Technical note: Effect of storage time and temperature on total calcium concentrations in bovine blood.

Our objectives were to examine the stability of (1) serum and plasma total calcium (tCa) after delayed separation of bovine whole blood stored at 4°C, and (2) frozen serum and plasma tCa stored at -80°C. Whole blood was collected from 19 cows into vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing no additives (serum) or lithium heparin (plasma). Baseline tCa concentrations (0-h tCa) were determined on serum and plasma harvested within 2 h of blood collection. To evaluate the effect of prolonged whole blood storage at 4°C, serum and plasma were harvested from samples after 6, 24, 48, 72 h, and 7 and 14 d of storage. For evaluation of the stability of frozen serum and plasma stored at -80°C, samples were stored for 7 d and 1, 3, 6, and 12 mo. The median 0-h tCa concentration for serum and plasma was 2.25 mmol/L (range: 1.58 to 2.60 mmol/L) and 2.28 mmol/L (range: 1.60 to 2.65 mmol/L), respectively. The average difference in tCa from 0-h samples ranged from -0.02 to 0.03 mmol/L for all samples stored at either 4 or -80°C. The median within-cow variability for whole blood samples stored at 4°C was 1.1 and 1.0% for serum and plasma, respectively, and for serum and plasma stored at -80°C was 1.1 and 1.3%, respectively. When whole blood was stored at 4°C, no differences in serum or plasma tCa concentrations were observed across the evaluated time points as compared with 0-h concentrations. Similarly, frozen serum and plasma stored at -80°C remained stable up to 12 mo. Therefore, our findings show that whole blood samples may be stored for at least 14 d at 4°C in vacutainer tubes containing no additives or lithium heparin without biologically important changes in tCa concentrations beyond expected analytical variation. Additionally, serum or plasma may be stored at -80°C for up to 12 mo with no effect on tCa concentrations.

Method comparison and validation of a prototype device for measurement of ionized calcium concentrations cow-side against a point-of-care instrument and a benchtop blood-gas analyzer reference method.

The objective of this study was to assess an optimized ion-selective electrode Ca-module prototype as a potential cow-side device for ionized Ca (iCa) measurements in bovine blood. A linearity experiment showed no deviation from linearity over a range of iCa concentrations compared with a commercial point-of-care (POC) device commonly used in the field (POCVS; VetScan i-STAT, Abaxis North America, Union City, CA) and a laboratory gold standard benchtop blood-gas analyzer [reference analyzer (RA); ABL-800 FLEX, Radiometer Medical, Copenhagen, Denmark]. Coefficient of variation on 3 samples with high, within-range, and low iCa concentrations ranged from 1.0 to 3.9% for the prototype. A follow-up validation experiment was performed, in which our objectives were to (1) assess the performance of the prototype cow-side against the POCVS (farm gold-standard) using fresh non-anticoagulated whole-blood samples; (2) assess the performance of the prototype and the POCVS against the RA in a diagnostic laboratory using blood collected in a heparin-balanced syringe; and (3) assess the agreement of the prototype and POCVS on-farm (fresh non-anticoagulated whole blood) against the RA on heparin-balanced blood. Finally, sensitivity and specificity of the results obtained by the prototype and the POCVS cow-side compared with the results obtained by the laboratory RA using 3 different iCa cut points for classification of subclinical hypocalcemia were calculated. A total of 101 periparturient Holstein cows from 3 dairy farms in New York State were used for the second experiment. Ionized Ca results from the prototype cow-side were, on average, 0.06 mmol/L higher than the POCVS. With heparin-balanced samples under laboratory conditions, the prototype and POCVS measured an average 0.04 mmol/L higher and lower, respectively, compared with the RA. Results from the prototype and POCVS cow-side were 0.01 mmol/L higher and 0.05 mmol/L lower, respectively, compared with results from the laboratory RA on heparinized blood. Sensitivity and specificity for the prototype and the POCVS under farm conditions at 3 potential subclinical hypocalcemia cut points were 100 and ≥93.5%, respectively. This novel ion-selective electrode Ca-module could become a rapid low-cost tool for assessing iCa cow-side, while qualitatively allowing classification of subclinical hypocalcemia on-farm.

