary followed by treatment with WFA in combination with CIS resulted a in suppression of tumor growth and metastasis . In contSecurin also known as a pituitary tumor transforming gene (PTTG) is a multi-domain and multifunctional oncogene, which was originally cloned from rat pituitary tumor , and theIn recent years, number of investigators have provided evidence about the existence of CSCs in various cancers -10. ThesAssessed from the staining pattern observed across tumor tissues versus normal ovary, ALDH1 protein appears to be in OSE as well as the in stroma Figure , C and DIn our studies, we showed that treatment of spheroids (tumorigenic characteristics of CSCs) formed by isolated ALDH1 positive CSCs with WFA alone or in combination with CIS significantly inhibit the spheroid formation, suggesting that combination of WFA with CIS is very effective in suppressing the tumorigenic function of ALDH1 Figure . SimilarTo determine the mechanisms by which WFA alone or in combination with CIS mediates its antitumor effect, we explored the possibility of involvement of securin which is a multi-domain and multifunctional oncogene. Overexpression of securin causes cellular transformation and development of tumors in nude mice , 53. Knoin vitro (spheroid formation) and tumor growth in vivo (tumors generated by injecting ovarian cancer cell line A2780). There appears to be a correlation between antitumor effects of WFA on ALDH1 expression, CSC population and regulation of securin expression . We obtained human ovarian tumor tissues from Brown Cancer Center’s repository under the University of Louisville Institution Review Board (IRB) approval.We obtained ovarian epithelial cancer cell line A2780 from Denise Connolly from FOX Chase Cancer Center, Philadelphia, PA. A2780 cell line was originally generated from human ovarian cancer patient prior to treatment . The cel6 cells/ml. The cells were incubated with Aldefluor substrate (1 μM) at 37°C for 45 min. Cells were centrifuged at 1,500 rpm for 3 min and resuspended in binding buffer to a concentration of 10X106/ml. Negative control samples were treated with 50 mmol/L of diethylaminobenzaldehyde  before adding Adelfluor as described by Ginestier et al [Aldehyde dehydrogenase 1 (ALDH1) is a major cancer stem cell population in ovarian cancer and has been shown to be highly tumorigenic when injected into nude mice . Therefoer et al . Highly 2 incubator for 3 to 5 days. Spheroids formed, were collected by low speed centrifugation and dispersed mechanically to a single cell. Approximately 1,000 cells were plated in 6 well ultralow attachment plates. After 24 h of plating, small spheroids (colonies) were formed which were treated with WFA and CIS both alone and in combination. After 72 h of treatment, spheroids with size > 50 mm were counted and photogrpahed. The experiments were repeated at least three times.To determine the tumorigenic function of ALDH1 positive cancer stem cells and effects of treatment with WFA and CIS both alone or in combination, we performed standard spheroid formation assays as described previously . The iso6 A2780 cells in 10 μl volume directly into left ovary of 5 to 6 weeks old nu/nu female mouse (NCI). After 10 days of injection of cells, animals were injected i.p with control vehicle, WFA and CIS both alone or in combination as described previously [+ population and its expression, we collected the tumor tissues from control animals (treated with vehicle) and animals treated with WFA and CIS both alone or in combination. The tissues were collected in 10% buffered formalin, and processed for embedding in paraffin blocks and further sectioned as per routine histology procedures.To determine the effect of WFA and CIS both alone and in combination, orthotopic ovarian tumor were generated by injecting 1X10eviously . The effeviously . To deteParaffin embedded human ovarian as well as animals’ tissue sections were deparaffinized in xylene and rehydrated in a decreasing graded series of ethanol as described previously . Section2O2 and probed with β-actin monoclonal antibody in order to serve as an internal control [The tumor tissues collected from animals were homogenized in chilled RIPA buffer (Sigma) supplemented with a Complete Mini Protease Inhibitor tablet . The homogenates were centrifuged at 10,000 rpm and supernatants were collected. The protein concentration for each sample was determined using Bradford reagent (BioRad Laboratories), according to supplier’s instructions. Forty μg of protein from each sample was separated on 7% polyacrylamide/SDS gel. The proteins were transferred to nitrocellulose membranes as described previously . The mem control .http:/www.graphpad.com) was used for data and graphic analysis.All experiments were repeated at least two to three times. All data were expressed as mean ± SEM. Statistical analysis was performed using Student’s t-test. A p-value < 0.05 was considered to indicate a statistical significance. Graphpad Prism® (version 4.03, GraphPad software, San Diego California USA,


