Expression of oestrogen receptor alpha during development of the skeleton in mice fetuses: immunohistochemical study

Sequential pattern of ossification and expression of oestrogen receptor alpha (ERalpha) during development of the skeleton in male and female mice fetuses was investigated. Twenty-seven mice fetuses of gestational age between 14.5 and 18.5 days post coitum (p.c.) were examined by haematoxylin-eosin and toluidine blue staining to determine the ossification. The presence of ERalpha was detected by immunostaining using ERalpha-specific antibodies. Ossification centres were determined in fetuses of 14.5 days p.c. of both sexes in the base of skull, ribs and front limbs, while in the mandible ossification was observed only in female fetuses at that age. ERalpha was found in all investigated tissues in which the occurrence of ossification centres was determined. ERalpha was first detected in some tissues involved in ossification at 14.5 days p.c. in fetuses of both genders. There were some minor gender differences in the pattern of ERalpha expression. ERalpha was localized in the metatarsal chondrogenic condensations at 14.5 days p.c. and in phalangeal chondrocytes at 17.5 and 18.5 days p.c. only in females. ERalpha-positive osteogenic cells at 14.5 days p.c. in the mandible were seen only in females. At 16.5 days p.c. male but not female fetuses expressed ERalpha in the vertebrae. Our findings support the view that ERalpha protein is found in the tissues that undergo bone formation and that ERalpha expression in these tissues shows only minor gender differences in mice fetuses.     

In utero infection of swine fetuses with infectious bovine rhinotracheitis virus (bovine herpesvirus-1)

The pathogenesis of infectious bovine rhinotracheitis (IBR) virus (bovine herpesvirus-1) was studied in porcine fetuses after in utero inoculation. Laparotomies were performed on 8 seronegative pregnant sows at 34 to 86 days of gestation, and all fetuses in 1 uterine horn of each sow were exposed to IBR virus via inoculation into the amniotic sacs. Fetuses in the other horn served as controls. Clinical signs of infection were not observed in the sows, except for 2 sows that aborted at postinoculation days (PID) 11 and 15. Fetuses of the remaining 6 sows were collected at slaughter on PID 15 to 28. Fetuses were examined for gross abnormalities, presence of IBR virus in tissues, and the formation of neutralizing antibodies to IBR virus. Of 33 inoculated fetuses from 6 sows, 10 were mummified, 11 were hemorrhagic and/or edematous, and 12 were alive. Necrotic lesions were observed on the skin and in the liver of dead and live fetuses. Virus was recovered from 29 of 33 inoculated fetuses. Infectious bovine rhinotracheitis virus was isolated from fetal skin, liver, lungs, kidney, spleen, stomach contents, brain, amniotic fluid, and placenta. Virus was isolated from 4 of 11 fetuses recovered from 1 aborting sow. Antibodies to IBR virus were not detected in sera from the sows. However, antibodies were detected in 6 of 15 fetuses inoculated at 63 to 86 days of gestation and collected at slaughter at 86 to 112 days of gestation. The youngest fetus with detectable IBR antibody was estimated to be 74 days of gestation by measuring crown-rump length of the fetus.     

Cellular and humoral responses of Brucella abortus-infected bovine fetuses

Fifty-nine bovine fetuses naturally and experimentally infected with Brucella abortus were studied. Lymphoid hyperplasia in multiple lymph nodes, lymphoid depletion in the thymic cortex, adrenal cortical hyperplasia, and disseminated inflammatory foci composed mainly of large mononuclear leukocytes were present in infected fetuses. Histopathologic changes in naturally infected fetuses were indistinguishable from those infected fetuses inoculated in utero. Fetuses inoculated with 1.0 X 10(3) to 1.0 X 10(5) colony-forming units of strain 2308 B abortus were aborted on postinoculation day (PID) 7 to 19. Fetuses obtained by PID 9 and 10 had increased immunoglobulin concentrations and antibody. Increased cortisol values were present in fetuses obtained as early as PID 6. The initial fetal inflammatory response was composed of large mononuclear leukocytes. In fetuses obtained by PID 9 to 10, moderate numbers of neutrophils mixed with mononuclear leukocytes were present in the inflammatory foci. This shift in the initial inflammatory reaction coincided with the appearance of agglutinating antibody.     

