Upon calving, all cows were treated similarly with access to cooling for their entire lactation. Six cows per treatment were randomly selected for mammary gland biopsies at 14, 42, and 84 days in milk. Tissues were sectioned and stained for histological analysis. During lactation, HT cows produced 4 kg less colostrum and 3.7 kg less milk compared with CL cows. Lactating mammary gland microstructure was impacted after exposure to dry period heat stress; HT cows had fewer alveoli and a higher proportion of connective tissue in the mammary gland relative to CL cows, however alveolar area was similar between treatments. Rates of mammary epithelial cell proliferation and apoptosis were similar between treatment groups. This suggests that heat stress exposure during the dry period leads to reductions in milk yield that could be caused, in part, by a reduction in alveoli number in the lactating mammary gland but not to dynamic alterations in cellular turnover once lactation is established.The bovine dry period is a non-lactating period between consecutive lactations characterized by mammary gland involution and redevelopment phases to replace senescent mammary epithelial cells with active cells primed for the next lactation. Dairy cows exposed to heat stress during the dry period experience milk yield reductions between 3–7.5 kg/d in the next lactation, partially attributed to processes associated with mammary cell growth and turnover during the dry period. However, the carry-over impact of dry period heat stress on mammary morphology during lactation has yet to be determined. In the current study, we hypothesized that exposure to heat stress during the dry period would alter alveolar microstructure and cellular turnover (i.e. proliferation and apoptosis) during lactation. Cows were either subjected to heat stress (HT, access to shade;  THI) of 68, forcing the cow to promote heat loss through sweating and panting and reduce heat production by limiting feed intake and decreasing milk production [Heat stress is a costly and wide-spread concern to the dairy industry. Physiologically, heat stress is induced when a dairy cow is pushed above her thermoneutral zone at a temperature-humidity index (oduction –3. Risinoduction –7.MEC) turnover, where senescent epithelial cells are replaced with fully-functional cells in preparation for the next lactation [Even when not directly exposed to heat stress during lactation, a dairy cow can experience negative consequences when heat stress occurs during the dry period. We have previously shown that cows exposed to heat stress during the dry period experience decreases in milk production by 3 to 7.5 kg/d and increased incidences of health disorders in the next lactation –11. Indeactation . Once laactation .In vitro heat shock in connective and epithelial tissue results in a number of morphological alterations including structure and function of the plasma membrane, disruption of the Golgi complex, mitochondrial swelling, and alterations in cytoskeletal elements [Heat stress impacts cellular morphology and processes such as apoptosis and proliferation in the mammary gland. elements ,17. Specelements –23. In aelements . Dry perelements ,25.While we understand some direct implications of heat stress on dry period mammary development in the dam and the long-lasting repercussions in the offspring, less is known about the carry-over effect of dry period heat stress on mammary morphology in the dam’s upcoming lactation. Thus, the objective of this study was to determine the impact of dry period heat stress on dairy cow mammary microstructure and cellular processes as they enter the next lactation. We hypothesized that dry period heat stress would alter alveolar architecture and impair MEC proliferation and apoptosis during lactation, thus reducing capacity for milk synthesis and storage which may further explain observed reductions in milk yield.HT; n = 12) or cooled . Mature-equivalent milk production in the previous lactation and parity were similar between treatments. For the entire dry period, both groups were housed in a shaded, sand-bedded free-stall barn; the CL group had access to water soakers and fans whereas the HT group did not. Fans ran continuously whereas water soakers were activated for 1.5 min in 6 min intervals when ambient temperature exceeded 21.1°C. Ambient temperature and relative humidity of the barn were recorded every 15 min for the entire dry period with Hobo Pro series Temp probes . The temperature humidity index (THI) was calculated for the HT and CL areas of the barn based on the equation by Dikmen et al. [TMR), formulated to the National Research Council recommendations . Daily dry matter intake (DMI) of individual cows was measured from dry-off to calving and from calving up to 42 days in milk (DIM) using a Calan gate system . After calving, all cows were treated similarly, housed in shaded, free-stall barns with water soakers and fans and fed lactating cow TMR . Cows were milked two times daily according to Dairy Unit standard operating procedure.This study was approved by the Institutional Animal Care and Use Committee at the University of Florida and was conducted from May to December 2016 at the University of Florida Dairy Unit  using a herd of multiparous Holstein cows. Cows were dried off approximately 46 days before expected calving date according to UF Dairy Unit standard operating procedures and randomly assigned to one of two treatment groups for the duration of the dry period; heat stressed  from birth to 84 or 280 DIM (for weight and milk yield respectively). Gestation length was calculated by subtracting calving date from the artificial insemination date. Calf birth weight was recorded prior to first colostrum feeding.n = 6 per treatment). Biopsies were collected according to the procedures from Farr et al. with slight modifications [Mammary biopsies were collected on 14, 42, and 84 DIM for a randomly selected subset of cows  and all incubations occurred at room temperature (~ 25°C). Sections were incubated with H2O2 for 15 min, washed, and incubated with UltraV block for 5 min. Slides were incubated for 30 min with primary antibody  previously diluted 1:100 in 1x TBSt. Upon washing, slides were incubated with Ultravision One HRP polymer for 30 min, washed again, then incubated with diluted DAB Plus chromogen for 5 min. Sections were washed with ddH2O and counterstained with hematoxylin for 1 min. Sections were dehydrated in an ethanol and xylene wash series and mounted with Permount . Positive and negative controls were established using calf jejunum (high proliferative rates) and bovine mammary tissue at 14 DIM without primary antibody incubation, respectively.The Ki67 assay was performed to detect the number of proliferating cells  and non-proliferating cells  in a section of mammary tissue. The protocol for Ki67 staining was previously described in a companion study  using thin situ Apoptosis Kit  according to manufacturer’s instructions. Tissues were deparaffinized and rehydrated, and antigen retrieval was performed by incubating slides in 20 μg/ml proteinase K  for 8 min. Unless otherwise stated, incubations occurred at room temperature. Slides were washed in ddH2O then quenched in 3% H2O2 for 10 min. Slides were incubated in equilibration buffer for 10 min then incubated in a 1:3 working dilution of TdT enzyme in reaction buffer for 60 min at 37°C. Sections were washed in stop/wash buffer and 1x PBS followed by antibody incubation of anti-digoxigenin-peroxidase for 30 min and a DAB incubation for 8 min. Counterstaining was applied using methyl green stain , then slides were washed 3x. The sections were dehydrated in butanol and xylene washes and mounted with Permount . Positive and negative controls were rat mammary tissue at 3–5 d involution (high apoptotic rates) and a section of bovine mammary tissue at 14 DIM incubated with TdT enzyme diluted in stop/wash solution instead of reaction buffer, respectively.The TUNEL assay was conducted to quantify the number of cells undergoing apoptosis  and those not undergoing apoptosis  in a section of mammary tissue. The procedure followed was previously described in a companion paper  Briefly,2 and at 40x is 96,820 μm2. Alveolar area was traced using the freehand tool, whereas connective tissue area was quantified by threshold measurement. Briefly, Masson’s trichrome images were converted to RGB stack color (black and white) and set at threshold of 55 for all sections. This converted connective tissue stain to a red color and all other tissue stain to white. The percentage area was calculated as the area of the red color as a proportion of the total image area.The EVOS XL Core imaging system  was used to capture images of H&E, Masson’s trichrome, Ki67, and TUNEL histological sections. ImageJ software was used for tissue quantification. The ImageJ Point Picker plugin was utilized to count alveoli number in H&E stained sections and the number of positive and negative cells in Ki67 and TUNEL sections. Area measurements for individual alveolar area (H&E) and connective tissue area (Masson’s trichrome) were calibrated to the image size and magnification (i.e. pixels to μm). Total tissue area for photomicrographs taken at 20x is 574,497 μmFor H&E stained sections, three photomicrographs were captured per section with a 10X objective for quantification of alveolar number and a 20X objective for alveoli area. The 10X magnification was chosen for alveolar number to ensure alveoli presence in each field. All photomicrographs captured were selected from random fields of view, taking care to avoid tissue edges. Alveoli were defined as structures with a lumen surrounded by a single layer of MEC, and alveolar area was quantified by measuring an individual alveoli’s luminal area excluding the surrounding MEC. Mammary ducts were not counted or measured. Care was taken to avoid all intralobular ducts and apparent interlobular ducts. Alveoli number and area were averaged across the three photomicrographs per section for each cow at each timepoint for statistical analysis.Sections from Masson’s trichrome staining were taken at 20X magnification with three randomly captured photomicrographs per section to measure connective tissue area. Connective tissue was defined as tissue stained blue, whereas all other remaining tissue  was stained pink or dark purple as a covariate. The remaining tissue area was not calculated. Connective tissue area was averaged across the three photomicrographs per section for each cow at each timepoint for statistical analysis.Histological sections from Ki67 and TUNEL assays were captured with three photomicrographs taken randomly per section at a 40X objective. In each capture, every cell was counted and defined as either positive (stained brown) or negative (stained blue). Cells were classified as either MEC or stromal cell. Mammary epithelial cells encompassed alveolar cells, ductal epithelial cells, and myoepithelial cells, whereas stromal cells included cells in the surrounding epithelium such as fibroblasts, immune cells, adipocytes, endothelial cells, and red blood cells. Results are expressed as proportion of positive cells per section. To calculate the proportion of cells either proliferating (Ki67) or undergoing apoptosis (TUNEL), the total number of positive cells per section were divided by the total number of cells per section (positive and negative) for each cell type .P ≤ 0.05 and tendency was declared at 0.05 < P ≤ 0.10. Data are presented as least squares means (LSM) ± standard error (SE) unless otherwise stated. The minimal data set can be found in Statistical analysis was conducted in SAS v. 9.4  with all data tested for normality. PROC GLM was used for one-way ANOVA analysis of colostrum yield, gestation length, and calf birth weight