An intramammary device (IMD) was adapted for use in ewes; this device was made of abraded poly. ethylene material (1.7 mm in diameter, 47 mm long) and formed a 15-mm-diameter loop in the gland cistern. The IMD was inserted in 1 gland in each of 43 ewes. A significant (P < 0.0001) increase in milk somatic cell count (SCC) was observed in glands provided with an IMD. This increase was attributable to an increase in neutrophil numbers and was observed during the first 12 weeks after insertion. The IMD had a protective effect against experimentally induced staphylococcal mastitis (Staphylococcus aureus and S epidermidis), although different milk SCC were required for protection from each bacterial species in most ewes (10(6) and 2 X 10(5) cells/ml, respectively). Histologic studies revealed that the IMD induced local squamous metaplasia in the glandular part of the lactiferous sinus. Erythrocytes were found in milk from glands provided with an IMD throughout the studied period (35 days of the 45-day lactation) and, in some cases, blood clots were observed during the first 2 weeks of lactation. Glands with IMD also had lower milk production and quality at 30 and 32 days of lactation. Eight ewes with IMD were studied throughout a subsequent lactation. Milk from the IMD-containing glands had an increase in SCC, as in the previous lactation period; did not contain blood clots or erythrocytes; and had normal composition (similar to that in glands without the IMD).

OBJECTIVE To assess clotting times, coagulation factor activities, sterility, and thromboelastographic parameters of liquid plasma (LP), thawed fresh frozen plasma (FFP-T), and 2 novel formulations of freeze-dried plasma (FDP) stored refrigerated over 35 days. SAMPLE 6 units of canine LP and FFP-T from a commercial animal blood bank and 5 units each of 2 formulations of canine FDP. PROCEDURES Prothrombin time; activated partial thromboplastin time; activities of coagulation factors II, V, VII, VIII, IX, X, XI, and XII; and thromboelastographic parameters were determined for each product on days 0 (baseline), 3, 7, 14, 21, 28, and 35. For each day, a sample of each product was also submitted for aerobic bacterial culture. RESULTS Small changes in coagulation factor activities and mild increased time to initial clot formation in LP and FFP-T were noted over the 35-day storage period. Activities of factor VIII in FDP1 and factor XII in FDP2 were < 50% at baseline but varied throughout. Compared with FFP-T, time to initial clot formation was increased and clot strength was preserved or increased for the FDPs throughout the study. One FDP had decreased pH, compared with other products. No plasma product yielded bacterial growth. CONCLUSIONS AND CLINICAL RELEVANCE Liquid plasma and FFP-T would be reasonable to use when stored refrigerated for up to 35 days. Both FDP products showed variability in coagulation factor activities. Studies investigating the usefulness of these plasma products (FDPs) in dogs and the variable days of refrigerated storage (all products) are warranted.

OBJECTIVE To assess feasibility of the use of a dynamic viscoelastic coagulometer on chicken blood and compare coagulation variables for fresh whole blood and sodium citrate–preserved whole blood as well as effects of 3 coagulation activators on blood from chickens. SAMPLE Blood samples from 30 hens. PROCEDURES Chickens were allowed to rest undisturbed for 1 hour. A blood sample was collected from an ulnar vein; 1.4 mL was analyzed immediately, and 1.8 mL was mixed with sodium citrate and subsequently recalcified and analyzed. A separate coagulation activator (glass beads, kaolin clay, or tissue factor) was in each of the 2 channels of the analyzer. Chickens were allowed a 1-hour rest period, and another blood sample was collected from the contralateral ulnar vein; it was processed in the same manner as for the first sample, except both channels of the analyzer contained the same coagulation activator. RESULTS Compared with fresh samples, citrated samples had higher values for activated clotting time and platelet function and lower clotting rates. Intra-assay coefficients of variation of coagulation profiles for citrated samples were markedly greater than the limit of 10%, whereas values for fresh samples were close to or < 10%. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that use of a dynamic viscoelastic coagulometer on chicken blood was feasible and that analysis of fresh whole blood from healthy chickens provided results with less variability than did analysis of citrated blood. Samples preserved with sodium citrate were associated with significant relative hypocoagulability, compared with results for fresh blood.

