Role of endochondral ossification of articular cartilage and functional adaptation of the subchondral plate in the development of fatigue microcracking of joints.

The mechanisms that regulate functional adaptation of the articular ends of long bones are poorly understood. However, endochondral ossification of articular cartilage and modeling/remodeling of the subchondral plate and epiphyseal trabeculae are important components of the adaptive response. We performed a histologic study of the distal end of the third metacarpal/metatarsal bone of Thoroughbreds after bones were bulk-stained in basic fuchsin and calcified sections were prepared. The Thoroughbred racehorse is a model of an extreme athlete which experiences particularly high cyclic strains in distal limb bones. The following variables were quantified: microcrack boundary density in calcified cartilage (N.Cr/B.Bd); blood vessel boundary density in calcified cartilage (N.Ve/B.Bd); calcified cartilage width (Cl.Cg.Wi); duplication of the tidemark; and bone volume fraction of the subchondral plate (B.Ar/T.Ar). Measurements were made in five joint regions (lateral condyle and condylar groove; sagittal ridge; medial condylar and condylar groove). N.Cr/B.Bd was site-specific and was increased in the condylar groove region; this is the joint region from which parasagittal articular fatigue (condylar) fractures are typically propagated. Formation of resorption spaces in the subchondral plate was co-localized with microcracking. N.Ve/B.Bd was also site-specific. In the sagittal ridge region, N.Ve/B.Bd was increased, Cl.Cg.Wi was decreased, and B.Ar/T.Ar was decreased, when compared with the other joint regions. Multiple tidemarks were seen in all joint regions. Cumulative athletic activity was associated with a significant decrease in B.Ar/T.Ar in the condylar groove regions. N.Cr/B.Bd was positively correlated with B.Ar/T.Ar (P < 0.05, r(s) = 0.29) and N.Ve/B.Bd was negatively correlated with B.Ar/T.Ar (P < 0.005, r2 = 0.14) and Cl.Cg.Wi (P < 0.05, r2 = 0.07). We conclude that endochondral ossification of articular cartilage and modeling/remodeling of the subchondral plate promote initiation and propagation of site-specific fatigue microcracking of the joint surface, respectively, in this model. Microcracking of articular calcified cartilage likely represents mechanical failure of the joint surface. Propagation of microcracks into the subchondral plate is a critical factor in the pathogenesis of articular condylar fatigue (stress) fracture. Functional adaptation of the joint likely protects hyaline cartilage from injury in the short-term but may promote joint degeneration and osteoarthritis with ongoing athleticism.

Carpal and fetlock conformation of the juvenile Thoroughbred from birth to yearling auction age.

REASONS FOR PERFORMING STUDY: There is little information available about conformational changes in the forelimbs of growing foals. OBJECTIVES: To describe the conformation of the carpus and fetlock of Thoroughbred foals from birth to yearling sale age. METHODS: Subjective assessments of the fetlock and carpal conformation of 119 Thoroughbred foals were made within the first month of life and then at 30 day intervals until at least age 120 days. At least 70 subjects were examined further at 60 day intervals until September of their second year. Conformation grades are reported for 5 age groups: first 7 days and at a mean of 46, 176, 362 and 525 days. The conformation of all available sires and dams of subjects was also graded. RESULTS: All subjects demonstrated carpal deviations, such as valgus, outward rotation and offset, and approximately 30% had fetlock deviations. Heavier birth weights were associated with carpal offset and fetlock inward conformation at most ages, and heavier yearlings were more likely to be carpal valgus. The carpal conformation of the sire (offset and outward rotation) was associated with similar yearling carpal conformation. During the study period, the carpal conformation of Thoroughbred foals became less valgus and more offset. Fetlock conformation became more inwardly deviated during the first 6 months of the study. CONCLUSIONS: Carpal and fetlock conformation change greatly in Thoroughbred foals up to age 18 months. The phenotype of the sire can be associated with yearling carpal conformation and bodyweight, particularly at birth and as yearlings, is associated with yearling fetlock and carpal conformation. POTENTIAL RELEVANCE: New factors associated with forelimb conformational deviations have been identified that may help breeders better to manage young racing stock.

Up-regulation of site-specific remodeling without accumulation of microcracking and loss of osteocytes.

