The purpose of this study was to implement full/half bowtie filter models in a commercial treatment planning system (TPS) to calculate kilovoltage (kV) cone‐beam CT (CBCT) doses of Varian On‐Board Imager (OBI) kV X‐ray imaging system. The full/half bowtie filter models were created as compensators in Pinnacle TPS using MATLAB software. The physical profiles of both bowtie filters were imported and hard‐coded in the MATLAB system. Pinnacle scripts were written to import bowtie filter models into Pinnacle treatment plans. Bowtie filter‐free kV X‐ray beam models were commissioned and the bowtie filter models were validated by analyzing the lateral and percent‐depth‐dose (PDD) profiles of anterior/posterior X‐ray beams in water phantoms. A CT dose index (CTDI) phantom was employed to calculate CTDI and weighted CTDI values for pelvis and pelvis‐spotlight CBCT protocols. A five‐year‐old pediatric anthropomorphic phantom was utilized to evaluate absorbed and effective doses (ED) for standard and low‐dose head CBCT protocols. The CBCT dose calculation results were compared to ion chamber (IC) and Monte Carlo (MC) data for the CTDI phantom and MOSFET and MC results for the pediatric phantom, respectively. The differences of lateral and PDD profiles between TPS calculations and IC measurements were within 6%. The CTDI and weighted CTDI values of the TPS were respectively within 0.25 cGy and 0.08 cGy compared to IC measurements. The absorbed doses ranged from 0 to 7.22 cGy for the standard dose CBCT and 0 to 1.56 cGy for the low‐dose CBCT. The ED values were found to be 36‐38 mSv and 7‐8 mSv for the standard and low‐dose CBCT protocols, respectively. This study demonstrated that the established full/half bowtie filter beam models can produce reasonable dose calculation results. Further study is to be performed to evaluate the models in clinical situations.PACS number(s): 87.57.uq With the advances in cone‐beam computed tomography (CBCT) technology, image‐guided radiation therapy (IGRT) has become the routine method to accurately deliver the therapeutic radiation dose to the target while minimizing unnecessary irradiation to normal tissues. By performing the CBCT imaging prior to radiation treatment, the treatment setup can be verified and fine‐tuned while the patient is on the treatment table. Despite these great benefits, there is concern about the CBCT dose if used frequently during the course of treatment. Therefore, the accurate estimation of the CBCT dose is essential to predict any potential detrimental effects.CTDIW) of the CBCT for both XVI and OBI systems. In their study, the CTDIW for OBI were found to be 8.3 cGy for a head scan and 5.4 cGy for a body scan in standard dose mode. Although these previous studies provided the level of the CBCT doses for various CBCT systems, all their data were acquired in the phantom geometries, which quite differ from the anatomies of patients.There have been many studies performed to estimate the CBCT doses. Islam et al.Alaei et al.To overcome the limitation of the wedge function, our current study employed a compensator function to implement both full and half‐bowtie filter models in Pinnacle. We focused on the flexible feature of the compensator model, which allows building the concave shape of the full bowtie filter. After implementing the full and half‐bowtie filter compensator models, we generated and commissioned the kV X‐ray beam models and also validated the bowtie filter models by comparing the calculation results with ion chamber (IC) measurements and Monte Carlo (MC) simulations. We also performed CBCT dose calculations using a CT dose index (CTDI) body phantom and compared the calculation results with IC and MC data. To explore the application of the established models further, we investigated absorbed doses and ED calculations for the CBCT protocols with a five‐year‐old pediatric anthropomorphic phantom and the results were compared with MOSFET measurements and MC data. It should be mentioned that this is the first study which implemented/commissioned the full‐bowtie filter model in any treatment planning system and applied it to the CBCT dose calculations completely. We expect that this study provides valuable information on CBCT dose estimations to patients.This study was performed by following these steps: 1) addition of the low energy deposition kernels to the Pinnacle TPS, 2) generation of full and half bowtie compensator models, 3) commissioning/validation of the full and half bowtie X‐ray beam models, 4) calculation of CBCT imaging doses in the CTDI phantom and CTDI/weighted CTDI estimations, and 5) calculation of CBCT doses in the anthropomorphic phantom and absorbed dose and ED estimations. The process of each step will be described in detail.The low‐energy deposition kernels created by Alaei et al.SSD=33 cm in the Pinnacle beam modifier section, which is different than the actual location of the bowtie filter (10 cm away from the source). Second, a Pinnacle script was written to load each bowtie filter compensator data into a Pinnacle treatment plan as a compensator. All the parameters for the compensators, such as physical density, location, output factor, and resolution, were stored in the script and loaded into the treatment plan when the script was executed.The full‐ and half‐bowtie filter beam models of a Varian OBI system were developed in the Pinnacle TPS. Both bowtie filter beam models were created as compensators in the Pinnacle using MATLAB software . First, the physical profiles and dimensions of both bowtie filters were acquired from the manufacturer under a nondisclosure agreement, imported into the MATLAB system, and hard‐coded into binary file format. Due to a restriction within Pinnacle, the compensator model must be located at the source‐surface distance (SSD) greater than 30 cm, so the profiles of the compensator models were interpolated by considering the geometric magnification effect. The compensators were set to be placed at the In the Pinnacle physics module, two kV X‐ray beam models, without the bowtie filters, were created and commissioned based on the lateral and percent depth‐dose (PDD) profile data obtained from MC simulations, which were benchmarked with IC measurements in a previous study.50×50×50 cm3) was generated in the TPS and an anterior/posterior static 125 kVp X‐ray beam was placed on the surface of the phantom at SSD=100 cm. For each beam model, corresponding bowtie filter compensator was loaded to the corresponding treatment plan by executing the Pinnacle script. Dose calculation for each bowtie filter was performed using collapsed cone convolution (CCC) dose calculation algorithm.After the commissioning of the two bowtie filter‐free kV X‐ray beam models, the validation of the bowtie filter compensator models was performed. A cuboid water phantom  with density Wri=tissue weighting factor of individual organ i, and Hi=equivalent dose of individual organ i.For the application of CBCT dose calculations in a more realistic geometry, a five‐year‐old pediatric anthropomorphic phantom  was employed and the absorbed doses from standard dose head and low‐dose head CBCT protocols (Varian OBI version 1.3) were calculated in Pinnacle TPS. The calculated data were compared with MOSFET measurements and MC simulation data obtained from previous work.The detailed calculation method of organ and ED values can be found in the previous study.Note that the treatment table was not included in both CTDI and CBCT dose calculations.CTDIW values from Pinnacle TPS, IC measurements and MC simulations in the CTDI body phantom are presented in CTDIW values of the three methods were relatively close; dose differences between IC measurement and Pinnacle's CCC dose calculations were within 0.03 cGy and 0.08 cGy for pelvis spotlight and pelvis CBCT protocols, respectively.We implemented the full/half bowtie filter models of the Varian OBI system in the Pinnacle TPS to enable the kV CBCT imaging dose assessments. As the initial geometric verification step, the shapes and orientations of the bowtie filter models were visually inspected as well as the dose distributions of the kV static X‐ray beams with full/half bowtie filters in the water phantom see ; these vFor the second verification step, the dosimetric properties of the static X‐ray beams with the bowtie filter models were investigated; the lateral and PDD profiles of the bowtie filter models were compared with IC measurements and MC simulations. As seen in CTDIW were calculated and compared with IC measurements and MC results. It was found that there was no significant difference in CTDI and CTDIW values among the three methods presented in To verify the bowtie filter models in the cylindrical geometry, the CTDI body phantom was introduced and the CTDI and To extend the application of the bowtie filter models, we employed the five‐year‐old pediatric anthropometric phantom to calculate the absorbed doses and ED from the CBCT imaging protocols. It was found that the maximum differences of the absorbed doses between MOSFET measurements and the Pinnacle's CCC dose calculations were 0.8 cGy and 0.3 cGy for the standard dose head and low‐dose head CBCT protocols, respectively. Additionally, the ED values between MOSFET measurements and the Pinnacle's CCC dose calculation were found to be within 5% and 11%. These results demonstrated that the full/half bowtie filter models in the Pinnacle TPS can produce reasonably accurate absorbed dose and ED values in the anthropomorphic phantom geometry.There is some extensive work to be done in the future. 1) This study only investigated two CBCT imaging protocols that are frequently used in the clinic. The kV beam models for other CBCT imaging protocols will be investigated in the future. 2) The bowtie filter models were only implemented for the certain models of Varian's OBI system  To be applicable to the TrueBeam kV imaging system, new full/half bowtie filter models for the TrueBeam kV X‐ray beam need to be established and commissioned, which is beyond the scope of this study and will be explored in future work. 3) The implemented bowtie filter models were applied only to the cylindrical and anthropomorphic phantoms and not to the real patient body habitus. Again, further study will be performed in the future.In this study, the full/half bowtie filter kV X‐ray beam models for CBCT imaging protocols were established and commissioned in the Pinnacle TPS. With the CTDI and five‐year‐old pediatric anthropomorphic phantoms, both bowtie models were also validated by comparing the calculation results with the IC, MOSFET measurements, and MC simulations. It was found that the established kV X‐ray beam models can provide reasonable accuracies for the CBCT dose calculations in water, CTDI, and five‐year‐old pediatric anthropomorphic phantoms. Implemented bowtie filter models can be applicable for the CBCT imaging dose assessments in radiation therapy patients.We thank Dr. Jeffrey Siebers of University of Virginia for discussion regarding Pinnacle scripting, Dr. Terry Yoshizumi of Duke University for the permission of ion chamber and MOSFET data usage, and Mr. Todd Racine of Advocate Condell Medical Center for the access of Pinnacle TPS system. This study was presented as a snap‐oral presentation at the AAPM 2015 annual meeting, Anaheim, CA.Creative Commons Attribution 4.0 International License.This work is licensed under a


