The ossification of the pectoral girdle and wing skeleton of the quail (Coturnix coturnix japonica)

The onset of ossification centres of the pectoral girdle and wing skeleton of the quail (Coturnix coturnix japonica) has been studied in embryos and juvenile birds, which were sampled daily from the 4th day of incubation up to the 12th day after hatching. Specimens, which were cleared and were stained with Alcian Blue and Alizarin Red S, were examined at the stereomicroscope. The furcula ossified with intramembranous ossification at the 7th day of incubation. The first centres of perichondral ossification in the scapula and the coracoid bones were observed at the 7th day of incubation. In the humerus, radius and ulna, perichondral ossification was observed at the 6th day of incubation. The carpal bones were ossified between the 8th and 10th day of the post-hatching period. The 2nd metacarpal bone began to ossify at the 6th day of the post-hatching period, whereas the 3rd and 4th metacarpal bones were ossified at the 7th day of incubation. At the 9th day of incubation, ossification was observed in the 1st phalanx of the alular digit and in the phalanges of the major digit. The 2nd phalanx of the alular digit began to ossify at the 12th, whereas the phalanx of the minor digit at the 14th day of incubation. The data presented here provide useful baseline information on the normal sequential pattern of ossification in the pectoral girdle and wing skeleton in this species.     

Skeletal development in blue‐breasted quail embryos

The blue‐breasted quail (Coturnix chinensis), the smallest species of quail with short generation interval and excellent reproductive performance, is a potential avian research model. A normal series of skeletal development of avian embryos could be served as a reference standard in the fields of developmental biology and teratological testing as well as in the investigation of mutation with skeletal abnormalities and in the study of the molecular mechanisms of skeletal development through genome manipulation. Furthermore, ossification sequence shows a species‐specific pattern and has potential utility in phylogeny. However, data on the skeletal development of blue‐breasted quail embryos are scarce. Here, we established a series of normal stages for the skeletal development of blue‐breasted quail embryos. Cartilage and ossified bones of blue‐breasted quail embryos were stained blue and red with Alcian blue 8GX and Alizarin red S, respectively. The time and order of chondrification and calcification of their skeletons were documented every 24 hr from 3 to 17 days of incubation, and a 15‐stage series of skeletal development was created. Moreover, a comparative study with the Japanese quail (Coturnix japonica) demonstrated that ossification sequence differed significantly between these two species.     

The Ossification of the Pelvic Girdle and Leg Skeleton of the Quail (Coturnix coturnix japonica)

The onset of ossification centres of the pelvic girdle and leg skeleton of the quail in embryos and juvenile birds were studied. Specimens, which were cleared and were stained with Alcian Blue and Alizarin Red S, were examined at the stereomicroscope. The ilium and the pubis began to ossify at the 8th day (E8), whereas the ischium at E9. Perichondral ossification was observed at E6 in the femur, tibia and fibula. A secondary ossification centre was detected in the proximal epiphysis of the tibiotarsus at the 15th post‐hatching day (P15). The patella began to ossify at P30. Regarding the tarsal bones tibiale, pre‐tibiale and fibulare, ossification was observed at the E15, E12 and E16, respectively. The metatarsals II, III, IV ossified at E7, whereas the metatarsal I at E11. The centres of ossification of the 1st phalanges of all digits were observed at E9. At the same day, the ossification centres of the 2nd phalanx of digits II and III as well as the 3rd phalanx of digit III appeared. At E10, ossification was observed in the 2nd phalanx of digit I, in the 3rd phalanx of digit II and in the 2nd and 3rd phalanx of digit IV. In the 4th phalanx of digit III and in the terminal phalanges of digit IV, ossification was observed at E11. The data presented here provide useful baseline information on the normal sequential pattern of ossification in the pelvic girdle and leg skeleton in this species.     

