Synthetic polymers as substrates for a DNA-sliding clamp protein.

The clamp protein (gp45) of the DNA polymerase III of the bacteriophage T4 is known to bind to DNA and stay attached to it in order to facilitate the process of DNA copying by the polymerase. As part of a project aimed at developing new biomimetic data-encoding systems we have investigated the binding of gp45 to synthetic polymers, that is, rigid, helical polyisocyanopeptides. Molecular modelling studies suggest that the clamp protein may interact with the latter polymers. Experiments aimed at verifying these interactions are presented and discussed.

Lack of correlation between predicted and actual off-target effects of short-interfering RNAs targeting the human papillomavirus type 16 E7 oncogene.

BACKGROUND: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. METHODS: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo. RESULTS: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types. CONCLUSION: These data do not support the assumption that actual OTEs correlate well with predicted OTEs.

A retrospective analysis of the histology of resected polyps and colonoscopy quality parameters in Belgium.

Background and aims: adenoma detection rate is a well known quality parameter for colonoscopy. However recently other quality parameters have emerged. We wanted to evaluate the histology of the resected polyps, different quality indicators of colonoscopy and post colonoscopy colorectal cancer (PCCRC) in Belgium and analyzed data about colonoscopies performed between 2008-2015. Methods: Reimbursement data on colorectal related medical procedures from the Intermutualistic Agency were linked with data on clinical and pathological staging of colorectal cancer and with histologic data of resected polyps available at the Belgian Cancer Registry over a period covering 8 years (2008-2015). Results: 298,246 polyps were resected in 294,923 colonoscopies, of which 275,182 were adenomas (92 %) and 13,616 were SSLs (4%). There was a significant but small correlation between the different quality parameters and PCCRC. Post colonoscopy colorectal cancer rate after 3 years was 7.29 %. There were marked geographic differences in Belgium concerning adenoma detection rate, sessile adenoma detection rate and post colonoscopy colorectal cancer. Conclusion: Most resected polyps were adenomas, only a small percentage involved sessile serrated lesions. There was a significant correlation between adenoma detection rate and other quality parameters, and a small but significant correlation between PCCRC and the different quality parameters. The lowest post colonoscopy colorectal cancer rate was reached with an ADR of 31.4 % and a SSL-DR of 1.2 %.

The influence of stocking density on broiler chicken bone quality and fluctuating asymmetry.

Because broiler chickens are juvenile animals undergoing physical development, stocking density during rearing may influence this development. Some of these physical changes may cause welfare problems, for example, decreased bone quality, which may lead to fracture during catching and transport. Others do not influence welfare directly but can be used as indicators of the animal's ability to cope with its environment (e.g., fluctuating asymmetry). The present study evaluates the effect of stocking density on bone quality and fluctuating asymmetry. Birds were stocked at densities of 2.4, 5.8, 8.8, 12.1, 13.6, 15.5, 18.5, and 21.8 birds/m(2) from 1 until 39 d of age. Increased stocking density had a negative effect on some aspects of bone quality (tibia curvature and shear strength). Tibias were shorter at high density, possibly due to increased curvature. Several other bone quality aspects (tibia weight, torsion, and dyschondroplasia, and femur curvature and epiphysis shape) remained unaffected. Middle-toe length was the only character that showed a significant increase with increasing density when each character was analyzed separately. Nevertheless, a composite index of fluctuating asymmetry, which combined data on all 11 measured characters, tended to increase with stocking density. Such increased fluctuating asymmetry may indicate decreased welfare. However, one of the assumptions of fluctuating asymmetry is that the animal is subjected to the same environmental influences on both sides. This assumption may not be fulfilled when leg deformations occur, as these may lead to asymmetric changes in bone growth by altering the division of force over the 2 legs. In addition, leg deformations decrease the accuracy of bone length measurements made in a straight line. This raises some concerns on the applicability of fluctuating asymmetry measurements on broiler chicken legs, especially because stocking density did not effect the asymmetry of the head.

The views of healthcare professionals on self-management of patients with advanced cancer: An interview study.