Kinetics and kinematics of the passage

Reasons for performing study: The load acting on the limbs and the load distribution between fore‐ and hindlimbs while performing specific dressage exercises lack objective assessment. Hypothesis: The greater a horse's level of collection, the more load is shifted to the rear and that during the passage the vertical load on the limbs increases in relation to the accentuated vertical movement of the centre of mass. Methods: Back and limb kinematics, vertical ground reaction force and time parameters of each limb were measured in 6 Grand Prix dressage horses performing on an instrumented treadmill at the trot and the passage. Horses were ridden by their own professional rider. Results: At the passage, horses moved at a slower speed (?43.2%), with a lower stride frequency (?23.6%) and, therefore, higher stride impulses (+31.0%). Relative stance duration of fore‐ and hindlimbs and suspension duration remained unchanged. While at the trot the diagonal limbs impacted almost simultaneously, the hindlimbs always impacted first at the passage; the time dissociation between landing and lift‐off remained unchanged. Because of the prolonged stride duration, stride impulse and consequently limb impulses were higher at the passage in the fore‐ as well as in the hindlimbs (+24.8% and +39.9%, respectively). Within the diagonal limb pair, load was shifted from the forehand to the hindquarters (percentage stride impulse carried by the forehand ?4.8%). Despite the higher impulses, peak vertical forces in the fore‐ and hindlimbs remained unchanged because of the prolonged absolute stance durations in fore‐ and hindlimbs (+28.1% and +32.2%, respectively). Conclusions: Based on the intralimb timing, the passage closely resembles the trot. Compared to other head‐neck positions, the higher degree of collection resulted in a pronounced shift in impulse towards the hindquarters. Despite the higher limb impulses, peak forces acting on the limbs were similar to those observed at the trot. Potential clinical relevance: An understanding of load distribution between fore‐ and hindlimbs in relation to different riding techniques is crucial to prevent wear‐and‐tear on the locomotor apparatus.     

Radiographic assessment of cattle hoofs (ungulae) of the Nellore breed: Reference standards

The purpose of this work was to determine a radiographic standard of normality for lateral and medial ungulae in 3‐year‐old bovine females of the Nellore breed. Twenty‐six cows underwent radiographic exposure of the medial and lateral ungulae of the hindlimbs and forelimbs; abaxioaxial projections were used. The images were analyzed; distances were measured between the dorsal surface of the distal phalanx and the surface of the hoof capsule. The two angles formed, i.e., an angle between the dorsal surface of the distal phalanx and a line parallel to the sole (inner), and an angle between the dorsal surface of the hoof and a line parallel to the sole (outer), were also recorded. The results suggested a standard of normality for the proximal, middle and distal distances of the ungulae of the forelimbs: 0.96 ± 0.12 cm. For the measurement of hindlimbs, except for the distal distances of the side ungulae, the suggested value was 1.02 ± 0.14 cm; for the distal measurement of the lateral ungulae of the hindlimbs, the value was 1.09 ± 0.12 cm. For inner and outer angles, the values were 36.45° ± 4.4° for the medial ungulae of the forelimbs and 42.50° ± 3.8° for other ungulae of the forelimbs and hindlimbs (standard deviations of the values suggested for the angles should be used as variations between different animals, limbs and ungulae; they should not be used between angles of the same ungula).     

Occurrence of Blood Cells and Serum Proteins in Bovine Fetuses and Calves

Hematological values of peripheral blood were determined for bovine fetuses and calves of various ages. Erythrocyte values increased through gestation. Fetuses 100 days or older had total values within the ranges of those reported for normal adult cattle. Mature erythrocytes were not observed in embryos and only a few were observed in fetuses 40 days of age. Fetuses 250 days or older had only a few rubricytes (<10/100 WBC). Leukocytes were first identified in the peripheral blood of a 45-day old fetus. Absolute leukocyte values increased through gestation and reached maximum values shortly before parturition. Granulocytes were first observed at 130 days of gestation and reached maximum values near parturition.