Effect of Maternal Lead Exposure on Craniofacial Ossification in Rat Fetuses and the Role of Antioxidant Therapy

Lead exposure during intrauterine life was found to result in reduced birth weight, impaired skeletal development and post‐natal neurotoxic effects. In this study, the effect of pre‐natal exposure to different doses of lead on the development of craniofacial skeleton in rat fetuses was investigated. Vitamin E was tested as a concomitant treatment, aiming to improve the fetotoxic effects of lead. Positively pregnant female rats were randomly divided into four groups; groups I and II (L250 and L500), exposed to lead acetate in doses of 250 and 500 mg/l respectively, group III (L500 + E), exposed to lead acetate (500 mg/l) wit concomitant vitamin E and group IV (Control) which was given sodium acetate only. All the treatments started from the first day of gestation till the 20th day, where all rats were sacrificed and the fetuses were recovered. Fetuses were processed to alizarin red staining for ossified components. Twenty‐seven bones of the craniofacial skeleton were studied in each fetus where the ossification was scored as being complete, delayed or absent. In all studied fetuses from all groups, changes were found only in eight bones while the remaining craniofacial bones were normally ossified. In affected bones there was a significant decrease in the number of completely ossified bones; associated with a significant increase of both partially ossified and absent bones in L₂₅₀ and L₅₀₀ treated groups when compared to the control group. These differences were more significant in the L₅₀₀ treated group. Giving vitamin E improved the percentage of completely ossified craniofacial bones and decreased the percentage of both partially ossified and absent bones. The most affected bone was presphenoid, then to a lesser extent supraoccipital, squamosal, parietal, interparietal and frontal bone respectively. In conclusion, lead exposure to rats during pregnancy led to varying degrees of fetal growth retardation as well as delayed ossification of some craniofacial bones which were dose dependent and the concomitant supplementation with vitamin E greatly improved the deleterious effect of lead.     

Osteological Development of the Garfish (Belone belone) Larvae

Garfish, Belone belone (Linnaeus, 1761) is an elongate, slander fish inhabiting the Eastern Atlantic and Mediterranean Sea. These fish from the Belonidae (Actinopterygii) family have important commercial value for Croatian fisheries. Samples for research were collected from experimental hatching in the Novigrad Sea. Preparation techniques included fixation in buffered formalin, trypsin clearing and staining with alcian blue and alizarin red. As little is known of garfish osteology and bone morphology, the main goal of this study was to describe ossification process in garfish fry. At hatching, no skeletal structure is present. Newly-hatched larvae also had no osteological elements. Ossification started at 7 day post-hatching (DPH) [total length (TL) 18 mm] with head bones and vertebral neural arch. Head skeleton continued to develop mostly over the period from 7 to 10 DPH. At 21 DPH (TL 49 mm), ossification process seemed to be finished, but it was not possible to distinguish borders of all bones. The primary interest of our research was to understand the growth dynamics as well as transformation of supporting body elements from cartilage to bone. At the end, developmental characteristics and functional aspects of this formation in different fish species are discussed.     

Mycoplasma meleagridis-induced lesions in the tarsometatarsal joints of turkey embryos

Mycoplasma meleagridis (MM) has the ability to cause bone deformity in turkey poults. However, few pathological lesions have been described and no evidence of MM-induced damage to the bones has been shown. In this study, 17-day-old turkey embryos were inoculated with MM into the allantoic cavity. On the 27th day, eight of the 22 embryos presented with curved toes. Scanning electron microscopy of the tarsometatarsal joints showed fissures in the cartilage. Histological sections of the joints revealed only the infiltration of cells with eosinophilic granules. Immunohistochemical staining (IHS) showed the presence of MM in the aggregates of the bone marrow cells and the cells with eosinophilic granules. Some of these cells were harvested by laser capture microdissection (LCM), lysed, and used as template DNA. With a pair of MM-specific primers in a conventional polymerase chain reaction (PCR), a gene product was amplified, and it comigrated with the MM DNA, which indicates that these captured cells contained MM DNA. Thus, this research shows that inoculation of MM into the turkey embryos produced joint lesions and caused cellular infiltration within the bones.     