OBJECTIVES: Self-management of patients with advanced cancer is challenging. Although healthcare professionals may play a crucial role in supporting these patients, scant scientific attention has been paid to their perspectives. Therefore, we examined healthcare professionals' views on self-management and self-management support in this population. METHODS: We conducted qualitative interviews with 27 purposively sampled medical specialists (n = 6), nurse specialists (n = 6), general practitioners (n = 8) and homecare/ hospice nurses (n = 7) in the Netherlands. Transcripts were analysed using thematic analysis. RESULTS: Healthcare professionals experienced self-management of patients with advanced cancer to be diverse, dynamic and challenging. They adopted instructive, collaborative and advisory roles in self-management support for this population. Whereas some professionals preferred or inclined towards one role, others indicated to switch roles, depending on the situation. CONCLUSIONS: Just like patients with advanced cancer, healthcare professionals differ in their views and approaches regarding self-management and self-management support in this population. Therefore, instructive, collaborative and advisory self-management support roles will all be useful under certain circumstances. PRACTICE IMPLICATIONS: Healthcare professionals can support self-management by being aware of their own views and communicating these clearly to their patients and colleagues. Education in self-management support should include self-reflection skills and discuss the relation between self-management and professional care.

The influence of historical geneflow, bathymetry and distribution patterns on the population genetics of morphologically diverse Galápagos' Opuntia echios.

Throughout history, remote archipelagos have repeatedly been designated natural laboratories to study evolutionary processes. The extensive, geographically structured, morphological variation within Galápagos' Opuntia cacti has been presumed to be another example of how such processes shape diversity. However, recent genetic studies on speciation and potential effects of plasticity within this system failed to confirm earlier classification and hypothesized radiation on both global and single island levels. Detailed population genetic information, however, is crucial in conserving these semi-arid ecosystem keystone species. In this article, we re-evaluate the genetics of Opuntia echios inhabiting one of the most taxon rich places on the archipelago: Santa Cruz and its surrounding satellite islands, using microsatellite data. Our analysis revealed high genetic variability within all sampled locations, providing little support for the hypothesis of clonal reproduction. Inter-island gene flow patterns appear to be largely influenced by bathymetry and sea levels during last ice ages. Although O. echios from Seymour Norte are morphologically recognized as being a separate taxon, Daphné Major's cacti are the most differentiated. In addition, we found a potential barrier for gene flow along the ring-like distribution of Opuntias at the western side of Santa Cruz, suggesting potential links with geology.

The normal distribution as appropriate model of developmental instability in Opuntia cacti flowers.

Developmental instability (DI) as measured by fluctuating asymmetry (FA) has been proposed to reflect fitness and stress. Furthermore, the associated developmental buffering may reduce morphological variation, conceal the expression of genetic variation and as such play an important role in evolutionary biology. However, observed associations between FA and various forms of stress and quality appear very heterogeneous. Presently it is difficult to interpret the biological relevance of this heterogeneity because little is known about the link between FA and the underlying process of DI, casting doubt whether DI can be viewed as an individual property and how closely FA reflects the underlying process of DI. Therefore, studies that explicitly test the validity of assumptions of the proposed theoretical models and estimate between-individual variations in DI are needed. We present data on Opuntia cacti floral traits confirming that the normal distribution can be viewed as an appropriate approximation of the distribution of DI and that the concept of hypothetical repeatability can provide useful insights into the interpretation of patterns in FA as a measure of DI. Furthermore, we detected significant between-individual variation in DI. Measuring petals from several flowers within individual plants allowed making inference of individual DI.

Sex and asymmetry in humans: what is the role of developmental instability?