A retrospective review of acute myeloid leukaemia in 35 dogs diagnosed by a combination of morphologic findings, flow cytometric immunophenotyping and cytochemical staining results (2007-2015).

Acute myeloid leukaemia (AML) is an uncommon, rapidly progressive neoplasm in dogs. The aim of this retrospective study was to characterize the clinical presentation, haematologic findings, diagnostic imaging results, treatment and survival time of a contemporary cohort of dogs with AML. Diagnosis was based on >20% blasts in bone marrow or blood identified as myeloid based on morphologic findings, flow cytometric immunophenotyping and cytochemical staining. Medical records of 35 dogs diagnosed with AML from 2007 to 2015 were included. Most dogs presented with inappetence (66%) and lethargy (57%) and physical examination findings of peripheral lymphadenopathy (74%) and tachypnea (62%). Common haematologic findings were quantifiable circulating blasts (85%; median blast count 35 700/µL; range: 300-276 500/µL), anaemia (median haematocrit 34%; range: 11%-52%) and thrombocytopenia (median 57 000/µL; range: 9000-252 000/µL). Bicytopenia and pancytopenia were each found in 44% of dogs. Follow-up information was available for 34 dogs. The overall median survival time from diagnosis was 19 days (range: 1-121 days). Clinical progression in some dogs was not as rapid as previously reported. Haematologic responses to various chemotherapeutics were documented in 3 dogs, with associated survival times of 62, 103 and 121 days. Dogs treated with prednisone or a combination of chemotherapy and prednisone had improved survival compared to dogs that received symptomatic care only (P < .0001). Our results show canine AML has an overlapping clinical presentation with lymphoma. The prognosis for canine AML remains extremely guarded. Further studies are needed to optimize therapeutic regimens for dogs with AML.

Effects of prepartum dietary cation-anion difference on aspects of peripartum mineral and energy metabolism and performance of multiparous Holstein cows.

The objectives of this study were to determine the effect of decreasing dietary cation-anion difference [DCAD; (Na+ + K+) - (Cl- + S2-)] of the prepartum diet on aspects of mineral metabolism, energy metabolism, and performance of peripartum dairy cows. Multiparous Holstein cows (n = 89) were enrolled between 38 and 31 d before expected parturition and randomized to treatments in a completely randomized design (restricted to balance for previous 305-d mature equivalent milk production, parity, and body condition score) at 24 d before expected parturition. Treatments consisted of a low-K ration without anion supplementation [CON; n = 30, DCAD = +18.3 mEq/100 g of dry matter (DM)]; partial anion supplementation to a low-K ration (MED; n = 30, DCAD = +5.9 mEq/100 g of DM); and anion supplementation to a low-K ration to reach a targeted average urine pH between 5.5 and 6.0 (LOW; n = 29, DCAD = -7.4 mEq/100 g of DM). Cows were fed a common postpartum diet and data collected through 63 d in milk. Urine pH (CON = 8.22, MED = 7.89, and LOW = 5.96) was affected quadratically by decreasing prepartum DCAD. A linear relationship between urine pH and urine Ca:creatinine ratio was observed (r = -0.81). Plasma Ca concentrations in the postpartum period (d 0 to 14; CON = 2.16, MED = 2.19, and LOW = 2.27 mmol/L) were increased linearly with decreasing prepartum DCAD. A treatment by parity (second vs. third and greater) interaction for postpartum plasma Ca concentration suggested that older cows had the greatest response to the low DCAD diet and older cows fed LOW had decreased prevalence of hypocalcemia after calving. A quadratic effect of decreasing DCAD on prepartum DMI was observed (CON = 13.6, MED = 14.0, and LOW = 13.2 kg/d). Milk production in the first 3 wk postpartum was increased linearly with decreasing DCAD (CON = 40.8, MED = 42.4, and LOW = 43.9 kg/d) and DMI in this period also tended to linearly increase (CON = 20.2, MED = 20.9, and LOW = 21.3 kg/d). Overall, effects on intake and milk yield analyzed over wk 1 to 9 postpartum were not significant. This study demonstrates that feeding lower DCAD diets prepartum improves plasma Ca status in the immediate postpartum period and results in increased DMI and milk production in the 3 wk after parturition. Compared with no anion supplementation or lower levels of anion supplementation, greater improvements were observed with the lower DCAD feeding strategy, in which an average urine pH of 5.5 to 6.0 was targeted.

