Morphological and acrosomal changes of canine spermatozoa during epididymal transit

Background

During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.

Results

After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes.

Conclusions

Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions.     

Transfer of MicroRNAs From Epididymal Epithelium to Equine Spermatozoa

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.     

Migration of protoplasmic droplets and phagocytosis of damaged sperm during their passage through the efferent ducts in boars and bulls

Seventeen gonad pairs of boars and ten gonad pairs of bulls were examined to evaluate the migration of protoplasmic droplets and the phagocytosis of defective spermatozoa. The material for a microscopic investigation of secretions was collected from two sites in the testis and from seven sites in the epididymis. The greatest motion of protoplasmic droplets was recorded in the caput epididymidis, although the migration of droplets from the proximal section of the connective part of the flagellum towards the distal parts could also be observed as far as in the cauda epididymidis in both animals. A proximally located droplet still occurred in the cauda epididymidis in 4.5% of the spermatozoa of boars and in 1.9% of those of bulls. Absent mitochondrial spirals or swollen connective parts were observed in the imprints of testicular tissue in almost 50% of the spermatozoa whereas in the secretion of efferent ducts they were observed only in 0.3% of bull spermatozoa and about 3% of boar spermatozoa. No such defects were recorded in the epididymis head and tail in either of the two species. The marked reduction in the number of defective spermatozoa without mitochondrial spirals in the secretion of efferent ducts and after passage through the caput epididymidis testifies to the phagocytic ability of the epithelium of this part of efferent ducts.     

Accuracy of open magnetic resonance imaging for guiding injection of the equine deep digital flexor tendon within the hoof

Lesions of the distal deep digital flexor tendon (DDFT) are frequently diagnosed using MRI in horses with foot pain. Intralesional injection of biologic therapeutics shows promise in tendon healing; however, accurate injection of distal deep digital flexor tendon lesions within the hoof is difficult. The aim of this experimental study was to evaluate accuracy of a technique for injection of the deep digital flexor tendon within the hoof using MRI‐guidance, which could be performed in standing patients. We hypothesized that injection of the distal deep digital flexor tendon within the hoof could be accurately guided using open low‐field MRI to target either the lateral or medial lobe at a specific location. Ten cadaver limbs were positioned in an open, low‐field MRI unit. Each distal deep digital flexor tendon lobe was assigned to have a proximal (adjacent to the proximal aspect of the navicular bursa) or distal (adjacent to the navicular bone) injection. A titanium needle was inserted into each tendon lobe, guided by T1‐weighted transverse images acquired simultaneously during injection. Colored dye was injected as a marker and postinjection MRI and gross sections were assessed. The success of injection as evaluated on gross section was 85% (70% proximal, 100% distal). The success of injection as evaluated by MRI was 65% (60% proximal, 70% distal). There was no significant difference between the success of injecting the medial versus lateral lobe. The major limitation of this study was the use of cadaver limbs with normal tendons. The authors conclude that injection of the distal deep digital flexor tendon within the hoof is possible using MRI guidance.     

Diffusion of radiodense contrast medium after perineural injection of the palmar digital nerves

Limited information exists on distribution of local anaesthetic solution following palmar digital nerve blocks. The aim of this study was to demonstrate potential distribution of local anaesthetic solution following perineural injection of the palmar digital nerves using 2 different volumes of contrast medium and 2 different injections sites. Twelve mature horses were used. Perineural injection of the palmar digital nerves were performed at the level of or 2 cm proximal to the proximal aspect of the ipsilateral ungular cartilage, using 1.5 or 2.5 ml radiopaque contrast medium. In total, 96 injections were performed. Four standard radiographic views of the pastern were obtained immediately after injections and 10 and 20 min later. Images were analysed subjectively and objectively. After distal injections, the contrast medium was more localised around the injection site; after proximal injections the contrast patch had greater proximal–distal length. The greatest proximal diffusion was to 31.7% of the length of the proximal phalanx (from the level of the proximal interphalangeal joint) after distal injections and to 70% after proximal injections. The larger volume resulted in significantly greater proximal diffusion than the smaller volume at the distal, but not at the proximal injection site (P<0.01). There was significant proximal diffusion with time after proximal and distal injections (P<0.01). In most limbs, numerous radiopaque lines of various thickness extended proximally from the contrast patches; subjectively, their number and thickness were greater at the distal injection site. In conclusion, palmar digital nerve blocks at the level of the ungular cartilage using ≤2.5 ml local anaesthetic solution may improve proximal interphalangeal joint and pastern region pain. If using a more proximal site, distal fetlock region pain may be improved. Due to diffusion into lymphatic vessels, too small a volume at the distal injection site may not provide sufficient analgesia.     