with calf sex as a covariate and a sex by treatment interaction was included for calf weight. Cell proliferation or apoptosis was analyzed by generalized linear mixed models using PROC GLIMMIX with a binomial distribution and logit link function. Fixed effects included treatment, DIM, and their interaction, and cow ID within treatment as random effect. Correlation between alveoli number and MEC number was assessed using Pearson’s correlation (PROC CORR). All other data were analyzed by generalized linear mixed models using PROC MIXED with fixed effects of treatment, DIM (repeated measures), and their interaction. The first-order autoregressive covariance structure (AR(1)) was used as the covariate structure. Significance was declared at P < 0.01) and rectal temperature in the morning  and afternoon  were higher for the HT cows relative to CL cows across the duration of the dry period, indicating abatement of heat stress in the cooled group during the dry period was successful (P = 0.02). Dry matter intake was not different between treatments during the dry period , yet respiration rate , but HT cows tended to weigh less over 84 DIM  = 0.06, P(INT) = 0.20). After calving, when both groups of cows were provided with active cooling, rectal temperature, respiration rates, and DMI were similar between treatment groups and averaged 38.71° C, 58 bpm, and 17.4 kg/d . There was an interaction between DIM and treatment on respiration rate for the lactating cows (P = 0.02); whereby respiration rate was significantly higher at 7 DIM in HT cows but was higher in CL cows at 28 DIM.Cows exposed to heat stress during the dry period had a shorter gestation length and gave birth to lower birthweight calves than did cows provided with active cooling during the dry period (P < 0.01). Milk yield changed over the course of lactation for both groups (P < 0.01), but there was no interaction between DIM and treatment .Cows that were cooled during the dry period produced 4 kg more colostrum and 3.7 kg/day more milk than HT cows across 280 DIM  across 84 DIM, but individual alveoli area was similar between treatment groups . There were no significant interactions between DIM and treatment for alveoli area and number (P > 0.59). Alveoli number was positively correlated with the number of MEC . Cows heat-stressed during the dry period had a significantly higher proportion of mammary connective tissue during lactation relative to cows cooled during the dry period . There was also a DIM effect whereby mammary tissue at 14 DIM had a greater percentage of connective tissue compared with 42 and 84 DIM . No interaction between treatment and DIM was observed (P = 0.63).There were fewer alveoli in the mammary gland of HT cows compared to CL cows  or proportion of MEC or stromal proliferating cells (P > 0.62) during lactation at 14, 42, or 84 DIM. There was a significant DIM effect where the percent of proliferating MEC and total cells  was decreased at 14 DIM compared to 84 DIM . Exposure to heat-stress during the dry period did not impact the proportion of total apoptotic cells  or proportion of MEC or stromal apoptotic cells (P > 0.18) during lactation at 14, 42, or 84 DIM. There were no treatment by time interactions for cell proliferation or apoptosis (P > 0.35).There was no difference between CL and HT dry period treatments in proportion of total proliferating cells  milk yield and milk components, (b) vitals  and growth, (c) mammary gland H&E, and Masons trichrome, (d) mammary gland proliferation and apoptosis (Ki67 and TUNEL).(XLSX)Click here for additional data file.


Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Detecting and enumerating circulating tumor cells (CTCs) in patients with colorectal cancer emerged as an important prognostic tool which provides a direct estimate of metastatic potential. Improving the turnaround time and decreasing sample volume is critical for incorporating this liquid biopsy tool into routine practice. The objective of the current study was to validate the clinical feasibility of a self-assembled cell array (SACA) chip, a CTC counting platform with less than 4 h turnaround time, in patients with newly diagnosed colorectal cancers. In total, 179 patients with newly diagnosed colorectal cancers from a single institute were enrolled. Epithelial cell adhesion molecule positive (EpCAM(+)), cluster of differentiation 45 negative (CD45(−)) cells were isolated and enumerated from 2 mL of peripheral vein blood (PB) and inferior mesenteric vein blood (IMV) samples obtained during surgery. We found that the CTC count in PB but not IMV correlates with disease stages. Neoadjuvant chemotherapy did not lead to decreased CTC count in both types of blood samples. With cutoffs of four CTCs per 2 mL of blood, and serum carcinoembryonic antigen (CEA) level of 5 ng/mL, patients with non-metastatic disease were more likely to experience recurrence if they had high PB CTC count and high serum CEA concentration . Our study demonstrates the feasibility of enumerating CTCs with a SACA chip in patients with colorectal cancer. Metastasis is the leading cause of cancer morbidity and mortality . CirculaColorectal cancer (CRC) is one of the leading causes of death in the world ,14,15. I9 blood cells) [5 cells [. In this study, we designed an observational clinical research to evaluate the feasibility of enumerating CTC with SACA from whole-blood samples of patients with CRC.CTC enumeration is one of the possible solutions for early detection of primary or recurrent CRC, since CTC can be isolated from the blood of patients with colorectal cancer . Howeverd cells) ,22, and d cells) ,25,26,27d cells) ,29. Meand cells) , we coulIn order to detect rare cells in a small amount of biological fluid samples, SACA chips are designed for a seamless microfluidic process to enrich and immunolabel CTC from whole blood. The SACA chip used in this experiment can detect one target cell spiked into 10,000,000 white blood cells WBCs; . In the In total, 132 patients were prospectively enrolled in the study between May 2015 and August 2018 . All patTwo milliliters of PB with or without IMV samples were collected from each patient included in this study. CTC detection and enumeration were carried out through the SACA chip system . CTCs weWith the system established, we were able to identify CTCs from 132 prospectively collected clinical samples. Buffy coat from 2 mL of whole blood was loaded onto the SACA chip and was allowed to form a tightly packed monolayer. The whole preparation was stained and imaged for quantification of CTCs. Representative images from five cases are shown in The CTC counts and their correlation to neoadjuvant chemotherapy and disease stages are analyzed in In addition, we found that the mean of CTC count in the PB NAC(−) group increased with disease stage  4.0 ±1.7 to 6.3 ± 1.8, p = 0.00081, OR = 4.00, p = 0.00046, OR = 2.68, p = 0.00085, OR = 7.16, n = 19), the recurrence rate was 50% for patients with CTC > 4, in contrast to 0% recurrence rate for CTC ≤ 4  for these biomarkers. Disease recurrence was defined as local recurrence, distal metastasis, or death at the time of last clinical contact. For CEA, the cutoff value was set to 5 ng/mL based on the clinical laboratory reference. For PB CTC, the cutoff value was set to four cells per 2 mL of blood, which was the median in this cohort. In non-metastatic patients (stage 0–III), the recurrence rate was 10% for patients with CTC > 4, in contrast to a 3% recurrence rate in patients with CTC ≤ 4  in non-metastatic patients were plotted. Subjects without these events at the time of their last visit were censored on that day. Cutoffs were set to four CTCs/2 mL of blood and 5 ng/mL CEA as described in the previous section. There was no statistical significance between PFS of patients with CTC > 4 and in patients with CTC ≤ 4 A. StratiIn this study, we showed that the SACA chip could be reliably used to enrich rare cancer cells . This deThe serum CEA level C showed The prospective trial was conducted at the General Clinical Center for colorectal surgery in Taipei, Taiwan. The objective was to evaluate the consistency of CTC number and to analyze the corresponding changes of different clinical stages via images and CTC counts. From May 2015 to August 2018, 179 subjects were enrolled with a median follow-up of 159 days. The protocol was approved by the Institutional Review Board of Taipei Veterans General Hospital and the research was registered in clinical trials . All patients agreed and signed their informed consent.®, Plymouth, UK) at room temperature and were processed within 24 h of collection. The isolation procedure began with the Leucosep TM tube  to remove red blood cells from whole blood. In this step, red blood cells were removed through Ficoll–Paque PLUS  by concentration gradient. The mononucleated cells containing CTC ) after centrifugation were collected, and the CTC cell retention rate was about 75–85% [PB was collected before the treatment began (baseline) and IMV was collected during surgery in order to conduct CTC evaluation. Samples were kept in plastic blood collection tubes with K2EDTA  staining at 37 °C for 30 min, and then EpCAM-FITC  and CD45-pecy7  antibody staining at 37 °C for 30 min. Hoechst staining is part of the family of blue fluorescent dyes used to stain cell nucleus DNA. CD45 is a 180–240-kDa glycoprotein, also known as leukocyte common antigen (LCA), which recognizes white blood cells in PBMC, and almost all cancers are expressed as EpCAM antigens; thus, EpCAM epithelial differentiation antibodies were used to identify CTCs, while FITC is a fluorescent dye with a molecular weight of 389 kDa. The SACA chip system was used in CTC enumeration the repeatability, reproducibility, accuracy, and technical details of the SACA chip were described previously .Fluorescence imaging was carried out using a normal vertical optical microscope and fluorescence microscope ((Olympus IX71) Olympus Taiwan Co., Ltd., Taichung, Taiwan). The exposure time for each sample was kept at 0.1 s for each appropriate wavelength for different dyes.®, CCL-2™, Building Construction Resource Center, Inc., Hsinchu, Taiwan)) involves human colon cancer cells, and they were injected into the white blood cells in order to simulate CTC in patients’ blood. HCT116 cell lines was cultured in a medium containing Roswell Park Memorial Institute (RPMI-1640) , 10% fetal bovine serum (FBS) , 1% penicillin/streptomycin (Pen-Strep)  at 37 °C and 95% air and 5% CO2.The HCT116 cell line  (GIBCO) was used to dissociate the link between the monolayer and the culture dish before spiking-in whole-blood samples.p < 0.05 (both sides). All statistical analyses were performed using SPSS version 12.0  and GraphPad Prism 6  for Windows. Progression-free survival (PFS) referred to the time between the day of blood sampling and the day of either local recurrence or distal metastasis or death. Subjects without these events at the time of their last visit were censored on that day.The values are expressed as means ± SEM. The significance level was set at Metastasis is a major cause of death from various solid tumors, and it is often associated with poor prognosis. CTC isolation and characterization emerged as an exciting field of study and provided us with unprecedented opportunities to detect early micrometastases for radical interventions. Our study successfully demonstrates the feasibility of SACA, a novel CTC enumerating platform, to obtain clinically meaningful information for CRC patients. We also provide evidence that combining CTC count from SACA and CEA levels is a strong risk stratification tool. Since SACA is a quick platform for reliable CTC detection, its potential to assist in clinical liquid biopsy in all spectra of CRC management warrants further investigation.