OBJECTIVE To evaluate effects of pneumoperitoneum created with warmed humidified CO2 (WHCO2) during laparoscopy on core body temperature, cardiorespiratory and thromboelastography variables, systemic inflammation, peritoneal response, and signs of postoperative pain in healthy mature dogs. ANIMALS 6 mature purpose-bred dogs. PROCEDURES In a randomized crossover study, each dog was anesthetized twice, and pneumoperitoneum was created with standard-temperature CO2 (STCO2; 22°C and 0% relative humidity) and WHCO2 (37°C and 98% relative humidity). Data were collected during each procedure, including core body temperature, cardiorespiratory and thromboelastography variables, and inflammatory biomarkers. Peritoneal biopsy specimens were collected and evaluated with scanning electron microscopy. Dogs were assessed for signs of postoperative pain. RESULTS Mean core body temperature was significantly lower (35.2°C; 95% confidence interval, 34.5° to 35.8°C) with WHCO2 than with STCO2 (35.9°C; 95% confidence interval, 35.3° to 36.6°C) across all time points. Cardiac index increased during the procedure for both treatments but was not significantly different between treatments. Thromboelastography variables did not differ significantly between treatments as indicated by the coagulation index. Subjective evaluation of peritoneal biopsy specimens revealed mesothelial cell loss with STCO2. There was no significant difference in circulating C-reactive protein or interleukin-6 concentrations. There was a significant increase in the number of postoperative pain scores > 0 for the WHCO2 treatment versus the STCO2 treatment. CONCLUSIONS AND CLINICAL RELEVANCE Analysis of these data suggested that effects on evaluated variables attributable to the use of WHCO2 for creating pneumoperitoneum in healthy mature dogs undergoing laparoscopy did not differ from effects for the use of STCO2.

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII–deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in factor VII–deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

OBJECTIVE To determine effects of oral administration of Yunnan Baiyao on platelet activation, coagulation, and fibrinolysis in healthy horses. ANIMALS 12 healthy adult horses. PROCEDURES In a randomized blinded crossover study that included a 4-week washout period between treatments, horses were orally administered a paste containing Yunnan Baiyao (15 mg/kg) or placebo at 12-hour intervals for 3 days. Blood samples were collected before start of treatment (time 0) and at 24 and 72 hours for a CBC, measurement of fibrinogen concentration, coagulation screening tests, and a panel of assays to assess platelet activation (including ADP- and collagen-induced aggregation and closure times, flow-cytometric variables of platelet-leukocyte aggregates, platelet membrane P-selectin and phosphatidylserine expression, and microparticle release), von Willebrand factor (vWF) concentration, and cofactor activity. In addition, thrombelastography was used to evaluate fibrin formation in tissue factor–activated whole blood and plasma and to assess tissue plasminogen activator–induced plasma fibrinolysis. For each treatment, values obtained before and 72 hours after start of administration were compared by use of Wilcoxon signed rank tests. RESULTS Yunnan Baiyao treatment had no significant effect on any hemostatic variable, compared with results for the placebo treatment. CONCLUSIONS AND CLINICAL RELEVANCE Administration of Yunnan Baiyao at a dosage typically used in clinical practice had no effect on in vitro measures of platelet or vWF function and no enhancement of fibrin-clot formation or stability. Any hemostatic actions of Yunnan Baiyao may require higher dosages or result from cell-surface interactions at sites of vascular and tissue injury not examined in this study.

OBJECTIVE To measure thrombin generation by high and low tissue factor (TF)–expressing canine cancer cell lines. SAMPLE Canine cell lines CMT25 (high TF–expressing mammary gland tumor cell line) and HMPOS (low TF–expressing osteosarcoma cell line). PROCEDURES Thrombin generation by cancer cells was measured in pooled normal canine plasma by use of calibrated automated thrombography without added trigger reagents. Results were expressed as lag time, time to peak thrombin concentration, peak thrombin concentration, and total thrombin concentration or thrombin generation potential. Corn trypsin inhibitor, hirudin, and annexin V were used to inhibit contact activation, thrombin formation, and phosphatidylserine activity, respectively. Pooled normal human plasma deficient in coagulation factors VII, VIII, IX, X, XI, or XII was used to assess the role of individual coagulation factors on thrombin generation. RESULTS CMT25 generated significantly more thrombin than did HMPOS (mean ± SD, 3,555 ± 604nM thrombin•min and 636 ± 440nM thrombin•min, respectively). Thrombin generation of CMT25 was dependent on factor VII and phosphatidylserine and was independent of contact activation. In contrast, thrombin generation of HMPOS was attributed to contact activation. CONCLUSIONS AND CLINICAL RELEVANCE High TF-expressing canine mammary cancer cells generated thrombin in a plasma milieu in vitro in a factor VII- and phosphatidylserine-dependent manner. These findings support a role for TF in hypercoagulability detected in dogs with mammary gland tumors and potentially for other tumors that strongly express TF.