Morphologic and morphometric studies of the vagus and recurrent laryngeal nerves in clinically normal adult dogs

Morphologic and morphometric studies were performed on the vagus nerve and its major branch, the recurrent laryngeal nerve (RLN): (1) to determine normal histologic data in myelinated fibers of clinically normal young adult dogs, (2) to establish reference values for mean fiber diameter in the vagus nerve and in the proximal and distal portions of the RLN, and (3) to delineate relative frequency distribution curves for each nerve. Few degenerative changes were observed in single teased-nerve fiber preparations. There was no statistical difference between left and right sides of the vagus nerve or between the proximal and distal portions of the RLN (right and left sides). In contrast to the unimodal distribution of fibers in the vagus nerve and the proximal portion of the RLN, the distribution of fibers in the distal portion of the RLN was bimodal. Mean (+/- SD) fiber diameters of the vagus nerve and in the proximal portion of the RLN (3.02 +/- 1.44 microns and 3.63 +/- 1.49 microns, respectively) were not significantly different, despite a shift to large-diameter fibers in the latter. However, mean fiber diameters of distal and proximal portions of the RLN (5.56 +/- 1.88 microns and 3.63 +/- 1.49 microns, respectively) were significantly (P less than 0.001) different. Approximately 86% of fibers in the vagus nerve and 76% of fibers in the proximal portion of the RLN, had a diameter less than 5 microns, and about 70% of fibers in the distal portion of the RLN had a diameter greater than 5 microns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparisons of computed tomography, contrast-enhanced computed tomography and standing low-field magnetic resonance imaging in horses with lameness localised to the foot. Part 2: Lesion identification

Reasons for performing study: No previous study compares computed tomography (CT), contrast‐enhanced computed tomography (CECT) and standing low‐field magnetic resonance imaging (LFMRI) to detect lesions in horses with lameness localised to the foot. This study will help clinicians understand the limitations of these techniques. Objectives: To determine if CT, CECT and LFMRI would identify lesions within the distal limb and document discrepancies with lesion distribution and lesion classification. Methods: Lesions in specific structures identified on CT and MR images of feet (31 limbs) from the same horse were reviewed and compared. Distributions of lesions were compared using a Chi‐squared test and techniques analysed using the paired marginal homogeneity test for concordance. Results: Lesions of the deep digital flexor tendon (DDFT) were most common and CT/CECT identified more lesions than LFMRI. Deep digital flexor tendon lesions seen on LFMRI only were frequently distal to the proximal extent of the distal sesamoid and DDFT lesions seen on CT/CECT only were frequently proximal to the distal sesamoid. Lesions identified on LFMRI only were core (23.3%) or splits (43.3%), whereas lesions identified only on CT were abrasions (29.8%), core (15.8%), enlargement (15.8%) or mineralisation (12.3%). Contrast‐enhanced CT improved lesion identification at the DDFT insertion compared to CT and resulted in distal sesamoidean impar ligament and collateral sesamoidean ligament vascular enhancement in 75% of cases. Low‐field MRI and CT/CECT failed to identify soft tissue mineralisation and bone oedema, respectively. Conclusions and potential relevance: Multiple lesions are detected with CT, CECT and LFMRI but there is variability in lesion detection and classification. LFMRI centred only on the podotrochlear apparatus may fail to identify lesions of the pastern or soft tissue mineralisation. Computed tomography may fail to identify DDFT lesions distal to the proximal border of the distal sesamoid.     