Candida tropicalis and mulberry leaf flavonoids) on intestinal microbiota of preweaned calves challenged with Escherichia coli K99. Sixty Holstein calves were randomly assigned to 5 treatments: fed a basal diet (N-CON); fed a basal diet and challenged with E.coli K99 (P-CON); fed a basal diet supplemented with C.tropicalis (CT), mulberry leaf flavonoids (MLF), and the combination of the two additives (CM), respectively, and challenged with E.coli K99. The MLF and CM groups had significantly higher average daily grain and feed efficiency, and significantly lower fecal scores compared with the P-CON group after E. coli K99 challenge. The supplementation groups increased the relative abundance, at the phylum level, of Bacteroidetes and Proteobacteria, whereas at the genus level, they increased the relative abundance of Prevotella, Lactobacillus, and Enterococcus. Quantitative PCR revealed that the CT, MLF, and CM groups had significantly lower copy numbers of E.coli K99 compared with the P-CON group. The CT, MLF, and CM treatments reduce days of diarrhea, improve intestinal health, and beneficially manipulate the intestinal microbiota in preweaned calves.The aim of this study was to investigate the effects of dietary supplementation with two alternatives to antibiotics ( Enterotoxigenic Escherichia coli expressing K99 fimbriae and heat-stable type Ia (STa) toxin is one of the major pathogens associated with neonatal calf diarrhea6. The K99 fimbrial adhesins promote attachment and colonization of bacterial cells to the surface of epithelial cells of the small intestines, while the STa toxin damages the epithelial cells and disrupts fluid homeostasis, resulting in fluid and electrolyte hypersecretion that leads to watery diarrhea, dehydration, and acidosis in neonatal calves9.Neonatal calf diarrhea is a serious health and welfare problem on dairy farms, with the resulting high mortality and morbidity contributing to considerable economic losses worldwide in the cattle industry13.Although antibiotics are given therapeutically after scours is observed, concerns have been raised regarding microbial resistance to antibiotics and increasing passage of laws banning the use of antibiotics in livestock production throughout the world. Therefore, alternatives to antibiotics for prevention and treatment of neonatal calf diarrhea are urgently needed to maintain the health of livestock. Recently recognized alternatives include yeasts and flavonoid-containing plant extracts, which are showing beneficial effects on animal intestinal health in an increasing number of studiesCandida tropicalis, a yeast of the Candida genus, is considered an important inhabitant of the healthy animal gut, and it can commonly be isolated from the gastrointestinal tracts of humans14, bovines15, birds16, and fish17. Previous ruminal fermentation studies demonstrated that C. tropicalis stimulated total and cellulolytic microbial populations, increased gas production, and activated in vitro ruminal fermentation, indicating its excellent potential for use as a feed additive in ruminants19. Long et al. reported C. tropicalis stimulated lactate uptake by Selenomonas ruminantium and increased the production of acetate and propionate and the ratio of propionate to acetate20. C. tropicalis isolated from some fish gastrointestinal tracts increased phytase and tannase production, as well as crude protein, lipid, and mineral contents, and reduced the antinutritional factors in different plant feedstuff21.Morus alba) leaves, have also shown health-promoting properties due to the alteration of the expression and activity of key enzymes in lipid and carbohydrate metabolism23, induction of protective effects against hydroxyl and superoxide radical damage24, antimicrobial activity26, antiparasitic activity27, and antioxidant activity28, among others. Previous studies have shown that flavonoids isolated from the leaves of many different plants exhibit antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi in in vitro antimicrobial assays34. Phytochemical investigations have shown that lipophilic flavonoids exert their antimicrobial activities through their ability to penetrate biological membranes35. Omosa et al.26 demonstrated that the antibacterial activity depended on the relative positions of the hydroxyl and methoxy groups on the flavone skeleton, and that a methoxy group at the C-3 position in the flavone skeleton was associated with good activity against E. coli. Several studies have demonstrated that probiotic yeasts might have inhibitory activity against specific pathogens38, but very few experimental and clinical trials have examined C. tropicalis as a possible probiotic. A considerable number of studies have shown that flavonoids isolated from different plants demonstrate antimicrobial activity, but most of these have been in vitro investigations. The aim of the present study was therefore to examine the effects of dietary supplementation with C. tropicalis and mulberry leaf flavonoids, singly or in combination, on the intestinal bacterial community composition in preweaned calves challenged with E. coli K99.Plant-derived flavonoids, such as those extracted from mulberry , dry matter intake (DMI), and feed efficiency were analyzed , mulberry leaf flavonoids (MLF), or the combination (CM) had lower fecal scores and experienced fewer days with fecal score >2 than that in the P-CON group  when compared with the P-CON group on d 2–5 after the E. coli K99 challenge. Dietary supplementation with C. tropicalis and mulberry leaf flavonoids, singly or in combination, improved fecal scores and reduced the number of days with mild or watery diarrhea.In order to observe whether dietary supplementation with the two alternatives to antibiotics would have effects on prevention of diarrhea in preweaned calves challenged with oup Fig. . Fecal sIllumina MiSeq sequencing analysis of the 24 jejunum digesta samples of preweaned calves generated a total of 1017,001 trimmed reads with an average of 42,622 ± 12,268 reads per sample after data filtering, quality control, and removal of primers, chimeras, and low-confidence singletons. For further analyses, all reads were classified into 512 operational taxonomic units (OTUs) based on ≥ 97% nucleotide sequence identity between reads.P < 0.05). The community diversity (Shannon index) differed significantly (P < 0.05) between the CM group and other groups in preweaned calves  in the MLF group than in the N-CON and P-CON groups. The phyla Bacteroidetes and Proteobacteria were abundant in samples taken from the MLF, CT, and CM groups when compared with the N-CON and P-CON groups, and the two phyla were more abundant in the MLF and CM groups (P < 0.05) than in the other groups. Among other minor