IPV) and other constraints to women's reproductive autonomy, and the influence of IPV on reproductive health.To understand intersections between intimate partner violence (aPRs) were calculated for associations between IPV experience and potential constraints to reproductive autonomy and health outcomes.A secondary analysis examined cross‐sectional data from a facility‐based sample of women seeking abortion care (for spontaneous or induced abortion) between March 1 and October 31, 2013. Women aged 18–49 years, who received abortion services and selected a short‐acting contraceptive method or no contraception completed an interviewer‐administered survey after treatment. Adjusted prevalence ratios . IPV was also associated with receiving post‐abortion care after an induced abortion compared with accessing legal menstrual regulation, and with the use of medication abortion compared with manual vacuum aspiration (both P<0.05).There were 457 participants included in the present analysis and 118 (25.8%) had experienced IPV. Ensuring women's reproductive freedom requires addressing IPV and related constraints.Intimate partner violence was associated with additional constraints on reproductive autonomy from husbands/partners, in‐laws, and religious communities. Seeking induced abortion unaccompanied and using medication abortion could be strategies to access abortion covertly among women experiencing  Intimate partner violence was associated with multiple threats to women's reproductive autonomy; the findings identify potential strategies to control fertility in the presence of violence. In the context of IPV, a diminished sense of reproductive agency could prompt women to perceive more limited access to contraception, with IPV victims less able to use contraception effectively owing to refusal by partners and contraceptive sabotage.The aim of the present study was to examine IPV experiences in the past year and other potential constraints to reproductive autonomy among women utilizing abortion services in Bangladesh, with the goal of identifying the influence of IPV on reproductive health outcomes.2The present study was a cross‐sectional analysis of data collected under a prospective parent study that aimed to understand short‐acting post‐abortion contraceptive use among women using public‐sector abortion services in Bangladesh (unpublished data). The parent study collected data between March 1 and October 31, 2013, using a stratified one‐stage cluster sampling approach. The sampling frame consisted of 47 public‐sector facilities that were participating in an intervention to improve the quality of abortion care; 16 were randomly selected for inclusion using probability proportional to size sampling within facility‐type strata. Women were eligible for inclusion in the parent study if they were presenting for abortion services (MR or PAC), were aged 18–49 years, and if they selected to use pills, injectable contraception, or condoms as post‐abortion contraception, or if they chose no contraception. The parent study was focused on women who chose to use short‐acting contraceptive methods or no contraception; women who selected long‐acting or permanent contraceptive methods were ineligible. The parent study protocol received ethical approval from the Bangladesh Medical Research Council in Dhaka, Bangladesh, and the Allendale Investigational Review Board in the USA; written informed consent was obtained from all participants.After recovering from abortion procedures, eligible women were introduced to female research assistants who were posted at each facility during all available clinic hours. Research assistants confirmed participants' eligibility and obtained written informed consent before participants completed a 30‐minute interviewer‐administered survey. Consistent with international ethical standards for research into violence against women,The exposure of interest, IPV experience during the preceding year, included experience of either physical or sexual violence that was perpetrated by the woman's husband or sexual partner. Standard questions from the Bangladesh Demographic and Health Survey were used;The outcomes assessed were individual perceptions about access to contraception, discordance in fertility intentions within the family, family‐level constraints to reproductive autonomy, and measures of women's reproductive health. Perceptions about access to contraception were assessed using three yes or no questions: (1) “Do you think that it is too difficult to obtain family planning methods, or that you would have to travel too far to obtain a method?”; (2) “Do you think that it is too expensive to obtain family planning methods?”; and (3) “Do you think that family planning methods are inconvenient to use?” Discordance in fertility intentions was assessed both for intentions regarding the index pregnancy (ending in abortion) and future pregnancies. Intentions regarding the index pregnancy were assessed by asking, “Right before you became pregnant, did you want to become pregnant then, did you want to wait until later, did you not want to have any (more) children, or did you not think about it?”.P<0.05 was considered statistically significant.Socio‐demographic characteristics were presented for the full study population and by IPV experience; F‐tests from simple logistic regression models were used to test bivariate associations. Each potential constraint to reproductive autonomy and reproductive health outcome was presented across the full sample and by IPV experience. Adjusted prevalence ratios (aPRs) were calculated using multinomial logistic regression models for categorical outcomes and generalized linear models using log‐binomial maximum likelihood estimators for dichotomous outcomes. The Poisson distribution was specified if the model failed to converge using the binomial distribution, a conservative approach that was expected to result in valid point estimates with confidence intervals wider than those from log‐binomial estimates.3A total of 555 eligible women were approached for participation in the parent study and 498 were enrolled. There were 41 women (8%) excluded from the present analysis owing to missing IPV‐experience data. Residence location was the only variable that was associated with missing IPV data owing to clustering in several facilities where privacy was difficult to obtain. This resulted in a sample size of 457 women with complete IPV data.The mean age of participants was 27 years range 18–45), 374 (81.8%) had at least one child, 251 (54.9%) had secondary or higher education, 406 (88.9%) were Muslim, and almost all were married at the time of the study (Table 8–45, 374Experiencing IPV in the preceding year was associated with multiple other constraints to reproductive autonomy Table . Women wAssociations were also observed between having experienced IPV in the previous year and reproductive health outcomes Table . A highe4The present study found that over 25% of women seeking abortion care reported experiencing IPV in the preceding year; in turn, this was associated with other potential constraints to reproductive autonomy and reproductive health outcomes. The findings suggest that husbands/partners, in‐laws, and religious communities play a role in women's reproductive lives, with women who experienced IPV reporting discordance in fertility intentions within the family and opposition to contraceptive use. Associations between IPV with PAC services for induced abortion rather than MR suggests that women who experienced IPV could be more likely to attempt induced abortion illegally outside the healthcare system. The findings suggest that women experiencing IPV could be taking active steps to control fertility when faced with IPV, including seeking abortion services unaccompanied and selecting medication abortion as an induced abortion procedure, which can be used to simulate spontaneous abortion and terminate pregnancy covertly.Women who experienced IPV perceived contraception to be difficult to access. Despite widespread availability of contraceptive methods such as oral contraceptive pills having been reported at the community level,Intimate partner violence was also associated with discordance in fertility intentions within the family and opposition to contraceptive use. Women who experience IPV could also experience fertility pressure or pregnancy coercion from husbands/partners and in‐laws. Ethnographic work in Bangladesh has demonstrated the interplay between fulfilling in‐laws' role expectations and IPV;The present study also provided potential insights into strategies used by women experiencing IPV to control their fertility. Women who reported IPV were more likely to attend unaccompanied, to seek care for PAC for an induced abortion, and to select medication abortion as an induced abortion procedure; all of which could indicate a propensity for accessing induced‐abortion services covertly, without the knowledge of their families. The higher prevalence of accessing PAC for induced abortions, compared with attending for care for legal MR could indicate that women experiencing IPV were more likely to access induced‐abortion services outside the healthcare system; however, the present study was not able to ascertain the safety or quality of care, which could range from induction using instruments such as sticks or roots to accessing medication abortion drugs through a pharmacy. The finding that women who reported IPV were more likely to select an induced medication abortion compared with manual vacuum aspiration was in line with reported use of induced medication abortion to simulate spontaneous abortion without the knowledge of abusive spouses.Overall, the present findings suggested that IPV intersects with other constraints to reproductive autonomy imposed by a husband/partner, in‐laws, and religious communities. The results suggested that women could respond to these threats by seeking multiple MR procedures over time and accessing induced abortion services covertly. The findings should be viewed in the context of macro‐level indicators such as the gender inequality index. In 2013, Bangladesh ranked 115 out of 151 countries, with a gender inequality index value of 0.529,The results of the present study should be viewed in light of the limitations. The study focused on an abortion care‐seeking population, and generalizing to the broader population of women experiencing IPV in Bangladesh could be inappropriate. The sample was limited to facilities participating in an intervention to improve the quality of abortion care in more urban areas of Bangladesh, and women younger than 18 years and those selecting long‐acting and permanent contraceptive methods were excluded. Consequently, the findings might not be generalizable to all patients seeking abortion services in Bangladesh. The findings were also limited by the available IPV data because information on the frequency and severity of violence and reproductive coercion was lacking. Finally, family‐member fertility intentions were based on the respondent's report, and could reflect family discord rather than true discordance in fertility preferences.Women experiencing IPV face additional constraints to reproductive autonomy, and IPV was associated with reproductive health outcomes, including accessing PAC for induced abortions compared with legal MR services. Further research is needed to improve the understanding of women's perspectives on access to contraception and potential strategies to control fertility in the context of IPV. Preliminary findings from the present study suggest that interventions are needed at multiple levels to mitigate the impact of violence and lack of reproductive autonomy on women's reproductive health.EP, KLA, KB, and RC designed the study with input from MRD. KB and RC provided input on the study tools and managed data collection. EP led the analysis and writing with input from all authors. All authors reviewed and approved the final version of the manuscript.The authors have no conflicts of interest.