Total serum protein and gamma-globulin concentrations of colostrum-deprived calves were similar to serum protein and gammaglobulin concentrations of fetuses older than 265 days and were lower than values for the colostrum-fed calves. The immunoelectrophoretic pattern of 59-day old fetuses, the earliest age at which serum samples were obtained, demonstrated albumin, an α1 globulin and a β globulin, possibly transferrin. Additional α and β globulins appeared in the older fetuses and by 175 days of gestation serum electophoretic patterns of the fetuses were similar to patterns normally found with adult bovine serum except for the absence of the gammaglobulins in fetal serum. Immunoglobulin M was detected in 39 of 95 fetal serum samples by radial diffusion and in 13 of 95 samples by immunoelectrophoresis. Immunoglobulin G was detected in ten of 95 fetal serum samples by radial diffusion and in six of 95 samples by immunoelectrophoresis.

     

Maternal and fetal toxicity of poison hemlock (Conium maculatum) in sheep

Poison hemlock (Conium maculatum) was toxic to pregnant ewes and their fetuses when fed during gestation days 30 through 60. Maternal effects included trembling, muscular weakness in the neck initially, then progressing to the limbs, ataxia, frequent urination and defecation, and death. Convulsive seizures were not observed. Fetotoxic effects included excessive flexure of the carpal joints with lateral deviation in the front limbs and kinked tails. At term, 7 of 11 lambs had varying degrees of the limb abnormalities, but all lambs appeared clinically normal at 8 weeks after parturition.     

Morphometric analysis of growth retardation in fetal lambs following experimental infection of pregnant ewes with Border Disease virus

The onset of growth retardation was investigated in fetal lambs following experimental infection of pregnant ewes with Border Disease virus ( ) on day 53 of pregnancy. Fetuses from control and infected ewes were harvested at weekly intervals between day 60 and day 95 of gestation and morphometric studies were completed on tibial radiographs and tibial growth cartilage metaphyseal junctions. Mean tibial areas were significantly reduced (P<0·01) in fetuses from infected ewes at 35 and 42 days after infection and growth cartilage metaphyseal junctions were less mature in fetuses from infected ewes at 42 days after infection. Positive immunostaining for BDV antigen was demonstrated in the brains of all fetuses from infected ewes between 14 and 42 days after infection. Attempts to demonstrate BDV antigen in bone proved unsuccessful. It is concluded that intrauterine growth retardation is an early manifestation of BDV infection in lambs and that the process is initiated shortly following infection of the fetus.     

The development of the inner layer of backfat in fetal and young pigs

The development of the innermost layer of backfat was studied histologically and histochemically in fetuses from Yorkshire sows at 45, 60, 65, 75, 85, 90, 105, 110, and 112 d of gestation. Samples were also obtained from fetuses at 110 d of gestation from Ossabaw sows and from sows selected for high (obese) or low (lead) backfat thickness. Fetuses from some sows were decapitated at 45 d of gestation and samples were obtained from these fetuses at 110 d of gestation. The inner layer develops before 75 d of gestation and has stromal-vascular characteristics that clearly distinguish it from the middle layer of backfat. For instance, a well organized and dense collagen matrix immediately surrounds small fat cell clusters in the inner layer. Most fat cell cluster capillaries (in the inner layer) reacted strongly for ATPase histochemically and are histologically immature. The inner layer morphology and cell size was similar in Ossabaw, obese and lean fetuses. The number of PAS (Periodic Acid Schiff) positive cells were dependent on fetal strain. The number was highest in obese fetuses and lowest in Ossabaws. Comparison of decapitated fetuses and weight-matched controls indicated that the inner layer was most affected by decapitation. A decreased organization and amount of collagen was associated with larger fat cell clusters in decapitated fetuses, and most fat cell cluster capillaries reacted little for ATPase histochemically and were histologically mature. The range in inner-layer fat cell diameter was wider in decapitated fetuses (11 to 55 micron) than in control fetuses (11 to 30 micron). These results demonstrate that the characteristics of the inner layer are clearly different from the other layers and that the amount and organization of collagen was inversely associated with the size of fat cell clusters and fat cells and with the maturity of the adipocyte-associated capillaries.     

Development of the skeleton in mouse fetuses of both sexes between 14.5 and 18.5 days post coitum

Ossification during embryogenesis of female and male mouse fetuses between 14.5 and 18.5 days post coitum (d.p.c.) was investigated with two methods. The Alizarin-red based method allows differential staining of cartilage and bone tissue on whole animal, however standard histochemical methods allow detailed identification of cells involved in ossification process. There were minor differences in the time of occurrence of some ossification centers between male and female mouse fetuses in a period of 14.5 - 18.5 d.p.c. At 14.5 d.p.c. ossification of female but not male mandible was observed and at 18.5 d.p.c. histochemistry demonstrated ossification in the phalanges in male but not in female.     