phyla, the TM7 and Verrucomicrobia were more prominent in the MLF, CT and CM groups, while the Tenericutes and Synergistetes were more prominent in the N-CON and P-CON groups.At the phylum level, 15 phyla were identified in the samples from the preweaned calf guts, and these were dominated by oup Fig. . Howeverps Table . The phyPrevotella, Enterococcus, and Lactobacillus made up the main bacterial species in MLF group, with Prevotella and Lactobacillus having the highest relative abundances in this group. The Enterococcus and Pseudomonas were the two most abundant genera in the samples from the CM group.At the genus level, 185 genera belonging to the 15 phyla were detected in the samples. In total, 25 most abundant shared genera with a relative abundance ≥ 0.1% Table  were preE.coli K99, and four selected bacterial species that showed statistically significant in sequencing results . Many studies indicated that diet supplemented with LAB could improve weight gain and feed efficiency, and reduced diarrhea incidence46. Furthermore, the structure of flavonoids is similar to estradiol, which can regulate the secretion of growth hormone by the hypothalamus-pituitary hormone axis47. Growth hormone directly accelerates protein synthesis or stimulates insulin-like growth factors 1 to promote muscle tissue growth and body weight gain48.In the present study, the MLF and CM groups had significantly higher ADG and feed efficiency, and significantly lower fecal scores compared with the P-CON group after C. tropicalis and mulberry leaf flavonoids, singly or in combination, improved fecal scores and reduced the number of days with mild or watery diarrhea, suggesting protective effects of both supplements in calves with a high risk of morbidity. Previous studies showed that a yeast culture supplement decreased the risk of diarrhea due to E. coli, because the E. coli adhered to the oligosaccharides present in the yeast cell walls rather than attaching to and invading the intestinal cells50. This might be the reason that the copy number of E. coli K99 in CT group was significantly lower than that in the P-CON group. A considerable number of studies have demonstrated that flavonoids isolated from the leaves of many different plants show good antimicrobial activity against E. coli34. The antimicrobial activity of flavonoids is apparently due to their ability to penetrate biological membranes35. The antimicrobial activity of flavonoids might be one of the reasons that the MLF group had significantly lower copy number of E. coli K99 compared with the P-CON group. As mentioned above, feeding C. tropicalis, mulberry leaf flavonoids, or their combination increased the relative abundance of LAB in the calf gut. The LAB can adhere to the intestinal tract and produce lactate and acetate, which reduces the attachment and colonization of E. coli on the surface of intestinal epithelial cells46. The increasing abundance of LAB in the calf gut might be another reason that the CT, MLF, and CM groups had significantly lower copy number of E. coli K99 compared with the P-CON group. The observed increase in LAB abundance would be expected to improve gut health and may explain the benefits seen in fecal scores and diarrhea observed in this study when calves were fed either of the two supplements or their combination.Dietary supplementation with C. tropicalis and mulberry leaf flavonoid supplements increased the intestinal community diversity in preweaned calves, whereas each supplement on its own did not have this effect. As mentioned above, flavonoids isolated from the leaves of many plants exhibit antimicrobial activity34, which means flavonoids might decrease the community diversity. However, in the current study, dietary supplementation with the combination of C. tropicalis and mulberry leaf flavonoid increased the intestinal community diversity. The reason might be the synergistic effect of C. tropicalis and mulberry leaf flavonoid. The action mechanism needs to be further studied in future.The community diversity index Fig.  and OTU 53. The gradual increase in consumption of large amounts of different solid feeds might be one reason for an age-dependent increase in bacterial diversity55. Another reason might be that the digesta samples were collected from different gastrointestinal tract regions in different studies. Malmuthuge et al. reported that the highest number of OTUs and the greatest bacterial community diversity indices were observed in the rumen, followed by the large intestine (cecum and colon), and then the small intestine (jejunum and ileum)56. The rumen and large intestine are regarded as fermentation tanks for microbial fermentation of indigestible dietary substrates, and the retention time of digesta is longer in the rumen and large intestine than in the small intestine, which would facilitate the growth of a more complex bacterial community56.The OTU number and community diversity of the gastrointestinal tracts of the calves in the present study were lower than previously reported in some studies. One reason might be that the previous studies examined weaned calves or adult cows, and that the species richness and diversity in the gut increases with ageFirmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. The groups shared 113 genera, and the 25 most abundant shared genera, with a relative abundance ≥ 0.1%, were present in all samples across the different groups. These dominant phyla and shared genera represented the core microbiome of the calves of this age, irrespective of the treatments. However, the relative abundance of the phyla and genera from this shared community varied considerably among the groups.The dominant phyla found in all groups were Firmicutes showed the highest overall relative abundance and dominated in all the treatment groups. Our findings are consistent with the studies of Oikonomou et al. and Malmuthuge et al., who