To date, epidemiological studies at the index site of the 2013–16 west African Ebola outbreak in Meliandou, Guinea, have been restricted in their scope. We aimed to determine the occurrence of previously undocumented Ebola virus disease (EVD) cases and infections, and to reconstruct transmission events.This cross-sectional seroprevalence survey of the adult population of Meliandou used a highly specific oral fluid test and detailed interviews of all households in the village and key informants. Each household was interviewed, with all members prompted to describe the events of the outbreak, any illness within the household, and possible contact with suspected cases. Information for deceased individuals was provided by relatives living in the same household. Symptoms were based on Ebola virus Makona variant EVD case definitions . For antibody testing, we used an Ebola virus glycoprotein IgG capture enzyme immunoassay developed from a previously validated assay. A maximum exposure level was assigned to every participant using a predetermined scale. We used a generalised linear model (logit function) to estimate odds ratios for the association of sociodemographic variables and exposure level with Ebola virus infection. We adjusted estimates for age and maximum exposure, as appropriate.Between June 22, and July 9, 2017, we enrolled 237 participants from 27 households in Meliandou. Two households refused to participate and one was absent. All adults in participating households who were present for the interview provided an oral fluid swab for testing, of which 224 were suitable for analysis. In addition to the 11 EVD deaths described previously, on the basis of clinical description and oral fluid testing, we found two probable EVD deaths and eight previously unrecognised anti-Ebola virus IgG-positive survivors, including one who had mild symptoms and one who was asymptomatic, resulting in a case fatality of 55·6% (95% CI 30·8–78·5) for adults. Health-care work  and level of exposure  were independent risk factors for infection.vs 11 cases). We show the first serological evidence of survivors in this population (eight anti-Ebola virus IgG seropositive) and report a case fatality lower than previously reported (55·6% vs 100% in adults). These data show the high community coverage achievable by using a non-invasive test and, by accurately documenting the beginnings of the west African Ebola virus outbreak, reveal important insight into transmission dynamics and risk factors that underpin Ebola virus spillover events.Ebola virus infection was more widespread in this spillover population than previously recognised (21 US Food and Drug Administration, Wellcome Trust, and German Research Council. We searched PubMed and Web of Science for articles published between Dec 1, 2013, and Dec 31, 2018, using the keywords “Ebola” AND “Meliandou” OR “Guinea”. We selected articles collecting data or describing events at the index site of the outbreak. No language restrictions were used. Despite extensive diagnostic, molecular, and phylogenetic research into the transmission of Ebola virus during the 2013–16 west African outbreak, no quantitative study or diagnostic methods have been employed at the suspected index location of Meliandou village in Guéckédou prefecture of south-eastern Guinea. Previous studies in this area, conducted during the outbreak, were necessarily limited and did not include serological investigation. Past studies on Ebola virus spillover events in other locations have relied on immunoassays with questionable specificity. During the west African outbreak, a new high specificity capture assay capable of detecting anti-Ebola virus immunoglobulins from oral fluid samples demonstrated the occurrence of mild and asymptomatic infections, and facilitated non-invasive serological studies with high population coverage.Added value of the studyThis study provides the first evidence of Ebola virus-infected survivors from the index site of the west African outbreak. A thorough retrospective epidemiological study done concomitantly among the resident adult population also greatly expands our understanding of the initiating events including transmission dynamics, probable transmission chains, lower case fatality rates, and the presence of both mild and asymptomatic cases.Implications of all the available evidenceOur conclusions highlight the importance and potential of deploying appropriate quantitative serological tools. The acceptability and specificity of this approach alongside careful epidemiological investigation provide comprehensive understanding of transmission dynamics in Meliandou. These data thoroughly characterise the initiating events of an Ebola virus outbreak and show that it spread further within this community than previously appreciated. They also show that it is possible to access a large proportion of a deeply affected community by building up trust and using an acceptable and non-invasive approach.Although severe clinical manifestations dominate during human EVD outbreaks, both minimally symptomatic and asymptomatic infections occur, particularly among contacts of cases.The true incidence of subclinical infections and their contribution to transmission dynamics are not fully understood.The Ebola virus Makona variant (Zaire species), which emerged from Guinea in 2013, caused the largest recorded outbreak of any Ebola virus species in humans (28 625 cases and 11 325 deaths notified)22We sought to enrol all family members residing in Meliandou who were aged 18 years or older at the time of the study. Meliandou is a rural village of 30 households living in 75 buildings, all belonging to the Kissi ethno-linguistic group. The village is encircled by a 100–200 m perimeter of forest and is about 12 km by uneven road from the nearest major urban centre (Guéckédou). All residents were checked by village leaders and local health-care workers before registration to confirm that they had been resident during the period of the outbreak in this area . Information on EVD exposure and symptoms was collected retrospectively. Interviews were done for all households on the basis of qualitative and quantitative approaches used in Sierra Leone, as described previously.Permission for the study was granted by the Guinea Comité National D'Ethique Pour La Recherche en Santé, the ethics committee of the London School of Hygiene & Tropical Medicine, and the UK National Health Service National Research Ethics Service. All participants gave written informed consent before interviewing and sample collection.Participants were initially denoted as being possible cases on the basis of symptoms reported during interviews . PossiblAfter each group interview, all adult participants provided an oral fluid swab using Oracol Plus collection devices . Swabbing was demonstrated by field staff and participants were directly observed firmly rubbing the sponge tip along the upper and lower gums for 90 s. Swabs were sealed and placed on ice in a cool box. The same day, swabs were centrifuged at 1500 g for 10 min to extract oral fluid and stored immediately at −20°C. Swabs were transported to Conakry, Guinea, at a maximum temperature of −15°C and shipped to the UK on dry ice for analysis at Porton Down, UK. Positive controls of oral fluid samples were provided by two PCR-confirmed survivors of Ebola virus infection (based in Guéckédou town) with persistent neutralising anti-Ebola virus IgG titres recorded as part of survivor studies. Serum samples were also acquired from the positive controls, one local negative control, and two suspected EVD survivors from Meliandou. Four UK-based volunteers also provided negative control samples.We detected human anti-Ebola virus (Zaire) IgG using an enzyme-linked immunosorbent anti-Ebola virus glycoprotein IgG capture assay  that was developed as a commercially available assay from the validated assay described previously.Epidemiological data were double-entered into EpiData  and exported into RStudio (version 1.0.13) for analysis. We used a generalised linear model (logit link function) to estimate odds ratios for the association of sociodemographic variables and exposure level with Ebola virus infection (defined as anti-Ebola virus IgG seropositive or died of suspected EVD during the outbreak period). We calculated p values via likelihood ratio tests . We adjusted estimates for age and maximum exposure, and assessed model assumptions for violations. For additional capture assay analysis to assess the association between sample volume or precipitate and normalised optical density we used unadjusted linear regression and Kruskall-Wallis non-parametric methods.The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Between June 22, and July 9, 2017, we enrolled 237 participants from 27 households in Meliandou. One household was absent and two refused. 237 adults (≥18 years) were interviewed and gave swabs, of whom 117 (49·4%) were men and 120 were women (50·6%), with a mean age of 29·8 years. 38 adults from participating families were absent for survey activities. The most common occupations were subsistence farmers (40·4%), housewives (19·8%), or students or unemployed (23·5%).32 possible cases were identified via the interviews on the basis of reported symptoms. Of these, 13 people had died and ten were confirmed by key informants to have had EVD-like symptoms at the time, resulting in 23 suspected cases (S1–23). The remaining possible cases with reported symptoms were not confirmed by the key informants (pre-fixed U01–09). Of the 23 suspected cases, three deaths were in children and one survivor with confirmed symptoms was younger than 18 years so no oral fluid sample was collected.After we checked sample integrity and the volume extracted after centrifugation, 224 of 237 oral fluid samples were available for testing. During assay optimisation, the magnitude of normalised optical density responses in the positive control oral fluid samples  fell below a priori defined cutoffs for seroconversion .7 An altEight of 224 oral fluid samples were seropositive  including six of the nine suspected cases in adults , table 1Among unconfirmed cases (U01–09), the median normalised optical density of oral fluid was 0·68 (95% CI 0·67–0·70), compared with 0·71 (0·65–0·94) among all other oral fluid samples. Three suspected cases in which symptoms were confirmed  remained under the seropositive cutoff with normalised optical density values of 1·0, 0·73, and 0·64, respectively.By use of interviews and a population of cases defined by immunoassay results, including eight anti-Ebola virus IgG positive survivors, all seronegative patients or suspected cases that were not tested, and 13 suspected EVD deaths (including the three children who died during the outbreak), we generated a transmission chain that affected ten households .Figure 2The index case arose in December, 2013, in a boy aged 2 years (S1) whose first contact with Ebola virus reservoirs was possibly insectivorous bats.While in hospital, S4 was cared for by several family members (S7–S10) and her body was returned to Meliandou for burial. The next patients, S5 and S6, were the first cases in individuals residing outside the home of