Ossification and Growth Rates of the Limb Long Bones during the Prehatching Period in the Quail (Coturnix coturnix japonica)

The timing of ossification and the growth of six long bones of the prehatching period in the quail was studied. Ninety-nine quail eggs were incubated and in total nine fetuses were selected daily from the sixth to the sixteenth day of incubation. The fetuses were stained with alizarine and alcian blue double colouration. The fetuses were studied under the stereoscopic microscope and linear measurements were obtained from the humerus, ulna, radius, femur, tibia and fibula. The first appearance of the primary ossification centres in the diaphysis of the studied bones was found to occur between the sixth and the seventh day of incubation. Different growth patterns between the bones of the leg and of the wing were observed. Humerus and tibia showed the greatest growth rate while the radius and fibula showed the lowest.     

Epiphyseal cartilage canal blood supply to the distal femur of foals

REASONS FOR PERFORMING STUDY: The developmental pattern of the cartilage canal blood supply to epiphyseal growth cartilage has been linked to osteochondrosis (OC) in the tarsus of foals. This pattern has not yet been described in the distal femur, another site frequently affected by OC. OBJECTIVE: To describe the developmental pattern of the blood supply to the distal femoral epiphyseal growth cartilage in 8 Standardbred foals age 0-7 weeks. METHODS: One foal was sacrificed weekly from birth to age 7 weeks (n=8) to undergo a barium perfusion procedure to demonstrate vessels within cartilage canals of one hindlimb. The distal end of the femur was cleared in methyl salicylate and perfused vessels were studied in the intact bones. Each distal femur was then sawed into 5 mm thick slabs in the transverse plane, and the slabs decalcified and radiographed. Finally, the lateral trochlear ridge was separated from each slab and examined histologically. RESULTS: The cartilage canal blood supply regressed with increasing age, but several regions remained vascularised in the oldest foal at age 7 weeks. Vessels arose from perichondrial and subchondral arterial sources, and coursed perpendicular or parallel to the ossification front. The midsection of parallel vessels became incorporated into the ossification front during growth. Anastomoses formed and vessels within the distal portion of canals with an original perichondrial source shifted to use subchondral vessels as their arterial source. Both parallel and perpendicular vessels therefore traversed the ossification front to enter cartilage canals. No histological lesions were observed in sections from any of the foals. CONCLUSION: The same anatomical feature (traversing the ossification front to enter cartilage canals) reported to render vessels vulnerable to failure in the tarsus was also present in the distal femur of foals. POTENTIAL RELEVANCE: OC may occur by the same pathogenetic mechanism in the distal femur as in the tarsus of foals.     

Radiographic studies of ossification of the hoof cartilage of horses and its clinical evaluation]

Radiographic examination is a valuable means for the identification of ossified lateral cartilage. The extent and stages of development of the ossification can be determined. The active stage of development of ossified lateral cartilage, which by itself can provoke lameness, can be defined. By means of the X-ray it is tried to differentiate the three kinds of ossification: the enchondral, the peri- and parachondral ossification of the lateral cartilage. Centres of ossification and fractures of the ossified lateral cartilage are described.     

Prolonged gestation in a Holstein cow: adenohypophyseal aplasia and skeletal pathology in the offspring

A Holstein fetus was delivered by Caesarean section at a gestational age of 441 days. The pituitary pars distalis was aplastic and the adrenal and thyroid glands were severely hypoplastic. Arrested or retarded cartilage cell maturation resulted in absence or minimal development of epiphyseal ossification centers, delayed ossification of carpal bones, and arrest of longitudinal growth of bones. The pathophysiology of prolonged gestation and of skeletal pathology is discussed.     

A study of ossification of carpal and tarsal bones in normal and hypothyroid foals

The degree of ossification of carpal and tarsal bones was determined in normal foals of various ages and in hypothyroid and thyroidectomized foals. In normal foals ossification occurred very rapidly in the last few weeks of gestation and less rapidly from birth to 33 days. The ulnar carpal bone was consistently less ossified than other carpal or tarsal bones. Foals with congenital hyperplastic goitre had retarded ossification of the cuboidal bones, especially the central and third tarsal bones. Thyroidectomized foals had retarded ossification of lesser degree.     

