Cloning and tissue expression of the equine transferrin receptor

Background: Characterization of anemia in horses presents a challenge, as they do not release reticulocytes into peripheral blood. Transferrin receptor (TfR) expression is highest on erythroid cells in people and rats, and measurement of a soluble serum form (sTfR) is used to quantify erythropoiesis in these species. We hypothesized that equine TfR (eTfR) expression is similar in quantity and distribution to that in these other species and thus has potential for characterization of the regenerative response in anemic horses. Objective: This study was conducted to clone and sequence the eTfR gene and measure expression levels using quantitative real‐time PCR and immunohistochemical (IHC) staining. Methods: Total RNA from equine bone marrow was used to produce cDNA. The eTfR gene was amplified using pooled gene‐specific primers, and PCR products were sequenced. Rapid amplification of cDNA ends was used to obtain the first 22 nucleotides of the coding sequence. Quantitative PCR was performed using eTfR gene‐specific primers, and IHC staining was used to localize TfR protein expression. Results: The deduced amino acid (aa) sequence (767 aa) of the eTfR was 75–83% identical with sequences of the receptor in several other mammals. As in people and rats, eTfR mRNA expression was highest in the bone marrow, and distribution in other tissues was also similar. Conclusion: The eTfR gene is similar to that of other mammals in structure and expression levels. We hypothesize that it is also similar in function and that, following development of an immunoassay, determining sTfR concentrations will be useful for identifying the regenerative response in anemic horses.     

Acute myeloid leukaemia (M6B: pure acute erythroid leukaemia) in a Thoroughbred foal

A 10-week-old Thoroughbred filly was referred for anaemia of 4 weeks' duration. Haematology revealed severe anaemia and panleucopenia. Cytological examination of bone marrow smears revealed a myeloid to erythroid ratio <0.02:1 (reference range 0.5-2.4:1.0) and an abundance of erythroid precursor cells. The erythroid cell population included rubriblasts, prorubricytes and rubricytes, with only scant numbers of metarubricytes present. There were numerous mitotic erythroid cells, some of which were atypical and megaloblastic. These cytomorphological changes are consistent with pure acute erythroid leukaemia. No treatment was instituted and the filly died three days after presentation. This case illustrates the need to consider both haematology and bone marrow findings to establish a diagnosis of pure erythroid leukaemia. To our knowledge, there is no documented case of acute myeloproliferative disease in horses involving cells of erythroid lineage, but this condition should be considered a differential diagnosis for horses presenting with anaemia.     

Hematopoietic Neoplasias in Horses: Myeloproliferative and Lymphoproliferative Disorders

Leukemia, i.e., the neoplasia of one or more cell lines of the bone marrow, although less common than in other species, it is also reported in horses. Leukemia can be classified according to the affected cells (myeloproliferative or lymphoproliferative disorders), evolution of clinical signs (acute or chronic) and the presence or lack of abnormal cells in peripheral blood (leukemic, subleukemic and aleukemic leukemia). The main myeloproliferative disorders in horses are malignant histiocytosis and myeloid leukemia, the latter being classified as monocytic and myelomonocytic, granulocytic, primary erythrocytosis or polycythemia vera and megakaryocytic leukemia. The most common lymphoproliferative disorders in horses are lymphoid leukemia, plasma cell or multiple myeloma and lymphoma. Lymphoma is the most common hematopoietic neoplasia in horses and usually involves lymphoid organs, without leukemia, although bone marrow may be affected after metastasis. Lymphoma could be classified according to the organs involved and four main clinical categories have been established: generalized-multicentric, alimentary-gastrointestinal, mediastinal-thymic-thoracic and cutaneous. The clinical signs, hematological and clinical pathological findings, results of bone marrow aspirates, involvement of other organs, prognosis and treatment, if applicable, are presented for each type of neoplasia. This paper aims to provide a guide for equine practitioners when approaching to clinical cases with suspicion of hematopoietic neoplasia.     

Reference values for the bone marrow aspirates in adult dogs

Reference ranges for each cell type and six different ratios (M : E ratio = quotient of number of myeloid cells/erythroid precursors; different maturation ratios) were calculated for bone marrow aspirates of adult dogs. These values were based on 2.5 and 97.5% percentiles of differentials of 1000 cells in bone marrow aspirates of 92 healthy 1-8-year-old dogs. The results of intact male and female dogs were compared. No distinct sex-related differences were found (P > 0.05). A wide physiological range was observed in almost all bone marrow cells resulting in broad reference ranges for the ratios. The clinically relevant M : E ratio varied between 0.45 and 2.87. The accurate cytological examination of bone marrow based on the reference ranges presented in this study requires preparation of high-quality bone marrow films with minimal blood contamination.     