A Macro‐Anatomical Investigation of the Some Skull Bones of Nehring's Blind Mole Rats (Spalacidae: Nannospalax nehringi)

This study was aimed to demonstrate the specific anatomical features of the skull bones of Nehring's blind mole rats. Eight skulls, belonging to animals of both sexes, were used. The occipital squama contributed to the formation of the caudal portion of the skull roof. The foramen magnum was quite large. The external occipital crest was present only in the males. The parietal bones formed the middle portion of the skull roof together with the well‐developed inter‐parietal bones. The zygomatic process of the temporal bone formed the zygomatic arch by extending to the temporal process of the zygomatic bone. The zygomatic process of the frontal bone and the frontal process of the zygomatic bone laterally bordered the orbit. There was a single septal process of the nasal bone. Each ramus of the mandibula had four processes. The mandibular ramus had an angular process on its caudal rim, which extended dorsolaterally. The dorsal free end of the mandibular ramus possessed a coronoid process. In the back, there were two other processes, situated medially and laterally. The medially situated process was referred to as a condylar process, and the laterally situated process was referred to as an alveolar process. The alveolar process detected on the mandibular ramus has not been reported in any rodent species, other than those of the family Spalacidae. Blind mole rats can be a real eye‐opener for evolutionary science. The burrowing rodents are key to answering a controversial question about how new species arise.     

The histology of developing porcine adipose tissue

At each of the following days after conception (45, 60, 75, 90 and 105), pig fetuses were removed from sows representing lean and fat stains. From two additional litters, postnatal pigs were sacrificed at 1, 3, 6, 9, 12, 15, 18 and 21 d. Pelikan dye was injected into fetuses and pigs. The whole of the dorsal subcutaneous tissue, including some underlying muscle, was removed. Tissue was fixed into paraffin blocks or was frozen. Paraffin and frozen sections were stained and examined for stromal-vascular and cellular changes during growth. Organized stromal-vascular changes occurred during a period of adipocyte formation from 45 d gestation until 9 d postnatally. At 45 d gestation, the subcutaneous tissue contained many short unorganized connective tissue fibers. Gradually, these fibers became more organized in a ventral to dorsal and caudal to cranial gradient, so that by 1 d postnatally, they formed complete lobules around all existing fat cell clusters. The presumptive adipose space of the complete lobules contained delicate strands of connective tissue and reacted metachromatically for mucin. Connective tissue around lobules became progressively thinner throughout the remaining postnatal ages. Vascularity of the subcutaneous tissue increased as the stromal became organized. Lipid was not present in the subcutaneous tissue at 45 d gestation, but some deposition was apparent in the inner layer at 60 d. Between 60 d gestation and 9 d postnatally, fat cells filled both subcutaneous layers in a ventral to dorsal formation. Presumptive adipose lobules were the source of adipocytes and capillaries of developing fat cell clusters. Adipocytes from fetuses through 1-d postnatal pigs were multilocular, while unilocular fat cells were first observed at 3 d. At 9 d, multilocular adipocytes were found singly or in groups within unilocular fat cell lobules.     

Radiographic features of the limbs of juvenile and subadult loggerhead sea turtles (Caretta caretta)

This study aimed to provide the normal radiographic anatomic appearance of the limbs of the loggerhead sea turtle, Caretta caretta. Dorsopalmar and dorsoplantar radiographs were taken of the forelimbs and hindlimbs of 15 juvenile and 15 subadult loggerhead sea turtles, 17 alive and 13 dead. For comparison, computed tomographic, gross anatomic, osteologic, and histologic studies were performed on the limbs of 5 of the sea turtles. Bones from the distal part of the fore and hind flippers were seen in detail with a mammographic film-screen combination. The pectoral and pelvic girdles, superimposed by the carapace, were better seen on standard radiographs with the use of rare-earth intensifying screens. Mammographic radiographs of the manus of 5 small juvenile turtles showed active growth zones. Visualization of bone contours in the distal part of the limbs was clearer than in mammals owing to the very few superimpositions. The presence of a substantial amount of cartilage in the epiphyses produced better visibility of limb ends. We conclude that use of a mammography film-screen combination is the best way to evaluate the bony and joint structures of the limbs of sea turtles. Radiography provides reliable images for clinical purposes. Considering the low cost and logistics of this technique, it is a practical ancillary test for marine animal rehabilitation centers to use.     