PMMA phantoms for three panoramic‐imaging devices: the panoramic mode on two CBCT machines (Carestream 9300 and i‐CAT NG) and the Planmeca ProMax 2D. A SEDENTEXCT dose index adult phantom and custom‐built adolescent and pediatric PMMA dosimetry phantoms were used. Panoramic radiographs were performed using a Planmeca ProMax 2D and the panoramic mode on a Carestream 9300 CBCT and an i‐CAT NG using the protocols used clinically. Point dose measurements were performed at the center, around the periphery and on the surface of each phantom using a thimble ionization chamber. Five repeat measurements were taken at each location. For each machine, single‐factor ANOVA was conducted to determine dose differences between protocols in each phantom, as well as determine the differences in absorbed dose when the same protocol was used for different‐sized phantoms. For any individual phantom, using protocols with lower kVp, mA, or acquisition times resulted in statistically significant dose savings, as expected. When the same protocol was used for different‐sized phantoms, the smaller phantom had a higher radiation dose due to less attenuation of x‐rays by the smaller phantom and differences in the positioning of the ion chamber relative to the focal trough. The panoramic‐mode on the CBCT machines produce images suitable for clinical use with similar dose levels to the stand‐alone panoramic device. Significant dose savings may result by selecting age‐ and size‐ appropriate protocols for pediatric patients, but a wider range of protocols for children and adolescents may be beneficial.The aim of this study is to measure the radiographic dose in adult, adolescent, and child head‐sized  Helmrot et al. have suggested using the dose area product (DAP) as a standardized dose metric for all dental radiographyAlthough dental imaging contributes less than 0.1% of the radiation dose the global population receives, radiation risk should always be considered when conducting panoramic radiography.The aim of this study was to measure and compare the absorbed dose in adult, adolescent, and child head‐sized PMMA phantoms for three panoramic‐imaging devices: the panoramic modes on the Carestream 9300 CBCT and i‐CAT Next Generation CBCT, and the Planmeca ProMax 2D panoramic machine. The study also aims to establish the importance of selecting patient‐appropriate protocols, particularly in pediatric patients.22.ACylindrical polymethyl methacrylate (PMMA) phantoms containing five holes drilled through the height of the cylinder for ionization chamber placement Fig.  were useA panoramic radiograph of a Pan DXTTR  was taken by each machine to assess their images. The DXTTR phantom is an anthropomorphic phantom comprised of a natural bone skull embedded in resin. The head has detailed facial features to enable positioning using anatomical landmarks and is mounted on a tripod that can articulate to enable angling the head to align with the laser positioning guides.2.BPanoramic radiographs were performed using the panoramic‐mode on two CBCT machines, the Carestream 9300  and i‐CAT NG , and using a stand‐alone panoramic machine, the ProMax 2D . Although panoramic images can be obtained by reformatting a 3D CBCT image, both machines used in this study have a separate panoramic‐imaging acquisition mode; it is the panoramic‐imaging acquisition mode that was used in this study. All of the devices studied were equipped with digital detectors, a TFT detector for the Carestream 9300, a‐Si flat panel detector with a CsI scintillator for the i‐CAT NG and a CCD detector for the Planmeca Promax 2D.A PMMA plate was mounted onto a tripod upon which the PMMA phantoms were situated. Positioning lasers were used to place each PMMA phantom within the field of view (FOV). Specifically, the front peripheral hole within the phantom was positioned in the imaging focal trough and the phantom was centered vertically within the field of view. Phantoms were exposed to acquisition protocols deemed clinically appropriate with regard to phantom dimensions. All protocols used in the study are pre‐set within their respective machines except the small child protocol (ProMax 2D) for which the exposure parameters  were manually adjusted to match the posted clinically accepted technique chart. Table The Pan DXTTR phantom was situated within each device using positioning lasers to ensure that the mandible of the phantom was in the focal trough. Panoramic radiographs were performed using the average adult protocol in the Carestream 9300 and ProMax 2D, and the large protocol in the i‐CAT NG. The set‐up of the adult PMMA and DXTTR phantoms are shown for the Planmeca ProMax 2D machine  measured the absorbed dose. The ion chamber was calibrated at the factory to be within ±5% over the range of energies used in diagnostic CT. Utilizing the high‐sensitivity setting improved the accuracy of the measurements. Five measurements for the absorbed dose were taken at each measurement location; measurements were obtained in the center of the phantom, and in four locations  around the periphery and the same four locations on the surface of the phantom for each protocol to provide nine measurement locations for each phantom.A thimble ion chamber , with an active volume of 0.6 cm2.D® . For each phantom, a single‐factor ANOVA (α = 0.05) and Sidak's multiple comparisons test was conducted using the absorbed dose values to determine statistically significant differences between protocols for each machine. An additional single‐factor ANOVA (α = 0.05) and Sidak's multiple comparisons test was conducted using the normalized absorbed dose value (μGy/mAs) to determine statistically significant differences between the Carestream 9300, i‐CAT and ProMax 2D for equivalent protocols used for each phantom.The mean and standard deviations of the five repeated dose measurements are reported in μGy and dose measurements normalized by the tube current in milliamps and exposure time are given as μGy/mAs. All statistical analysis was done using GraphPad Prism3P<0.01), as expected. For each imaging device, when the same protocol was used for different‐sized phantoms, the smaller phantom generally had a higher dose measured at every location within the phantom for each imaging modality (P<0.0001).The absorbed dose in adult‐, adolescent‐, and child‐sized phantoms was measured for the panoramic‐mode of the Carestream 9300 Table , the panP<0.001) except as indicated on the graphs. The adolescent setting on the ProMax 2D system seemed similar to the small adult setting on the CS 9300 machine, with a few measurements in the peripheral regions showing no difference between the machines  resulted in significant dose increases, whereas decreasing exposure parameters resulted in significant dose savings at all locations within a phantom, as expected. The one exception was the measurements taken on the left side of the adolescent phantom imaged using the child protocol and the small adult protocol on the CS9300; in this case, the small adult protocol showed slightly lower doses which could be due to a slight difference in positioning the phantom within the focal trough of the imaging system or could mean that the dose levels are similar in that location between the two protocols. Imaging protocols pre‐set within each device must be chosen appropriately for each patient based on size and age. Although protocols with lower exposure parameters result in significant dose savings, the diagnostic quality of images should not be compromised. Studies have observed image quality deterioration as a result of a reduction in exposure parameters.4.CFor identical imaging parameters, the smaller phantom generally has a higher radiation dose. A couple of exceptions were observed for the left and/or right sides of the phantoms; in a few cases, the smaller phantom showed higher doses under the same imaging conditions . One possible explanation is the smaller phantoms had their measurements in different locations within the x‐ray beam due to the curvature of the phantom and fixed hole positions, which positioned the ion chamber in a different location relative to the machine's focal trough. The smaller diameter allows x‐rays to directly reach more areas on the phantom during any point of the x‐ray tube's trajectory. Less attenuation of x‐rays through the PMMA also increases the radiation dose measured at the center and front of the smaller phantoms. Clearly, children may be exposed to unnecessary radiation dose if exposure parameters are not adjusted appropriately. Minimizing radiation exposure to children is of primary concern due to the increased radiosensitivity of their tissues. Children also have a longer life span, increasing the risk of cancer induction during their lifetime.4.DDifferences in kVp, mA, and acquisition time result in differences in absorbed dose between the devices. In order to compare the effect of different imaging modalities on dose within a phantom, dose measurements were normalized for mAs. The i‐CAT NG generally had the highest normalized doses within the child phantom, and the Carestream 9300 generally had the lowest normalized doses. Consequently, manufacturing differences  between the Carestream 9300, i‐CAT, and ProMax 2D may also contribute to the differences in doses at each location. For example, the kVp for each protocol within the Carestream 9300 changes throughout an exposure potentially causing differences in dose distribution and absolute dose within the phantom relative to the other machines. Image quality is invariably dependent on equipment; however, the CBCT machines produce panoramic images that are of comparable diagnostic quality to the ProMax 2D Fig. .The pediatric protocols available within the Carestream 9300, i‐CAT NG, and ProMax 2D decrease the radiation exposure within children. However, the devices used in the study only had one pre‐set child protocol available, and only the ProMax 2D had an adolescent setting. For the ProMax 2D, manually inputted exposure parameters based on the University of British Columbia Dentistry guidelines for panoramic radiographs were used to expand the imaging protocols available for children. The importance of providing a spectrum of pediatric (and adolescent) imaging protocols prevents clinicians from over‐ and under‐estimating the radiation exposure received by the variety of children seen in a dental clinic. From our dosimetric comparisons, it is clear that the stand‐alone panoramic‐imaging system had a clear advantage over the combination units, because we had the flexibility to manually adjust the imaging parameters for smaller children, and there were more pre‐set protocols for the pediatric population available.The use of PMMA phantoms in this study allows for measurements of the absorbed dose; however, measurements within a uniform phantom do not accurately represent the dose distribution within a patient. To more accurately reflect patient dose, phantoms with bony anatomy representing each of the age demographics  should be developed for future studies. The bony structures will alter the absorption and scatter properties of the phantom, giving a more realistic dose distribution. Furthermore, having tissue equivalent material will also enable estimates of the radiation risk using ICRP weighting factors.5The panoramic‐mode on the CBCT machines studied produced diagnostic quality images with comparable radiographic dose to a stand‐alone panoramic‐imaging device. Smaller phantoms receive more radiation when imaging protocols are identical for each device. The study demonstrates that pediatric protocols reduce the radiographic dose to children, but the combination units had a limited number of protocols available. All panoramic‐imaging devices, both stand‐alone and combination units, will benefit from including a larger range of pre‐set options representing the pediatric and adolescent populations.The authors have no conflicts of interest to disclose.