Erythrocyte volume distribution analysis and hematologic changes in two horses with immune-mediated hemolytic anemia

Immune-mediated hemolytic anemia was diagnosed in two horses on the basis of regenerative anemia, increased erythrocyte fragility in hypotonic saline, autoagglutination, and a positive direct antiglobulin (Coomb's) test. During steroid therapy partial resolution of the anemia was indicated by rising packed cell volume, macrocytosis, and bone marrow erythroid hyperplasia. Using erythrocyte volume distribution histograms (erythrograms), the regenerative response was characterized by analysis of macrocytic and normocytic erythrocyte subpopulations. In both horses, a gradual net increase of about 2 X 10(6) macrocytes/microliter occurred over a four- to five-week period. Over the same interval there was a gradual decrease in the number of normocytes. We suggest that the macrocytes remained large through this period rather than contributing to normocyte population growth. Erythrograms may provide an additional means of evaluating erythrocyte regeneration in horses.     

Isolation and characterization of bone marrow-derived equine mesenchymal stem cells

OBJECTIVE: To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. SAMPLE POPULATION: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. PROCEDURES: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. RESULTS: Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.     

Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity

OBJECTIVES: To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. STUDY DESIGN: In vitro experimental study. ANIMALS: Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years). METHODS: Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob). RESULTS: Initial MSC isolates had a lag phase with a significantly longer CD time (DT=4.9+/-1.6 days) compared with the average DT (1.4+/-0.22 days) of subsequent MSC passages. Approximately 1 in 4224+/-3265 of the total nucleated BM cells displayed fibroblast colony-forming activity. Primary MSCs differentiated in response to adipogenic and osteogenic inductive conditions and maintained their differentiation potential during subsequent passages. CONCLUSIONS: The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of foals and young adult horses are similar to those documented for BM MSCs of other mammalian species. CLINICAL RELEVANCE: The results have direct relevance to the use of BM as a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine.     

92匹马骨髓细胞正常值的测定

通过对92匹三河马（其中成年马67匹，育成马25匹）骨髓内各种细胞的形态学观察，计算出成年马和育成马骨髓细胞的正常值，为马血液疾病的诊断提供了参考依据。     

Using red blood cell creatine concentration to evaluate the equine erythropoietic response

Red blood cell creatine concentration was examined to determine its association with the equine erythropoietic response. Studies were conducted on 9 healthy horses, 4 healthy ponies, 24 anemia horses, and 2 horses in which anemia was experimentally induced. A modified Jaffe reaction was used to measure RBC creatine concentration. The mean RBC creatine concentration of the 9 healthy horses was 5.72 +/- 0.42 mg/dl, and that of the 4 healthy ponies was 2.59 +/- 0.31 mg/dl. Density-separation of erythrocytes from the healthy horses revealed significantly higher (P less than 0.001) creatine content (7.72 +/- 0.57 mg/dl) in the young RBC populations than in the old RBC populations (4.03 +/- 0.27 mg/dl). The RBC creatine content was assayed in 19 hot-blooded horses which were anemic due to a variety of causes. Of these anemic horses, 12 with PCV between 25% and 30% had a mean RBC creatine concentration of 6.12 +/- 0.46 mg/dl. The 7 other anemic horses with PCV less than 25% had a mean RBC creatine value of 6.07 +/- 0.12 mg/dl. Bone marrow films were examined from 5 anemic horses and in the 2 horses in which anemia was experimentally induced. The RBC creatine concentration correlated positively (P less than 0.001) with the reticulocyte count in the bone marrow and negatively with the myeloid-erythroid ratio (P less than 0.001).     