Proximal radial and distal humeral osteosarcoma in 12 dogs

Twelve dogs were diagnosed with osteosarcoma of the proximal radius or distal humerus from 1990 to 2002, representing 1.0% of all dogs diagnosed with appendicular osteosarcoma. The median body weight (29.8 kg) was significantly less than that of dogs with appendicular osteosarcoma at other sites. Ten dogs were treated with amputation and chemotherapy. These dogs had a metastatic rate of 60%, a median metastasis-free interval of 356 days, and a median survival time of 824 days. There were no significant differences in metastasis-free interval or survival time between dogs with osteosarcoma of the proximal radius or distal humerus and dogs with appendicular osteosarcoma at other sites.     

The Arterial Supply of the Metacarpophalangeal Joint in the Bactrian Camel (Camelus bactrianus)

The arterial supply of the six metacarpophalangeal joints was studied in Bactrian camels. The arterial branches supplying the metacarpophalangeal joints were derived from the metacarpal distal perforating, medial and lateral branches of the palmar metacarpal artery III, medial and lateral branches of the palmar common digital artery III and the abaxial palmar proper digital arteries III and IV. These arterial branches were the proximal dorsoaxial distal metacarpal, distal dorsoaxial distal metacarpal, abaxial distal metacarpal, palmar distal metacarpal, interosseous distal metacarpal, dorsoaxial proximal proximal phalangeal, palmoaxial proximal proximal phalangeal, palmoabaxial proximal proximal phalangeal and dorsoabaxial proximal proximal phalangeal branches. They linked with each other around the metacarpophalangeal joint.     

In vitro comparison of proximal ulnar osteotomy and distal ulnar osteotomy with release of the interosseous ligament in a canine model

Objective: To determine the effect of proximal ulnar osteotomy (PUO), distal ulnar osteotomy (DUO), and DUO with release of the interosseous ligament (DOLR) on displacement of the proximal ulna at the radioulnar joint. Study Design: Experimental mechanical study. Sample Population: Cadaveric, skeletally mature canine thoracic limb pairs (n=11). Methods: Thoracic limbs disarticulated at the elbow were randomly assigned to 1 of 2 groups: (A) limbs were tested with no treatment (NOTX), then with PUO; (B) limbs were tested with DUO followed by DOLR. A distraction force was applied to the proximal ulna with the distal limb secured in a frame, and displacement of the proximal ulnar segment at the radioulnar joint was recorded. Results: Mean displacement was 0.36 mm (95% confidence interval [CI]: 0?1.12 mm) in the NOTX group, 4.68 mm (95% CI: 3.82–5.55 mm) for PUO limbs, 0.95 mm (95% CI: 0.52–1.39 mm) for the DUO group, and 4.36 mm (95% CI: 3.71–5.02 mm) for the DOLR group. Mean ulnar displacement of the DOLR group was significantly different compared with the DUO group, but there was no significant difference between the PUO and DOLR groups. Conclusions: No significant difference was observed in displacement of the proximal ulnar segment after DOLR compared with PUO.     

COMPUTED TOMOGRAPHIC DETERMINATION OF TIBIAL TORSION IN THE DOG

The goal of this study was to develop a method for computed tomographic (CT) measurement of tibial torsion, and to compare this technique with direct anatomic measurement of tibial torsion in cadaveric canine tibiae. Paired hind limbs of 10 cadaveric dogs were mounted on a custom-designed limb holding apparatus. One-millimeter thick, contiguous, transverse CT slices were obtained from the distal femur to the proximal tibia and 2 mm CT slices were obtained from the distal tibia to the proximal tarsus. The tibiae were freed of soft tissues and digital photographic images of the proximal and distal articular surfaces were obtained with the camera lens aligned perpendicular to the long axis of the tibia. Multiple proximal and distal tibial axes were identified on the images; two proximal and two distal axes were found to be repeatable in all specimens in both the direct and CT methods. The torsion angle was calculated by determining the difference between the axis angles for each pair of proximal and distal axes. There was no significant difference in torsion angle identified between the direct photographic and CT method for any pair of proximal and distal axes. CT determination of tibial torsion is a rapid and accurate method, and warrants investigation in clinical patients.     