A likely consequence of this ‘intralocus caste antagonism’ should be the maintenance of genetic variation for fitness and maladaptation within castes (termed ‘caste load’), analogous to the result of intralocus sexual antagonism. The aim of this review is to create a research framework for understanding caste antagonism, drawing in part upon conceptual similarities with sexual antagonism. By reviewing both the social insect and sexual antagonism literature, we highlight the current empirical evidence for caste antagonism, discuss social systems of interest, how antagonism might be resolved, and challenges for future research. We also introduce the idea that sexual and caste antagonism could interact, creating a three‐way antagonism over gene expression. This includes unpacking the implications of haplodiploidy for the outcome of this complex interaction.The breeding and non‐breeding ‘castes’ of eusocial insects provide a striking example of role‐specific selection, where each caste maximises fitness through different morphological, behavioural and physiological trait values. Typically, queens are long‐lived egg‐layers, while workers are short‐lived, largely sterile foragers. Remarkably, the two castes are nevertheless produced by the same genome. The existence of inter‐caste genetic correlations is a neglected consequence of this shared genome, potentially hindering the evolution of caste dimorphism: alleles that increase the productivity of queens may decrease the productivity of workers and  I.et al., Individuals within a population often take different routes to maximise fitness, which they follow by having distinct morphological, behavioural and physiological trait values. Familiar examples include males and females Trivers, , as wellIntralocus antagonism was a concept initially developed in relation to the antagonism between males and females. Analogous to caste antagonism, ‘sexual antagonism’ is defined by a negative inter‐sexual genetic correlation for fitness, where individuals of one sex with high fitness tend to have relatives of the opposite sex with low fitness. This conflict arises because the divergent reproductive roles of the two sexes favour sex‐specific phenotypes Trivers, , but theII.versus a non‐breeder – the hallmark of caste antagonism. Although inter‐caste genetic correlations for fitness itself have not yet been measured in any social system, inter‐caste genetic correlations for fitness‐related traits have been identified that are indicative of caste antagonism. For example, a positive inter‐caste genetic correlation for ovarian development was found in the ant Lasius niger, such that especially fecund queens tended to produce more fecund workers Caste‐specific selection selects for divergent trait values, suggesting that caste dimorphism can represent at least partially resolved caste antagonism Fig. . Here, wet al., et al., et al., et al., et al., via neutral processes. However, neutrality could be difficult to demonstrate et al., et al., et al., et al., Vg) genes that are linked to egg production and caste determination are recent duplicates that differ in level of expression between queens and workers  caused worker‐destined larvae to develop queen‐like traits  that carry the alternate forms , which benefit only the heterogametic sex might be selected to move to the Y (or W) chromosome, resulting in adaptive, sex‐limited expression Rice, . Sex chrr et al.  for a reet al., et al., et al., et al., et al., et al., et al., via simple modifications to pre‐existing systems Although caste conflict can lead to caste dimorphism, the use of caste dimorphism as a proximate measure of caste conflict across species is problematic. This is because the relationship between caste dimorphism and conflict is not simply positive or negative, as the genetic architecture of caste‐specific traits and the nature of caste‐specific selection can change in complex ways as dimorphism evolves.et al., Despite this complex relationship, caste polymorphism can mitigate intralocus caste antagonism over specific phenotypic traits within a population, just as sexual dimorphism can reduce intralocus sexual antagonism Fig. . For exaet al., et al., et al., et al., et al., Studies of sexual antagonism suggest that multiple factors will conspire to make the evolution of caste dimorphism incomplete, such that caste antagonism will often be strong in practice. For example, high standing levels of caste antagonism might result from pleiotropic and epistatic constraints on the evolution of caste‐specific gene regulation [see Van Doorn , PennellIV.(1)L. niger males have a very short lifespan and also short telomeres relative to queens and workers, while the female castes differ in lifespan but not telomere length  are diploid, and so all alleles are fully exposed to selection in males, while in females recessive alleles can ‘hide’ from selection in heterozygotes. Haplodiploidy thus causes a difference in the efficacy of selection between the sexes, which might skew the average phenotype towards the male optimum. The versus indirect transmission of its genes. Selection on worker traits becomes weaker (relative to selection on queen traits) when workers gain little or no fitness from direct reproduction, and when the average relatedness between workers and the recipients of their help is low Pheidole ants are categorised into minor and major workers, with minor workers taking on general ‘housekeeping’ duties within the nest, and major workers specialising in colony defence, food storage and seed milling  will have an additional source of antagonistic selection acting on males, where divergent selection operates between males with helper and reproductive roles. This contrasts with Hymenoptera, where the function of a male is purely reproductive. The dynamic of antagonism becomes increasingly complicated to predict when so many polymorphisms are involved.V.(1)Measurement of caste antagonism presents a number of obstacles that are not present when measuring sexual antagonism. But as we discuss here, caste antagonism is empirically tractable and social systems provide novel ways to quantify role‐specific selection Table .et al., et al., et al., et al., In some social systems, it may be difficult to measure inter‐caste genetic correlations using quantitative genetics because of the confounding effects of the common environment shared by queens and workers. Although workers can often be maintained separately in the laboratory, normal colony functioning typically requires the castes to be kept together. Maternal and sib‐social effects can also strongly affect the phenotype, and must be considered when estimating the additive effects of genes  by 1.0, while their daughters are related to one another by 0.75. One way to explore the link between sexual antagonism and caste antagonism is to partition variance in offspring phenotype within and between colonies of the same and different patrilines. By knowing the relatedness between individuals it is possible to explore genetic effects on sex and caste‐specific fitness. Indeed, some social insect species normally exhibit multiple patrilines within colonies, whereby different workers share a maternal genome but inherit different paternal genes [e.g. Acromyrmex leaf‐cutting ants et al.  could also answer fundamental questions regarding the genetic constraints on antagonism resolution. For instance, are genes that are embroiled in caste antagonism also under pleiotropic or epistatic constraints preventing complete caste‐specific expression? This has been explored in relation to sexual antagonism by uncovering links between sex‐biased gene expression and proximate measures of pleiotropy, such as the tissue specificity and network connectivity of genes As well as being of interest in its own right, understanding the involvement of specific traits in caste antagonism could help to answer general questions about social group dynamics. One example is exploring caste antagonism over egg production and its effect on the partitioning of reproduction between the members of a social group Polistes paper wasps i) the movement of genes to sex chromosomes offers a potential resolution to intralocus sex and caste antagonism, and (ii) diploidy in males reduces the exposure of alleles to selection, which can alter the evolutionary trajectory of traits shared between sexes and castes. Levels of sexual dimorphism are also low in termites relative to social Hymenoptera Some social insects have unusual genetic systems, in which we hypothesise that caste antagonism should shape genomic architecture in different ways. In at least three ant species, queens are produced asexually and workers sexually, while males are genetic clones of the queen's mate versus daughters produced depending on whether the offspring will carry male‐ or female‐beneficial alleles – has been proposed to occur in dioecious species  when threats from competitors are detected  depending on whether her genotype would produce offspring best suited to a queen or worker role. In a similar way, sex ratio adjustment in response to sexual antagonism – where females might alter the number of sons (2)et al., et al., Caste antagonism could be responsible for maintaining variation in ‘personality’ – i.e. repeatable inter‐individual differences in behaviour – which have been documented in social insects (e.g. Jandt (3)Plodia interpunctella, which together resulted in the sexes being displaced from their respective phenotypic optima. Berg & Maklakov et al., et al., et al., et al., A fourth possibility is that caste antagonism could contribute to the occurrence of disease. An example is the disease outbreaks that occur in commercial honeybee populations  Caste antagonism is a conflict between social castes over gene expression. The conflict arises because castes share a genome but have divergent phenotypic optima. The fittest allele at a given locus will then often not be the same across the different castes. Given the varied and contrasting roles that queens and workers occupy across social insect taxa, this conflict is likely to be widespread in nature. Despite this prediction, caste antagonism is a greatly understudied field of research in comparison with sexual antagonism, an analogous conflict between males and females, which is now thought to be ubiquitous.(2) Evidence for a positive inter‐caste correlation for fecundity suggests that queens and workers are constrained from reaching their phenotypic optima in ants and bees. Quantifying other physiological and behavioural phenotypes associated with this correlation, such as how it affects worker productivity, would be useful for further exploration of the potential for caste antagonism. Additionally, very few studies have estimated inter‐caste correlation, and it is not clear if it is similarly strong throughout the social insects.(3) An increasing number of studies of the molecular biology of social insects have revealed dimorphism in gene expression between queens and workers, suggesting that caste antagonism has at least been partially resolved. There is mixed evidence for the involvement of gene duplication, gene splicing, and methylation in caste differentiation, paralleling routes involved in the evolution of sexual dimorphism. In contrast to intralocus sexual conflict, caste antagonism cannot be resolved by the movement of genes to sex chromosomes, since these are absent in social Hymenoptera.(4) Given that social taxa have different sexes as well as different castes, antagonism over gene expression is likely to extend further than just the conflict that exists between castes – creating a three‐ (or more) way conflict. Although unexplored so far, this could alter the dynamics of the conflict and its resolution in unexpected ways. The net evolution of any trait will be dependent on the specific selection pressures on each individual, and it is unlikely that evolution will optimise all phenotypes. The outcome will also be affected by haplodiploidy, where the efficiency of selection will be greater in males. In the same vein, relatedness structure and co‐variation between direct and indirect genetic effects that occur within social groups will add an additional layer of complexity. Moreover, the conflict is likely to extend beyond a three‐way interaction, as it will be affected by selection from breeders that have different reproductive tactics, as well as workers that take on different roles within the colony.(5) Detecting caste antagonism is empirically tractable, and our understanding of it could be aided by borrowing concepts from sexual antagonism research, but also through the use of empirical methods that are unique to social systems. For example, genetic and environmental effects on caste fitness‐related traits can be disentangled through quantitative genetic breeding designs and by investigating wild populations where pedigrees are known. The clonal sperm of hymenopteran males might also be exploited to explore the additive effects of genes on caste‐specific fitness (see Table (6) Caste antagonism can help to explain why we see variation for fitness‐related traits within castes in natural populations. This is expected to impact trait evolution, with far‐reaching effects on biological processes, including altering caste ratios within social groups, and creating the genetic variation underlying personality traits, lifespan and disease. By exploring caste antagonism in more detail, our understanding of adaptive processes of evolution could be greatly improved.(7) Sexual antagonism research provides inspiration and the initial building blocks for outstanding questions in caste antagonism research. There is ample opportunity for these two research streams to complement one another, since they involve analogous conflicts that lend themselves to contrasting experimental approaches. Understanding caste antagonism also has the potential to shed new light on questions about the evolution of sociality, such as the genetic mechanisms that promote and prevent caste differentiation, and the processes underlying reproductive skew.