the index patient. Both were buried in Meliandou within 2 days of each other. S6's son  later died with EVD-like symptoms. The funerals of S4–S6 are believed to have spread the infection to other local villages.The next cases arose in the family of those caring for S4 during her stay in hospital and burial (S7–S10). S7, who also had high exposure to S3 and lived in the index household, was cared for in the home of a friend (S13) while symptomatic. S7 died and was buried in Meliandou. The asymptomatic seropositive patient (A01) attended the funeral of S7 but was not involved in the preparations of the body. The other family members who cared for S4 in hospital (S8–S10) all subsequently developed EVD symptoms. S8 and S10 returned to their home village (Dawa), where they were buried. S9 travelled to Conakry, where he developed symptoms and died.Although S11 and S12, who were health-care volunteers, were not previously reported as EVD cases, both developed EVD-like symptoms . S11's son (S19) cared for her and developed unreported EVD-like symptoms; he subsequently left Meliandou to seek traditional remedies and survived. During the same week, S13 developed symptoms after caring for S7, including contact with body fluids, but survived without admission to hospital.The local health-care worker (S18) developed EVD-like symptoms, including fever, vomiting, diarrhoea, red eyes, and blurry vision, but survived. He was involved in the care of all affected patients and in several burials. S18 was cared for exclusively by his wife (S23), who was incapacitated with EVD-like symptoms but survived.The last two suspected EVD deaths in Meliandou (S21 and S22) had safe burials by health authorities and external non-governmental organisations . S21 was probably exposed to Ebola virus outside Meliandou, while preparing for burial the body of a patient with suspected EVD in another village. Patient U01, who reported mild nausea, vomiting, and diarrhoea, cared for S21 and participated in her funeral. S22, who was the last individual to die, had several possible routes of infection: providing care in the same compound as S6 and S17, interacting with her symptomatic brother (S19), and assisting in the funeral of S7.None of the first ten patients (S1–10) survived, after which only three patients died and eight survived , 92 reported high-level exposure to suspected cases , with a further 45 sharing residence or providing care for a symptomatic patient without direct contact level 3; . All EVDIn the univariable analysis, Ebola virus infection was most common among women, with increasing age, and in those with a formal or informal health-care responsibility in the community . After avs 11) and the identification of eight seropositive survivors. Although initial zoonotic and human-to-human transmission within the index household have already been documented,This cross-sectional seroprevalence survey used detailed investigation and non-invasive immunological tools to document Ebola virus transmission at the index site of the largest recorded outbreak of human EVD. Although an outbreak investigation was done in 2014, no diagnostic or immunological methods have been previously used in Meliandou.Identifying survivors enhances our understanding of the transmission dynamics of Ebola virus during this spillover event. After the initial cases in the index family, the virus propagated across numerous households, stemming from a series of high-level exposures to cases in the absence of any preventive interventions. Both traditional funeral practices and contact with symptomatic patients or their body fluids were important in the dissemination of Ebola virus infection, as was also reported in rural areas during the course of the wider outbreak.We found evidence of asymptomatic and minimally symptomatic cases of Ebola virus infection in Meliandou, adding to the evidence on the prevalence of these disease states during the west Africa outbreak.The case fatality  in Meliandou was lower than previously reported (100%).The use of a non-invasive immunoassay based on oral fluid to detect anti-Ebola virus IgG was acceptable to the community and ensured high participation. Although the assay was previously validated in a comparable setting,This study has important limitations. Owing to the time that has elapsed since the outbreak, recall bias is likely, and is only partly offset by using group interviews and key informants. Serology was restricted to adults (aged ≥18 years), so children who had mild or no symptoms might have been missed, which underestimates these disease states.Our findings from Meliandou provide important documentation of the initiating events of the 2013–16 outbreak of Ebola virus in west Africa. Future research into Ebola virus and other emerging diseases will benefit from the use of acceptable non-invasive sampling to further our knowledge of mild and asymptomatic infection and transmission among populations at risk of Ebola virus spillover events. Such information can improve our understanding of the natural history of Ebola virus and contribute to establishing appropriate and sustainable surveillance systems to prevent communities like Meliandou from suffering the long-term effects of Ebola virus and related outbreaks.


Aristeus antennatus is a demersal marine species harvested by bottom trawling in the Mediterranean Sea, the adjacent Atlantic Ocean (AO) waters, and the Mozambique Channel in the Indian Ocean (IO). As it is considered to be a priority species for sustainable fishing, identification of its genetic stocks and the connectivity between them is essential. Using 12 microsatellite loci we detected at least four genetic stocks distributed in the Western Mediterranean (WM), Eastern Mediterranean (EM), AO, and IO and signals for a possible fifth stock in the Alborán Sea. We detected no additional population structuring within the WM. Thus, although the Almería-Orán Front exerts some isolating effect, high genetic homogeneity and gene flow are present within the WM Basin. The IO stock is genetically closer to the AO stock than to the others; thus, the species dispersion route is more likely via the Atlantic Ocean than via the Red Sea. Large effective population sizes suggest population sustainability, but moderate genetic diversity values indicate to proceed with caution. Our genetic results serve as a basis for species conservation to ensure long-term sustainability of this marine resource.The blue and red shrimp  Aristeus antennatus   is a demersal marine decapod that inhabits the muddy bottoms of the continental slope along submarine canyons1. The species is distributed throughout the Mediterranean Sea, in the Atlantic Ocean (AO) waters adjacent to the Strait of Gibraltar from Portugal to Cape Verde, and in the western Indian Ocean (IO) from South Africa across the Mozambique Channel, Zanzibar, and the Maldives Islands; more recently, its presence off the coast of Brazil has been suggested2. Biological studies indicate that this species is the most eurybathic in the Mediterranean Sea, present at depths ranging from 80 to 3,300 m, with a greater abundance in the first 1,000 m depth, where fishing occurs3.The blue and red shrimp A. antennatus is among the most important demersal resources for bottom-trawling fishery in the Mediterranean Sea, with 1,782 tonnes collected in 20144. In fact, since 2006, the General Fisheries Commission for the Mediterranean Sea included A. antennatus on the list of priority species for which fishing regulation plans should be developed5. It is highly appreciated gastronomically and retail market prices are quite high ($70–160/kg) in the Western Mediterranean (WM), where it has been fished most intensely. Outside the Mediterranean Sea, other fishing zones of lesser importance extend to the south of Portugal6, and a new fishing ground was established recently in the Mozambique Channel7.A. antennatus exists at the international level yet. Long-term species sustainability requires adequate fishing and conservation strategies that depend heavily on the identification of the species’ population structure. One of the primary tools for this purpose is genetic analysis, which involves the identification of genetic stocks and connectivity among them8. Such analysis is particularly important to study population processes of marine organisms from inaccessible habitats, such as deep-sea species9.However, no sustainable fishing management policy for A. antennatus in the range of distribution where the species is exploited: the AO, WM, Eastern Mediterranean (EM), and IO2. However, no mtDNA marker study to date has been able to discern genetic structuring at a more regional level between proximal fishing zones12.A previous study using two mitochondrial DNA (mtDNA) markers (16S rDNA and COI) detected four genetic stocks of A. antennatus and the degree of connectivity between stocks. Microsatellite analysis has enabled the identification of stocks in other penaeoid marine shrimp species . However, very few microsatellite markers are available for A. antennatus , and they have not been proven to be useful for the detection of genetic stocks15. This situation led us to develop and characterise 35 new microsatellite loci for A. antennatus using next-generation sequencing13 and in the present study we used 12 of them.A priori, the examination of more variable molecular markers, such as microsatellites, which are sensitive to small but significant genetic differences, should reveal genetic structure at a finer regional scale in populations of 16, affect the population configuration of marine organisms, other factors such as marine geology and ocean currents create many geographic and oceanographic discontinuities that can reduce connectivity, and strongly influence population structure affecting effective species conservation strategies, as has been observed in numerous Mediterranean species . In the Mediterranean Sea, they include the following The Strait of Gibraltar, which forms the dividing zone between the denser Mediterranean waters and the Atlantic Ocean waters.(ii)The Almería-Orán Front, formed at the eastern end of the anticyclonic Eastern Alborán Gyre, between the coasts of the Iberian Peninsula and Algeria.(iii)The Balearic Front and Ibiza Channel, which act in the area between the Balearic Islands and the Iberian Peninsula.