Because asymmetric individuals are less attractive and may suffer from reduced fitness, bilateral asymmetry is widely believed to affect human sexual selection. Its evolutionary significance is based on the presumed relationship with developmental instability (DI). Yet, relationships between DI and bilateral asymmetry are often weak and possibly confounded by asymmetric mechanical loadings because of handedness. We related asymmetry in hands and faces to degrees of handedness and sexual behaviour in 100 humans. Handedness correlated to levels of asymmetry, thereby likely invalidating the use of asymmetry to estimate DI. For facial asymmetry, applying existing theoretical models refuted a link between asymmetry and DI. Explicit statistical modelling at the level of DI confirmed the absence of a link between DI and aspects of sexual behaviour. Nevertheless, asymmetries in both hands and face correlated significantly with sexual behaviour. We conclude that bilateral asymmetry per se, rather than its presumed link with DI, more likely relates to measures of human sexual behaviour. Because lateralization of behaviour appears widespread, evaluating the role of DI in evolution and ecology relies on a very critical selection of traits whose asymmetry can reliably reflect DI.

Evolutionary history shapes the association between developmental instability and population-level genetic variation in three-spined sticklebacks.

Developmental instability (DI) is the sensitivity of a developing trait to random noise and can be measured by degrees of directionally random asymmetry [fluctuating asymmetry (FA)]. FA has been shown to increase with loss of genetic variation and inbreeding as measures of genetic stress, but associations vary among studies. Directional selection and evolutionary change of traits have been hypothesized to increase the average levels of FA of these traits and to increase the association strength between FA and population-level genetic variation. We test these two hypotheses in three-spined stickleback (Gasterosteus aculeatus L.) populations that recently colonized the freshwater habitat. Some traits, like lateral bone plates, length of the pelvic spine, frontal gill rakers and eye size, evolved in response to selection regimes during colonization. Other traits, like distal gill rakers and number of pelvic fin rays, did not show such phenotypic shifts. Contrary to a priori predictions, average FA did not systematically increase in traits that were under presumed directional selection, and the increases observed in a few traits were likely to be attributable to other factors. However, traits under directional selection did show a weak but significantly stronger negative association between FA and selectively neutral genetic variation at the population level compared with the traits that did not show an evolutionary change during colonization. These results support our second prediction, providing evidence that selection history can shape associations between DI and population-level genetic variation at neutral markers, which potentially reflect genetic stress. We argue that this might explain at least some of the observed heterogeneities in the patterns of asymmetry.

scRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation.

BACKGROUND: The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design. RESULTS: This study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types. We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific. CONCLUSIONS: In conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.

Population, environmental, and community effects on local bank vole (Myodes glareolus) Puumala virus infection in an area with low human incidence.

In this study, the distribution of Puumala hantavirus (PUUV) infection in local bank vole Myodes glareolus populations in an area with low human PUUV infection (nephropathia epidemica [NE]) incidence in northern Belgium was monitored for 2 consecutive years. Bank voles were trapped in preferred habitat and tested for anti-PUUV IgG. Infection data were related to individual bank vole features, population demography, and environmental variables. Rare occurrence of PUUV infection was found and PUUV prevalence was low compared with data from the high NE incidence area in southern Belgium. Small-scale climatic differences seemed to play a role in PUUV occurrence, vegetation index and deciduous forest patch size both influenced PUUV prevalence and number of infected voles in a positive way. The data suggested a density threshold in vole populations below which PUUV infection does not occur. This threshold may vary between years, but the abundance of bank voles does not seem to affect the degree of PUUV seroprevalence further. We found indications for a dilution effect on PUUV prevalence, dependent on the relative proportion of nonhost wood mice Apodemus sylvaticus in a study site. In conclusion, we regard the combination of a dilution effect, a possible threshold density that depends on local conditions, and a higher fragmentation of suitable bank vole habitat in our study area as plausible explanations for the sparse occurrence of PUUV infection and low prevalence detected. Thus, beside human activity patterns, local environmental conditions and rodent community structure are also likely to play a role in determining PUUV infection risk for humans.

Parameters influencing the quality of colonoscopy in Belgium : a critical evaluation.