detected a significantly higher relative abundance of Firmicutes in the gut digesta of preweaned Holstein calves56. Malmuthuge et al. implied that Firmicutes tended to more readily colonize the small intestinal digesta of preweaned calves, while Bacteroidetes tended to more readily colonize the rumen and large intestinal contents56. The regional variations in the gastrointestinal tract of preweaned calves revealed differences in the dominant bacterial community56.In the present study, Lactobacillus and Enterococcus, have known beneficial effects on feed efficiency and animal health46, and both Lactobacillus and Enterococcus have been used as direct-fed microbials for a long time. Studies have reported that direct feeding of calves or dairy cows with microbials consisting of Lactobacillus and Enterococcus prevented declines in ruminal pH and decreased the risk of metabolic acidosis. These effects occurred due to facilitation of the growth of ruminal microorganisms adapted to the presence of lactic acid and by stimulation of the utilization of lactic acid by lactic acid utilizing bacteria58. Calves fed Lactobacillus and Enterococcus also showed an improved abundance of ruminal cellulolytic bacteria, such as Butyrivibrio fibriosolvens and Eubacterium ruminantium58. In our study, the relative abundance of Lactobacillus increased in the CT and MLF groups, and the relative abundance of Enterococcus increased in the CT, MLF, and CM groups.The LABs, such as Bacteroidetes was significantly more abundant in the MLF and CM groups, and especially in the MLF group, when compared to the N-CON and P-CON groups. Bacteroidetes was composed mainly of the genera Alistipes, Myroides, and Prevotella in all groups, and the genus Prevotella was significantly more abundant in the MLF group, accounting for up to 15.22% of the total reads. The Prevotella genus contains several ruminal species that are capable of utilizing starches, other non-cellulosic polysaccharides, and simple sugars as energy sources59, and this genus was more abundant in the CT, MLF, and CM groups than in the N-CON and P-CON groups.The phylum Bacteroidetes found in the rumens of preweaned calves fed whole milk or milk replacer contain more Bacteroides than Prevotella60, whereas the rumens of adult cattle contain almost exclusively Prevotella51. A recent study showed that the relative abundance of Bacteroides in the feces of adult cattle was negatively associated with a high fiber diet54. The diet of preweaned calves, which mainly includes whole milk or milk replacer, is rich in protein, fat, and sugar, whereas the adult cattle’s diet is composed mainly of plant fiber. In the present study, the small intestinal digesta of the 9-week-old preweaned calves contained more Prevotella than Bacteroides and the diet of the calves consisted of milk replacer, calf starter, and hay, with the calf starter and hay dominating, suggesting that, during calf development, increased fiber ingestion and decreased milk consumption decreases the relative abundance of Bacteroides55.Previous studies have reported that the Actinobacteria and Proteobacteria, were also found in high proportion in all treatment groups. The relative abundance of Actinobacteria decreased and Proteobacteria increased in the MLF, CT, and CM groups, and especially in the MLF group, when compared with the N-CON and P-CON groups, indicating that the dietary supplements helped to encourage the growth of Proteobacteria and to inhibit the growth of Actinobacteria in the preweaned calves.Other phyla, such as the C. tropicalis and mulberry leaf flavonoids), and their combination on the intestinal microbiota in preweaned calves challenged with E. coli K99. The MLF and CM groups had significantly higher ADG and feed efficiency compared with the P-CON group after E. coli K99 challenge. Dietary supplementation with the two alternatives to antibiotics, singly or in combination, improved fecal scores and reduced days with mild or watery diarrhea. Dietary supplementation with the combination of C. tropicalis and mulberry leaf flavonoids significantly increased the number of OTUs and the community diversity. Dietary supplementation with the two alternatives to antibiotics, singly or in combination, increased the relative abundance, at the phylum level, of Bacteroidetes and Proteobacteria and decreased the relative abundance of Actinobacteria, while at the genus level, this supplementation increased the relative abundance of Prevotella, Lactobacillus, and Enterococcus. Quantitative real-time PCR revealed that dietary supplementation with mulberry leaf flavonoids significantly increased the copy numbers of total bacteria and of the genera Prevotella and Lactobacillus in jejunum digesta. The CT, MLF, and CM groups had significantly lower copy numbers of E.coli K99 compared with the P-CON group. Our results establish a strong foundation for evaluating the potential of C. tropicalis and mulberry leaf flavonoids as feed additives for the reduction of diarrhea and improvement of intestinal health in preweaned calves challenged with E. coli K99.The results presented here provide new information regarding the effects of dietary supplementation with two alternatives to antibiotics  positive control (P-CON): fed a basal diet and orally challenged with E. coli K99 ; 3) C. tropicalis treatment (CT): fed a basal diet supplemented daily with C. tropicalis  and then orally challenged with E. coli K99 ; 4) mulberry leaf flavonoid treatment (MLF): fed a basal diet supplemented daily with mulberry leaf flavonoids  and then orally challenged with E. coli K99 ; and 5) combined C. tropicalis and mulberry leaf flavonoid treatment (CM): fed a basal diet supplemented daily with both C. tropicalis  and mulberry leaf flavonoids  and then orally challenged with E. coli K99 . The basal diet  negative control (N-CON) treatment: fed a basal diet and not challenged with