(iv)The Strait of Sicily separating the Western Mediterranean Basin from the Eastern Mediterranean Basin.Although human impacts, particularly through fishingOn the other hand, the Red Sea is a possible connection between the Indian Ocean and the Mediterranean Sea, but the Bab-el-Mandeb Strait and the Suez Canal are possible barriers along this route Fig. .A. antennatus and the effect of putative geographic and oceanographic barriers, indicated before, to prevent connectivity among stocks.By using genetic variation at 12 microsatellite loci, the aim of this study is the identification of genetic stocks across the distribution of n = 30) were private alleles present at single locations. Among five sampled regions, Mozambique contributed the largest number of alleles  alleles per locus, of which 19%  of 0.458  and 0.628 , respectively. Mozambique sample presented the highest values in all cases  alleles per locus, average allelic richness (Ar) value of 7.5 , and mean observed and expected heterozygosities  using NeEstimator software often yielded negative values that were interpreted as very large , although even in these cases the upper boundaries of the 95% confidence intervals were infinite  and not significant , rather than within groups (FSC). The first and fourth hypotheses yielded the most significant FCT values and no significant divergence within groups  due to the low homogeneity within the groups created  revealed significant genetic structuring in all hypothesised grouping scenarios, with significant ps Table . For theed Table . These rDiscriminant analysis of principal components (DAPC) enabled differentiation of four groups: IO (Mozambique), AO (Faro), EM (Ionian Sea), and AS + WM Fig. . The horr = 0.67579, p = 0.00842) and the Red Sea route . When we excluded Mozambique and Faro (AO), which is located on the other side of the Strait of Gibraltar, but retained the EM sample, again a significant correlation was obtained , indicating a pattern of isolation by distance (IBD) along the Mediterranean Sea.We detected significant correlation between genetic and geographical distances for both: the Atlantic route  Fig. , which aet al.13, likely because of higher sample size in the present study. Our overall median Ho and He values 20. The Mozambique sample presented the highest levels of diversity , and to a much lesser degree from the Red Sea21.Our analysis yielded higher average Na values  lower limit of CI, the risk of demographic reduction due to inbreeding is low. Nevertheless, as estimates of genetic diversity at our study were not particularly high, caution is warranted because overfishing could result in the loss of essential alleles for adaptation to environmental changes16.The dy Table , to avoiFST values obtained in our study may appear to be low, but they were significant among the principal groups detected  >2 weeks] are significantly affected by oceanographic discontinuities that reduce gene flow between populationsA. antennatus between the AO and Mediterranean Sea  in A. antennatus, as they reported no significant genetic variation between the Atlantic and the Mediterranean populations. Considering that the depth of maximum abundance of A. antennatus adults (600–800 m)3 falls below the maximum sill depth in the Strait of Gibraltar (284 m at a width of ~30 km)21, the dispersal of adults is restricted, although possible planktonic larval drift by surface currents would not36.The observed genetic differentiation of ea Table , 3, 4 fiA. antennatus2. However, our results indicated a slight isolation of the AS sample from the other WM samples  and Palaemon elegans (decapod)39, although it seems to cause no effect on other marine decapod species, including Perapenaeus longirostris (Penaoidea)26.Previous mtDNA analyses failed to identify the Almería-Orán Front as a barrier between populations of es Table , despitees Table . The sizLiocarcinus depurator26 and littoral fish40, respectively, they had no effect on A. antennatus genetic structuring, confirming the results obtained using mtDNA12 and other microsatellite loci15. The detection of several larval stages of A. antennatus in this area36 reinforce the hypothesis that larvae are passively transported by oceanic currents in most of the marine decapods33, and justify the observed high degree at gene flow in the Western Mediterranean  kerathurus30. Our analysis showed genetic differentiation between the WM and EM  and wider (130 km) than the latter21. IBD signals along the WM–EM axis also have been detected in the decapod P. longirostris42. Thus, the EM stock of A. antennatus might be affected by adaptations to the Eastern Mediterranean Basin due to differences in environmental conditions from the Western Mediterranean Basin21.The Strait of Sicily acts as a weak geographic barrier to gene flow in many marine speciesEM Table , Fig. 2,44, our findings indicate that the IO stock of A. antennatus is genetically closer to the AO stock than to the Mediterranean ones 45, prevent connectivity between the Mediterranean Sea and Indian Ocean populations, and the dispersal route of A. antennatus is more likely via the Atlantic Ocean than via the Red Sea. On the other hand, an open question is raised, applicable also to other demersal species for which little information is available, of how we may explain the scant presence of A. antennatus along the Atlantic Coast of Africa2. The species may have a “patchwork” distribution, or demersal populations may remain undiscovered.The IO stock presented the greatest genetic distance Table  and was es Table , 4. ThesA. antennatus, we were able to detect genetic differentiation and identify at least four genetic stocks and the signal for a fifth stock along the species’ distribution. Our results support efforts to achieve sustainability of this resource for future generations via genetic assessment. Currently, overfishing strongly affects marine natural resources16. Taking into account genetic connectivity are fundamentally important to provide a basis for the study of A. antennatus resilience and adaptation to possible environmental changes from human impact46.In conclusion, using a set of sensitive microsatellite loci in A. antennatus is currently exploited across the following five regions: AO, AS, WM, EM, and IO. As the species is traditionally harvested in waters of the WM4, more locations from this region were included in the sampling design  and one singleplex PCR for optimal amplification, following standard procedures with an annealing temperature of 50 °C or 60 °C, as described in Planella et al.22. PCR product sizing was performed with the GeneScanTM 500 LIZ® size standard (Applied Biosystems) on an ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems). Allele scoring was performed with Geneious v. 7.1.9 software47.We performed genomic DNA extraction following the standard phenol-chloroform procedure, and we carried out genotyping for 12 microsatellites developed for Na) and richness (Ar) per locus and per population with FSTAT v. 2.9.3.2 software48, and computed the number of private alleles (Ap) with GenAlEx v. 6.503 software49. We estimated observed and expected heterozygosities (Ho and He), and inbreeding coefficient FIS50 and tested for linkage and HW equilibria using Genepop v. 4.4.3 software51. We assessed test significance using exact tests  and Fisher’s method for global tests. Significance values were adjusted for multiple comparisons using Bonferroni correction. Micro-Checker software52 was employed to identify scoring errors due to large allele drop-out, stuttering or presence of null alleles.We calculated allele number (Ne) for each location using NeEstimator v. 2.1 software19 with the Linkage Disequilibrium method under a random mating model, and we determined 95% confidence intervals using the non-parametric jack-knife method. Given the minimum sample size of 38  was inferred. We estimated the frequency of null alleles and computed FST values that were and were not corrected for the presence of null alleles using 1,000 bootstrap replicates.The FreeNA programme53, computing F-statistics derived from hierarchical partitions. Four different a priori grouping scenarios were independently analyzed to test whether geographic and oceanographic discontinuities effectively reduced gene flow across the species distribution  additional groupings with modification of the effectiveness of various barriers. Statistical significance of F-statistics for each scenario was tested with 10,000 permutations. In such approach, the best grouping scenario maximizes FCT by reducing FSC.We conducted a global locus-by-locus AMOVA analysis with Arlequin v. 3.5.1.2 software55 to construct an NJ tree based on Latter’s FST genetic distance56. The robustness of the tree was tested using bootstrap analysis with 1,000 replicates. DAPC is a multivariate model-free type of analysis conducted with no assumption of HW or linkage equilibrium57. According to Pometti et al.58, it allows more rapid and accurate inference of K populations than do Bayesian model–based clustering methods. We conducted DAPC with adegenet v. 2.1.059 to identify genetic clusters among sampled individuals. We also computed membership probabilities based on the retained discriminant functions, and we used a compoplot function to interpret group memberships in DAPC and to infer admixture between clusters.We used Populations v. 1.2.32 software51. We used the Mantel test based on 100,000 permutations and correlated the logarithm of the pairwise geographical distances between locations and pairwise estimates of linearized FST [FST/(1 − FST)], obtained with FreeNA. The geographical distances between sampling sites were measured as the shortest marine map distance (in kilometers) in Google Earth based on two hypothesised dispersal routes: (i) that connecting Mozambique with Atlantic populations through the Cape of Good Hope and along the Atlantic coast of Africa, and (ii) passage through the Red Sea and Suez Canal, and then through the Mediterranean Sea.IBD was tested with the ISOLDE programme in Genepop v. 4.4.360 (https://popgen.shinyapps.io/divMigrate-online/) using Nm61 as a measure of genetic distance. Population samples (or groups of samples) are represented by nodes in the networks. In addition, to test whether the gene flow among regions was asymmetrically significant, 1,000 bootstrap replicates were performed (α = 0.05).Finally, we generated a network graph representing relative directional migration rates between regions, considering the locations adjoining the oceanographic barriers, with divMigrate-onlineSupplementary Information