Prevalence and some clinical characteristics of equine cheek teeth diastemata in 471 horses examined in a UK first-opinion equine practice (2008 to 2009)

Cheek teeth (CT) diastemata are now recognised as a clinically significant equine disorder, but their prevalence in the general equine population is unknown. There is also limited information on the signalment of affected horses; the more commonly affected Triadan sites; and the shape and clinical characteristics of CT diastemata. During the 12-month study period (2008 to 2009), standardised records were obtained during routine dental examinations performed by five veterinarians in a first-opinion equine practice. Cheek teeth diastemata were identified in 49.9 per cent of all horses (n=471) of mean age 11 years (range one to 30 years), with 83.5 per cent of all diastemata affecting mandibular CT and 16.5 per cent affecting maxillary CT. The mean number of diastemata per case was 1.7 (range one to 20) and the mandibular 07 to 08 position was most commonly affected. Valve diastemata were more common (72.1 per cent prevalence) than open diastemata (27.9 per cent). Food trapping was present in 91.4 per cent of diastemata, with gingivitis and periodontal pockets adjacent to 34.2 per cent and 43.7 per cent, respectively. Halitosis was present in 45.5 per cent of affected horses. There was an age-related increase in both the prevalence of diastemata, and in the numbers of diastemata per affected horse, and horses over 15 years old had a significantly increased proportion of open diastemata.     

Comparison of Chondrogenic Potential in Equine Mesenchymal Stromal Cells Derived from Adipose Tissue and Bone Marrow

Objective— To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). Study Design— In vitro experimental study. Animals— Adult Thoroughbred horses (n=11). Methods— BM (5 horses; mean [±SD] age, 4±1.4 years) or adipose tissue (6 horses; mean age, 3.5±1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis±transforming growth factor‐3 (TGFβ₃) and bone morphogenic factor‐6 (BMP‐6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross‐sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays. Results— Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline‐like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. Conclusion— Equine MSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species. Clinical Relevance— Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.     

Hematologic effects of subcutaneous administration of recombinant human granulocyte colony-stimulating factor (filgrastim) in healthy alpacas

OBJECTIVE: To determine the effects of SC administration of filgrastim on cell counts in venous blood and bone marrow of healthy adult alpacas. ANIMALS: 10 healthy alpacas. PROCEDURES: Alpacas were randomly assigned to receive treatment with filgrastim (5 microg/kg, SC; n=5) or an equivalent volume of physiologic saline (0.9% NaCl) solution (5) once a day for 3 days. Blood samples were obtained via jugular venipuncture 1 day prior to treatment and once a day for 5 days commencing 24 hours after the first dose was administered. Complete blood counts were performed for each blood sample. Bone marrow aspirates were obtained from the sternum of each alpaca 48 hours before the first treatment was administered and 72 hours after the third treatment was administered. Myeloid-to-erythroid cell (M:E) ratio was determined via cytologic evaluation of bone marrow aspirates. RESULTS: In filgrastim-treated alpacas, substantial increases in counts of WBCs and neutrophils were detected within 24 hours after the first dose was administered. Band cell count and percentage significantly increased 24 hours after the second dose. Counts of WBCs, neutrophils, and band cells remained high 48 hours after the third dose. Red blood cell counts and PCV were unaffected. The M:E ratio also increased significantly after treatment with filgrastim. CONCLUSIONS AND CLINICAL RELEVANCE: Filgrastim induced rapid and substantial increases in numbers of circulating neutrophils and M:E ratios of bone marrow in healthy alpacas. Therefore, filgrastim may be useful in the treatment of camelids with impaired bone marrow function.     

Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study

Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP labelled cells located mainly within injected lesions, but with a small proportion integrated into the crimp pattern of adjacent healthy areas of tendon. Furthermore, there was no visible cell mediated immune response to allogeneic MPCs in either of the host horses.     

Validation of a centrifugation/flotation technique for the diagnosis of equine cestodiasis.

A centrifugation/flotation technique for the identification of equine tapeworm eggs is described. It was validated by using faeces samples from 80 horses of known tapeworm status, and had a sensitivity of 61 per cent and a specificity of 98 per cent. The exclusion of false negative results in animals with less than 20 tapeworms increased the sensitivity to 92 per cent. No significant correlation was found between the number of eggs observed and the number of tapeworms present in the horses.     

