Effects of lighting schedule during incubation of broiler chicken embryos on leg bone development at hatch and related physiological characteristics.

Providing a broiler chicken embryo with a lighting schedule during incubation may stimulate leg bone development. Bone development may be stimulated through melatonin, a hormone released in darkness that stimulates bone development, or increased activity in embryos exposed to a light-dark rhythm. Aim was to investigate lighting conditions during incubation and leg bone development in broiler embryos, and to reveal the involved mechanisms. Embryos were incubated under continuous cool white 500 lux LED light (24L), continuous darkness (24D), or 16h of light, followed by 8h of darkness (16L:8D) from the start of incubation until hatching. Embryonic bone development largely takes place through cartilage formation (of which collagen is an important component) and ossification. Expression of genes involved in cartilage formation (col1α2, col2α1, and col10α1) and ossification (spp1, sparc, bglap, and alpl) in the tibia on embryonic day (ED)13, ED17, and at hatching were measured through qPCR. Femur and tibia dimensions were determined at hatch. Plasma growth hormone and corticosterone and pineal melatonin concentrations were determined every 4h between ED18.75 and ED19.5. Embryonic heart rate was measured twice daily from ED12 till ED19 as a reflection of activity. No difference between lighting treatments on gene expression was found. 24D resulted in higher femur length and higher femur and tibia weight, width, and depth at hatch than 16L:8D. 24D furthermore resulted in higher femur length and width and tibia depth than 24L. Embryonic heart rate was higher for 24D and 16L:8D in both its light and dark period than for 24L, suggesting that 24L embryos may have been less active. Melatonin and growth hormone showed different release patterns between treatments, but the biological significance was hard to interpret. To conclude, 24D resulted in larger leg bones at hatch than light during incubation, but the underlying pathways were not clear from present data.

Light-dark rhythms during incubation of broiler chicken embryos and their effects on embryonic and post hatch leg bone development.

There are indications that lighting schedules applied during incubation can affect leg health at hatching and during rearing. The current experiment studied effects of lighting schedule: continuous light (24L), 12 hours of light, followed by 12 hours of darkness (12L:12D), or continuous darkness (24D) throughout incubation of broiler chicken eggs on the development and strength of leg bones, and the role of selected hormones in bone development. In the tibiatarsus and femur, growth and ossification during incubation and size and microstructure at day (D)0, D21, and D35 post hatching were measured. Plasma melatonin, growth hormone, and IGF-I were determined perinatally. Incidence of tibial dyschondroplasia, a leg pathology resulting from poor ossification at the bone's epiphyseal plates, was determined at slaughter on D35. 24L resulted in lower embryonic ossification at embryonic day (E)13 and E14, and lower femur length, and lower tibiatarsus weight, length, cortical area, second moment of area around the minor axis, and mean cortical thickness at hatching on D0 compared to 12L:12D especially. Results were long term, with lower femur weight and tibiatarsus length, cortical and medullary area of the tibiatarsus, and second moment of area around the minor axis, and a higher incidence of tibial dyschondroplasia for 24L. Growth hormone at D0 was higher for 24D than for 12L:12D, with 24L intermediate, but plasma melatonin and IGF-I did not differ between treatments, and the role of plasma melatonin, IGF-I, and growth hormone in this process was therefore not clear. To conclude, in the current experiment, 24L during incubation of chicken eggs had a detrimental effect on embryonic leg bone development and later life leg bone strength compared to 24D and 12L:12D, while the light-dark rhythm of 12L:12D may have a stimulating effect on leg health.

Proteomics analysis of the zebrafish skeletal extracellular matrix.