Canine diffuse idiopathic skeletal hyperostosis

The radiographic and necropsy findings in a case of canine diffuse idiopathic skeletal hyperostosis are reported. Radiographically, the disease was characterized by progressive development of para-articular mineral densities along joint surfaces of the appendicular skeleton; progressive osseous fusion of the ilium, pubis, femur, and ischium; and progressive flowing ossification of the dorsal spinous processes of cervical and thoracic vertebrae. The para-articular lesions were considered to develop as a consequence of fibrocartilaginous proliferation followed by endochondral ossification. Progression of the ossification process led to para-articular joint fusion and almost complete ossification of the metaplastic cartilage. A distinguishing feature was the sparing of intra-articular structures.     

EVALUATION OF THE VARIABLY OSSIFIED COLLATERAL CARTILAGES OF THE DISTAL PHALANX AND ADJACENT ANATOMIC STRUCTURES IN THE FINNHORSE WITH COMPUTED TOMOGRAPHY AND MAGNETIC RESONANCE IMAGING

Six Finnhorse cadaver forefeet were selected to represent radiographically different types and grades of ossification of the collateral cartilages of the distal phalanx. These cartilages and adjacent tissues were evaluated with computed tomography (CT) and high field magnetic resonance imaging (MRI). In CT the internal structure of the cartilages was consistent, but in MRI some differences were noted. The shape of the collateral cartilages and their ligamentous attachments varied. The border between ossified and non-ossified cartilage appeared distinct, with considerable variation in the extent of the ossified area in regard to the cross-sectional area of the cartilage. Ossification originating from the palmar processes and extending in the proximaVpalmaroproximal direction, without separate centers of ossification, generally appeared smooth and inactive. Palmar ossification followed the irregular shape of the cartilage. Separate centers of ossification had a medullary cavity or were sclerotic. Presence ofamedullary cavity or sclerosis were also found at the base of the cartilages. The incomplete fusion lines between separate centres of ossification and the ossified base of the cartilage varied from congruent and inactive to reactive with marked sclerosis, flared margins and parachondral changes. Incomplete fusion may be clinically significant. Local conformational adaptations of the hoof were also documented with extensive ossification of the collateral cartilage.     

Postnatal Osteological Development of the Hyoid Bone in the New Zealand White Rabbit

This study augments knowledge of bone growth by observing the development of the hyoid bone in the New Zealand White rabbit. Preserved hyoid bones representing five different age periods, each period including five individuals and the total number of animals being 25, were fixed in 3.5% formaldehyde solution and 95% ethanol, followed by a pure acetone bath. They were then stained with an alcian blue–alizarin red combination. The mode of bone formation was intracartilaginous type ossification. While the basihyoideum and thyrohyoideum were observed to start ossifying first at prenatal stage, indicating that they are the main skeletal structures of the hyoid apparatus, the ceratohyoideum and lingual process began to ossify in the second and third periods. The separately occurring primary ossification centres fused completely among themselves in the fifth period. Because no further ossification centers were observed and the ossified parts continued the development and growth, the research was terminated after 10 weeks of age.     

Is there an association between ossification of the cartilages of the foot and collateral desmopathy of the distal interphalangeal joint or distal phalanx injury?

Reasons for performing study: Associations between degree of ossification of the cartilages of the foot and injuries to other structures of the foot have been suggested, but have not been investigated by large scale studies. Objectives: To describe the frequency of grade >3 ossification of the cartilages of the foot (possibly significant ossification, PSO), mediolateral symmetry of ossification and left‐right symmetry between feet; and to investigate associations between PSO and injury of either the collateral ligaments (CLs) of the distal interphalangeal (DIP) joint or the distal phalanx. Hypotheses: Possibly significant ossification of the cartilages of the foot is associated with CL and distal phalanx injury. Distal phalanx injury is associated with a mediolateral difference in ossification grade of ≥2. Methods: Horses were examined for lameness localised to the foot by perineural analgesia, and underwent radiographic and magnetic resonance imaging examinations. Age, breed, occupation, duration of lameness, lame(st) limb, primary cause of lameness, and presence or absence of CL injury were recorded. Dorsopalmar (dorsoplantar) radiographs were examined and ossification of the cartilages of the foot graded using a modification of a previously published scale. Results: One foot from each of 462 horses was included for analysis. There was left‐right symmetry of ossification between feet, and significant association between grades of each foot, with lateral ≥medial cartilages. Possibly significant ossification occurred in the maximally ossified cartilage in 59 (12.8%) feet. There were significant associations between PSO of the maximally ossified cartilage of the foot and injuries of both the CLs of the DIP joint and the distal phalanx. There was no association between distal phalanx injury and marked asymmetry of the ossified cartilages of the foot. Conclusions and clinical relevance: Extensively ossified cartilages of the foot are significantly associated with CL or distal phalanx injury. Markedly asymmetric ossification did not increase the likelihood of distal phalanx injury and should be considered at a prepurchase examination.     