A 365 million year‐old trilobite moult‐carcass assemblage was described by Błażejowski et al. (2015) as the oldest direct evidence of moulting in the arthropod fossil record. Unfortunately, their suppositions are insufficiently supported by the data provided. Instead, the morphology, configuration and preservational context of the highly fossiliferous locality  suggest that the specimen consists of two overlapping, queued carcasses. The wider fossil record of moulting actually extends back 520 million years, providing an unparalleled opportunity to study behaviour, ecology and development in early animals. Taking cues from modern analogues, it is possible to quantify precise details about moulting behaviour to determine broad‐scale evolutionary trends, ontogenetic sequences and morphological selection pressures. In this review, we argue that this rich source of data has been underused in evolutionary studies, though has great potential for investigating the life history and evolution of arthropods in deep time. The extensive fossil record of Arthropoda provides a fascinating insight into the evolution of the most diverse and abundant animal phylum, upon its appearance amongst the earliest animals 541 million years ago The fossil record of moulting is largely composed of exuviae, and fossils preserved during moulting are extremely rare because these events take place on a relatively rapid time scale Trimerocephalus chopini, which they interpret as in situ preservation of an individual having just emerged from its moulted exuvia, located directly above the carcass  was illustrated emerging anteriorly from its exoskeleton, and therefore represents the earliest example of an arthropod preserved mid‐moult T. chopini described by Błażejowski et al. Błażejowski et al. Many arthropods have distinct exoskeletal arrangements upon moulting, which are found preserved in the fossil record due to their reinforced cuticle. These moult assemblages can usually be recognised by a number of criteria, described by Henningsmoen T. chopini figured by Błażejowski et al. Paciphacops preserved ‘immediately following moulting’ (p. 2). They (and we) reject the idea that this assemblage represents a specimen preserved during or immediately before moulting (with the ‘carcass’ still enclosed in the soon‐to‐be moulted exoskeleton) because the two individuals are ‘separated by a layer of sediment’ , which occurs through an anterior gape suture created by ventral disarticulation of the cephalon and thorax, causing the cephalon to hinge dorsally at an angle of nearly 90° . Adult trilobites may have grown indeterminately, meaning they increased in size with each adult moult until death while maintaining the same morphology Błażejowski et al. H. primitivus specimen figured by Dean , Crustacea  and Hexapoda (insects). Many myriapods and crustaceans show continuous growth and moulting during adulthood Crenatolimulus from the Late Jurassic Owadów‐Brzezinki Quarry in Central Poland, to examine both preservation and behaviour. The proposed wrinkles on the exoskeletons of both the horseshoe crab and the trilobite are taken as evidence for these being freshly moulted individuals that have not yet undergone post‐moulting ‘decompression’  Kowala Quarry of central Poland, that they argued consisted of a freshly moulted individual preserved ventral to its recently moulted exoskeleton. This supposition is based on a suggested increase in the thoracic segment number and wrinkling of the ‘carcass’. On consideration of the preservation, morphology and arrangement of the specimen, and in comparison to other trilobite material from the locality, it is more likely that this specimen represents two separate trilobite individuals, which are queueing in a manner similar to that described for Trimerocephalus by Radwański et al. Błażejowski et al. With every new discovery of moulting preserved in the fossil record, we are more able to discuss important aspects of the evolution of development and behaviour. The rare and exquisite preservation of caught‐in‐the‐act moulting is informative for snapshot views of ecdysial behaviour The authors have declared no conflicts of interest.


EU accession. The aim of this pilot study was to: (1) test the feasibility of a protocol for monitoring farm animal welfare in Serbia, (2) ascertain preliminary data on animal‐based outcomes of farm welfare and (3) gain insight into Serbian farmers’ understanding of animal welfare as part of a wider project working towards inclusion of animal‐based assessments in a ‘higher welfare’ voluntary assurance scheme. This study encompasses the first national survey of farm animal welfare in which animal‐based outcomes were tested on 105 farms by a total of ten trained assessors. Data on the views and aspirations of the farmers from these 105 farms were also systematically gathered during face‐to‐face interviews. Existing animal‐based measures for pigs, sheep, dairy cows and broilers that have been successfully applied and identified as valid, reliable and feasible measures in other countries, were found to be largely transferable. However, some on‐farm protocols, previously used in other countries, had to be shortened for logistical reasons when used in Serbia. Our findings suggest that further refinement may be needed in order to allow local application of all measures. While the term ‘animal welfare’ has only recently been introduced into the Serbian language, seventy‐three percent of farmers had heard of it. Overall, few positive associations were found between farmer satisfaction with animals’ living conditions and animal‐based data. Many farmers had aspirations to develop and expand their farms, which may potentially enhance animal welfare, but these farmers identified that financial and technical advice and support would be needed in order to achieve these goals.The selection and field application of animal‐based welfare measures for pigs, sheep, dairy cows and broilers was the first step towards the future development of welfare improvement schemes for Serbia – a country that is applying for  Serbia is currently undergoing the process of accession for approval and entry as a member state of the European Union (EU), having applied for membership in December 2009, and confirmed as a candidate country in March 2012. An important priority in the accession pathway is the harmonization of animal welfare regulations  a national pilot survey of the welfare of cattle, sheep, pigs and poultry on farms in Serbia was conducted. The ultimate aim was to work towards inclusion of animal‐based assessments in a ‘higher welfare’ voluntary assurance scheme. Prior to this project some animal welfare assessments of cattle had been made in Serbia based on the Animal Needs Index (ANI) . Our ambition was to gain an insight into changing perceptions – particularly important in a country where the overall concept of animal welfare, and farm animal welfare assessment schemes (including farm assurance schemes) that aim to promote and support animal welfare, are relatively new concepts , provided by species‐specific animal welfare scientists. Training involved 1 day of interactive discussions and seminars on animal‐based welfare measures and assessment protocols, and 1 day of on‐farm practical training. During classroom training, scoring exercises using images and videos were undertaken to promote standardization of outcome assessments. Assessors also carried out practice interviews with farmers to pilot test the questionnaire prior to the national survey. The on‐farm training for each of the species included visits to five farms to include one pig, one broiler, one sheep and two cattle farms. During on‐farm training, aspects of the protocol were demonstrated and trainees tested the application of the animal‐based measures under the guidance of the trainer. At the end of each training visit, the group of assessors discussed their individual assessment results to assist in alignment of scoring attributes. Following completion of all the farm training visits, the assessors also attended a 1‐day meeting in which they examined reference photographs and discussed scoring methods to encourage standardization, and to help reach scoring consensus  of holdings are less than 5 hectares (12.4 acres), and are usually mixed farms rearing a variety of livestock species NPRD . The SerHave you heard of animal welfare?Questionnaire data collected from each farm included; animal numbers, general husbandry and productivity information, mortality records, routine use of mutilations (castration and tail docking), and bulk milk somatic cell count for dairy herds . The assessor conducted a short (approximately 15 min) semi‐structured face‐to‐face interview with the farmer. The same open and closed questions were used on all farms to ascertain background information relating to farmer understanding and perceptions of animal welfare. These questions were drafted by the Serbian partners. Translated into English, the questions were:What does animal welfare mean to you?If so,What does animal care mean to you?If not,Are you satisfied with living conditions for your animals?Is there anything you want to change ?Do you need support for improving conditions on your farm?If yes, what kind of support?What are your plans for your farm?All respondents were then asked:The questionnaires were conducted in Serbian, and the farmers’ responses were recorded on paper by the interviewer at the time of the interview.Data from individual animal‐based outcome measures were used to provide a group‐level prevalence (percentage observed) for each of the recorded measures for each farm. The median and range for the measure values across all farms were determined.The terms or words used by the farmers when responding to the questions on ‘What does animal welfare mean to you?’ and ‘What does animal care mean to you?’ were allocated to the following ‘term categories’ at the point of data entry: Good care, Good food, Good housing, Good environment, Good health, Good treatment, Good hygiene, Five freedoms, Better production. The features of their farms, or systems or aspects which farmers wanted to change, and the plans they had for the farm, were allocated to broad thematic categories by one researcher and checked by a second researcher following data entry and prior to analysis.P = 0.05.The proportions of farmers who had, and had not, heard of the term ‘animal welfare’ were calculated per species group. For those who ‘had’, the elements included in their description of what ‘animal welfare’ meant to them were categorized, and the proportion of farmers including each ‘category term’ was then calculated. This was repeated for those who ‘had not’ heard of animal welfare, but described welfare indirectly in terms of animal care . Within