Risk factors associated with postpartum subclinical hypocalcemia in dairy cows.

Our study objectives were to evaluate the association of prepartum plasma Mg concentrations with subclinical hypocalcemia (SCH) classification at parturition and to evaluate the association of other cow-level risk factors with SCH classification at calving or at 2 d in milk (DIM). A total of 301 animals from 2 dairy herds located in New York were enrolled in a cohort study. Blood samples were collected at approximately 1 wk before the expected calving date, within 4 h of calving, and at 2 DIM. Prepartum samples had plasma macromineral concentrations (Ca, K, Mg, P), albumin, and ß-hydroxybutyrate analyzed. Samples collected at calving were analyzed for Ca only, and samples from 2 DIM had macromineral and albumin concentrations determined. Postpartum SCH was defined as Ca concentrations ≤2.1 mmol/L. The prevalence of SCH at calving was 2, 40, and 66% for first, second, and third or greater parities, respectively. Only 4% of cows could be classified with prepartum subclinical hypomagnesemia (Mg concentrations <0.8 mmol/L), which did not provide enough power to appropriately determine the association of plasma Mg with postpartum Ca concentrations and its effect on SCH classification. Multiparous cows with Ca concentrations ≤2.4 mmol/L in the prepartum period and third or greater parity cows had a higher risk of being categorized as SCH at calving [relative risk (RR) = 1.4 and 1.7, respectively]. The risk of SCH at 2 DIM was associated with the interaction of Ca status at calving and lameness score. Nonlame cows with Ca concentrations ≤2.1 mmol/L (RR = 3.2) and normocalcemic lame cows at parturition (RR = 3.4) were more likely to be SCH at 2 DIM compared with nonlame normocalcemic cows. In conclusion, we identified a prepartum Ca cut-point for identification of cows that are more likely to be classified as SCH at calving. Different risk factors were associated with SCH depending on the timing of diagnosis relative to parturition.

Holoprosencephaly and Pure Red Cell Aplasia in a Feline Leukaemia Virus-Positive Kitten.

A 9-month-old, female, domestic longhair cat with severe anaemia tested positive for feline leukaemia virus (FeLV) and was humanely destroyed and submitted for necropsy examination. Gross findings included a non-divided rostral telencephalon, consistent with semilobar holoprosencephaly. Histological examination of the bone marrow revealed an almost complete absence of erythroid precursor cells, consistent with pure red cell aplasia, and mild to moderate myelofibrosis. This case demonstrates a very unusual central nervous system defect, as well as an atypical presentation of pure red cell aplasia, in a FeLV-positive kitten.

HUS1 regulates in vivo responses to genotoxic chemotherapies.

Cells are under constant attack from genotoxins and rely on a multifaceted DNA damage response (DDR) network to maintain genomic integrity. Central to the DDR are the ATM and ATR kinases, which respond primarily to double-strand DNA breaks (DSBs) and replication stress, respectively. Optimal ATR signaling requires the RAD9A-RAD1-HUS1 (9-1-1) complex, a toroidal clamp that is loaded at damage sites and scaffolds signaling and repair factors. Whereas complete ATR pathway inactivation causes embryonic lethality, partial Hus1 impairment has been accomplished in adult mice using hypomorphic (Hus1(neo)) and null (Hus1(Δ1)) Hus1 alleles, and here we use this system to define the tissue- and cell type-specific actions of the HUS1-mediated DDR in vivo. Hus1(neo/Δ1) mice showed hypersensitivity to agents that cause replication stress, including the crosslinking agent mitomycin C (MMC) and the replication inhibitor hydroxyurea, but not the DSB inducer ionizing radiation. Analysis of tissue morphology, genomic instability, cell proliferation and apoptosis revealed that MMC treatment caused severe damage in highly replicating tissues of mice with partial Hus1 inactivation. The role of the 9-1-1 complex in responding to MMC was partially ATR-independent, as a HUS1 mutant that was proficient for ATR-induced checkpoint kinase 1 phosphorylation nevertheless conferred MMC hypersensitivity. To assess the interplay between the ATM and ATR pathways in responding to replication stress in vivo, we used Hus1/Atm double mutant mice. Whereas Hus1(neo/neo) and Atm(-/-) single mutant mice survived low-dose MMC similar to wild-type controls, Hus1(neo/neo)Atm(-/-) double mutants showed striking MMC hypersensitivity, consistent with a model in which MMC exposure in the context of Hus1 dysfunction results in DSBs to which the ATM pathway normally responds. This improved understanding of the inter-dependency between two major DDR mechanisms during the response to a conventional chemotherapeutic illustrates how inhibition of checkpoint factors such as HUS1 may be effective for the treatment of ATM-deficient and other cancers.