The extracellular matrix of the immature and mature skeleton is key to the development and function of the skeletal system. Notwithstanding its importance, it has been technically challenging to obtain a comprehensive picture of the changes in skeletal composition throughout the development of bone and cartilage. In this study, we analyzed the extracellular protein composition of the zebrafish skeleton using a mass spectrometry-based approach, resulting in the identification of 262 extracellular proteins, including most of the bone and cartilage specific proteins previously reported in mammalian species. By comparing these extracellular proteins at larval, juvenile, and adult developmental stages, 123 proteins were found that differed significantly in abundance during development. Proteins with a reported function in bone formation increased in abundance during zebrafish development, while analysis of the cartilage matrix revealed major compositional changes during development. The protein list includes ligands and inhibitors of various signaling pathways implicated in skeletogenesis such as the Int/Wingless as well as the insulin-like growth factor signaling pathways. This first proteomic analysis of zebrafish skeletal development reveals that the zebrafish skeleton is comparable with the skeleton of other vertebrate species including mammals. In addition, our study reveals 6 novel proteins that have never been related to vertebrate skeletogenesis and shows a surprisingly large number of differences in the cartilage and bone proteome between the head, axis and caudal fin regions. Our study provides the first systematic assessment of bone and cartilage protein composition in an entire vertebrate at different stages of development.

A 3D analysis of flight behavior of Anopheles gambiae sensu stricto malaria mosquitoes in response to human odor and heat.

Female mosquitoes use odor and heat as cues to navigate to a suitable landing site on their blood host. The way these cues affect flight behavior and modulate anemotactic responses, however, is poorly understood. We studied in-flight behavioral responses of females of the nocturnal malaria mosquito Anopheles gambiae sensu stricto to human odor and heat. Flight-path characteristics in a wind tunnel (flow 20 cm/s) were quantified in three dimensions. With wind as the only stimulus (control), short and close to straight upwind flights were recorded. With heat alone, flights were similarly short and direct. The presence of human odor, in contrast, caused prolonged and highly convoluted flight patterns. The combination of odor+heat resulted in longer flights with more landings on the source than to either cue alone. Flight speed was greatest (mean groundspeed 27.2 cm/s) for odor+heat. Odor alone resulted in decreased flight speed when mosquitoes arrived within 30 cm of the source whereas mosquitoes exposed to odor+heat maintained a high flight speed while flying in the odor plume, until they arrived within 15 cm of the source. Human odor evoked an increase in crosswind flights with an additive effect of heat at close range (<15 cm) to the source. This was found for both horizontal and vertical flight components. However, mosquitoes nevertheless made upwind progress when flying in the odor+heat generated plume, suggesting that mosquitoes scan their environment intensively while they progress upwind towards their host. These observations may help to improve the efficacy of trapping systems for malaria mosquitoes by (1) optimizing the site of odor release relative to trap entry and (2) adding a heat source which enhances a landing response.

Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio).

Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.

Contribution of postnatal collagen reorientation to depth-dependent mechanical properties of articular cartilage.

The collagen fibril network is an important factor for the depth-dependent mechanical behaviour of adult articular cartilage (AC). Recent studies show that collagen orientation is parallel to the articular surface throughout the tissue depth in perinatal animals, and that the collagen orientations transform to a depth-dependent arcade-like structure in adult animals. Current understanding on the mechanobiology of postnatal AC development is incomplete. In the current paper, we investigate the contribution of collagen fibril orientation changes to the depth-dependent mechanical properties of AC. We use a composition-based finite element model to simulate in a 1-D confined compression geometry the effects of ten different collagen orientation patterns that were measured in developing sheep. In initial postnatal life, AC is mostly subject to growth and we observe only small changes in depth-dependent mechanical behaviour. Functional adaptation of depth-dependent mechanical behaviour of AC takes place in the second half of life before puberty. Changes in fibril orientation alone increase cartilage stiffness during development through the modulation of swelling strains and osmotic pressures. Changes in stiffness are most pronounced for small stresses and for cartilage adjacent to the bone. We hypothesize that postnatal changes in collagen fibril orientation induce mechanical effects that in turn promote these changes. We further hypothesize that a part of the depth-dependent postnatal increase in collagen content in literature is initiated by the depth-dependent postnatal increase in fibril strain due to collagen fibril reorientation.