Influence of maternal obesity on fetal development in pigs

Fetuses from linebred lean (L) and linebred obese (O) and reciprocal crossmatings were examined at 110 d of gestation for line, maternal and heterotic effects. There was no significant heterotic effect for any trait measured. A significant maternal effect was observed for adipose tissue lipoprotein lipase (LPL) activity and for serum triglycerides. The enzyme activity and triglycerides concentration were higher in fetuses from O dams than in fetuses from L dams. In a lipid clearance test, no maternal effect was observed for changes in serum concentrations of triglycerides and free fatty acids or in optical density (associated with the disappearance of injected Liposyn). Linebred O fetuses exhibited higher LPL activity in both the biceps femoris muscle and sc adipose tissue compared with linebred L fetuses. The LPL activity of the adipose tissue was higher than that of the skeletal muscle. The percentage of dry matter, percentage of triglycerides and protein/DNA were higher in the muscle of linebred O fetuses than in that of linebred L fetuses. Based on tissue LPL activity and on muscle compositional traits, linebred O fetuses were more mature at 110 d of gestation than were the linebred L fetuses. Maternal obesity had little detectable influence on fetal development of the pig when measured at 110 d of gestation.     

Fetal protection against bovine viral diarrhea virus types 1 and 2 after the use of a modified-live virus vaccine

The objective of this study was to demonstrate the efficacy of a modified-live virus (MLV) vaccine in protecting fetuses from infection with type 1 or type 2 Bovine viral diarrhea virus (BVDV) when pregnant heifers were challenged at approximately 170 d of gestation with noncytopathic field isolates. The 83 pregnant heifers had been bred naturally 4 wk after vaccination. Fetuses were collected 60 d after BVDV type 2 challenge, and newborn calves were collected before colostrum intake after BVDV type 1 challenge. Protection was determined by measuring the serum neutralizing (SN) antibody response in the fetus or calf and by virus isolation from thymus, lung, spleen, and kidney tissue samples. There was a measurable SN antibody response to BVDV in all the fetuses and calves of the control heifers, which had received a placebo vaccine. However, only 4 of 22 calves and 7 of the 28 fetuses of the MLV-vaccinated heifers demonstrated SN antibody after BVDV challenge. Type 1 BVDV was isolated from tissue samples of 5 of the 12 calves of control heifers and none of 22 calves of the MLV-vaccinated heifers challenged with type 1 BVDV. Type 2 BVDV was isolated from tissue samples of 17 of the 18 fetuses of the control heifers and 2 of the 28 fetuses of the MLV-vaccinated heifers challenged with type 2 BVDV. The results of this study demonstrate that the MLV vaccine reduces the fetal infection rate by at least 82% for BVDV type 1 and by 75% for BVDV type 2 when heifers are exposed to highly fetotrophic BVDV at 170 d of gestation.     

Radiographic measurements of the hindlimbs in crab‐eating foxes (Cerdocyon thous,Linnaeus, 1766)

Life expectancy of crab‐eating foxes in captivity is approximately 11 years, which favours the development of diseases including orthopaedic disorders. However, to understand disorders that may affect these animals, it is worth determining a normal range of measurements. Therefore, this study aimed to evaluate radiographic measurements of joint angles of the hindlimbs and the ratio of patellar ligament length to patellar length in healthy crab‐eating foxes. Fifteen captive crab‐eating foxes (Cerdocyon thous), eight males and seven females, aged more than 1 year old, weighing 5.5–7.9 kg were used. Anatomic and mechanical femoral joint angles were assessed in ventrodorsal radiographic views, and mediolateral views were taken to measure patellar ligament length and patellar length, and the tibial plateau angle. The right and left hindlimbs were evaluated. All measurements were conducted in triplicate by the same investigator. No statistically significant differences were observed between sides in the radiographic measurements. The anatomic lateral proximal femoral angle and the anatomic lateral distal femoral angle recorded mean values of 95.43° and 92.75°, respectively. The mechanical lateral proximal femoral angle and the mechanical lateral distal femoral angle recorded mean values of 93.39° and 96.75°, respectively. Inclination angles measured according to the Tomlinson, Hauptman A and Hauptman B methods were 133.80°, 147.32° and 128.63°, respectively. The tibial plateau recorded 18.77°, and the ratio of the patellar ligament length to the patellar length was 1.99. In conclusion, the collected data are important to characterize the normal values of crab‐eating fox limbs.     