OBJECTIVE To compare stability of hemostatic proteins in canine fresh-frozen plasma (FFP) thawed with a modified commercial microwave warmer (MCM) or warm water bath (37°C; WWB) or at room temperature (22°C). SAMPLE Fresh-frozen plasma obtained from 8 canine donors of a commercial blood bank. PROCEDURES A commercial microwave warmer was modified with a thermocouple to measure surface temperature of bags containing plasma. The MCM and a WWB were each used to concurrently thaw a 60-mL bag of plasma obtained from the same donor. Two 3-mL control aliquots of FFP from each donor were thawed to room temperature without use of a heating device. Concentrations of hemostatic proteins, albumin, and D-dimers; prothrombin time (PT); and activated partial thromboplastin time (aPTT) were determined for all samples. RESULTS Significant decreases in concentrations of factors II, IX, X, XI, fibrinogen, von Willebrand factor, antithrombin, protein C, and albumin and significant increases in PT and aPTT were detected for plasma thawed with the MCM, compared with results for samples thawed with the WWB. Concentrations of factors VII, VIII, and XII were not significantly different between plasma thawed with the MCM and WWB. Concentrations of D-dimers were above the reference range for all thawed samples regardless of thawing method. No significant differences in factor concentrations were detected between control and WWB-thawed samples. CONCLUSIONS AND CLINICAL RELEVANCE Significant differences in hemostatic protein concentrations and coagulation times were detected for plasma thawed with an MCM but not between control and WWB-thawed samples. Clinical importance of these changes should be investigated.

The effects of heparin administration, by the oral route, were evaluated in dogs. In single and multiple dose studies (single 7.5 mg/kg, multiple 3 × 7.5 mg/kg per 48 h), plasma, urine, and fecal samples were collected at various times up to 120 h after oral administration of unfractionated heparin. Changes in plasma and urine anti-Xa activity, plasma and urine anti-IIa activity, plasma activated partial thromboplastin time (APTT) and antithrombin (ATIII), and chemical heparin in urine and feces were examined with time. There was support for heparin absorption, with significant differences in APTT, heparin in plasma as determined by anti-Xa activity (Heptest) in the single dose study and plasma anti-Xa activity, anti-IIa activity and ATIII; and chemical heparin in urine in the multiple dose study. No clinical evidence of bleeding was detected in any dog during the studies. Oral heparin therapy may be applicable for thromboembolic disease in animals. Further studies are warranted to determine the effects of oral heparin at the endothelial level in the dog.

Objective: To investigate the hemostatic response to surgery and compare the response for ovariohysterectomy with that for ovariectomy and to evaluate the usefulness of thromboelastography on plasma samples. Animals: 42 female dogs. Procedures: Dogs were assigned to undergo ovariohysterectomy or ovariectomy. Blood samples were collected immediately before and 1, 6, and 24 hours after surgery and stored at −80°C for subsequent analysis. Plasma samples were subjected to thromboelastography after thawing. In addition, coagulation variables were measured, including concentrations of von Willebrand factor antigen, fibrinogen, antithrombin, and protein C; activity of factor VIII; activated partial thromboplastin time; prothrombin time; and thrombin time. The fibrinolytic response was assessed via concentrations of D-dimer, plasminogen, and α-2-antiplasmin (plasmin inhibitor). Results: Substantial hemostatic and fibrinolytic activation was evident after surgery in both groups, as characterized by significantly increased global clot strength and an overall hypercoagulable state at 4 hours after surgery in addition to decreases in von Willebrand factor antigen and factor VIII concentrations and shortened prothrombin and thrombin times. The dogs also typically had activation of the fibrinolytic system, as evidenced by increased postoperative concentrations of D-dimer, plasminogen, and plasmin inhibitor. Differences between the 2 groups could not be detected for any variables. Conclusions and Clinical Relevance: Elective surgery with limited tissue trauma induced hemostatic activation in dogs, which led to hypercoagulability after surgery. A difference between the ovariohysterectomy and ovariectomy groups was not detected. Thromboelastography can be used on plasma samples and may be useful for evaluating patterns over time.