Use of serum amyloid A in equine medicine and surgery

Serum amyloid A (SAA) has become an indispensable part of the management of equine patients in general practice and specialized hospital settings. Although several proteins possess acute phase properties in horses, the usefulness of SAA exceeds that of other acute phase proteins. This is due to the highly desirable kinetics of the equine SAA response. SAA concentrations exhibit a rapid and pronounced increase in response to inflammation and a rapid decline after the resolution of inflammation. This facilitates the detection of inflammatory disease and real-time monitoring of inflammatory activity. SAA may be used in all stages of patient management: (1) before diagnosis (to rule in/rule out inflammatory disease), (2) at the time of diagnosis (to assess the severity of inflammation and assist in prognostication), and (3) after diagnosis (to monitor changes in inflammatory activity in response to therapy, with relapse of disease, or with infectious/inflammatory complications). By assessing other acute phase reactants in addition to SAA, clinicians can succinctly stage inflammation. White blood cell counts and serum iron concentration change within hours of an inflammatory insult, SAA within a day, and fibrinogen within 2–3 days; the interrelationship of these markers thus indicates the duration and activity of the inflammatory condition. Much research on the equine SAA response and clinical use has been conducted in the last decade. This is the prerequisite for the evidence-based use of this analyte. However, still today, most published studies involve a fairly low number of horses. To obtain solid evidence for use of SAA, future studies should be designed with larger sample sizes.     

The Kinetics of Hematopoiesis in the Light Horse I. The Lifespan of Peripheral Blood Cells in the Normal Horse

Three Standardbred horses were given 0.2 mg (1 mCi) of ⁷⁵selenomethionine intravenously and a second group of three were given 10 mCi of tritiated diisopropylfluorophosphate (0.5 mg) intravenously. Observations on labeled cells were continued for 250 days after radioselenium injection and 160 days after tritium injection.

The lifespan of erythrocytes using ⁷⁵selenmethionine was 155 ± 10 days and 148 ± 7.8 days using tritiated diisopropylfluorophosphate. There was no significant difference at the 10% level between the lifespans, using these labels. The uptake of radioselenium into erythrocytes reached a mean maximum of 11.5% at 82 ± 18.9 days after injection of the label. There was an elution of from 17.6% of the injected dose of tritium label down to 7.5% eight days after injection of this isotope. From this study both of these labels appear to be satisfactory for determining the erythrocyte lifespan of the horse.

The mean time of the curve of mean radioselenium activity of peripheral blood leukocytes for the three horses was 5.54 days and the lifespan of these cells was seven days. The mean lifespan of peripheral blood leukocytes of the three horses after in vivo labelling with tritiated diisopropylfluorophosphate was 17.3 hours. No specific labelling was found in bone marrow or peripheral blood cells at this level of tritium labelling by dipping emulsion autoradiography.

The mean lifespan of equine platelets for three horses using radioselenomethionine was 9.2 days and the mean lifespan of equine platelets using tritiated diisopropylfluorophosphate was 6.6 days in a group of three horses.

     

Differentiation of dogs with regenerative and non-regenerative anaemia on the basis of their red cell distribution width and mean corpuscular volume

The red blood cell distribution width (RDW), which provides a quantitative measure of the heterogeneity of the red cell population (anisocytosis) in the peripheral blood, the mean corpuscular volume (MCV) and a regression model combining both variables were used to assess their predictive accuracy in differentiating 51 dogs with regenerative anaemia from 92 dogs with non-regenerative anaemia, which had been diagnosed on the basis of the corrected reticulocyte count A classification tree analysis was constructed to generate an optimum set of diagnostic rules to differentiate between the two types of anaemia. Seventy-four dogs with a normal haemogram were used as controls. An increase of 1 per cent in the RDW and of 1 fl in the MCV increased the odds of an anaemic dog suffering from regenerative anaemia by factors of 1.3 and 1.14, respectively. By the classification tree, 78 per cent of anaemic dogs with a RDW of 16.25 per cent or less would be expected to have non-regenerative anaemia. With a RDW over 16.25 per cent, an MCV of 68.2 fl was the cut-off between dogs expected to have regenerative (71 per cent) or non-regenerative (75 per cent) anaemia. The RDW and MCV are measured by most automatic haematology analysers and may give the first indication of the bone marrow response of an anaemic dog. However, different electronic counters give different normal values of the RDW and MCV.     