Effect of crofelemer extract on severity and consistency of experimentally induced enterotoxigenic Escherichia coli diarrhea in newborn Holstein calves.

The objective of this study was to evaluate the effect of a standardized botanical extract of Croton lechleri, named crofelemer extract, on fecal dry matter and fecal scores on diarrheic newborn Holstein bull calves induced by enterotoxigenic Escherichia coli. A double-blinded randomized clinical trial was performed in which 60 newborn Holstein bull calves were clean caught and transported to an isolation facility where calves were individually housed and randomly assigned to 1 of 3 treatment groups: placebo (control), enteric-coated formulation of crofelemer extract (ECROF), and nonenteric-coated formulation of crofelemer extract (CROF). Diarrhea was induced at first feeding with an inoculum of the enterotoxigenic Escherichia coli (ATCC 31616) administered with a third of the recommended dose of a colostrum replacer. All calves enrolled in this study received treatments starting on the second feeding (diarrhea onset) and treatments were administered before feeding time (0600 and 1600h) for 6 feedings consecutively. All calves in this study had failure of passive transfer. The only cause of death in this study was due to septicemia, accounting for 1 death out of each treatment group. All the calves were examined twice daily, within 2h after feeding, from d 1 (prechallenge) until 10, on d 15, and a last examination on d 25 of life. Five parameters were evaluated during each examination; rectal temperature, clinical assessment of dehydration status, fecal scores, attitude, and appetite. No differences were observed between treatment groups for rectal temperature, attitude, and appetite. Fecal dry matter was analyzed as prechallenge fecal dry matter, dry matter during treatment, and fecal dry matter after treatment cessation. No difference in prechallenge fecal dry matter was observed and prechallenge fecal dry matter was used as a covariate in the models. Fecal dry matter during treatment was significantly higher for ECROF calves when compared with control calves and CROF calves. Additionally, ECROF fecal dry matter after treatment cessation had a statistical tendency to be higher when compared with control calves. Together, these results suggest that enteric-coated formulation of the standardized crofelemer extract, a natural-product with antisecretory properties, can significantly increase fecal dry matter of neonatal calves with experimentally induced enterotoxigenic Escherichia coli diarrhea. More research is needed to test the efficacy of enteric-coated crofelemer on incidence and severity of secretory diarrhea on calves under natural challenge conditions.

Stalled cerebral capillary blood flow in mouse models of essential thrombocythemia and polycythemia vera revealed by in vivo two-photon imaging.

BACKGROUND: Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) that share the JAK2(V617F) mutation in hematopoietic stem cells, leading to excessive production of predominantly platelets in ET, and predominantly red blood cells (RBCs) in PV. The major cause of morbidity and mortality in PV and ET is thrombosis, including cerebrovascular occlusive disease. OBJECTIVES: To identify the effect of excessive blood cells on cerebral microcirculation in ET and PV. METHODS: We used two-photon excited fluorescence microscopy to examine cerebral blood flow in transgenic mouse models that mimic MPNs. RESULTS AND CONCLUSIONS: We found that flow was 'stalled' in an elevated fraction of brain capillaries in ET (18%), PV (27%), mixed MPN (14%) and secondary (non-MPN) erythrocytosis (27%) mice, as compared with controls (3%). The fraction of capillaries with stalled flow increased when the hematocrit value exceeded 55% in PV mice, and the majority of stalled vessels contained only stationary RBCs. In contrast, the majority of stalls in ET mice were caused by platelet aggregates. Stalls had a median persistence time of 0.5 and 1 h in ET and PV mice, respectively. Our findings shed new light on potential mechanisms of neurological problems in patients with MPNs.