BACKGROUND AND STUDY AIMS: In relation to recent implementation of colorectal cancer screening programs at the regional level, quality assessment of colonoscopy gains more interest in Belgium. In order to evaluate quality indicators of colonoscopies in Belgium, we retrospectively analysed data about colonoscopies performed between 2002-2010. PATIENTS AND METHODS: Coded data concerning number of medical procedures and polypectomy were provided by the Intermutualistic Agency (IMA). This database was used to calculate different quality indicators such as polyp detection rate (PDR), use of sedation, amount of procedures and time interval according to physician and center type. RESULTS: Considerable differences in polyp detection rate (PDR) exist between different physicians and centers. Mean PDR significantly correlated with the number of colonoscopies performed each year. A minimum of 106 colonoscopies per year was identified to maintain competence. Recuperation rate for polyps was low, and time intervals between colonoscopies were generally too short in comparison to European and international guidelines. CONCLUSIONS: In absence of a central colonoscopy registry in Belgium, our results were based on reimbursement data. Other quality parameters, although accuracy is questionable (eg. bowel cleansing and withdrawal time) are not systematically registered. Despite these difficulties, we were able to demonstrate that a minimum amount of 106 colonoscopies per year is necessary to maintain competence. The results from this large database can be used as a foundation to work out a quality colonoscopy bundle.

Transfection by cationic gemini lipids and surfactants.

Diseases that are linked to defective genes or mutations can in principle be cured by gene therapy, in which damaged or absent genes are either repaired or replaced by new DNA in the nucleus of the cell. Related to this, disorders associated with elevated protein expression levels can be treated by RNA interference via the delivery of siRNA to the cytoplasm of cells. Polynucleotides can be brought into cells by viruses, but this is not without risk for the patient. Alternatively, DNA and RNA can be delivered by transfection, i.e. by non-viral vector systems such as cationic surfactants, which are also referred to as cationic lipids. In this review, recent progress on cationic lipids as transfection vectors will be discussed, with special emphasis on geminis, surfactants with 2 head groups and 2 tails connected by a spacer.

Cost-effectiveness of home-based vs. in-hospital treatment of paediatric tuberculous meningitis.

SETTING: Cape Town, South Africa, 2014. OBJECTIVE: To assess the societal costs and cost-effectiveness of home-based vs. in-hospital treatment of paediatric tuberculous meningitis. DESIGN: This was an economic evaluation from a societal perspective using probabilistic analysis. Health care, informal care, lost productivity costs and costs in other sectors, health-related quality of life (HRQoL) and family impact were assessed during interviews with care givers, children, medical staff and management. RESULTS: Societal costs for home-based treatment were USD3857, and USD28 043 for in-hospital treatment. Home-based vs. in-hospital treatment HRQoL scores were 90.9% vs. 84.5%, while family impact scores were 94.8% vs. 73.1%. The point estimate of the incremental cost-effectiveness ratio indicated that improving HRQoL and family impact by 1% was associated with a saving of respectively USD3726 and USD1140 for home-based vs. in-hospital treatment. The probability that home-based treatment was less expensive and more effective than in-hospital treatment was 96.3% for HRQoL and 100% for family impact. CONCLUSION: Societal costs of home-based treatment were lower than for in-hospital treatment. Children treated at home had a better HRQoL and family impact scores. Home-based treatment was a cost-effective alternative to in-hospital treatment of drug-susceptible tuberculous meningitis.

Fluctuating asymmetry as an animal welfare indicator -- a review of methodology and validity.

It has been suggested that fluctuating asymmetry (FA) reflects an animal's ability to cope with the sum of challenges during its growing period and, thus, is a potential welfare indicator. In this review we investigate the evidence of associations between FA and other welfare indicators measured at the level of the individual and of effects of welfare-relevant environmental conditions on FA in populations of captive birds and mammals including humans. As the question of validity cannot be treated independently from the quality of the available data, first a checklist for the proper measurement and analysis of FA is drafted and used to evaluate the methodological quality of the various studies. We recommend this checklist to be used as a standard for future FA studies. We found 17 relevant studies on associations between FA and other welfare indicators, and 36 studies on effects of welfare-relevant factors on FA. Frequent methodological shortcomings or insufficient methodological information allow for only cautious conclusions. The proportion of significant results supporting the link between higher FA and poorer welfare is only moderately high. Independent from statistical significance, almost all studies found the relationship between FA and welfare to be prevailingly in the expected direction. FA is a promising measure of animal welfare, despite a great number of open questions, e.g. relating to the ontogeny of FA or its sensitivity to various stressors. The considerable potential of FA as a welfare indicator makes it worthwhile to pursue more intensely validation studies as well as applied studies. These studies should pay particular attention to an appropriate methodological approach.