Functional adaptation of bone normally protects the skeleton from fracture during daily activity. Accumulation of microcracking and loss of osteocytes have been implicated in the regulation and initiation of targeted (reparative) remodeling of bone and, in certain situations, the development of fatigue or stress fracture. We performed a histologic study of the dorsal cortex of the mid-diaphysis of the third metacarpal (Mc-III) bone of Thoroughbred racehorses after bones were bulk-stained in basic fuchsin and transverse calcified sections were prepared. The Thoroughbred racehorse is an extreme athlete whose Mc-III bone experiences particularly high cyclic strains during training and racing. A group of non-athletic horses was also included in the experiment. The following variables were quantified: activation frequency (Ac.f); bone formation rate (BFR); resorption space density (Rs.N/T.Ar); microcrack density (Cr.Dn); microcrack mean length (Cr.Le); microcrack surface density (Cr.S.Dn); osteocyte density (Ot.N/T.Ar; Ot.N/B.Ar); and bone volume fraction (B.Ar/T.Ar). Ac.f and BFR were estimated using a mathematical algorithm. Using confocal microscopy, bones were examined for fine microcracks, diffuse matrix injury, and disruption of the osteocyte syncytium. Low values for Cr.Dn (#/mm2) were found in both groups (0.022+/-0.008 and 0.013+/-0.006 for racing Thoroughbreds and non-athletic horses, respectively). There was no significant relationship between Cr.Dn and Ot.N/T.Ar; Ot.N/B.Ar, B.Ar/T.Ar, and Ot.N/T.Ar; Ot.N/B.Ar, and remodeling (Ac.f, Rs.N/T.Ar) and Ot.N/T.Ar; Ot.N/B.Ar. Intense remodeling of the Mc-III dorsal cortex was found in the racing Thoroughbreds (Ac.f 12.8+/-7.4 #/mm2/year; BFR 31.5+/-15.6%; Rs.N/T.Ar 0.19+/-0.09 #/mm2) and was significantly increased compared with non-athletic horses. Overall, remodeling was weakly correlated with Cr.Dn (r2=0.15, P<0.05). Subtle matrix injury, not detectable by bright-field microscopy, was particularly evident adjacent to resorption spaces in Thoroughbred bone. In non-athletic horses, disruption of the dendritic cell processes of osteocytes associated with cement lines and interstitial fragments was more evident. Taken together, these findings suggest that site-specific (targeted) induction of remodeling during functional adaptation of bone in a high-strain skeletal site is not dependent on accumulation of microcracking or loss of osteocytes. We hypothesize that athleticism can directly influence bone turnover in this extreme athlete through pathways that do not involve classical linear microcracks.

Incidence of the endothelin receptor B mutation that causes lethal white foal syndrome in white-patterned horses.

OBJECTIVE: To determine incidence of the Ile118Lys endothelin receptor B (EDNRB) mutation responsible for overo lethal white syndrome (OLWS) and its association with specific types of white patterning. ANIMALS: 945 horses of white-patterned bloodlines and 55 solid-colored horses of other breeds. PROCEDURE: Horses were genotyped by use of allele-specific polymerase chain reaction to determine incidence of the Ile118Lys EDNRB mutation. RESULTS: Genotypes detected were homozygous Ile118, homozygous Lys118, and heterozygous. All foals with OLWS were homozygous for the Ile118Lys EDNRB mutation, and adults that were homozygous were not found. White patterning was strongly associated with EDNRB genotype. Color patterns with highest incidence (> 94%) of heterozygotes were frame overo, highly white calico overo, and frame blend overo. White-patterned bloodlines with lowest incidence of heterozygotes (< 21 %) were tobiano, sabino, minimally white calico overo, splashed white overo, nonframe blend overo, and breeding-stock solid. The mutation was not detected in solid-colored horses from breeds without white patterning. CONCLUSIONS AND CLINICAL RELEVANCE: In homozygotes, the Ile118Lys EDNRB mutation causes OLWS. In heterozygotes, the mutation is usually responsible for a frame overo phenotype. The frame pattern can be combined with other white patterns, making accurate estimation of EDNRB genotype by visual inspection difficult. Wide range of incidence of heterozygotes in various subtypes of white-patterned horses indicates different genetic control of these color patterns. Determination of EDNRB genotype by use of a DNA-based test is the only way to determine with certainty whether white-patterned horses can produce a foal affected with OLWS.