The body weight of each calf was recorded at d 28, 56, and 64 of age. Feed intake and fecal scores of each calf was recorded daily. Five calves were selected from each group and euthanized at 64 d of age. Jejunum digesta samples were collected from the middle of the jejunum and at the same site consistently for all the animals. Jejunum digesta samples were snap frozen in liquid nitrogen and kept at −80 °C until further analysis. The flow scheme of this trial was shown in Fig. 9 CFU/g) was purchased from Beijing Huanong Biological Engineering Co., Ltd., Beijing, China. Commercial mulberry  leaf extracts were purchased from Xi’an Feida Biotechnology Co. Ltd., Xi’an, China. The mulberry leaf extracts were vacuum-dried and contained 5.0% flavonoids (w/w). The CT and MLF were mixed with the milk replacer and administered with the morning feeding.A commercial CT probiotic . A growth curve was constructed for E. coli K99 to determine the appropriate incubation time required to reach the target challenge dosage of approximately 1.0 × 109 CFU/mL. E. coli K99  was mixed with milk replacer and fed to the calves on d 57.The et al.50. Briefly, fecal consistency was scored as 1 when firm, 2 when soft or of moderate consistency, 3 when runny or mild diarrhea, and 4 when watery and profuse diarrhea. Daily fecal scores were generated for individual calves for statistical analyses. Calves with fecal score >2 were used for analysis of incidence of diarrhea.Fecal consistency scoring was performed daily before the morning milk feeding using a 1 to 4 scale, as described by Magalhaes 61, where barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate 20 μl mixtures containing 4 μl 5×FastPfu Buffer, 2 μl 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl FastPfu Polymerase, and 10 ng template DNA.Microbial DNA was extracted from jejunum digesta samples using the OMEGA E.Z.N.A.® digesta DNA Kit , according to the manufacturer’s protocols. The quality and quantity of the DNA were measured using an ND1000 spectrophotometer . The V3–V4 regions of the bacterial 16S ribosomal RNA gene were amplified by PCR  using primers 338 F 5′-barcode-ACTCCTACGGGAGGCAGCAG-3′ and 806 R 5′-barcode-GGACTACHVGGGTWTCTAAT-3′Amplicons were extracted from 2% agarose gels, purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions, and quantified using the QuantiFluor™ -ST system . Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300 bp) on an Illumina MiSeq PE300 platform  according to the standard protocols.62 following the criteria: (i) the 300-bp reads were truncated at any site receiving an average quality score of < 20 over a 50-bp sliding window, and truncated reads shorter than 50 bp were discarded; (ii) reads containing any mismatch in the barcode region, two or more nucleotide mismatches in the primer sequence, or ambiguous characters were removed; and (iii) only sequences with an overlap of > 10 bp and < 10% mismatches were assembled and reads that could not be assembled were discarded. The assembled sequences were then trimmed of primers and barcodes. Chimeric sequences were identified and removed using usearch63. After quality control, the assembled sequences were assigned to operational taxonomic units (OTUs) at a 97% identity threshold using UPARSE64. Alpha diversity index, including ACE, Chao1, Shannon, and Simpson were calculated by normalizing the number of reads in all samples to 7668 sequences using mothur65. Rarefaction curves were analyzed with mothur and plotted using R. The taxonomy of each 16S rRNA gene sequence was assigned against the SILVA bacteria alignment database66 using RDP classifier67 with a confidence threshold of 70%. Sequences were aligned against the PyNAST68, and a phylogenetic tree was built using FastTree69. The sequencing data obtained in this study were deposited in the NCBI Sequence Read Archive (SRA) under accession numbers SRR5406973 to SRR5406996.After raw FASTQ files demultiplexed, sequences were filtered using TrimmomaticE. coli K99, and four selected bacterial species in the jejunum digesta using the amplification primers shown in Supplementary Table E.coli K99, and each individual bacterial strain selected using universal primers. Real-time PCR was performed in a 20 μl reaction mixture containing 10 μl 2 × SG Green qPCR Mix , 0.5 μl of each primer (10 μM), 0.5 μl 10 ng DNA templates, and 8.5 μl nuclease-free water. Amplification involved one holding cycle at 95 °C for 10 min for initial denaturation and then 40 cycles at 95 °C for 20 s for denaturation followed by annealing at 60 °C for 30 s and extension at 72 °C for 20 s. The copy numbers of the total bacteria, E.coli K99, and four bacterial species per gram of jejunum digesta were then calculated. The relative abundances of four bacterial species were calculated by dividing the gene copy number of each bacterial species by the gene copy number of total bacteria.Absolute quantitative real-time PCR analysis was performed to estimate the copy numbers of the total bacteria, E. coli K99, and the four selected bacterial species were analyzed by one-way ANOVA using SAS . Statistical differences among the means of the treatments were compared using the Duncan’s Multiple Range Test. The relative abundances of communities do not fit normal distribution and arcsine transformation function were performed before analyses. The transformed data of the abundances of communities at the phylum and genus levels were analyzed by one-way ANOVA using SAS. Daily fecal scores were analyzed by ANOVA using the MIXED procedure of SAS, fitting a Poisson distribution50 and square root transformation function with repeated measures for count data. The MIXED procedure model included the fixed effects of treatment and day, interaction between treatment and day, and the random effect of the individual nested within treatment. Treatment differences with P < 0.05 were considered statistically significant, and 0.05 ≤ P < 0.10 was designated as a tendency.The body weight and feed intake of calves, the alpha diversity indices, and the quantification of total bacteria, Supplementary Information