Ex vivo comparison of one versus two distal screws in 8 mm model 11 interlocking nails used to stabilize canine distal femoral fractures

OBJECTIVE: To compare the structural properties of an 8 mm model 11 interlocking nail (IN) with 2 proximal and 2 distal screws (2/2) to 2 proximal and 1 distal screws (2/1) in an unstable canine fracture model. STUDY DESIGN: Ex vivo biomechanical investigation. SAMPLE POPULATION: Eight pairs of adult canine femurs. METHODS: A simple transverse distal metaphyseal femoral fracture with a 1 cm gap was created. The unstable fracture in 1 femur was repaired with a nail with 2 distal and 2 proximal screws and the paired femur with a nail with 1 distal and 2 proximal screws. Cyclic mechanical testing in torsion was performed to assess fatigue life, peak torque, stiffness, and mode of failure. RESULTS: All 2/1 IN-femoral constructs, but only 2 of eight 2/2 constructs, failed before completion of 50,000 loading cycles. The 2/2 constructs had significantly greater peak torque to failure (P = .002) and longer fatigue life (P = .00003) compared with 2/1 constructs. There were no significant differences in stiffness between 2/2 and 2/1 constructs when the non-failed constructs were compared (P > .5). All constructs failed by screw deformation. CONCLUSIONS: An 8 mm model 11 IN used for fixation of unstable canine distal femoral fractures has a longer fatigue life and is stronger under torsional loads when 2 rather than 1 distal screws are placed. CLINICAL RELEVANCE: When repairing unstable canine distal femoral fractures with an IN system, 2 distal screws should be inserted to avoid catastrophic implant failure before bone healing is achieved.     

Carpal Joint Arthrodesis as a Treatment for Chronic Septic Carpitis in Calves and Cattle

Chronic septic carpitis in 72 calves and adult cattle was treated by arthrodesis of the carpus (n = 24), proximal or distal carpal row resection with arthrodesis (n = 31), or resection of both the proximal and distal carpal bones and radio-metacarpal arthrodesis (n = 17). Painfree arthrodesis permitting full weight-bearing was obtained in 69% of the cases. Arthrodesis without resection of carpal bones was successful in 87% of the cases. With resection of one carpal row, 72% of the cases were treated successfully. Radio-metacarpal arthrodesis with removal of both carpal rows resulted in fusion in 35% of the animals.     

High field magnetic resonance imaging contributes to diagnosis of equine distal tarsus and proximal metatarsus lesions: 103 horses

Injury of the distal tarsus and proximal metatarsus commonly causes lameness. Magnetic resonance imaging (MRI) allows concurrent assessment of both the distal tarsal joints and suspensory ligament origin, and aids identification of lesions that may otherwise go undetected by other modalities. In this retrospective observational study, the medical records of a veterinary imaging center were searched for MRI exams of the distal tarsus and proximal metatarsus for the years 2012 through 2014. Studies for 125 limbs of 103 horses were identified and retrospectively evaluated by two board‐certified veterinary radiologists. Soft tissue and osseous changes were characterized and graded by degree of severity. The patients’ signalment, lameness severity, and results of diagnostic analgesia were recorded. Osteoarthritic changes of the distal intertarsal and tarsometatarsal joints were the most common findings. Other findings included bone marrow lesions, degenerative changes of the small cuboidal bones, subchondral cystic lesions, and intertarsal desmopathy. Suspensory ligament desmopathy was found in 53% of limbs. Fourty‐seven percent of limbs that responded to analgesia of the proximal suspensory ligament had more severe lesions in the distal tarsus. Bone marrow lesions of the third tarsal bone were the only MRI finding that correlated with grade of lameness in patients for which lameness grade was reported. The grade of lameness has a poor correlation with the severity of lesions found on MRI. The findings support the use of MRI for simultaneous evaluation of the proximal metatarsus and distal tarsus, particularly given the difficulty of lesion localization with diagnostic analgesia.     