Postnatal development of depth-dependent collagen density in ovine articular cartilage.

BACKGROUND: Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Adult AC is characterised by a depth-dependent composition and structure of the extracellular matrix that results in depth-dependent mechanical properties, important for the functions of adult AC. Collagen is the most abundant solid component and it affects the mechanical behaviour of AC. The current objective is to quantify the postnatal development of depth-dependent collagen density in sheep (Ovis aries) AC between birth and maturity. We use Fourier transform infra-red micro-spectroscopy to investigate collagen density in 48 sheep divided over ten sample points between birth (stillborn) and maturity (72 weeks). In each animal, we investigate six anatomical sites (caudal, distal and rostral locations at the medial and lateral side of the joint) in the distal metacarpus of a fore leg and a hind leg. RESULTS: Collagen density increases from birth to maturity up to our last sample point (72 weeks). Collagen density increases at the articular surface from 0.23 g/ml ± 0.06 g/ml (mean ± s.d., n = 48) at 0 weeks to 0.51 g/ml ± 0.10 g/ml (n = 46) at 72 weeks. Maximum collagen density in the deeper cartilage increases from 0.39 g/ml ± 0.08 g/ml (n = 48) at 0 weeks to 0.91 g/ml ± 0.13 g/ml (n = 46) at 72 weeks. Most collagen density profiles at 0 weeks (85%) show a valley, indicating a minimum, in collagen density near the articular surface. At 72 weeks, only 17% of the collagen density profiles show a valley in collagen density near the articular surface. The fraction of profiles with this valley stabilises at 36 weeks. CONCLUSIONS: Collagen density in articular cartilage increases in postnatal life with depth-dependent variation, and does not stabilize up to 72 weeks, the last sample point in our study. We find strong evidence for a valley in collagen densities near the articular surface that is present in the youngest animals, but that has disappeared in the oldest animals. We discuss that the retardance valley (as seen with polarised light microscopy) in perinatal animals reflects a decrease in collagen density, as well as a decrease in collagen fibril anisotropy.

Postnatal development of collagen structure in ovine articular cartilage.

BACKGROUND: Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries) as our model animal. RESULTS: Predominant collagen orientation is parallel to the articular surface throughout the tissue depth for perinatal cartilage. This remodels to the Benninghoff structure before the sheep reach sexual maturity. Remodelling of predominant collagen orientation starts at a depth just below the future transitional zone. Tissue retardance shows a minimum near the articular surface at all ages, which indicates the presence of zonal differentiation at all ages. The absolute position of this minimum does change between birth and maturity. Between different anatomical sites, we find differences in the dynamics of collagen remodelling, but no differences in adult collagen structure. CONCLUSIONS: The collagen network in articular cartilage remodels between birth and sexual maturity from a network with predominant orientation parallel to the articular surface to a Benninghoff network. The retardance minimum near, but not at, the articular surface at all ages shows that a zonal differentiation is already present in the perinatal animals. In these animals, the zonal differentiation can not be correlated to the collagen network orientation. We find no difference in adult collagen structure in the nearly congruent metacarpophalangeal joint, but we do find differences in the dynamics of collagen network remodelling.

Modeling optical behavior of birefringent biological tissues for evaluation of quantitative polarized light microscopy.

Quantitative polarized light microscopy (qPLM) is a popular tool for the investigation of birefringent architectures in biological tissues. Collagen, the most abundant protein in mammals, is such a birefringent material. Interpretation of results of qPLM in terms of collagen network architecture and anisotropy is challenging, because different collagen networks may yield equal qPLM results. We created a model and used the linear optical behavior of collagen to construct a Jones or Mueller matrix for a histological cartilage section in an optical qPLM train. Histological sections of tendon were used to validate the basic assumption of the model. Results show that information on collagen densities is needed for the interpretation of qPLM results in terms of collagen anisotropy. A parameter that is independent of the optical system and that measures collagen fiber anisotropy is introduced, and its physical interpretation is discussed. With our results, we can quantify which part of different qPLM results is due to differences in collagen densities and which part is due to changes in the collagen network. Because collagen fiber orientation and anisotropy are important for tissue function, these results can improve the biological and medical relevance of qPLM results.

Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton.

Retinoic acid (RA) plays important roles in diverse biological processes ranging from germ cell specification to limb patterning. RA ultimately exerts its effect in the nucleus, but how RA levels are being generated and maintained locally is less clear. Here, we have analyzed the zebrafish stocksteif mutant, which exhibits severe over-ossification of the entire vertebral column. stocksteif encodes cyp26b1, a cytochrome P450 member that metabolizes RA. The mutant is completely phenocopied by treating 4 dpf wild-type embryos with either RA or the pharmacological Cyp26 blocker R115866, thus identifying a previously unappreciated role for RA and cyp26b1 in osteogenesis of the vertebral column. Cyp26b1 is expressed within osteoblast cells, demonstrating that RA levels within these cells need to be tightly controlled. Furthermore, we have examined the effect of RA on osteoblasts in vivo. As numbers of osteoblasts do not change upon RA treatment, we suggest that RA causes increased activity of axial osteoblasts, ultimately resulting in defective skeletogenesis.

A functional analysis of myotomal muscle-fibre reorientation in developing zebrafish Danio rerio.

The fast muscle fibres in the anterior trunk of teleost fish are primarily responsible for large amplitude undulatory swimming motions. Previous theoretical studies suggested that the near-helical arrangement of these fibres results in a (fairly) uniform distribution of fibre strain and work output during swimming. However, the underlying simplifications of these studies precluded unequivocal support for this hypothesis. We studied the fast muscle-fibre reorientation and the concomitant myotomal strain variance in a body segment near the anus during larval and juvenile development in the zebrafish. From 2 to 4 days post fertilization (d.p.f.), the measured angles between the muscle fibres and the longitudinal axis of the zebrafish were small. Yet, onset of a near-helical muscle-fibre arrangement was recognized. Juveniles of 51 d.p.f. have larger mean fibre angles and already possess the near-helical pattern of adult teleosts. We present a model that computes the distribution of the strain along the muscle fibres from measured muscle-fibre orientations, body curvature and prescribed tissue deformations. We selected the most extreme body curvatures, which only occur during fast starts and turning manoeuvres. Using the model, we identified the (non-linear) tissue deformations that yield the least variance in the muscle-fibre strain. We show that simple beam theory cannot reliably predict the strain distribution: it results in very small strains and negligible work output of the most medial fibres. In our model, we avoided these functional limitations by adding a shear deformation to the simple beam deformation. At 2 d.p.f., the predicted variance in the muscle-fibre strain for the shear deformation optimized for strain uniformity is fairly small, due to the small variation in the fibre distances to the medial plane that is caused by the relatively large spinal cord and notochord. The predicted minimal strain variance increases sharply from 2 d.p.f. to 3 d.p.f., remains relatively large at 4 d.p.f., but decreases again considerably at 15 and 39 d.p.f. The 51 d.p.f. stage exhibits the smallest variance in the fibre strains (for the identified optimal deformation), in spite of the widely varying muscle-fibre distances to the medial plane. The non-linear nature of the body deformations with the least strain variance implies an interesting optimization constraint: the juvenile muscle-fibre arrangement results in small predicted spatial strain variations at large-amplitude body curvatures, at the (modest) expense of a large coefficient of variation for small curvatures. We conclude that larval fish rapidly change their muscle-fibre orientations (probably in response to mechanical signals). Within the theoretically examined plausible range of deformations, the closest correspondence to a uniform strain field was found for the juvenile stage.

Automated visual tracking for studying the ontogeny of zebrafish swimming.