Long-term outcome of horses with a slab fracture of the central or third tarsal bone treated conservatively: 25 cases (1976-1993).

OBJECTIVE: To determine clinical features of horses with a slab fracture of the central or third tarsal bone and to report outcome of horses in which treatment did not include surgery. DESIGN: Retrospective study. ANIMALS: 25 horses (14 Standardbreds, 6 Thoroughbreds, 5 Quarter Horses). PROCEDURE: Medical records of horses with a slab fracture of the central (n = 9) or third (16) tarsal bone were reviewed. Only horses for which treatment consisted of confinement to a stall were included in this study. Clinical features and radiographic findings were recorded and summarized. Outcome was determined for racing breeds by obtaining official lifetime race results. Outcome for Quarter Horses was determined by phone survey of the owners. RESULTS: 16 (64%) horses had a successful outcome. Ten of 14 (71%) Standardbreds and 2 of 6 Thoroughbreds returned to racing and started at least 5 races after injury. Four of 5 Quarter Horses for which follow-up information was available successfully returned to their previous activity. Sex, age, limb affected, or gait was not associated with final outcome. Percentage of racehorses with central tarsal bone fractures that had a successful outcome (2/7) was significantly less than percentage with third tarsal bone fractures that did (10/13). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that enforced rest without surgical fixation can be an effective therapeutic option for horses with a slab fracture of the central or third tarsal bone, even if athletic function is expected.

Pharmacokinetics of gentamicin in mares in late pregnancy and early lactation.

The disposition of drugs may differ between pregnant and nonpregnant animals, necessitating a change in dosage. We hypothesized that volume of distribution or clearance may be different for aminoglycoside antibiotics in pregnant mares vs. nonpregnant lactating mares. To examine this hypothesis, we administered gentamicin sulfate to seven Thoroughbred and Quarterhorse mares on two occasions, followed by plasma drug gentamicin assay and pharmacokinetic analysis. The first dose was administered 1-4 weeks before parturition (mean weight 578 kg) and the second dose was administered in the period 1-4 weeks after parturition (mean weight 518 kg). The dose administered at each time was approximately 6.6 mg/kg, intravenously (i.v.). Plasma gentamicin concentrations were determined using fluorescence polarization immunoassay and pharmacokinetic analysis was performed using a two-compartment open model. The plasma concentration vs. time profiles and total area-under-the-curve were almost identical for mares at late gestation vs. early lactation. Mean volume of distribution at steady-state was 0.15 (+/-0.02) and 0.16 (+/-0.03) L/kg, systemic clearance was 1.06 (+/-0.17) and 1.11 (+/-0.17) mL/kg/min, and mean (harmonic) elimination half-life was 2.2 and 2.1 h, for pregnant and nonpregnant mares, respectively. We concluded that there were no differences in drug distribution and clearance between pregnant and nonpregnant lactating mares. Gentamicin was also assayed in plasma of newborn foals after an injection of 6.6 mg/kg to three of the mares within 60 min of parturition. Gentamicin was undetectable in plasma samples from these foals and, therefore, apparently does not cross the placenta of mares at term.

Colic surgery in 206 juvenile thoroughbreds: survival and racing results.

The purpose of our study was to determine the types of lesions that cause colic in juvenile Thoroughbreds, factors associated with these lesions and the subsequent survival and athletic performance of the juveniles. The age of juvenile Thoroughbreds requiring surgical exploration for abdominal pain has an influence on the type of lesion causing colic. The short-term survival rate (discharge from the hospital) after colic surgery for foals was 85% and was strongly influenced by the lesion causing colic. Thirteen percent of juveniles recovered from the first surgery experienced another severe colic episode requiring additional surgery or euthanasia. Eight percent of foals recovered from the first celiotomy developed adhesions. Adhesion formation was related to the initial lesion causing colic and the foals' age at the first surgery. Foals being suckled (15 days to 6 months) were at greatest risk for adhesions and more frequently required multiple surgeries. Juvenile Thoroughbreds that had a celiotomy were significantly less able to race (63%) than their unaffected siblings (82%), and age at the initial surgery was associated with the percentage of horses that raced. However, affected foals able to race won as much money, raced as often, and made as many starts as their siblings. Colic and surgical treatment have a negative impact on athletic performance, but the majority of foals discharged from the hospital after colic surgery will perform athletically as adults.