each species, the herd or flock sizes of the farmers who ‘had’ and ‘had not’ heard of animal welfare were statistically examined. To test the hypothesis that the farmers’ degree of satisfaction with farm conditions might be related to animal outcome measures, a Mann–Whitney test was used to compare the prevalence of selected welfare outcomes between farms where farmers were and were not satisfied with their animals’ living conditions . Significance testing was set as The results of the key animal‐based welfare outcome assessments are summarized in Table A total of 701 sows from 91 pens on 16 farms, and 3801 fattening pigs from 303 pens on 18 farms were assessed. Fattening pig farms had a mean of 1608 pigs and 18 pigs per pen, and farms with sows had a mean of 310 sows, 18.8 piglets per sow per year and a 33 day weaning age. Enrichment was observed on three fattening pig farms (chains or plastic tubes) and five sow farms . Thirty‐five percent (%) and 11% of fattening pig and sow farms respectively had slatted floors. Fattening pig farms had a mean of 100% of castrated male pigs and 95% of pigs were tail‐docked. Breeding farms had a mean of 94% of castrated male pigs, and 83% of piglets were tail‐docked and tooth‐clipped. Only one farm reported using anaesthesia for docking, castration and tooth clipping. There was an average annual mortality of 3.0% of sows on farms, and 0.3% of animals were culled. Overall 11% of farms reported cannibalism occurring in sow units. Fattening pig farms had an average annual mortality of 3.7% (range 0–10%) and 2.4% of fattening pigs were culled. Overall 15% of fattening pig farms observed cannibalism.The most common lesion observed during the farm assessment visits was bursitis (swellings on the legs), with a median incidence of 57% (range 0–100%) of sows and 70% (range 17–99%) of fattening pigs per herd affected. No sows were observed interacting with any enrichment provided; however one fattening pig farm showed 83% of observed pigs with their mouth and snout in contact with the chain enrichment (as opposed to pen fittings) in contrast to the other two farms where the enrichment contact rate was reported to be <5%.A total of 1009 sheep, and 217 lambs were assessed on 27 farms in mountainous and hill (44%) and lowland areas (56%). Just over half (56%) of these meat‐producing sheep flocks were reportedly housed indoors year‐round, a further 11% were housed indoors with paddock access, and 33% of flocks were grazed (i.e. outdoor) throughout the production cycle. Just under 20% of flocks underwent seasonal migration to new pastures. Most (67%) flocks lambed indoors, 11% lambed outdoors, and the remaining 22% practised both. Tail docking, using a rubber ring or knife was reportedly practised by 63% of farms, and 19% routinely castrated approximately week‐old lambs by rubber ring (37%), burdizzo (26%), or surgical (37%) methods. Most sheep flocks (63%) were slaughtered at an abattoir, fewer (15%) performed home slaughter and the remaining 22% sold live sheep at markets. The pilot study period coincided with the lambing period, and with the housing of 20 out of 27 flocks. The most prevalent conditions observed were wool loss , followed by breech  soiling , and lameness . The recorded median prevalence of all young lamb indicators was zero (Table −1 (range 48 000–600 000). Four farms had an average SCC over 400 000. The average age of a cow in production was 5.25 years (range 4–7 years). Dirtiness was the parameter recorded with the widest range and highest prevalence by assessor pairs. Apparent prevalence ranged from 0 to 76% (median 19%) for dirty flanks, 0 to 90% (median 28%) for dirty hindlegs, and 0 to 87% (median 13%) for dirty udders.A total of 776 cattle were assessed across 28 dairy herds. All were managed under intensive conventional management with the exception of one ‘calf‐at‐foot’ completely pasture‐based herd. Six herds had access to pasture, and 16 herds had access to outdoor space, for at least part of the year. Six herds were kept in tie‐stalls, 18 in loose housing, and four farms included both housing types. Straw was the most common type of bedding used. The number of cows per farm ranged from 7 to 683 with a median of 45. Reported median daily milk yield was 20 L (range 10–30 L). Annual average somatic cell count (SCC) data was available for 18 farms and values were relatively high, with a mean of 294 000 cells mL2. All were indoor intensive flocks, and none had outdoor access. Fifty‐four percent of flocks had straw litter, 13% were provided with wood shavings, and 29% had both types of litter. Thirty‐eight percent of flocks had some form of environmental enrichment; of those 78% were provided with straw bales and 11% (one flock) had perches. The mean mortality reported by farm staff was 3.1% per cycle, and morbidity was 2.0% per cycle. The mean mortality during the flock cycle, as recorded by assessors was 4.1%. On average 0.1% of the flock was culled during the flock cycle. The mean age for assessment in this study was 30 days, (approximately 13 days prior to average slaughter age), and a median of 25% (range 0–51%) of birds had a gait score of 3 (out of a maximum score of 5) or higher.A total of 3620 birds from 24 broiler farms were individually assessed for mobility and 1807 for other individual bird assessment; 55% of flocks were Cobb genotype, 36% Ross, and the remaining 9% were Hubbard. The assessed farms had a mean of 24,182 birds per farm, and 11 396 (range 1500–45 000) birds per house. The average house size was 44 by 13 metres, giving a mean stocking density of 19.9 birds per mHave you heard of animal welfare? What does animal welfare mean to you? or What does animal care mean to you?The majority of farmers (73%) had heard of animal welfare but there were some in each farm type group who had not. Only 17 sheep farmers (63%) had heard of animal welfare compared with 76% of each of the other groups of farmers Table . The per‘Good food’ was the factor most commonly mentioned, either alone, or in conjunction with other factors, in explanations of both animal welfare Fig.  and goodAre you satisfied with living conditions for your animals?Is there anything you want to change ?Seventy‐four percent of farmers were satisfied with the living conditions of the animals on their farm Table . The perDo you need support for improving conditions on your farm?If yes, what kind of support?Eighty‐seven percent of farmers suggested they would need some support to improve their farm. The majority of these farmers reported that they needed financial support (76%), with the remainder needing both financial and expert technical support, or improved farm infrastructure. Twenty‐three percent said they would need expert advice either with or without financial support.P = 0.17) and body lesions . It is possible that there was confounding between prevalence and herd size, since there was a trend for farmers who had heard of animal welfare to have more pigs . Farmers who had heard of welfare tended to have smaller flocks of sheep . In this group there was a trend for fewer sheep with wool loss . Dairy herd size did not differ between farmers who had or had not heard of welfare . A trend was observed for dirty flanks in cows, which appeared to be more prevalent where farmers had heard of the term ‘animal welfare’ .The comparison of welfare measures on farms where the farmer had or had not heard of welfare showed no significant differences. A few measures demonstrated weak trends. For sows, there was a weak trend for a higher median prevalence of two indicators if the farmer had heard of welfare: this was for bursitis . For sows, statistical comparisons were not made as only five farmers were not satisfied with living conditions. However, a trend was observed in that the median prevalences of bursitis, body soiling and body lesions were numerically higher where farmers were dissatisfied.Table P = 0.1). The only animal‐based sheep welfare measure with a high enough prevalence to be included in the analysis was wool loss. However, a significant association between the reported level of farmer satisfaction and the recorded prevalence of wool loss was not identified. In contrast, there were no significant differences in the dairy herd sizes between farmers who were and were not satisfied . When farmers were satisfied, there was a trend for fewer tarsal abnormalities . As only four broiler farmers expressed any dissatisfaction with living conditions for their birds, no comparison of satisfaction levels was made for broiler farms.Farmers who were satisfied with conditions for their sheep tended to have smaller flocks , especially given the slightly older slaughter age of broiler birds examined in other EU countries , which is an important and relevant welfare concern for Serbian sheep flocks. Given the importance of body condition ‐ globally accepted as a key welfare outcome for sheep including extensively‐managed animals  should be aware of this. Immink et al.  mapped set al. et al. The relatively limited number of components of the Serbian farmers’ concept of animal welfare, which tended to be based on physical conditions rather than mental state, might be expected from farmers facing economic difficulties and without experience of value‐enhanced welfare‐based products  examined was based on feasibility, given the timing and funding restrictions, the logistics of visiting farms, and the stated aim to collect preliminary information regarding the four main types of livestock farms. Therefore the findings do not claim to give a detailed picture of the precise welfare state in Serbia, but rather, should be viewed as a preliminary informative data set on which to build, and a necessary starting point in the process of moving towards farm animal assurance schemes. It is clear that for future developments and training, standardization of practices must be firmly established, and evaluation of observer agreement should be prioritized.This pilot project created ten trained farm animal welfare assessors with increased experience and capacity in farm animal welfare assessment. The number of farms  of all assessors would have been tested prior to their pilot application in Serbia, but this was not feasible given the study resources. However, the issue of testing IOR is not uncommon to other animal welfare studies that have undertaken national or regional surveys or during the preliminary steps towards the development of farm animal welfare standards. The approach taken here is consistent with that of other researchers who did not test IOR but instead paid particular attention to the detailed training of assessors in order to reach a consensus and agreed standardization in observer scores ahead of on‐farm assessments . It is likely that the protocols adopted in this