The aim of this study was to investigate and compare the effect of wheat bread fortification with varied levels  of chia seed powder (full fat) and cakes . Chia flour was added to whole wheat bread rich in vital wheat gluten for the first time. The breadcrumbs were assessed for their antioxidant activity, nutritional content, textural properties, color, and sensory profiles. The addition of chia seed powder, particularly in high levels, was more effective in improving antioxidant activity compared to bread fortified with chia cakes. Bread supplementation with chia flour improves its nutritional value, especially in the case of chia cakes. A higher moisture content and lower hardness were observed after bread fortification, the influence was more evident with the defatted cake than with seed powder. Fortification with chia flour led to darker breads without significantly affecting their global acceptability. However, the fortified bread showed better values than control in terms of sensory profile. These results suggest that the addition of chia seed powder and defatted cake can enhance the overall whole wheat bread quality. Our results also highlight that bread making could be an unconventional alternative for the exploitation of defatted chia seed. Food technologists are always working forward to ensure the perfect food formulations which please consumer’s growing requests for more natural, functional food with multiple health benefits . Cereal Salvia hispanica, a Lamiaceae species, commonly known as chia, has a long history of use as a food since antiquity. It was a main component in the diet of pre-Colombian people in South and Central America. Chia seeds are a well-known food, their global production is renowned nowadays for their potential health properties. Recently, they have been largely studied because of their increasing popularity and acceptance as a healthy food choice [d choice . They ard choice . They ard choice . Moreoved choice . Consumpd choice .After oil extraction, residual meals of chia are reported to have a high content of fiber (19–23%), protein (33.9–39.9%), and antioxidant compounds which are comparable to other oilseeds used in the food industry . In ordeBesides, since the opinion given by the European Food Safety Authority about the safety of chia as a novel food ingredient ,16,17, aThe objective of this work was to study the influence of the supplementation of different levels of full fat and defatted chia seed powder on the bread quality. Whole wheat bread with added vital wheat gluten was used. The technological and nutritional characteristics of all formulations were assessed.Chia seeds were screw pressed using a single-screw  press equipped with an 18 cm screw with pitch screws of 1.8 cm, an internal diameter of 1.4 cm, and a channel depth of 0.5 cm. The screw rotation speed was 40 rpm and the feed rate was 0.9 kg/h. The nozzle diameter and the nozzle/screw distance used in the pressing of chia seeds were 5 mm and 3 cm, respectively. The residual cake from the extrusion process was collected after passing through a nozzle and used for further investigation.Whole wheat flour, wheat protein isolate Arise 5000 , sugar, salt, and dry yeast were procured from the local market of Jelgava, Latvia; chia seeds were brought at a local market in Toulouse, France.In order to assess the effect of chia addition on whole wheat bread quality and chemical composition, chia seed powder and cakes were added at 2%, 4%, and 6% of the whole wheat flour amount . All ing−1; protein: 4 kcal·g−1; Fat: 9 kcal·g−1, and Fibre: 2 kcal·g−1. The contents of carbohydrates, protein, fat, and fiber of the protein bread samples were determined according to EU Regulation No. 1169/2011 on the provision of food information to consumers using the following converting actors: Carbohydrates (except polyols): 4 kcal·gThe moisture content of the protein bread was determined using standard method ISO 712:2009 . MeasureAfter cooling for at least 2 h, hardness analyses were performed using a TA-XT plus Texture Analyzer  equipped with a cylindrical probe of 25 mm in diameter . All values are given as an average of six measurements. L*), redness (a*), and yellowness (b*). Measurements were done at five different points within the crumb region; mean values were reported for each sample.The CIE Lab coordinate of bread samples was determined using Color Tec-PCM/PSM  reporting luminosity (ΔE) were calculated using Minolta equations as follows:L, a and b—measured values of protein bread samples with chia flour; L0, a0, and b0—the values of the protein bread (control).The total color differences . Coded samples of the same size were served to participants in identical containers each in a panel cupboard.v/v/v) was used to extract protein bread samples. Ultrasonic extraction was done in an ultrasonic bath YJ5120-1  at 35 KHz for 10 min at 20 ± 1 °C and then centrifuged using a CM-6MT  centrifuge at 3500 rpm for 5 min [A mixture solution consisting of ethanol/acetone/water (7/7/6 or 5 min . A simil−1) solution was added to the mixture. The reaction was kept in the dark for 30 min, after which the absorbance was read at 765 nm. Gallic acid was used to construct the standard curve. The results were mean values + standard error of mean and expressed as expressed as Gallic acid equivalents (GAE) 100 g−1 dry weight (DW) of the samples. Measurements were made in three biological triplicates for each sample.TPC of the protein bread extract was estimated by the Folin–Ciocalteu method  with som−1 dry weight (DW) of the samples. Measurements were made in three different biological triplicates for each sample.The 2,2-diphenyl-1-picrylhydraziyl (DPPH) method  with slip < 0.05. All experiments were carried out in triplicate  and the mean values were expressed.Analysis of variance was conducted using one-way ANOVA and Tukey test by pairwise analysis with significance defined at The calculated nutrient content of all enriched breads is given in The moisture content significantly augmented by increasing the level of fortification with chia seed powder and cakes a. This cTextural properties are an important feature in defining bread quality, softness, and strength characteristics . The harL* and b* decreased while those of a* and ΔE* significantly increased with the augmentation of chia level in the bread formulation ; results were expressed as Gallic Acid (GAE) per 100 g dry weight (DW). Chia addition had a positive impact on the levels of these phytochemicals in all samples as TPC values showed significant variation with respect to chia seed powder and cakes level d. HoweveThe total antioxidant activities (TEAC) determined by DPPH of whole wheat breads were expressed as mM of Trolox per 100 g dry weight e. ResultThis study has shown that chia seeds can be used as a raw material to improve the overall quality of whole wheat bread containing a high percentage of vital gluten. These findings may help in the development of enhanced functional commercial whole wheat bread and for the effective valorization of chia seeds and the related residues. This species could be utilized for a variety of food uses as well as being valuable in bio-refining processes thus using several products and residues.


The aim of the present study was to quantitatively assess changes in enamel roughness parameters before and after lingual bracket debonding. The lingual surface of 25 sound premolars extracted for orthodontic reasons was studied by 3D optical interferometric profilometry before and after debonding of lingual brackets following enamel finishing (with fine diamond) and polishing (with 12- and 20-fluted carbide burs). The roughness parameters tested were the amplitude parameters Sa and Sz, the hybrid parameter Sdr, and the functional parameters Sc and Sv. The parameter differences (after debonding-reference) were calculated, and statistical analysis was performed via a Wilcoxon signed-rank test. Statistically significantly higher values were observed in all the surface roughness parameters of enamel surfaces after finishing and polishing, with the mostly affected parameter being the Sdr. Under the conditions of the present study, the finishing and polishing instruments used after debonding of lingual noncustomized brackets created a surface texture rougher than the control in all the tested roughness parameters. In the last decade, the demand for invisible orthodontics and aesthetic considerations, primarily across adult patients, has been increasing. Various different aesthetic approaches such as ceramic or polycarbonate brackets, thermoplastic aligners, and fixed lingual orthodontic appliances have been developed, the latter being the only literally invisible appliance system available.Since the introduction of fixed lingual appliances in 1979 by Fujita , patientBonding techniques in lingual brackets may differ slightly from conventional buccal brackets. As access and visibility are compromised, indirect bonding is popular especially among complex procedures, mainly due to the demand for precision in bracket positioning due to the irregular shape of the lingual surface, which strongly influences the spatial orientation of the crown. Direct bonding might also be an option mainly for two-dimensional corrections and sectional appliances . RegardlTungsten carbide burs in conjunction with a multistep Sof-Lex disc system for polishing are recommended, as they can achieve a smooth surface . FurtherTo the authors’ knowledge, there is no study evaluating the lingual enamel surface roughness after debonding lingual brackets. The aim of the present study was to quantitatively assess the changes in enamel roughness after the debonding of lingual brackets, finishing, and polishing. The null hypothesis was that the finishing and polishing procedures after debonding do not increase the enamel surface roughness in comparison with the native control.Twenty-five sound premolars extracted for orthodontic reasons and stored in 0.5% chloramine-T at 8 °C were used in this study. The teeth were free of caries or periodontal problems, with intact enamel and no history of exposure to bleaching agents. The roots were embedded in silicon holders to facilitate handling during bonding and polishing. Each tooth was labelled for identification purposes. The teeth were cleaned with a nonfluoride paste , rinsed with tap water and air-dried. The central region of each lingual surface was marked with a permanent ink marker (Ø: 4 mm), and the surface morphology was examined under a stereomicroscope  operated under reflected light at 20× magnification.2 light intensity at standard mode. After storage in distilled water at 37 °C for a week, all brackets were debonded by a single trained operator using a lingual debonding plier . The gross amount of resin remnants was removed using an extra fine finishing diamond , and the surface located residual resin was finally removed with 12-fluted  and 20-fluted  tungsten carbide burs attached to an air-rotor handpiece with water spray coolant, until no macroscopically (5×) visible adhesive remnants could be found. The finished and polished surfaces were examined again under a stereomicroscope, and the roughness parameters were determined at the same regions under the same conditions.Thereafter, roughness measurements were obtained from each surface. These regions were acid-etched for 30 s with a 35% phosphoric acid gel , rinsed with water for 10 s and dried with oil-free and moisture-free compressed air for 5 s. Metal lingual brackets  were then bonded with Transbond XT (3M Unitek) on these surfaces by firmly pressing the brackets, removing excess resin with a dental explorer, and light-curing from incisal and cervical directions  with an LED unit  emitting 1200 mW/cm2 analysis area), tilt correction, 5 µm Gaussian high-pass filter to remove surface waviness, and 0.1 nm (z-axis) and 0.2 µm  resolution. The following roughness parameters were tested:Roughness analysis was performed by optical interferometric profilometry. A 3D optical profiler  was used under the following conditions: vertical scanning mode, Mirau lens, 20× magnification (231.1 × 303.8 μm(a) The amplitude parameters Sa (the absolute profile deviation versus the average over a 3D surface) and Sz .