Simultaneous recording of pituitary oxytocin secretion and myometrial activity in oestrous mares exposed to various breeding stimuli.

The aim of this study was to investigate the effect of different breeding stimuli on uterine contractility and pituitary oxytocin release in five oestrous mares, in order to design better treatments for mares with defective physical uterine clearance mechanisms. Electrodes and strain gauges were implanted surgically on the uterine myometrium and myometrial activity was monitored with a Grass polygraph. A catheter was placed non-surgically in the intercavernous sinus of each oestrous mare to sample pituitary venous blood and a second catheter was placed in the jugular vein. Continuous sampling was performed for 2 h to determine the baseline value and during sequentially applied stimuli of: (i) stallion call; (ii) visual contact with a stallion; (iii) active teasing; and (iv) artificial insemination. No association was observed between uterine contraction and pituitary oxytocin release episodes during baseline recording. Exposure of the mares to any of the breeding stimuli was associated with rapid onset of myometrial contractions (P < 0.0001). The application of a stimulus significantly increased pituitary oxytocin release (P < 0.02) and contraction duration (P < 0.05), and the response to artificial insemination was greater than the responses to the other treatments (P < 0.05). The onset of oxytocin secretion was generally simultaneous with the increase in uterine contraction, rather than preceding it, and in some instances the responses were dissociated, making a cause and effect relationship less likely. In conclusion, interactions between stallions and mares are important components of the uterine clearance mechanism around the time of breeding. Knowledge of these physiological responses may be useful in designing therapeutic regimens for mares with endometritis due to defective uterine contractions.

Dimeric inhibin concentrations in mares with granulosa-theca cell tumors.

OBJECTIVE: To determine whether concentrations of dimeric inhibin (CaCA) are greater in plasma and tumor fluid from mares with granulosa-theca cell tumors (GTCT), compared with concentrations in plasma and equine follicular fluid (eFF) from control mares. ANIMALS: 6 mares with GTCT and 12 clinically normal mares. PROCEDURE: The alphabetaA immunoradiometric assay used 2 antibodies, one against each subunit of inhibin (alpha and betaA subunits). Tumor tissue, tumor fluid, and a single blood sample were collected at the time of surgical removal of the GTCT. A single blood sample was collected from 7 control mares during various stages of the estrous cycle. Five other control mares were ovariectomized when their ovaries contained growing follicles of 25 to 35 mm in diameter. A blood sample and eFF from the largest follicle were collected at the time of ovariectomy. RESULTS: Mares with GTCT had significantly greater plasma concentrations of betabetaA (mean +/- SEM, 0.86 +/- 0.53 ng of recombinant human-alphabetaA/ml), compared with control mares (0.14+/-0.02 ng/ml). Concentrations of alphabetaA in tumor fluid and eFF were similar. Concentrations of alphabetaA were significantly lower after ovariectomy. CONCLUSIONS AND CLINICAL RELEVANCE: Dimeric inhibin concentration was higher in plasma from mares with GTCT than in plasma from control mares. Increased granulosa cell mass and loss of mechanisms regulating alphabetaA release in mares with GTCT likely accounted for the increase in plasma concentrations. Measurement of alphabetaA concentrations may be useful for identifying mares with GTCT.

Antibacterial properties of a silver chloride-coated nylon wound dressing.