Experimental stress does not increase fluctuating asymmetry of broiler chickens at slaughter age.

Fluctuating asymmetry (FA) is increasingly applied as a putative indicator of animal welfare. Yet its sensitivity to measure welfare of ad libitum-fed farm animals (that presumably have little or no energy allocation constraints) remains largely untested. This study was conducted to examine whether FA is sensitive to experimentally induced stress in broiler chickens and whether effect sizes differ between emotional and physical stressors. Broiler chickens were randomly assigned to emotional stress treatments (pain or frustration), physical stress treatments (wet litter or high temperature and density), or no stress treatment (control). Both physical stressors, unlike the emotional stressors, were known to affect a number of conventional welfare indicators measured at slaughter age. Left-right asymmetry of 14 bilateral traits was measured at slaughter age and compared between treatments. Seven of the 14 bilateral traits proved unsuitable for the study of FA, either due to the presence of directional asymmetry or high measurement error. Fluctuating asymmetry tended to be lowest in the control group and highest in the high temperature and density treatment. However, either when modeling traits as repeated measures at individual broiler level or when performing trait-by-trait analysis, no significant differences between treatments were detected. This negative result may indicate that FA is not a suitable indicator to detect variations of welfare status in fast-growing broiler chickens because of strong past selection for increased BW and improved feed efficiency, which can mask additional stress effects on developmental processes. Alternatively, FA is not a sensitive indicator of welfare in ad libitum-fed animals because of absence of energy allocation constraints. Finally, FA may still be a suitable indicator of welfare under such conditions, but differences between treatments may remain undetected due to insufficient statistical power, which was estimated at 35% for our study.

Fluctuating asymmetry in broiler chickens: a decision protocol for trait selection in seven measuring methods.

Nonidentical development of bilateral traits due to disturbing genetic or developmental factors is called fluctuating asymmetry (FA) if such deviations are continuously distributed. Fluctuating asymmetry is believed to be a reliable indicator of the fitness and welfare of an animal. Despite an increasing body of research, the link between FA and animal performance or welfare is reported to be inconsistent, possibly, among other reasons, due to inaccurate measuring protocols or incorrect statistical analyses. This paper reviews problems of interpreting FA results in poultry and provides guidelines for the measurement and analysis of FA, applied to broilers. A wide range of morphological traits were measured by 7 different techniques (ranging from measurements on living broilers or intact carcasses to X-rays, bones, and digital images) and evaluated for their applicability to estimate FA. Following 4 selection criteria (significant FA, absence of directional asymmetry or antisymmetry, absence of between-trait correlation in signed FA values, and high signal-to-noise ratio), from 3 to 14 measurements per method were found suitable for estimating the degree of FA. The accuracy of FA estimates was positively related to the complexity and time investment of the measuring method. In addition, our study clearly shows the importance of securing adequate statistical power when designing FA studies. Repeatability analyses of FA estimates indicated the need for larger sample sizes, more repeated measurements, or both, than are commonly used in FA studies.

An efficient algorithm for large-scale detection of protein families.

Detection of protein families in large databases is one of the principal research objectives in structural and functional genomics. Protein family classification can significantly contribute to the delineation of functional diversity of homologous proteins, the prediction of function based on domain architecture or the presence of sequence motifs as well as comparative genomics, providing valuable evolutionary insights. We present a novel approach called TRIBE-MCL for rapid and accurate clustering of protein sequences into families. The method relies on the Markov cluster (MCL) algorithm for the assignment of proteins into families based on precomputed sequence similarity information. This novel approach does not suffer from the problems that normally hinder other protein sequence clustering algorithms, such as the presence of multi-domain proteins, promiscuous domains and fragmented proteins. The method has been rigorously tested and validated on a number of very large databases, including SwissProt, InterPro, SCOP and the draft human genome. Our results indicate that the method is ideally suited to the rapid and accurate detection of protein families on a large scale. The method has been used to detect and categorise protein families within the draft human genome and the resulting families have been used to annotate a large proportion of human proteins.