The zebrafish Danio rerio is a widely used model organism in studies of genetics, developmental biology, and recently, biomechanics. In order to quantify changes in swimming during all stages of development, we have developed a visual tracking system that estimates the posture of fish. Our current approach assumes planar motion of the fish, given image sequences taken from a top view. An accurate geometric fish model is automatically designed and fit to the images at each time frame. Our approach works across a range of fish shapes and sizes and is therefore well suited for studying the ontogeny of fish swimming, while also being robust to common environmental occlusions. Our current analysis focuses on measuring the influence of vertebra development on the swimming capabilities of zebrafish. We examine wild-type zebrafish and mutants with stiff vertebrae (stocksteif) and quantify their body kinematics as a function of their development from larvae to adult (mutants made available by the Hubrecht laboratory, The Netherlands). By tracking the fish, we are able to measure the curvature and net acceleration along the body that result from the fish's body wave. Here, we demonstrate the capabilities of the tracking system for the escape response of wild-type zebrafish and stocksteif mutant zebrafish. The response was filmed with a digital high-speed camera at 1500 frames s(-1). Our approach enables biomechanists and ethologists to process much larger datasets than possible at present. Our automated tracking scheme can therefore accelerate insight in the swimming behavior of many species of (developing) fish.

Adaptive bone formation in acellular vertebrae of sea bass (Dicentrarchus labrax L.).

Mammalian bone is an active tissue in which osteoblasts and osteoclasts balance bone mass. This process of adaptive modelling and remodelling is probably regulated by strain-sensing osteocytes. Bone of advanced teleosts is acellular yet, despite the lack of osteocytes, it is capable of an adaptive response to physical stimuli. Strenuous exercise is known to induce lordosis. Lordosis is a ventrad curvature of the vertebral column, and the affected vertebrae show an increase in bone formation. The effects of lordosis on the strain distribution in sea bass (Dicentrarchus labrax L.) vertebrae are assessed using finite element modelling. The response of the local tissue is analyzed spatially and ontogenetically in terms of bone volume. Lordotic vertebrae show a significantly increased strain energy due to the increased load compared with normal vertebrae when loaded in compression. High strain regions are found in the vertebral centrum and parasagittal ridges. The increase in strain energy is attenuated by a change in architecture due to the increased bone formation. The increased bone formation is seen mainly at the articular surfaces of the vertebrae, although some extra bone is formed in the vertebral centrum. Regions in which the highest strains are found do not spatially correlate with regions in which the most extensive bone apposition occurs in lordotic vertebrae of sea bass. Mammalian-like strain-regulated bone modelling is probably not the guiding mechanism in adaptive bone modelling of acellular sea bass vertebrae. Chondroidal ossification is found at the articular surfaces where it mediates a rapid adaptive response, potentially attenuating high stresses on the dorsal zygapophyses.

Lordotic vertebrae in sea bass (Dicentrarchus labrax L.) are adapted to increased loads.

Lordosis in fish is an abnormal ventral curvature of the vertebral column, accompanied by abnormal calcification of the afflicted vertebrae. Incidences of lordosis are a major problem in aquaculture and often correlate with increased swimming activity. To understand the biomechanical causes and consequences of lordosis, we mapped the morphological changes that occur in the vertebrae of European sea bass during their development from larva to juvenile. Our micro-CT analysis of lordotic and non-lordotic vertebrae revealed significant differences in their micro-architecture. Lordotic vertebrae have a larger bone volume, flattened dorsal zygapophyses and extra lateral ridges. They also have a larger second moment of area (both lateral and dorso-ventral) than non-lordotic vertebrae. This morphology suggests lordotic vertebrae to be adapted to an increased bending moment, caused by the axial musculature during increased swimming activity. We hypothesize the increase in swimming activity to have a two-fold effect in animals that become lordotic. The first effect is buckling failure of the axial skeleton due to an increased compressive load. The second effect is extra bone deposition as an adaptive response of the vertebrae at the cellular level, caused by an increased strain and strain rate in these vertebrae. Lordosis thus comprises both a buckling failure of the vertebral column and a molecular response that adapts the lordotic vertebrae to a new loading regime.