The histological characteristics of the periosteum allow it to possess a remarkable capacity to induce bone growth and remodeling, making it suitable as an in vivo bioreactor strategy for bone graft prefabrication. The present study was designed to prefabricate vascularized bone grafts using pedicled periosteal flaps and decellularized bone matrix (DBM) scaffolds in a rabbit model. The muscular pouches created in the femoral muscle were acted as a control. Our histological results revealed that both the periosteal flap group and muscular pouch group induced bone tissue formation on the DBM surface at both 8 and 16 weeks postoperatively. However, micro-computed tomography (microCT) scanning, biomechanical, and histomorphometric findings indicated that bone grafts from the periosteal flap group showed larger bone mass, faster bone formation rates, higher vascular density, and stronger biomechanical properties than in the muscular pouch group. We suggest that using the pedicled periosteal flap as an in vivo bioreactor is a promising approach for functional bone graft prefabrication.The  Historically, different strategies, including bone transport methods (distraction osteogenesis), implantation of biomaterials , or bone grafting , have been used for bone defect reconstruction, but none of the currently available bone substitutes has all the ideal characteristics3, has seen tremendous advances over the years and gives hope to solving this problem. Strategies of BTE have relied on one of two approaches: in vitro or in vivo tissue engineering. The in vitro BTE strategy attempts to create functional bone grafts by culturing osteogenic cells on bioactive scaffolds in an in vitro environment3. However, when in vitro engineered bone grafts are transferred in vivo, they lack the defined vascular and nerve networks to support cell survival and matrix synthesis and thus must rely on the ingrowth of neo-vascular structures from their surroundings, resulting in limited long-term outcomes in clinical therapeutic studies6. An emerging trend to circumvent these problems follows the in vivo bioreactor principle, which uses the body as a bioreactor to cultivate the traditional triad  and to leverage the self-regenerative capacity of the body to regenerate new bone tissue. We previously used a vessel bundle in combination with the muscularis membrane to act as an in vivo bioreactor and wrap a BMSC-seeded beta tricalcium phosphate (β-TCP) construct in a rabbit model. The results, as demonstrated by new cartilage and bone tissue formation in the β-TCP construct, proved that it is possible to engineer a bone graft following the in vivo bioreactor principle7. To date, the in vivo bioreactor principle concept has been applied in clinical conditions for bone defect reconstruction13. Although limited clinical successes have been reported, this concept provides a promising and translatable approach for bone regeneration.Bone tissue engineering (BTE), which aims to regenerate new biological bone tissue with similar properties and functions as natural occurring bone rather than building new spare partsin vivo bioreactor strategies for regenerating bone tissue, such as the muscular pouch14, arteriovenous bundle (AVB)15, and arteriovenous loop (AVL)16, have been extensively investigated. Due to limited bone regeneration efficiency and clinical transition feasibility, research is ongoing to develop an ideal in vivo bioreactor strategy for clinical application. The periosteum is a thin but highly vascularized and innervated tissue with a bilayer structure, the outer fibrous layer and the inner cambium layer, which consists primarily of skeletal progenitor cells and osteoblasts that possess a remarkable capacity to allow appositional bone growth as well as cortical bone modeling and remodeling17. Considering these findings, surgeons have harnessed this osteogenic capacity to induce bone regeneration for more than two centuries. Tatara AM et al. generated large volumes of mineralized tissue for mandibular reconstruction using the rib periosteum in a sheep model18. However, current periosteum-based strategies are still limited by the lack of a large-sized periosteum donor site and a feasible surgical procedure for clinical translation.Different 19. A typical prefabrication procedure begins with the implantation of a vascular pedicled flap to an avascular receipt territory/construct. After a period of in vivo cultivation, the vascular pedicled flap will revascularize and even regenerate new tissue in the receipt territory/construct, allowing the prefabricated new flap to be transferred to any specified recipient site, and greatly expanding the armamentarium of reconstructive options20. Therefore, we hypothesized that a functional bone graft could be regenerated by using a pedicled periosteal flap as an in vivo bioreactor and a DBM as a bioscaffold. Here, we here report our results in the form of histologic, microCT, and biomechanical review, and we discuss the feasibility of translating this promising bone graft prefabricating strategy into clinical conditions.The prefabrication technique is one of the most exciting areas in plastic surgery because of its bridging role between conventional reconstructive surgery and tissue engineeringAfter approximately 10 days of processing, the decellularized femoral head bones were tailored into cylinders and submitted for scanning electron microscopy (SEM) examination. The SEM images revealed that soft tissue was removed completely, and there was no obvious cellular debris residue on the surface of the DBM scaffold. The cylindrical DBM scaffolds were identified as a porous material (a size range of 200 ~ 400 μm), with a similar