OBJECTIVE: A silver chloride-coated nylon wound dressing (Ag-WD) was evaluated in vitro for antimicrobial activity against five common equine wound pathogens. STUDY DESIGN: Bacterial susceptibility study. SAMPLE POPULATION: Equine wound pathogens: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus equi subspecies zooepidemicus, and Staphylococcus aureus. METHODS: An inoculum of each pathogen was incubated directly with Ag-WD and quantitated after 24 to 48 hours of incubation. To determine if bactericidal activity of Ag-WD was contact dependent, an inoculum of E. coli and Staphylococcus aureus was incubated separately from Ag-WD by a filter and quantitated after 18 hours of incubation. Inductively coupled plasma emission spectrometry (ICP) determined the silver concentration of Mueller-Hinton broth containing Ag-WD after 24 hours of incubation. To establish if the rate of bacterial killing by Ag-WD differed from a constant silver concentration, pathogens were exposed to a silver concentration of 6.45 microg/mL and quantitated after 18 hours. RESULTS: Direct exposure to Ag-WD significantly reduced bacterial numbers after 15 minutes for K. pneumoniae, 30 minutes for E. coli, 1 hour for P. aeruginosa, and 2 hours for S. equi subspecies zooepidemicus and Staphylococcus aureus. Indirect exposure to Ag-WD resulted in > or =99.9% and > or =90% kill of the inoculum doses of E. coli at 2 hours and Staphylococcus aureus at 18 hours, respectively. Incubation of the pathogens at the constant silver concentration resulted in bacterial killing rates similar to those obtained by incubation with Ag-WD. CONCLUSIONS: In vitro, equine pathogens are effectively killed when exposed to Ag-WD, and the rate of bacterial killing by Ag-WD is similar to a constant silver concentration of 6.45 microg/mL. CLINICAL RELEVANCE: The in vitro antimicrobial properties of this silver-coated nylon wound dressing are promising for future prevention of equine wound infections.

Heritability of recurrent exertional rhabdomyolysis in Thoroughbred racehorses.

OBJECTIVE: To determine the likely mode of inheritance and identify probable foundation horses for recurrent exertional rhabdomyolysis (RER) in Thoroughbred (TB) racehorses. ANIMALS: 4 families of TB racehorses with a high prevalence of RER, consisting of 3 to 53 horses/family, were used to determine mode of inheritance. Sixty-two TB horses with RER and 34 control TB racehorses without RER were used to identify probable foundation horses for the RER trait. PROCEDURE: RER was diagnosed by a veterinarian and verified by detecting high serum creatine kinase activity. Pedigrees dating from 1930 for all horses were entered into a database. Pedigrees of horses in 4 families were visually inspected for a pattern of inheritance and used for calculation of foundation horse contributions and inbreeding coefficients. The Markov chain Monte Carlo technique was used to analyze pedigrees of the 62 affected and 34 control horses for the conditional probability of foundation genotypes. A dominant mode of inheritance with variable expression model was used. RESULTS: Pedigree analysis supported an autosomal dominant mode of inheritance with variable expression. All affected horses from the 4 families shared a common ancestor. This ancestor and 5 other stallions had a conditional probability of 1.00 for being affected. All 6 stallions shared a common male ancestor within 3 to 5 generations. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of this study, the RER trait has been in TB racehorses for more than 70 years and may be inherited as an autosomal dominant trait with variable expression.

Identification and radiation hybrid mapping of members of the porcine proteasome/ubiquitin system.

We report the identification and radiation hybrid mapping of members of the proteasome/ubiquitin system in pigs that, so far, have only been identified in humans and cattle. Expressed sequence tags (ESTs) were constructed from ten oligo(dT)-primed individually tagged, directionally cloned and normalized cDNA libraries from peripheral blood cells (PBC), spleen (Sp), thymus (Th), lymph node (LN) and bone marrow (BM) from immunologically naive and challenged pigs as part of an implant-associated orthopedic infection model. The ESTs mapped using the 7000 rad IMpRH panel (Hawken et al., 1999) were ubiquitin fusion-degradation 1 like protein (UFD1L), ubiquitin activating enzyme E1 and ubiquitin-S27a fusion protein which mapped to porcine chromosomes 14, 7 and X, respectively.

Endothelin receptor B polymorphism associated with lethal white foal syndrome in horses.

Overo lethal white syndrome (OLWS) is an inherited syndrome of foals born to American Paint Horse parents of the overo coat-pattern lineage. Affected foals are totally or almost totally white and die within days from complications due to intestinal aganglionosis. Related conditions occur in humans and rodents in which mutations in the endothelin receptor B (EDNRB) gene are responsible. EDNRB is known to be involved in the developmental regulation of neural crest cells that become enteric ganglia and melanocytes. In this report we identify a polymorphism in the equine EDNRB gene closely associated with OLWS. This Ile to Lys substitution at codon 118 is located within the first transmembrane domain of this seven-transmembrane domain G-protein-coupled receptor protein. All 22 OLWS-affected foals examined were homozygous for the Lys118 EDNRB allele, while all available parents of affected foals were heterozygous. All but one of the parents also had an overo white body-spot phenotype. Solid-colored control horses of other breeds were homozygous for the Ile118 EDNRB allele. Molecular definition of the basis for OLWS in Paint Horses provides a genetic test for the presence of the Lys118 EDNRB allele and adds to our understanding of the basis for coat color patterns in the horse.