Sialolithiasis in horses: A retrospective study of 25 cases (2002–2013)

This study evaluated outcomes of surgical treatment of equine sialolithiasis, highlighting cases involving the proximal parotid salivary duct. Sialoliths in the proximal parotid duct were difficult to identify radiographically and more frequently associated with draining tracts and sialadenitis compared with sialoliths in the distal parotid duct. Ultrasonography confirmed the diagnosis of sialolithiasis in all cases in which there was no radiographic evidence of a sialolith. All cases of proximal parotid duct sialolithiasis required transcutaneous removal. A longer duration of illness was observed in cases of proximal parotid duct sialolithiasis compared with cases involving the distal parotid duct, and in cases requiring transcutaneous removal compared with cases requiring transoral removal. Recurrence of sialolithiasis was documented in 24% of cases, all of which were located in the distal parotid duct. The average time to recurrence was 2.8 years.     

The influence of temperature and age on the T1 relaxation time of the equine distal limb

The extent of fat suppression using short tau inversion recovery (STIR) imaging is variable between horses. Our aim was to determine if patient's age and/or hoof temperature have an influence on the T(1) relaxation time of bone marrow in the equine distal limb, thereby affecting the suppression of fat signal. Magnetic resonance imaging was conducted on standing horses and cadaver samples using a low-field magnet (0.27 T). The hoof temperature was measured at the lateral side of the coronary band. A modified inversion recovery fast spin-echo (IR-FSE) sequence was used to measure the signal intensity for a range of inversion times (TIs) at six different regions of interest (ROI): (1) distal aspect of the proximal phalanx, (2) proximal aspect of the middle phalanx, (3) distal aspect of the middle phalanx, (4) navicular bone, (5) proximal aspect of the distal phalanx, and (6) distal aspect of the distal phalanx. The T(1) of the bone marrow in the equine distal limb was calculated from the results and was found to increase by 3.13 ± 0.08 (SE) ms/°C. There was no significant effect of age (2-16 years) but the T(1) values measured from the limbs of young (< 1 year) animals were considerably longer (32.6 ± 1.7 (SE) ms). Similar effects of temperature and age were found for all measured ROIs but there were significant differences in the mean values of T(1) , ranging from +7.7 (distal aspect of the distal phalanx) to -13.2 ms (distal aspect of the proximal phalanx).     

Is there a characteristic distal tarsal subchondral bone plate thickness pattern in horses with no history of hindlimb lameness?

REASONS FOR PERFORMING STUDY: Osteoarthritis (OA) is a common cause of distal tarsal pain, but disease development is poorly understood. Awareness of normal tarsal structure and function is important in order to understand the pathogenesis of OA. Thickening of the subchondral bone (SCB) plate has been related to the development of OA, but SCB plate patterns in the equine tarsus have not been documented. HYPOTHESES: There is a repeatable pattern of SCB thickness across the distal tarsal joints, and specifically that thickness would be greatest dorsally and laterally. METHODS: Twenty cadaver tarsi were collected from mature horses that had undertaken low-level exercise only with no history of hindlimb lameness. Magnetic resonance images were acquired using a high-resolution sagittal 3-dimensional T1-weighted spoiled gradient echo sequence. Subchondral bone thickness was measured on sagittal images at dorsal and plantar locations on the proximal and distal aspects of the central (CT) and third (T3) tarsal bones and proximal aspect of the third metatarsal bone (Mt3). RESULTS: On the proximal aspect of CT, medial and lateral SCB thickness were significantly greater than midline. On the distal aspect of CT and T3 and proximal Mt3, lateral SCB thickness was significantly greater than medial and midline sites. Dorsal SCB thickness was greatest on the proximal and distal aspects of CT and proximal Mt3. Subchondral bone accounted for a greater proportion of CT and T3 on the dorsal aspect than the plantar. CONCLUSIONS: There is a repeatable pattern of SCB thickness in the distal tarsal bones of horses with no history of hindlimb lameness. This reflects the pattern of loading across the joints. POTENTIAL RELEVANCE: This study provides evidence of a consistent osteochondral pattern in the equine tarsus for reference in identification of osteoarticular pathologies.     