Hematopoiesis in the equine fetal liver suggests immune preparedness.

We investigated how the equine fetus prepares its pre-immune humoral repertoire for an imminent exposure to pathogens in the neonatal period, particularly how the primary hematopoietic organs are equipped to support B cell hematopoiesis and immunoglobulin (Ig) diversity. We demonstrated that the liver and the bone marrow at approximately 100 days of gestation (DG) are active sites of hematopoiesis based on the expression of signature messenger RNA (mRNA) (c-KIT, CD34, IL7R, CXCL12, IRF8, PU.1, PAX5, NOTCH1, GATA1, CEBPA) and protein markers (CD34, CD19, IgM, CD3, CD4, CD5, CD8, CD11b, CD172A) of hematopoietic development and leukocyte differentiation molecules, respectively. To verify Ig diversity achieved during the production of B cells, V(D)J segments were sequenced in primary lymphoid organs of the equine fetus and adult horse, revealing that similar heavy chain VDJ segments and CDR3 lengths were most frequently used independent of life stage. In contrast, different lambda light chain segments were predominant in equine fetal compared to adult stage, and surprisingly, the fetus had less restricted use of variable gene segments to construct the lambda chain. Fetal Igs also contained elements of sequence diversity, albeit to a smaller degree than that of the adult horse. Our data suggest that the B cells produced in the liver and bone marrow of the equine fetus generate a wide repertoire of pre-immune Igs for protection, and the more diverse use of different lambda variable gene segments in fetal life may provide the neonate an opportunity to respond to a wider range of antigens at birth.

Acute pancreatitis in two five-day-old Appaloosa foals.

Severe acute pancreatitis (SAP) in foals is rare and treatment has not been documented. This paper describes the clinical, haematological and ultrasonographic findings as well as attempted treatment of SAP in two 5-day-old Appaloosa fillies. Clinical signs, including colic, diarrhoea and coma, may be mistaken for sepsis or neonatal encephalopathy. Hyperlipaemic serum and peritoneal fluid, and elevated serum and peritoneal fluid amylase and lipase activities aided the diagnosis. Severe acute pancreatitis should be included as a differential in an acutely ill foal with diarrhoea, colic, cerebral cortica dysfunction and hyperlipaemia.

Distribution of the feline calicivirus receptor junctional adhesion molecule a in feline tissues.

Junctional adhesion molecule A (JAM-A) is an immunoglobulin superfamily protein that plays an important role in the assembly and maintenance of tight junctions and the establishment of epithelial cell polarity. The feline JAM-A (fJAM-A) is a functional receptor for feline calicivirus (FCV). Among natural diseases associated with FCV infection, isolates that cause oral vesicular disease are detected in epithelial cells; however, isolates that cause systemic disease are detected in multiple cell types. The distribution of an FCV receptor or receptors in feline tissues is relevant to viral pathogenesis in that it should reflect the wide latitude of clinical sequelae associated with FCV infection. The authors examined the expression of feline JAM-A in the cat by using confocal immunofluorescence localization on normal tissues, with special regard to tissue targets of naturally occurring FCV. As described in the human and the mouse, fJAM-A was widely distributed in feline tissues, where it localized at cell-cell junctions of epithelial and endothelial cells. fJAM-A was highly expressed on feline platelets, with lower levels of expression on feline peripheral blood leukocytes. Additionally, FCV infection of a feline epithelial cell monolayer causes redistribution of fJAM-A to the cytosol of infected cells. It is reasonable to propose that the spectrum of lesions caused by FCV reflects disruption of intercellular junctions that rely on fJAM-A function and tight junctional integrity.