microstructure to the native cancellous bone tissue  reconstructive images were harvested from two dimensional (2D) microCT scanning images. Representative preoperative and 16 weeks postoperative images of two DBM scaffolds, which were assigned to the periosteal flap group and the muscular pouch group, respectively, are shown in Fig. in vivo cultivation resulted in significantly increased bone mass in a time-dependent course manner. In the muscular pouch group, the BV increased 1.40-fold at 8 weeks and 1.79-fold at 16 weeks, and the BMC increased 1.94-fold at 8 weeks and 2.73-fold at 16 weeks. In the periosteal flap group, the BV increased 1.62 folds at 8 weeks and 2.56 folds at 16 weeks, and the BMC increased 2.34-fold at 8 weeks and 4.11-fold at 16 weeks. More importantly, the increases of BV, BMC, and BMD were statistically significant in both the muscular pouch group and periosteal flap group , indicating that the in vivo bioreactor strategy induced a considerable amount of new bone formation on the DBM scaffolds. To compare the bone regeneration efficiency of the two in vivo bioreactor strategies, the bone morphological data were analyzed between the two groups. Compared with the muscular pouch group, the periosteal flap group showed superiority in terms of a larger BV  and higher BMC . Furthermore, the bone grafts from the periosteal group showed more Tb.N and higher Tb.Th but smaller Th.Sp. than that from the muscular pouch group at 16 weeks. These data suggest that the periosteal flap induced more mineral tissue formation on the surface of the DBM scaffold.To quantitatively evaluate the increased bone mass, bone morphological parameters, including bone volume (BV), bone mineral content (BMC), bone mineral density (BMD), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), were analyzed. As shown in Fig. p < 0.001) and 1.45-fold larger at 16 weeks (p = 0.026) than that in the muscular pouch  staining images demonstrated the microstructure of the bone trabeculae and the shape of the generated bone grafts Fig. , and theuch Fig. , indicatin vivo cultivation . The MAR was 0.57 μm/day in the muscular pouch group and 1.07 μm/day in the muscular pouch group at 14–15 weeks  and Calcein (CA) were intramuscularly injected into rabbits one and two weeks before bone graft explanation, respectively, and then, confocal laser scanning microscopy (CLSM) was applied to observe sequential fluorochrome-labeling Fig. . As showion Fig. . Howevereks Fig. . These dp = 0.002 at 8 weeks, p = 0.016 at 16 weeks). Quantitative analysis of the blood vessels showed that the blood vessel densities in the muscular pouch group were 2.30 per field at 8 weeks and 6.07 per field at 16 weeks, and the blood vessel densities in the periosteal flap group were 3.87 per field at 8 weeks and 8.62 per field at 16 weeks  staining images, in which the blood vessel walls were identified as blue collagen fibers and red blood cells were stained orange. The bone graft sections from both groups showed a considerable amount of blood vessels Fig. . Howevereks Fig. . These dp = 0.805) and height (p = 0.692) of each sample were examined and showed no significant differences . The periosteal flap strategy considerably enhanced the biomechanical properties of the prefabricated bone grafts over in the muscular pouch strategy.To evaluate the biomechanical characteristics of the prefabricated bone grafts, freshly harvested bone grafts were submitted for comparison with autologous native cancellous (n = 5) and cortical bone samples (n = 5) by Young’s modulus Fig. . Before ces Fig. . Young’sces Fig. . More prin vivo bioreactor to cultivate a functional bone graft is a feasible and promising approach for bone graft prefabrication.The regeneration of vascularized functional bone grafts with minimum donor site morbidity and maximum size for large bone defect reconstruction would be a tremendous advancement for reconstructive surgery. This study offers a proof of concept that a vascularized functional bone graft can be efficiently prefabricated with a pedicled periosteal flap and a DBM scaffold. The pedicled periosteal flap strategy yielded higher osteoinductive and angioinductive properties and considerable enhancement of biomechanical properties of the prefabricated bone grafts compared with the muscular pouch strategy. Thus, the technique using a pedicled periosteal flap as an in vivo bioreactor principle was already proposed since 200522. Following the in vivo bioreactor principle, the in vivo bioreactor is a vascular territory as well as a regenerative niche for vascularization, regeneration, and remodeling of prefabricated bone grafts23. Here, we describe the use of a pedicled periosteal flap as an in vivo bioreactor to regenerate functional bone grafts, in contrast to the use of a muscular pouch or flap, which is one of the most commonly used strategies due to its rich capillary and nerve network25. Theoretically, the most important consideration for an in vivo bioreactor strategy is the tissue type surrounding the bioreactor, which may directly affect the interaction with the implanted construct, recruitment of autologous cells, reestablishment of a functional neurovascular network, and the results of bone graft prefabrication. Compared with the muscle tissue, several significant features of the periosteum support this as an excellent strategy for bone graft prefabrication: a niche for skeletal progenitor/stem cells26; an abundance of molecular signals27; increased nutrient-rich blood supply and acting as a natural barrier membrane to prevent soft-tissue invasion28. Therefore, the rabbit skull periosteal flap pedicled on the supraorbital vessel was chosen as an in vivo bioreactor, and the femoral muscle was chosen as a control strategy. To quantificationally compare osteoinductive capacity of the pedicled periosteal flap strategy and the muscular pouch strategy, bone mass