(b) The hybrid parameter Sdr . (c) The functional parameters Sc  and Sv .p < 0.05. Statistical analyses were performed with STATA version 15.1 software .Normality assumptions were checked through Shapiro–Wilk tests and q–q plots. Due to the non-normal distribution of the residuals, nonparametric statistics were used. Descriptive statistics were used to present actual reference and debonded values as well as differences (Δ = debonded-reference) for roughness parameters. The following parameters were explored: Sa, Sz, Sc, Sv, and Sdr. A Wilcoxon signed-rank test for paired data was used to check similarity between debonded and initial roughness values for each parameter. The level of statistical significance was pre-specified at All analyses were undertaken in Stata version 15.1 software .Representative reflected light stereomicroscopic images of intact and polished surfaces after bracket debonding finishing and polishing are illustrated in Representative 3D profilometric images of the reference and the debonded finished and polished surface are illustrated in p-value < 0.001; Sz: p-value = 0.004). The most thoroughly affected parameter was Sdr with a 1.7× increase, whereas all the other parameters ranged from a 1.2× to 1.4× increase after debonding, finishing, and polishing.The reference surfaces were smooth with an amplitude deviation ranging from 7 to 9 μm, whereas the corresponding numbers after debonding finishing and polishing ranged between 25 and 27 μm, indicating increased amplitude deviation. The descriptive statistics for surface roughness parameters Sa, Sz, Sc, Sv, and Sdr for the reference and the debonded surfaces separately are presented in Enamel reduction that might occur during debonding may compromise tooth resistance, creating enamel cracks and fractures in combination with increasing surface roughness ,13. ThisThe measured roughness parameters of natural surfaces may be influenced by the measurement device and the magnification. Only a quantitative assessment of roughness parameters allows for direct comparisons between treated and nontreated enamel surfaces. Scanning electron microscopy (SEM) is unreliable and subjective, and the surfaces cannot be quantitatively evaluated. 3D noncontact optical profilometry is superior to the contact surface roughness measuring devices, as 3D noncontact optical devices are not dependent on the stylus tip diameter . In addiThe null hypothesis was rejected as all enamel surface roughness parameters  were significantly higher after debonding and polishing. The statistically significant difference found in the amplitude parameters Sa and Sz implies that the enamel surface after debonding and polishing has higher peaks and/or deeper valleys compared to the reference enamel surface.A number of studies have examined the buccal enamel surface roughness after debonding buccal brackets analyzing different debonding and polishing protocols ,19,20,21None of the existing studies evaluated the lingual enamel surface. There might be some differences between the debonding procedure on lingual and buccal enamel surface. Wang et al. showed similar bond strength on lingual and buccal surfaces of premolars , but BroIn the present study, the analyzed area was standardized to a preliminary limited size of a 4 mm diameter. When debonding lingual brackets, the extension of the grinded and damaged area depends essentially on the bracket design. As such, brackets with customized metal bracket bases may lead to a more extensive ground enamel areas due to their increased basal profile. On top of the usual careful consideration on patient selection in conjunction with individual oral hygiene characteristics, clinical procedures that allow better the identification of bonding materials at debonding should be considered ,26,27,28Debonding lingual brackets from enamel results in an increase of a number of roughness parameters. The most affected parameter was Sdr, associated with the enamel surface area exposed. Depending on the type of lingual bracket type used, this increased roughness may involve a significant area of enamel surface with the potential of enhancing the plaque-retaining capacity of teeth.


A significant negative correlation between femoral bone density and CTx-1 was found. The results of this study suggest that the combination of OVX and glucocorticoids induces bone loss in a short period of time in sheep.The aim of this study was to validate the combination of ovariectomy and glucocorticoid treatment in sheep as a large animal model for osteoporosis by measuring the concentration of specific biomarkers in the blood of the sheep and measuring bone loss over five months. Aged Merino ewes were randomly allocated into four groups: control, ovariectomy (OVX), and two OVX groups receiving glucocorticoids—one group once-monthly for five months (OVXG), and the other for two months followed by no treatment for three months (OVXG2). Parameters measured were biochemical markers of bone turnover, areal bone mineral density, volumetric bone mineral density, and total and trabecular bone parameters. Ovariectomy increased the concentrations of bone resorption marker C-terminal telopeptides of type 1 collagen (CTx-1) and bone turnover marker serum osteocalcin (OC) concentrations in the OVX group compared to control sheep. The combination of ovariectomy and glucocorticoid treatment increased the concentrations of CTx-1 and decreased serum OC concentrations in the OVXG group compared to OVXG2. Femur and lumbar spine bone density were lower in experimentally treated groups when compared with the control group. Total and trabecular  •Optimise and characterise a sheep model for osteoporosis over five months•Combining OVX and glucocorticoids induced BMD loss in sheep over five months.•Bone turnover markers resulted in a reduction of OC and increased CTx-1 markers. In womeLarge animal models are approved by the US Food and Drug Administration for mimicking human osteoporosis in orthopaedic research . A well-The OVX sheep is an established large animal model of osteoporosis. Sheep have the advantage that their bone structure and bone metabolism is similar to humans . The modIt was hypothesised that ovariectomising sheep in combination with monthly injections of glucocorticoids would result in decreased BMD and increased plasma bone remodelling marker concentration over a shorter period of time than previously reported, while eliminating the negative side effects. The aims of this study were to: 1) validate the OVX sheep model by measuring the concentration of serum biomarkers and bone loss over a five-month period; and 2) investigate the effects of short- and long-term glucocorticoid administration on BMD in the lumbar spine, femur and tibia, as well as bone biomarkers, in the OVX sheep.22.1n = 28) were obtained from a commercial farm in the Whanganui region, New Zealand. The animals were housed in a barn and adapted to indoor pens with rubber mat flooring. Sheep had free access to water and were fed the control diet described below for 20 days. After adaptation, sheep were randomly allocated into groups: control group (n = 10), the OVX group (n = 12), and two OVX groups receiving glucocorticoids, one group once a month for five months (OVXG) (n = 3) and the other group once a month for two months followed by no treatment for three months (OVXG2) (n = 3)  .Fig. 1Flad libitum throughout the study.Control and low-calcium sheep pellet concentrate diets were formulated from soya, brollard and wheat to contain 11% protein, 34% starch, and 29% nondigestible fibre, as well as >2000 IU/kg vitamin D3/cholecalciferol and <200 IU/kg vitamin D2 , which are required for calcium absorption. While 2–4 g/kg calcium is sufficient for sheep under normal circumstances, the animals in the current study were housed indoors and had limited exposure to sunlight and no exposure to sun-cured forage. Under these circumstances, calcium absorption was likely to be impaired. It was considered undesirable to risk vitamin D toxicity by increasing the vitamin D levels to aid calcium absorption in the gut, so the dietary calcium levels were increased to compensate. Ewes were fed at approximately 200 g/sheep/day. Wheat straw and water were available to all sheep via a ventral abdominal incision. The linea alba and subcutaneous tissues were closed using 1 USP polydioxanone  and the skin with 1 USP nylon suture , and the sutures were removed 14 d after the surgeries. Prior to extubation, each sheep was given 20 mg/kg meloxicam  for pain. At the same time, the 10 sheep in the control group were anaesthetised but no surgery was performed. Post-surgery, until suture removal, sheep were examined twice daily. After suture removal, sheep were checked twice daily and food, water, activity and hair condition monitored.Sheep were anaesthetised using 0.5 mg/kg diazepam  and 10 mg/kg ketamine  intravenously, followed by endotracheal intubation and maintenance of anaesthesia with halothane. The distal tibia from each sheep was pQCT-scanned under general anaesthesia. The sheep, except for the control group, underwent bilateral OVX. The OVX was performed , Vetoquinol SA, Lure Cedex, France) subcutaneously in the neck. Methylprednisolone treatment started before the sutures were removed. Three of the sheep were given four further treatments at the same dose once a month (OVXG). The remaining three sheep received the initial treatment and then an additional treatment was given one month later, after which the treatment was discontinued in order to observe the effect on bone after glucocorticoid withdrawal (OVXG2).On day two after OVX, six OVX sheep were administered an injectable suspension of 400 mg methylprednisolone  and performed according to the Code of Ethical Conduct for the use of live animals for research at Massey University, Palmerston North, New Zealand.2.2via jugular venepuncture into 10 mL plain (serum), heparin and EDTA tubes  on the day of surgery (month 0) and then monthly for the duration of the study until the animals were euthanised. Blood samples were taken between 08:00 and 10:00 h to limit any circadian effect.A blood sample was collected from all sheep g for 10 min and the plasma removed and stored at −80 °C for later analysis. The tubes with no anticoagulant were left at room temperature for 45 min to clot prior to being processed as above.The EDTA and heparin blood tubes were centrifuged at 20002.3n = 5) and half from the OVX group (n = 6) were euthanised using a captive bolt followed by exsanguination at two months. At the end of the study (five months), the remaining ewes from the control group (n = 5) and the OVX group (n = 6), and all of the ewes from the OVXG (n = 3) and the OVXG2 (n = 3) groups, were similarly euthanised. After euthanasia, the femurs, lumbar spines (LSs) and tibias were collected and stored at 4 °C. Samples of ileum, liver, kidney, heart and lung were collected and stored in 10% neutral buffered formalin until processing for histology. Microscopic examination of 3 μm paraffin-embedded haematoxylin- and eosin-stained sections confirmed the absence of unrelated disease.Half the ewes from the control group (2.4de novo) OC in serum. The MicroVue immunoassay used has cross-reactivity with sheep species  was measured in serum. Samples were tested in duplicate with an immunoassay kit  which uses a monoclonal mouse antibody that recognises only intact  were measured in heparinised plasma. Samples were tested in duplicate using the Serum CrossLaps ELISA kit , which uses two highly specific monoclonal antibodies. The IDS immunoassay has been used for sheep .x 808 ultramicroplate reader  was used to generate a standard curve from each ELISA kit and measure samples. Then, sample absolute concentrations were extrapolated from the standard curve. OC and CTx-1 were measured in the sheep at baseline (month zero), two months and five months of the trial.EL2.52.5.1Ex vivo measurements of bone mineral component (BMC) and areal BMD (aBMD) for the lumbar spine and femur were determined using the Hologic Discovery A Bone Densitometer .Lumbar spine and femur were thawed at room temperature. 