A Case of Bronze Turkey Twins Developing from Hatching

A double‐yolked egg that was laid by an 11‐month‐old bronze turkey hen on 2 April, 2013 was reported. The presented case of developing turkey twin embryos is the first ever described case for this species. The egg weighed 125 g and had 74.6 mm in length and 53.0 mm in width. The embryos died between the 23rd and 24th day of incubation. Both twin 1 and twin 2 were fully developed with body weights of: 37.7 g and 40.3 g, respectively. The likely causes of death include inappropriate arrangement of embryos and insufficient gas exchange in the last period of incubation. The embryos were arranged along the long axis of the egg, one underneath the other, and were closely adhering to one another. The twins were different in plumage pigmentation. In twin 1, a spontaneous mutation of plumage pigmentation (B) was observed, whereas twin 2 showed appropriate pigmentation (b+) in respect to parents.     

Some observations on the development of the long bones of ratite birds

Observations were made on the long bones of 16 young ratite birds (10 rheas, 3 emus and 3 ostriches), from 1-day-old to 12 weeks of age.
At hatching all long bones contained large cartilaginous cones which were continuous with the growth plates, and an osseous cortex. At one week of age ossification had commenced on the periphery of these embryonic cones and in some bones the cones had become separated from the growth plates. At 3 weeks of age the embryonic cones of cartilage were still present in the proximal and distal tibio-tarsi and narrow cartilaginous bridges connected the cones to the growth plates. Embryonic cones were not present in other long bones of this 3-week-old rhea nor in the long bones of ratites 6, 8 and 12 weeks of age. Other praecocial birds such as turkey poults and chickens have cones of embryonic cartilage in their long bones at hatching and these persist in the tibio-tarsi till one to 2 weeks of age. The presence of large cones of embryonic cartilage in the tibio-tarsal bones of 3-week-old ratite birds is probably a normal phenomenon. Awareness of this feature is necessary for the correct differential diagnosis of the prevalent musculoskeletal disorders of ratite birds.     

FRACTURES OF THE DISTAL PHALANX AND ASSOCIATED SOFT TISSUE AND OSSEOUS ABNORMALITIES IN 22 HORSES WITH OSSIFIED SCLEROTIC UNGUAL CARTILAGES DIAGNOSED WITH MAGNETIC RESONANCE IMAGING

Ungual cartilage ossification in the forelimb is a common finding in horses. Subtle abnormalities associated with the ungual cartilages can be difficult to identify on radiographs. Magnetic resonance (MR) imaging findings of 22 horses (23 forelimbs) with a fracture of the distal phalanx and ossified ungual cartilage were characterized and graded. All horses had a forelimb fracture. Eleven involved a left forelimb (seven medial; four lateral), and 12 involved a right forelimb (five medial; seven lateral). All fractures were nonarticular, simple in configuration, and nondisplaced. The fractures were oriented in an axial proximal to abaxial distal and palmar to dorsal direction, and extended from the base of the ossified ungual cartilage into the distal phalanx. The fracture involved the fossa of the collateral ligament on the distal phalanx in 17 of 23 limbs. The palmar process and ossified ungual cartilage was abnormally mineralized in all horses. Ligaments and soft tissues adjacent to the ossified ungual cartilages were affected in all horses. The routine site of fracture in this study at the base of the ossified ungual cartilage extending into the distal phalanx suggests a biomechanical cause or focal stress point from cycling. The ligamentous structures associated with the ungual cartilages were often affected, showed altered signal intensity as well as enlargement and were thought to be contributing to the lameness. In conclusion, ossified ungual cartilages may lead to fracture of the palmar process of the distal phalanx and injury of the ungual cartilage ligaments.     