of the same decellularized bone was evaluated by microCT scanning at reoperation and postoperation  scanning was performed to determine DBM microstructure and porosity as previously described39. Finally, the prepared scaffolds were sterilized by gamma-radiation  before use.The DBM scaffolds were derived from eleven donated, human femoral head bones (n = 11 donors) during artificial femoral head replacement, and informed consent was obtained in accordance with the Declaration of Helsinki. DBM scaffolds were prepared as previously reportedn = 30) and the periosteal flap group (n = 30). The rabbits were anesthetized by face mask induction with 3.5% halothane followed by intubation with 3.5% halothane in oxygen. The parietal and femoral fur was shaved, the operative area was sterilized with 10% povidone-iodine, and the animal was placed in a supine position. For the periosteal flap group, a 4-cm vertical incision was made on the top of the skull to expose the skull periosteum. By careful dissection and hemostasis, a pedicled periosteal flap (1.5 × 1.5 cm) based on the supraorbital vessels was elevated using a periosteal detacher. Then, a DBM scaffold was wrapped in this periosteal flap using 5–0 nylon to form a periosteum-DBM construct. The construct was sutured to adjunctive tissue to minimize movement. The incision was closed with 4–0 nylon. For the muscular pouch group, a 3-cm vertical incision was made on the right femur to expose the femoral muscle. By careful dissection and hemostasis, a muscular pouch was made in the femoral muscle, and a DBM scaffold was inserted into the muscular pouch. The muscular pouch was closed with 5–0 nylon, and the incision was closed using 4–0 nylon. Overall, 60 DBM scaffolds were used in 60 rabbits. Each rabbit was administered 20 mg of enrofloxacin intraperitoneally. The animals were monitored daily for postoperative complications.All animal procedures in this study were approved and conducted in accordance with the Laboratory Animal Research Committee of Shanghai Jiao Tong University School of Medicine. In total, 60 New Zealand white rabbits with an average body weight of 2.5 ± 0.4 kg  were randomly allocated to two groups: the muscular pouch group  and ARS S , respectively. Mineralized tissues were observed by means of sequential fluorescent labeling using a CLSM . Excitation/emission wavelengths were as follows: 488/517 nm  and 543/617 nm . The bone formation indices were evaluated using Image-Pro 5.0 software , and the MAR (μm/day) was measured.39. Meanwhile, the other half of the specimens was dehydrated in a graded alcohol series and then embedded in polymethylmethacrylate. After hardening, longitudinal sections were cut into 150–200 μm slices using a microtome (Leica), glued onto a plastic support, and then polished to a final thickness of approximately 50 μm. First, the sections were examined for fluorescent labeling. Then, the sections were stained with VG picrofuchsin to evaluate new bone formation. The area of new bone formation was quantitatively evaluated in four random sections using Image-Pro 5.0 software (Media Cybernetics).The bone specimens harvested at 8 and 16 weeks postoperatively were used for histological and histomorphometric analyses. Half of the specimens were fixed in 4% paraformaldehyde for 2 days. Then, the specimens were decalcified in 20% ethylenediaminetetraacetic acid for 30 days and dehydrated, embedded in paraffin, and sectioned. Sections were stained with HE and AM for the evaluation of bone structures and blood vessels as previously describedAll bone graft specimens were imaged and analyzed with a high-resolution microCT  with 80 kV source voltage, 100 mA source current, 35 μm pixel size, 180° rotation, 0.2 seconds exposition time, frame averaging 20, and aluminum-copper filters. On average, 1260 slides per sample were reconstructed using NRecon software . To compare the changes of morphological parameter data, the DBM scaffolds were scanned preoperatively and postoperatively. To measure newly formed bone, a circular area of pre-defined size was selected as the region of interest (ROI) in the two-dimensional images. The pixel zone representing ossification in the defined ROI was then reconstructed in 3D by creating a volume of interest in the lower and upper ranges of the threshold using grayscale units. After using CTAn 1.8 on each reconstructed BMP file, BV, BMD, Tb.Th, Tb.N, and Tb.Sp were obtained using a CT-analyzer in direct 3D based on a surface-rendered volume model. In addition, total BMC was calculated by multiplying BV and BMD.39.Four months after bone graft prefabrication, compression tests were performed on six prefabricated bone grafts from the periosteal flap group, six prefabricated bone grafts from the muscular pouch group, eight native cancellous bone samples from the iliac crest, and eight native cortical bone samples from the radius to evaluate the biomechanical properties. These samples were chosen as controls because of their similar cross-sectional area and the fact that both bone qualities  were used for bone defect reconstruction. Prior to mechanical testing, the test samples were cut into cylinders with the same diameter as the prefabricated bone grafts (4 mm) and a height of 8 mm. Then, the cross-sectional area and height of the test samples were measured using Image-Pro 5.0 software (Media Cybernetics). The samples were tested under uniaxial compression using a biomechanical analyzer  according to a previously described protocolP values < 0.05 were considered statistically significant. IBM SPSS Statistics V20 was used for statistical analyses.All the above data were analyzed as the means ± standard deviations. One-way analysis of variance (ANOVA) and Tukey’s post-test multiple comparison tests were used to determine significance between experimental groups. Student’s t test was used to determine significance between two groups.