Comparison of Holding Power of Three Different Pin Designs for External Skeletal Fixation in Avian Bone: A Study in Common Buzzard (Buteo buteo)

Objective— (1) To evaluate resistance to axial extraction of 3 pin designs in avian humerus and tibiotarsus; (2) to assess the effect of pin location within the bone on holding power; and (3) to assess the influence of thread pitch on holding power. Study Design— Resistance of pins to axial extraction was measured immediately after insertion. Animals— Adult common buzzards (Buteo buteo; n=9). Methods— Different pin designs (1 smooth; 2 threaded pins, differing in pitch) were inserted into the proximal and distal metaphysis and the proximal, middle, and distal diaphysis of the humerus and tibiotarsus. Maximum force required for axial extraction of pins was recorded. Results— Smooth pins had the lowest extraction force (P<.05). Pins inserted into the diaphysis (proximal, middle and distal) of the humerus and the distal metaphysis of the tibiotarsus had a greater pullout strength than pins in other locations. Pins with a smaller pitch inserted into the proximal diaphysis and distal metaphysis of the humerus, and the proximal metaphysis of the tibiotarsus had significantly greater holding power than pins with a larger pitch (P<.05). Conclusions— Pins inserted into the diaphysis of humerus and the distal metaphysis of the tibiotarsus are better at resisting extraction. Pins with a smaller pitch possess greater holding power than pins with a larger pitch in avian humerus and tibiotarsus. Clinical Relevance— Consideration should be given to pin location and thread pitch, when choosing external skeletal fixation to repair an avian humeral or tibiotarsal fracture.     

The Bacteria Isolated from the Skin of 20 Horses at a Veterinary Teaching Hospital

The objective of this study was to isolate bacteria on the skin of the proximal to distal equine limb to guide the practitioner in the selection of prophylactic antimicrobial protocols. This prospective study involved 20 client-owned horses that were admitted to the Veterinary Teaching Hospital for routine elective surgery. Each horse spent between 12 and 36 hours at our hospital before sampling. Samples were collected from the skin of the left mid-thorax and the dorsal aspect of nine joints on the left side of each horse: front and hind coffin and fetlock joints, carpi, elbows, shoulders, hocks, and stifles. Samples were cultured aerobically and speciated when possible. When evaluating bacterial composition by location, a 40% difference was considered clinically significant. When comparing proximal sites above the fetlock to distal sites, the odds of isolating gram-positive bacteria were 1.23 times (P = .0124) higher at proximal sites; the odds of isolating coliform bacteria were 1.32 times (P = .023) higher at distal sites; and the odds of isolating a common septic arthritis pathogen were 1.16 times (P = .018) higher at distal sites. Coagulase-positive Staphylococcus was not isolated in this study. All comparisons between sites and between the proximal and distal limb were <40%, and thus were not considered clinically significant. No coagulase-positive Staphylococcus was isolated from any of the 200 sites in this study, suggesting that iatrogenic infections by that organism may not be because of preexisting flora. These data suggest that antibiotic prophylaxis targeting preexisting normal flora should be similar regardless of the area of interest on the limb.