Transferrin receptor expression in canine lymphoma.

Transferrin receptor 1 (TfR1) expression was measured by immunohistochemistry in 78 archived cases of canine B-cell and T-cell lymphoma with an anti-human TfR1 monoclonal antibody that was validated in the dog by immunohistochemistry and immunoblotting. The canine lymphoma cases were initially classified on the basis of World Health Organization criteria and then subdivided into the following 4 subgroups: low-grade B-cell (LGB), high-grade B-cell (HGB), low-grade T-cell (LGT), and high-grade T-cell (HGT). A visual scoring system and densitometric analysis of the proportion and intensity of positive staining were used to quantify TfR1 expression. TfR1 expression was also correlated to mitotic rate. TfR1 expression was significantly lower in the LGT tumors compared to all other lymphoma subgroups (LGB, HGB, and HGT). LGB tumors showed a TfR1 expression similar to those of the high-grade tumors (HGB and HGT). Significant correlations were found between mitotic rate and densitometric TfR1 variables in the T-cell tumors but not in the B-cell tumors. Further studies are needed to investigate the underlying molecular basis of the high TfR1 expression in LGB lymphomas and its pathological relevance. The anti-human TfR1 monoclonal antibody is a useful tool for measurement of total cellular transferrin receptor expression in the dog; however, an antibody with specificity for the canine TfR1 ectodomain is needed to investigate the potential of this receptor as an oncolytic target.

Association between the proportion of sampled transition cows with increased nonesterified fatty acids and beta-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level.

In this study the herd alarm level was defined as the proportion of sampled transition cows per herd with increased prepartum nonesterified fatty acid (NEFA), postpartum beta-hydroxybutyrate (BHBA), or NEFA concentrations that were associated with herd-level incidence of displaced abomasum (DA) or clinical ketosis (CK), pregnancy rate (PR), and milk production. The objectives were to 1) identify the herd alarm level for excessive negative energy balance and 2) describe the herd-level prevalence of this proportion. This was a prospective cohort study of 60 free-stall herds fed total mixed rations in the northeast United States. Two cohorts of approximately 15 animals were assessed for prepartum NEFA and postpartum BHBA and NEFA. The herd alarm level (i.e., the proportion of sampled animals above a certain metabolite threshold) was as follows: 15% had prepartum NEFA of 0.27 mEq/L; 15 and 20% had BHBA of 10 and 12 mg/dL, respectively; and 15% had postpartum NEFA of 0.60 and 0.70 mEq/L. The different herd alarm levels correspond to differences between the metabolites and respective herd-level effect. The herd-level effects for herds above the herd alarm level for prepartum NEFA were 3.6% increase in DA and CK incidence, 1.2% decrease in PR, and 282 kg decrease in average mature equivalent 305-d (ME 305) milk. For BHBA, the herd-level effects were a 1.8% increase in DA and CK, 0.8% decrease in PR, and 534 and 358 kg decrease in projected ME 305 milk yield for heifers and cows, respectively. For postpartum NEFA, the herd-level effects were 1.7% increase in DA and CK, 0.9% decrease in PR, and 288 and 593 kg decrease in projected ME 305 milk yield for heifers and cows, respectively. The prevalence of herds in which more than 15% of animals sampled had prepartum NEFA concentration >or=0.30 mEq/L was 75%, BHBA >or=12 mg/dL was 40%, and postpartum NEFA >or=0.70 mEq/L was 65%. This study showed that there were detrimental herd-level effects if a large enough proportion of cows had increased metabolite concentrations, and further demonstrated that a high prevalence of herds have opportunity for improvement.

Associations of elevated nonesterified fatty acids and beta-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.