Survival of an adult Quarter Horse gelding following bacterial meningitis caused by Escherichia coli

Bacterial meningitis in the mature horse is a rare and typically fatal condition. This report describes a 7‐year‐old Quarter Horse gelding that initially presented following suspected trauma to the left eye but subsequently developed bacterial meningitis, and a nasal and palpebral abscess, all of which cultured the same isolate of Escherichia coli. The entry site of infection in bacterial meningitis is often related to a breach in the calvarium and extension of bacteria residing in the paranasal sinuses. This case is unique as there were multiple pathways through which bacteria may have entered the central nervous system including haematogenous spread from a nasal abscess, local extension of periocular infection or an undetected skull fracture. Aside from persistent blindness in the left eye, the horse made a full recovery.     

MAGNETIC RESONANCE IMAGING,GROSS POSTMORTEM,AND HISTOLOGICAL FINDINGS FOR SOFT TISSUES OF THE PLANTAR ASPECT OF THE TARSUS AND PROXIMAL METATARSAL REGION IN NON‐LAME HORSES

Injuries of the plantar soft tissues of the tarsus and proximal metatarsus can be a source of lameness in horses, however published information is lacking on high field MRI characteristics of these tissues. Objectives of the current anatomic study were to (1) describe high‐field MRI features of the plantar tarsal and proximal metatarsal soft tissues; and (2) compare MRI findings with gross and histological appearances of selected structures for a sample of cadaver limbs from non‐lame horses. Single hindlimbs for 42 horses, and right and left hindlimbs for eight horses were scanned using high‐field MRI. The MRI findings were described for the 50 single limbs; and the MRI, gross postmortem and histological findings were compared for the eight pairs of hindlimbs. The superficial digital flexor tendon had uniform low signal intensity, surrounded by the flexor retinaculum of intermediate to high signal intensity on all sequences. The lateral digital flexor tendon had slightly higher signal intensity, enclosed on the plantaromedial aspects by the low signal intensity metatarsocalcaneal ligament. The accessory ligament of the deep digital flexor tendon varied in size and signal intensity. The proximal and distal plantar ligaments, accessory ligament of the suspensory ligament, and calcaneoquartal ligament had low signal intensity. The long plantar ligament comprised a number of related parts, separated by lines of high signal intensity corresponding with fibrous septae seen in gross anatomical specimens. The plantar aspect of the ligament had uniform low signal intensity in all sequences, but the dorsal half was more heterogeneous with multifocal spots or lines of higher signal intensity.     

Is a single force plate adequate for stabilographic analysis in horses?

Reasons for performing study: Postural balance can be quantified using stabilographic variables derived from force plate data that describe movements of the horse's centre of pressure (COP) in the horizontal plane. Most force plates are not large enough to accommodate all 4 limbs of a standing horse, so the study was designed to assess whether representative stabilographic data could be collected from the forelimbs or hindlimbs. Objective: To determine whether stabilographic data from either the forelimbs or the hindlimbs were representative of data for the total body. Methods: Stabilographic data (960 Hz) were collected from 17 sound horses standing with the forelimbs and the hindlimbs on separate force plates. To increase variability in the data, horses were of different sizes, the recording duration was varied (15–60 s) and data were collected under sighted and blindfolded conditions. Results: Correlation matrices indicated that total body stabilographic variables were highly significantly correlated with both forelimb and hindlimb data but correlation coefficients were higher for forelimb data. Forward stepwise regression selected forelimb data for inclusion in the model for 15/16 variables, the exception being mean absolute mediolateral velocity. Conclusions: Ground reaction force data from a pair of limbs, preferably the forelimbs, can be used to measure variables that represent total body postural balance in sound standing horses. Potential relevance: Stabilographic data from either the forelimbs or hindlimbs may be useful for detecting and quantifying deficiencies in postural balance in ataxic horses.