study would similarly need to be reduced in time if they are to be incorporated into Serbian farm animal welfare standards schemes. Animal welfare assessments in existing farm assurance schemes in the UK have usually been limited to 20–30 min due to time and financial constraints  for animal‐based assessments within their program of annual statutory farm inspections conducted by government veterinary surgeons. While there remain challenges, this provides an example of how an accession country can go beyond base legislative requirements and common practices for animal welfare in the EU, by utilizing the infrastructure already in place for farm inspections – in this case official regulatory inspections, rather than relying solely on private voluntary assurance schemes.Introducing assessment frameworks for the first time in a country undergoing EU accession can stimulate positive actions and training processes directed towards improvements in animal welfare. For EU accession countries, this process of conducting a survey of the welfare of farm animals may be of real value to highlight areas for focused or supported welfare improvement and to assist in achieving EU legislation compliance. It may also serve to raise awareness of welfare assessment among farmers, increase capacity and experience in assessment methods, and increase understanding of regulatory and voluntary mechanisms for welfare improvement among stakeholders. This pilot project has already expanded the sphere of influence with regard to animal welfare in Serbia, by working directly with veterinary surgeons, students, agricultural graduates and farmers, involving them practically in assessments and discussions of direct indicators of welfare. Training has increased the pool of skills in animal‐based assessment. However, there remain clear challenges for trainers, assessors, producers and policy‐makers due to the limited resources and support infrastructure that are currently available.As a pilot study, this first national survey has captured some preliminary, baseline data for setting improvement targets. Pilot study results and experiences suggest that there are areas where further species‐specific training, and improved knowledge and competencies of assessors and farmers in identifying animal health and welfare conditions could inform improvements in farm animal welfare standards. Improved communication and planning of visits for optimal timing, and closer engagement of farmers in animal‐based assessments, could further facilitate adherence to and feasibility of the described protocols and animal handling. While ‘animal welfare’ (‘dobrobit zivotinja’) has only recently been introduced into the Serbian language, seventy‐three percent of farmers had heard of this term. Among those who have heard of animal welfare, there is scope to expand their understanding of what ‘welfare’ incorporates, although it is encouraging that many farmers described ‘welfare’ in positive terms. Many farmers (70%) reported they would like to make changes to their farms, many of which could be beneficial to animal welfare. Expansion, modernization and structural alterations to farms may offer animal welfare benefits, and this study may assist in planning for this due to more refined information on farmer and animal needs. This study supports the view that opportunities for welfare improvement exist, if technical advice and financial support are available, to facilitate change and progress the improvement aspirations of these farmers.This project was part of an EU‐financed project “Farm Animal Welfare Standards in Serbia” (FAWSS). The project was contracted by the Austrian Development Agency from the combined funds of the IPA 2011 programme, the Republic of Austria's assistance programme and Serbian national co‐financing (grant number 526‐00/2011/Grant69).The authors declare no conflict of interest.The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. This on‐farm study involved non‐invasive measures and minimal handling of animals managed on commercial farms, as per routine stockperson assessments and veterinary physical examinations. The study received approval from the Veterinary Office of the Ministry of Agriculture, Forestry and Water Management of Serbia.


Although parenteral nutrition and whole organ allogeneic transplantation have improved the survival of these patients, current therapies are associated with a high risk for morbidity and mortality. Development of methods to propagate adult human intestinal stem cells (ISCs) and pluripotent stem cells raises the possibility of using stem cell‐based therapy for patients with monogenic and polygenic forms of intestinal failure. Organoids have demonstrated the capacity to proliferate indefinitely and differentiate into the various cellular lineages of the gut. Genome‐editing techniques, including the overexpression of the corrected form of the defective gene, or the use of CRISPR /Cas9 to selectively correct the monogenic disease‐causing variant within the stem cell, make autologous ISC transplantation a feasible approach. However, numerous techniques still need to be further optimized, including more robust ex vivo ISC expansion, native ISC ablation, and engraftment protocols. Large‐animal models can to be used to develop such techniques and protocols and to establish the safety of autologous ISC transplantation because outcomes in such models can be extrapolated more readily to humans. S The field of intestinal stem cell biology has exploded over the past 5 years with discoveries related to in vivo and in vitro stem cell identity and function. The goal of this review article is to highlight the potential use of these cells to treat various epithelial disorders of the gut and discuss the various roadblocks that will be encountered in the coming years.Intestinal failure (IF) is a rare multifactorial clinical condition that results in patients' inability to sustain normal growth and nutritional and hydration status without the use of parenteral nutrition (PN) In contrast, patients with the non‐SBS form of IF have normal or reduced small bowel surface area despite a normal‐length bowel. A growing number of congenital disorders of the gut epithelium result in a generalized loss of nutrient absorption capacity . Among tCurrent management of patients with IF involves close dietary modulation of PN and enteral feeds, medical therapy, and surgical intervention. Parenteral nutrition is the mainstay of therapy for patients with IF; however, its long‐term use is associated with various significant limitations, including IF‐associated liver disease Intestinal stem cell (ISC) research has made significant strides in improving the understanding of the unique cell populations involved in self‐renewal and differentiation during homeostatic and stressed conditions Concurrent development of gene‐editing techniques has also made treatment of monogenic disorders more likely. Indeed, the first functional restoration experiments have been performed in ISCs derived from patients with cystic fibrosis using CRISPR /Cas9 genome editing Here we review the current models of stem cell‐driven intestinal epithelial regeneration, with particular emphasis on human models; discuss how advancements in ISCs and gene editing can be applied in management of IF; and address foreseeable obstacles and limitations.The small bowel epithelium is organized into two fundamental structures: villi and crypts . Villi f3H‐thymidine uptake in the intestinal epithelium suggested two models of multipotent ISCs: CBCCs and +4 label‐retaining cells in mouse models Lgr5 locus demonstrate that CBCCs are actively cycling and are long‐lived cells that self‐renew and differentiate into the full assortment of epithelial cells + cells were subsequently shown to produce enteroid structures in an in vitro setting, indicating their stem‐like function Early observations with + cells had been established as the "workhorse stem cells" fueling self‐renewal of the intestine, the cells are by no means hard‐wired; they require niche signals and therefore are particularly prone to injury Bmi1, Lrig1, Hopx, and mTert, suggests that quiescent cells, or a reserve cell population located at the +4 position, are required to reconstitute the CBCCs after injury + cells were ablated, crypts remained intact for at least a week through repopulation of CBCCs with quiescent cells Drosophila models, and a significant gap exists in our understanding of human ISCs.Although Lgr5Ligand‐induced signaling pathways required for intestinal epithelial homeostasis emanate from numerous cell types that constitute the ISC niche. The dynamic interplay between the ISCs and the other cells within the niche creates the system necessary for tissue generation, maintenance, and repair, and for the ultimate design of stem cell therapeutics In the intestine, canonical Wnt signals from Paneth cells (Wnt3a) and the surrounding mesenchyme  act as essential short‐range signals to maintain the ISC compartment INK4BIn contrast, several β‐catenin‐independent noncanonical signaling pathways have been proposed wherein Wnt signaling is mediated through Frizzled proteins and receptor tyrosine kinases, such as Ror2, and are independent from the LRP5/6 coreceptor + cells in in vivo and in vitro settings R‐spondins (Rspo) are a family of cysteine‐rich, thrombospondin type I repeat containing proteins that have an essential role in survival and proliferation of Lgr5Interaction of a Notch receptor with its cell‐bound ligands, such as Dll1 or Dll4, results in proteolytic release of the Notch intracellular domain (NICD) through the actions of the γ‐secretase protease. NICD translocate to the nucleus, where it binds the transcription factor CBF1/Suppressor of Hairless/LAG‐1 (CSL), thus activating transcription of target genes. Notch signaling activates expression of Hes1, which transcriptionally represses Atoh1, the gatekeeper of entry into the secretory lineage in the gut . SimultaThe bone morphogenetic protein (BMP) signaling pathway acts as a negative regulator of crypts, although the exact mechanism remains unknown. BMP‐2 and ‐4 ligands are expressed in the mesenchyme along the villus Epidermal growth factor (EGF) signals exert strong mitogenic effects on ISCs and TA cells by activation of the Ras/Raf/Mek/Erk signaling axis . LuminalIn conclusion, both intrinsic and extrinsic signaling controls self‐renewing or asymmetric division of ISCs. Gene‐targeting mice experiments have led to remarkable progress in the understanding of intercellular mechanisms, especially elucidation of the role of the niche in regulating ISC proliferation and directed differentiation. Wnts are provided by Paneth cells and surrounding mesenchyme, whereas Rspo and BMP inhibitors are distinctly provided from nonepithelial sources