The objectives were to evaluate the effects of elevated pre- and postpartum nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) concentrations during the transition period on reproductive performance and milk production in dairy cattle. In a prospective cohort study of 91 freestall, total mixed ration-fed herds in the northeastern United States, blood samples were collected from approximately 15 prepartum and 15 different postpartum transition animals in each herd. All samples were stratified based on pre- or postpartum status at the time of sample collection, and 2,259 and 2,290 animals were used to evaluate reproductive and milk production performance, respectively. Reproductive performance was assessed by time to conception within 70 d post-voluntary waiting period (VWP) and milk production was assessed using mature-equivalent 305-d (ME305) milk yield estimated at 120 d in milk. While controlling for body condition score (BCS), calving season, median ME305 milk production, and parity, NEFA and BHBA concentrations were evaluated with time to event analysis to investigate reproductive performance. These same predictor variables were used to determine the effects of elevated NEFA and BHBA concentrations on ME305 milk yield with herd as a random effect. Heifers and cows were grouped in the final analyses if the results between groups were similar. In all animals sampled prepartum, the risk of pregnancy within 70 d post-VWP was reduced by 19% when NEFA concentrations were >or=0.27 mEq/L. In all animals sampled postpartum, those with NEFA concentrations >or=0.72 mEq/L had a 16% decrease in risk of pregnancy and those with BHBA concentrations >or=10mg/dL had a 13% decrease in risk. In cows and heifers, ME305 milk yield was decreased by 683 kg when prepartum NEFA concentrations were >or=0.33 mEq/L. In heifers sampled postpartum, ME305 milk yield was increased by 488 kg when NEFA concentrations were >or=0.57 mEq/L and increased by 403 kg when BHBA concentrations were >or=9 mg/dL. In cows sampled postpartum, ME305 milk yield was decreased by 647 kg when NEFA concentrations were >or=0.72 mEq/L and decreased by 393 kg when BHBA concentrations were >or=10mg/dL. With the exception of milk production in heifers, this study indicates that increased concentrations of serum NEFA and BHBA had a detrimental effect on reproductive performance and milk production.

Evaluation of nonesterified fatty acids and beta-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.

The objectives of this study were to 1) establish cow-level critical thresholds for serum concentrations of nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) to predict periparturient diseases [displaced abomasa (DA), clinical ketosis (CK), metritis and retained placenta, or any of these three], and 2) investigate the magnitude of the metabolites' association with these diseases within 30 d in milk. In a prospective cohort study of 100 freestall, total mixed ration-fed herds in the northeastern United States, blood samples were collected from approximately 15 prepartum and 15 different postpartum transition animals in each herd, for a total of 2,758 samples. Serum NEFA concentrations were measured in the prepartum group, and both NEFA and BHBA were measured in the postpartum group. The critical thresholds for NEFA or BHBA were evaluated with receiver operator characteristic analysis for all diseases in both cohorts. The risk ratios (RR) of a disease outcome given NEFA or BHBA concentrations and other covariates were modeled with multivariable regression techniques, accounting for clustering of cows within herds. The NEFA critical threshold that predicted any of the 3 diseases in the prepartum cohort was 0.29mEq/L and in the postpartum cohort was 0.57mEq/L. The critical threshold for serum BHBA in the postpartum cohort was 10mg/dL, which predicted any of the 3 diseases. All RR with NEFA as a predictor of disease were >1.8; however, RR were greatest in animals sampled postpartum (e.g., RR for DA=9.7; 95% CI=4.2 to 22.4. All RR with BHBA as the predictor of disease were >2.3 (e.g., RR for DA=6.9; 95% CI=3.7 to 12.9). Although prepartum NEFA and postpartum BHBA were both significantly associated with development of clinical disease, postpartum serum NEFA concentration was most associated with the risk of developing DA, CK, metritis, or retained placenta during the first 30 d in milk.

Hypercoagulability in cats with cardiomyopathy.