Diaphragmatic hernia repair in three young horses.

OBJECTIVE: This clinical report describes surgical correction of diaphragmatic hernia in three young horses. STUDY DESIGN: Retrospective investigation of medical records and subsequent racing performance. ANIMALS: Three young horses with diaphragmatic hernia. RESULTS: Three young horses with signs of abdominal pain had diaphragmatic hernia causing small intestinal strangulation. The strangulated small intestine was resected and an end-to-end jejuno-jejunal (two horses) or a side-to-side jejuno-cecal anastomosis (one horse) was performed. Diaphragmatic hernias were closed with a continuous suture pattern. All horses recovered and raced. No difference in race records was found between the subject horses and their siblings. One subject horse died of colic at 5 years of age, but the cause of the colic was undetermined. The remaining two horses are in use as broodmares and have produced multiple foals without recurrence of signs of diaphragmatic hernia. CONCLUSIONS: Diaphragmatic hernias can be repaired in horses. These horses can achieve race records similar to their siblings and can produce foals without recurrence of signs of diaphragmatic hernia.

Ovariohysterectomy in six mares.

Six mares had ovariohysterectomy performed for chronic pyometra associated with cervical abnormalities, uterine neoplasia, or removal of a macerated fetus. Ovariohysterectomy was performed through a ventral midline incision with access to the ovarian and uterine vessels aided by traction on the uterus and retraction of abdominal viscera. Abdominal pain, the most common complication after surgery, occurred in four mares but resolved within 36 hours. Peritonitis occurred in two mares; one mare was subsequently euthanatized. Other complications that resolved with treatment included infection of the uterine stump (two mares), abdominal hemorrhage (one mare), diarrhea (one mare), and incisional infection (one mare). Complications after surgery can be reduced by removing as much of the uterus as possible, minimizing peritoneal contamination with uterine contents, and providing a secure closure of the caudal reproductive tract.

Successful pregnancy after partial hysterectomy in two mares.

Infertility was associated with a uterine mass in 2 Thoroughbred mares. Both mares had produced live foals, but had been barren for 2 years, despite repeated breedings. Each mass was removed through a right ventral paramedian incision. Histologic examination of the masses revealed a leiomyoma and an ovarian abscess. In 1 mare, approximately 30% of 1 uterine horn was removed because of tumor attachment; in the other mare, approximately 50% of 1 uterine horn was removed because of adhesions to the ovarian abscess. Pregnancy information was available for both mares for the 3 years after surgery; each mare had produced 2 healthy foals and was pregnant with a third.

Use of ultrasound in horses for diagnosis of left dorsal displacement of the large colon and monitoring its nonsurgical correction.

During a 28 month period, 82 horses with clinical signs of abdominal pain were examined for left dorsal displacement of the large colon (LDDLC) using percutaneous ultrasound. Left dorsal displacement of the large colon was diagnosed when a gas echo dorsal to the spleen obliterated the dorsal splenic border, or when the colon was observed lateral to the spleen. In 42 horses, ultrasound confirmed a diagnosis of LDDLC and 40 horses had no evidence of LDDLC. There were five false negative results and no false positives. In four horses with LDDLC, the colon was displaced between the spleen and body wall; three of these colic episodes resolved with medical therapy and the fourth required a celiotomy to relieve a sand impaction. The remaining 38 horses had a renosplenic entrapment; surgical correction was elected in 4 horses, 21 horses were corrected by a nonsurgical rolling procedure, 12 were corrected at surgery after an unsuccessful rolling attempt, and one was corrected by rolling but required surgery later because of an additional lesion. Percutaneous abdominal ultrasound was a valuable aid in the diagnosis of LDDLC and in confirming correction of the displacement after a nonsurgical rolling procedure.