Scintigraphic examination of the cartilages of the foot

REASONS FOR PERFORMING STUDY: Radiographic examination of the cartilages of the foot is well documented; however, there is limited information about their scintigraphic assessment. OBJECTIVES: To evaluate the scintigraphic appearance of the cartilages of the foot using subjective and quantitative image analysis and to correlate radiographic and scintigraphic findings. HYPOTHESES: An ossified cartilage would have similar radiopharmaceutical uptake (RU) to the ipsilateral aspect of the distal phalanx; RU would extend throughout the length of the ossified cartilage; a separate centre of ossification (SCO) would be identified on a scintigraphic image; and fracture or trauma to an ossified cartilage would manifest as increased RU (IRU). METHODS: Front feet (n = 223) of horses (n = 186) that had dorsopalmar radiographic views and dorsal scintigraphic images were included in the study. The cartilages of the foot were graded radiographically and scintigraphically. Quantitative evaluation of the scintigraphic images was carried out using region of interest (ROI) analysis. For statistical analysis RU ratios were used. Correlations between a radiographically detected SCO and focal RU and between IRU and radiographic abnormalities were assessed. RESULTS: There was a good correlation and an excellent agreement between radiographic and scintigraphic grades. ROI analysis showed a proximal to distal increase in RU ratios within each cartilage of the foot. A radiographically identified SCO could be detected scintigraphically in 12/17 feet (70.6%). Thirty-eight feet had IRU in the region of a cartilage, 25 of which (65.8%) had corresponding radiographic abnormalities. Fracture of an ossified cartilage was associated with IRU in all horses. CONCLUSIONS AND POTENTIAL RELEVANCE: Scintigraphy may give information about the potential clinical significance of ossification of the cartilages of the foot and associated lesions, therefore prompting further investigation by use of a uniaxial ipsilateral palmar nerve block and imaging, using either magnetic resonance imaging and/or computed tomography.     

Closed fluoroscopic-assisted spinal arch external skeletal fixation for the stabilization of vertebral column injuries in five dogs

Objective— To evaluate outcome after closed fluoroscopic-assisted application of spinal arch external skeletal fixators in dogs with vertebral column injuries.
Study Design— Retrospective case series.
Animals— Dogs with traumatic vertebral column injuries (n=5).
Methods— Medical records of dogs with vertebral column fractures and/or luxations stabilized with spinal arch external skeletal fixator frames applied using a closed fluoroscopic-assisted technique were reviewed. Owners were contacted to obtain long-term clinical outcomes.
Results— Five dogs (age range, 6–72 months; weight, 10–54 kg) had traumatic vertebral column injuries stabilized with spinal arch external skeletal fixators applied in closed fashion. Injuries involved vertebral segments of the thoracolumbar junction, lumbar spine, and lumbosacral junction. Immediately postoperatively, 4 dogs had anatomic alignment of their vertebral fracture/luxation; 1 dog had 1 mm of vertebral canal height compromise. Time to fixator removal ranged from 65 to 282 days (141±87 days). All dogs had regained satisfactory neurologic function by 3 months. At long-term follow-up (range, 282–780 days; mean 445±190 days) all dogs were judged to have good to excellent return of function by their owners.
Conclusion— Successful closed fluoroscopic-assisted application of external skeletal fixators using spinal arches provided satisfactory reduction with few complications in 5 dogs. Return to function was judged to be good to excellent in all dogs at long-term evaluation.
Clinical Relevance— Closed fluoroscopic-assisted application of ESF using spinal arches provided satisfactory reduction and effective stabilization of spinal fractures with few complications and should be considered as a treatment approach.