Evaluation of the erythroid regenerative response in two different models of experimentally induced iron deficiency anemia

Anemia was induced in weanling Sprague Dawley rats either by feeding an iron-deficient diet or by chronic phlebotomy. The erythroid regenerative response was then evaluated before and after a hemolytic event, and results were compared with those of a third group of control nonphlebotomized rats fed an iron-replete diet. Diet and phlebotomy groups developed a similar degree of anemia (mean hemoglobin concentration 7.9 g/dL and 7.8 g/dL, respectively; controls, 13.9 g/dL) and hypoferremia (mean serum iron concentration 25.4 microgram/dL and 34.9 microgram/dL, respectively; controls, 222.0 microgram/dL). However, the anemia in diet rats was nonregenerative (reticulocyte count, 83.1 X 10(3) cells/microliter) and associated with bone marrow erythroid hypoplasia; whereas the anemia in phlebotomy rats was regenerative (reticulocyte count, 169.6 X 10(3) cells/microliter) and associated with bone marrow erythroid hyperplasia. Thrombocytosis was seen in diet rats (1,580 X 10(3) cells/microliter) but not phlebotomy rats (901 X 10(3) cells/microliter) when compared with controls (809 X 10(3) cells/microliter). To further evaluate the regenerative capability, phenylhydrazine (PHZ) was administered to induce hemolysis. Erythrocyte mass declined approximately 25% in all groups, including controls. The reticulocytosis (265.3 X 10(3) cells/microliter) seen in phlebotomy rats was earlier and significantly greater than that seen in either diet or control rats. Hemoglobin concentration returned to pre-PHZ concentrations (7.9 g/dL) in phlebotomy rats within 4 days posthemolysis. In diet rats, the maximal regenerative response (176.3 X 10(3) cells/microliter) was not seen until 8 days posthemolysis, and hemoglobin (7.5 g/dL) did not return to pre-PHZ concentrations during the 8-day study. In many aspects, the anemia seen following diet- or phlebotomy-induced iron deficiency was similar. However, the erythroid regenerative capability varied depending on the mechanism by which anemia was induced and furthermore altered the efficiency of hemoglobin production following a hemolytic event. These results suggest that the availability of iron in the diet may modulate the pathogenesis of iron deficiency anemia.     

Serum amyloid A in equine health and disease

Serum amyloid A (SAA) is the major acute phase protein in horses. It is produced during the acute phase response (APR), a nonspecific systemic reaction to any type of tissue injury. In the blood of healthy horses, SAA concentration is very low, but it increases dramatically with inflammation. Due to the short half-life of SAA, changes in its concentration in blood closely reflect the onset of inflammation and, therefore, measurement of SAA useful in the diagnosis and monitoring of disease and response to treatment. Increases in SAA concentration have been described in equine digestive, reproductive and respiratory diseases and following surgical procedures. Moreover, SAA has proven useful for detection of some subclinical pathologies that can disturb training and competing in equine athletes. Increasing availability of diagnostic tests for both laboratory and field use adds to SAA's applicability as a reliable indicator of horses’ health status. This review article presents the current information on changes in SAA concentrations in the blood of healthy and diseased horses, focussing on clinical application of this biomarker.     

Comparison of bone marrow aspiration at the sternum and the tuber coxae in middle-aged horses

The objective of this study was to compare bone marrow (BM) aspirates from the sternum and the tuber coxae of middle-aged horses. Bone marrow was obtained from the sternum and both tubera coxae of 12 healthy, 13-year-old geldings. Two different puncture techniques were used for the tuber coxae. The 2 syringes used for sternal sampling were evaluated separately. The mononuclear cell (MNC) fraction of the BM was isolated and the mesenchymal stem cells (MSCs) were culture-expanded. At the sternum, BM aspiration was always possible. Bone marrow aspiration at the tuber coxae required straight and deep needle penetration combined with high negative pressure. With this technique a median sample amount of 11.0 mL with large individual variation was obtained. A median of 3.06 × 10(6) MNC/mL BM (1st syringe) and 2.46 × 10(6) MNC/mL BM (2nd syringe) was isolated from sternal samples. In contrast, the tuber coxae yielded a median of 0.27 × 10(6) MNC/mL BM. The first passage yielded a median of 2.19 × 10(6) MSC (1st syringe) and 1.13 × 10(6) MSC (2nd syringe) from sternal samples, compared to a significantly lower median number of MSC from tuber coxae BM (0.06 × 10(6) MSC). The number of MNC and MSC obtainable from the BM aspirates taken from the tuber coxae is significantly lower than that obtained from the sternal BM aspirates. Autologous BM for the equine athlete is particularly clinically relevant at an advanced age. Based on our findings, the tuber coxae cannot be recommended for BM aspiration in middle-aged horses.