http://www.corning.com) assists dynamic patterning and structural self‐formation of complex organ buds Long‐term maintenance and propagation of murine intestinal epithelial cells were first reported when the cells were cocultured with native mesenchymal cellular elements in collagen gels in a growth factor‐independent fashion The reconstituted small bowel enteroids recapitulate in vivo intestinal epithelial structures: single stem cells initially form thin‐walled, balloon‐like cystic structures (spheroid) and subsequently form multiple‐budding, crypt‐like budding structures (enteroid) Although murine small bowel epithelium is culturable with the addition of specific niche factors (ENR), additional factors are required for growth of similar human cells. We successfully grew human small bowel epithelium in 3D Matrigel seeded on a feeder layer of ISEMFs with ENR Fetal intestinal epithelium is more acquiescent to in vitro culture than is its adult counterpart. The differentiation of fetal intestinal epithelium depends on developmental age and intestinal region Recently, a novel mesenchymal‐feeder layer‐based culture condition for human fetal intestinal epithelium has been reported to establish homogeneous clonogenic ISC populations, referred to as ground‐state ISCs Recent advancements with pluripotent stem cells, embryonic (ESCs), or iPSCs have defined the timing of various soluble factors required to generate human intestinal organoids (HIOs) Stem cell therapy is a promising alternative strategy for overcoming the current limitations of IF treatment, but genetic modification of ISCs will be necessary to fully exploit their potential. Through use of recent culture technology, gene editing of ISCs is enabled by the highly amenable and reproducible culture of intestinal epithelium. The clonogenic capacity and rapid expansion of spheroids should permit culture of genetically identical populations of intestinal epithelium. However, optimal methods to expand human ISCs, ablate native in situ ISCs while maintaining a receptive niche, and optimally engraft genetically manipulated ISCs have yet to be described. If successful engraftment of modified ISCs into the ablated small bowel niche of IF patients is possible, autologous ISC transplantation will be a promising treatment option.Although numerous viral and nonviral gene‐editing systems have been developed, optimization has not been described for the use of human ISCs in gene therapy CFTR locus by homologous recombination in cultured ISCs of cystic fibrosis patients Recently, genome engineering with site‐specific nucleases to edit DNA allowed targeted gene knockouts, generated tissue‐specific cell lineage reporters, overexpressed genes from defined loci, and introduced and repaired point mutations in stem cells The prerequisite for ISC therapy for intestinal disorders is successful engraftment of implanted cells. The proof‐of‐concept that donor ISCs can reconstitute a new intestinal epithelial layer after ablation was first demonstrated in 2005 when transplantation of ileal organoids containing crypts and surrounding mesenchyme was successfully engrafted into a denuded jejunal segment + ISC‐derived colonoids were transplanted into dextran sulfate sodium (DSS)‐ablated colonic epithelium of Rag2−/− immunodeficient mice More recently, single murine Lgr5The replacement of diseased epithelium with gene‐corrected autologous ISCs or human leukocyte antigen‐matched allogenic ISCs would be feasible therapeutic approaches for IF with severe monogenic enteropathies. Overexpressing the wild‐type form of the defective gene may be sufficient for treatment. MVID and CTE are among several monogenetic disorders that would be feasible to pursue.MYO5B gene, which codes for a nonconventional motor protein; the loss‐of‐function variants result in abnormal intracellular protein trafficking and defects in recycling of apical vesicles and have abnormalities of cell polarity. Recent whole‐exome sequencing from patients with a milder form of MVID showed biallelic loss‐of‐function mutations in STX3, which is the encoded cellular receptor for transport vesicles in enterocytes. In vitro assessment of patient‐derived enteroids and overexpression cells of truncated STX3 recapitulated most characteristics of variant MVID MVID is an autosomal recessive disorder characterized by chronic profuse watery secretory and malabsorptive diarrhea EpCAM gene, which encodes an epithelial cell adhesion molecule protein, resulting in isolated congenital diarrhea without associated extra digestive symptoms. Specific founder mutations associated with consanguineous pairing of families from the Arabic peninsula and Mexico are particularly common in patients with CTE and is in contrast to MVID, which is associated with significant allelic variant heterogeneity Similarly, patients with CTE present with persistent neonatal diarrhea that is both secretory and malabsorptive in nature. Biopsy of the small bowel demonstrates focal epithelial “tufts,” with villus atrophy and crypt expansion. CTE is associated with biallelic loss‐of‐function mutations of the An obvious approach for ISC gene correction of monogenic disorders, such as MVID or CTE, would be ex vivo isolation, correction, and expansion of ISCs and subsequent implantation . SpecifiAnother promising approach may the use of gene transfer mediated by a recombinant adenovirus‐associated viral (AAV) vector The use of AAV for gene therapy is illustrated by its efficacy in many clinical trials, including those for hemophilia B Although rodent models have demonstrated the feasibility of intestinal stem cell‐based therapies, several gaps and hurdles need to be addressed to optimize the clinical utility of these therapies, particularly for the treatment of monogenetic disorders. For instance, we need to improve our understanding of human ISC and progenitor cell biology, while establishing optimal ablation and engraftment techniques in a large‐animal model, as we continue to collect preclinical evidence of safety and therapeutic efficacy.First, many of the lessons learned from rodent studies do not necessarily translate to human systems. Because most studies of ISCs and their niche were conducted in mouse models, we have a relative incomplete understanding of the human ISCs/progenitor and niche cells. Certainly compared with the mouse, in vitro culture for human cells requires additional growth factors and small molecules, suggesting that the biological properties of human cells are more complex. Among the shortcomings are the absence of selective markers that identify the various nondifferentiated cell populations, both for fluorescence‐activated cell sorting and immunofluorescence Second, to date, regenerative approaches for diffuse intractable diseases have focused on the development of stem cell transplantation. Certainly a prerequisite is a detailed procurement plan to obtain a sufficient number of stem cells because a robust expansion that is sufficient to repopulate the immense surface area of the small bowel has not been described for adult human ISCs. Furthermore, although ISCs can be expanded ex vivo in the presence of growth factors, recombinant proteins are exceedingly expensive. Certainly it may be worth adapting recently methods described for in vitro expansion of human pancreatic β cells generated from iPSCs that use a renewable cell source and a scalable method Third, efficient methods to enrich for subtypes of ISCs are lacking, including those isolated from different regions of the gut or from different developmental ages that might exhibit varying responses to signaling cues that could affect their expandability.Fourth, robust methods to integrate engrafted ISCs into existing intestinal mucosa are still underdeveloped. Previous reported experimental ISC transplantation models in rodents have applied different techniques, and for use in humans these methods need to be both safe and carefully standardized. The first step of transplantation is preparing the diseased ISC for ablation and generating a receptive niche to engraft healthy ISCs. EDTA sequesters divalent cations, resulting in the separation of epithelium from its underlying basement membrane, and can be used with soft serosal compressions or luminal brushings to enhance the extent and uniformity of epithelial separation along the gut Fifth, ensuring the safety and efficacy of stem cell‐based products is a major challenge. Cells manufactured in large quantities outside their natural environment can potentially become ineffective and induce adverse effects, such as tumors or immune responses Finally, the FDA will not allow the use of cells that were maintained in Matrigel because it is a poorly defined extract from an Engelbreth‐Holm‐Swarm tumor in mice. Of course, Matrigel mimics the native basal membrane composed of ECM molecules, such as laminin, collagen type IV, entactin, and heparan sulfate proteoglycans, as well as some growth factors, such as TGF‐β and fibroblast growth factor Therefore, among the current limitations of using ISCs for regenerative medicine is the need to establish a large‐animal model in which the techniques and safety of transplantation methods may be established, including methods of mucosal ablation, and the dosing of implanted cell, optimal ex vivo expansion of ISCs, and the routes of administration may be extrapolated readily to humans.IF is a rare but life‐threatening condition resulting from the inability to maintain the growth and development with nutrition obtained via the enteral route. Although PN and allogeneic intestinal transplantation improve the survival of patients with IF, current therapies have significant long‐term limitations. The recent development of methods for expansion of human ISCs and iPSCs has made it possible to reconstitute complex cellular structures that mimic the native bowel in an in vitro setting. Moreover, when intestinal enteroids were transplanted into suitable donor sites, they gave rise to a complete mucosal epithelium with all cell lineages, and the appropriate ECM enables the growth potential of the neo‐mucosa. Finally, the development of genome editing techniques, such as CRISPR/Cas9, has made genetic engineering of stem cells possible and provides new opportunities to perform autologous stem cell transplantation for IF with severe monogenic enteropathies. Although the methods used to ablate diseased mucosa, dosing and optimal growth of ISCs, and the route of administration still require optimization, the replacement of diseased epithelium with corrected epithelium originating from gene editing of autologous stem cells is a feasible therapeutic approach.S.N.H., J.C.Y.D., M.S., and M.G.M.: manuscript writing, final approval of the manuscript.The authors indicated no potential conflicts of interest.