2.5.2In vivo measurements were made of total volumetric BMD (vBMD), bone area, bone mineral content (BMC), cortical/subcortical and trabecular vBMD, cortical/subcortical and trabecular bone area, cortical/subcortical and trabecular BMC, and geometric variables of the mid-metaphysis 10 mm from the distal surface of bone and at the mid-diaphysis 70 mm from the distal end.Three-dimensional analyses of the left tibial bone were performed using pQCT. The first initial baseline scan was obtained after the sheep were anaesthetised for surgery. Ex vivo measurements were made of total vBMD, bone area, BMC, cortical/subcortical and trabecular vBMD, cortical/subcortical and trabecular bone area, cortical/subcortical and trabecular BMC and geometric variables of the proximal tibia, the mid-metaphysis and at the mid-diaphysis using the XCT2000 peripheral quantitative computed tomography (pQCT) scanner . The measurement site was located at the proximal metaphysis 15 mm from the proximal surface of bone. The pQCT scan for the in vivo and ex vivo measurements had a voxel size of 0.3 mm.Left tibias were thawed at room temperature. 3 to separate soft tissue from the outer edge of bone. For cortical bone, the threshold was set at 710 mg/cm3.Norland/Stratec XCT 5.50 software was used for scan imaging analysis. The trabecular compartment was obtained at the central 45% of bone at the trabecular sites. The manufacturer's contour threshold was set at 280 mg/cm2.6p-values <0.05 were considered statistically significant.Data for bone marker concentrations were analysed to report the effects of treatments at specified time points. For analysis of percentage of change from baseline, baseline results were included as covariate and the analysis was based on the results from month two and month five. Data for lumbar spine, femoral and tibial parameters were analysed to test the effect of the OVX plus glucocorticoid treatments after sacrifice at month two and month five. Data are expressed as mean and standard deviation (SD). Statistical analyses were performed with the nonparametric Kruskal–Wallis test to assess whether the treatment means were significantly different. The nonparametric Kruskal–Wallis test was followed by post-hoc comparisons of treatment means using Dunn's post-hoc test. Statistical analyses on data were conducted using R . We performed the Spearman rank-order correlation to assess the association between BMDs of the lumbar spine, femur and tibia with bone biomarker concentrations. This test was done using Minitab 17.2.1 . In all statistical analyses, 33.1During bone loss in osteoporosis, measurable metabolites are released in the blood, including OC and CTx-1. OC is a noncollagenous bone protein, which is released from osteoblasts during bone formation and from bone matrix during bone resorption. CTx-1 is a collagen breakdown product, which is released during bone resorption .p-value = 0.337) within two months and by 43%  at five months compared to baseline in OVX sheep. On the other hand, serum OC concentration decreased by 24%  at two months and remained at this decreased level  at five months compared to baseline in the OVXG group. However, when the glucocorticoids were discontinued in the OVXG2 group at two months, the serum OC concentrations observed at five months increased by 26% , compared to baseline in the OVXG2 group.p-value = 0.002) from baseline, increasing to 115%  at five months compared to baseline. Further, serum CTx-1 concentrations increased 37%  from baseline within two months of glucocorticoid treatment in the OVXG group. Surprisingly, these concentrations decreased by 20%  after five months of glucocorticoid treatment when compared to baseline. Discontinuing glucocorticoids at two months decreased serum CTx-1 concentrations by 52%  at five months from baseline in the OVXG2 group.The experimental treatment had a significant effect on the bone resorption marker, serum CTx-1 concentration . Sheep e3.2p-value = 0.36) after OVX, but lumbar spine BMC was not affected two months after OVX. In addition, femoral aBMD did not vary between the control group and OVX group at two months.The results of DXA scans of the lumbar spine and femur are shown in p-value = 0.06) in the OVX, OVXG and OVXG2 groups, respectively  in the OVX, OVXG and OVXG2 groups, respectively  in the OVX, OVXG and OVXG2 groups, respectively   D.p-value >0.05). Total vBMD was reduced by 8% after two months of treatment . Bone area and BMC did not vary between groups at two months. No difference could be found in trabecular vBMD , trabecular area  and trabecular BMC .The results of pQCT scans of the proximal metaphysis of the tibia post-cull are shown in p-value = 0.052), 27%  and 13%  in the OVX, OVXG and OVXG2, respectively , 28%  and 1%  in the OVX, OVXG and OVXG2, respectively  and 30%  in the OVX and OVXG, respectively  and 20%  in the OVX and OVXG, respectively .The data were pooled from all the treatment groups at two months and five months, and further analyses performed to investigate any possible associations between parameters. Relationships of BMD of femur and tibia with OC and CTx-1 concentrations in sheep at two months were not significant using Spearman's rank-order correlations . Howeverr = −0.505; p-value = 0.039).The correlations between biochemical markers and BMD of lumbar spine, femur and tibia at five months were positive for OC and negative for CTx-1, but not significant using Spearman's rank-order correlation . However4In this study, we applied a combination of advanced age, OVX and glucocorticoid treatment to determine bone mass quantity, quality and bone markers using sheep as a large animal model for postmenopausal osteoporosis. A period of five months caused a decreased BMD in the glucocorticoid OVX sheep with a significant reduction of aBMD in the lumbar spine and femur as measured by DXA, and a significant decrease in tibial cortical and trabecular vBMD as measured by pQCT. The decreases in aBMD and vBMD were greater in the glucocorticoid-treated ovariectomised sheep compared with the control groups and those that only underwent OVX. Interestingly, after cessation of glucocorticoid administration, there was a recovery of bone mass in sheep treated with glucocorticoids for only two months. Serum biomarkers were increased in the ovariectomised sheep; however, those same serum markers were reduced in the glucocorticoid-treated OVX sheep.4.1Bone turnover markers can be used as measures of the status of bone remodelling. The bone remodelling depends on the synchronization of bone resorption cells—osteoclasts and subsequent bone formation cells—osteoblasts. During menopause, bone resorption increases as a result of oestrogen deficiency as well as bone formation to fill the bone cavities that have been destroyed . In thisIn this study, a reduction of lumbar and femur BMD was found, as assessed by DXA, when compared with the control group that was not ovariectomised and was fed a diet with plentiful calcium. These results are similar to those reported by previous studies; with a decrease of aBMD reported in the spine at six months  and 12 mIn this study, tibia vBMD, trabecular vBMD and cortical/subcortical vBMD as assessed by pQCT showed a decrease in bone mass after five months of OVX. However, published reports are controversial, as two of the few studies published to date reported no significant differences in trabecular vBMD in the proximal tibia after twelve months of OVX in sheep . However4.2The combination of OVX, minimal dietary calcium, and glucocorticoid treatment caused a transient increase in the serum CTx-1 concentrations at two months, after which concentrations were lowered by the end of the study. This result is supported by previous studies, where serum CTx-1 concentrations increased during the first months in OVXG sheep but decreased thereafter . The bonIn contrast, the serum OC concentration of sheep treated with glucocorticoids for five months decreased until the end of this study. This result is similar to some previous studies, where serum OC concentration rapidly decreased  and remaAn unexpected outcome of the current study is that the serum OC and CTx-1 concentrations in the group that received only two months of glucocorticoids were closer to the control group after cessation of glucocorticoid administration. This finding is supported by In the present study, BMD decreased after five months of the combination of OVX and glucocorticoids in the lumbar spine, femur and tibia, although this reduction varied according to the skeletal site. Previous research reported decreased cortical and trabecular vBMD in the lumbar spine and distal radius with OVX plus glucocorticoids in sheep over a period of six months , tibia  at 12 moSheep is recognised as a large animal model for studying human osteoporosis. The OVX sheep model is useful to understand the mechanisms that contribute to bone loss. However, there has been controversy about the optimal period of time for achieving bone loss due to there is no agreement within the literature of the time necessary to establish an osteoporotic (osteopenic) bone in OVX sheep. There have been several hypotheses proposed on the time required to induce measurable bone loss in sheep , althougThe limitations of this study include the small sample size and that bone mineral density was determined as a function of treatment evaluated in OVX sheep. Future studies such as mechanical properties of bone and changes in its extracellular matrix components are required to fully investigate bone quality in OVX sheep. However, the strengths are the induction of bone loss in a short period of time and the assessment of bone mass during and after the cessation of glucocorticoids combined with OVX. Therefore, this present project should be considered a pilot study, where a combination of OVX and glucocorticoids was used to induce bone loss over a short period of time in sheep. A reduction in time may allow the sheep model to be used more frequently, to test novel therapies for postmenopausal women.5This pilot study suggested that the interaction of OVX and glucocorticoids plays a significant role and induces bone loss in sheep over five months, and this bone loss is similar to that seen in humans. The combination of OVX and length of glucocorticoid treatment should be carefully considered when designing studies using OVX sheep. Further, the use of experimental animal models for studying bone loss to test new experimental drug therapies and orthopaedic implants offers opportunities to enhance our understanding of bone diseases, particularly postmenopausal osteoporosis.Transparency document.Image 1The authors have no conflict of interest.