BACKGROUND: Arterial thromboembolism (ATE) is a common complication of feline cardiomyopathy; however, the pathogenesis of ATE is unknown. HYPOTHESIS: Systemic activation of the coagulation cascade (hypercoagulability) and endothelial injury promote ATE in cardiomyopathic cats. ANIMALS: Healthy cats (n = 30) and 3 groups of cardiomyopathic cats: Group (1) left atrial enlargement only (LAE [n = 11]), ie, left atrial to aortic ratio >1.4; Group (2) LAE with spontaneous echocardiographic contrast, atrial thrombi or both (SEC-T [n = 16]); and Group (3) acute ATE with LAE (n = 16). METHODS: Hypercoagulability was defined by 2 or more laboratory abnormalities reflecting coagulation factor excess (high fibrinogen concentration or Factor VIII coagulant activity), inhibitor deficiency (low antithrombin activity), or thrombin generation (high thrombin-antithrombin complex [TAT] and d-dimer concentrations). High von Willebrand factor antigen concentration (vWF : Ag) was considered a marker of endothelial injury. Data were analyzed using nonparametric statistics. RESULTS: The 3 groups of cats with cardiac disease had higher median fibrinogen concentrations than did the healthy cats. Criteria of hypercoagulability were found exclusively in cats with SEC-T (50%) and ATE (56%). Hypercoagulability was not associated with left atrial size or congestive heart failure (CHF). ATE cats had significantly higher median vWF : Ag concentration than did the other groups. CONCLUSION AND CLINICAL IMPORTANCE: Systemic hypercoagulability is evident in many cardiomyopathic cats, often without concurrent CHF or overt ATE. Hypercoagulabilty may represent a risk factor for ATE. High vWF : Ag in ATE cats was attributed to downstream endothelial injury from the occlusive thrombus.

Effect of anticoagulant and storage conditions on bovine nonesterified fatty acid and beta-hydroxybutyrate concentrations in blood.

The objective of this study was to determine if nonesterified fatty acid (NEFA) and beta-hydroxybutyrate (BHBA) concentrations were affected by anticoagulants or gel and clot activator tubes (serum separator tubes, SST), storage of samples as whole blood, separated plasma or serum at 24 degrees C or 4 degrees C for 24 to 72 h, or storage as serum at -40 degrees C for 1 mo. Blood was collected from dairy cows into EDTA, heparin, nonanticoagulant tubes, and SST, and analyzed immediately to obtain baseline NEFA and BHBA concentrations. Portions were stored as whole blood or separated plasma or serum at 4 or 24 degrees C and assayed daily for 24 (whole blood) and 72 (separated samples) h. Serum samples were frozen at -40 degrees C and assayed at 24 h or weekly for 1 mo. Baseline NEFA concentrations were unaffected by anticoagulants; however, they were significantly higher in SST compared with nonanticoagulant tubes. Concentrations of NEFA were stable in all samples at 4 degrees C, whereas they sequentially increased from 24 to 48 h at 24 degrees C. Changes were more dramatic in heparinized samples. Serum could be stored frozen for up to 1 mo with minimal changes in NEFA concentrations. Concentrations of BHBA were stable under all conditions evaluated. Our results indicated that blood for NEFA testing should be collected into EDTA or nonanticoagulant tubes (but not SST), separated promptly from cells, and maintained at 4 degrees C until analysis.

Effect of citrate concentration on coagulation test results in dogs.

OBJECTIVE: To determine the effect of citrate concentration (3.2 vs 3.8%) on coagulation tests in dogs. DESIGN: Original study. ANIMALS: 30 clinically healthy dogs and 12 dogs with hereditary hemostatic disorders. PROCEDURE: Blood was collected from all dogs directly into collection tubes containing 3.2 or 3.8% buffered citrate. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration were measured by use of 3 clot-detection assay systems (2 mechanical and 1 photo-optic). Factor VIII and factor IX coagulant activities (FVIII:C and FIX:C, respectively) were determined by use of a manual tilt-tube method and a mechanical clot-detection device. RESULTS: Significant differences were not detected in median PT, fibrinogen concentration, FVIII:C, or FIX:C between 3.2 and 3.8% citrate for any assay system. A significant prolongation in aPTT for 3.2% citrate, compared with 3.8% citrate, was found in 1 mechanical system. CONCLUSIONS AND CLINICAL RELEVANCE: Citrate concentration does not significantly affect results of most coagulation assays, regardless of assay system. The aPTT was mildly influenced by the citrate concentration, although this was animal-, instrument-, and reagent-dependent. The choice of 3.2 or 3.8% citrate as an anticoagulant for coagulation tests has minimal influence on assay results in healthy dogs or dogs with hereditary hemostatic disorders.