Comparison of the inter- and intra-observer repeatability of three gait-scoring scales for sows.

Most gait-scoring scales for pigs have a limited number of categories, supposedly to improve repeatability. However, reducing the number of categories could lead to loss of information if the observers' discriminative capacities are underused. With a recently estimated within-herd prevalence of sow lameness of 8.8% to 16.9% in the European Union and the associated losses, the availability of reliable tools for the timely detection of initial cases warrants attention. This study investigated the intra- and inter-observer repeatability (intra-OR and inter-OR) of three gait-scoring scales for sows: a continuous 'tagged' visual analogue scale (tVAS, measured in mm), a 5-point and a 2-point ordinal scale (5P and 2P), all with the same descriptors. Veterinary medicine students (n=108) were trained to use the scales and then asked to score 90 videos (30 per scale) of sows with normal and abnormal gait. Thirty-six videos were shown once and 18 were randomly shown three times, of which one mirrored horizontally. The students' opinions on the scales were also collected. Intra- and inter-OR were higher with the tVAS than the 2P scale (inter-OR: 0.73 v. 0.60; P<0.05. Intra-OR: 0.80 v. 0.67; P<0.05). Intra-OR was higher with the 5P (0.81) than the 2P scale (0.67; P<0.05). For all three scales, repeatabilities were lower (P<0.05) for non-lame sows (gait score of ⩽45 mm on the tVAS) than for sows showing some signs of lameness (gait score>45 mm). Video order (first 45 v. last 45 clips), mirroring, users' opinions on the scales, and previous declared experience in handling pigs or scoring lameness in other species had no effect on repeatabilities. Correlations between the students' and experts' scores were high (tVAS=0.92; 5P=0.91; 2P=0.88) but the association for the 2P was not linear and the frequency distribution showed lower correlations for a group of students. This study confirms recent evidence that it is possible to design high-resolution gait-scoring scales that do not reduce observer repeatability. Visual gait-scoring scales with fewer than five categories are likely to entail loss of information on lameness in individual sows.

Cage size and enrichment effects on the bone quality and fluctuating asymmetry of fattening rabbits.

We studied the effect of increased cage size on different aspects of bone quality (bone strength, diameter, weight, and length) and fluctuating asymmetry (FA). Such characteristics may indicate improved animal welfare, as greater bone quality may decrease fracture incidence during handling, whereas decreased FA has been suggested to signal decreased stress. As stress is likely not only influenced by the quantity of space, but also by its quality, we also studied the effect of environmental enrichment. Groups of 8 rabbits were housed in wire open-top cages of 0.40, 0.46, 0.53, 0.64, 0.80, 1.07, and 1.60 m(2) from weaning until slaughter. All cages of 0.40 and 0.46 m(2) (12 cages/size) were left barren to allow sufficient mobility. One-half of the larger cages were enriched with a wooden-enrichment structure that could be used to gnaw on, hide in, or lie in (6 cages per size × enrichment treatment). Increased cage size led to an increase in tibiofibula diameter (P = 0.008), a tendency for increased tibiofibula weight (P = 0.051), and decreased FA (P = 0.010), suggesting improved welfare. Bone length and strength were not affected by cage size (P > 0.1). Enrichment did not affect FA (P > 0.1), in contrast with our expectations based on previous glucocorticoid analysis. This discrepancy between indicators may be due to sensitivity to other types of stressors or different sensitive periods. In summary, rabbits housed in larger cages had wider, heavier bones, but the absence of changes in bone strength indicate that this is unlikely to result in decreased fracture incidence. In larger cages, FA was lower, suggesting a favorable effect on welfare, whereas no effect of enrichment was shown.